A strain of Akkermansia myxophilus and its application in weight management products

Abstract

This invention, entitled "A strain of Akkermansia myxophilus and its application in weight management products," belongs to the field of microbial technology. The technical problem to be solved is to provide a strain of Akkermansia myxophilus that can effectively alleviate obesity. The key technical solution is a strain of Akkermansia myxophilus (… Farmhouse mucinophila The accession number for the myxotrophic Akkermansia HHAKKα is GDMCC No:68013.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Akkermansia myxophilus and its application in weight management products. Background Technology

[0002] Obesity is a prevalent chronic metabolic disease worldwide. Its essence is a metabolic disorder syndrome caused by an imbalance between the body's energy intake and consumption, leading to excessive accumulation of adipose tissue. It not only causes obesity but also significantly induces various metabolic complications such as insulin resistance, type 2 diabetes, hyperlipidemia, and non-alcoholic fatty liver disease. It is also accompanied by a series of pathological problems such as intestinal barrier damage, low-grade chronic inflammation, and immune disorders, and has become a public health problem that urgently needs to be addressed.

[0003] Currently, clinical and everyday interventions for obesity mainly fall into three categories: lifestyle interventions, pharmacological interventions, and surgical interventions. Among these, lifestyle interventions such as diet control and exercise suffer from poor adherence, long weight loss cycles, and are prone to rebound after cessation, making long-term stable metabolic regulation difficult. Weight loss drugs have toxic side effects such as gastrointestinal irritation and endocrine disorders, limiting their applicability. Furthermore, most weight loss drugs only control energy intake and cannot fundamentally repair the intestinal metabolic damage caused by obesity. Weight loss surgery is highly invasive, carries a high risk of postoperative complications, and is only suitable for severely obese patients, with very low general applicability. Overall, existing obesity intervention technologies generally suffer from limited efficacy, poor safety, high rebound rates, and an inability to target and regulate metabolic homeostasis, resulting in significant bottlenecks in clinical application.

[0004] Akkermansia myxophilus ( Akkermansia muciniphila Akk bacteria, as a core beneficial bacterium in the human gut, specifically colonizes the intestinal mucus layer. It can play a role in repairing the intestinal barrier, inhibiting low-level inflammation, regulating glucose and lipid metabolism, and promoting lipid metabolism remodeling by degrading intestinal mucoprotein and secreting active substances such as short-chain fatty acids. It can intervene in the pathogenesis of obesity from the perspective of intestinal microecology and is a high-quality functional strain for green intervention in obesity, with extremely high development and application value.

[0005] Relevant patent documents retrieved: The country of origin is China, publication number CN120555307A, publication date August 29, 2025. This document discloses Akkermansia myxophilus (… Akkermansia muciniphilaThe Akk-101 and AMY001 strains in this invention differ from standard strains in nucleotide consistency and physiological and biochemical indicators. They exhibit superior acid-base tolerance and colonization ability compared to standard strains, more effectively inhibiting lipid accumulation in liver cells, reducing fat deposition in nematodes, and promoting weight loss and improved glucose and lipid metabolism.

[0006] Relevant non-patent literature retrieved: The journal or book title is "China Food and Nutrition", the article title is "Effects of AKK bacteria on glucose and lipid metabolism in high-fat diet-induced obese rats", the publication date is 2022. This article discloses that AKK bacteria can significantly reduce body weight, blood glucose, blood lipids and transaminase levels in high-fat diet-induced obese rats, and may improve liver function by increasing insulin sensitivity in rats.

[0007] While some existing technologies have explored the use of Akkermansia myxophilus to improve metabolism, current strains generally suffer from weak obesity-relieving effects and poor targeting. Therefore, developing an Akkermansia myxophilus strain that can effectively alleviate obesity and improve metabolic homeostasis is a pressing technical challenge in this field. Summary of the Invention

[0008] The purpose of this invention is to provide: A strain of Akkermansia myxophilus and its application in weight management products, and related technologies to address technical issues such as providing a strain of Akkermansia myxophilus that can effectively alleviate obesity, or combinations thereof.

[0009] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0010] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0011] Definitions of standard chemical terms can be found in the references "Principles and Identification Techniques of Bacterial and Archaea Systematic Taxonomy, Higher Education Press, Chief Editors Li Wenjun, Liu Lan, Jiao Jianyu, and Fang Baozhu, 2025-01"; "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Science Press, 4th Edition, 2017; and "Microbiology Experiments", Higher Education Press, 4th Edition, 2016.

[0012] Unless otherwise stated, conventional methods within the scope of the art, such as bacterial culture preparation, shall be used.

[0013] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0014] The term “Ackermania glutinis” used in this article refers to a strictly anaerobic intestinal commensal bacterium of the phylum Verrucomicrobia, which can degrade intestinal mucin and participate in maintaining the intestinal barrier, regulating the body's metabolism and intestinal immunity.

[0015] The term "white adipose tissue" used in this article refers to the largest proportion of adipose tissue in the adult body. It is mainly composed of unilocular white adipocytes, which contain a single large lipid droplet and have a small number of mitochondria. It is mainly distributed in the subcutaneous tissue and around internal organs. Its core function is to store triglycerides as energy reserves. At the same time, it acts as an endocrine organ to secrete adipokines such as leptin and adiponectin, which participate in the body's energy homeostasis, glucose and lipid metabolism and inflammation regulation. Excessive accumulation is associated with metabolic diseases such as obesity and insulin resistance.

[0016] The term "brown adipose tissue" used in this article refers to the thermogenic adipose tissue unique to mammals, composed of multilocular brown adipocytes. These cells contain a large number of mitochondria and highly express uncoupling protein 1 (UCP1). They mainly perform non-shivering thermogenesis, consuming glucose and lipids to generate heat. At the same time, they secrete a variety of adipokines to participate in the regulation of systemic glucose and lipid metabolism homeostasis. They are abundant in newborns, while in adults they are mainly distributed in the supraclavicular, paravertebral, and cervical regions.

[0017] The term "short-chain fatty acids" used in this article refers to saturated fatty acids with 2-6 carbon atoms produced by gut microbiota fermentation of dietary fiber and other carbohydrates. These mainly include acetic acid, propionic acid, and butyric acid. They can serve as energy substrates for intestinal epithelial cells, regulate intestinal barrier function, immune responses, and glucose and lipid metabolism, and participate in intestinal... Organ axis signal transmission.

[0018] In a first aspect, the present invention provides a strain of Akkermansia myxophilus ( Akkermansia muciniphilaHHAKKα, with accession number GDMCC No:68013.

[0019] Specifically, the 16S sequence of the myxotropic Akkermansia hHAKKα is shown in SEQ ID NO:1.

[0020] SEQ ID NO:1:

[0021] In a second aspect, the present invention provides a preparation of Akkermansia xylophilus HHAKKα, including fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria, dead bacteria, and lyophilized powder.

[0022] Specifically, the fermentation broth refers to the liquid produced after inoculating the microbial strain into a culture medium and culturing it.

[0023] Specifically, the supernatant of the fermentation broth refers to the clear liquid at the top of the fermentation broth after centrifugation; it contains abundant metabolic products, secretions, and some bacterial fragments from the bacterial growth and reproduction process.

[0024] Specifically, the fermentation broth precipitation refers to the liquid precipitate after centrifugation, including free proteins, residual bacterial cells, broken cells, and culture medium residues.

[0025] Specifically, the dead bacteria refer to microorganisms that have lost their vitality and are unable to grow and reproduce.

[0026] Specifically, the method for preparing freeze-dried powder can employ methods currently used in the art, or other methods that may emerge in the future to obtain a product in solid form of microorganisms or microbial cultures. It should be clarified that the method for preparing freeze-dried powder should not be a condition limiting the scope of protection of this invention.

[0027] Thirdly, the present invention provides a method for preparing Akkermansia myxophilus HHAKKα, comprising inoculating Akkermansia myxophilus HHAKKα into a culture medium and culturing it.

[0028] According to some embodiments of the present invention, the method for culturing Akkermansia xylophilus HHAKKα provided by the present invention is as follows: HHAKKα is cultured in a brain heart extract culture medium supplemented with 0.05% type II mucin at 37°C under specific anaerobic conditions (containing 10% hydrogen, 10% carbon dioxide and 80% nitrogen).

[0029] Fourthly, the present invention provides a microbial agent comprising the above-mentioned Akkermansia xylophilus HHAKKα and / or the above-mentioned preparations.

[0030] Specifically, the bacterial agent is a powder or liquid preparation.

[0031] Specifically, the microbial agent may be a solid, liquid, semi-solid, or any physical form containing the necessary active ingredients to achieve any of the applications, functions, or effects described in this invention, which can be obtained by those skilled in the art through current or future technologies.

[0032] In some implementations, the bacterial agent is in solid form.

[0033] Fifthly, the present invention provides the use of the above-mentioned Akkermansia xylophilus HHAKKα, the above-mentioned preparations and / or the above-mentioned bacterial agents in the preparation of products for relieving obesity.

[0034] Specifically, the products mentioned include pharmaceuticals and health supplements.

[0035] Furthermore, when the product is a health supplement, it helps control body fat.

[0036] Specifically, the health products also include conventional excipients for health products, including but not limited to fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

[0037] Furthermore, the health products mentioned include emulsion products, solution products, powder products, and solid products.

[0038] Specifically, the product contains 1 × 10⁻⁶ Akkermansia xylophilus HHAKKα. 6 -1×10 12 CFU / unit or 1×10 6 -1×10 12 TFU / unit, for example, 1×10 6 CFU / unit (TFU / unit), 5×10 6 CFU / unit (TFU / unit), 1×10 7 CFU / unit (TFU / unit), 5×10 7 CFU / unit (TFU / unit), 1×10 8 CFU / unit (TFU / unit), 5×10 8 CFU / unit (TFU / unit), 1×10 9 CFU / unit (TFU / unit), 5×10 9 CFU / unit (TFU / unit), 1×10 10 CFU / unit (TFU / unit), 5×10 10 CFU / unit (TFU / unit), 1×10 11 CFU / unit (TFU / unit), 5×10 11 CFU / unit (TFU / unit), 1×10 12 CFU / unit (TFU / unit), etc., and other point values ​​within this range can be selected.

[0039] Specifically, the medicine includes a pharmaceutically acceptable carrier.

[0040] Furthermore, the pharmaceutically acceptable carrier is selected from one or more excipients, stabilizers, diluents, binders, preservatives, and lubricants.

[0041] Specifically, the dosage form of the drug is drops, mixture, tincture, injection, tablet, powder, oral liquid, capsule, granule, ointment, suspension, powder, emulsion, solution, drop pill, pill, lozenge, lyophilized powder for injection, gel, suppository or aerosol.

[0042] Specifically, the product has at least one of the following functions: (1) Inhibit weight gain; (2) Improves excessive fat deposition; (3) Improve glucose metabolism abnormalities caused by obesity; (4) Improves dyslipidemia and leptin levels caused by obesity; (5) Downregulate the levels of inflammatory factors and improve the chronic inflammatory state caused by obesity; (6) Improves hepatic steatosis and reduces fat cell volume; (7) Improves intestinal barrier dysfunction caused by obesity; (8) Enhances gut microbial diversity and reverses gut microbiota dysbiosis; (9) Relieve metabolic disorders.

[0043] In a sixth aspect, the present invention provides a product for relieving obesity, the product comprising the above-mentioned Akkermansia xylophilus HHAKKα, the above-mentioned preparation, and / or the above-mentioned bacterial agent.

[0044] Specifically, the products mentioned include pharmaceuticals and health supplements.

[0045] Furthermore, when the product is a health supplement, it helps control body fat.

[0046] Specifically, the health products also include conventional excipients for health products, including but not limited to fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

[0047] Furthermore, the health products mentioned include emulsion products, solution products, powder products, and solid products.

[0048] Specifically, the product contains 1 × 10⁻⁶ Akkermansia xylophilus HHAKKα. 6 -1×10 12 CFU / unit or 1×10 6 -1×10 12 TFU / unit, for example, 1×10 6CFU / unit (TFU / unit), 5×10 6 CFU / unit (TFU / unit), 1×10 7 CFU / unit (TFU / unit), 5×10 7 CFU / unit (TFU / unit), 1×10 8 CFU / unit (TFU / unit), 5×10 8 CFU / unit (TFU / unit), 1×10 9 CFU / unit (TFU / unit), 5×10 9 CFU / unit (TFU / unit), 1×10 10 CFU / unit (TFU / unit), 5×10 10 CFU / unit (TFU / unit), 1×10 11 CFU / unit (TFU / unit), 5×10 11 CFU / unit (TFU / unit), 1×10 12 CFU / unit (TFU / unit), etc., and other point values ​​within this range can be selected.

[0049] Specifically, the medicine includes a pharmaceutically acceptable carrier.

[0050] Furthermore, the pharmaceutically acceptable carrier is selected from one or more excipients, stabilizers, diluents, binders, preservatives, and lubricants.

[0051] Specifically, the dosage form of the drug is drops, mixture, tincture, injection, tablet, powder, oral liquid, capsule, granule, ointment, suspension, powder, emulsion, solution, drop pill, pill, lozenge, lyophilized powder for injection, gel, suppository or aerosol.

[0052] Specifically, the product has at least one of the following functions: (1) Inhibit weight gain; (2) Improves excessive fat deposition; (3) Improve glucose metabolism abnormalities caused by obesity; (4) Improves dyslipidemia and leptin levels caused by obesity; (5) Downregulate the levels of inflammatory factors and improve the chronic inflammatory state caused by obesity; (6) Improves hepatic steatosis and reduces fat cell volume; (7) Improves intestinal barrier dysfunction caused by obesity; (8) Enhances gut microbial diversity and reverses gut microbiota dysbiosis; (9) Relieve metabolic disorders.

[0053] The present invention has at least the following beneficial effects: 1. Compared with the prior art, the present invention has better technical effects in alleviating weight gain.

[0054] According to experimental tests, this invention reduces weight gain from about 7g in existing technologies to less than 5.1g.

[0055] A. muciniphila HHAKKα intervention effectively improved metabolic disorders induced by a high-fat diet in obese mice, specifically manifested as weight loss, improved lipid profile, and reduced hepatic lipid deposition. Mechanistically, this strain can regulate key signaling pathways related to lipid metabolism (including the FASN and C / EBPα pathways). Furthermore, it can promote the expression of adipogenic factors such as leptin, maintain gut microbiota balance, and enhance the production of short-chain fatty acids, thereby further improving metabolic function.

[0056] Considering the possibility of this invention entering other countries, this invention also provides the following technical solutions: A method for alleviating obesity includes administering to a subject the above-mentioned Akkermansia muciniphila HHAKKα, the above-mentioned preparation, and / or the above-mentioned bacterial agent.

[0057] Preservation Instructions Preserved strains: Akkermansia muciniphila HHAKKα; Category Naming: Akkermansia muciniphila ; Accession number: GDMCC No:68013; Preservation period: March 27, 2026; Preservation institution: Guangdong Provincial Center for Microbial Culture Collection; Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0058] Figure 1 To evaluate the effects of *Akermansia myxophilus* on body weight and obesity in HFD-induced mice. (A) Experimental flowchart; (B) Body weight changes during the experiment; (C) Average daily energy intake; (D) Body composition of each group (including intraperitoneal white adipose tissue iWAT, extraperitoneal white adipose tissue eWAT, brown adipose tissue BAT, and liver); (E) Body weight on day 42; (FI) Weight of inguinal white adipose tissue (iWAT), epididymal white adipose tissue (eWAT), brown adipose tissue, and liver. Data are expressed as mean ± standard deviation (SD). Statistical significance was determined as follows: p<0.05, p<0.01, p<0.001, p<0.0001.

[0059] Figure 2 This study investigated the effects of *Akermansia myxophilus* on blood glucose and lipid-related parameters induced by high-frequency epidemiology (HFD) in mice. (A) Fasting blood glucose (FBG); (B) Two-hour postprandial blood glucose (PBG); (C) Serum leptin level (LEP); (D) Serum total cholesterol (TC); (E) Serum triglycerides (TG); (F) Serum low-density lipoprotein cholesterol (LDL-C). Data are expressed as mean ± standard deviation (SD). Statistical significance was determined as follows: p<0.05, p<0.01, p<0.001, p<0.0001.

[0060] Figure 3 The effects of *Akermansia myxophilus* on inflammatory factors induced by HFD in mice were investigated. (A) Serum tumor necrosis factor-α (TNF-α); (B) Serum interleukin-1β (IL-1β); (C) Serum mononuclear cell chemokine-1 (MCP-1); (D) Serum interleukin-10 (IL-10). Data are expressed as mean ± standard deviation (SD). Statistical significance was determined as follows: p<0.05, p<0.01, p<0.001.

[0061] Figure 4 Histopathological analysis of liver and adipose tissue. (A) Image of HE-stained section of mouse liver tissue; (B) Image of fat deposition in Oil Red O-stained section of mouse liver tissue; (C) Image of fat volume and adipocyte area in HE-stained section of white adipose tissue (eWAT and iWAT) of mouse viscera. Scale bar: 0.050 mm. Data are expressed as mean ± standard deviation (SD). Statistical significance criteria are as follows: p<0.01, p<0.001, p<0.0001.

[0062] Figure 5 Immunofluorescence images and quantitative analysis of fluorescence intensity in colon tissue were performed. (A) Immunofluorescence staining image of ZO-1; (B) Immunofluorescence staining image of closure protein; Scale bar: 0.050 mm. Data are expressed as mean ± standard deviation (SD). Statistical significance was determined as follows: p<0.001.

[0063] Figure 6 For α diversity analysis.

[0064] Figure 7 The figures show β diversity analysis (PCoA) (left) and β diversity analysis (NMDS) (right).

[0065] Figure 8 The abundance distribution of the top 20 species at the phylum level.

[0066] Figure 9 This is a distribution map of the abundance of the top 20 species at the genus level.

[0067] Figure 10 Heatmap of species composition at the genus level (clustering results of the top 50 species).

[0068] Figure 11 To analyze the dominant taxa of the HFD and HHAKKα groups using linear discriminant analysis (LEfSe), taxa with an LDA value > 2 and p < 0.05 were identified as significant distinguishing marker species.

[0069] Figure 12 Predicted abundance distribution of KEGG secondary functional pathways.

[0070] Figure 13 Cluster heatmap of total metabolites (seven short-chain fatty acids).

[0071] Figure 14 for A. muciniphila Effects on short-chain fatty acids in feces. (A) Total amount of short-chain fatty acids; (B) Butyric acid level; (C) Propionic acid level; (D) Isobutyric acid level; (E) Valeric acid level; (F) Isovaleric acid level; (G) Acetic acid level; (H) Octanoic acid level. p<0.05, p<0.01, p<0.001.

[0072] Figure 15 This diagram compares the HFD group with the HHAKKα group. Blue dots represent metabolic pathways, while other dots represent metabolite molecules. The size of the pathway dots indicates the number of metabolite molecules linked to that pathway, and the gradient color scale reflects the log2 (FC) value of the metabolite molecules.

[0073] Figure 16 Spearman correlation analysis was performed on the gut microbiota, short-chain fatty acids (SCFAs), and metabolic / inflammatory markers. (A) Correlation analysis between biochemical markers and differentially expressed microbiota; (B) Correlation analysis between gut microbiota and short-chain fatty acids (SCFAs); (C) Correlation analysis between biochemical markers and SCFAs. FBG: Fasting blood glucose; PBG: 2-hour postprandial blood glucose; TC: Total cholesterol; TG: Triglycerides; HDL-C: High-density lipoprotein cholesterol; LDL-C: Low-density lipoprotein cholesterol; TNF: Tumor necrosis factor; IL: Interleukin; MCP-1: Monocyte chemoattractant protein-1; GLP-1: Glucagon-like peptide-1; ADP: Adiponectin; LEP: Leptin. p<0.05, p<0.01.

[0074] Figure 17 The effect of *Akermansia myxophilus* on liver protein expression in obese HFD mice. (A) Protein expression levels of PGC-1α, C / EBPα, FASN, p-AMPKα, AMPKα, P-ACC, ACC, PPARγ, and SREBP-1 in the liver; (B) Relative protein expression levels (n=3). Data are expressed as mean ± standard deviation (SD). Statistical significance was determined as follows: p<0.01.

[0075] Figure 18 The effect of *Akermansia myxophilus* on serum biomarkers in HFD-induced mice was investigated. (A) Serum GLP-1 level; (B) Serum adiponectin (ADP) level; (C) Serum high-density lipoprotein cholesterol (HDL-C) level. Data are expressed as mean ± standard deviation (SD). Statistical significance was determined as follows: p<0.05. Detailed Implementation

[0076] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0077] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0078] Basic Implementation Separation from fresh stool samples of healthy infants Akkermansia Factosome strain. Fecal samples were added to pre-reduced sterile diluent and thoroughly mixed. After 10-fold serial dilutions, the mixture was inoculated onto a culture medium containing 0.4% mucin. Akkermansia The culture was carried out in anaerobic medium at 37°C under strictly anaerobic conditions for 48–72 h. Morphologically uniform candidate single colonies were selected and purified three times by streak plating to obtain pure culture strains. The strains were then identified by 16S rRNA gene sequencing and whole genome sequence analysis. Akkermansia muciniphila The strains obtained from the initial isolation were used as parent strains, and then targeted domestication was carried out.

[0079] The first step was gastrointestinal environment adaptation. The parental strain was inoculated into a medium containing 0.4% mucin and cultured strictly anaerobically at 37°C until the stable growth phase. The cells were then subjected to short-term acidic treatments at pH 5.0, 4.5, 4.0, and 3.5, each for 30 minutes. After each level of acid stress, the cells were transferred to a normal pH medium for recovery before proceeding to the next level of stress. Simultaneously, stepwise acclimatization was performed using 0.05%, 0.10%, and 0.20% ox bile salts, each concentration for 60 minutes. The surviving cells were then transferred to a bile-free medium for recovery. Acid and bile salt stress were cycled 10 times. In each cycle, viable cell counts were used to evaluate the survival rate and recovery ability of the strains, retaining strains with strong gastrointestinal environment tolerance and stable recovery.

[0080] The second step was targeted acclimatization to the mucin niche. The candidate strains obtained in the first step were inoculated into a culture system with mucin as the main nutrient source. The mucin concentration was successively reduced from 0.4% to 0.2%, 0.1%, and 0.05%, with each concentration followed by three consecutive anaerobic passages, for a total of 12 passages. Cultures with good growth and high viable cell counts from each generation were selected for the next generation, allowing the strains to gradually adapt to the low mucin supply environment. After acclimatization, the candidate strains were uniformly restored to a culture condition containing 0.4% mucin, and their growth capacity, viable cell count, and recovery ability were compared to screen for candidate strains with strong adaptability to the mucin niche.

[0081] The third step is Akkermansia Targeted screening for specific functions. Candidate strains acclimated to the mucin niche were cultured in the same medium, with the same inoculum size and culture time. Cells were collected and total RNA was extracted. The relative transcription levels of Amuc_1100 and Amuc_1631 (P9) were detected by RT-qPCR. Using unacclimated parental strains as controls, candidate strains exhibiting stable increases in the expression levels of Amuc_1100 and Amuc_1631 while maintaining good growth performance were screened.

[0082] The fourth step is targeted screening of cells related to obesity and metabolic regulation. The candidate strains obtained in the third step are subjected to standardized culture, and bacterial cells and sterile culture supernatants are prepared with the same number of viable cells. The results are then compared using cell models related to obesity and metabolic regulation.

[0083] First, an enteroendocrine cell model was used to detect the ability of different candidate strain cultures to promote GLP-1 secretion. The parent strain was used as a control to screen candidate strains that could significantly increase the level of GLP-1 secretion in cells.

[0084] Secondly, the Caco-2 intestinal epithelial cell model was used to evaluate the effects of different candidate strains on intestinal barrier function. The expression levels of tight junction proteins such as ZO-1, Occludin, and Claudin-1 were detected and evaluated in conjunction with cell barrier integrity to screen candidate strains that can enhance or maintain intestinal epithelial barrier function.

[0085] The lipid metabolism regulation capacity of candidate strains was further evaluated using the 3T3-L1 adipocyte model. During 3T3-L1 cell differentiation and lipid accumulation, sterile culture supernatant or cell-treated material from different candidate strains were added. The degree of lipid accumulation was evaluated by Oil Red O staining and intracellular triglyceride content detection, and candidate strains capable of reducing lipid accumulation in adipocytes were screened.

[0086] By combining the expression levels of Amuc_1100 and Amuc_1631, the promoting effect of GLP-1, the improving effect of intestinal epithelial barrier, and the inhibiting effect of lipid accumulation in adipocytes, the candidate strain with the best metabolic regulatory function was screened.

[0087] The fifth step is strain stability acclimation. The final selected candidate strains were removed from the selection pressures of acid, bile salts, and low-viscosity proteins, and continuously passaged anaerobically for 20 generations under standard culture conditions. Growth curves, viable cell counts, and relative transcription levels of Amuc_1100 and Amuc_1631 were measured every 5 generations. GLP-1 secretion, intestinal barrier function, and lipid accumulation-related cellular function were repeatedly measured at the 10th and 20th generations. Strains that maintained stable growth characteristics and major functional phenotypes after 20 generations were identified as the final artificially acclimated strains.

[0088] Ultimately, through the separation of feces from healthy infants, adaptation to the gastrointestinal environment, and directional adaptation to the mucus niche, Akkermansia Specific function screening, screening of cells related to obesity and metabolic regulation, and validation of stability through continuous passage resulted in a strain with good environmental adaptability, high expression levels of Amuc_1100 and Amuc_1631, and the ability to promote GLP-1 secretion, improve the intestinal epithelial barrier, and reduce lipid accumulation in adipocytes. Akkermansia muciniphila The artificially domesticated strain was named HHAKKα and was deposited at the Guangdong Provincial Microbial Culture Collection Center with accession number GDMCC No:68013.

[0089] Example 1 1. Materials and Methods 1.1 Strain preparation A. muciniphila HHAKKα was initially isolated from the feces of healthy individuals. A. muciniphila The 16S sequence of HHAKKα is shown in SEQ ID NO:1.

[0090] SEQ ID NO:1:

[0091] A. muciniphila HHAKKα was cultured in brain and heart extract (BHI) medium supplemented with 0.05% type II mucin at 37°C under specific anaerobic conditions (containing 10% hydrogen, 10% carbon dioxide, and 80% nitrogen). After 72 hours of incubation, bacterial growth was analyzed and identified. The bacterial suspension was then resuspended in phosphate-buffered saline (PBS) and diluted to a concentration of 5 × 10⁻⁶. 9 CFU / mL.

[0092] 1.2 Animal Experiment Design This study used 28 five-week-old male C57BL / 6J mice, provided by Beijing HFK Biotechnology Co., Ltd. (Beijing, China). The mice were housed in a controlled environment with a temperature of 25±2 °C, humidity of 50±5%, and a 12-hour light / dark cycle. All animal experiments followed the guidelines for laboratory animal care and use of Jilin Agricultural University and were approved by the university's Animal Ethics Committee (Approval No.: 20250609002).

[0093] After a one-week acclimatization period, C57BL / 6J mice were randomly assigned to two experimental groups: a control group (ND, n=7) and a high-fat diet group (HFD, n=21). The control group was fed a standard diet (XTCON50J, 3.83 kcal / g), while the HFD group received a high-fat diet (XTHF60, 5.22 kcal / g; both diets were purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd.). After eight weeks of feeding, an obesity model was established when the body weight of the HFD group increased by more than 20% compared to the control group.

[0094] The HFD group was further randomized into three subgroups (n=7): the high-fat diet subgroup (HFD), the HHAKKα subgroup, and the orlistat subgroup (ORL; Delpham Milan Ltd., Italy). All subgroups continued to receive the high-fat diet.

[0095] The control group and the HFD group received daily oral PBS (10 mg / kg / day), while the probiotic intervention group received daily oral HHAKKα (5 × 10⁻⁶ mg / kg / day). 9 The CFU / kg / day group received an oral dose of 24 mg / kg body weight daily in the ORL group. The study lasted for six weeks, with body weight and food intake monitored weekly.

[0096] One week prior to the end of the study, mice were fasted for 12 hours, and fasting blood glucose and blood glucose levels two hours after a meal were measured using a glucometer. Mice were then euthanized under isoflurane anesthesia, and feces, blood, liver, colon, white adipose tissue (epididymis and groin region), and brown adipose tissue were collected, weighed, and analyzed. All tissue samples were rapidly frozen in liquid nitrogen and stored at -80°C for subsequent analysis.

[0097] 1.3 Serum Biochemical Analysis The concentrations of serum triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured using commercially available biochemical assay kits (Nanjing Jiancheng Biotechnology Research Institute Co., Ltd., China).

[0098] 1.4 Quantitative determination of serum adipokines and inflammatory cytokines This study used enzyme-linked immunosorbent assay (ELISA) to detect the concentrations of adiponectin (ADP; SMK3283B), leptin (LEP; SMK2895B), glucagon-like peptide-1 (GLP-1; SMK2763B), interleukin-1β (IL-1β; SMK2776B), monocyte chemoattractant protein-1 (MCP-1; SMK2818B), interleukin-10 (IL-10; SMK2912B), and tumor necrosis factor-α (TNF-α; SMK2868B) in serum. All assay procedures were strictly performed in accordance with the instructions of the ELISA kit (Jiangsu Sunk Biotechnology Co., Ltd.).

[0099] 1.5 Histopathological Analysis 1.5.1 H&E staining and Oil Red O staining Liver and epididymal white adipose tissue (eWAT) samples, along with inguinal white adipose tissue (iWAT) samples, were fixed in 4% paraformaldehyde for 24 hours. The fixed samples were then continuously dehydrated using a series of gradient ethanol solutions and aseptically embedded in paraffin. Paraffin-embedded tissues were sectioned into 4 μm thick sections using a microtome. Hematoxylin and eosin (H&E) staining was used to assess pathological changes in liver tissue and morphological changes in adipocytes. For lipid analysis, fresh liver samples were rapidly frozen in liquid nitrogen, dehydrated, embedded in OCT resin, and sectioned into 8 μm thick frozen sections. Oil Red O staining was used to quantitatively determine liver lipid accumulation. Stained sections were imaged using CaseViewer software at 20x magnification, and the adipocyte area in H&E-stained fat sections was quantitatively analyzed using ImageJ software.

[0100] 1.5.2 Immunofluorescence assay Colon tissue sections were sequentially hydrated using environmentally friendly dewaxing solution (3 times, 10 minutes each), anhydrous ethanol (3 times, 5 minutes each), and distilled water. Antigen retrieval was performed using microwave heating in EDTA buffer (pH 8.0), with the following steps: medium power heating for 8 minutes, intermittent pause for 8 minutes, and low to medium power heating for 7 minutes. After natural cooling, sections were washed three times with phosphate-buffered saline (PBS, pH 7.4), 5 minutes each time. Bovine serum albumin (BSA) was then added for blocking for 30 minutes. Sections were incubated overnight at 4°C with primary antibody; after three 5-minute PBS washes, they were incubated with secondary antibody at room temperature in the dark for 50 minutes; after three more 5-minute PBS washes, they were stained with DAPI for 10 minutes in the dark, and endogenous fluorescence was quenched for 5 minutes. Finally, sections were mounted with anti-quenching mounting medium, and images were acquired under a fluorescence microscope.

[0101] 1.6 16S rRNA sequencing of the gut microbiome Fecal samples were collected under sterile conditions and immediately frozen in liquid nitrogen, then stored at -80°C. Nucleic acid extraction was performed on pre-collected fecal samples (n=5 per group) using the MagBeads Fast DNA Soil Extraction Kit (catalog number 116564384, MPBiomedicals, USA). The extracted DNA was analyzed by 0.8% agarose gel electrophoresis to determine molecular size, followed by quantitative DNA detection using a Nanodrop instrument. PCR amplification was performed using specific primers targeting the V3-V4 region of bacterial 16S rRNA: 338F (5′-[barcode]-ACTCCTACGGGAGGCAGCA-3′, SEQ ID NO:2) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′, SEQ ID NO:3). Quantitative analysis of the PCR products was performed using the Quant-iT PicoGreen Double-Stranded DNA Detection Kit on a microplate reader (BioTek FLx800). Library construction was performed using the TruSeq Nano DNA Library Preparation Kit from Illumina; qualified libraries were sequenced using the NovaSeq 6000 SP kit on an Illumina NovaSeq 6000 SP sequencer (500 cycles in total).

[0102] Microbiome data analysis was performed using QIIME 2 version 2024.5 software, strictly following the official tutorial. Raw sequences were decoded using the demux plugin, followed by primer trimming using the cutadapt plugin; quality filtering, noise removal, sequence assembly, and chimeric sequence removal were all performed using DADA2. An ASV feature table and representative sequences were generated through 100% similarity clustering; ASV species annotation was based on the Greengenes database, and rare ASVs were excluded from subsequent analyses. Bar charts of species composition at each taxonomic level were generated using R software. Alpha diversity analysis was performed using QIIME 2: sequencing depth was assessed using sparse curves; after scaling the data to 95% of the minimum sample sequencing depth, the Chao index, observed species number, Shannon index, Simpson index, Pielou evenness, and Good coverage index were calculated, and box plots were generated. β diversity analysis was performed using both R and QIIME 2: principal component analysis (PCoA) and non-metric multidimensional scaling (NMDS) were performed based on Bray-Curtis distance, and PCA was performed in conjunction with genus-level species composition data; Permanova tests were used to analyze community differences between groups; differentially enriched taxa were identified using linear discriminant analysis effect size (LEfSe); and the prediction of metabolic functions of the microbiome was performed using the PICRUSt2, MetaCyc, and KEGG databases.

[0103] 1.7 Short-chain fatty acid analysis Seven short-chain fatty acids in fecal samples (n=5 per group) were quantitatively analyzed using liquid chromatography-mass spectrometry (LC-MS / MS). All standards were purchased from Shanghai Yuye Biotechnology Co., Ltd.

[0104] Sample preparation procedure: Mix 20 mg of fecal sample with 800 μL of extraction reagent (containing internal standard) and 2 steel balls, and vortex at 55 Hz for 60 seconds in a tissue homogenizer. Centrifuge at 4000 g for 10 minutes at 10°C. Collect 40 μL of supernatant and mix with 20 μL of 200 mM 3-NPH solution; add 20 μL of 120 mM (EDC) HCl-6% pyridine solution and mix thoroughly. Incubate in a metal bath at 40°C and 1200 rpm for 30 minutes, then centrifuge at 12000 g for 10 minutes at 4°C to obtain the supernatant for detection.

[0105] A Kinetex C8 column (Type 100A, 2.6 µm, 2.1 mm × 100 mm) was used at a flow rate of 0.3 mL / min, a column temperature of 40°C, and an autosampler temperature set to 4°C. The injection volume was 1 μL. Mobile phase A consisted of 0.1% formic acid in water; mobile phase B was a 50% methanol-isopropanol mixture (containing 0.1% formic acid). The mobile phase gradient was set as follows: 0–1 min: A / B = 80:20 (V / V); 6 min: A / B = 60:40 (V / V); 9–10 min: A / B = 80:20 (V / V). A SCIEX CitrineTriple Quad™ mass spectrometer was used, operated in ESI (electrospray ionization) negative ion mode using OS software (version 4.7, SCIEX), and multiple reaction monitoring (MRM) data acquisition was employed. The ion source parameters were set as follows: ion ejection (IS) negative ion mode voltage 4500 V; spray gas (GS1) and auxiliary heating gas (GS2) pressures both 50 psi; desolvation gas temperature 550°C; curtain gas (CUR) pressure 30 psi; collision gas (CAD) pressure 10 psi. All formal samples and quality control (QC) samples were analyzed using the above chromatographic and mass spectrometric methods. Two to four QC samples were tested before formal sample injection to verify instrument performance stability. During the injection process, one QC sample was injected every 12 samples processed for subsequent data evaluation and quality control.

[0106] Quantitative data integration was performed using OS software (version 4.7, SCIEX). After acquiring mass spectrometry analysis data from different samples, the chromatographic peaks of all target compounds were integrated. A standard curve was constructed using reference standards for quantitative analysis to obtain the detection concentration values.

[0107] 1.8 Western blotting (WB) 0.1 g of liver tissue was homogenized in RIPA lysis buffer containing a mixture of protease / phosphatase inhibitors for 5 minutes, followed by centrifugation at 12,000 rpm for 6 minutes at 4°C. The total protein concentration in the supernatant was determined using a BCA protein quantification kit (Shanghai Yas Biomedical Technology Co., Ltd., Shanghai, China). After heat denaturation, equal volumes of samples were separated by 10% SDS-PAGE gel electrophoresis, and the separated proteins were transferred to a PVDF membrane (Merck Millipore GmbH, Germany). The membrane was then incubated at room temperature for 1.5 hours in a blocking solution containing 5% TBST (skim milk powder), followed by overnight incubation at 4°C with primary antibodies. The primary antibodies used included: anti-AMPK (Wanlei Life Sciences Co., Ltd.), anti-P-AMPK (Wuhan Service Biotechnology Co., Ltd.), anti-C / EBPα (Wanlei Life Sciences Co., Ltd.), anti-FASN (Wanlei Life Sciences Co., Ltd.), anti-P-ACC (Taizhou Baijia Biotechnology Co., Ltd.), anti-ACC (Taizhou Baijia Biotechnology Co., Ltd.), anti-PGC-1α (Wanlei Life Sciences Co., Ltd.), anti-SREBP-1 (Wuhan Service Biotechnology Co., Ltd.), anti-PPARγ (Wuhan Service Biotechnology Co., Ltd.), and anti-β-actin (Suzhou Sanying Biotechnology Co., Ltd.). After washing with Tris-buffered saline (TBST) containing 0.05% Tween-20, the samples were incubated at room temperature for 1.5 hours with the corresponding enzyme-labeled goat anti-rabbit secondary antibody (1:5000). The reaction signal was then detected using ECL chemiluminescence detection reagents (Seven Innovation Biotechnology Co., Ltd., Beijing), and protein bands were visualized using a chemiluminescence imaging system (GelView9000 Lite, Guangzhou Boluten Biotechnology Co., Ltd.). Protein signal intensity was quantitatively analyzed using ImageJ software (Free Software Foundation, Boston, Massachusetts, USA).

[0108] 1.9 Statistical Analysis Statistical analysis and graphical presentation of experimental data were performed using GraphPad Prism software (version 10.1.2). One-way ANOVA was used to assess differences among groups, and unpaired Student's t-test was used for comparisons between two groups. A p-value <0.05 was considered statistically significant. All data are expressed as mean ± standard deviation (SD).

[0109] 2 Results 2.1 A. muciniphila It can improve obesity caused by a high-fat diet. like Figure 1As shown in A, this animal experiment aimed to investigate the anti-obesity effect of *Akermansia myxophilus* HHAKKα on mice fed a high-fat diet (HFD). During the 42-day intervention period, compared with the HFD group, administration of HHAKKα significantly inhibited weight gain and significantly reduced the weight change curve. This intervention effect was comparable to that of the positive control drug orlistat (ORL) group. Figure 1 (B in the text). On day 42, the final body weight of the HHAKKα group was significantly lower than that of the HFD group by 16.46% ( ). Figure 1 In addition, the average daily energy intake of this group was significantly reduced by 31.13% compared with the HFD group (E). Figure 1 The C in the figure indicates that the weight loss effect of HHAKKα is related to the regulation of energy intake.

[0110] Macroscopic observation showed that the HFD group mice exhibited significant obesity and excessive fat deposition, while HHAKKα treatment significantly improved these conditions. Figure 1 (D in the text). Further tissue weighing results confirmed that, compared with the HFD group, the HHAKKα group showed significantly reduced inguinal white adipose tissue, epididymal white adipose tissue, and liver weight by 56.92%, 35.89%, and 22.67%, respectively. Figure 1 F in Figure 1 G in Figure 1 (I); there was no significant difference in the weight of brown adipose tissue among the groups ( Figure 1 (H in H). These results indicate that HHAKKα can effectively alleviate obesity induced by a high-fat diet in mice.

[0111] 2.2 A. muciniphila It can improve glucose and lipid metabolism disorders and related hormone abnormalities induced by a high-fat diet in mice.

[0112] like Figure 2 As shown, compared with the HFD group, the fasting blood glucose of mice treated with HHAKKα ( Figure 2 (A) and 2-hour postprandial blood glucose levels ( Figure 2 The levels of B) in the samples were significantly reduced by 30.36% and 29.66%, respectively, indicating that the strain can effectively improve glucose metabolism abnormalities caused by a high-fat diet.

[0113] Serum metabolic hormone testing showed that the leptin level in the HHAKKα group was significantly lower than that in the HFD group by 10.49% ( Figure 2 (C); while there were no significant differences in GLP-1 and adiponectin levels among the groups (C). Figure 18 A and Figure 18(B in the text). Serum lipid profile analysis showed that the levels of triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C) in the HHAKKα group were significantly lower than those in the HFD group by 29.03% and 20.12%, respectively. Figure 2 E and Figure 2 (F in the text); total cholesterol (TC) showed a decreasing trend but did not reach statistical significance ( Figure 2 In the D), while high-density lipoprotein cholesterol (HDL-C) showed an upward trend but no significant difference. Figure 18 (C in the text). These results collectively demonstrate that HHAKKα can effectively improve glucose metabolism disorders, dyslipidemia, and abnormal leptin levels in mice induced by a high-fat diet.

[0114] 2.3 A. muciniphila It can alleviate chronic inflammatory responses caused by a high-fat diet. Lipid metabolism disorders are often associated with chronic inflammation; therefore, this study further examined the levels of inflammatory factors in serum. Compared with the HFD group, the serum levels of pro-inflammatory cytokines interleukin-1β (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) in mice fed HHAKKα were significantly reduced by 10.16% and 8.93%, respectively. Figure 3 B- Figure 3 In contrast, there were no statistically significant differences in serum levels of tumor necrosis factor-α (TNF-α) and the anti-inflammatory cytokine interleukin-10 (IL-10). Figure 3 A in Figure 3 (D in the text). These results indicate that HHAKKα can improve the systemic chronic inflammatory state induced by a high-fat diet by downregulating certain key pro-inflammatory mediators.

[0115] 2.4 A. muciniphila It can improve hepatic steatosis and reduce fat cell volume. To further investigate the effects of this intervention on improving mouse tissue morphology, we examined liver lipid deposition and morphological changes in adipocytes. H&E staining of liver tissue showed numerous lipid droplet vacuoles and significant fatty degeneration in the HFD group hepatocytes; after HHAKKα intervention, the number of hepatocyte vacuoles was significantly reduced, and fatty degeneration was significantly improved; however, the ORL group still showed some degree of vacuolation. Figure 4 A in the text). Oil Red O staining results were consistent with HE staining: lipid droplet deposition in the liver tissue was significantly increased in the HFD group, while HHAKKα intervention significantly reduced lipid droplet accumulation and improved liver fat deposition; however, obvious red lipid droplets were still visible in the orlistat group ( Figure 4(B in the text). H&E staining and morphological analysis of epididymal and inguinal white adipose tissue showed that the adipocyte volume was significantly increased in the HFD group, while the adipocytes were significantly reduced after HHAKKα intervention. Cell area statistical analysis also showed that the cell area was significantly reduced in the HHAKKα group compared with the HFD group. Figure 4 (C in the middle).

[0116] 2.5 A. muciniphila It can improve intestinal barrier dysfunction caused by a high-fat diet. To investigate the protective effect of this intervention on the intestinal barrier, researchers performed immunofluorescence staining and quantitative analysis of intestinal tight junction-related proteins. The results showed that compared with the HFD group, the fluorescence intensity of ZO-1 protein in the colon tissue of mice treated with HHAKKα showed an increasing trend, but the difference was not statistically significant. Figure 5 (A in the text); while the fluorescence intensity of the key tight junction protein Occludin was significantly higher in the HHAKKα group than in the HFD group ( Figure 5 (B in the text). These results indicate that HHAKKα can effectively improve intestinal barrier dysfunction caused by a high-fat diet by upregulating the expression of the tight junction protein Occludin.

[0117] 2.6 A. muciniphila It can reshape the structure and function of the gut microbiota damaged by a high-fat diet. To investigate the microbial mechanism by which HHAKKα improves obesity and metabolic disorders, this study performed high-throughput 16S rRNA sequencing analysis on the mouse gut microbiota. α diversity results ( Figure 6 The results showed that, compared with the HFD group, the Shannon index and Goods coverage of the gut microbiota were significantly increased in the HHAKKα group. Although there were no statistically significant differences in Chao1, Simpson index, Pielou-e index, and observed species index, they all showed a trend towards normalization compared with the normal diet group, indicating that HHAKKα intervention can enhance gut microbiota diversity and reverse gut microbiota dysbiosis. PCoA analysis based on Bray-Curtis distance (…) Figure 7 ) and NMDS analysis ( Figure 7 This revealed significant clustering differences in the gut microbiota composition among the groups. Changes in the relative abundance of phylum and genus-level microorganisms ( Figure 8 and Figure 9 The results showed that after probiotic supplementation, the number of Firmicutes increased while the number of Verrucous microorganisms decreased in the HHAKKα group; simultaneously, the numbers of *Lactobacillus*, *Lactobacillus*, *Laurella*, *Lactobacillus*, and *Femtobacter* increased, while *Clostridium erythropoiesis* decreased. (Horizontal species composition clustering heatmap) Figure 10Further, it was shown that HHAKKα intervention significantly reversed gut microbiota dysbiosis caused by a high-fat diet, enriched specific beneficial bacterial genera, and reduced the abundance of harmful bacterial genera associated with metabolic disorders. LEfSe analysis ( Figure 11 This further revealed the differences among the various microbial communities at the phylum, class, order, family, and genus levels. The results showed that... A. muciniphila The dominant bacterial genera in the HHAKKα group include Kineothrix, Lactobacillus, Acutalibacter, and Adlercreutzia. Kineothrix and Lactobacillus have anti-inflammatory and metabolic-improving effects, while research on Acutalibacter and Adlercreutzia is still relatively limited. In the HFD group, Helicobacter, Clostridium, Streptococcus, and Horadorrhena are the main species, all exhibiting pro-inflammatory and metabolic-disrupting effects. Furthermore, the KEGG secondary pathway function prediction results ( Figure 12 The results indicate that probiotic intervention significantly modulates functional metabolic pathways of the gut microbiota, particularly those related to lipid metabolism, carbohydrate metabolism, and energy metabolism. These findings confirm that HHAKKα can improve obesity and metabolic disorders induced by a high-fat diet by enhancing gut microbial α diversity, reshaping the microbial community structure, and regulating key differentially expressed species and functional pathways related to energy and lipid metabolism.

[0118] 2.7 A. muciniphila It can restore the functional impairment of short-chain fatty acids and related metabolic pathways in mouse feces induced by HFD.

[0119] To elucidate the metabolic role of HHAKKα in regulating the gut microbiota, this study analyzed the levels of seven short-chain fatty acids (SCFAs) in mouse feces. SCFAs clustering heatmap ( Figure 13 The results showed that a high-fat diet significantly altered the overall metabolic profile of SCFAs in feces, while HHAKKα intervention restored the SCFA metabolic pattern to the level of the normal control group. Statistical analysis of total SCFA content indicated that the SCFA level in the intervention group was significantly higher than that in the HFD group. Figure 14 Quantitative analysis of each SCFA type showed that, compared with the HFD group, the levels of butyrate, propionate, isobutyrate, and valerate in the intervention group were significantly increased by 170.76%, 125.76%, 142.37%, and 109.78%, respectively. Figure 14 Although the levels of isovaleric acid, acetic acid, and decanoic acid did not show statistically significant differences, they all exhibited varying degrees of upward trends. Figure 14These results suggest that HHAKKα helps alleviate metabolic disorders induced by a high-fat diet by regulating gut microbial metabolite levels by promoting the production of key SCFAs, particularly butyrate and propionate.

[0120] KEGG metabolic pathway analysis showed that short-chain fatty acids (SCFAs) are mainly involved in the absorption and digestion of carbohydrates and proteins. Compared with the HFD group, the HHAKKα group showed higher levels of metabolic pathway-related metabolites, suggesting that HHAKKα may improve obesity by regulating protein and carbohydrate metabolic pathways. Figure 15 ).

[0121] 2.8 Correlation between gut microbiota and short-chain fatty acids (SCFAs) and metabolic and inflammatory markers in the HFD and HHAKKα groups To investigate the correlation between the gut microbiota and its metabolites (short-chain fatty acids, SCFAs) and biochemical markers, we performed Spearman correlation analysis. Figure 16 A- Figure 16 B in the HFD group. The main microbial communities (including Helicobacter spp., Clostridium melioides spp., Streptococcus spp., and Horadric spp.) Figure 11 The HHAKKα group showed a positive correlation with serum lipid levels and inflammatory markers, but a negative correlation with SCFAs; conversely, the main microbial communities of the HHAKKα group (including Lactobacillus, Averrhoa, Duncanie, and Acute Ribavirin) were positively correlated with these levels. Figure 11 ) was negatively correlated with serum lipid levels and inflammatory markers, but positively correlated with GLP-1, ADP, and SCFAs. These results indicate that the differences in the correlation between the major microbial communities and serum metabolism and inflammatory markers between the HFD group and the HHAKKα group are consistent with their respective SCFA profiles: SCFAs were negatively correlated with serum lipid levels and inflammatory marker indices, but positively correlated with GLP-1 and ADP. Figure 16 The C in the figure is consistent with the microbial characteristics of the HHAKKα group.

[0122] 2.9 A. muciniphila It can regulate the expression of key proteins involved in hepatic lipid metabolism. To investigate the molecular mechanism by which HHAKKα improves hepatic lipid deposition, this study used Western blotting to detect the expression of proteins related to hepatic lipid metabolism. Figure 17 A in Figure 17As shown in Figure B, compared with the HFD group, after HHAKKα intervention, the expression levels of p-AMPK / AMPK and PGC-1α proteins showed an increasing trend, while the expression levels of PPARγ and SREBP-1 showed a decreasing trend. Simultaneously, the expression of fatty acid synthesis-related proteins FASN and C / EBPα was significantly reduced, with the overall expression level of C / EBPα returning to normal. These results indicate that HHAKKα intervention can effectively inhibit the expression of proteins involved in hepatic lipidogenesis and demonstrates a potential regulatory role in activating the AMPK / PGC-1α energy metabolism axis, thereby improving hepatic lipid metabolism disorders induced by a high-fat diet.

[0123] Comparative Example 1 The experimental method disclosed in Example 8 of patent document CN117965391A was adopted, and the myxotrophic Akkermansia hHAKKα of the present invention was used.

[0124] Experimental Design: 4-5 week old, 17-19 g SPF-grade male C57BL / 6J mice were used. After 7 days of acclimatization, the mice were randomly divided into 5 groups according to body weight: normal control group, model control group, *Ackermannii myxotropic* HHAKKα group, and *Ackermannii myxotropic* HHAKKα inactivated group, with 6 mice in each group. The normal control group was fed a standard maintenance diet, while the other groups were fed a high-fat diet (HFD) for 8 consecutive weeks. During the experiment, the normal control group and model control group were administered 0.2 mL / mouse via gavage with lyophilized preservative, and *Ackermannii myxotropic* HHAKKα was administered 0.2 mL / mouse via gavage with 5×10⁻⁶ lyophilized preservative. 9 The group was given a bacterial suspension of CFU / mL, and the inactivated group was given 0.2 mL of the prepared inactivated bacterial solution by gavage. All groups were given the medication once a day for 8 consecutive weeks.

[0125] During the experiment, mice were weighed weekly, and their weight gain was calculated; weight gain = measured weight - initial weight. The results are shown in Table 1 below: Table 1

[0126] Note: Compared with the normal control group, ### p <0.001; compared with the model control group, p <0.01, p <0.001.

[0127] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A strain of Akkermansia myxophilus ( Akkermansia muciniphila HHAKKα, characterized in that, The preservation number of the myxotrophic Akkermansia hHAKKα is GDMCC No:68013.

2. The *Ackermania* HHAKKα according to claim 1, characterized in that, The 16S sequence of the myxotropic Akkermansia hHAKKα is shown in SEQ ID NO:

1.

3. The preparation of Akkermansia xylophilus HHAKKα according to any one of claims 1-2, characterized in that, This includes fermentation broth, fermentation broth supernatant, fermentation broth sediment, live bacteria, dead bacteria, and freeze-dried powder.

4. The method for preparing the Akkermansia xylophilus HHAKKα preparation according to claim 3, characterized in that, This includes inoculating Akkermansia xylophilus HHAKKα into a culture medium and culturing it.

5. A microbial agent, characterized in that, The bacterial agent comprises the *Ackermania xylophilus* HHAKKα as described in any one of claims 1-2 and / or the preparation as described in claim 3.

6. The use of the *Ackermania xylophilus* HHAKKα according to any one of claims 1-2, the preparation according to claim 3, and / or the bacterial agent according to claim 5 in the preparation of products for relieving obesity.

7. The application according to claim 6, characterized in that, The products mentioned include pharmaceuticals and health supplements.

8. The application according to claim 6, characterized in that, The product contains 1 × 10⁻⁶ Akkermansia xylophilus HHAKKα. 6 -1×10 12 CFU / unit or 1×10 6 -1×10 12 TFU / unit.

9. The application according to claim 6, characterized in that, The product has at least one of the following functions: (1) Inhibit weight gain; (2) Improves excessive fat deposition; (3) Improve glucose metabolism abnormalities caused by obesity; (4) Improves dyslipidemia and leptin levels caused by obesity; (5) Downregulate the levels of inflammatory factors and improve the chronic inflammatory state caused by obesity; (6) Improves hepatic steatosis and reduces fat cell volume; (7) Improves intestinal barrier dysfunction caused by obesity; (8) Enhances gut microbial diversity and reverses gut microbiota dysbiosis; (9) Relieve metabolic disorders.

10. A product for relieving obesity, characterized in that, The product comprises the *Ackermania xylophilus* HHAKKα as described in any one of claims 1-2, the preparation as described in claim 3, and / or the bacterial agent as described in claim 5.

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