Acanthamoeba polyphaga and application thereof in reducing trimethylamine-n-oxide

Abstract

The present application relates to a strain of Akkermansia muciniphila and its application, and belongs to the technical field of microbial strains. Specifically, the strain is Akkermansia muciniphila AUSA023, and its preservation number in Guangdong Microbial Culture Collection Center is GDMCC No. 65716. Akkermansia muciniphila AUSA023 can reduce the content of trimethylamine in human intestinal tract and oxidized trimethylamine in blood, and is used for preventing or adjuvant treating related diseases.

Description

Technical Field

[0001] This invention relates to a strain of Akkermansia myxophilus AUSA023 and its use in preparing products for reducing trimethylamine oxide, belonging to the field of microbial technology. Background Technology

[0002] Trimethylamine N-oxide (TMAO) is a phosphatidylcholine and carnitine metabolite produced by the gut microbiota. It is generated in the liver through a chemical reaction between trimethylamine and oxygen. Trimethylamine itself is produced in the intestines from components of certain foods (such as red meat, egg yolks, and certain fish), then enters the bloodstream and is converted into trimethylamine N-oxide by the liver.

[0003] Recent studies have shown that TMAO is associated with the development and progression of diabetes, cardiovascular disease, obesity, chronic kidney disease, metabolic diseases, neurological disorders, and cancer. From a pathological perspective, TMAO metabolic imbalance may affect cellular metabolism and inflammatory responses. Therefore, TMAO, as a novel detection indicator, is gradually demonstrating its important value in clinical medicine and may become a potential biomarker for disease diagnosis, prognostic assessment, and treatment intervention. Among trimethylamine metabolism disorders, trimethylamineuria (TMAU) is a typical example. Patients have a deficiency in enzyme genes, preventing the liver from effectively converting trimethylamine (TMA) into TMAO. Currently, clinical treatment mainly focuses on reducing TMA production by limiting the intake of foods rich in TMA precursors (choline, carnitine, etc.). Simultaneously, elevated TMA levels are also associated with various odor problems. Bacterial metabolism in the vagina, on the skin surface, and in the mouth produces TMA, leading to abnormal vaginal odor, body odor, and halitosis, respectively. Furthermore, elevated TMAO levels in the blood during pregnancy (late pregnancy and postpartum) are significantly associated with an increased risk of thrombosis; therefore, reducing TMA and TMAO levels is of great significance.

[0004] Currently, there are two main approaches to lowering TMAO levels: medication and dietary adjustments. TMA lysin inhibitors, such as 3,3-dimethyl-1-butanol (DMB), iodomethylcholine (IMC), and flumethylcholine (FMC), can target and inhibit TMA-producing bacteria, and statins can also lower TMAO levels. However, there are currently no approved drugs specifically for lowering TMAO. The safety and efficacy of DMB in humans remain to be verified, and statins have side effects such as liver damage and rhabdomyolysis. Therefore, exploring safe and effective strategies for lowering TMAO may open up new avenues for the treatment of related diseases.

[0005] Given the regulatory role of probiotics in the gut microbiota, they may be an effective strategy for addressing TMAO-related problems. Akkermansia muciniphila (AKK) constitutes a significant portion of the human gut microbiota, approximately 3%–5%, and is one of the most promising next-generation probiotics developed based on specific symbiotic bacteria in the human gut. AKK has been shown to effectively improve obesity, type 1 / 2 diabetes, hyperuricemia, hepatic steatosis, intestinal inflammation, and cognitive impairment in mice, demonstrating promising clinical applications. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing product technologies and provide Akkermansia mycotoxin-loving bacteria for reducing the content of trimethylamine in the human intestine and trimethylamine oxide in serum.

[0007] The technical solution adopted in this invention is:

[0008] The first objective of this invention is to provide a strain of Akkermansia muciniphila, AUSA023, which was deposited on January 3, 2025, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 65716), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Taxonomically, it is named Akkermansia muciniphila.

[0009] In this invention, the Ackermansia muciniphila AUSA023 was isolated from infant feces.

[0010] In this invention, the Akkermansia muciniphila AUSA023 includes one or more of the following: live bacteria, dead bacteria, cell fragments, fermentation supernatant, modified bacteria, mutant bacteria, and / or induced mutant bacteria.

[0011] The second objective of this invention is to provide a culture medium for *Akermansia myxophilus*, comprising: 5-20 g / L yeast extract, 5-20 g / L tryptone, 5-30 g / L glucose, 2-10 g / L threonine, 1-10 g / L N-acetylglucosamine, and vitamin B1. 12 50-200 μg / L, Vitamin B9 50-200 μg / L.

[0012] In the culture medium provided by the present invention, as a preferred embodiment, the culture medium composition is: yeast extract 10 g / L, tryptone 10 g / L, glucose 15 g / L, threonine 5 g / L, N-acetylglucosamine 3 g / L, and vitamin B1. 12 100 μg / L, Vitamin B9 100 μg / L.

[0013] The present invention also provides the use of the culture medium in culturing Akkermansia myxophilus.

[0014] A third objective of this invention is to provide a microbial inoculant containing Akkermansia myxophilus AUSA023 or Akkermansia myxophilus AUSA023 cultured in the above-mentioned culture medium.

[0015] As a preferred embodiment, the microbial agent provided by the present invention contains prebiotics, which are selected from one or more of the following: mannose oligosaccharides, fructooligosaccharides, maltodextrin, isomaltitol, isomaltooligosaccharides, galactooligosaccharides, xylooligosaccharides, inulin, etc. As a more preferred embodiment, the prebiotics are a mixture comprising fructooligosaccharides, isomaltitol, and isomaltooligosaccharides.

[0016] In this invention, the microbial agent can be prepared into tablets, capsules, powders, granules, semi-solid preparations, liquid preparations, etc. The semi-solid preparations include gels, and the liquid preparations include oil drops, aqueous solutions, etc.

[0017] In the microbial agent provided by this invention, the viable count of *Ackermania pseudomallei* AUSA023 is 1 × 10⁻⁶. 8 ~1×10 11 CFU / g. Preferably, the viable count of the *Ackermania* AUSA024 is 1 × 10⁻⁶. 9 ~1×10 10 CFU / g.

[0018] A fourth object of the present invention is to provide the use of Akkermansia myxophilus AUSA023 or an agent containing Akkermansia myxophilus AUSA023 in the preparation of a product for reducing serum trimethylamine oxide.

[0019] A fifth object of the present invention is to provide the use of Akkermansia myxophilus AUSA023 or an agent containing Akkermansia myxophilus AUSA023 in the preparation of a product for reducing trimethylamine.

[0020] A sixth object of the present invention is to provide the use of Akkermansia myxophilus AUSA023 or an agent containing Akkermansia myxophilus AUSA023 in the preparation of products for treating diseases associated with elevated serum trimethylamine oxide.

[0021] A seventh object of the present invention is to provide the use of Akkermansia myxophilus AUSA023 or an agent containing Akkermansia myxophilus AUSA023 in the preparation of products for treating trimethylamine metabolism disorders.

[0022] In this invention, diseases associated with elevated serum trimethylamine oxide include diabetes, cardiovascular disease, obesity, chronic kidney disease, metabolic diseases, neurological diseases, and cancer caused by elevated trimethylamine levels. The disorder of trimethylamine metabolism is trimethylamineuria (TMAU).

[0023] The *Ackermannii myxophilus* AUSA023 strain provided by this invention was isolated from infant feces. This strain has the advantage of utilizing mucin in the intestine for growth, exhibiting strong colonization ability and being difficult to eradicate. The *Ackermannii myxophilus* AUSA023 culture medium provided by this invention is prepared by adding vitamin B to the culture medium. 12 In conjunction with vitamin B9, high-density growth and enrichment of Akkermansia myxophilus strains, including Akkermansia myxophilus AUSA024, can be achieved. Therefore, the culture medium provided by this invention is also suitable for other Akkermansia myxophilus strains. The Akkermansia myxophilus AUSA023 or a bacterial agent containing Akkermansia myxophilus AUSA023 provided by this invention non-obviously reduces intestinal TMA-producing microorganisms, thereby lowering the levels of trimethylamine and serum trimethylamine oxidase in the human body, achieving the purpose of preventing and adjuvant treatment of diseases, with significant effects. Attached Figure Description

[0024] Figure 1 Image: This is a fluorescence quantitative PCR detection image of Akkermansia AUSA023, a myxotrophic strain, provided by the present invention.

[0025] 1. Akkermansia myxophilus AUSA023; 2. Water

[0026] Figure 2 Gram staining image of Akkermansia AUSA023, a myxotrophic strain provided by this invention.

[0027] Figure 3 : An phylogenetic tree diagram of Ackermania AUSA023 and other Ackermania species provided by this invention. Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments, but this is not intended to limit the present invention. Any equivalent substitutions made in the art in accordance with the content of the present invention shall fall within the protection scope of the present invention.

[0029] Example 1: Isolation and identification of Akkermansia AUSA023, a myxotrophic strain provided by the present invention.

[0030] 1. Strain isolation:

[0031] The separation steps are as follows:

[0032] (1) Sample collection: Feces were collected from a healthy infant volunteer in Shenzhen.

[0033] (2) High-throughput screening of strains: 1g of collected infant fecal samples were added to 9mL of sterile physiological saline and mixed thoroughly to prepare a sample suspension. 1mL of the sample suspension was added to 9mL of sterile physiological saline and diluted a series of 10-fold values. The resulting 10-fold concentrations were then collected. -5 10 -6 and 10 -7 0.1 mL of the diluted solution was spread onto solid plates of AKK strain-specific selection medium (Table 1), with three replicates for each dilution. The plates were anaerobically incubated at 37°C for 72 h. Round, transparent, small (<1 mm) single colonies were then picked up with a sterile toothpick and inoculated into 96-well plates for further culture. 200 μL of AKK strain-specific selection liquid medium was added to each well of the 96-well plate. The 96-well plates were then anaerobically incubated at 37°C for 48 h.

[0034] Table 1: Formulation of Specific Screening Culture Medium for Akkermansia myxophilus (AKK)

[0035]

[0036] (3) Initial screening of strains: 1 μL of bacterial culture from a 96-well plate was used as a DNA template, and specific primers for AKK strains (F: CAATCATGCGGAAGATATC, R: GAAGGTTTTGGATGCAAA) were used for DNA screening.

[0037] Using qPCR (quantitative real-time polymerase chain reaction) (Table 2), the bacteria in the wells showing fluorescent signals were potential AKK bacteria. Figure 1 These bacteria were then subjected to further streak purification culture.

[0038] Table 2: qPCR system for screening Akkermansia myxophilus

[0039]

[0040] (4) Observation of strain morphology: The selected strains were Gram-stained and their morphology was observed under a microscope. Figure 2 ).

[0041] 2. Strain identification:

[0042] (1) 16S rDNA sequencing analysis:

[0043] Strain DNA was extracted using a bacterial genomic DNA extraction kit, and the full-length 16S rDNA was amplified by PCR technology. Sequencing analysis was then performed to identify the species of the strains isolated in the above steps.

[0044] The 16S rDNA gene sequence of the *Akermansia obliterans* AUSA023 is as follows:

[0045]

[0046] The obtained 16S rDNA full-length sequence was searched and compared for similarity using the BLAST webpage of the National Center for Biotechnology Information (NCBI) (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The results showed greater than 99% identity with the 16S rDNA sequences of the first 100 reported Akkermanis muciniphila strains, thus identifying it as Akkermanis muciniphila. However, the identity with the 16S rDNA of the first 100 reported Akkermanis muciniphila strains is not completely identical, indicating that the Akkermanis muciniphila AUSA023 strain provided in this invention is original. (See attached...) Figure 3 The diagram shows the phylogenetic tree of Akkermanisa muciniphila AUSA023 and other Akkermania species.

[0047] The bacterial culture medium was mixed with 50% glycerol at a ratio of 2:1 and stored at -80℃ for later use.

[0048] (2) Whole genome sequence analysis

[0049] DNA was extracted from the bacterial strain using a bacterial genomic DNA extraction kit, and whole-genome sequencing analysis was performed to further identify the genomic characteristics of the strain. The whole genome sequence of the *Akkermanisa muciniphila* AUSA023 has been uploaded to the National Center for Biotechnology Information (NCBI) in the United States, with Accession No. CP186329.

[0050] Example 2: High-density culture method of Akkermansia myxophilus AUSA023

[0051] Traditional Akkermansia myxophilus cultivation requires the addition of a mixture of animal-derived mucin and vitamins (as shown in Table 1). By analyzing the metabolic pathways of the Akkermansia myxophilus genome, carbon, nitrogen, and vitamin sources were prepared according to Table 3 to replace the mucin and vitamin mixture in the control medium. After cultivation, the OD600 values ​​were measured and compared with those of the medium formulated in Table 1 as a control to determine whether each candidate medium component could replace the mucin and vitamin mixture and achieve high-density growth of Akkermansia myxophilus.

[0052] The specific steps are as follows: Akkermansia muciniphilis AUSA023, preserved at -80℃, was activated for 48 hours in the control medium (Table 1) mentioned above. Then, it was inoculated at a 1% inoculum and cultured anaerobicly (10% H2, 10% CO2, 80% N2) for 48 hours. The OD600 (200 μL / well) was then measured using a microplate reader, and the results are shown in Table 3. The results showed that without the addition of mucin, ATCC vitamin Mix, and ATCC trace minerals mix, the optimal concentration of yeast extract (10 g / L), tryptone (10 g / L), glucose (15 g / L), threonine (5 g / L), N-acetylglucosamine (3 g / L), and vitamin B1 was significantly higher than that of other bacteria. 12 100 μg / L, Vitamin B9 100 μg / L, Akkermansia myxophilus AUSA023 OD 600 It can reach 1.6, and the viable count can reach 1×10⁶. 9 CFU / ml.

[0053] Table 3: Optimization of High-Density Culture of Akkermansia myxophilus

[0054]

[0055] Example 3: Preparation of freeze-dried bacterial powder containing the myxotrophic Akkermansia AUSA023 described in this invention

[0056] Preparation process of Akkermansia muciniphila AUSA023 powder:

[0057] (1) The *Ackermania myxophilus* strain AUSA023 was activated by streaking onto solid culture medium prepared according to the optimized formula in Table 3 of Example 3, and incubated at 37°C for 72 h. After activation, single colonies were picked and cultured anaerobically in 10 ml of liquid culture medium at 37°C for 48 h, with an inoculum size of 5%.

[0058] (2) The culture was expanded again to 100 ml under the same culture medium and conditions to serve as the seed culture for fermentation. 5% of the seed culture was inoculated into 4 L of fermentation medium. The fermentation conditions were 37°C, pH 6.5, nitrogen flow rate of 0.5 L / min, and fermentation time of 44–48 h. After fermentation, the mycelial sludge was collected by centrifugation at 6000 rpm for 10 min.

[0059] (3) Freeze-drying. The mycelial sludge and a preservative (preservative preparation method: mix 12g skim milk powder, 5g sucrose, 3g lactose, 3g trehalose, and 5g fructooligosaccharides in 100ml water) were mixed at a weight ratio of 1:2, and freeze-dried for 30-48 hours. The prepared freeze-dried mycelial powder had a viable count greater than 1.0 × 10⁻⁶. 9 CFU / g.

[0060] Example 4: In vitro inhibition experiment of trimethylamine (TMA)-producing bacteria

[0061] I. Methods

[0062] Preparation of bacterial suspension and bacterial cell fragments: Akkermansia myxophilus strain AUSA023 was selected and cultured according to its culture medium and conditions. The preparation method of the bacterial agent was as follows: Akkermansia myxophilus strain AUSA023 was prepared into solid plates using the optimized formula in Table 3 of Example 2, and cultured under anaerobic conditions at 35–37℃ for 24–48 h until the strain concentration reached 1 × 10⁻⁶. 9 CFU / mL. Inoculate 2% of the culture medium into 500 mL of the appropriate liquid medium and culture. After culture, centrifuge at 4°C, 6000 rpm for 10 min. Collect the supernatant and bacterial cells. Store the bacterial culture for later use or freeze at -80°C. Resuspend the bacterial cells in 50 mL of sterile water or 1×PBS buffer and centrifuge twice. Resuspend the washed bacterial cells again and sonicate at 25 kHz, 450 W for 20 min. Mix with lysozyme at a 1:1 ratio and incubate at 37°C for 30 min before centrifugation. Finally, store for later use or freeze at -80°C.

[0063] Prepare Akkermansia ATCC BAA-835 bacterial suspension and cell disruption according to the above method.

[0064] In vitro inhibition of TMA bacteria: The experiment was divided into 5 groups, namely 4 experimental groups and 1 control group. The experimental groups included culture supernatant of Akkermansia myxophilus and bacterial cell fragments, namely culture supernatant of Akkermansia myxophilus AUSA023, culture supernatant of Akkermansia myxophilus ATCC BAA-835, bacterial cell fragments of Akkermansia myxophilus AUSA023, and bacterial cell fragments of Akkermansia myxophilus ATCC BAA-835. The control group used 1×PBS buffer. The bacteria containing TMA lysin (CntA or cutC / D gene cluster), such as Proteus mirabilis, Clostridium saccharolyticum, or Escherichia coli, were co-cultured at 35-37℃ for 24 h at a 1:1 volume ratio. The OD of the culture medium was measured using an enzyme-linked immunosorbent assay (ELISA) reader. 600 Monitor the growth of TMA-producing bacteria and determine the OD value after 24 hours. 600 Size determination of the inhibitory effect of Akkermansia muciniphila AUSA023 on TMA-producing intestinal bacteria.

[0065] II. Results

[0066] As shown in Table 4, negative values ​​represent the percentage reduction compared to the blank control; that is, the larger the value after the negative sign, the better the antibacterial effect. Compared with the blank control (1×PBS) group, the OD values ​​of ATCC BAA-835 culture supernatant and ATCC BAA-835 bacterial cell fragments group were significantly higher. 600 No significant differences were found in the OD values ​​of the AUSA023 culture supernatant group, the AUSA023 cell fragment group, and the [other groups]. 600 All values ​​were significantly reduced (P < 0.05), indicating that Akkermansia myxophilus AUSA023 produced a significant reduction in OD. 600 The effect of in vitro inhibition of trimethylamine (TMA)-producing bacteria is significant.

[0067] Table 4: Inhibitory effect of the bacterial agent of the present invention on TMA-producing intestinal bacteria

[0068]

[0069] Note: Compared with the blank control group, a P<0.05; compared with the ATCC BAA-835 culture supernatant group, b P<0.05; compared with the ATCCBAA-835 bacterial cell fragment group, c P<0.05.

[0070] Example 5: In vitro experiment on inhibition of choline to trimethylamine (TMA) conversion

[0071] I. Methods

[0072] The preparation of bacterial suspensions and cell fragments of Akkermansia myxophilus AUSA023 and Akkermansia myxophilus ATCCBAA-835 were the same as in Example 4 above.

[0073] Prepare Akkermansia ATCCBAA-835 bacterial suspension and cell disruption according to the above method.

[0074] In vitro inhibition of TMA bacteria: The experiment was divided into 5 groups: 4 experimental groups and 1 control group. The experimental groups included probiotic culture supernatant and probiotic cell fragments (specifically, Akkermansia myxophilus AUSA023 culture supernatant and fragments of Akkermansia myxophilus AUSA023 cells). The control group used 1×PBS buffer. These were then co-cultured at a 1:1 volume ratio with bacteria containing TMA lysin (CntA or cutC / D gene clusters), such as Proteus mirabilis, Clostridium saccharolyticum, or Escherichia coli, at 35–37°C for 24 h. Choline chloride (CC) (1M stock solution) was added to the culture medium to achieve a final concentration of 0.5 mmol / L. After cultivation, 600 μL of a mixed solution of acetonitrile:methanol:water (V:V:V, 40:40:20) was added to each sample to precipitate proteins and other substances. Simultaneously, d9-TMA at a final concentration of 10 μmol / L was added as an internal standard. The mixture was shaken and incubated at -80℃ for 2 h. After centrifugation at 12000×g for 15 min at 4℃, the supernatant was transferred to a sample vial and stored at -80℃. TMA concentration was determined using high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS). The concentration of TMA produced during cultivation was determined by constructing regression curves using the response (peak area) versus concentration for each standard, using TMA and d9-TMA calibration standards.

[0075] II. Results

[0076] As shown in Table 5, compared with the blank control (1×PBS) group, there was no significant difference in TMA content between the ATCCBAA-835 culture supernatant group and the ATCCBAA-835 cell fragment group. However, the TMA content in both the AUSA023 culture supernatant group and the AUSA023 cell fragment group was significantly reduced (P<0.05). This indicates that Akkermansia myxophilus AUSA023 produced a significant effect in reducing TMA content, that is, it significantly inhibited the conversion of choline to trimethylamine (TMA) in vitro.

[0077] Compared with the ATCC BAA-835 culture supernatant group, the TMA content of the AUSA024 culture supernatant group was significantly lower (P<0.05), and the TMA content of the AUSA024 cell fragments group was also significantly lower than that of the ATCC BAA-835 cell fragments group (P<0.05).

[0078] Table 5: Effect of the bacterial agent of the present invention on the in vitro inhibition of choline to trimethylamine (TMA) conversion.

[0079]

[0080] Note: Compared with the blank control group, a P<0.05; compared with the ATCC BAA-835 culture supernatant group, b P<0.05; compared with the ATCCBAA-835 bacterial cell fragment group, c P<0.05.

[0081] Example 6: Effects of the composition of the present invention on TMAO in rats

[0082] I. Methods

[0083] Experimental animals and grouping: SD rats, half male and half female, weighing 200±20g, were purchased from Guangdong Provincial Medical Laboratory Animal Center. They were housed in an environment with room temperature of 18–28℃ and relative humidity of 40%–70%, with free access to food and water. After one week of acclimatization feeding with standard diet, they were randomly divided into groups as shown in Table 6 below, with 10 rats in each group.

[0084] Model building and probiotic administration: References and guidelines recommend a probiotic dose of 1 × 10⁻⁶ for rats. 8- 1×10 10 CFU / day was used, therefore three doses (low, medium, and high) were designed (Table 6). Except for the model control group, rats in other groups were administered the corresponding test samples by gavage at the doses specified in Table 6 (samples were diluted with physiological saline to the corresponding viable bacterial content). The model control group rats were administered an equal volume of phosphate-buffered saline by gavage daily. Simultaneously, all rats were administered 53 mg / kg choline chloride by gavage daily, and all rats had free access to a high-fat diet (purchased from Guangdong Provincial Medical Laboratory Animal Center). On day 14 of the experiment, plasma was collected from all rats using ocular vein sampling (after a 12-hour fast). All plasma samples were then centrifuged at 3000g for 20 minutes at 4°C, and serum was collected. The concentration of trimethylamine oxide (TMAO) was detected by liquid chromatography-mass spectrometry.

[0085] II. Results

[0086] As shown in Table 6, compared with the model control group, there was no significant difference in serum TMAO content in the ATCC BAA-835 group (P>0.05), while the plasma TMAO content in the low-dose AUSA023 group, medium-dose AUSA023 group, and high-dose AUSA023 group was significantly reduced (P<0.05), indicating that Akkermansia myxophilus AUSA023 has a significant effect on reducing serum TMAO content.

[0087] Compared with the ATCC BAA-835 group, the serum TMAO levels in the low-dose AUSA023 group, the medium-dose AUSA023 group, and the high-dose AUSA023 group were significantly different (P<0.05).

[0088] Table 6: Effects of the composition on serum TMAO levels in rats ( n = 9 to 10)

[0089]

[0090] Note: Compared with the model control group, a P<0.05; compared with the ATCC BAA-835 group, b P<0.05.

Claims

1. A strain of Akkermansia muciniphila AUSA023 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on January 3, 2025, with accession number GDMCC No. 65716.

2. The Akkermansia muciniphilis AUSA023 according to claim 1, characterized in that, The strain was isolated from infant feces.

3. A culture medium for *Akermansia myxophilus*, comprising: yeast extract 5–20 g / L, tryptone 5–20 g / L, glucose 5–30 g / L, threonine 2–10 g / L, N-acetylglucosamine 1–10 g / L, and vitamin B1. 12 50-200 μg / L, Vitamin B9 50-200 μg / L; as a preferred embodiment, the culture medium composition is: yeast extract 10 g / L, tryptone 10 g / L, glucose 15 g / L, threonine 5 g / L, N-acetylglucosamine 3 g / L, Vitamin B9 50-200 μg / L; 12 100 μg / L, Vitamin B9 100 μg / L.

4. Use of the culture medium according to claim 3 in culturing Akkermansia myxophilus.

5. A microbial agent containing Akkermansia myxophilus AUSA023 as described in claim 1 or Akkermansia myxophilus AUSA023 cultured in accordance with claim 3.

6. The microbial agent according to claim 5, characterized in that, The microbial agent contains prebiotics, which are selected from one or more of the following: mannose oligosaccharides, fructooligosaccharides, maltodextrin, isomaltitol, isomaltooligosaccharides, galactooligosaccharides, xylooligosaccharides, inulin, etc.

7. The microbial agent according to claim 5, characterized in that, The microbial agent can be prepared into tablets, capsules, powders, granules, semi-solid preparations, liquid preparations, and other formulations.

8. The microbial agent according to claim 5, characterized in that, The viable count of the *Ackermania* strain was 1 × 10⁻⁶. 8 ~1×10 11 CFU / g; preferably, the viable count of the myxotrophic Akkermansia is 1×10⁻⁶. 9 ~1×10 10 CFU / g.

9. Use of the Akkermansia myxophilus AUSA023 of claim 1 or the microbial agent of claim 5 in the preparation of a product for reducing serum trimethylamine oxide or treating diseases related to elevated blood trimethylamine oxide levels.

10. Use of the Akkermansia myxophilus AUSA023 of claim 1 or the bacterial agent of claim 5 in the preparation of a product for reducing trimethylamine or treating trimethylamineuria.

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