M. moricola for improving obesity and glucose and lipid metabolism

Abstract

The application belongs to the field of intestinal microbiology, and discloses a new mucinophilic Akkermansia with improved obesity and glycolipid metabolism, wherein the mucinophilic Akkermansia strain is Akk-101 and AMY001. The preservation number of Akk-101 is CGMCC No. 40786, and the preservation number of AMY001 is CGMCC No. 46141. The Akk-101 and AMY001 strains in the application are different from standard strains in nucleotide consistency and physiological and biochemical indexes, have better acid-base tolerance and colonization capacity than the standard strains, can more effectively inhibit the accumulation of liver cell lipids, reduce the fat deposition in nematodes, and have the effects of weight loss and improvement of glycolipid metabolism, so that related products can be effectively developed based on the strains.

Description

Technical Field

[0001] This invention relates to the field of gut microbiology, specifically to a novel Akkermansia muciniphila strain and its application in improving obesity and glucose and lipid metabolism. Background Technology

[0002] In recent years, the important role of the gut microbiota in glucose and lipid metabolism, obesity, and related metabolic diseases has been extensively studied. As a crucial regulator of host health, the gut microbiota's role in metabolic diseases has attracted widespread attention. Studies have found that gut microbiota imbalance (also known as "microbiota dysbiosis") is closely related to various metabolic diseases, including diabetes, obesity, and fatty liver. Specifically, alterations in the gut microbiota may affect the host's metabolic state, thereby participating in the occurrence and development of glucose and lipid metabolism disorders and obesity.

[0003] *A. muciniphila* (Akk bacteria), initially isolated from the feces of healthy individuals in Denmark in 2004, is an important member of the gut microbiota, accounting for 0.5%-5% of the total flora. Studies have shown that Akk bacteria have significant positive regulatory effects on intestinal barrier function, metabolic regulation, and inflammatory responses. In clinical and animal studies, the abundance of this bacterium is generally correlated with a healthy metabolic state, leading journals such as *Nature* to haile it as the "King of Bacteria." In studies on obese patients, the abundance of Akk bacteria was significantly lower than in healthy individuals, and its levels were negatively correlated with body weight and body fat percentage. Furthermore, Akk bacteria supplementation significantly improved insulin sensitivity in obese mice. Akk bacteria is currently considered one of the "next-generation probiotics." The European Food Safety Authority evaluated its safety in 2021, deeming Akk bacteria a well-defined, non-toxin-producing, non-pathogenic microorganism, part of the normal gut microbiota, and suitable for specific medical purposes. The development and industrialization of Akk strains are currently underway.

[0004] While Akk bacteria have demonstrated potential for improving metabolism to some extent, their application still faces certain challenges. On the one hand, the actual effects of different strains vary from person to person; on the other hand, their high requirements for culture conditions limit their widespread applicability. Furthermore, the functions of probiotics vary significantly at the strain level, necessitating specific strain development at the individual strain level. Currently, most Akk strains on the market are either the standard strain ATCC-BAA 835 or very similar. Since the standard strain originates from the Danish population, which differs from the Chinese population, and there is a strain monopoly, identifying a locally sourced Akk strain that is suitable for the Chinese physique and possesses superior efficacy would bring significant economic and social benefits, benefiting the health of the Chinese people and holding broad application prospects. Summary of the Invention

[0005] To address the aforementioned issues, this invention screened Akkermansia muciniphila strains Akk-101 and / or Akkermansia muciniphila AMY001 from the intestines of centenarians in Hainan, China, which are suitable for the physique of Chinese people and have excellent efficacy.

[0006] According to one aspect of the present invention, the Akk-101 myxophilus strain has the accession number CGMCC No. 40786, and the AMY001 myxophilus strain has the accession number CGMCC No. 46141.

[0007] According to one aspect of the invention, a culture of Akk-101 and / or AMY001 of Akk-101 and / or AMY001 is provided, the culture comprising fermentation broth, fermentation broth precipitate, fermentation broth supernatant, live bacteria, dead bacteria, lyophilized powder and lysate of Akk-101 and / or AMY001.

[0008] According to one aspect of the invention, a microbial agent is provided, the microbial agent containing *Ackermania pseudomallei* Akk-101 and / or *Ackermania pseudomallei* AMY001. Preferably, the microbial agent is a solid or liquid formulation.

[0009] According to one aspect of the present invention, a microbial composition is provided comprising *Ackermania pseudomallei* Akk-101 and *Ackermania pseudomallei* AMY001. Preferably, the microbial composition contains 1 × 10⁻⁶ ppm. 9 Total bacterial count of CFU or higher.

[0010] According to one aspect of the invention, the use of a composition comprising Akk-101 and / or AMY001, the culture, the microbial agent, or the microbial composition as an active ingredient in the preparation of a product for the prevention and improvement of overweight, obesity, type II diabetes, metabolic syndrome, hypoglycemia, hypolipidemia, or improvement of glucose intolerance is provided.

[0011] According to one aspect of the present invention, a method for preparing a product composition is provided, the method comprising: adding the Akk-101 and / or AMY001 Akk-101, the culture, the bacterial agent, or the microbial composition to a product matrix to obtain the product composition.

[0012] According to one aspect of the present invention, a product is provided comprising the Akk-101 and / or AMY001 Akk-101, the culture, the inoculum, or the microbial composition.

[0013] The Akk-101 and AMY001 strains in this invention differ from the standard strains in terms of nucleotide consistency and physiological and biochemical indicators. They have superior acid-base tolerance and colonization ability compared to the standard strains, and more effectively inhibit lipid accumulation in liver cells, reduce fat deposition in nematodes, and have the effects of weight loss and improving glucose and lipid metabolism. Therefore, related products can be effectively developed based on these strains.

[0014] Biological Preservation

[0015] Bacterial species name: Akkermansia myxophilus ( Akkermansia muciniphila Akk-101

[0016] Latin name: Akkermansia muciniphila

[0017] Classification and nomenclature: Akkermansia myxophila Akkermansia muciniphila

[0018] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0019] Collection institution abbreviation: CGMCC

[0020] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0021] Date of preservation: September 13, 2023

[0022] CGMCC Registration Number: 40786

[0023] Bacterial species name: Akkermansia muciniphila AMY001

[0024] Latin name: Akkermansia muciniphila

[0025] Classification and nomenclature: Akkermansia myxophila Akkermansia muciniphila

[0026] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0027] Collection institution abbreviation: CGMCC

[0028] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0029] Deposit date: August 26, 2024

[0030] CGMCC Registration Number: 46141 Attached Figure Description

[0031] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0032] Figure 1 This is a comparison of the genome similarity of Akk-101 and AMY001 of the present invention with other Akk strains. (A) FastANI heatmap; (B) Evolutionary clade diagram.

[0033] Figure 2 The effects of the fermentation supernatant of Akk-101 and AMY001 of the present invention on lipid accumulation in HepG2 liver cancer cells are shown. n=6; *, p<0.05; **, p<0.01; ***, p<0.001.

[0034] Figure 3 The effects of Akk-101 and AMY001 bacterial suspensions of the present invention on lipid accumulation in nematodes are shown. n=30; *, p<0.05; **, p<0.01; ***, p<0.001.

[0035] Figure 4The effects of Akk-101 and AMY001, the myxotrophs of this invention, on the body weight of obese mice are shown. (A) Body weight curve of mice, (B) Weight gain of mice, (C) White adipose tissue weight, and (D) Liver weight. n=8,*, p<0.05;**, p<0.01; ***, p<0.001.

[0036] Figure 5 The effects of Akk-101 and AMY001, the myxotrophs of this invention, on blood lipid levels in obese mice are shown. (A) Serum total cholesterol (TC) content, (B) Serum total triglyceride (TG) content, (C) Serum low-density lipoprotein cholesterol (LDL-c) content, (D) Serum high-density lipoprotein cholesterol (HDL-c) content, (E) Liver HE staining. n=8, *, p<0.05; **, p<0.01; ***, p<0.001.

[0037] Figure 6 The effects of Akk-101 and AMY001 *Acetobacter* strains of the present invention on glucose metabolism in obese mice are illustrated. (A) Oral glucose tolerance test (OGTT) in mice, (B) AUC statistics of OGTT, (C) Fasting blood glucose level at 8 weeks, (D) Insulin level, (E) Serum GLP-1 level. n=8, *, p<0.05; **, p<0.01; ***, p<0.001.

[0038] Figure 7 The effects of the Akk strain of *Ackermania*, a myxophile strain of the present invention, on gene expression of important molecules in the glycolipid metabolic pathway are shown. (A) AMPK, (B) AKT1, (C) GSK3b, (D) GCK, (E) PEPCK, (F) GLUT4, (G) GLP-1R, (H) GPR41, and (I) PC1 / 3. n=8, *, p<0.05; **, p<0.01; ***, p<0.001.

[0039] Figure 8 The effects of the Akk strain of Akkermansia muciniphila of the present invention on glucosidase (A), amylase (B), and cellular glucose consumption (C) are shown. n=3, *, p<0.05; **, p<0.01; ***, p<0.001. Detailed Implementation

[0040] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0041] Acquisition and characteristics of strains:

[0042] The Akk-101 and AMY001 strains were isolated from intestinal samples of centenarians in Hainan, China, and after rigorous screening, they were confirmed to have significant effects superior to the standard strain in anti-obesity and improving glucose and lipid metabolism. Both strains differ from the Akk bacterium standard strain in nucleotide identity and possess unique physiological and biochemical characteristics.

[0043] Example 1. Identification of the myxophilic Akkman strains Akk-101 and AMY001 of the present invention

[0044] The *Ackermania* strain used in this invention was isolated from fecal samples of centenarians in Hainan Province. The subjects had not taken antibiotics within the two months prior to collection and had no history of gastrointestinal diseases. The culture conditions for the strain were as described in the previous patent (CN 117701423A).

[0045] (1) Strain identification

[0046] Frozen bacterial strains stored at -80℃ were removed, inoculated into liquid culture medium, and incubated in an anaerobic incubator at 37℃ for 48 hours. The bacterial pellet was then collected by centrifugation, and bacterial DNA was extracted and sent to a sequencing company. After sequencing, the genomes of Akk-101 and AMY001 were assembled and separated to obtain the genome sequences. Alignment with the software gtdbtk (https: / / github.com / Ecogenomics / GTDBTk) revealed that the Akk-101 genome closely matched the reference strain Akkermansia muciniphilaATCC BAA-835. T The average nucleotide identity comparison value (ANI) for (GCF_000020225.1) is 97.26. Figure 1 A), AMY001 genome and reference strain Akkermansia muciniphila ATCC BAA-835 T The average nucleotide identity comparison value (ANI) for (GCF_000020225.1) is 97.5. Figure 1 A) It can be identified that both strains belong to the Akkermansia species, and the ANI value of Akk-101 and AMY001 strains is 98.1. In order to further determine the phylogenetic relationship between the newly discovered Akk strain and the standard strain, its gene sequence was clustered with several representative species and strains of Akkermansia in the NCBI database ( Figure 1B). The Akk-101 and AMY001 strains clustered in different branches from the standard strain BAA-835, indicating that the Akk-101 and AMY001 strains described in this invention are Akkermansia muciniphila and are significantly different from the standard strain BAA-835. The ANI values ​​of the two strains differ by 2.74 and 2.5, respectively, which is higher than that of the Akkermansia muciniphila strain disclosed in other patents (such as Chinese invention patent with publication number CN118421503A), and the similarity with the standard strain is 99.71%.

[0047] (2) Preservation of strains

[0048] The Akk-101 myxophilic Akkman strain described in this invention was deposited on September 13, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 40786.

[0049] The Akkermansia myxophilus AMY001 described in this invention was deposited on August 26, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46141.

[0050] Example 2. Assessment of the colonization ability of Akk-101 and AMY001 strains of Akkermansia myxophilus.

[0051] The ability of probiotics to automatically aggregate and their surface hydrophobicity are key indicators of probiotic adhesion to the host's gut, directly affecting their colonization ability in the gut.

[0052] ① Autoaggregation Assay: Normally cultured Akk-101, AMY001, and BAA-835 bacterial suspensions were centrifuged to obtain bacterial precipitates. The precipitates were then washed three times with PBS, and the OD600 was adjusted to approximately 0.60 before being added to sterile test tubes. The test tubes were placed at 37°C and allowed to stand for a period of time to allow autoaggregation. After standing, the test tubes were gently shaken to evenly disperse the non-aggregated cells in the liquid. The absorbance of the supernatant in the test tube was measured using a spectrophotometer to reflect the number of non-aggregated cells. Simultaneously, the absorbance of the original bacterial suspension was measured as a control. Autoaggregation percentage = 1 - (Supernatant absorbance / Original bacterial suspension absorbance) × 100%.

[0053] According to Table 1, the autoaggregation rates of Akk-101 after 2h, 6h, and 24h of static incubation were 52.31%, 71.79%, and 92.55%, respectively; the autoaggregation rates of AMY001 after 2h, 6h, and 24h of static incubation were 44.84%, 63.52%, and 88.36%, respectively, which were higher than the autoaggregation rates of the BAA-835 standard strain at the same time (40.98%, 62.32%, and 85.15%, respectively).

[0054] Table 1. Autoaggregation rate of strains

[0055]

[0056] ② Hydrophobicity test: Centrifuge the overnight cultured bacterial suspension and collect the bacterial cells. Wash the bacterial cells twice with physiological saline (or PBS buffer) and adjust the OD600 to 1.00±0.05. Take 2 mL of the adjusted bacterial suspension and add 400 μL of xylene. Shake for 30 s, pause for 10 s, shake for another 30 s, and let stand for 5, 15, 30, and 60 min before separation. Carefully aspirate the upper xylene layer and then measure the OD value of the lower aqueous phase. Calculate the hydrophobicity based on the measured OD value. Hydrophobicity H% = (1 - A / A0) × 100%, where A0 is the OD value of the bacterial suspension before adding xylene, and A is the OD value of the aqueous phase after adding xylene.

[0057] According to Table 2, the hydrophobicity of Akkermansia myxophilus strains increased with time. During the detection period, Akk-101 reached its highest hydrophobicity at 60 min, with a hydrophobicity of 45.19%; AMY001 had a highest hydrophobicity of 31.82%; while the BAA-835 standard strain had a hydrophobicity of 29.94% at 60 min. Based on the commonly used judgment range [hydrophobicity is divided into low (0~29%), medium (30%~59%), and high (60~100%)], it is indicated that the hydrophobicity of Akk-101 and AMY001 strains provided by this invention is moderately high, while the hydrophobicity of BAA-835 strain is low.

[0058] Table 2. Surface hydrophobicity of strains

[0059]

[0060] The results of the autoaggregation and surface hydrophobicity experiments of the strains showed that the adhesion and colonization potential of the Akk-101 and AMY001 strains of Akkermansia mycotoxin provided by the present invention was higher than that of the standard strain BAA-835.

[0061] Example 3. Gastrointestinal tolerance assessment of Akk-101 and AMY001 strains of Akkermansia myxophilus.

[0062] (1) Preparation of artificial stomach, intestinal fluid and bile salt solution

[0063] Artificial gastric fluid: Accurately measure 20 ml of 1 mol / L hydrochloric acid solution, add distilled water or deionized water to adjust the pH of the hydrochloric acid solution to 2.5, then add pepsin (1 g / 100 ml) to the adjusted hydrochloric acid solution, and use it after it is fully dissolved.

[0064] Artificial intestinal fluid: Weigh 6.8 g of potassium dihydrogen phosphate and 10 g of pancreatic enzyme, add distilled water or deionized water to dissolve them completely, adjust the pH value to 6.8 with 0.1 mol / L sodium hydroxide solution, and dilute to 1000 ml before use.

[0065] Bile salt solution: Weigh out 0, 0.1, 0.2 and 0.3 g of bile salts respectively, add them to distilled water or deionized water, stir with a magnetic stirrer or glass rod until the bile salts are completely dissolved, and bring the volume to 100 mL before use.

[0066] (2) Experimental methods

[0067] Viable cells of Akk-101, AMY001, and BAA-835 after culture were collected and centrifuged at 13,000 rpm and 4°C for 15 min. The supernatant was discarded, and the bacterial pellet was collected and resuspended in physiological saline. The viable count was 10⁻⁶ cells / mL. 9 CFU / mL was inoculated into artificial gastric / intestinal fluid and bile salt solution (0.1%, 0.2%, 0.3%), respectively. After incubation at 37℃ for 0, 1, 2, and 4 hours, samples were taken for viable cell count detection. The logarithmic value of viable cell count at 0 hours was used as a control. The survival rate was calculated as follows: survival rate (%) = (live cell count at 1 hour / 2 hours / 4 hours / live cell count at 0 hours) * 100%.

[0068] Table 3 shows that the survival rates of strain Akk-101 remained at 84.4% and 83.7% after incubation in simulated gastric and intestinal fluids for up to 4 hours, respectively. Akk-101 also exhibited high tolerance to bile salts, but this tolerance gradually decreased with increasing bile salt concentration and incubation time. Its survival rate in high-concentration (0.3%) bile salts after 4 hours was still 68.6%. The survival rates of strain AMY001 after 4 hours of incubation in simulated gastric and intestinal fluids were 79.2% and 81.1%, respectively, while the survival rate in high-concentration (0.3%) bile salts after 4 hours was still 56.5%. These results indicate that strains Akk-101 and AMY001 possess excellent acid-base tolerance, allowing them to withstand the highly acidic and alkaline environment of the gastrointestinal tract after ingestion, maintaining a high bacterial count in the intestines, which is beneficial for maximizing the efficacy of probiotics. In comparison, the BAA-835 standard strain exhibited lower tolerance to acid and alkali (artificial gastric juice: 78.4%, artificial intestinal juice: 79.7%) and bile salts (4h, 53.9%) than the Akk-101 and AMY001 strains provided in this invention. The assessment results of gastric acid and bile salt tolerance indicate that the Akk-101 and AMY001 strains have strong survival ability and good adaptability in the gastrointestinal tract, possessing the potential for long-term survival and probiotic effects. This lays the foundation for the development and application of the Akk-101 and AMY001 strains.

[0069] Table 3. Survival rate of Akk strain in simulated gastrointestinal fluid and bile salt solution

[0070]

[0071] Example 4. Akk-101 and AMY001 strains of Akkermansia myxophilus inhibit cellular lipid accumulation.

[0072] This embodiment was used to evaluate the effects of Akk-101 and AMY001 strains of Akkermansia myxophilus on intracellular lipid accumulation. The strain samples involved were culture supernatants. The obtained strain working solutions were centrifuged at 12000 r / min for 10 min at 4°C, and the supernatant was collected as the strain culture supernatant sample, which was used in this experiment.

[0073] The human hepatocellular carcinoma HepG2 cells used in this invention were purchased from the National Biomedical Experimental Cell Resource Bank and cultured using conventional methods in the field. The human hepatocellular carcinoma HepG2 cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2. During the experiment, HepG2 cells in good logarithmic growth phase were seeded at an appropriate concentration in 96-well plates. When the cells reached 80% confluence, the original medium was discarded, and fresh medium containing oleic acid (final concentration 100 µM) was added. The oleic acid was used to construct the lipid accumulation model, and 30% (v / v) of Akk-101 / AMY001 / ATCC BAA-835 standard strain culture supernatant was added. After culturing for 20-24 h, the medium was discarded, and the cells were fixed with 4% paraformaldehyde at room temperature for 30 min. After washing with PBS, Oil Red O staining solution was added to each well for 30 min at room temperature. After staining, the cells were rinsed three times with PBS. After the final rinse, the PBS was discarded, and 100 µL of DMSO was added to each well to fully dissolve the Oil Red. The absorbance at 358 nm was then measured to assess the intracellular lipid accumulation. Each sample was prepared in quadruplicate wells, with an equal volume of blank culture medium added as a negative control.

[0074] according to Figure 2 As shown, the OD358nm reading of cells treated with oleic acid after Oil Red O staining was 0.396 (average), significantly higher than that of the control group NC (0.308). The average absorbance of OD358nm after treatment with Akk-101 supernatant was approximately 0.329, with a lipid reduction rate of approximately 16.9%. The average absorbance of OD358nm after treatment with AMY001 supernatant was approximately 0.341, with a lipid reduction rate of approximately 13.8%. The OD358nm reading of cells treated with the BAA-835 standard strain was approximately 0.354, with a lipid reduction rate of approximately 10.6%, significantly lower than the effect brought by the Akk-101 strain. This indicates that the Akk-101 strain of Acermetia gravidarum provided by this invention has a significantly stronger ability to inhibit lipid accumulation in HepG2 cells than the standard strain BAA-835.

[0075] Example 5. Akk-101 and AMY001 strains of Akkermansia myxophilus inhibit lipid accumulation in nematodes.

[0076] This example was used to evaluate the effects of Akk-101 and AMY001 strains of *Ackermania pseudomallei* on lipid accumulation in the model organism *Nematodea*. The strain samples involved were live bacterial suspensions and pasteurized dead bacteria. The obtained working solutions of the strains were centrifuged at 12000 r / min for 10 min at 4°C, the bacterial pellet was collected, resuspended in sterile PBS, and adjusted to a suitable viable count for the experiment (e.g., 5 × 10⁻⁶). 9(CFU / mL). The pasteurized dead bacteria can be obtained by heating live bacteria at a temperature of 65-85℃ for 30 minutes to kill them.

[0077] Take Akk-101 / BAA-835 and Escherichia coli OP50 bacterial suspensions (both with a concentration of 10). 7 CFU / mL was plated on nematode growth medium (NGM) and cultured overnight. Akk-101, AMY001, and BAA-835 strains were cultured normally in liquid medium. Escherichia coli OP50 was cultured in LB liquid medium under the following conditions: temperature 37℃, time 20h, and rotation speed 200rpm. The NGM medium formulation was: 3g sodium chloride, 17g agar powder, 2.5g peptone, 1 mL 1M CaCl2, 1 mL 1M MgSO4, 25 mL 1M KPO4 buffer, and 1 mL 5mg / mL cholesterol. This experiment used L4-stage nematodes, randomly divided into seven groups: a control group (OP50), a model group, a live Akk-101 group, a dead Akk-101 group (pAkk-101), a live AMY001 group, a dead AMY001 group (pAMY001), and a live BAA-835 group, with 30 nematodes in each group. The day the L4-stage nematodes were collected was designated as day 0. During the experiment, the nematodes were cultured in NGM medium containing OP50 or OP50 + Akk-101 / pAkk-101 / AMY001 / pAMY001 / BAA-835 every two days. After one week of culture, the nematodes were stained with Oil Red stained gel and photographed according to the manufacturer's instructions. ImageJ software was used to analyze the staining intensity of each group.

[0078] The results are as follows Figure 3As shown, the average staining intensity of nematodes fed a normal diet was approximately 10.7, while the staining intensity in the group fed only cholesterol-rich nematodes was 17.7. When fed various Akk strain suspensions, the Oil Red staining intensity of lipid droplets in the nematodes was lower than that in the model group. The staining intensity after treatment with live bacterial suspension was weaker than that after treatment with dead bacterial suspension, indicating that the optimal form for lipid reduction by the two Akk strains was live bacterial treatment. However, pasteurized dead bacteria still showed a significant inhibitory effect on lipid droplet accumulation. Specifically, among the two strains, the staining intensities of the Akk-101 live and inactivated bacteria provided by this invention were 12.4 and 13.9, respectively, with lipid reduction rates of 29.9% and 21.5%, significantly better than the BAA-835 treatment (staining intensity 14.0, lipid reduction rate 20.9%). After treatment with live and dead strains of AMY001, the lipid droplet staining intensity in zebrafish was 13.8 and 14.5, respectively, with lipid reduction rates of 22.0% and 18.1%. The live effect of AMY001 was slightly higher than that of the standard strain BAA-835 (22.0% vs 20.9%), but the effect after inactivation was slightly lower than that of the standard strain (18.1% vs 20.9%). Nevertheless, it still showed a significant inhibitory effect on lipid accumulation. These results indicate that both Akk strains (Akk-101 and AMY001), including both live and inactivated forms, can effectively reduce lipid droplet accumulation in nematodes, with significant lipid-reducing effects. Among them, strain Akk-101 showed a significant lipid-reducing effect, significantly better than known standard strains, and has strong development potential.

[0079] Example 6. Akk-101 and AMY001 of Akkermansia myxophilus inhibited weight gain in mice.

[0080] This example was used to evaluate the effect of Akk-101 strain of Akkermansia myxophilus on improving obesity in mice. The strain samples involved were live bacterial suspensions and pasteurized dead bacteria.

[0081] The mice used were purchased from Vital River Pharmaceuticals Beijing. They were 8-week-old male C57BL / 6 mice. After one week of acclimatization, they were randomly divided into 6 groups of 8 mice each: ① Normal diet group (NC group): fed a standard diet without probiotics; ② High-fat diet group (Model group): fed a high-fat diet without probiotics; ③ Akk-101 group (Akk-101 group): drinking a diet containing Akk-101 strain (5×10⁻⁶). 9 ④ Live bacterial suspension containing Akk-101 strain (CFU / mL / animal / day); 9 ⑤ Dead bacterial suspension containing CFU / mL / animal / day; ⑤ AMY001 group: Drinking water containing AMY001 strain (5×10⁻⁶ CFU / mL / animal / day) 9⑥ Live bacterial suspension (CFU / mL / animal / day); ⑥ AMY inactivated bacterial group (pAMY001 group): drinking water containing AMY001 strain (5×10 9 ⑦ Dead bacterial suspension containing CFU / mL / animal / day; ⑦ Akk-standard strain group (BAA-835 group): Drinking water containing BAA-835 standard strain (Akk, 5×10) 9 Live bacterial suspension (CFU / mL / mouse / day) was administered. Except for the normal diet group, all other groups were fed a high-fat diet for 8 weeks. Mice in the bacterial suspension group were gavage with 0.2 mL of the suspension daily, while the NC and Model groups received an equal volume of physiological saline. Mouse weight was measured weekly during the experiment. At the end of the experiment, all mice underwent a final weight measurement, and blood samples were collected for blood lipid and blood glucose determination. An oral glucose tolerance test (OGTT) was also performed to assess insulin sensitivity. After euthanasia, mouse liver and colon tissue were collected and cryopreserved for subsequent analysis.

[0082] Animal experiment results such as Figure 4 As shown, the weight curve ( Figure 4 A) It was observed that, compared with the mice fed a normal diet, the mice fed a high-fat diet for 8 weeks showed a gradual increase in body weight, indicating that a high-fat diet led to weight gain and obesity. Compared with the model group of obese mice, the weight gain of mice in each group treated with the strain was slowed down, indicating that Akk strains, including the Akk-101 live / pasteurized inactivated bacteria, AMY001 live / pasteurized inactivated bacteria, and the standard strain BAA-835 live bacteria of this invention, can inhibit weight gain induced by a high-fat diet, and the live bacteria of Akk-101 and AMY001 are more effective than their pasteurized inactivated dead bacteria. Specifically, based on the statistical analysis of weight gain ( Figure 4 (B) The weight gain of mice in the Akk-101, AMY001, and BAA-835 live bacteria groups was 5.28g, 5.89g, and 6.52g, respectively, which was lower than the weight gain of the model group mice (8.27g). Based on the average initial weight of the experimental mice (approximately 21.6g), it can be concluded that the three Akk live bacteria gavage treatments on high-fat diet mice resulted in a weight loss of approximately 10.01%, 8.03%, and 5.86%, respectively. The average weight gain of mice in the Akk-101 and AMY001 inactivated bacteria groups was approximately 5.97g and 6.39g, respectively, with a weight loss of approximately 7.7% and 6.29%, respectively. The results show that the Akk-101 live bacteria were the most effective in inhibiting weight gain, followed by the AMY001 live bacteria. The inactivated Akk-101 and AMY001 strains were still more effective than the standard strain BAA-835. Regarding visceral fat accumulation, the inguinal white fat of obese mice treated with all Akk strains ( Figure 4 C) and liver weight ( Figure 4D) were significantly lower than those in the model group, indicating that these mice had less visceral fat accumulation than the obese model mice, showing a similar trend to weight gain, and Akk-101 live bacteria had the best effect.

[0083] Serum tests showed that, compared with the normal NC mice, the obese model group had significantly increased levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C), and decreased levels of high-density lipoprotein cholesterol (HDL-C), exhibiting a clear hyperlipidemia phenotype. Figure 5 AD). Treatment with the Akk strain showed an improving trend in blood lipids. Specifically, after gavage with Akk-101 live bacteria, mice showed a 25.11% decrease in serum TC (3.46 mmol / L vs 4.62 mmol / L); a 35.18% decrease in serum TG (1.29 mmol / L vs 1.99 mmol / L); a 26.52% decrease in LDL-c (0.97 mmol / L vs 1.32 mmol / L); and a 15.1% increase in HDL-c (3.28 mmol / L vs 2.85 mmol / L). With the AMY001 strain, the decrease rates of TC, TG, and LDL-c were 15.58%, 23.61%, and 12.88%, respectively, while HDL-c increased by 7.37%. After treatment with the standard strain BAA-835, the serum TC level in mice decreased by 16.70%, TG decreased by 20.52%, LDL-c decreased by 14.84%, and HDL-c increased by 10.88%. Compared with the model group, inactivated strains of Akk-101 and AMY001 could alleviate dyslipidemia in mice, but the effect was weaker than that of the corresponding live bacterial intervention. Therefore, feeding obese mice with Akk-101 and AMY001 strains is beneficial in improving the hyperlipidemia phenotype, and their effect is superior to that of the standard strain. HE staining results of liver tissue showed (…). Figure 5 (E) Compared with normal mice, the model group mice had more fat deposition and irregular cell morphology in their liver cells. This condition was improved and alleviated after treatment with the strain, and fat was reduced. In summary, the Akk-101 and AMY001 strains of Akkermansia mycotoxin provided by this invention have better effects than the standard strain BAA-835 in inhibiting weight gain, reducing hyperlipidemia, and improving obesity.

[0084] Example 7. Akk-101 strain of Aktmanella myxophilus improves abnormal blood glucose levels in obese mice.

[0085] Obesity is not only associated with dyslipidemia but also often with disordered glucose metabolism. This embodiment of the invention systematically evaluated the effect of the Akk-101 strain of the present invention on glucose homeostasis regulation in obese mice by conducting a series of tests on glucose-related indicators in mice. According to... Figure 6 OGTT experimental results ( Figure 6A) and B) showed that at the time of testing, the blood glucose level of the model group mice was around 12.5 mmol / L, higher than the average of 7.0 mmol / L in the normal NC group, indicating that obesity did indeed lead to hyperglycemia. After glucose intake, the blood glucose level of the model mice rose rapidly, reaching an average of 19.7 mmol / L at 30 min. After treatment with Akk strain, the overall OGTT curve was between that of the model group and the normal group, and the blood glucose rise trend was significantly lower than that of the model group. At the end of the experiment (120 min), the average blood glucose level of the model group was 15.1 mmol / L, the Akk-101 live and inactivated bacteria groups were 9.8 mmol / L and 10.7 mmol / L, the AMY001 live and inactivated bacteria groups were 11.0 mmol / L and 12.5 mmol / L, and the BAA-835 live bacteria group was 11.9 mmol / L. It can be seen that Akk strain treatment, including live and inactivated bacteria, improved the glucose intolerance caused by obesity and made blood glucose regulation more sensitive. After the completion of the 8-week animal experiment, there were also significant differences in fasting blood glucose levels in the mice. Figure 6 (C) The blood glucose level of mice in the NC group was approximately 7.27 mmol / L, while that in the model group was 13.80 mmol / L. The fasting blood glucose levels in the Akk-101 live bacteria and Pasteurella groups were 9.09 mmol / L and 10.21 mmol / L, respectively, representing a glucose reduction of approximately 34.13% and 26.01%. The fasting blood glucose levels in the AMY001 live bacteria and Pasteurella groups were 10.52 mmol / L and 10.88 mmol / L, respectively, representing a glucose reduction of approximately 23.77% and 21.16%. The fasting blood glucose level in the BAA-835 live bacteria group was 10.89 mmol / L, representing a glucose reduction of 21.09%. These results indicate that the Akk-101 and AMY001 strains of Akkermansia mycophila provided in this invention can improve hyperglycemia and glucose intolerance caused by a high-fat diet.

[0086] Furthermore, the Akk strain also exhibited a similar regulatory trend on insulin levels in mice. The obese model mice had low insulin levels, and treatment with the strain significantly increased insulin levels. Compared to the model (197.9 mIU / L), treatment with Akk-101, AMY001, and BAA-835 live bacteria increased the average insulin level to 219.3 mIU / L, 211.2 mIU / L, and 210.2 mIU / L, respectively. Figure 6D), approximately 10.87%, 6.78%, and 6.27% higher, respectively; inactivated strains also showed significant effects. After inactivation treatment of Akk-101 and AMY001, serum insulin levels increased by 8.75% and 4.56%, respectively. Among them, the effect of Akk-101 inactivated strains was only weaker than its live form, and higher than that of AMY001 and BAA-835 strains. Glucagon-like peptide-1 (GLP-1), as a glucose metabolism regulatory hormone secreted by intestinal cells, showed a trend in its content changes that were basically consistent with those of insulin. Figure 6 Compared to the model (51.79 pmol / L), treatment with live Akk-101, AMY001, and BAA-835 bacteria increased GLP-1 levels by approximately 23.74%, 14.10%, and 14.58%, respectively. Inactivated Akk-101 and AMY001 treatments also increased serum insulin levels in mice, with increases of 16.53% and 12.65%, respectively. These results indicate that the Akk-101 and AMY001 strains of Akkermansia mycophila provided in this invention can significantly improve glucose metabolism and reduce abnormal blood glucose symptoms caused by obesity, with overall performance superior to the standard strain BAA-835. Furthermore, the inactivated dead bacteria of both strains retained the regulatory effects of live bacteria on glucose and lipid metabolism; the Akk-101 strain also showed better results than the AMY001 strain.

[0087] Example 8. Akkermansia myxophilus strains regulate glucose and lipid metabolism via the molecular pathway of "AMPK activation – GLP-1 promotion – enhanced glucose uptake".

[0088] This embodiment is used to study the molecular mechanism of glucose and lipid metabolism regulation by the Akk strain of the present invention. The expression level of relevant genes in mouse target tissues was detected by real-time quantitative PCR (RT-qPCR) technology. The genes include, but are not limited to, AMPK, AKT1, GCK, GLP-1R, PC1 / 3, etc.

[0089] Total RNA was extracted from the small intestine, liver, and ileum of mice in each group using the TRIzol method. cDNA was then synthesized via reverse transcription using a commercial reverse transcription kit (Takara PrimeScript™ RT reagent Kit) according to the manufacturer's instructions. qPCR was subsequently performed using the SYBR Green method. GAPDH was used as an internal control gene, and the relative expression levels of each gene were calculated using the 2⁻ΔΔCt method. All experiments were performed in triplicate, and statistical analysis was conducted using GraphPad Prism 8.

[0090] AMPK is a key component of cellular energy metabolism. Its phosphorylation activation promotes AKT phosphorylation, a crucial molecule in glucose metabolism. AMPK activation triggers the activation of downstream genes involved in glucose and lipid metabolism, such as GSK3 (regulating glycogen synthesis), GCK (glucokinase), and PEPCK (phosphoenolpyruvate carboxylkinase). These two genes encode enzymes that are key enzymes in glycolysis and gluconeogenesis, respectively. GLP-1R (GLP-1 receptor), GPR41 / 43 (amino acid receptors), and PC1 / 3 (prohormone convertase) encode proteins that control the synthesis and secretion of GLP-1. Figure 7 The expression of glucose metabolism-related genes in the liver tissue of model mice was significantly different from that in the normal group. AMPK, AKT1, GCK, and GLUT4 were significantly decreased, while GSK3 and PEPCK were significantly increased. This indicates that the glucose-consuming metabolic pathway is downregulated in type 2 diabetic mice, activating regulatory genes that increase blood glucose concentration. Intervention with Akk strains, including live and inactivated strains, could restore the expression of these genes, bringing them closer to that of normal mice. Akk-101 showed the most significant regulatory effect on glucose metabolism genes (up / down regulation of these genes by 1.5-3.5 times compared to normal mice). The regulation after live bacterial treatment was superior to that of the standard strain (up / down regulation of these genes by 1.2-1.9 times compared to normal mice), and the effect of Akk-101 inactivated strain (up / down regulation of these genes by 1.3-2.1 times compared to normal mice) was also comparable to that of the standard strain. Both live and dead forms of the AMY001 strain showed significant gene expression reversion. Compared with normal mice, the live strain upregulated / downregulated these genes by 1.3-2.6 times, while the dead strain upregulated / downregulated them by 1.1-1.8 times. Regarding the regulation of GLP-1 synthesis, the expression levels of GLP-1, GPR41, and PC1 / 3 in the ileum of mice in the strain intervention groups (Akk-101, AMY001) were significantly higher than those in the model group, and the live strain showed better results than the dead strain (p<0.05), indicating that the Akk strain can also upregulate GLP-1 secretion-related pathways. Combined with the upregulation of AMPK, AKT1, and GLUT4 gene expression in liver tissue, this suggests that Akk-101 and AMY001 function through the "AMPK activation–GLP-1 promotion–enhanced glucose uptake" pathway.

[0091] Example 9: Ackermania strains with myxotropin inhibit glycosidase activity and promote cellular glucose consumption.

[0092] This embodiment is used to study the biochemical mechanism of the Akk strain in regulating glucose and lipid metabolism. In vitro enzyme activity assays were used to detect the inhibitory effect of the strain on the activity of key enzymes in carbohydrate decomposition, and cell experiments were used to evaluate the promoting effect of the Akk strain on glucose consumption by hepatocytes.

[0093] (1) α-Amylase inhibition experiment

[0094] Take 2 ml of α-amylase solution with a mass concentration of 2 mg / ml (prepared with 50 mM, pH 7.0 PBS buffer), add 2 ml of sample solution (the bacterial suspension of the Akkermansia myxophilus strain in this invention), react at 37°C for 30 min, then add 2 ml of 1% soluble starch, react at 37°C for 15 min, add iodine solution for color development, and measure the absorbance (OD660) at 660 nm using an ELISA reader. Acarbose is used as a positive control.

[0095] The experimental group showed that the live and dead bacterial suspensions of strain Akk-101 inhibited α-amylase at rates as high as 63.3% and 44.1%, respectively. Figure 8 A) The live and dead bacterial suspensions of strain AMY001 showed inhibition rates of 52.4% and 37.2% against α-amylase, respectively, while the live bacterial suspension of the standard strain BAA-835 showed an inhibition rate of only 11.5%, and the positive control group acarbose showed an inhibition rate of 72.3%. This indicates that the Akk strain of the present invention has strong α-amylase inhibitory activity, and both live and dead strains showed higher inhibitory effects than the standard strain. Among them, the inhibition rate of Akk-101 was 4-6 times that of BAA-835, and the inhibition effect of AMY001 was 3-5 times that of BAA-835.

[0096] (2) α-glucosidase inhibition experiment

[0097] Each 30 µL suspension of *Akkermansia myxophilus* strain was mixed with 30 µL of α-glucosidase enzyme solution (0.1 U / mL) and incubated at 37 °C for 10 min. Then, 60 µL of substrate PNPG (0.5 mM) was added, and the reaction was continued at 37 °C for 20 min. The reaction was terminated by adding 100 µL of 2 M sodium carbonate solution. The absorbance (OD) at 405 nm was then measured using a microplate reader. 405 Acarbose was used as a positive control.

[0098] Experimental results are as follows Figure 8As shown in Figure B, the average α-glucosidase inhibition rates of Akk-101 live and dead bacterial suspensions were approximately 78.9% and 49.1%, respectively, and their viability was comparable to that of the positive control acarbose (81.7%). The average α-glucosidase inhibition rates of AMY001 live and dead bacterial suspensions were approximately 59.8% and 40.7%, respectively, which were much higher than the effect of the standard strain (17.1%), indicating that the two Akk strains in this invention have high α-glucosidase inhibitory activity.

[0099] (3) Experiment on promoting cellular glucose consumption

[0100] Human hepatocellular carcinoma HepG2 cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum at 37℃ and 5% CO2. The cells were then seeded into 96-well plates at an appropriate concentration and cultured for 24 h until complete cell adhesion. Before the experiment, the original medium was discarded, and each well was replaced with high-glucose, phenol red-free incomplete medium (8 wells per group). Corresponding treatments were then performed: 20% volume of the bacterial suspension was added to each well in the sample group; the control group received the same volume of the culture medium (MRS) used to culture the bacterial strain; and the positive control group received insulin solution (final concentration 10 µmol / L) prepared in incomplete medium. After 24 h of culture, the absorbance at 505 nm was measured using a microplate reader according to the Nanjing Jiancheng glucose oxidase kit, and the glucose consumption of each group was calculated.

[0101] according to Figure 8 The results of experiment C showed that the glucose consumption of cells in the control group was 4.7 mmol / L. Adding the suspensions of the *Bifidobacterium adolescentis* strains (Akk-101, AMY001, and BAA-835) increased glucose consumption. Akk-101 showed the most significant promoting effect on glucose consumption, with live bacteria treatment resulting in 7.9 mmol / L glucose consumption and dead bacteria treatment in 6.2 mmol / L. AMY001 was next (6.7 mmol / L for live bacteria and 5.3 mmol / L for dead bacteria), while BAA-835 had the lowest effect (4.7 mmol / L). Compared with the standard strains, Akk-101 increased glucose consumption by 68.1% (live bacteria) and 31.9% (dead bacteria). However, compared with the control group, BAA-835 had no significant effect on promoting glucose consumption. This indicates that the fermentation supernatant of *Bifidobacterium adolescentis* strain BUAD can significantly promote glucose consumption in HepG2 liver cancer cells.

[0102] Since α-glucosidase and amylase are key rate-limiting enzymes in the body's metabolism of carbohydrates into glucose, substances that inhibit the activity of these enzymes can effectively inhibit the absorption of glucose in the small intestine, limit the rise in blood glucose levels, and thus reduce hyperglycemia and related phenotypes. In addition, the active utilization of glucose by liver cells can also rapidly metabolize glucose, reducing blood glucose accumulation. Therefore, the Akk strain provided by this invention, in addition to regulating important factors in glucose and lipid metabolism at the molecular level and improving abnormal glucose and lipid metabolism, can also directly act on processes affecting glucose production and utilization at the biochemical level. These regulatory mechanisms are significant in Akk-101 and AMY001, but not obvious in the standard strain BAA-835.

[0103] In summary, the Akk-101 and AMY001 strains provided by this invention are two novel functional Akkermansia muciniphila strains, different from the standard strain (BAA-835), screened from intestinal samples of long-lived elderly people in China. These two strains differ from the standard strain BAA-835 in their physiological and biochemical characteristics, exhibiting stronger adhesion and colonization abilities and acid-base tolerance. Physiologically, compared to the Akk standard strain, both Akk-101 and AMY001 significantly inhibit fat accumulation in human liver cells and nematodes, and also demonstrate superior effects in reducing obesity in mice, inhibiting weight gain, and improving glucose and lipid metabolism. Mechanistically, Akk-101 and AMY001 of this invention can regulate important molecules in glucose and lipid metabolism pathways, exerting their blood glucose-improving effects through a combined mechanism of "AMPK activation—increased GLP-1 secretion—enhanced glucose uptake" molecular axis and "inhibition of glucosidase / amylase and promotion of glucose consumption." In practical applications, the two strains may exert their effects through complementary or substitution mechanisms. These results provide strong support for the potential applications of Akk-101 and AMY001 strains of Akkermansia myxophilus in metabolic diseases such as obesity and diabetes.

[0104] The Akk-101 and AMY001 strains provided by this invention have at least the following applications: (1) intervention for obesity: Akk-101 and AMY001 can be used to regulate fat metabolism and can be used for the intervention and improvement of obesity; (2) improvement and prevention of diabetes and metabolic syndrome; (3) Akk-101 and / or AMY001 can be developed into probiotic preparations as functional probiotics to improve the health level of consumers. In addition, the Akk-101 and / or AMY001 strains provided by this invention can increase the basal metabolic rate of mammals, preferably humans, but can also be cats, dogs, pigs, rats, cattle, sheep and other animals.

[0105] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Akkermansia myxophilus ( Akkermansia muciniphila AMY001, where, The accession number of the Ackermansia amyloliquefaciens AMY001 is CGMCC No. 46141.

2. The culture of Akkermansia amyloliquefaciens AMY001 according to claim 1, characterized in that, The culture includes the fermentation broth, fermentation broth precipitate, fermentation broth supernatant, live bacteria, dead bacteria, lyophilized powder, and lysate of Akkermansia muciniphila AMY001.

3. A microbial agent containing Akkermansia amyloliquefaciens AMY001 as described in claim 1.

4. The microbial agent according to claim 3, wherein, The microbial agent can be a solid or liquid preparation.

5. A microbial composition, characterized in that, The microbial composition comprises Akkermansia myxophilus AMY001 and Akkermansia myxophilus Akk-101 as described in claim 1, wherein the Akkermansia myxophilus Akk-101 has the accession number CGMCC No. 40786.

6. The microbial composition according to claim 5, wherein, The microbial composition contains 1×10 9 Total bacterial count of CFU or higher.

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