Bacteroides multipara and application thereof for relieving metabolic dysfunction-related fatty liver disease

Abstract

The application discloses Bacteroides multipara and application thereof for relieving metabolic dysfunction related fatty liver disease, and belongs to the technical field of microorganisms. Phocaeicola dorei The Bacteroides multipara N02 can inhibit the increase of liver and the increase of liver weight / body weight of a mouse with metabolic dysfunction related fatty liver disease without affecting the food intake, can regulate the lipid metabolism disorder of the mouse with metabolic dysfunction related fatty liver disease and relieve the liver tissue damage, and can improve the intestinal barrier function of the mouse with metabolic dysfunction related fatty liver disease. The Bacteroides multipara N02 has a very wide application prospect when used for preparing a medicine composition for relieving metabolic dysfunction related fatty liver disease and a fermented food.

Description

Technical Field

[0001] This invention relates to a strain of *Dorhizium anisopliae* that alleviates fatty liver disease associated with metabolic dysfunction and its applications, belonging to the field of microbial technology. Background Technology

[0002] Metabolic dysfunction-associated steatotic liver disease (MAFLD), formerly known as non-alcoholic fatty liver disease (NAFLD), is a chronic liver disease closely related to metabolic disorders. It is a chronic liver disease closely associated with metabolic syndrome, including insulin resistance, obesity, type 2 diabetes, and dyslipidemia. Its disease spectrum includes simple fatty liver, steatohepatitis, liver fibrosis, cirrhosis, and even hepatocellular carcinoma. The global prevalence has exceeded 30% and continues to rise. The pathogenesis of MAFLD is complex, involving multiple factors such as hepatic lipid metabolism disorders, insulin resistance, oxidative stress, inflammatory responses, and gut microbiota imbalance. There are currently no approved specific drugs, and clinical treatment primarily focuses on lifestyle interventions. Because the diagnostic criteria have removed alcohol restriction and emphasized metabolic abnormalities, MAFLD has a broader coverage and has become the fastest-growing cause of cirrhosis and hepatocellular carcinoma, as well as an independent risk factor for cardiovascular events.

[0003] Gut microbiota dysbiosis is one of the key environmental factors in the development and progression of MAFLD. When the gut microbiota is imbalanced, it can lead to impaired intestinal barrier function, making it easier for microbial products such as endotoxins (e.g., LPS) to enter the portal circulation, migrate to the liver, trigger chronic low-grade inflammation, interfere with insulin signaling, and promote hepatic steatosis, directly driving the progression of MAFLD. MAFLD patients often have gut microbiota dysbiosis. *Bacteroides dorebren*, as a commensal bacterium in the human gut, has certain beneficial potential. *Bacteroides dorebren* is a Gram-negative anaerobic bacterium, formerly classified under the genus *Bacteroides* (…). Bacteroides ), now reclassified as Seal This bacterium is abundant in healthy individuals and possesses probiotic properties such as polysaccharide degradation, short-chain fatty acid production, and maintenance of intestinal barrier function. Recent studies have found that its abundance is significantly reduced in obese, diabetic, and fatty liver patients, suggesting a potential protective effect against metabolic diseases.

[0004] Patents CN117919283A and CN112105371B disclose *Dorbacterium dorsiflorum* that plays a therapeutic role in an obesity-related fatty liver model induced by a high-fat diet. Existing technologies have also reported *Dorbacterium dorsiflorum* that can alleviate symptoms associated with a Western diet (WD)-induced fatty liver model. However, no *Dorbacterium dorsiflorum* model has been found to alleviate non-alcoholic fatty liver disease (NAFLD) caused by a methionine-choline deficiency diet (MCD). The fatty liver corresponding to different disease models differs significantly in its pathogenesis, symptoms, and treatment methods, belonging to different disease subtypes. For example, the disease caused by a Western diet is mainly characterized by obesity, insulin resistance, and high-fat, high-glucose metabolic disorders, making it closer to obesity-related fatty liver; while the disease caused by a methionine-choline deficiency diet (MCD) is not accompanied by obesity, and its core pathological changes are rapid and significant hepatocellular steatosis, inflammatory response, and liver damage, mainly reflecting fatty liver disease driven by nutritional deficiencies, abnormal lipid metabolism, and direct hepatocellular damage. Therefore, the treatment effects of different diseases are unpredictable.

[0005] Therefore, there is an urgent need for a probiotic that can effectively improve fatty liver disease associated with metabolic dysfunction caused by methionine-choline deficiency diet (MCD) and alleviate its associated problems such as glucose and lipid metabolism disorders and liver damage. Summary of the Invention

[0006] To alleviate dietary-induced metabolic dysfunction-related fatty liver disease, and the accompanying glucose and lipid metabolism disorders and liver damage, this invention screened out a strain of *Dorhizium anisopliae* that can alleviate metabolic dysfunction-related fatty liver disease. It has been demonstrated that this strain comprehensively improves liver function impairment and metabolic dysfunction-related fatty liver disease, while also further preventing metabolic disorders associated with metabolic dysfunction-related fatty liver disease. This has significant implications and broad prospects for dietary intervention in the early stages of metabolic dysfunction-related fatty liver disease.

[0007] This invention provides a strain of *Dorbacterium doretrix* ( Golden seal No. N02 was deposited on December 30, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 67580.

[0008] The Dorary bacterium ( Golden seal N02 was isolated from the fermentation broth of human fecal samples from normal individuals in Jiangxi Province. Sequencing analysis revealed its 16S rDNA sequence to be shown in SEQ ID NO.1. The sequence was then compared with the nucleic acid sequence of *Bacteroides dorebrenica* using NCBI, showing a 99.90% similarity. Therefore, it was named *Bacteroides dorebrenica*. Golden seal NO2, the main text of this invention uses *Bacteroides dorebrenella* ( Golden seal The naming of N02.

[0009] The *Dorbacterium doretrix* GDMCC N02 strain described herein possesses the following properties: Cell characteristics: Gram-positive rod-shaped bacteria, non-spore-forming, non-flagellated, with a cell width of approximately 0.5-1.5 µm and a length of 1.5-4.0 µm. Colony characteristics: Forms distinct colonies on culture media, with a diameter between 0.5-2 mm. The colonies are round on the upper surface, convex in the center, with neat edges, slightly whitish, opaque, and moist and smooth. Growth characteristics: This strain is an obligate anaerobe. The optimal growth temperature is 36℃-38℃, and the optimal growth pH is 6.6-7.0. It grows well in glucose-containing media and enters the late logarithmic growth phase or the early stationary phase within 12-24 hours.

[0010] The present invention also provides a microbial agent containing the above-mentioned *Dorbacterium doracetam* (…). Golden seal )N02.

[0011] In one embodiment of the present invention, the microbial agent contains *Dorbacterium doracetam* (… Seal dorei The cell count of NO2 is not less than 1×10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

[0012] The present invention also provides a product containing the above-mentioned *Dorrebacterium* ( Seal dorei )N02.

[0013] In one embodiment of the present invention, the product contains *Dorbacterium doretrix* (… Golden seal NO2 has a viable bacterial count of not less than 1×10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

[0014] In one embodiment of the present invention, the product is food, medicine, health product or feed additive.

[0015] In one embodiment of the present invention, the food includes beverages, dairy products, or other products containing the aforementioned *Doracetamia* (…). Golden seal Foods containing NO2.

[0016] In one embodiment of the present invention, the drug comprises Dorex bacillus ( Golden seal NO2, drug carriers and / or pharmaceutical excipients.

[0017] In one embodiment of the present invention, the dosage form of the medicine or health product includes granules, capsules, tablets, pills or oral liquids.

[0018] In one embodiment of the present invention, the pharmaceutical excipient is a pharmaceutically acceptable excipient.

[0019] In one embodiment of the present invention, the acceptable excipients include one or more commonly used thickeners, antioxidants, pH adjusters, emulsifiers, preservatives, fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents.

[0020] In one embodiment of the present invention, the filler is starch, sucrose, lactose, calcium sulfate and / or microcrystalline cellulose.

[0021] In one embodiment of the present invention, the adhesive is a cellulose derivative, alginate, gelatin, and / or polyvinylpyrrolidone.

[0022] In one embodiment of the present invention, the wetting agent is water, ethanol, starch and / or syrup.

[0023] In one embodiment of the present invention, the disintegrant is sodium carboxymethyl starch, carboxypropyl cellulose, croscarmellose, agar, calcium carbonate and / or sodium bicarbonate.

[0024] In one embodiment of the present invention, the lubricant is talc, calcium stearate, magnesium stearate, micronized silica gel, and / or polyethylene glycol.

[0025] In one embodiment of the present invention, the flavoring agent is a simple syrup, sucrose, lecithin, orange peel syrup, cherry syrup, lemon, fennel, peppermint oil, sodium alginate, gum arabic, gelatin, methylcellulose, sodium carboxymethyl cellulose, citric acid, tartaric acid and / or sodium bicarbonate.

[0026] The present invention also provides the above-mentioned *Dorbacterium doretrix* ( Golden seal The use of NO2, or the above-mentioned microbial agents, in the preparation of medicines for the prevention and / or treatment of fatty liver disease associated with metabolic dysfunction.

[0027] In one embodiment of the present invention, the pharmaceutical product contains *Dorasis bacillus* (…). Golden seal NO2 has a viable bacterial count of not less than 1×10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

[0028] In one embodiment of the present invention, the drug comprises Dorex bacillus ( Golden seal NO2, drug carriers and / or pharmaceutical excipients.

[0029] In one embodiment of the present invention, the dosage form of the medicine includes granules, capsules, tablets, pills, or oral liquids.

[0030] In one embodiment of the present invention, the pharmaceutical excipient is a pharmaceutically acceptable excipient.

[0031] In one embodiment of the present invention, the acceptable excipients include one or more commonly used thickeners, antioxidants, pH adjusters, emulsifiers, preservatives, fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents.

[0032] In one embodiment of the present invention, the filler is starch, sucrose, lactose, calcium sulfate and / or microcrystalline cellulose.

[0033] In one embodiment of the present invention, the adhesive is a cellulose derivative, alginate, gelatin, and / or polyvinylpyrrolidone.

[0034] In one embodiment of the present invention, the wetting agent is water, ethanol, starch and / or syrup. In one embodiment of the present invention, the disintegrant is sodium carboxymethyl starch, carboxypropyl cellulose, croscarmellose, agar, calcium carbonate and / or sodium bicarbonate.

[0035] In one embodiment of the present invention, the lubricant is talc, calcium stearate, magnesium stearate, micronized silica gel, and / or polyethylene glycol.

[0036] In one embodiment of the present invention, the flavoring agent is a simple syrup, sucrose, lecithin, orange peel syrup, cherry syrup, lemon, fennel, peppermint oil, sodium alginate, gum arabic, gelatin, methylcellulose, sodium carboxymethyl cellulose, citric acid, tartaric acid and / or sodium bicarbonate.

[0037] The present invention also provides the above-mentioned *Dorbacterium doretrix* ( Golden seal NO2, or the above-mentioned microbial agents, are used in the preparation of health products that help maintain healthy blood lipid levels.

[0038] Beneficial effects 1. This invention screened out a strain of *Dorbacterium doracetam* (… Golden seal NO2 has the effect of alleviating fatty liver disease related to metabolic dysfunction, specifically manifested in: 1) It can reduce liver weight and inhibit liver weight-to-body weight ratio in mice with metabolic dysfunction-related fatty liver disease without affecting their food intake; 2) It can improve the levels of total cholesterol and triglycerides in the liver and the levels of alanine aminotransferase and aspartate aminotransferase in the plasma of mice with MCD-induced metabolic dysfunction-related fatty liver disease; 3) It can alleviate liver fibrosis and lipid deposition in mice with MCD diet-induced metabolic dysfunction-related fatty liver disease; 4) It can reduce the expression of pro-inflammatory factor-related genes in the liver of mice with MCD diet-induced metabolic dysfunction-related fatty liver disease.

[0039] 5) It can increase the expression of genes related to intestinal barrier function in mice with MCD diet-induced metabolic dysfunction-related fatty liver disease.

[0040] 2. Doracetam ( Golden seal Doracetam is a potentially valuable probiotic, and its various probiotic effects have been extensively reported. Through numerous inventive experimental studies, the inventors of this invention have discovered that Doracetam NO2 can effectively alleviate fatty liver disease associated with impaired intestinal barrier function and metabolic dysfunction caused by diet, and can be used in drugs or health products for the prevention and treatment of fatty liver disease and related diseases.

[0041] Preservation of biological materials A strain of *Dorbacterium doraceae* ( Golden seal N02 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 30, 2025, and its taxonomic name is: Golden seal The accession number is GDMCC No: 67580, and the accession address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Provincial Center for Microbial Culture Collection. Attached Figure Description

[0042] Figure 1 Effects of Doracetamibacterium NO2 intervention on liver weight and liver weight / body weight in mice with metabolic dysfunction-related fatty liver disease; where A represents the liver weight of mice at the end of week 8 of the experiment; and B represents the liver weight / body weight of mice at the end of week 8 of the experiment.

[0043] Figure 2 Effects of Doracetam N02 intervention on liver function indicators in mice with metabolic dysfunction-related fatty liver disease; where A represents total cholesterol level in the liver, B represents triglyceride level in the liver, C represents plasma alanine aminotransferase level, and D represents plasma aspartate aminotransferase level.

[0044] Figure 3 Effects of *Dorbacterium doracetam* NO2 intervention on liver morphology in mice with metabolic dysfunction-related fatty liver disease; Image showing liver tissue H&E staining (left); Oil Red O staining (middle); Sirius Red staining (right); Scale bar: 50 μm.

[0045] Figure 4Effects of *Dorbacterium doracetam* NO2 intervention on liver injury pathological scores in mice with metabolic dysfunction-related fatty liver disease; where A represents liver steatosis score, B represents liver inflammation score, C represents liver ballooning degeneration score, D represents total score of liver metabolic dysfunction-related fatty liver disease, and E represents percentage of liver fibrosis area. Among them, the model group was the metabolic dysfunction-related fatty liver disease group of MCD diet, and the dorebacterium group was the MCD diet + dorebacterium NO2 group. "*" indicates a significant difference from the model group (*: p <0.05; **: p <0.01; ***: p <0.001; ****: p <0.0001); some data results in the tables are expressed as averages. Data analysis was performed using SPSS 24 with one-way ANOVA and Tukey's HSD post-hoc test. Different letters in the same column. Detailed Implementation

[0046] The mice used in the following examples were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. They were housed at 25±2℃, constant humidity 50±5%, and light for 12 hours (8:00-20:00), with soundproofing, and free access to food and water. The experiment began after one week of acclimatization. The total cholesterol (TC) kit (catalog number: 100000180), triglyceride (TG) kit (catalog number: 100000220), alanine aminotransferase (ALT) kit (catalog number: 100000010), and aspartate aminotransferase (AST) kit (100000020) involved in the following examples were purchased from Sinopharm Biotechnology Co., Ltd.; the Trizol reagent (catalog number: 15596026) involved in the following examples was purchased from Thermo Fisher Scientific, Inc.; the reverse transcription kit (catalog number: RR092A) and real-time quantitative kit (catalog number: RR820A) involved in the following examples were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.; all culture medium components involved in the following examples were purchased from Shanghai Yuanye Co., Ltd.; and the L-amino acid diet (MCD diet) containing 60 kcal% fat and 0.1% methionine without added choline involved in the following examples was purchased from ResearchDiets, Inc. (catalog number: A06071302). The primers used in the following examples were all ordered from Shanghai Sangon Biotech Co., Ltd.

[0047] The following examples involve culture media: Preparation of activation medium (g / L): The components include carbon sources: pectin 0.047, xylan 0.047, arabinogalactan, amylopectin 0.04, soluble starch 0.392; nitrogen sources: bacterial peptone 24, tryptone 24; inorganic salts: magnesium sulfate heptahydrate 0.5, potassium dihydrogen phosphate 2.5, sodium chloride 4.5, calcium chloride dihydrate 0.45, ferric sulfate heptahydrate 0.005; bile salts 0.4, cysteine ​​hydrochloride 0.2, and acid-base buffer (MES) 19.52. First, the above components were prepared, and the pH was adjusted to 6 before deoxygenation and sterilization (121℃, 15 min). After sterilization, the culture medium was transferred to an anaerobic glove box. 1 μg of heat-sensitive heme, 1 μg of vitamin K3 (VK3), and 0.1 mL of a vitamin mixture (Wolfe's Vitamin Solution) were added to 1 L of the culture medium and filtered through a 0.22 μm filter membrane. The medium was then deoxygenated overnight in the anaerobic glove box to obtain the activated liquid culture medium.

[0048] Preparation of enrichment medium: Each liter of enrichment medium is composed of 350 mL of solution A, 150 mL of solution B, 500 mL of solution C, 1 mL of solution D, and 0.08 mL of Wolfe's Vitamin Solution. The formula (g / L) includes: Solution A: bacterial peptone 68.57, tryptone 68.57, bile salts 1.14, anaerobic agent cysteine ​​hydrochloride 1.43, magnesium sulfate 1.14, potassium monohydrogen phosphate 5.48, sodium chloride 12.86, calcium chloride 0.97, ferric sulfate heptahydrate 0.014; Solution B: acid-base buffer (MES) 130; Solution C: Dendrobium officinale polysaccharide 10; Solution D: heme 10 mg, vitamin K3 (VK3) 8 mg. First, the autoclaved components (A and B solutions) are prepared, the pH is adjusted to 6, and deoxygenation is performed, followed by sterilization (121℃, 15 min). After sterilization, the culture medium was transferred to an anaerobic glove box and left overnight. Finally, solution D and Wolfe's Vitamin Solution were filtered through a 0.22 μm membrane and added to the culture medium in the specified proportion to obtain the enrichment medium.

[0049] BHI liquid medium (g / L): 10.0g peptone, 17.5g ox heart extract, 5.0g sodium chloride, 2.0g glucose, and 2.5g disodium hydrogen phosphate are dissolved in 1L of distilled water. 0.5g cysteine ​​hydrochloride is added and mixed thoroughly. The pH is then adjusted to 7.2-7.6. The mixture is sterilized at 115-121℃ for 15-20 min to obtain the BHI liquid medium.

[0050] Preparation of BHI solid medium: Add 1.5-2% agar to BHI liquid medium. Mix well, then adjust the pH to 7.2-7.6, and sterilize at 115-121℃ for 15-20 min to obtain the BHI solid medium.

[0051] BHI selective medium (g / L): BHI solid medium is prepared by adding 5 mg / L of heme (sterilized by membrane filtration), 10 mg / L of vitamin K1, 7.5 mg / L of vancomycin, and 100 mg / L of kanamycin.

[0052] The detection methods involved in the following embodiments are as follows: Mouse body weight and liver tissue weight determination: After the 8th week of the experiment, the mice were weighed, then anesthetized and sacrificed. The complete livers of the mice were separated, weighed and recorded.

[0053] Liver weight / body weight (%) = (liver weight (g) / mouse body weight (g)) × 100 Liver function assay in mice: After week 8 of the experiment, mice were anesthetized and sacrificed. Blood was collected from the orbital region of the mice, centrifuged at 3000 rpm for 15 min, and the resulting plasma was obtained. The levels of total cholesterol (TC) and triglycerides (TG) in the mouse liver, which reflect liver function, were measured using a kit. At the same time, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the mouse plasma were measured.

[0054] Observation of mouse liver tissue morphology: (1) H&E staining: The tissue was embedded in paraffin. Before staining, the paraffin sections were dewaxed and stained with hematoxylin and eosin respectively. After dehydration, the sections were mounted and observed under a microscope. (2) Oil Red O staining: The tissue was embedded in paraffin, frozen and cut into sections. The sections were then immersed in Oil Red staining solution and counterstained with hematoxylin. After mounting with glycerol gelatin, the sections were observed under a microscope.

[0055] (3) Sirius red staining: The tissue was embedded in paraffin, the paraffin sections were dewaxed before staining, stained with Sirius red staining solution, counterstained with hematoxylin, and then mounted with neutral resin for microscopic observation.

[0056] Disease activity index assessment: Mouse liver tissue morphology was scored according to the scoring criteria described in Table 1. Histological score = fatty degeneration score + ballooning degeneration score + inflammation score.

[0057] Table 1. Mouse NASH scoring system (0-8 points)

[0058] RNA extraction and gene expression determination: First, RNA was extracted from the tissue using the trizol method, following the kit instructions. RNA concentration and purity were determined using NanoDrop and agarose gel electrophoresis. For further reverse transcription, 1 μg of total RNA from the tissue was added to 2 μL of 5×gDNA Buffer, 1 μL of gDNA Eraser, and RNase-Free ddH2O to a final volume of 10 μL. The mixture was briefly mixed, centrifuged, and incubated at 42°C for 2 min to remove genomic DNA. Then, 1 μL of PrimeScript RT Enzyme Mix I, 1 μL of RT Primer Mix, 4 μL of 5×PrimeScript Buffer 2, and RNase-Free ddH2O were added to a final volume of 20 μL. The mixture was incubated at 37°C for 15 min, followed by incubation at 85°C for 5 s to complete the reverse transcription.

[0059] The reverse transcription product was diluted 10-fold and used as a template for real-time quantitative PCR. The qPCR reaction system (20 μL) consisted of: 2 μg cDNA template, 10 μL SYBR dye, 0.8 μL primer, with the remaining volume made up with ddH2O. Reaction conditions: 25-50℃, 1.6 ℃ / s, 50℃ for 2 min; 50-95℃, 1.6 ºC / s, 95℃ for 30 s; amplification (95℃ for 20 s, 60℃ for 1 min, for a total of 40 cycles). Two auxiliary wells were set up for each gene. Data processing was performed using 2... -ΔΔCT The primers used for relative quantitative analysis are shown in Table 2.

[0060] Table 2 Primers used for real-time quantitative PCR

[0061] Example 1: Isolation and screening of Bacteroides doretrix NO2 1. Sample collection Fecal samples were collected from normal individuals in Jiangxi Province. The samples were placed in preservation tubes and 5 times their weight of protective solution were added (preparation of protective solution: weigh 1 g / L cysteine ​​hydrochloride and 200-300 g / L glycerol, dissolve them evenly in PBS (1×), and sterilize at 115-121℃ for 15-20 min). The samples were then stored in an insulated box containing dry ice and brought back to the laboratory. The samples were then quickly placed in a -80°C freezer for separation and screening.

[0062] 2. Accumulation of fecal bacteria The above fecal microbiota solution was taken out of the -80℃ freezer, thawed, and centrifuged at low speed (500 g, 5 min, 4℃) to obtain the supernatant. Then, it was passed through a 100 μm sterile cell sieve to remove impurities from the supernatant. The supernatant fecal microbiota solution was inoculated into activation medium (fecal microbiota solution: activation medium = 1:9, (v / v)) and cultured at 37℃ and 140 rpm for 16 h. Then, it was inoculated into enrichment medium at an inoculation ratio of 10% (v / v) and cultured at 37℃ and 140 rpm for 24 h.

[0063] 3. Isolation and purification of Bacteroides The enriched fecal microbial solution was serially diluted in a sterile anaerobic environment. The diluted microbial solution was then spread onto BHI solid medium and incubated at 37°C under anaerobic conditions for 48 h to obtain diluted spread plates. Single colonies with neat edges, slightly white color, opacity, moist and smooth surfaces, and uniform morphology were selected from plates with appropriate colony counts and inoculated into 5 mL of liquid BHI selective medium. These were then incubated at 37°C under anaerobic conditions for 24 h to obtain purified culture.

[0064] 4. Preservation and Identification of Microbial Strains The purified culture medium with the best viability from step 3 was used as an amplification template, and PCR amplification was performed using 16S universal primers (see Table 3). Amplification was carried out on a PCR instrument according to the following procedure: pre-denaturation at 95℃ for 5 min; followed by 29 cycles of 95℃ for 15 s, 60℃ for 15 s, and 72℃ for 45 s; extension at 72℃ for 5 min; and termination of the reaction by cooling to 4℃. The amplified products were analyzed by 1% agarose gel electrophoresis and then subjected to first-generation sequencing. High-quality sequences were extracted from the sequencing data and submitted to NCBI for BLAST alignment to find relevant sequence annotation information. The results showed that the sequence had more than 99% homology with the 16S rDNA sequence of *Bacteroides dorebrenella*. This strain is now named *Bacteroides dorebrenella*. Golden seal )N02, and is deposited at the Guangdong Provincial Center for Microbial Culture Collection.

[0065] Table 3 Primer Names

[0066] Example 2: Effects of Doracetamibacterium NO2 on body weight, liver weight, and diet in mice with metabolic dysfunction-associated fatty liver disease (MCD). The specific steps are as follows: 1. Preparation of Doracetam NO2 cryopreservation medium: (1) Cultivation method: In a sterile anaerobic environment, streak Bacteroides NO2 strain on BHI solid medium and culture under anaerobic conditions for 48 h. After single colonies are formed, pick single colonies and inoculate them into BHI liquid medium. Culture under anaerobic conditions at 37℃ for 12-24 h to reach the stationary phase. At this time, the OD value is 0.8-1.0, and the seed liquid is prepared.

[0067] (2) Preparation of protective agent: Weigh 1 g / L cysteine ​​hydrochloride and 200-300 g / L glycerol, dissolve them evenly in distilled water, and sterilize at 115-121℃ for 15-20 min.

[0068] (3) Preparation of cryoprotectant: After centrifuging the seed culture of Doraemonas NO2 cultured to the stable period in step (1) (8000 rpm, 10 min, 4℃), wash it 1-2 times with sterile phosphate buffer (pH 7.2), and then resuspend the bacterial culture with the protectant prepared in step (2) to obtain Doraemonas NO2 cryoprotectant, which is stored at -80℃ for later use.

[0069] 2. Preparation of Bacteroides dorebrenella NO2 inoculum: (1) Activation of strains: The Dorebrella NO2 cryopreservative prepared in step 1 was streaked on BHI solid medium and cultured under anaerobic conditions for 48 h. After single colonies were formed, they were inoculated into BHI liquid medium and cultured anaerobically at 37℃ for 16-24 h to reach the stationary phase (OD value: 0.8-1.1).

[0070] (2) Preparation of bacterial agent: Take 100 μL of the culture medium obtained in step (1) at different dilution ratios and spread it on BHI solid medium. Count the number of colonies on the BHI solid plate and calculate the number of viable bacteria in the liquid medium of step (1). After washing 1-2 times with sterile phosphate buffer (pH 7.2), resuspend the bacterial solution in PBS to a concentration of 1×10⁻⁶. 9 For formulations with a concentration of CFU / mL, the gavage volume is 0.2 mL.

[0071] 3. Test methods: Intervention and treatment trial process: This invention uses an MCD diet to induce metabolic dysfunction-associated fatty liver disease (MDF) in mice. Twelve healthy male C57BL / 6J mice aged 6 weeks were randomly divided into two groups (n=6 per group): the MCD diet-induced MDF group (model group) and the Doraemonas NO2 intervention group (Doraemonas group).

[0072] The experimental procedure is shown in Table 4. After a one-week adaptation period: Model group: During the intervention period (0-8 weeks), the patient was given the MCD diet, 0.2 mL of sterile phosphate buffer was administered by gavage four times a week, and free access to water was allowed.

[0073] Doracetam group: During the intervention period (0-8 weeks), patients were given the MCD diet and simultaneously administered 0.2 mL of a 1×10⁻⁶ solution by gavage on Mondays, Wednesdays, Fridays, and Sundays. 9 CFU / mL of Doracetamibacterium N02 bacterial suspension, available in drinking water.

[0074] At the end of week 8 of the experiment, mice were euthanized after anesthesia. Blood was collected from the orbital sinus of the mice, and the blood was centrifuged at 3000 rpm for 15 min to obtain mouse plasma. Plasma, colon, and liver were preserved in [location missing]. 80℃ is used for subsequent analysis.

[0075] Table 4 Test Procedure

[0076] 4. Effects of Doracetamibacterium NO2 on liver weight, liver weight-to-body weight ratio, and diet in mice with metabolic dysfunction-related fatty liver disease (MCD) induced by diet. The specific experimental procedure is the same as steps 1-3. During the intervention period, the mice in each group were weighed weekly. After 8 weeks of intervention, the mice were anesthetized and sacrificed.

[0077] (1) Effects of Doracetamibacterium NO2 on liver weight and liver weight-to-body weight ratio in mice with metabolic dysfunction-related fatty liver disease induced by MCD diet. The livers of euthanized mice were weighed. Liver weight can be used to assess the degree of fatty liver and steatosis in animals. Results are as follows: Figure 1 A. The liver weight-to-body weight ratio of the sacrificed mice was measured, and the results are as follows: Figure 1 B. The results showed that after intervention with *Dorbacterium doracetam* NO2, the liver weight of mice in the *Dorbacterium doracetam* group (B) was 1.69 g, while the liver weight of mice in the model group was 1.83 g. The liver weight of mice treated with *Dorbacterium doracetam* NO2 was significantly reduced compared to the model group. p <0.05%, a decrease of 7.6%. The liver weight / body weight ratio in the *Dorbacterium doracetam* group was 7.40%, while that in the model group was 7.97%. After intervention with *Dorbacterium doracetam* NO2, the liver weight / body weight ratio in mice was significantly reduced compared to the model group. p <0.05), a decrease of 0.57%.

[0078] (2) Effects of Doracetamibacterium NO2 on the diet of mice with metabolic dysfunction-related fatty liver disease (MCD) Table 5 shows the changes in the diet of mice during the intervention period (weeks 0-8). Compared with the model group, there was no significant difference in food intake among mice in the *Dorbacterium doracetam* group. pThe value >0.05 indicates that the effect of Dorexobacterium NO2 on liver weight and liver weight-to-body weight ratio in mice is not caused by affecting the amount of food consumed by the mice.

[0079] Table 5. Feed intake (g / day / mice) of mice in each group during the intervention period (weeks 0-8) of *Dorbacterium doracetam* intervention.

[0080] The above results indicate that the Dorexobacterium NO2 of the present invention can inhibit the increase in liver weight and liver weight / body weight ratio in MCD diet-induced metabolic dysfunction-related fatty liver disease, and this control of liver weight is not caused by reducing the food intake of mice.

[0081] Example 3: Effects of Doracetamibacterium NO2 on lipid metabolism in mice with MCD diet-induced metabolic dysfunction-related fatty liver disease The specific steps are as follows: The specific experimental method was the same as in Example 2. After the 8th week of the experiment, mouse plasma and liver were collected. The levels of total cholesterol (TC) and triglycerides (TG) in the mouse liver were measured using a kit, while the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the mouse plasma were measured simultaneously. The results are as follows: Figure 2 As shown.

[0082] like Figure 2 As shown in A-2B, after intervention with Doraemonas N02, compared with the model group, the liver TC and TG levels of mice in the Doraemonas group (7.01 mg / g and 15.37 mg / g, respectively) decreased by 9.49% and 16.05%, respectively.

[0083] like Figure 2 As shown in C-2D, after intervention with Doraemonas NO2, compared with the model group, the plasma ALT and AST levels in the Doraemonas group mice (492.89 U / L and 251.83 U / L, respectively) decreased by 14.43% and 25.60%, respectively.

[0084] The above results indicate that the *Dorbacterium doracetam* NO2 of this invention can reduce the levels of total cholesterol (TC) and triglycerides (TG) in the liver of mice with metabolic dysfunction-related fatty liver disease, and reduce the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the plasma of mice, thus significantly improving lipid metabolism in mice with metabolic dysfunction-related fatty liver disease.

[0085] Example 4: Effects of Doracetamibacterium NO2 on Liver Injury in Mice with MCD Diet-Induced Metabolic Dysfunction-Associated Fatty Liver Disease The specific steps are as follows: The specific experimental method was the same as in Example 2. At the end of the experiment (week 8), the mouse livers were weighed, photographed, and stained to observe the morphological changes of the livers in each group. The livers of mice with metabolic dysfunction-related fatty liver disease were scored according to the NASH system score. The results are as follows: Figure 3-4 As shown.

[0086] Figure 3 The results showed that the livers of the model group exhibited significant diffuse steatosis (vacuolation) and fibrosis. After treatment with *Dorbacterium doracetam* NO2, the morphology, structure, lipid droplet accumulation, collagen fiber deposition, and degree of fibrosis in the mouse livers were significantly improved. This indicates that *Dorbacterium doracetam* can alleviate liver fibrosis and lipid deposition in mice with MCD diet-induced metabolic dysfunction-related fatty liver disease.

[0087] like Figure 4 As shown in A-4D, after intervention with *Dorbacterium doracetam* NO2, compared with the model group, the steatosis score, inflammation score, and ballooning degeneration score of the liver in mice in the *Dorbacterium doracetam* group (1.67, 1.33, and 0.33, respectively) decreased by 31.82%, 40.00%, and 50.00%, respectively. The total score of metabolic dysfunction-related fatty liver disease in mice (3.33) also decreased by 37.50%.

[0088] like Figure 4 As shown in E, after intervention with Doraemonas NO2, the liver fibrosis area in the Doraemonas group mice (1.47%) decreased by 69.36% compared with the model group.

[0089] The above results indicate that the Doraemonas NO2 of the present invention can significantly reduce the accumulation of lipid droplets, deposition of collagen fibers and degree of fibrosis in the liver of mice with metabolic dysfunction-related fatty liver disease, and significantly improve liver damage.

[0090] Example 5: Effects of Doracetamibacterium NO2 on liver inflammation in mice with MCD diet-induced metabolic dysfunction-related fatty liver disease The specific experimental method is the same as in Example 2, except that mouse liver tissue was collected after the experiment, and inflammation-related genes in the mouse liver tissue were measured. TNFα , IL1β , Cc12 The expression levels of ) were measured. The results are shown in Table 6.

[0091] Table 6 shows the results for the *Dorbacterium doraceae* group. TNF-α, IL1β and Cc12 The expression level was significantly lower than that of the model group. Compared with the model group, TNF-α, IL1β and Cc12 mRNA levels decreased by 23.47%, 20.68%, and 15.20%, respectively, indicating that *Dorbacterium doracetam* can effectively reduce liver inflammation levels.

[0092] Table 6. Effects of *Dorbacterium doracetam* on the expression levels of liver inflammation-related genes in mice with metabolic dysfunction-associated fatty liver disease.

[0093] The above results indicate that *Dorbacterium doracetam* NO2 of the present invention can significantly reduce the levels of metabolic dysfunction-related fatty liver disease in mice. TNF-α、IL1β and Cc12 The expression of [the substance] effectively reduced the level of inflammation in the liver of mice with metabolic dysfunction-related fatty liver disease.

[0094] Example 6: Effects of Doracetamibacterium NO2 on intestinal barrier function in mice with MCD diet-induced metabolic dysfunction-related fatty liver disease The specific experimental method is the same as in Example 2, except that mouse colon tissue was collected after the experiment, and intestinal barrier-related genes in the mouse colon tissue were measured. Zo1、Claudin1 , Claudin3、Muc2 The expression levels of ) were measured. The results are shown in Table 7.

[0095] Table 7 shows the results for the *Dorbacterium doraceae* group. Zo1、Claudin1 , Claudine3 and Muc2 mRNA expression levels were significantly higher in the mRNA group than in the model group. Zo1、Claudin1 , Claudine3 and Muc2 mRNA levels increased by 19.30%, 27.77%, 35.18%, and 39.08%, respectively, indicating that *Dorbacterium doracetam* can effectively improve intestinal barrier function.

[0096] Table 7. Effects of *Dorbacterium doracetam* on the expression levels of intestinal barrier-related genes in mice with metabolic dysfunction-associated fatty liver disease.

[0097] The above results indicate that *Dorbacterium doracetam* NO2 of the present invention can significantly upregulate the colonic tissue of mice with metabolic dysfunction-related fatty liver disease. Zo1、Claudin1 , Claudine3 and Muc2 The expression of mRNA effectively improves intestinal barrier function in mice with fatty liver disease associated with metabolic dysfunction.

[0098] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of *Dorbacterium doracetam* ( Phocaeicola dorei Accession number N02 was deposited on December 30, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No:67580.

2. A microbial inoculant, characterized in that, Contains *Dorbacterium doretrix* NO2 as described in claim 1.

3. The microbial agent as described in claim 2, characterized in that, In the microbial agent, the number of *Dorbacterium doracetam* NO2 cells is not less than 1 × 10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

4. A product containing *Dorbacterium doracetam* NO2 as described in claim 1, characterized in that, The product is food, medicine, health product, or feed additive.

5. The product as described in claim 4, characterized in that, In the product, the viable count of *Dorbacterium doracetam* NO2 is not less than 1 × 10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

6. The product as described in claim 4, characterized in that, The food products mentioned include beverages and dairy products.

7. The product as described in claim 4, characterized in that, The drug comprises *Dorbacterium doretrix* NO2 as described in claim 1, and also contains a drug carrier and / or pharmaceutical excipients.

8. The product as described in claim 4, characterized in that, The dosage forms of the product include granules, capsules, tablets, pills, or oral liquids.

9. The use of *Dorbacterium doretrix* NO2 as described in claim 1, or the microbial agent as described in claim 2 or 3, in the preparation of a medicament for the prevention and / or treatment of fatty liver disease associated with metabolic dysfunction.

10. The use of *Dorbacterium doretrix* NO2 as described in claim 1, or the microbial agent as described in claim 2 or 3, in the preparation of health products that help maintain healthy blood lipid levels.

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