Multifunctional ackermania muci-3 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to multifunctional Ackermania muci-3 and application thereof. The preservation number of the ackermania muci-3 is CCTCC (China Center For Type Culture Collection) NO: M 2024048, and the ackermania muci-3 has multiple functions of reducing cholesterol, inhibiting fat synthesis, controlling body weight, resisting tumors and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a multifunctional muciniphilic Akkermansia Amuci-3 and an application thereof. Background Art

[0002] Obesity is a common chronic disease characterized by abnormal or excessive accumulation of body fat that may impair health. It is usually caused by a variety of factors, including genetics, eating habits, lifestyle, psychological factors, and social environment. Obesity not only affects appearance, but more importantly, it is associated with many serious health problems, such as heart disease, diabetes, high blood pressure, certain cancers, sleep apnea, and osteoarthritis.

[0003] Obesity has become a global health problem. The Body Mass Index (BMI) is usually used as a standard to judge the degree of obesity. BMI ≥ 25 is considered overweight, and BMI ≥ 30 is considered obese. According to a study by the World Obesity Federation, it is predicted that by 2035, more than 4 billion people in the world will be obese or overweight, accounting for 51% of the world's population.

[0004] The main treatment methods for obesity are: ① Lifestyle and behavioral treatment: improving physical activity, arranging a reasonable diet, and changing environmental factors. This method is simple and easy to implement, but it requires high patient compliance, and has a slow onset and unstable treatment effect. ② Drug treatment: When BMI exceeds 30kg / m 2 When accompanied by obesity complications, drugs are needed for auxiliary treatment. Currently, the obesity treatment drug approved by the State Food and Drug Administration in China is orlistat. However, long-term use of the drug will produce adverse reactions and drug resistance. ③Surgical treatment: Suitable for severe obesity (BMI between 30 and 35 kg / m 2 For patients with severe complications, it can more effectively reduce and maintain the weight after weight loss for a long time, but this method is costly, has greater risks and side effects, and has a negative impact on the patient's mental health. ④ Target therapy, gene-directed therapy, etc.: There are certain risks and uncertainties, there are no sufficient clinical trials, and the safety needs to be further verified.

[0005] Therefore, it is still necessary to further develop new substances that can be used to prevent and / or treat obesity. The latest research reports reveal the complex relationship between intestinal flora and obesity. Studies have shown that there is an imbalance in the structure of intestinal flora and a decrease in bacterial diversity in obese people. Some "harmful" bacteria may translocate across the intestinal epithelium, triggering a low-grade inflammatory response in the body, leading to the destruction of pancreatic β-cell function and abnormal blood sugar. There is a causal relationship between intestinal flora and metabolic diseases. For example, Bacteroides ovatus has been found to be associated with the progression of obesity-related type 2 diabetes (T2D). It may affect pancreatic β-cell function by activating specific immune cells (such as MAIT cells) and triggering an inflammatory response. The content of a species called Bacteroides thetaiotaomicron in obese people is significantly lower than that in normal people. Bacteroides thetaiotaomicron may reduce the concentration of glutamate in blood samples by affecting the metabolism of glutamate into γ-aminobutyric acid (GABA), increase the process of cell fat analysis and fatty acid oxidation, and help lose weight. By regulating the intestinal flora, such as by supplementing "probiotics", it may affect the body's metabolism and become one of the popular ways to lose weight. However, the current understanding of which flora is "beneficial" or "harmful" to metabolism is still limited, and more targeted intervention of specific intestinal flora is needed to drive weight loss and improve body metabolism.

[0006] Akkermansia muciniphila is a strictly anaerobic Gram-negative bacterium in the genus Verrucomicrobia of the phylum Verrucomicrobia. It can colonize in the human intestine and survive on mucin in the intestinal mucus layer. Its abundance in the healthy intestine is 3% to 5%. In the past decade, more and more studies have found that the abundance of Akkermansia muciniphila is reduced in patients with obesity, diabetes, intestinal diseases, neurological diseases and other diseases, indicating that its colonization in the intestine is closely related to the health of the host.

[0007] Patent application CN104918626 A discloses that the standard strain of Akkermansia muciniphila MucT (ATTC BAA-835) can affect three potential dysfunctions of obesity and related diseases, namely metabolic dysfunction, low-grade inflammatory state associated with higher blood lipopolysaccharide (LPS) levels, and impaired intestinal barrier dysfunction. Patent application CN108289918A found that the standard strain of Akkermansia muciniphila MucT (ATTC BAA-835) can more effectively increase barrier function and treat metabolic dysfunction associated with obesity and related diseases after pasteurization than non-pasteurization. Patent application CN117050914A proves that a specific strain of Akkermansia muciniphila can improve GLP-1 secretion and increase basal metabolic rate. Patent CN116200312A discloses a new strain of Akkermansia with antioxidant, tumor suppression and fat-reducing potential. Patent CN114250179A shows that the weight loss effect of the combined use of Lactobacillus gasseri and Akkermansia muciniphila is better than that of either bacterium alone. Patent CN113330109A provides a specific Akkermansia muciniphila strain that may increase GLP-1 secretion through the B2UM07 protein and thus inhibit weight gain. CN110964650B discloses an Akkermansia strain that has preventive and therapeutic prospects mainly in weight control and sugar metabolism.

[0008] Although multiple strains of Akkermansia muciniphila that improve obesity have been reported, due to the strain specificity, further research is necessary to obtain Akkermansia muciniphila strains with better efficacy or more different efficacy. Summary of the invention

[0009] The technical problem to be solved by the present invention is to provide a multifunctional mucinotropic Akkermansia strain with good safety and better efficacy.

[0010] To achieve the purpose of the present invention, the present invention first provides an Akkermansia muciniphila Amuci-3 strain or its pure culture or inactivated bacteria, and the deposit number of the strain is CCTCC NO: M2024048.

[0011] The present invention also provides a method for culturing the aforementioned Akkermansia muciniphila strain, which comprises inoculating the Akkermansia muciniphila into a culture medium, performing proliferation culture, and obtaining proliferated Akkermansia muciniphila.

[0012] The present invention also provides a food, a health product or a pharmaceutical composition, which contains the aforementioned Akkermansia muciniphila strain or the Akkermansia muciniphila strain obtained by the aforementioned culture method.

[0013] In some specific embodiments, the composition further contains a lyoprotectant or a formulation excipient.

[0014] In some embodiments, in the pharmaceutical composition, the Akkermansia muciniphila strain is the only active ingredient.

[0015] In some specific embodiments, the pharmaceutical composition further contains one or more of metformin, semaglutide, orlistat, loperamide, lovastatin, simvastatin, atorvastatin, rosuvastatin, ezetimibe, bezafibrate, fenofibrate and gemfibrozil.

[0016] In some embodiments, a single preparation of the composition contains 10 6 ~10 15 CFU of Akkermansia muciniphila strain or an equivalent amount of killed bacteria.

[0017] In addition, the present invention also provides the use of the aforementioned Akkermansia muciniphila strain in the preparation of drugs for lowering cholesterol, inhibiting fat synthesis, controlling body weight or anti-tumor.

[0018] In some embodiments, the formulation is administered to subjects with hypercholesterolemia, obesity and / or tumors.

[0019] In some embodiments, the subject includes humans and other mammals.

[0020] In some specific embodiments, provided is the use of the inactivated Akkermansia muciniphila in preparing a health product or medicine for controlling weight.

[0021] The Akkermansia muciniphila of the present invention has no virulence factors and drug-resistant genes, has good safety, and simultaneously has multiple functions of lowering cholesterol, inhibiting fat formation and resisting tumors, and is a potential probiotic strain.

[0022] Microbiological deposit information

[0023] Strain name: Akkermansia muciniphila Amuci-3

[0024] Storage time: January 9, 2024

[0025] Depository: China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, Hubei, China, Postal Code: 430072, Tel: 027-68754052

[0026] Deposit number: CCTCC NO: M 2024048. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 These are the results of a test on the cholesterol degradation ability of Akkermansia muciniphila Amuci-3.

[0028] Figure 2 The oil red-O staining was used to detect the effect of strain Amuci-3 on 3T3-L1 cell adipogenesis. A is the oil red O staining picture of each group; B is the relative amount of lipid accumulation of each group of cells based on the model group.

[0029] Figure 3 qPCR was used to detect the effect of Akkermansia muciniphila Amuci-3 on the relative expression level of acox1 gene during adipogenic induction of 3T3-L1 cells.

[0030] Figure 4 This is a graph showing the inhibitory effect of inactivated Akkermansia muciniphila Amuci-3 on zebrafish fat synthesis.

[0031] Figure 5 This is a 21-day subacute toxicity test on live and killed bacteria of Akkermansia muciniphila Amuci-3. Body weight chart: A. Body weight of male mice; B. Body weight of female mice.

[0032] Figure 6 This is a graph showing the effect of live Akkermansia muciniphila Amuci-3 on improving the body weight of DIO mice. A is the body weight result at the end of the experiment; B is the body weight gain result at the end of the experiment.

[0033] Figure 7 This is a graph showing the effect of live Akkermansia muciniphila Amuci-3 on improving fat mass in DIO mice. A: fat mass; B: lean meat mass.

[0034] Figure 8This is a graph showing the effect of live Akkermansia muciniphila Amuci-3 on improving glucose tolerance in DIO mice. A. Blood glucose concentration curve at different time points after intragastric glucose administration; B. Area under the curve (AUC); C. Blood glucose concentration 30 minutes after intragastric glucose administration; D. Blood glucose concentration 60 minutes after intragastric glucose administration.

[0035] Fig. 9 This is a graph showing the effect of inactivated Akkermansia muciniphila Amuci-3 on improving the body weight of DIO mice. A is the body weight result at the end of the experiment; B is the body weight gain result at the end of the experiment.

[0036] Fig.10 This is a graph showing the effect of inactivated Akkermansia muciniphila Amuci-3 on improving fat mass in DIO mice. A: fat mass; B: lean meat mass.

[0037] Fig.11 This is a graph showing the effect of inactivated Akkermansia muciniphila Amuci-3 on improving glucose tolerance in DIO mice. A. Blood glucose concentration curve at different time points after intragastric glucose administration; B. Area under the curve (AUC); C. Blood glucose concentration 30 minutes after intragastric glucose administration; D. Blood glucose concentration 60 minutes after intragastric glucose administration.

[0038] Fig.12 The effect of live Akkermansia muciniphila Amuci-3 on the body weight of mice transplanted with CT-26 tumors.

[0039] Fig.13 The effect of live Akkermansia muciniphila Amuci-3 on the growth of CT-26 transplanted tumors in BALB / c mice. A is a curve of tumor volume changes after oral administration; B is a histogram of tumor volume on the 18th day; C is a histogram of tumor weight on the 18th day. DETAILED DESCRIPTION

[0040] The specific accession number for which the present invention is claimed strain , which means but is not limited to:

[0041] (1) strains with specific deposit numbers deposited in the above depository;

[0042] (2) a strain having the same genome as the strain described in (1);

[0043] (3) A subculture strain without mutation based on the above (1) or (2);

[0044] (4) strains based on (1), (2) or (3) above that have accumulated minor mutations during subculture but have no substantial changes in toxicity, immunogenicity or biological activity;

[0045] As is known in the art, minor mutations are inevitably introduced into strains through passage and application. When mutations occur in non-coding sequence regions or synonymous mutations in coding regions or mutations that do not affect the toxicity, immunogenicity and biological activity of the strain (for example, it may be a connecting amino acid residue between two domains, or a residue with minor mutations located inside the higher-level structure of a protein that does not contact immune cells and does not affect toxicity, immunogenicity and biological activity), it can be reasonably expected that when these minor changes do not significantly affect the toxicity, immunogenicity and biological activity of the offspring strains, the purpose of the present invention can still be achieved, and they are derived from the strains contributed by the present invention, and therefore are still within the scope of the substantial technical contribution of the present invention. These minor mutations are still non-substantial mutations and should be regarded as mutant strains with no changes in toxicity, immunogenicity and biological activity.

[0046] There is no substantial change in toxicity, immunogenicity and biological activity, including but not limited to, within the limits of detection technology such as detection sensitivity and detection limit and acceptable or unavoidable errors, the toxicity, immunogenicity and biological activity are considered to be the same. When the toxicity, immunogenicity and biological activity of the offspring of the strain are measured using cells and animals, there is no substantial change due to differences in cell strains, animal species, age, sex, health status, culture conditions, etc., as well as foreseeable or unavoidable systematic errors.

[0047] Pure culture A pure culture is a culture consisting of a single species of microorganism. A pure culture means that all the microorganisms growing on the culture medium are descendants of the same microorganism and have the same genetic characteristics.

[0048] Live bacteria It refers to microorganisms that have the ability to carry out life activities, metabolism, growth and reproduction, and can form colonies under suitable environmental conditions. These colonies can be confirmed by visible growth on the culture medium.

[0049] Inactivated bacteria It refers to microorganisms that have lost their ability to survive and are unable to metabolize, grow or reproduce due to heat treatment, chemical disinfectants, radiation or other physical or chemical methods.

[0050] The present invention also provides a food, a health product or a pharmaceutical composition, which contains the aforementioned Akkermansia muciniphila strain or the Akkermansia muciniphila strain obtained by the aforementioned culture method, or an inactivated bacterial cell thereof.

[0051] In some specific embodiments, a single administration of a food, health product or pharmaceutical composition to a subject contains 10 2 ~10 15CFU, 10 3 ~10 14 CFU, 10 4 ~10 13 CFU, 10 5 ~10 12 CFU, 10 6 ~10 12 CFU, 10 7 ~10 11 CFU, 10 8 ~10 10 CFU, or 10 8 ~10 10 When inactivated bacteria are used, it contains an equivalent number of inactivated bacteria as described above.

[0052] The present invention provides a composition in the form of a common food, health product, or medicine, which contains the muciniphilic Akkermansia of the present invention. These common foods, health products, or medicines contain various exemplary embodiments of the composition of the present invention. These common foods, health products, or medicines can be made or provided as probiotic powders, capsules, cereals, baby food, health food or food for specific health purposes, and can also be pharmaceutical capsules, tablets, powders, etc.

[0053] The probiotic composition of the present invention may further contain other beneficial ingredients, such as another functional probiotic, prebiotic or medicine, etc. Examples of other beneficial ingredients include, but are not limited to, Bacillus licheniformis, Bifidobacterium, Clostridium butyricum, fructooligosaccharides, galacto-oligosaccharides, isomaltooligosaccharides, xylo-oligosaccharides, manno-oligosaccharides, inulin, stachyose, soybean oligosaccharides, β-glucan, lactofructooligosaccharides, etc.

[0054] The specific temperature parameters in the present invention, unless otherwise specified, should be understood as constant temperature treatment, and are allowed to fluctuate within a certain temperature range, such as within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C.

[0055] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work should fall within the scope of protection of the present invention.

[0056] The culture medium used in the following examples is prepared as follows. If not otherwise specified, it can be purchased commercially or prepared according to a common method in the art:

[0057] Preparation of anaerobic BHI & mucin liquid culture medium: weigh 38.5 g of BHI broth powder (Qingdao Haibo Biotechnology Co., Ltd., HB8297-1) and 2 g of mucin (Sigma-Aldrich Trading Co., Ltd., M2378-100G), dissolve in 1 L of distilled water, deoxygenate with N2 replacement and package, sterilize at 121°C for 15 min, and store in a cool and dry place.

[0058] Preparation of anaerobic BHI & mucin liquid culture medium containing cholesterol: Weigh 0.5 g of cholesterol (Sigma-Aldrich Trading Co., Ltd., C8667), add it to 25 mL of Tween 80 (Shanghai MacLean Biochemical Technology Co., Ltd., T6336), heat it in a 90°C water bath until the cholesterol is dissolved (about 40 to 60 minutes), then add this mixture to the packaged anaerobic BHI & mucin liquid culture medium at a ratio of 2.5% (v / v), sterilize it at 121°C for 15 minutes, and store it in a cool and dry place.

[0059] Preparation of anaerobic saline: Weigh 8.5g of sodium chloride and dissolve it in 1L of pure water, stir and boil. After boiling, use a Hungate apparatus to pass N2 for 20 minutes. After cooling to room temperature, divide the culture medium into anaerobic bottles that have been deoxygenated with N2 for 121 minutes and sterilize at high temperature for 15 minutes.

[0060] Preparation of culture medium for bacterial powder: weigh 10g of anhydrous glucose, 2g of N-acetylglucosamine, 20g of tryptone, 10g of casein, 5g of yeast extract, 5g of soy peptone, 2g of threonine, 1g of serine, 2g of potassium dihydrogen phosphate, 2g of dipotassium hydrogen phosphate, 0.5g of sodium bicarbonate, 0.1g of manganese sulfate, 0.05g of calcium chloride, 0.5mL of Tween 80, and 0.5g of monohydrated cysteine ​​hydrochloride, dissolve in 1L of distilled water, deoxygenate with N2, dispense, and sterilize at 121℃ for 20min. Store in a cool and dry place.

[0061] Example 1 Isolation and identification of strains

[0062] Fresh fecal samples from healthy volunteers were collected, resuspended in appropriate amount of anaerobic PBS, shaken, and filtered. Under N2 protection, the samples were gradient diluted and anaerobically cultured for 5-9 days. The cultured strains were deduplicated by MALDI-TOF mass spectrometry and then amplified and sequenced by 16S rDNA gene (Beijing Qingke Biotechnology Co., Ltd. Chengdu Branch) for identification. The sequencing results were compared by BLAST, and a strain of Akkermansia muciniphila was isolated and named Amuci-3.

[0063] The Amuci-3 strain was deposited in the China Center for Type Culture Collection (CCTCC, Wuhan University Collection Center) on January 9, 2024, with the deposit number: CCTCC NO: M 2024048.

[0064] Example 2 Whole genome analysis

[0065] Amuci-3 was inoculated into 5 mL of anaerobic BHI & mucin liquid medium and cultured until the late logarithmic growth period. The whole genome DNA of the strain was extracted for whole genome sequencing. After quality control of the offline data, it was assembled and annotated using conventional methods, and the obtained protein sequence was compared with the VFDB (Virulence Factor Databases) and CARD (The ComprehensiveAntibiotic Resistance Database) databases to conduct virulence factor and drug resistance gene analysis. The results showed that the three strains were safe at the genetic level.

[0066] The novelty of the strains was analyzed using Average Nucleotide Identity (ANI) and SNP-based Variant analysis. By searching in Genbank, 307 publicly available Akkermansia muciniphila-related whole genomes were found. By comparing with fastANI (v1.33) and snippy (v4.6.0), the strains closest to the Amuci-3 whole genome ANI and related Variant information are shown in Table 1 below:

[0067] Table 1 Strain genome ANI and variant information

[0068] strain Genome assembly name ANI (%) Total Variant Amuci-3 ASM1022905v1 99.96 569

[0069] Therefore, it can be determined that Amuci-3 is a new strain.

[0070] Example 3 Cholesterol degradation experiment

[0071] The preserved Amuci-3 was inoculated into 5 mL of anaerobic BHI & mucin liquid medium, and the standard strain of Akkermansia muciniphila (CICC24917, China Industrial Microbiological Culture Collection Management Center, same as ATCC BAA-835) was used as the reference strain. All strains were cultured anaerobically at 37°C until the late logarithmic growth period to obtain activated strains. Each activated strain was inoculated into 1 mL of anaerobic BHI & mucin liquid medium containing cholesterol, with 3 parallels set for each group, and the cholesterol content was detected after anaerobic culture at 37°C for 72 hours.

[0072] Take 1mL of culture solution and centrifuge at 5000rpm for 10min, and take the supernatant to obtain the test sample. Use the total cholesterol (TC) content detection kit (Beijing Solebow Technology Co., Ltd., BC1985) to draw a standard curve and detect the total cholesterol content according to the instructions of the kit. The cholesterol degradation rate is calculated as follows: cholesterol degradation rate % = (cholesterol content in blank culture medium - cholesterol content in experimental group) / cholesterol content in blank culture medium × 100%. For the cholesterol degradation ability results of the reference strain and the test strain, see Figure 1 The results were expressed as Mean±SD, and T-test was used for comparative analysis. ** represents p<0.01.

[0073] Depend on Figure 1 It can be seen that the cholesterol degradation ability of strain Amuci-3 is higher than that of standard strain CICC24917, indicating that Amuci-3 has a stronger cholesterol degradation ability.

[0074] Example 4 Detection of the effect of strain Amuci-3 on adipogenesis of 3T3-L1 cells by oil red-O staining

[0075] Oil Red O staining is a method for measuring the amount of fat produced in cells by staining differentiated 3T3-L1 cells with Oil Red O reagent.

[0076] Strain culture: Inoculate Amuci-3 and standard strain CICC24917 from the bacterial storage into 15mL of bacterial powder preparation medium, and culture anaerobically in a 37℃ electric constant temperature incubator for 48h to obtain activated strains. Transfer the activated strain to 360mL of bacterial powder preparation medium and culture anaerobically for 48h. Collect the bacterial solution, centrifuge at 7000rpm / min for 15min, and wash the bacteria once with sterile PBS. After pasteurization, place at 4℃ for use.

[0077] Cell inoculation: 3T3-L1 cells (Wuhan Pronocell Life Science Co., Ltd., CL-0006) with good growth status and appropriate density were digested with trypsin (Beijing Lanjieke Technology Co., Ltd., BL501A), collected by centrifugation, and diluted to 3×10 with DMEM complete medium (Thermo Fisher Scientific, C11995500BT) containing 10% FBS and 1% PS (Thermo Fisher Scientific, 15140-122). 5 Each well was inoculated with 2 mL of cells / mL in a 6-well plate. The day of inoculation was recorded as Day 0.

[0078] Preparation of adipogenic inducing agent:

[0079] Inducer I: weigh 25 mg of dexamethasone (Sigma-Aldrich (Shanghai) Trading Co., Ltd., D1756), resuspend and mix with 12.74 mL of DMSO to make the concentration 5 mM; weigh 100 mg of rosiglitazone (Kaman Chemical Company, 71740), resuspend and mix with 139.89 mL of DMSO to make the concentration 2 mM; weigh 250 mg of IBMX (Sigma-Aldrich (Shanghai) Trading Co., Ltd., I5879), resuspend and mix with 2.25 mL of DMSO to make the concentration 0.5 M; weigh 25 mg of insulin (MCE Biotechnology Co., Ltd., USA, HY-P0035), resuspend and mix with 2.5 mL of DMSO to make the concentration 10 mg / mL. Dilute the above drugs 1000 times and add them to 100 mL of DMEM complete medium to make the final concentrations 5 μM, 2 μM, 0.5 mM and 10 μg / mL respectively.

[0080] Inducer II: Pipette 100 μL of 2 mM rosiglitazone and 100 μL of 10 mg / mL insulin, and add them to 100 mL of DMEM complete medium to make the final concentrations 2 μM and 10 μg / mL, respectively.

[0081] Cell adipogenesis induction and drug administration: This experiment was divided into the following groups: model group, standard strain CICC24917 group, and Amuci-3 group. The treatment methods for different groups were as follows: On Day 3, the model group was added with the inducer I prepared in advance, and the other groups were added with the inactivated bacteria solution resuspended with the inducer I to a concentration of 5×10 8 / mL. Placed in a 5% carbon dioxide incubator and cultured at 37°C. On Day 5, 7, 9, 11, and 13, the model group was added with the inducer II prepared in advance, and the other groups were added with the inactivated bacteria solution resuspended with the inducer II to a concentration of 5×10 8 The cells / mL were placed in a 5% carbon dioxide incubator and cultured at 37°C.

[0082] 3T3-L1 cell staining treatment: On Day 15, the cell supernatant was discarded, 4% paraformaldehyde (Beijing Lanjieke Technology Co., Ltd., BL539A) was added to fix for 30 minutes, and sterile PBS (Wuhan Boster Biotechnology Co., Ltd., PYG0021) was used to wash twice, 60% isopropanol (Sangon Biotechnology (Shanghai) Co., Ltd., A507048-0500) solution was added to stand for 3 minutes, and Oil Red O dye solution (Beijing Solebold Technology Co., Ltd., G1260) was added for staining. After the staining, 60% isopropanol solution was added to observe the intercellular space under a microscope. The intercellular space was clear, and sterile PBS was added to wash twice, and the cells were photographed under a microscope. After the end, isopropanol was added to dissolve the lipid droplets stained with Oil Red O solution, and the absorbance at 500nm was measured using an enzyme reader. The results are expressed as Mean±SD, and One-way ANOVA was used for multiple comparison analysis. ***, compared with the model group, p<0.001; **, compared with the model group, p<0.01; *, compared with the model group, p<0.05; #, compared with the standard strain CICC24917, p<0.05.

[0083] Experimental results: Figure 2 As shown, through microscopic observation, compared with the model group, the Amuci-3 group significantly reduced intracellular lipid droplets and fat accumulation.

[0084] Example 5 qPCR detection of the relative expression level of acox1 gene during adipogenesis induction of 3T3-L1 cells

[0085] Acox1 is a key protein in the β-oxidation process of fatty acids and plays a key role in the oxidative degradation of fatty acids. Detecting the relative expression level of the acox1 gene can reflect the level of fatty acid degradation to a certain extent.

[0086] Strain culture: Inoculate the preserved Amuci-3 into 15mL of bacterial powder preparation medium and culture anaerobically in a 37℃ electric constant temperature incubator for 48h. Use the standard strain CICC24917 as the reference strain. Transfer the activated strain to 360mL of bacterial powder preparation medium and culture anaerobically for 48h. Collect the bacterial solution, centrifuge at 7000rpm / min for 15min, and wash the bacteria once with sterile PBS. After pasteurization, place at 4℃ for use.

[0087] Cell inoculation: The cells used were 3T3-L1 cells (Wuhan Pronocell Life Science Co., Ltd., CL-0006). 3T3-L1 cells are adherent cells and need to be digested with trypsin (Beijing Lanjieke Technology Co., Ltd., BL501A). After digestion and centrifugation, they were diluted to 3×10 with DMEM complete medium (Thermo Fisher Scientific, C11995500BT) containing 10% FBS and 1% PS (Thermo Fisher Scientific, 15140-122). 5 Each well was inoculated with 2 mL of cells / mL in a 6-well plate. The day of inoculation was recorded as Day 0.

[0088] Preparation of adipogenic inducing agent: same as Example 4.

[0089] Cell adipogenesis induction and administration: Grouping, adipogenesis induction and administration methods refer to Example 4. On Day 13, the cell supernatant was discarded and RNA extraction, reverse transcription and qPCR experiments were performed.

[0090] qPCR: On Day 13, the cell supernatant was discarded and RNA was extracted according to the instructions of RNAsimple total RNA extraction kit (TIANGEN, DP419). After RNA extraction, reverse transcription was performed according to the instructions of Thermo cDNA reverse transcription kit (Thermo Fisher Scientific, K1622). After reverse transcription, qPCR experiments were performed using the SYBR method (Bio-Rad Biomedical Products (Shanghai) Co., Ltd., 1725124). The CT value obtained by detection was 2 -ΔΔCt The calculation formula was: gene expression level = 2^-[ΔCT (target gene CT-corresponding internal reference gene CT)-ΔCT (control group target gene CT-control internal reference gene CT)], target gene: acox1, internal reference gene: β-actin. After inducing adipogenic differentiation, the expression level of each gene in the model group was set as 100%, and the relative expression levels of the remaining groups were calculated based on the model group. The results are expressed as Mean±SD, and one-way ANOVA was used for multiple comparison analysis. ***, compared with the model group, p<0.001; **, compared with the model group, p<0.01; #, compared with the standard strain CICC24917, p<0.05.

[0091] Test results: Figure 3 As shown, compared with the model group, the Amuci-3 group significantly increased the mRNA level of acox1, and the effect was significantly better than that of the standard strain CICC24917.

[0092] Example 6 Cultivation and preparation of Akkermansia muciniphila

[0093] Lyophilization protective agent preparation:

[0094] Solution A: sucrose 5g, trehalose 5g, mannitol 5g, skim milk powder 8g, purified water 62g; sterilize at 115℃ for 20min.

[0095] Solution B: 3g of sodium vitamin C, 12g of purified water. Filter and sterilize for later use.

[0096] When using, mix in a mass ratio of A:B=17:3.

[0097] Preparation of bacterial powder: Inoculate the preserved Akkermansia muciphila Amuci-3 or standard strain into the bacterial powder preparation medium to obtain the first-level seed liquid. Subsequently, transfer it once more to obtain the second-level seed liquid. Pump the second-level seed liquid into the fermentation tank, set the fermentation parameters (37°C, pH 6.0, 100rpm, 0.06MPa), and ferment and culture. After the fermentation reaches the end point, collect the bacteria by centrifugation. Add lyophilization protective agent at a weight ratio of 1:1 to 1:2 to mix the emulsified bacterial mud. The emulsified bacterial suspension is freeze-dried and crushed to obtain bacterial powder.

[0098] Example 7 Inactivation of Akkermansia muciniphila Amuci-3 inhibits zebrafish fat synthesis

[0099] Preparation of inactivated bacterial suspension: Take 2×10 Amuci-3 powder of Akkermansia muciniphila in Example 6. 9 CFU, fully dissolved in 2 mL of 0.9% saline to make a concentration of 1×10 9 CFU / mL bacterial suspension was then pasteurized in a water bath at 80°C for 30-60 min to obtain an inactivated bacterial suspension.

[0100] Experimental grouping and drug administration: 5dpf melanin allele mutant Albino strain zebrafish (purchased from Hangzhou Huante Biotechnology Co., Ltd.) were selected and randomly divided into 5 groups, namely normal control group, model control group, orlistat group, inactivated CICC24917 group, and inactivated Amuci-3 group, with 30 fish in each group. The normal control group and the model control group were given PBS solution, the positive control orlistat group was given orlistat at a concentration of 15.0μg / ml (purchased from Shandong New Era Pharmaceutical Co., Ltd.), and the inactivated muciniphilic Akkermansia was prepared by adding the above-mentioned inactivated bacterial suspension to 18mL PBS, that is, the concentration of 1×10 8CFU / mL of Akkermansia muciniphila, the final volume of zebrafish solution in each group was 20mL. After incubation at 28℃ for 1h, except for the normal control group, all other groups were fed with high-fat food (egg yolk powder, 38% glyceride, 19% lecithin, 30% protein, a small amount of cholesterol, sugar, minerals, etc.), and then incubated at 28℃ for 24h. After the experiment, oil red O fat staining was performed (Oil Red O is a strong lipid solvent and lipid dye that can combine with neutral fat droplets such as triglycerides to form an orange-red color, which can quantitatively reflect the fat content in the intestine and blood vessels of zebrafish). Ten zebrafish were randomly selected from each group and photographed under a dissecting microscope to analyze the fat staining intensity of the intestine and tail blood vessels. The results are expressed as Mean±SEM, and one-way ANOVA was used for multiple comparison analysis. Compared with the model group, *p<0.05; ***p<0.001.

[0101] Test results: The results are as follows Figure 4 As shown, orlistat, inactivated CICC24917 and inactivated Amuci-3 can all significantly reduce the fat content in zebrafish, and the effect of inactivated Amuci-3 in improving zebrafish obesity is better than that of inactivated CICC24917.

[0102] Example 8 21-day subacute toxicity test of Akkermansia muciniphila Amuci-3 strain

[0103] Strain sample preparation:

[0104] Transfer the Amuci-3 strain of Akkermansia muciniphila from the glycerol tube to 3 ml of anaerobic BHI & mucin liquid culture medium and culture at 37 °C for 48 hours to activate the strain. Inoculate the activated strain into 360 mL of anaerobic BHI & mucin liquid culture medium, culture at 37 °C for about 60 hours, transfer to a 500 mL centrifuge tube, centrifuge at 8000 rpm / min for 10 minutes, and discard the supernatant. Resuspend the bacteria three times with 35 mL of anaerobic saline, take the resuspended bacterial solution and spot it on the hemocytometer for microscopic counting and record the data to ensure that the concentration of the bacterial solution is not less than 1.25×10 9 CFU / mL. Aliquot the bacterial solution after turbidity adjustment and counting into 10mL anaerobic tubes, 5mL per tube, for a total of 6 tubes.

[0105] Preparation of inactivated bacterial solution: Select 3 tubes of each bacterial solution and place them in a 70℃ water bath for 30 minutes for pasteurization.

[0106] Experimental Design:

[0107] The pathogenicity of the inactivated Akkermansia muciniphila Amuci-3 strain was evaluated with reference to the "Technical Guidelines for Safety Inspection and Evaluation of Bacteria for Health Food Raw Materials (2020 Edition)" issued by the State Administration for Market Regulation. 4-5 week-old ICR mice, male 14-16g, female 15-17g, were purchased from Sichuan Weitong Lihua Experimental Animal Technology Co., Ltd. After 3 days of feeding during the adaptation period, mice weighing 18-22g were selected and divided into 3 groups, namely the control group, the live bacteria Amuci-3 group and the inactivated Amuci-3 group, with 10 mice in each group, half male and half female. The experimental period was 21 days. The day of grouping was D0. After grouping, mice in each group were fasted overnight (16 h). The day of administration was D1. From D1 to D3, the test substance was gavaged at a dosage volume of 20 mL / kg for 3 consecutive days. The control group was gavaged with normal saline, and the live bacteria Amuci-3 group was gavaged with a concentration of 5.60×10 10 CFU / mL, the intragastric concentration of the inactivated Amuci-3 group was 2.12×10 10 CFU / mL (the guidelines require that the concentration of the test substance should not be less than 1.25×10 9 CFU / mL), and the food was fed 3-4h after the first oral administration. The observation was continued for 21 days starting from the first day of administration.

[0108] Clinical observation: Observe and record any abnormalities in the mouse's skin and fur, eyes and mucous membranes, breathing, limb movements, behavior, etc. Observe whether there are any tremors, convulsions, diarrhea, lethargy, drooling, and coma.

[0109] The results showed that in the 21-day observation of female and male mice in each group, each mouse had clean skin, smooth and shiny fur, normal eye mucosa, stable and normal breathing, normal limb movement, and normal behavior. No toxic reactions such as tremor, convulsion, diarrhea, lethargy, drooling, and coma occurred.

[0110] Body weight test: Weigh and record the body weight of all mice before and after the experiment. The results are expressed as Mean ± SEM and analyzed by T test.

[0111] The results are as follows Figure 5 As shown in the figure, the body weight of male or female mice in each group increased steadily during the experiment. At 21 days, there was no significant difference in the body weight of male or female mice in the live and inactivated Akkermansia muciniphila groups compared with the control group.

[0112] Result judgment criteria: If the animals in the test group do not show symptoms of poisoning or die during the test, and there is no statistically significant difference in indicators such as body weight compared with the control group, the strain can be determined to be non-pathogenic; if the animals in the test group show symptoms of poisoning or die during the test, or there is a significant difference in indicators such as body weight compared with the control group during the test, the strain can be determined to be pathogenic.

[0113] Conclusion: All vital signs of male or female mice in each group were normal during the experiment, weight gain was stable, and no toxic reactions or deaths occurred. At 21 days, there was no statistical difference in body weight between the two groups. In summary, both live and inactivated Akkermansia muciniphila Amuci-3 strains were non-pathogenic.

[0114] Example 9 Effect of live Akkermansia muciniphila on improving obesity in DIO mice

[0115] Experimental grouping and administration: 4-5 weeks old, 17-19g SPF male C57BL / 6J mice were purchased from Sichuan Weitong Lihua Experimental Animal Technology Co., Ltd. and raised in an SPF animal room. After 7 days of adaptive feeding, the mice were randomly divided into normal control group, model control group, orlistat group, CICC24917 (standard strain of Akkermansia muciniphila, purchased from the German Collection of Microorganisms and Cell Cultures) group, and Amuci-3 group according to their body weight, with 8 mice in each group. The normal control group was fed with ordinary maintenance feed, and the other groups were fed with high-fat feed (60kcal% fat; D12492, purchased from Research Diets Inc) for 8 consecutive weeks. During the experiment, the normal control group and the model control group were gavaged with 0.2 mL of freeze-dried protective agent per rat; the positive control orlistat group was gavaged with 10 mL / kg body weight of orlistat (purchased from Zhien Biotechnology Co., Ltd.), with a dose of 40 mg / kg; the CICC24917 group and the Amuci-3 group were gavaged with 1×10 9 CFU / 0.2mL / mouse, each group was given once a day. The experimental results were expressed as Mean±SEM, and one-way ANOVA was used for multiple comparison analysis. Compared with the model group, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0116] Body weight detection: All mice were weighed every Monday, and the body weight gain was calculated, body weight gain = detection body weight - initial body weight.

[0117] Body fat content detection: After the experimental period, the body composition analyzer (purchased from Suzhou Newmai Analytical Instrument Co., Ltd.) was used to detect the fat and lean meat content of mice.

[0118] Oral glucose tolerance test: To explore the effect of Akkermansia muciniphila on glucose tolerance in DIO mice, an OGTT test was performed 7 weeks after the experiment. After all mice were fasted overnight, blood was collected from the tip of their tails, and the fasting blood glucose concentration was measured using a blood glucose meter (purchased from Jiangsu Yuyue Medical Equipment Co., Ltd.). Subsequently, glucose (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was gavaged at a dose of 2 g / kg. The blood glucose concentration of the mice was measured at 30 min, 60 min, 90 min and 120 min, and the time-blood glucose concentration curve was drawn.

[0119] Test results: Figure 6 The weight at the end of the experiment was significantly lower in the orlistat group and the Amuci-3 group than in the model control group ( Figure 6 A), Orlistat group, CICC24917 group, Amuci-3 group can significantly reduce the weight gain of DIO mice ( Figure 6 B).

[0120] Figure 7 The results of body fat test showed that Amuci-3 can reduce the fat mass of obese mice ( Figure 7 A) but no significant difference was found. Amuci-3 had no effect on lean meat mass ( Figure 7 B).

[0121] Figure 8 The results of the oral glucose tolerance test (OGTT) showed that the blood glucose concentration of mice in each group increased sharply within 30 minutes after oral administration of glucose. Compared with the model control group, the blood glucose concentration of the CICC24917 group was significantly reduced at 30 minutes ( Figure 8 C); At 60 minutes, the blood glucose concentration in the Amuci-3 group was significantly lower than that in the model control group ( Figure 8 D), the Amuci-3 group had a better effect on reducing blood glucose concentration at 60 minutes than the standard strain CICC24917 group. The area under the curve (AUC) analysis of the OGTT curve showed that the Amuci-3 group had a significantly lower AUC than the model control group ( Figure 8 B), indicating that CICC24917 and Amuci-3 can effectively improve glucose intolerance in DIO mice.

[0122] Example 10: Effect of inactivating Akkermansia muciniphila on improving obesity in DIO mice

[0123] Experimental grouping and administration: 4-5 weeks old, 17-19g SPF male C57BL / 6J mice were purchased from Sichuan Weitong Lihua Experimental Animal Technology Co., Ltd. and kept in an SPF animal room. After 7 days of adaptive feeding, the mice were randomly divided into 5 groups according to their body weight, namely normal control group, model control group, orlistat group, inactivated CICC24917 group, and inactivated Amuci-3 group, with 8 mice in each group. The normal control group was fed with ordinary maintenance feed, and the other groups were fed with high-fat feed (60kcal% fat; D12492, purchased from Research Diets Inc) for 8 consecutive weeks. During the experiment, the normal control group and the model control group were gavaged with 0.2mL / mouse lyophilized protective agent; the positive control orlistat group was gavaged with 10mL / kg body weight of orlistat (purchased from Zhien Biotechnology Co., Ltd.) at a dose of 40mg / kg; the inactivated CICC24917 group and the inactivated Amuci-3 group were gavaged with 1×10 9 CFU / 0.2mL / mouse, each group was given once a day. The experimental results were expressed as Mean±SEM, and one-way ANOVA was used for multiple comparison analysis. Compared with the model group, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0124] Body weight test, body fat test, oral glucose tolerance test are the same as in Example 9

[0125] Test results: Fig. 9 The body weight at the end of the experiment, the body weight and weight gain of the orlistat group, inactivated CICC24917 group, and inactivated Amuci-3 group were significantly lower than those of the model control group ( Fig. 9 A, 9B).

[0126] Fig.10 The results of body fat test showed that the fat mass of the orlistat group, inactivated CICC24917 group, and inactivated Amuci-3 group was significantly lower than that of the model control group ( Fig.10 A) and had no significant effect on lean meat mass ( Fig.10 B).

[0127] Fig.11 The results of the oral glucose tolerance test (OGTT) showed that the blood glucose concentration of mice in each group increased sharply within 30 minutes after oral administration of glucose. Inactivated CICC24917 and inactivated Amuci-3 could significantly reduce the blood glucose concentration at 30 minutes ( Fig.11 C); At 60 minutes, the blood glucose concentrations in the inactivated CICC24917 and inactivated Amuci-3 groups were significantly lower than those in the model control group ( Fig.11D). The area under the curve (AUC) analysis of the OGTT curve showed that the AUC of the inactivated CICC24917 group and the inactivated Amuci-3 group were significantly lower than those of the model control group ( Fig.11 B), indicating that both inactivated CICC24917 and inactivated Amuci-3 can effectively improve glucose intolerance in DIO mice, and the overall improvement effect of inactivated Amuci-3 is slightly better than that of the inactivated standard strain CICC24917 group.

[0128] Example 11 Antitumor effect of live Akkermansia muciniphila Amuci-3 in a Balb / c mouse model of colon cancer cell CT-26 xenograft tumor

[0129] Female BALB / c mice were purchased from Sichuan Weitonglihua Experimental Animal Technology Co., Ltd. and housed in an SPF-grade animal room IVC system with an ambient temperature of 20-25°C, a relative humidity of 40-70%, and a 12-hour light-dark cycle.

[0130] Experimental plan: Specific grouping and dosing information are shown in Table 2.

[0131] Table 2 Grouping and dosing regimen

[0132]

[0133] The preparation of CICC24917 and Amuci-3 is described in Example 9.

[0134] Cell culture: Mouse colon cancer CT-26 cells were purchased from ATCC (American Type Culture Collection) and cultured in 75 cm 2 The cells were cultured in 1640 medium (Servicebio, catalog number: G4532-500ML) with 10% fetal bovine serum and penicillin and streptomycin (Servicebio, catalog number: G4003-100ML) at 37°C in an incubator containing 5% CO2. When the cells were in the exponential growth phase, they were digested with trypsin, collected, and resuspended in PBS to 3×10 7 Pieces / ml.

[0135] Modeling: After the mice were adapted to the laboratory environment for 7 days, CT-26 cell suspension was inoculated subcutaneously in the right rib of the mice, 3×10 6 The inoculation volume was 0.1 mL.

[0136] Grouping and administration: Six days after inoculation, the tumor diameter was measured and the tumor volume was calculated. The tumor volume of 100-300 mm was selected. 3The mice were randomly divided into 4 groups according to the tumor size, including model control group, anti-mouse PD1 (purchased from Bioxcell), CICC24917 group and Amuci-3 group, with 7 mice in each group. The first day of administration was defined as D1, and the 18th day was the experimental endpoint recorded as D18.

[0137] During the experiment, the model control group received intragastric administration of normal saline once a day, 0.2 mL / mouse, for a total of 17 times; the anti-mouse PD-1 group received intraperitoneal injection of anti-mouse PD-1 twice a week, with a dose of 10 mg / kg and a volume of 10 ml / kg, for a total of 5 times; the CICC24917 group received intragastric administration of CICC24917 live bacterial suspension every day, with a dose of 1×10 9 CFU / mouse, 0.2 mL / mouse, 17 times in total; the Amuci-3 group was treated with Amuci-3 live bacteria suspension intragastrically once a day, with a dose of 1×10 9 CFU / mouse, 0.2 mL / mouse, 17 times in total.

[0138] Index detection: During the trial, general health observations were performed every day, including but not limited to the animals' physical signs, behavioral activities, respiration, glandular secretions, fecal characteristics, and death. Body weight was monitored at least twice a week. Tumor diameter was measured with a vernier caliper at least twice a week, tumor volume was calculated, and tumor growth curves were drawn. At the end of the trial, tumor tissue was collected for tumor weight measurement.

[0139] At the end of the experiment, the relative tumor proliferation rate T / C (%) and tumor inhibition rate TGI (%) were calculated. The calculation formula is: relative tumor proliferation rate T / C (%) = TRTV / CRTV × 100%, tumor inhibition rate TGI (%) = 1-T / C; TRTV: treatment group RTV, CRTV: negative control group RTV; RTV = Vt / V0; V0 is the tumor volume measured when the cage is administered (i.e. D1), and Vt is the tumor volume at each measurement. The calculation formula for tumor volume (V) is: V = 1 / 2 × a × b 2 , where a and b represent the long diameter and short diameter of the tumor, respectively. The experimental data are expressed as Mean ± SEM, and one-way ANOVA was used for multiple comparison analysis. Compared with the model group, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0140] Test results:

[0141] In terms of weight, Fig.12 As shown, no mice in the Amuci-3 group experienced weight loss or death, showed no obvious drug toxicity, and were well tolerated during the treatment period.

[0142] In terms of anti-tumor effects, Fig.13 As shown, the tumor volume of each experimental group continued to grow from D1 ( Fig.13 A). At the end of the experiment (D18), the tumor volume and weight of the Amuci-3 group were significantly lower than those of the model control group ( Fig.13 B, C). As shown in Table 3, the relative tumor inhibition rates of D18 in the anti-mouse PD-1 group, CICC24917 group, and Amuci-3 group were 42.08%, 17.90%, and 30.73%, respectively.

[0143] Table 3D18 relative tumor inhibition rate

[0144] Group D18 relative tumor proliferation rate D18 tumor inhibition rate Model control group / / Anti-mouse PD-1 57.92％ 42.08％ CICC24917 82.10％ 17.90％ Amuci-3 69.27％ 30.73％

[0145] It can be seen that Amuci-3 has a significant inhibitory effect on the growth of colon cancer cell CT-26 xenograft tumors in BALB / c mice.

Claims

1. A kind of Akkermansia muciniphila Amuci-3 or its pure culture or inactivated bacteria, wherein the deposit number of Akkermansia muciniphila is CCTCC NO: M2024049.

2. The method for culturing Akkermansia muciniphila according to claim 1, comprising inoculating the Akkermansia muciniphila into a culture medium, performing proliferation culture, and obtaining proliferated Akkermansia muciniphila.

3. A food composition, a health product composition or a pharmaceutical composition, comprising the Akkermansia muciniphila according to claim 1 or a pure culture thereof or an inactivated bacterial cell thereof.

4. The food composition, health product composition or pharmaceutical composition according to claim 3, characterized in that: The composition further contains a freeze-drying protective agent or a preparation auxiliary material.

5. The food composition, health product composition or pharmaceutical composition according to claim 3, characterized in that: A single preparation of the composition contains 10 6 ~10 15 CFU of Akkermansia muciniphila or killed bacteria.

6. Use of Akkermansia muciniphila or a pure culture thereof according to claim 1 in the preparation of drugs for lowering cholesterol, controlling body weight or preventing tumors.

7. Use of the inactivated bacteria of Akkermansia muciniphila according to claim 1 in the preparation of health products or medicines for controlling weight.

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