A new strain of Akkermansia muciniphila and its application

Abstract

The present invention discloses a novel strain of Akkermansia muciniphila and its application, and relates to the field of microbial technology. The Akkermansia muciniphila is AKK ONE, with a deposit number of CGMCC No. 20954, and was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on October 26, 2020. The strain has good tolerance to gastric juice and intestinal juice, weak biofilm formation ability, and no mutagenicity. It has good safety, plays an important role in improving enteritis, regulating lipid metabolism, improving diabetes, anti-infection, and improving respiratory diseases, and has broad application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a novel Akkermansia muciniphila and application thereof. Background Art

[0002] Akkermansia muciniphila belongs to the phylum Verrucomicrobia, class Verrucomicrobiae, and order Verrucomicrobiales. It is an abundant intestinal bacterium that can use mucin as its only carbon, nitrogen, and energy source. Akkermansia muciniphila is an oval, strictly anaerobic Gram-negative bacterium. It is a representative of the phylum Verrucomicrobia and the only representative and in vitro cultivable member of the phylum Verrucomicrobia in the mammalian intestine. It can release amino acids or monosaccharides while degrading host mucin, produce short-chain fatty acids, and provide nutrition for the symbiotic flora in the intestine. While degrading mucin, Akkermansia muciniphila can also stimulate the production of mucin and increase the thickness of the mucus layer, further enhancing the integrity of the intestinal epithelium and reducing the leakage of lipopolysaccharide into the circulation.

[0003] Akkermansia muciniphila was originally isolated from healthy human feces and is an important member of the intestinal microbiota, accounting for 1%-5% of the total microbiota. It has the function of mediating key physiological activities such as energy metabolism and nutritional homeostasis, immune response, and brain activity of the host body. Akkermansia muciniphila is closely related to metabolic syndromes such as obesity, diabetes, cardiovascular disease, chronic low-grade inflammatory response, and enteritis, and has a certain effect on improving diseases and improving the health of the body.

[0004] Chinese patent CN117100768A discloses the use of Akkermansia muciniphila in improving and treating diarrhea. The Akkermansia muciniphila of the invention is selected from at least one of Akkermansia muciniphila AM06 with a preservation number of CGMCC No. 22793, Akkermansia muciniphila AM02 with a preservation number of CGMCC No. 22794, and Akkermansia muciniphila ATCC BAA-835. It has the effect of improving and treating diarrhea, has a good effect on improving infectious diarrhea or non-infectious diarrhea, has no side effects on the body, and has a good prospect for consumption and application.

[0005] The therapeutic effects of Akkermansia muciniphila in the prior art on diseases are relatively single, and there is much room for improvement in the therapeutic effect. Screening out a strain with high safety and good therapeutic effects on multiple diseases will bring certain economic and social benefits and have broad application prospects.

[0006] The present invention is the downstream application technology development carried out by the inventor for the independently developed microbial resources: Akkermansia muciniphila with the deposit number of CGMCC No.20954 (other patents have been submitted on the same day). After obtaining Akkermansia muciniphila, the inventor conducted application experiments in multiple fields including diabetes, infection, respiratory diseases, metabolic diseases, inflammatory diseases, etc. and obtained positive experimental results in some fields. Taking into account the relevant provisions of the unity of the patent law, protection is requested separately for each indication that does not have unity. The present invention is for the protection of Akkermansiamuciniphila strains. Summary of the invention

[0007] The invention aims to provide a novel Akkermansia muciniphila, whose deposit number is CGMCC No.20954. The strain has high tolerance, good safety and wide indications.

[0008] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0009] In one aspect, the present invention provides an Akkermansia muciniphila, with a deposit number of CGMCC No. 20954. Specifically, the Akkermansia muciniphila is AKK ONE.

[0010] Specifically, the 16S sequence of Akkermansia muciniphila is shown in SEQ ID NO:1.

[0011] Specifically, the Akkermansia muciniphila was cultured anaerobically in BHI medium at 37° C. for 7 days, and the colonies were light yellow, round, moist, opaque, and had neat edges.

[0012] Specifically, the Akkermansia muciniphila is cultured anaerobically at 37° C. for 3 days in a reinforced clostridial medium, and the bacteria are rod-shaped, 0.6-0.8 μm×0.8-4.1 μm, arranged singly or in pairs, and are Gram-negative.

[0013] In another aspect, the present invention provides a method for culturing Akkermansia muciniphila, wherein the method comprises inoculating Akkermansia muciniphila into a culture medium for culturing. Specifically, the inoculation amount may be 0.1%-20%.

[0014] Specifically, the culture medium is selected from any one of a solid culture medium, a semi-solid culture medium or a liquid culture medium. Further, the culture medium is a BHI culture medium, including but not limited to a BHI solid culture medium and a BHI liquid culture medium. Further, the BHI culture medium includes but is not limited to: peptone, dehydrated calf brain extract powder, dehydrated ox heart extract powder, glucose, sodium chloride and disodium hydrogen phosphate. Further, the pH of the BHI culture medium is 7.0-8.0; further, the pH is 7.4±0.2.

[0015] According to some embodiments of the present invention, the formula of the BHI medium is: 10 g / L peptone, 12.5 g / L dehydrated calf brain extract powder, 5.0 g / L dehydrated buffalo heart extract powder, 2.0 g / L glucose, 5.0 g / L sodium chloride and 2.5 g / L disodium hydrogen phosphate.

[0016] The Akkermansia muciniphila culture temperature of the present invention is 35-42° C., and further, the culture temperature is 37° C. The Akkermansia muciniphila culture conditions of the present invention are anaerobic culture, and the oxygen content in the culture conditions can be 0-8%.

[0017] In yet another aspect, the present invention provides a preparation of Akkermansia muciniphila.

[0018] Specifically, the preparation includes but is not limited to the fermentation broth of Akkermansia muciniphila, the fermentation broth precipitate of Akkermansia muciniphila, the fermentation broth supernatant of Akkermansia muciniphila, the live bacteria of Akkermansia muciniphila, the dead bacteria of Akkermansia muciniphila, the lyophilized powder of Akkermansia muciniphila, and the lysate of Akkermansia muciniphila.

[0019] Furthermore, the preparation can be prepared by the culture obtained by the above-mentioned culture method. The Akkermansia muciniphila fermentation broth can be obtained by the above-mentioned culture method. The Akkermansia muciniphila fermentation broth precipitate and the Akkermansia muciniphila fermentation broth supernatant are obtained by centrifuging the above-mentioned fermentation broth. The live Akkermansia muciniphila bacteria can obtain living cells by centrifuging, filtering or other means of the culture. The dead Akkermansia muciniphila bacteria are inactivated bacteria. The Akkermansia muciniphila freeze-dried powder is obtained by freeze-drying the above-mentioned culture broth. The Akkermansia muciniphila cell lysate is obtained by lysing the bacteria obtained by culturing the above-mentioned culture, and the lysis includes physical lysis and chemical lysis.

[0020] In another aspect, the present invention provides the use of the above-mentioned Akkermansia muciniphila or the above-mentioned preparation in the preparation of food, medicine, health care product, bacterial agent, food additive, health care product additive.

[0021] Specifically, the medicine is used to treat, prevent or assist in the treatment of diseases such as diabetes, infection, respiratory disease, abnormal lipid metabolism, inflammation, or complications of the above diseases.

[0022] Furthermore, the diabetes includes type 1 diabetes and type 2 diabetes.

[0023] Furthermore, the infection includes latent infection, overt infection and bloody state.

[0024] Furthermore, the respiratory diseases include but are not limited to acute upper respiratory tract infection, influenza, obstructive sleep apnea-hypopnea syndrome, chronic obstructive pulmonary disease, bronchial asthma, bronchiectasis, acute bronchitis, pneumonia, tuberculosis, pulmonary fibrosis, tuberculous pleurisy, bacterial pleurisy, malignant pleural effusion, and pulmonary embolism.

[0025] Furthermore, the abnormal lipid metabolism includes, but is not limited to, hyperlipoproteinemia, lipid storage disease, obesity, fatty liver, ketosis, scleredema neonatorum, and proteinopenia.

[0026] Furthermore, the inflammation includes but is not limited to otitis media, pneumonia, hepatitis, nephritis, stomatitis, rhinitis, pelvic inflammatory disease, cervicitis, arthritis, gastroenteritis, fasciitis, cystitis, conjunctivitis, and tracheitis.

[0027] Furthermore, the health care product is used for maintaining blood lipids, maintaining blood sugar, maintaining blood pressure, anti-oxidation, losing weight, regulating intestinal flora growth, and protecting memory.

[0028] In another aspect, the present invention provides a product comprising the Akkermansia muciniphila or the preparation thereof. Further, the product includes but is not limited to food, medicine, health care product, bacterial agent, food additive, health care product additive.

[0029] Specifically, when the product is a medicine, the medicine also includes pharmaceutically acceptable excipients.

[0030] Furthermore, the pharmaceutically acceptable excipients include, but are not limited to, excipients, stabilizers, diluents, adhesives, preservatives, lubricants, and antioxidants.

[0031] Furthermore, the dosage form of the medicine is tablets, powders, granules, capsules, oral liquids, powders, pills, syrups, effervescents, emulsions, pastes, sprays, liniments, suppositories, lotions, foams, and gels.

[0032] Preferably, the viable count of Akkermansia muciniphila in the product is 1×10 3 -1×10 14 CFU / g or 1×10 3 -1×10 14 CFU / mL; the number of dead Akkermansia muciniphila in the product is 1×10 3 -1×10 14 TFU / g or 1×10 3 -1×10 14 TFU / mL.

[0033] Furthermore, 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; the number of dead Akkermansia muciniphila in the drug is 1×10 3 -1×10 14 TFU / g or 1×10 3 -1×10 14 TFU / mL.

[0034] The beneficial effects of the present invention are:

[0035] The present invention provides a novel strain of Akkermansia muciniphila, AKKONE, wherein the hydrophobicity of the strain reaches more than 30% at 60min, the self-aggregation tends to be stable at 20h, and is maintained at about 52%. The strain has good tolerance to gastric juice and intestinal juice, weak biofilm formation ability, and the strain has no mutagenicity and good safety. The feasibility of clinical application of AKK ONE of the present invention is shown in the DSS-induced mouse colitis model. Related physiological abnormalities (food intake, body weight and colon length) can be improved. AKK ONE can improve the blood lipid level of hypercholesterolemia mice, significantly reduce the total TC and LDL-C content of serum and liver of hypercholesterolemia mice, increase HDL-C content, improve liver damage, and improve fasting blood sugar level and glucose tolerance. AKK ONE can significantly reduce the number of coughs in mice, downregulate the content of pro-inflammatory factors IL-6 and IL-8 in mouse serum, and upregulate the content of anti-inflammatory factor IL-10. AKK ONE significantly reduced the levels of IL-8 and TNF-α in the serum of mice infected with Helicobacter pylori.

[0036] Collection Instructions

[0037] Strain name: AKK ONE;

[0038] Taxonomic nomenclature: Akkermansia muciniphila;

[0039] Deposit number: CGMCC No.20954;

[0040] Deposit date: October 26, 2020;

[0041] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0042] Address of the preservation unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In the attached figure, the names of each group refer to the following meanings:

[0044] Saline group was normal saline group, Low dosage group was low dosage AKK ONE (1×10 9 CFU / day), Medium dosage is medium dose AKK ONE (5×10 10 CFU / day), High dosage is high dosage AKK ONE (5×10 11 CFU / day).

[0045] Figure 1The colony morphology (top) and microscopic morphology (bottom) of AKK ONE.

[0046] Figure 2 This is the phylogenetic tree of AKK ONE (neighbor joining method, NJ method).

[0047] Figure 3 This is the hydrophobicity verification result of AKK ONE.

[0048] Figure 4 This is the self-aggregation verification result of AKK ONE.

[0049] Figure 5 is the survival rate of AKK ONE in artificial gastric fluid.

[0050] Figure 6 is the survival rate of AKK ONE in artificial intestinal fluid.

[0051] Figure 7 These are the results of changes in mouse body weight, food intake, and blood sugar in the acute toxicity experiment.

[0052] Figure 8 These are the test results of triglyceride (TG), cholesterol (TC) and bile acid (TBA) content in mice in acute toxicity experiments.

[0053] Fig. 9 These are pathological sections of mouse liver and kidney in acute toxicity experiments.

[0054] Fig.10 The activity of alanine aminotransferase in mouse liver and serum was used for subchronic toxicity test.

[0055] Fig.11 The activity of aspartate aminotransferase in mouse liver and serum was used for subchronic toxicity test.

[0056] Fig.12 The effect of AKK ONE on blood glucose homeostasis in mice, where A is the fasting blood glucose level (mM); B is the glucose content (mM); C is the area under the curve of glucose tolerance (mM 120min).

[0057] Fig.13 The figure shows the effect of AKK ONE on the liver lipid level of mice, where A is the total TC in the liver (mmol / g); B is the total TG in the liver (mmol / g); C is the LDL-C in the liver (mmol / g); and D is the HDL-C in the serum (mmol / g).

[0058] Fig.14The effect of AKK ONE on mouse liver degeneration, where A is the color of mouse liver; B is the pathological section of mouse liver tissue.

[0059] Fig.15 The results of AKK ONE alleviating physiological damage in colitis mice. B is the statistical result of food intake. C is the statistical result of body weight. D is the statistical result of colon length. E is the disease activity index (DAI) score. F and G are liver index and spleen index, respectively. The data are the mean ± standard deviation of 8 independent samples. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0060] Fig.16 The number of coughs of mice within 5 minutes, where the same letters indicate no significant difference, and different letters indicate significant difference.

[0061] Fig.17 is the content of TNF-α in serum.

[0062] Fig.18 This is the blood glucose change curve of mice OGTT.

[0063] Fig.19 The effect of AKK ONE on insulin in T2DM mice.

[0064] Fig. 20 The effect of AKK ONE on serum AST and ALT in T2DM mice, where A is the AST content in serum; B is the ALT content in serum.

[0065] Fig.21 This is a histopathological section of mouse liver.

[0066] Fig. 22 This is a histopathological section of mouse pancreas. DETAILED DESCRIPTION

[0067] Example 1 Isolation and Identification of Akkermansia muciniphila ONE (AKK ONE)

[0068] 1. Isolation of strains

[0069] 1. Reagent preparation

[0070] The mucin culture medium is prepared by solution 1 and solution 2, wherein: solution 1: calcium chloride 1.1 g, magnesium chloride 1.0 g, disodium hydrogen phosphate 5.3 g, sodium chloride 3 g, dipotassium hydrogen phosphate 4 g, sodium bicarbonate 2 g, sodium sulfide 2.5 g, dilute to 100 mL, and sterilize by high pressure steam for later use;

[0071] Solution 2: Mucin (Sigma, Product No. M1778, CAS No. 84082-64-4) solution.

[0072] Preparation of mucin liquid culture medium: Take 200 mL of sterile distilled water and put it in a high-pressure wide-mouth bottle. Add 2 mL of solution 1 and 30-40 mL of solution 2 in an ultra-clean workbench. Use an electric pipette to draw 5 mL of liquid and divide it into 15 mL centrifuge tubes. Loosen the tube cap and place it in an anaerobic jar for 3 days, then tighten the tube cap and store it at 4°C.

[0073] Preparation of mucin solid culture medium: Add 20g / L agar powder to the mucin liquid culture medium; pour into sterile plates, 20mL per plate; after cooling and solidification, place in a sterile plastic bag and store in a sealed container at 4°C.

[0074] Pour the solid culture medium of Brain Heart Infusion (BHI, OXOID, Catalog No. CM1032B) into a sterile plate, place it in a sterile plastic bag, and store it at 4°C in a sealed state.

[0075] 2. Sample dilution and enrichment culture

[0076] Collect 1-2 g of fecal specimens from healthy individuals and immediately inoculate them into 9 mL of saline solution, which is recorded as 10 -1 ; Continue to dilute down 5 gradients, 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 Inoculate mucin liquid culture medium in the same way, inoculate 2 tubes for each gradient, and place the enrichment tubes in an anaerobic constant temperature incubator at 37°C for 3 days.

[0077] 3. Solid plate separation and culture

[0078] Select the turbid enrichment tube; select the relatively uniform, suspended, and free of precipitation, agglomeration, or filamentous material enrichment tube from the turbid liquid tube for separation and culture of the solid plate, and 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.

[0079] 2. Physiological and biochemical characteristics

[0080] Physiological and biochemical identification was carried out using VITEK ANC identification card (purchased from BioMérieux China, item number 21347). The identification results are as follows:

[0081] Table 1.

[0082]

[0083]

[0084] Note: “+”, positive; “-”, negative.

[0085] The 16S rDNA sequence of AKK ONE is SEQ ID NO: 1. After comparison with NCBI, the strain was classified and named Akkermansia muciniphila. It was deposited in the General Microbiology Center of China Microbiological Culture Collection on October 26, 2020, with the deposit number CGMCC No. 20954. The comparison results with some existing strains are as follows:

[0086] Table 2.

[0087]

[0088] Phylogenetic tree of 16S rDNA sequences of AKK ONE and related species Figure 2 The MEGA software was used to display the phylogenetic tree of 16S rDNA sequences of "AKK ONE" and related species by the neighbor joining method. The similarity was calculated 1000 times. Only the Bootstrap values ​​greater than 50% were displayed in the developmental tree nodes in the figure. The superscript "T" indicates the model strain.

[0089] AKK ONE was cultured anaerobically in BHI medium at 37℃ for 7 days. The colonies were light yellow, round, moist, opaque, and had neat edges. AKK ONE was cultured anaerobically in enhanced Clostridium medium at 37℃ for 3 days. The bacteria were rod-shaped, 0.6-0.8μm×0.8-4.1μm, arranged singly or in pairs, and were Gram-negative ( Figure 1 ).

[0090] Example 2 Safety Evaluation of Akkermansia muciniphila ONE (AKK ONE)

[0091] (1) Hydrophobicity

[0092] The bacterial adhesion test to hydrocarbons (BATH) was performed using xylene with some modifications. Specifically, the strains were 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 (OD600nm) was adjusted to 0.5 to standardize the number of bacteria. Then, an equal volume of xylene was added and vortexed for 5min to mix the two-phase system thoroughly. 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:

[0093]

[0094] Where P0 and P1 are the absorbance values ​​before and after extraction with hydrocarbons, respectively.

[0095] The results are as follows Figure 3 , the hydrophobicity of AKK ONE increased with time and reached more than 30% at 60 min.

[0096] (2) Self-aggregation

[0097] A cell suspension of strain AKK ONE was prepared as described above and incubated at 37°C. The absorbance (OD600nm) was adjusted to 0.5. The bacterial suspension was then cultured at 37°C and its absorbance at 600nm was measured at 4, 8, 12, 16, 20 and 24 hours. The percentage of self-aggregation was calculated using the following formula:

[0098]

[0099] The results are as follows Figure 4 , the self-aggregation of AKK ONE increased with time and stabilized at around 50% at 20 h.

[0100] (3) Gastrointestinal fluid tolerance

[0101] Prepare gastric juice (5M HCl, 2g / L NaCl, 3.2g / L pepsin, pH 2.0) and intestinal juice (6.8g / L KH2PO4, 10g / L pancreatin, 0.1M NaOH, pH 8.0), and then filter and sterilize them using a 0.22μm sterile filter. Centrifuge AKK ONE, which has been activated twice in 5 mL of BHI liquid medium, at 4000 rpm for 10 min to collect the bacterial cells, wash them twice with sterile normal saline, and resuspend them in 5 mL of normal saline. Mix the bacterial suspension with sterile artificial gastric juice or intestinal juice at a ratio of 1:1 (v / v), shake well, and place it in an incubator at 37°C for cultivation. Determine the viable cell count at 0 h and 3 h respectively, and calculate the survival rate of AKK ONE in artificial gastric juice or intestinal juice according to the following formula.

[0102]

[0103] The results are as Figure 5-Figure 6 shown. The survival rate of AKK ONE in gastric juice shows a downward trend with the extension of time, and the survival rate is about 80% at 240 min. The survival rate of AKK ONE in intestinal juice generally shows a downward trend, and the survival rate is above 80% at 240 min.

[0104] (4) Biofilm formation ability

[0105] AKK ONE is cultured overnight in tryptone soy broth (TSB, Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd., product number: HB4114) at 37°C. Then, harvest the bacterial cells by centrifuging at 8000 rpm for 10 min at 4°C, wash them twice with PBS, and resuspend them in the following media: TSB medium without glucose and TSB medium supplemented with 0.25%, 1%, and 2.5% glucose. Take three aliquots of 200 μL of each bacterial suspension and place them in a 96-well plate. The uninoculated medium is used as a negative control and cultured at 37°C for 24 h. Aspirate the medium in each well and wash it three times with sterile normal saline to remove the unattached cells. Fix the remaining attached cells with 200 μL of methanol per well. After 15 minutes, aspirate the liquid in the wells and let it dry. Then stain with 200 μL of 2% crystal violet per well for 5 minutes. Then wash it three times with sterile normal saline to remove the excess dye. After air-drying the plate, resuspend the adherent cells in 160 μL of 33% (v / v) glacial acetic acid, and then measure the absorbance at 600 nm. The critical value (ODC) is defined as the average OD value of the negative control. According to the OD value, the strains are 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).

[0106] Table 3.

[0107] Strain TSB 0% TSB 0.25% TSB 1% TSB 2.5% AKK ONE WA WA WA WA

[0108] By measuring the biofilm forming ability of the strain, it was determined that the biofilm forming ability of the strain was weak.

[0109] (5) Toxicological evaluation

[0110] 5.1 Cell reverse mutation assay

[0111] 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). In order to prepare the dosage formulation, a suspension (100 mg / mL) of the test item was prepared using distilled water as a carrier. The test item (strain AKK ONE) has the potential to affect the test results in a variety of ways, thereby invalidating the study. Therefore, the bacteria in the test item sample were removed by mechanical means. During the mixing of the preparation, any soluble components of the test item were dissolved in the carrier, and the residual bacteria were removed by centrifugation (4°C; 8000rpm; 10min). 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.

[0112] Based on the results of the preliminary experiments performed, initial mutation studies (plate incorporation method) and confirmatory mutation studies (pre-culture procedure) were performed. Confirmatory mutation studies were tested 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 dixon, sodium azide, methyl methanesulfonate, 2-aminofluorene dissolved in distilled water or dimethyl sulfoxide. The viability of the test bacterial cells was checked by plate experiments in each test. 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 for at least one dose group in at least one strain with or without metabolic activation. If the number of reversals in Salmonella typhimurium TA98, TA100 and Escherichiacoli WP2 uvrA strains was more than two times higher than the reversal rate of the negative control, the increase was considered biologically relevant; in Salmonella typhimurium TA97a and TA1535 strains, the number of reversals was more than three times higher than the reversal rate of the negative control. The results of the bacterial reverse mutation test showed that the strain was not mutagenic.

[0113] Table 4 AKK ONE bacterial reverse mutation test

[0114]

[0115]

[0116] Note: The drugs of positive control 1-4 are: dichlorodiphenylmethane, methyl methanesulfonate, sodium azide, and 2-aminofluorene.

[0117] 5.2 Acute toxicity

[0118] To evaluate the acute toxicity of strain AKK ONE, 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 CFU / day of strain AKK ONE suspension (0.5mL, with saline as solvent) or 0.5mL / day saline for 1 day, respectively recorded as sodium chloride solution group (Saline), low dosage group (Low dosage), medium dosage group (Medium dosage), high dosage group (High dosage), and general condition and body weight were observed every day for 7-10 days. At the end of the experimental period, blood samples were collected for hematological and serum biochemical analysis. Histopathological examination of major organs and tissues was performed on all animals in each group.

[0119] The results of the changes in mouse body weight, food intake, and blood sugar are shown in Figure 7 . The results showed that there was no death in mice after acute gavage of high, medium and low doses of AKK ONE. There was no significant difference in the daily weight change between the test group mice and the control saline group. During the acute gavage period, there was no significant difference in the daily food intake change between the test mice and the control saline group mice. During the acute gavage period, there was no significant difference in the daily blood sugar change between the test mice and the control saline group mice.

[0120] The results of mouse blood routine test are shown in the table below.

[0121] Table 5 Blood routine test of mice given high, medium and low doses of AKK ONE by oral administration

[0122]

[0123]

[0124] The above table shows that there are no significant differences in the blood parameters between the acute oral gavage group and the control saline group.

[0125] The results of the mouse triglyceride (TG), cholesterol (TC) and bile acid (TBA) content tests are shown in Figure 8 There were no significant differences in the levels of triglycerides and bile acids in the serum and liver between the test mice and the control group mice. There were extremely significant differences in the cholesterol levels in the liver between the mice given high doses of oral administration and the control group mice, but there were no significant differences in the serum and liver between the other groups.

[0126] There were no significant differences in the levels of glucose (GLU), total protein (TP), alanine aminotransferase (GPT) and aspartate aminotransferase (GOT) in serum and liver between the test mice and the control mice. There were no significant differences in the levels of creatinine (GRE) in serum between the test mice and the control mice. There were no significant differences in the concentrations of urea nitrogen (BUN) in serum between the test mice and the control mice.

[0127] The results of organ index comparison showed that there was no significant difference in the weight of the heart, liver, spleen, kidney, thymus, brain, testis, lung, stomach and intestine of the test mice and the control mice. There was no significant pathological damage in the liver and kidney of the test mice and the control mice ( Fig. 9 ).

[0128] 5.3 Subchronic toxicity test

[0129] 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) (9.2×108 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 new bacterial suspension was prepared every day. Oral gavage was performed for 90 consecutive days with 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 analyses. Autopsies were performed on all animals and the weights of major organs were recorded. Histopathological examinations were performed on the major organs and of all animals in each group.

[0130] Mouse glucose tolerance test method: Before the experiment, mice need to fast for 12-16 hours, but still have water supply. Glucose solution preparation: Prepare glucose solution, generally use 25% glucose solution at 2g / kg body weight, and calculate the required amount of glucose according to the weight of the mouse.

[0131] Determine basal blood glucose: Before the experiment, measure the basal blood glucose level of the mouse using a blood glucose meter or other blood glucose determination method. Usually measured in the tail vein blood of the mouse.

[0132] Glucose loading: Glucose solution was administered to mice by oral gavage.

[0133] Blood glucose measurement: 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.

[0134] Mouse insulin tolerance test method: Insulin solution preparation: Prepare an insulin solution of appropriate concentration, generally using insulin injection solution. Calculate the required amount of insulin for each mouse based on 0.5U / kg body weight. Insulin injection: The insulin solution is administered to the experimental group mice by subcutaneous injection. The control group mice are not treated in this way. Blood glucose measurement: At a certain time interval after administration, at 0, 15, 30, 60, 90 and 120 minutes, take blood from the tail vein of the mouse and use the blood glucose meter to measure the mouse blood glucose level.

[0135] Experimental results: Mice were gavaged with high, medium and low doses of AKK ONE for 90 consecutive days without death. There was no significant difference in the weekly weight change between the test group mice and the control saline group. During the continuous gavage period, there was no significant difference in the weekly food intake change between the test mice and the control saline group mice, and there was no significant difference in the weekly blood sugar change between the medium-dose gavage mice and the control saline group mice. There was no significant difference in the various blood indicators between the gavage group and the control saline group mice (see Table 6 below). There were no significant differences in the triglyceride levels, cholesterol levels, and bile acid levels in the liver and serum between the test mice and their control group mice. There were no significant differences in the glucose levels and total protein content in the serum and liver between the test mice and their control group mice. There were significant differences in the serum alanine aminotransferase enzyme activity and alanine aminotransferase enzyme activity between the medium-dose gavage mice and their control group mice, and there were no significant differences in the serum and liver in the other groups ( Figure 10-11 ). There were no significant differences in serum creatinine and urea nitrogen concentrations between the test mice and the control mice. There were no significant differences in the weights of the heart, liver, spleen, kidney, thymus, brain, testis, lung, pancreas, stomach and intestine between the test mice and the control mice. There were no significant pathological damages in the liver and kidneys of the test mice and the control mice. There were no significant differences in the blood sugar regulation ability (glucose tolerance and insulin tolerance) between the test mice and the control mice.

[0136] Table 6 Blood routine test of mice given high, medium and low doses of AKK ONE by oral administration

[0137]

[0138]

[0139] (6) Drug sensitivity analysis

[0140] The drug sensitivity of Akkermansia muciniphila ONE was determined according to the Clinical and Laboratory Standards Institute (CLSI) method. The quality control strain was Bacteroides fragilis ATCC25285.

[0141] Brucella agar medium and Brucella broth medium: purchased from BD Company; anaerobic gas production bag: purchased from Mitsubishi Corporation; sheep blood: purchased from Shanghai Kangrun Biotechnology Co., Ltd.; hemin: purchased from Sinopharm; vitamin K1: purchased from Sigma; ampicillin, ceftriaxone, cefotaxime, meropenem, tetracycline: purchased from China National Institute for Food and Drug Control; moxifloxacin: purchased from European Pharmacopoeia; chloramphenicol: purchased from European Pharmacopoeia.

[0142] The susceptibility of Akkermansia muciniphila ONE 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 ATCC25285 was determined using the microbroth dilution method recommended by CLSI.

[0143] The results of the drug sensitivity test of Akkermansia muciniphila ONE are shown below. The tested strain Akkermansiamuciniphila ONE is sensitive to ampicillin, ceftriaxone, cefotaxime, meropenem, tetracycline, moxifloxacin, and chloramphenicol.

[0144] Table 7 Results of drug sensitivity test of Akkermansia muciniphila ONE

[0145]

[0146] Example 3 Role of AKK ONE in regulating lipid metabolism

[0147] 1. Experimental Materials and Instruments

[0148] Ordinary maintenance feed for mice, Beijing Huafukang Biotechnology Co., Ltd.; high cholesterol feed for mice (ASHF4), Deci Biotechnology Co., Ltd.; simvastatin, Shandong Lukang Pharmaceutical Group Saite Co., Ltd.; blood glucose meter and blood glucose test strips, Sino Biosensor Co., Ltd.; total cholesterol (total TC) determination kit (A111-1-1), triglyceride (TG) determination kit (A110-1-1), low-density lipoprotein (LDL-C) determination kit (A113-1-1), high-density lipoprotein (HDL-C) determination kit (A112-1-1), Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.

[0149] 2. Experimental Methods

[0150] 2.1 Strain culture conditions: AKK ONE was cultured in BHI broth medium and placed in an anaerobic incubator (80% N2, 15% CO2, 5% H2) at 37°C for 48-72h.

[0151] 2.2 Animal experiments

[0152] 40 male C57BL / 6 mice (Beijing Huafukang Biotechnology Co., Ltd. (SCXK (Beijing) 2019-0008) aged 5 weeks were used for the experiment. All mice were raised in an SPF-grade, room temperature (25°C ± 1°C) environment with a 12 / 12h light-dark cycle. After one week of adaptive culture in the animal room, the 40 mice were divided into 4 groups, 10 mice in each group, namely the normal group (ND), the high cholesterol group (HCD), the simvastatin group (Sim), and the AKK ONE group. The normal group was fed with maintenance feed, and the other three groups were fed with high cholesterol feed. At the same time, the Sim group and the AKK ONE group were gavaged with simvastatin solution (3.8 mg / kg BW) and AKK ONE bacterial solution (1.1×10 10 CFU / mL), the control group and the high cholesterol group were gavaged with the same amount of 0.9% NaCl, once a day, for 4 consecutive weeks, and the food intake and weight changes of the mice were recorded during the experiment. The day before the end of the experiment, the mouse feces were collected under sterile conditions, the mice were fasted overnight before being killed, and the serum, liver, etc. were collected and stored in a -80℃ refrigerator for later use.

[0153] 2.3 Oral glucose tolerance test

[0154] One day after the experiment, the oral glucose tolerance of mice in each group was measured. The mice were fasted overnight before the measurement. The fasting blood glucose level of mice was measured before oral glucose administration, and the tail vein blood glucose content was measured at 15, 30, 60, and 120 minutes after oral glucose administration (2g / kg BW). The area under the curve of blood glucose concentration and time was calculated.

[0155] 2.4 Determination of serum and liver physicochemical indicators

[0156] The collected serum was used to measure the levels of total TC, TG, LDL-C, HDL-C, TBA, AST, and ALT in mouse serum according to the kit. The liver was accurately weighed, mixed with anhydrous ethanol at a ratio of 1:9, homogenized, and the supernatant was taken to measure the levels of total TC, TG, LDL-C, and HDL-C in mouse liver according to the kit.

[0157] 2.5 Oil Red O staining of liver tissue

[0158] The liver tissue was embedded and frozen, fixed with tissue fixative for 15 minutes, washed and dried. The slices were added with oil red dye and covered in light-proof staining for 8-10 minutes. Take out the slices, stay for 3 seconds, and then immerse them in two cylinders of 60% isopropanol for differentiation, 3 seconds and 5 seconds respectively. And then immerse them in 2 cylinders of pure water for 10 seconds each. Take out the slices and stay for 3 seconds, then immerse them in hematoxylin staining solution for 3-5 minutes, and then immerse them in 3 cylinders of pure water for 5 seconds, 10 seconds, and 30 seconds respectively. Differentiation solution for 2-8 seconds, and then immerse them in 2 cylinders of distilled water for 10 seconds. Then, blueing solution was used for 1 second, and the slices were gently immersed in 2 cylinders of tap water for 5 seconds and 10 seconds respectively, and the staining effect was examined under a microscope. Then, the slices were sealed with glycerol gelatin sealing agent, and the images were collected under a microscope.

[0159] 3. Experimental results

[0160] Effect of AKK ONE on blood glucose homeostasis in hypercholesterolemia mice

[0161] High cholesterol diet disrupts glucose levels in mice Fig.12 As shown in A, the fasting blood glucose level of mice in the HCD group was significantly higher than that in the ND group. After oral administration of AKK ONE, the fasting blood glucose level of mice was reduced and was similar to that of the ND group. The oral glucose tolerance test is used to determine the body's ability to regulate glucose homeostasis. After the HCD group mice ingested the glucose solution, the blood glucose level rose rapidly and then slowly dropped to the initial state ( Fig.12 B), while after oral administration of AKK ONE, the AUC values ​​corresponding to the glucose tolerance of mice decreased by 22.26% and 16.80% ( Fig.12 C), indicating that AKK ONE can improve the fasting blood glucose level and oral glucose tolerance of hypercholesterolemia mice and improve blood glucose homeostasis in mice.

[0162] Effects of AKK ONE on liver lipid levels and liver degeneration in hypercholesterolemia mice

[0163] As an important organ for lipid metabolism, the liver is closely related to blood lipid levels, and the homeostasis of liver lipid metabolism is crucial to maintaining normal liver metabolism. Fig.13 As shown in A, B, and C, a high cholesterol diet significantly increased the levels of total TC, TG, and LDL-C in the liver of mice. After oral administration of AKK ONE, the levels of these three lipids were significantly reduced. The intake of AKK ONE also significantly increased the level of HDL-C in the liver of mice, even exceeding the level of HDL-C in the liver of mice in the normal group ( Fig.13 D). The liver of mice on a high cholesterol diet was grayish yellow and enlarged, which was consistent with the liver index in the table. After oral administration of AKK ONE, the grayish yellow color of the liver decreased and its color was close to the reddish brown color of the liver in the ND group ( Fig.14 A in the figure). Next, the liver tissue pathological sections of the mice in the AKK ONE group were observed. Oil red O staining observed ( Fig.14 In B), the liver lipid droplets of mice fed a high cholesterol diet were stained red by Oil Red O and were obviously accumulated in flakes, but the liver lipid droplets of the AKK ONE group were significantly reduced and the fat volume was reduced. The above results show that AKK ONE can improve the liver degeneration of mice induced by a high cholesterol diet and improve the lipid level of the mouse liver.

[0164] Example 4 Effect of AKK ONE in improving enteritis

[0165] 1. Bacterial strain and preparation method: AKK ONE was cultured in BHI medium supplemented with 0.05% type II mucin under anaerobic conditions of 10% H2, 10% CO2 and 80% N2 for 3 days. Subsequently, the bacterial suspension was resuspended in PBS and diluted to 1×10 10 CFU / mL.

[0166] 2. Animal experiment and design: A total of 24 male C57BL / 6J mice (6-8 weeks old, weighing 20-22 g) were obtained from Beijing Huafukang Biotechnology Co., Ltd. The mice were housed under standard conditions (humidity, 50% ± 5%; temperature, 25 ± 2°C) and maintained in a 12 h light / 12 h dark cycle.

[0167] Mice were randomly divided into three groups, with 8 mice in each group: control group, dextran sodium sulfate (DSS) group, and AKK ONE group. From day 1 to day 7, all three groups of mice were given DSS (36-50 kDa) solution (3.5%, wt / vol) to establish an inflammatory bowel disease (IBD) model. From day 1 to day 14, mice in the DSS group and AKK ONE group were given phosphate buffered saline (PBS) and AKKONE (1×10 10 cfu / kg / day). The mice were weighed regularly during the experiment, and at the end of the experiment, the immune organs (spleen, liver), colon, feces and cecum contents of the mice were collected.

[0168] 3. Evaluation of colitis symptoms and disease activity index (DAI) scoring: Physiological indicators were used to evaluate the health of mice to evaluate the disease activity index (DAI), including parameters such as body weight, food intake and feces. A scoring system of 0-4 points was implemented for each parameter, with a maximum score of 12 points. The scoring criteria for each item are as follows:

[0169] Weight score: Compared with the initial weight, according to the degree of weight loss, 0-5%, 0 points; 5-10%, 1 point; 10-15%, 2 points; 15-20%, 3 points; more than 20%, 4 points. Food intake score: Compared with the initial food intake, according to the amount of food intake reduction, 0-0.5 grams, 0 points; 0.5-1 grams, 1 point; 1-1.5 grams, 2 points; 1.5-2 grams, 3 points; more than 2 grams, 4 points. Stool score: Normal stool 0 points; Loose stool 1 point; Watery diarrhea 2 points; Mild diarrhea 3 points; Severe watery diarrhea 4 points.

[0170] After euthanasia, the colon length, spleen weight, liver weight and kidney weight of 8 mice in each group were measured. The immune organ index was calculated as follows: spleen index = spleen weight (mg) / body weight (g), liver index = liver weight (mg) / body weight (g). The results are as follows:

[0171] AKK ONE ameliorates physiological abnormalities associated with DSS-induced colitis in mice. The food intake, body weight, and colon length of mice induced with DSS for 14 days (DSS group) were significantly reduced. In contrast, oral administration of AKK ONE (AKK ONE group) significantly alleviated these decreases (P<0.05, Fig.15 in BD).

[0172] The disease activity index (DAI) score is a standard indicator for evaluating the severity of colitis. The DSS group had a significantly higher score than the control group ( Fig.15 E in the figure). AKK ONE treatment significantly improved the adverse reactions caused by DSS (P<0.05). Increased immune organ indexes are usually associated with the occurrence of inflammation. The results of the present study showed that the DSS group had a significant increase in immune organ indexes in both the liver and spleen (P<0.01). However, after the use of AKK ONE, this increase was significantly attenuated (P<0.05, Fig.15 F and G in ).

[0173] Example 5 Effect of AKK ONE in improving respiratory diseases

[0174] Preparation of AKK ONE live bacterial resuspension: inoculate the frozen strain into BHI liquid culture medium, culture at 37℃ for 72h, centrifuge at 4℃ and 5000r / min for 5min, discard the supernatant, obtain bacterial sludge, inoculate the bacterial sludge into BHI liquid culture medium at 3% inoculation amount, and activate at 37℃ for 72h. Use an inoculation loop to take a small amount of bacterial liquid and make three-zone lines on BHI solid culture medium, culture at 37℃ for 48h, pick a single colony and culture it in BHI liquid culture medium for 15h, and use it as bacterial suspension after three consecutive activations. Count the live bacteria by flow cytometry, and the live bacteria concentration is 10 9 CFU / mL.

[0175] Preparation of AKK ONE dead bacteria resuspension: The AKK ONE live bacteria suspension obtained above was pasteurized (80° C., 30 min, water bath) to obtain a dead bacteria suspension.

[0176] Culture of Mycoplasma pneumoniae: Mycoplasma pneumoniae (ATCC 15531) was revived in a sterile environment, cultured in a 37°C, 5% CO2 incubator, and subcultured every 6 days.

[0177] 1.1 Animal model construction and drug administration

[0178] 6-week-old SPF BALB / c mice, weighing 20-22 g, were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., with the catalog number: hnslkjd004; ambroterol oral solution, manufactured by Beijing Hanmei Pharmaceutical Co., Ltd., with the approval number: National Medicine Standard H20040317.

[0179] Thirty-five BALB / c mice were randomly divided into a control group, a model group, an ambroxol oral solution group, an AKK ONE live bacteria resuspension group, and an AKK ONE dead bacteria resuspension group, with 7 mice in each group. Except for the control group, mice in each group were anesthetized with ether and 20 μL of 10 6 Mycoplasma pneumoniae, once a day, for 5 days. After modeling, each group was given medication for 6 consecutive days, once a day. Among them, the ambroxol oral solution group (1.5 mL / Kg), the AKK ONE live bacteria resuspension group (10 9 CFU / Kg), AKK ONE dead bacteria resuspension (10 9 TFU / Kg); the control group and the model group were given the same volume (1.5mL / Kg).

[0180] 1.2 Detection indicators

[0181] 1.2.1 Cough induction experiment

[0182] 40 minutes after the last administration, a beaker was placed upside down in the clean bench. A cotton ball containing 0.5 mL of ammonia was attached to the top of the beaker. The mouse was placed in the beaker and the timer was started. The number of coughs of the mouse within 5 minutes was observed and recorded. After the coughing experiment was completed, the mouse was killed and its lung tissue was taken for subsequent experiments.

[0183] 1.2.2 Enzyme-linked immunosorbent assay to determine the expression of related factors in serum

[0184] IL-6 ELISA test kit, purchased from Shanghai Saipeison Biotechnology Co., Ltd., catalog number: SPS-16497;

[0185] IL-8 ELISA test kit, purchased from Hefei Lyle Biotechnology Co., Ltd., catalog number: LE-B1155;

[0186] IL-10 ELISA detection kit, purchased from Shanghai Zeye Biotechnology Co., Ltd., item number: ZY72237H.

[0187] Enzyme-linked immunosorbent assay was used to determine IL-6, IL-8 and IL-10 in mouse serum. The experiment of the present invention was carried out strictly according to the steps in the operating instructions of the ELISA kit.

[0188] 1.3 Test results and analysis

[0189] 1.3.1 Cough induction experiment

[0190] Results Fig.16 Compared with the control group, the number of coughs within 5 minutes in the model group mice increased significantly (p<0.01). After drug intervention, the number of coughs within 5 minutes in the mice decreased significantly. The effects of the ambroxol oral solution group and the AKK ONE live bacteria resuspension group were comparable, and there was no significant difference between the two groups and the control group. The effect of the AKK ONE dead bacteria resuspension group was not as good as that of the AKK ONE live bacteria resuspension group (p<0.05).

[0191] 1.3.2 Enzyme-linked immunosorbent assay to determine the expression of related factors in serum

[0192] The test results are shown in Table 8.

[0193] Table 8.

[0194] Group IL-6 (pg / mL) IL-8 (pg / mL) IL-10 (pg / mL) Control group 20.32±3.53 15.81±1.82 384.56±91.42 Model Group 87.81±7.83 59.17±6.37 410.22±101.33 Positive drug control group 37.43±4.81 24.69±3.76 779.42±125.86 AKK ONE live bacteria resuspension group 36.15±3.47 27.45±4.01 836.57±130.44 AKK ONE dead bacteria resuspension group 49.94±5.95 37.84±2.69 695.46±109.85

[0195] From the data in Table 8, it can be seen that compared with the control group, the expression levels of IL-6 and IL-8 in the serum of the model group mice were significantly increased (p<0.01), and drug intervention could significantly downregulate the expression levels of IL-6 and IL-8. Among them, there was no significant difference between the positive drug control group and the AKKONE live bacteria resuspension group, and the downregulation degree of the two groups was the largest and the effect was the best. The downregulation effect of the AKK ONE dead bacteria resuspension group on pro-inflammatory factors was poorer than that of the AKK ONE live bacteria resuspension group.

[0196] The positive drug control group, AKK ONE live bacteria resuspension group and AKK ONE dead bacteria resuspension group could all upregulate the expression of the anti-inflammatory factor IL-10, and the upregulation effect of the AKK ONE live bacteria resuspension group was better than that of the positive drug group (p<0.05), but the AKK ONE dead bacteria resuspension group was worse than that of the positive drug group (p<0.05).

[0197] Example 6 The role of AKK ONE in anti-infection

[0198] Preparation of AKK ONE live bacterial resuspension: inoculate the frozen strain into BHI liquid culture medium, culture at 37℃ for 72h, centrifuge at 4℃ and 5000r / min for 5min, discard the supernatant, obtain bacterial sludge, inoculate the bacterial sludge into BHI liquid culture medium at 3% inoculation amount, and activate at 37℃ for 72h. Use an inoculation loop to take a small amount of bacterial liquid and make three-zone lines on BHI solid culture medium, culture at 37℃ for 48h, pick a single colony and culture it in BHI liquid culture medium for 15h, and use it as bacterial suspension after three consecutive activations. Count the live bacteria by flow cytometry, and the bacterial concentration is 10 9 CFU / mL.

[0199] Preparation of AKK ONE dead bacteria resuspension: The AKK ONE live bacteria suspension obtained above was pasteurized (80° C., 30 min, water bath) to obtain a dead bacteria suspension.

[0200] Cultivation of Helicobacter pylori: Under sterile conditions, the Helicobacter pylori culture liquid frozen at -80°C was quickly thawed and revived according to the cell resuscitation method, 100 μL was taken and spread on a COLUMBIA-agar blood plate containing 7% defibrinated sterile sheep blood, and placed in a three-gas incubator (O2:CO2:N2=5:10:85, 37°C) for 48-72 hours. After a single colony is generated, continue to pick a single colony and streak it on a new blood plate for culture. The Helicobacter pylori after revival for 3 generations was cultured on a COLUMBIA-agar blood plate at 37°C for 72 hours, and a bacterial suspension was prepared with BHI-broth and adjusted to 10 8 TFU / mL.

[0201] 1.1 Animal model construction and drug administration

[0202] Six-week-old SPF BALB / c mice, weighing 18-22 g, were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., catalog number: hnslkjd004.

[0203] Potassium bismuth citrate, manufacturer: Livzon Pharmaceutical Factory of Livzon Group, approval number: National Medicine Standard H10900084; Omeprazole, manufacturer: AstraZeneca Pharmaceuticals Co., Ltd., approval number: National Medicine Standard J20080097; Amoxicillin, manufacturer: Shijiazhuang Pharmaceutical Group Zhongnuo Pharmaceutical (Shijiazhuang) Co., Ltd., approval number: National Medicine Standard H13024512; Clarithromycin manufacturer: Jiangsu Hengrui Medicine Co., Ltd., approval number: National Medicine Standard H20031041.

[0204] Thirty-five BALB / c mice were randomly divided into control group, model group, positive drug control group, AKK ONE live bacteria resuspension group and AKK ONE dead bacteria resuspension group, with 7 mice in each group. Except for the control group, 1.5×10 8 TFU / mL, 8 weeks after infection, gastric tissue was histologically and tissue cultured to count Helicobacter pylori. The infection status of chronic atrophic gastritis in rats and chronic atrophic gastritis induced by Helicobacter pylori were confirmed, and the model was successfully established. Each group was given corresponding drugs by gavage, the positive drug control group (bismuth quadruple therapy, dose converted according to human and animal surface area: potassium citrate bismuth 50mg / Kg + omeprazole 5mg / Kg + amoxicillin 250mg / Kg + clarithromycin 65mg / Kg, fully mixed with purified water) and the AKK ONE live bacteria resuspension group (10 9 CFU / Kg), AKK ONE dead bacteria resuspension (10 9 TFU / Kg); the control group and the model group were given an equal amount of normal saline. The mice were killed 30 days later, and the serum and gastric tissue samples of the mice were collected and stored at -80℃ for subsequent detection.

[0205] 1.2 Test results and analysis

[0206] 1.2.1 Levels of IL-8 and TNF-α in serum

[0207] IL-8 ELISA test kit, purchased from Hefei Lyle Biotechnology Co., Ltd., catalog number: LE-M2500;

[0208] TNF-α ELISA detection kit, purchased from Shanghai Kanglang Biotechnology Co., Ltd., product number: KL-TNFa-Si.

[0209] The levels of IL-8 and TNF-α in mouse serum were determined by enzyme-linked immunosorbent assay. The experiment was carried out strictly according to the steps in the manufacturer's operating instructions of the ELISA kit. The results are shown in Table 9.

[0210] Table 9.

[0211]

[0212] From the results in Table 9, it can be seen that the levels of IL-8 and TNF-α in the serum of the model group were significantly increased, and the difference was statistically significant compared with the normal group (p<0.01). After drug intervention, the positive drug, AKK ONE live bacteria resuspension and AKK ONE dead bacteria resuspension could significantly reduce the levels of IL-8 and TNF-α, which was significantly different from the model group (p<0.01). Compared with the positive drug control group, the serum IL-8 level of the AKK ONE live bacteria resuspension group was higher (p<0.01), and the serum IL-8 level of the AKK ONE dead bacteria resuspension group was higher (p<0.05); the serum TNF-α level of the AKK ONE live bacteria resuspension group was higher (p<0.01), and the serum TNF-α level of the AKK ONE dead bacteria resuspension group was lower (p<0.05), with the best effect, see Fig.17 .

[0213] Example 7 Effect of AKK ONE in diabetes

[0214] 1 Experimental methods

[0215] 1.1 Preparation of experimental strain resuspension

[0216] The frozen strain was inoculated into BHI liquid culture medium, cultured at 37°C for 15 hours, centrifuged at 4°C and 5000r / min for 5 minutes, and the supernatant was discarded to obtain bacterial sludge. The bacterial sludge was inoculated into BHI liquid culture medium at a 3% inoculum and activated at 37°C for 15 hours. A small amount of bacterial liquid was taken with an inoculation loop to make three-zone lines on BHI solid culture medium, cultured at 37°C for 48 hours, and a single colony was picked and cultured in BHI liquid culture medium for 15 hours. After three consecutive activations, it was used as a bacterial suspension. The bacterial suspension was diluted and coated on a plate for counting, and the concentration of the bacterial suspension was adjusted to 10 with sterilized PBS liquid culture medium. 9 CFU / mL.

[0217] BHI medium: BHI (Brain Heart Infusion, OXOID, catalog number: CM1032B)) + 5% sheep blood.

[0218] Culture conditions: Completely anaerobic culture on 8 layers of gauze.

[0219] 1.2 Experimental Animal Grouping and Dosing

[0220] Forty male C57BL / 6J 6-week-old mice (average weight 21.1±0.3 g) were purchased from the Animal Experiment Center of Chongqing Medical University. After one week of adaptive feeding, the mice were randomly divided into a normal group, a model group, a metformin group, and an AKK ONE group (N=8). The mice in the normal group were fed with a standard diet (Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd., catalog number: 1010001), and the mice in the model group and the experimental group were fed with a 60% high-fat diet (Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd., catalog number: XTHF60). During this period, all mice had free access to water, and the experiment lasted for 4 weeks. Starting from the first day of the fifth week, 50 mg / kg·bw of STZ (Sigma-Aldrich, catalog number: V900890-1G) was dissolved in sodium citrate buffer, and the mice in the model group and the experimental group were intraperitoneally injected for 5 consecutive days. At the same time, the mice in the normal group were used as controls and only injected with sodium citrate buffer. One week later, the fasting blood glucose (FBG) of the mice was measured (fasting for 8 hours). If it exceeded 11.1 mmol / L, the mice were considered to have type 2 diabetes (T2DM) mice. Subsequently, one group of mice in the experimental group was gavaged with fresh AKK ONE bacterial solution (1×10 10 CFU / kg·bw), one group was gavaged with metformin (100mg / kg·bw; Shandong Sibonde Pharmaceutical Co., Ltd., catalog number: H20060230) daily for intervention, and the rest of the mice were gavaged with 10mL / kg·bw normal saline once a day for 4 weeks. During the experiment, the body weight of the mice was measured every week. During the treatment period, the food and water intake of the mice was recorded every 2 days. In the 10th week, the mice were fasted overnight, and the FBG and oral glucose tolerance test (OGTT) of the mice were measured. At the end of the 10th week, the experiment was terminated, the mice were fasted for 12h, and the mice were killed. Blood samples were collected, and the serum was separated by centrifugation at 4℃ and 3000r / min for 10min, and then transferred to -80℃ for use. Tissue samples (including liver, spleen, and kidney) were quickly processed and weighed, and some tissues were fixed in 4% paraformaldehyde for 48h, and the remaining tissues were frozen in liquid nitrogen and then transferred to -80℃ for storage.

[0221] 1.3 Oral glucose tolerance test (OGTT)

[0222] At week 0, mice were subjected to OGTT, fasted for 12 h, and allowed to drink water freely. The mice were weighed before the experiment, and their blood glucose was measured immediately (recorded as the blood glucose value at 0 min). Subsequently, 2 g / kg·bw glucose solution was gavaged, and the blood glucose level was measured once at 0 min, 30 min, 60 min, and 120 min, and the blood glucose curve was drawn.

[0223] 1.4 Determination of serum indicators

[0224] The collected blood samples were placed in a refrigerator for 30 minutes to prevent blood coagulation, and then centrifuged in a 4°C low-temperature centrifuge. The centrifugation conditions were: 3000r / min, 10min. The upper serum was taken and the Nanjing Jiancheng blood lipid determination kit was used to determine the contents of AST (Cat. No.: C010-2-1), ALT (Cat. No.: C009-2-1), INS (Cat. No.: E-EL-M1382c), TC (Cat. No.: A111-1-1), TG (Cat. No.: A110-1-1), LDL-C (Cat. No.: A113-1-1) and HDL-C (Cat. No.: A113-1-1) in mouse serum.

[0225] 1.5 HE pathological analysis of liver and pancreatic tissues

[0226] The size of mouse liver and pancreas tissue was about 1cm. 2 The tissue was fixed in a tissue fixative for 24 hours, then cleared, waxed, sliced, and finally stained with HE to observe the morphology and structure using a biological microscope.

[0227] 1.6 Data Analysis

[0228] The experimental data were expressed as mean ± standard deviation (SD), and Graph Pad Prism 8.0 software was used for statistical analysis and plotting.

[0229] 2 Experimental results

[0230] Effect of AKK ONE on oral glucose tolerance in T2DM mice

[0231] The blood glucose change curve in the oral glucose tolerance test (OGTT) experiment of mice is as follows Fig.18 As shown. 30 minutes after oral administration of glucose, the blood glucose levels of mice in each group reached the highest value. Subsequently, during the period of 30-120 minutes, the blood glucose levels of mice in each group showed a downward trend to varying degrees. Among them, compared with the normal group, the blood glucose levels of mice in the model group increased rapidly 30 minutes after oral administration of glucose, and maintained a high blood glucose level within 30-120 minutes. 120 minutes after oral administration of glucose, the blood glucose levels of mice in the model group failed to return to the blood glucose level before oral administration of glucose, indicating that diabetic mice had impaired glucose tolerance. The blood glucose levels of mice in the metformin and AKK ONE groups were significantly lower than those in the model group within 30 minutes after oral administration of glucose, especially the blood glucose level of the AKK ONE group at 120 minutes was slightly lower than that of the metformin group. The results showed that AKK ONE has the ability to improve blood glucose and glucose tolerance in T2DM.

[0232] 2.2 Effect of AKK ONE on insulin in T2DM mice

[0233] like Fig.19 The results showed that the serum insulin levels of the model group mice were significantly higher than those of the normal group mice, indicating that the abnormal islet cell function of diabetic mice led to excessive secretion of insulin. After 4 weeks of intervention with the probiotic AKK ONE and metformin, the serum insulin content decreased.

[0234] Effect of AKK ONE on serum AST and ALT in T2DM mice

[0235] In case of liver damage, ALT and AST can be used as key markers. Fig. 20 The results showed that compared with the normal group of mice, the ALT and AST levels in the serum of the model group mice were significantly higher, indicating that long-term T2DM would cause damage to the liver tissue of mice. After the intervention of AKK ONE and metformin, the levels of both were significantly reduced, but there was no significant difference between the groups, indicating that the strain can alleviate the liver damage of mice caused by HFD / STZ.

[0236] Effect of AKK ONE on blood lipids in T2DM mice

[0237] The effect of AKK ONE on blood lipid indexes of mice is shown in Table 10. The levels of TG, TC and LDL-C in the model group mice fed with a high-fat diet were significantly higher than those in the normal group mice, while the levels of HDL-C were significantly lower than those in the normal group mice, indicating that diabetes can cause lipid disorders in the serum. Oral administration of AKK ONE and metformin significantly inhibited the increase in blood lipids induced by a high-fat diet. After oral administration of AKK ONE and metformin, the levels of TG, LDL-C and TC in the serum of diabetic mice were significantly reduced, indicating that AKKONE and metformin can improve the phenomenon of increased lipids in diabetic mice.

[0238] Table 10 TG, TC, HDL-C and LDL-C levels in mouse serum

[0239]

[0240] 2.5 Pathological analysis of AKK ONE in mouse liver and pancreas

[0241] like Fig.21As shown in the figure, the liver tissue of the normal group mice showed no fatty degeneration, no fat infiltration, abundant hepatocyte cytoplasm, clear cell membrane, neat arrangement, large and normal nucleus in the center, and clear tissue structure. The hepatocytes of the model group mice induced by high fat were swollen, with obvious fatty degeneration, and most of the hepatocytes contained lipid droplets. After intervention with AKK ONE and metformin, the fat droplets in the hepatocytes were improved to varying degrees, among which the number and density of fat droplets in the AKK ONE group were less, and the fatty degeneration in the liver was significantly improved. This shows that AKK ONE improves the accumulation of lipids in the liver and prevents the absorption of fat in the body.

[0242] like Fig. 22 As shown. The pancreatic islets of mice in the normal group were complete in structure and full in morphology, and the number of pancreatic β cells was abundant and evenly distributed. The fat droplets in the pancreas of mice in the model group were saturated, the size of the pancreatic islets was significantly reduced, and the number of pancreatic β cells was also significantly reduced compared with the normal group. Compared with the model group, metformin and AKK ONE intervention for 4 weeks can significantly alleviate the atrophy of pancreatic islets in diabetic mice and reduce the phenomenon of massive apoptosis of pancreatic β cells. The above results show that metformin and AKK ONE can effectively improve pancreatic tissue damage caused by HFD and STZ and protect the pancreatic islet morphology of diabetic mice.

Claims

1. A muciniphilic Akkermansia Akkermansia muciniphila ), characterized in that, The deposit number is CGMCC No.20954.

2. according to claim 1 Akkermansia muciniphila , characterized in that, The 16S sequence is shown in SEQ ID NO:

1.

3. The method according to any one of claims 1 to 2 Akkermansia muciniphila The culturing method is characterized in that The culture method is to Akkermansia muciniphila Inoculate into culture medium for cultivation.

4. The culture method according to claim 3, characterized in that The culture medium is BHI culture medium, the culture temperature is 35-42° C., and the culture condition is anaerobic culture.

5. The method according to any one of claims 1 to 2 Akkermansia muciniphila A preparation comprising Akkermansia muciniphila of fermentation broth, Akkermansia muciniphila The fermentation broth precipitate, Akkermansia muciniphila Live bacteria 、Akkermansia muciniphila Freeze-dried powder, Akkermansia muciniphila Resuspended live bacteria Akkermansia muciniphila of dead bacteria resuspension.

6. The method according to any one of claims 1 to 2 Akkermansia muciniphila or the culture method according to any one of claims 3 to 4 Akkermansia muciniphila Or use of the preparation according to claim 5 in the preparation of food or bacterial agent.

7. The method according to any one of claims 1 to 2 Akkermansia muciniphila or the culture method according to any one of claims 3 to 4 Akkermansia muciniphila Or use of the preparation according to claim 5 in the preparation of health products.

8. Comprising any one of claims 1-2 Akkermansia muciniphila or the culture method according to any one of claims 3 to 4 Akkermansia muciniphila Or a product of the preparation according to claim 5.

9. The product according to claim 8, characterized in that The products are food and bacterial agents.

10. The product according to claim 8, characterized in that The product described is a health product.

11. The product according to any one of claims 8 to 10, characterized in that: The products described Akkermansia muciniphila The number of viable bacteria is 1×10 3 -1×10 14 CFU / g or 1×10 3 -1×10 14 CFU / mL; in the product Akkermansia muciniphila The number of dead bacteria is 1×10 3 -1×10 14 TFU / g or 1×10 3 -1×10 14 TFU / mL.

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