Ackermania muciniphila strain and application thereof

Abstract

The invention belongs to the field of microorganisms and medicine health care, and relates to an ackermania muciniphila strain and application thereof, the preservation number of the ackermania muciniphila strain is CGMCC (China General Microbiological Culture Collection Center) No.34370, and a 16SrDNA (Deoxyribose Nucleic Acid) sequence of the ackermania muciniphila strain has 93% similarity with a sequence shown as SEQ ID NO: 1. The new ackermania muciniphila strain (AKKBC300) is separated from a healthy human body and has good safety; good tolerance to low pH value and bile salt is shown in vitro, and it is indicated that the compound can reach the intestinal tract through the upper digestive tract and play a role; according to the present invention, genomics analysis results prove that the bacterial strain contains the gene Akkermansia BC00573 with high homology with the known probiotic functional protein Amuc1100, such that the molecular basis is provided;

Description

Technical Field

[0001] This invention relates to the fields of microbiology and pharmaceutical technology, and in particular to a strain of Akkermansia myxophilus and its applications. Background Technology

[0002] The gut microbiota is closely related to host health, and its dysbiosis is significantly associated with various metabolic diseases, such as obesity, diabetes, and non-alcoholic fatty liver disease. Akkermansia is an important commensal bacterium in the gut and is considered a next-generation probiotic. Multiple studies have shown that the abundance of *A. muciniphila* is positively correlated with the host's healthy metabolic state, and its abundance is typically lower in the guts of obese patients and those with type 2 diabetes.

[0003] Studies have shown that *A. muciniphila* can improve intestinal barrier function, regulate immune responses, and affect energy homeostasis. It may also exert anti-inflammatory and metabolic-improving effects through the interaction of its outer membrane proteins (such as Amuc_1100) with the host Toll-like receptor 2 (TLR2). In recent years, inactivated probiotics (also known as "postbiotics") have attracted widespread attention due to their higher safety, better stability, and efficacy comparable to or even superior to live bacteria. Pasteurized *A. muciniphila* has been shown to prevent diet-induced obesity, insulin resistance, and other metabolic disorders in mice.

[0004] However, A. muciniphila strains from different sources may exhibit significantly different probiotic effects due to differences in their genomes and functional proteins. Therefore, isolating and identifying novel A. muciniphila strains with excellent probiotic properties (such as high gastric acid / bile salt tolerance, strong colonization ability, efficient induction of GLP-1 secretion, and significant improvement of the intestinal barrier) and exploring the specific applications of their live and inactivated forms is of great significance for the development of related probiotic preparations, postbiotic products, and functional foods. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention provides a strain of Akkermansia myxophilus, the strain having the accession number CGMCC No. 34370, whose 16S rDNA sequence has 93% similarity to the sequence shown in SEQ ID NO: 1.

[0006] Preferably, the Akkermansia myxophilus strain is obtained by a method comprising the following steps: a) Collect fresh stool samples from healthy individuals; b) The sample was suspended in a sterile synthetic liquid culture medium containing 3.5%-4.0% brain heart extract BHI powder, 1.5%-1.7% soybean peptone, 0.3%-0.5% threonine, 20-30 mM glucose, 20-30 mM N-acetylglucosamine, 0.1%-0.3% ascorbic acid, and a pH of 7.2-7.4; c) Homogenize the suspension for 2-4 minutes; d) Perform a 10-fold serial dilution on the homogenized suspension; e) Take 0.08-0.12 mL of the appropriate dilution solution and spread it on a selective agar plate, wherein the selective agar plate is BHI agar supplemented with 3-5 g / L mucin and 4-6 μg / mL vancomycin; f) First, pre-culture at 37°C for 2 hours, then culture at 36.5-37.5°C under anaerobic conditions in an atmosphere of 83%-87% N2, 9%-11% CO2 and 4%-6% H2 for 5-7 days; g) Select single colonies with different morphologies and repeatedly streak them on fresh vancomycin BHI agar plates for purification until a pure culture is obtained.

[0007] On the other hand, the present invention provides a microbial preparation prepared by a method comprising the following steps: a) Culturing the above-mentioned Akkermansia myxophilus strain to obtain bacterial cells, wherein the culture is carried out in a liquid medium containing BHI medium, 1-10% fetal bovine serum and 0.1-0.5 μg / mL insulin, and statically cultured at 36-38°C under anaerobic conditions for 48-96 hours. b) Inactivate the bacterial cells obtained in step a), and / or process them into bacterial cell fragments, and / or collect their metabolites, wherein the inactivation treatment is pasteurization, and the specific conditions are: water bath treatment at 60-80°C for 15-45 minutes, followed by immediate ice bath cooling for 5 minutes; c) Mix one or more of the bacterial cells obtained in step a) and / or the inactivated bacterial cells, bacterial cell fragments, and metabolites obtained in step b) with a pharmaceutically or food-grade acceptable carrier, wherein the carrier comprises mucin-chitosan microcapsules or pH-sensitive colon-targeting microspheres. The culture in step a) is carried out in a liquid medium containing BHI medium and 1-10% fetal bovine serum, and is statically cultured at 36-38°C under anaerobic conditions for 48-96 hours. Furthermore, the inactivation treatment in step b) is pasteurization, specifically under the following conditions: water bath treatment at 60-80°C for 15-45 minutes.

[0008] Preferably, the inactivated bacterial cells are pasteurized inactivated bacterial cells.

[0009] On the other hand, the present invention provides the use of the above-mentioned Akkermansia myxophilus strain or the above-mentioned microbial preparation in the preparation of articles for the prevention and / or treatment of obesity or weight reduction.

[0010] On the other hand, the present invention provides the use of the above-mentioned Akkermansia myxophilus strain or the above-mentioned microbial preparation in the preparation of products for lowering blood lipids and / or improving fasting blood glucose.

[0011] On the other hand, the present invention provides the use of the above-mentioned Akkermansia myxophilus strain or the above-mentioned microbial preparation in the preparation of an article for promoting the secretion of glucagon-like peptide-1.

[0012] On the other hand, the present invention provides the use of the above-mentioned Akkermansia myxophilus strain or the above-mentioned microbial preparation in the preparation of articles for enhancing intestinal barrier function and / or upregulating the expression of intestinal tight junction proteins, wherein the tight junction proteins include one or more of ZO-1, Occludin and Claudin-1.

[0013] On the other hand, the present invention provides the use of the above-mentioned Akkermansia myxophilus strain or the above-mentioned microbial preparation in the preparation of articles for regulating immune responses, wherein the regulation of immune responses includes reducing the levels of pro-inflammatory factors TNF-α and / or IL-6, and / or increasing the levels of anti-inflammatory factor IL-10.

[0014] On the other hand, the present invention provides the use of the above-mentioned Akkermansia myxophilus strain or the above-mentioned microbial preparation in the preparation of products for improving hepatic steatosis and / or regulating the levels of hepatic transaminases ALT and AST.

[0015] Compared with the prior art, the present invention has the following main advantages: (1) The novel strain of Akkermansia myxophilus (AKKBC300) provided was isolated from healthy human subjects and has good safety. (2) The strain showed good tolerance to low pH and bile salts in vitro, indicating that it can effectively reach the intestine through the upper digestive tract and exert its effects. (3) Genomic analysis confirmed that the strain contains a gene (AkkermansiaBC_00573) that is highly homologous to the known probiotic functional protein Amuc_1100, providing a molecular basis for its function; (4) In vitro cell experiments have shown that both live and pasteurized forms of this strain can significantly promote the secretion of GLP-1 by human enteroendocrine cells NCI-H716. (5) In vitro cell experiments have shown that both live and pasteurized forms of this strain can significantly upregulate the expression of tight junction proteins (ZO-1, Occludin, Claudin-1) and TLR2 / TLR4 in human intestinal epithelial cells Caco-2, and downregulate the expression of pro-inflammatory factor TNF-α. (6) Animal experiments have shown that oral administration of live bacteria or pasteurized inactivated form of this strain can effectively prevent excessive weight gain in mice on a normal diet, improve their lipid profile (such as reducing total cholesterol and low-density lipoprotein), reduce fasting blood glucose, and improve liver fat accumulation. (7) Animal experiments have shown that the live strain and pasteurized inactivated form of this strain can regulate the level of cytokines in mouse plasma and upregulate the expression of GLP-1 and PYY genes in the colon and GLUT2 gene in the liver. (8) For the first time, a systematic comparison and confirmation were made that the live and pasteurized forms of this specific strain have significant probiotic effects in vitro and in vivo, providing a solid foundation for the development of microecological preparations in different forms (live bacteria, postbiotics). Attached Figure Description

[0016] To more clearly illustrate the solutions in this invention, the accompanying drawings used in the description of the embodiments of this invention will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0017] Figure 1 This is a schematic diagram of the colony morphology (A) and Gram staining microscopic examination results (B) of strain AKK BC300 (showing it as a Gram-negative short bacillus); Figure 2 This is a schematic diagram of the growth curve of strain AKK BC300 in BHI liquid medium. Figure 3 Schematic diagram showing the effect of live strain AKK BC300 and pasteurized inactivated form on GLP-1 secretion in NCI-H716 cells (*p<0.05, **p<0.01 compared with the control group); Figure 4A This diagram illustrates the effects of live and inactivated AKK BC300 on genes in the Caco-2 cell line, showing that both live and inactivated bacteria significantly upregulated the gene expression of ZO-1, Occludin, and Claudin-1. Figure 4B The diagram illustrates the effects of live and inactivated AKK BC300 on genes in the Caco-2 cell line. Both live and inactivated bacteria significantly upregulated the expression of TLR2 and TLR4 and significantly downregulated the expression of TNF-α. Figure 4C The effect of live and inactivated AKK BC300 on genes in the Caco-2 cell line is illustrated in the diagram. The expression of ANGPTL4 was upregulated by live and inactivated bacteria (*p<0.05, **p<0.01 compared with the control group). Figure 5 A schematic diagram showing the histopathological sections (H&E staining) of (A) colon and (B) liver of mice after 5 weeks of treatment with live strain AKK BC300 and pasteurized inactivated form. Figure 6 The diagram illustrates the relative gene expression levels of live and pasteurized strain AKK BC300 on mouse liver tissue GLUT2 gene and colon tissue GLP-1 and PYY gene after treatment with PBS, AKK BC300, and inactivated AKK BC300. The rpl-19 gene is shown as the relative expression level of the internal reference gene. Detailed Implementation

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein in the specification is for the purpose of describing particular embodiments only and is not intended to limit the invention; the terms "comprising" and "having," and any variations thereof, in the specification, claims, and foregoing drawings are intended to cover non-exclusive inclusion. The terms "first," "second," etc., in the specification, claims, or foregoing drawings are used to distinguish different objects and not to describe a particular order.

[0019] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of the invention. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0020] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0021] [Information on the Preservation of Biological Materials] The Akkermansia muciniphila strain AKKBC300 of this invention was deposited on April 27, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCCNo. 34370. The address of the depository is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. This deposit was completed before the application date, the specimen is viable, and the deposit period is 30 years, calculated from the date of deposit. It is available to third parties upon request.

[0022] Example 1: Isolation and purification of Akkermansia myxophilus strains Fresh fecal samples were collected from healthy individuals and suspended in sterile synthetic liquid culture medium (containing 3.8% BHI powder, 1.6% soybean peptone, 0.4% threonine, 25 mM glucose, 25 mM N-acetylglucosamine, pH 7.2-7.4) and homogenized for 3 minutes. After 10-fold serial dilutions, 0.1 mL of the appropriately diluted sample was plated onto BHI agar plates supplemented with 4 g / L mucin and 5 μg / mL vancomycin, and incubated at 37°C under anaerobic conditions (85% N2, 10% CO2, 5% H2) for 5-7 days. Single colonies with different morphologies were picked and repeatedly streaked on fresh vancomycin agar plates for purification until pure cultures were obtained. Purified colonies were small, round, slightly white, translucent colonies with smooth surfaces and regular edges.

[0023] Example 2: Preliminary morphological identification of the strain The purified colonies from Example 1 were Gram-stained and observed under an optical microscope (100x oil immersion). The results showed that the strain was a red, short, rod-shaped bacterium, and was determined to be a Gram-negative bacterium (see [link to original text]). Figure 1 , Figure 1 This is a schematic diagram of the colony morphology (A) and Gram staining microscopic examination results (B) of strain AKKBC300 (showing it as a Gram-negative short bacillus).

[0024] Example 3: Molecular biological identification of the strain PCR amplification of the isolated strain was performed using specific primers for *Akkermansia muciniphila* (forward: 5'-GCATATCAATAAGCGGAGGAAAAG-3', reverse: 5'-GGTCCGTGTTTCAAGACGG-3'), and the result was positive. Further amplification and sequencing of the strain's 16S rDNA using universal primers 27F and 1492R yielded its 16S rDNA sequence (denoted as SEQ ID NO: 1). BLAST alignment showed 93% similarity to the 16S rDNA sequence of a known *Akkermansia muciniphila* standard strain, thus identifying the isolated strain as *Akkermansia muciniphila* and naming it AKKBC300.

[0025] Example 4: Cultivation and Preservation of Strains High-concentration bacterial suspensions can be obtained by inoculating AKKBC300 strain into BHI liquid medium containing 5% fetal bovine serum and incubating it statically at 37°C under anaerobic conditions for 72 hours. The bacterial suspensions are then mixed with a cryoprotectant and stored long-term in an ultra-low temperature freezer at -80°C or in liquid nitrogen.

[0026] Example 5: Determination of the growth curve of the strain The AKKBC300 strain was transferred to fresh BHI liquid medium at a 1% inoculum and anaerobically cultured at 37°C. Samples were taken every 12 hours over 96 hours, serially diluted, and plated onto BHI agar plates. After 72 hours of anaerobic incubation, colony-forming units (CFU) were counted. A growth curve was plotted with incubation time on the x-axis and the logarithm of CFU / mL on the y-axis. (See [reference needed]). Figure 2 , Figure 2 This is a schematic diagram of the growth curve of strain AKKBC300 in BHI liquid medium. The results show that the strain enters the stationary phase after about 60 hours of culture.

[0027] Example 6: Gastrointestinal environment tolerance test of the strain (1) Acid tolerance: The strain was inoculated into synthetic liquid culture media with pH values ​​of 2.0, 3.0, 4.0, 5.0 and 7.0 (control), and after anaerobic culture for 2 hours, the strain was plated and counted. The survival rate under each pH condition was calculated (survival rate = CFU of each pH group / CFU of pH 7.0 group). The results (Table 1A) showed that the strain maintained a high survival rate at pH 3.0 and 4.0, demonstrating a certain degree of gastric acid tolerance.

[0028] The experimental results are shown in Tables 1A and 1B.

[0029] Table 1A: Survival rate of AKKBC300 under different pH conditions. Survival rate = CFU (at different pH values) / CFU (pH=7): (2) Bile salt tolerance: The strain was inoculated into synthetic liquid culture media containing 0.1%, 0.2%, 0.3%, 0.4% bile salt and no bile salt (control), and after anaerobic culture, it was spread and counted. The survival rate at each concentration was calculated (survival rate = CFU of each concentration group / CFU of the 0% concentration group). The results (Table 1B) showed that the strain still had a certain survival rate at a bile salt concentration of 0.3%, and had a certain bile tolerance.

[0030] Table 1B: Survival rate of AKKBC300 under different bile salt concentrations. Survival rate = CFU (different bile salt concentrations) / CFU (bile salt concentration = 0): Example 7: Genome sequencing and annotation of the strain Genomic DNA was extracted from strain AKKBC300, a library was constructed, and PE150 sequencing was performed using Illumina or MGI platforms. After quality control of the raw data, de novo assembly was performed using SPAdes software. The results showed that the genome size of this strain was 3,128,216 bp, with a GC content of 57.80%, and a total of 42 scaffolds were obtained. 2,570 genes were predicted using Prokka software. Functional annotation of the predicted genes was performed by comparing with databases such as NR, Swiss-Prot, KEGG, COG, NOG, and CAZy, obtaining comprehensive gene functional information for this strain.

[0031] Genomic DNA extraction: Genomic DNA was extracted using a DNA extraction kit (ALFA mini-extraction kit). DNA integrity and purity were assessed using 1% agarose gel electrophoresis. DNA concentration and purity were also assessed using Qubit4.0 (Thermo Fisher Scientific, Waltham, USA) and NanoDropOne (Thermo Fisher Scientific, Waltham, USA).

[0032] DNA samples that pass quality inspection are used to construct a library using the ALFA-SEQDNA Library Prep Kit, following the accompanying instructions. The main steps include: a) DNA fragmentation; b) End repair and 3' end A addition; c) Adapter ligation, fragment selection, and purification; d) PCR amplification and purification. The size of the library fragments was evaluated using a Qsep400 high-throughput nucleic acid and protein analysis system (e.g., Hangzhou Houze Biotechnology Co., Ltd., China), and the concentration of the library was measured using Qubit4.0 (Thermo Fisher Scientific, Waltham, USA).

[0033] e) Perform PE150 sequencing on the constructed DNA genomic library using the Illumina or MGI platform. Data quality control: The raw data undergoes quality control, and the resulting high-quality sequences are used for downstream data analysis. The specific processing steps are as follows: (1) Remove low-quality bases (quality value <20) from both ends of the reads and remove reads that are too short (default setting is 50bp). (2) Remove N bases to achieve a certain proportion of reads (default setting is 10bp). (3) Remove reads whose overlap with the Adapter exceeds a certain threshold (default is 15bp); (4) Remove duplication contamination.

[0034] Genome assembly: Reads with low sequencing quality values ​​are filtered through quality control steps. The clean data after data filtering and quality control is then de novo assembled using the tool SPAdesv 4.0.0 to obtain high-quality Scaffold fragments.

[0035] Genome characteristics: AkkermansiamuciniphilaAkkBC300 genome size: 3,128,216 bp, GC content: 57.80; Scaffoldnum: 42.

[0036] Gene prediction and functional annotation: Gene prediction was performed on the assembled genome using the gene prediction tool Prokka, resulting in 2,570 genes.

[0037] Functional annotation information was obtained by comparing the following databases using the Blast tool: NR (Non-Redundant Protein Database), Swiss-Prot, GO (Gene Ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes), COG (Clusters of Orthologous Groups), NOG (Non-supervised Orthologous Groups Database), Trembl (Translated EMBL Nucleotide Sequence Database), and SwissProt.

[0038] dbCAN (http: / / csbl.bmb.uga.edu / dbCAN / ) was used to predict carbohydrate-active enzymes.

[0039] The experimental results are shown in Table 2.

[0040] Table 2: The percentage of genes corresponding to different databases is shown below: Example 8: Identification and analysis of homologous genes of the key functional gene Amuc_1100 The reference sequence (WP_012420141.1, SEQ ID NO:2) of the Amuc_1100 protein was obtained from strain AkkermansiamuciniphilaATCCBAA-835. SEQ ID NO:2 was compared with the protein sequence encoded by the AKKBC300 genome using BLAST. The results showed that the reference sequence was 93% identical to the protein sequence encoded by the AkkermansiaBC_00573 gene in the AKKBC300 genome, indicating that AkkermansiaBC_00573 is a homolog of Amuc_1100. Further phylogenetic analysis confirmed that the AkkermansiaBC_00573 protein clustered with Amuc_1100 proteins from other strains within the same evolutionary branch, further confirming their homology.

[0041] The Amuc_1100 protein has been shown to bind to the host's Tol-like receptor 2 (TLR2), activating the immune system and promoting anti-inflammatory responses. Intestinal barrier protection: It enhances tight junctions in intestinal epithelial cells, thereby improving intestinal barrier function. In animal models, the Amuc1100 protein has shown the potential to improve metabolic disorders such as obesity and diabetes.

[0042] The target gene sequence was obtained by retrieving the reference sequence of the Amuc_1100 gene from the known “AkkermansiamuciniphilaATCCBAA835 (NC_010655.1)”.

[0043] Protein sequence of WP_012420141.1-Amuc_1100.Akkermansia.muciniphila.ATCC.BAA.835: MSNWITDNKPAAMVAGVGLLLFLGLSATGYIVNSKRSELDKKISIAAKEIKSANAAEITPSRSSNEELEKELNRYAKAVGSLETAYKPFLASSALVPTTPTAFQNELKTFRDSLISSCKKKNILITDTSSWLGFQVYSTQAPSVQAASTLGFELKAINS LVNKLAECGLSKFIKVYRPQLPIETPANNPEESDEADQAPWTPMPLEIAFQGDRESVLKAMNAITGMQDYLFTVNSIRIRNERMMPPPIANPAAAKPAAAQPATGAASLTPADEAAAPAAPAIQQVIKPYMGKEQVFVQVSLNLVHFNQPKAQEPSED.

[0044] Gene alignment was performed using the BLAST tool to compare the reference sequence with the sequenced genome. The Amuc_1100 gene was found to be aligned with the AkkermansiaBC_00573 gene of AkkermansiamuciniphilaAkkBC300, with a similarity of 93%.

[0045] Phylogenetic analysis was performed using MEGA12 software with Maximum Likehood and Bootstrap 1000. Phylogenetic analysis of the Akkermansia BC_00573 protein sequence with homologous genes from other strains further confirmed that Akkermansia BC_00573 is the Amuc_1100 gene.

[0046] Example 9: Effects of bacterial strains on GLP-1 secretion in enteroendocrine cells The NCI-H716 human enteroendocrine cell line was cultured and seeded in collagen-coated 96-well plates. After starvation with HBSS containing 0.2% BSA for 2 hours, cells were treated with 20 μL of viable AKKBC300 bacterial suspension (approximately 10^8 CFU), pasteurized AKKBC300 bacterial suspension (equivalent bacterial count), or HBSS (negative control) for 2 hours, respectively. Cell supernatants were collected, and GLP-1 concentration was detected using a GLP-1 ELISA kit. Results are shown below. Figure 3 show, Figure 3 This diagram illustrates the effects of live and pasteurized forms of strain AKKBC300 on GLP-1 secretion in NCI-H716 cells (*p<0.05, **p<0.01 compared to the control group). Both the live and pasteurized forms significantly promoted GLP-1 secretion in NCI-H716 cells compared to the control group.

[0047] Example 10: Effects of bacterial strains on gene expression in intestinal epithelial cells Caco-2 cells were cultured until they differentiated into a dense monolayer (21 days). Cells were treated for 24 hours with either active AKKBC300, pasteurized inactivated AKKBC300 (MOI = 100), or an equal volume of PBS (control). Total RNA was extracted from the cells and reverse transcribed into cDNA. The mRNA expression levels of tight junction proteins (ZO-1, Occludin, Claudin-1), Toll-like receptors (TLR2, TLR4), tumor necrosis factor-α (TNF-α), and angiopoietin-like protein 4 (ANGPTL4) were detected using quantitative real-time PCR (qRT-PCR), with GAPDH as an internal control gene. Results are as follows: Figure 4A , Figure 4B and Figure 4C show: Both live and inactivated bacteria significantly upregulated the gene expression of ZO-1, Occludin, and Claudin-1 (e.g., Figure 4A As shown, Figure 4A This diagram illustrates the effects of live and inactivated AKKBC300 on the genes of the Caco-2 cell line. Both live and inactivated bacteria significantly upregulated the gene expression of ZO-1, Occludin, and Claudin-1. Both live and inactivated bacteria significantly upregulated the expression of TLR2 and TLR4, and significantly downregulated the expression of TNF-α (e.g., ...). Figure 4B As shown, Figure 4B The diagram illustrates the effects of live and inactivated AKKBC300 on genes in the Caco-2 cell line. Both live and inactivated bacteria significantly upregulated the expression of TLR2 and TLR4, and significantly downregulated the expression of TNF-α. Live and inactivated bacteria also showed an upregulation trend in ANGPTL4 expression (e.g., [image of AKKBC300]). Figure 4C As shown, Figure 4CThe effect of live and inactivated AKKBC300 on genes in the Caco-2 cell line is illustrated in the diagram. Live and inactivated bacteria showed an upregulation trend in ANGPTL4 expression (*p<0.05, **p<0.01 compared with the control group).

[0048] These results indicate that the strain can enhance intestinal barrier function and regulate local immune inflammatory responses.

[0049] Example 11: Animal Experiment Design and Treatment Eight-week-old male C57BL / 6J mice (n=39) were housed under standard laboratory conditions. After one week of acclimatization, they were randomly divided into three groups (n=13 / group): Control group (C): 200 μL PBS was administered by gavage daily.

[0050] Live bacteria group (AKK): 200μL containing 10 bacteria was administered by gavage daily. 9 CFU-active AKKBC300 in PBS suspension.

[0051] Inactivated bacteria group (inactivated AKK): 200μL containing 10 μg / L of AKK administered by gavage daily. 9 CFU equivalent of pasteurized inactivated AKKBC300 in PBS suspension. All mice had free access to normal feed and water for 5 weeks. Body weight and food intake were recorded weekly.

[0052] Example 12: Effects of the strain on mouse body weight and plasma biochemical parameters After treatment, mice were fasted for 12 hours, and blood was collected via cardiac puncture to separate plasma. The concentrations of fasting blood glucose (Glu), total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in the plasma were measured using commercial kits. The results (summarized in Table 3) showed that, compared with the control group, the weight gain of mice in both the live bacteria group and the inactivated bacteria group was inhibited, and plasma TC, LDL, and fasting blood glucose levels were significantly reduced, while ALT and AST levels also showed an improving trend. This indicates that the strain has weight-loss, lipid-lowering, and liver-function-improving effects. The experimental results are shown in Table 3.

[0053] Table 3: Results of mouse growth and various blood indicators in animal experiments: Example 13: Effects of bacterial strains on mouse plasma cytokines The concentrations of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10) in mouse plasma collected in Example 12 were detected using an ELISA kit. The results (summarized in Table 3) showed that, compared with the control group, the levels of pro-inflammatory factors IL-6 and TNF-α in the plasma of mice in both the live bacteria group and the inactivated bacteria group showed a decreasing trend, while the level of anti-inflammatory factor IL-10 showed an increasing trend, indicating that this strain has a systemic anti-inflammatory effect.

[0054] Example 14: Effects of the strain on mouse histopathology After euthanizing the mice, colon and liver tissues were harvested, fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). The tissues were then examined and evaluated under a light microscope by an uninformed pathologist. Results are as follows: Figure 5 As shown, Figure 5 Schematic diagram of histopathological sections (H&E staining) of (A) colon and (B) liver of mice after 5 weeks of treatment with live and pasteurized strain AKKBC300: Colon: The colonic mucosa structure of each group was intact, and no obvious inflammatory cell infiltration or tissue damage was observed.

[0055] Liver: Obvious lipid droplet vacuoles were observed in the livers of mice in the control group (indicated by arrows in the figure), while lipid droplet accumulation was significantly reduced in the livers of mice in the live bacteria group and the inactivated bacteria group, indicating that this strain can improve hepatic steatosis.

[0056] Example 15: Effects of bacterial strains on gene expression in mouse tissues Colon and liver tissues were collected from mice, and total RNA was extracted using the Trizol method. After reverse transcription into cDNA, the expression of related genes was detected by qRT-PCR.

[0057] Colon tissue: mRNA expression of glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) was detected, with Rpl-19 as an internal reference gene.

[0058] Liver tissue: The mRNA expression of glucose transporter 2 (GLUT2) was detected, with Rpl-19 as an internal reference gene.

[0059] The results are as follows Figure 6 As shown, Figure 6This diagram illustrates the effects of live and pasteurized strain AKKBC300 on the relative expression levels of GLP-1 and PYY genes in (A) colon tissue and GLUT2 gene in (B) liver tissue of mice (compared to the control group, *p<0.05, **p<0.01). Compared to the control group, the expression of GLP-1 and PYY genes in the colon of mice in both the live and pasteurized groups was significantly upregulated, and the expression of GLUT2 gene in the liver was also significantly upregulated. This indicates that this strain can regulate the expression of genes related to intestinal hormones and liver glucose metabolism.

[0060] All in vitro and in vivo experimental data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups; if significant differences were found, pairwise comparisons were further performed using Tukey's post-hoc test. Relative quantification of gene expression was performed using... Method calculation. , which represents the difference in CT values ​​between the target gene and the internal reference gene. , which represents the difference in ΔCT values ​​between the experimental group and the control group. The formula is used to calculate the fold change in gene expression between the experimental group and the control group. A p < 0.05 is considered statistically significant.

[0061] Example 16: Validating the effect of extraction method on strain survival rate and tolerance The extraction method described in Example 1 was used to isolate Akkermansia myxophilus strains from fresh fecal samples from healthy individuals. The specific steps included: suspending the sample in sterile synthetic liquid medium (containing 3.8% BHI powder, 1.6% soybean peptone, 0.4% threonine, 25 mM glucose, 25 mM N-acetylglucosamine, 0.2% ascorbic acid, pH 7.2-7.4), homogenizing at 10,000 rpm for 3 minutes, performing 10-fold serial dilutions, and spreading the culture onto BHI agar plates supplemented with 4 g / L myxophilus and 5 μg / mL vancomycin. The culture was first pre-incubated at 37°C for 2 hours, then incubated anaerobically at 37°C (85% N2, 10% CO2, 5% H2) for 5-7 days. Single colonies were picked and purified to obtain pure culture AKKBC300.

[0062] To verify the effectiveness of the modified method, a comparative example was set up: the original method (without ascorbic acid, without pre-culturing, homogenization speed 5000 rpm) was used for isolation. The survival rate and tolerance of the strains under the two methods were compared. The experimental results are shown in Table 4.

[0063] Table 4: Effects on strain survival and tolerance: This method significantly improved the survival rate of the strain under low pH and bile salt conditions (p<0.05), indicating that the addition of ascorbic acid and pre-culture can enhance the strain's tolerance.

[0064] The strains extracted using this method exhibited good tolerance to low pH and bile salts in vitro. As shown in Example 6, the survival rates were 0.20% and 2.15% at pH 3.0 and 4.0, respectively, and 2.12% at a bile salt concentration of 0.3%. This indicates that the extraction method can screen for strains with good tolerance to the gastrointestinal environment, suggesting that they can reach the intestine and exert their effects through the upper digestive tract. The strains extracted using the method in this example exhibited even better tolerance to low pH and bile salts in vitro. This indicates that the extraction method can screen for strains with even better tolerance to the gastrointestinal environment, suggesting that they can reach the intestine and exert their effects more effectively through the upper digestive tract.

[0065] Example 17: Verification of the effect of extraction method on strain functionality The Akkermansia myxophilus strain AKKBC300, isolated using the extraction method described in the examples, was used for in vitro functional experiments. A strain isolated using the comparative method was used as a control. As shown in Examples 9 and 10, both the live and pasteurized forms of this strain significantly promoted the secretion of GLP-1 by human intestinal endocrine cells NCI-H716, upregulated the expression of tight junction proteins (ZO-1, Occludin, Claudin-1) in human intestinal epithelial cells Caco-2, and downregulated the expression of the pro-inflammatory factor TNF-α. The experimental results are shown in Table 5.

[0066] Table 5: Effects on strain functionality: The strain obtained in this embodiment was significantly superior to the comparative strain in promoting GLP-1 secretion and upregulating tight junction protein expression (p<0.05), and was also more effective in downregulating TNF-α expression. This indicates that this method can obtain strains with stronger functionalities. This extraction method can obtain strains with significant probiotic functions, including promoting GLP-1 secretion, enhancing intestinal barrier function, and anti-inflammatory effects. This demonstrates that the extraction method not only ensures the survival and purity of the strain but also guarantees its functionality.

[0067] Example 18: Verification of the effect of culture conditions on cell yield.

[0068] Following the extraction method described in Example 1, *Ackermania pseudomallei* strain AKKBC300 was cultured. Specifically, it was statically cultured at 37°C under anaerobic conditions for 72 hours in a liquid medium containing BHI medium, 5% fetal bovine serum, and 0.3 μg / mL insulin. Samples were taken every 24 hours to determine the bacterial concentration. A comparative example was also set up using a medium without insulin, with all other conditions remaining the same. The experimental results are shown in Table 6.

[0069] Table 6: Effects of culture conditions on cell yield Adding insulin significantly increased cell yield, doubling the yield of the control group at 72 hours (p<0.01).

[0070] The results showed that after 72 hours of cultivation, the bacterial concentration reached more than 10^9 CFU / mL (see the growth curve in Example 5).

[0071] These optimized culture conditions resulted in high-concentration bacterial cultures, providing an efficient culture protocol for large-scale production of microbial preparations. The addition of fetal bovine serum significantly increased bacterial yield, ensuring the effectiveness of the preparations.

[0072] In the formulation preparation, the characteristic of *Ackermania mucinosa* naturally colonizing the intestinal mucus layer was further utilized. In step c), the obtained bacterial cells were mixed with a carrier (such as PBS), and then encapsulated into microcapsules composed of mucin and chitosan using spray drying technology. This design simulates the intestinal mucus environment, providing protection for the bacterial cells against the erosion of gastric acid and bile salts. Furthermore, by utilizing the natural affinity of *Ackermania mucinosa* for mucin, targeted colonization and sustained release of the bacterial cells into the intestinal mucus layer are achieved, thereby significantly enhancing its residence time and absorption efficiency in the intestine.

[0073] Example 19: Verification of the effect of pasteurization conditions on bacterial cell stability and function Following the extraction method of Example 1, the obtained bacterial cells were pasteurized: treated in a 70°C water bath for 30 minutes, followed immediately by ice cooling for 5 minutes. After inactivation, the viability of the bacterial cells was checked (to confirm the absence of live bacteria), and their function was evaluated. A comparative example was also included: no ice cooling was performed after inactivation. As shown in Examples 9 and 10, pasteurized bacterial cells, like live bacterial cells, significantly promoted GLP-1 secretion and upregulated tight junction protein expression. The experimental results are shown in Table 7.

[0074] Table 7: Effects of pasteurization inactivation conditions on bacterial cell function: Immediate ice bath cooling after inactivation better preserves bacterial function and is significantly superior to the control group in promoting GLP-1 secretion and upregulating tight junction protein expression (p<0.05).

[0075] These inactivation conditions ensure complete inactivation of the bacteria while preserving their beneficial functions. Inactivated bacteria exhibit higher safety and stability, making them suitable for preparing postbiotic formulations and facilitating storage and transportation.

[0076] To further enhance the persistence of inactivated bacterial cell efficacy in the gut, in step c), during formulation preparation, pasteurized inactivated bacterial cells are mixed with a pH-sensitive material (such as Eudragit® FS30D) to prepare microspheres. These microspheres remain intact in gastric juice and the proximal small intestine, while dissolving and releasing their contents in the specific pH environment of the terminal ileum and colon. This design mimics the natural distribution characteristics of Akkermansia myxophilus as the dominant colonic flora, enabling precise and sustained-release delivery of inactivated bacterial cells and their fragments to the target intestinal segment, prolonging their interaction time with intestinal epithelial cells and immune cells, thereby more effectively promoting gut health.

[0077] Example 20: Verifying the overall efficacy of microbial preparations The microbial preparation was prepared according to the method for preparing microbial preparations: live or pasteurized bacterial cells obtained from culture were mixed with a pharmaceutically acceptable carrier (such as PBS). The live bacterial preparation was encapsulated using mucin-chitosan microcapsules, and the inactivated bacterial preparation was encapsulated using pH-sensitive colon-targeting microspheres. A comparative example was also set up: an unencapsulated preparation was used (directly mixed with PBS). Animal experiments (Examples 11-15) demonstrated that this preparation effectively prevented weight gain in mice, improved lipid profiles, reduced fasting blood glucose, improved hepatic steatosis, and modulated immune responses. The experimental results are shown in Table 8.

[0078] Table 8: Effects of microbial agents on mouse body weight and plasma parameters: Formulations using targeted delivery systems were significantly superior to unencapsulated formulations in terms of weight loss, blood glucose reduction, blood lipid reduction, and improvement of liver function (p<0.05).

[0079] The microbial preparations produced by this method (whether in live or inactivated form) not only exhibit significant in vivo probiotic effects, but also achieve intestinal-targeted delivery and sustained release through advanced dosage form design, significantly improving the bioavailability of the bacteria and their active ingredients. This demonstrates the reliability of the preparation method and the effectiveness of the formulation. This provides technical support for the development of highly efficient microecological preparations in different forms.

[0080] Obviously, the embodiments described above are merely some embodiments of the present invention, not all embodiments. The accompanying drawings show preferred embodiments of the present invention, but do not limit the patent scope of the present invention. The present invention can be implemented in many different forms; rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing specific embodiments, or make equivalent substitutions for some of the technical features. Any equivalent structures made using the content of this specification and drawings, directly or indirectly applied to other related technical fields, are similarly within the patent protection scope of this invention.

Claims

1. A strain of Akkermansia myxophilus, characterized in that, The strain has the accession number CGMCC No. 34370, and its 16S rDNA sequence has 93% similarity to the sequence shown in SEQ ID NO:

1.

2. The Akkermansia strain according to claim 1, characterized in that, The Akkermansia strain with myxotropin was obtained by a method comprising the following steps: a) Collect fresh stool samples from healthy individuals; b) The sample was suspended in a sterile synthetic liquid culture medium containing 3.5%-4.0% brain heart extract BHI powder, 1.5%-1.7% soybean peptone, 0.3%-0.5% threonine, 20-30 mM glucose, 20-30 mM N-acetylglucosamine, 0.1%-0.3% ascorbic acid, and a pH of 7.2-7.4; c) Homogenize the suspension at 10,000 rpm for 2-4 minutes; d) Perform a 10-fold serial dilution on the homogenized suspension; e) Take 0.08-0.12 mL of the appropriate dilution solution and spread it on a selective agar plate, wherein the selective agar plate is BHI agar supplemented with 3-5 g / L mucin and 4-6 μg / mL vancomycin; f) First, pre-culture at 37°C for 2 hours, then culture at 36.5-37.5°C under anaerobic conditions in an atmosphere of 83%-87% N2, 9%-11% CO2 and 4%-6% H2 for 5-7 days; g) Select single colonies with different morphologies and repeatedly streak them on fresh vancomycin BHI agar plates for purification until a pure culture is obtained.

3. A microbial preparation, characterized in that, It is prepared by a method including the following steps: a) Cultivate the Akkermansia myxophilus strain according to claim 1 or 2 to obtain bacterial cells, wherein the cultivation is carried out in a liquid medium containing BHI medium, 1-10% fetal bovine serum and 0.1-0.5 μg / mL insulin, and is statically cultured at 36-38°C under anaerobic conditions for 48-96 hours. b) Inactivate the bacterial cells obtained in step a), and / or process them into bacterial cell fragments, and / or collect their metabolites, wherein the inactivation treatment is pasteurization, and the specific conditions are: water bath treatment at 60-80°C for 15-45 minutes, followed by immediate ice bath cooling for 5 minutes; c) Mix one or more of the bacterial cells obtained in step a) and / or the inactivated bacterial cells, bacterial cell fragments, and metabolites obtained in step b) with a pharmaceutically or food-grade acceptable carrier, wherein the carrier comprises mucin-chitosan microcapsules or pH-sensitive colon-targeting microspheres. The culture in step a) is carried out in a liquid medium containing BHI medium and 1-10% fetal bovine serum, and is statically cultured at 36-38°C under anaerobic conditions for 48-96 hours. Furthermore, the inactivation treatment in step b) is pasteurization, specifically under the following conditions: water bath treatment at 60-80°C for 15-45 minutes.

4. The microbial preparation according to claim 3, characterized in that, The inactivated bacterial cells are pasteurized inactivated bacterial cells.

5. The use of the Akkermansia myxophilus strain of claim 1 or 2 or the microbial preparation of claim 3 or 4 in the preparation of articles for the prevention and / or treatment of obesity or weight loss.

6. The use of the Akkermansia myxophilus strain of claim 1 or 2 or the microbial preparation of claim 3 or 4 in the preparation of articles for lowering blood lipids and / or improving fasting blood glucose.

7. The use of the Akkermansia myxophilus strain of claim 1 or 2 or the microbial preparation of claim 3 or 4 in the preparation of articles for promoting the secretion of glucagon-like peptide-1.

8. The use of the Akkermansia myxophilus strain of claim 1 or 2 or the microbial preparation of claim 3 or 4 in the preparation of articles for enhancing intestinal barrier function and / or upregulating the expression of intestinal tight junction proteins, wherein the tight junction proteins include one or more of ZO-1, Occludin and Claudin-1.

9. The use of the Akkermansia myxophilus strain of claim 1 or 2 or the microbial preparation of claim 3 or 4 in the preparation of articles for modulating immune responses, wherein the modulation of immune responses includes reducing the levels of pro-inflammatory factors TNF-α and / or IL-6, and / or increasing the levels of anti-inflammatory factor IL-10.

10. The use of the Akkermansia myxophilus strain of claim 1 or 2 or the microbial preparation of claim 3 or 4 in the preparation of articles for improving hepatic steatosis and / or regulating hepatic transaminase ALT and AST levels.

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