Application of Lactobacillus mucosae vesicles in the preparation of drugs for treating MAFLD

Abstract

This invention discloses the application of *Lactobacillus mucosa* vesicles in the preparation of drugs for treating MAFLD, belonging to the field of microbial medicine. The *Lactobacillus mucosa* strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.37136, and its 16S rDNA gene sequence is shown in SEQ ID NO: 1. The *Lactobacillus mucosa* strain was isolated from fecal samples of healthy adults. This strain exhibits significant antioxidant, anti-inflammatory, and lipid metabolism regulating activities in vitro. Its secreted EVs are non-cytotoxic and non-immunogenic in vivo, and animal experiments have verified its good in vivo safety and liver-targeted enrichment characteristics.

Description

Technical Field

[0001] This invention relates to the field of microbial medicine, specifically to the use of *Lactobacillus mucosae* vesicles in the preparation of drugs for treating MAFLD. Background Technology

[0002] Metabolic dysfunction-associated fatty liver disease (MAFLD), formerly known as nonalcoholic fatty liver disease (NAFLD), is a chronic, progressive liver disease caused by overnutrition and insulin resistance in genetically susceptible individuals. It has a high global prevalence and is now the most common chronic liver disease and a major cause of elevated serum transaminase levels in healthy individuals undergoing routine checkups. Early MAFLD may only present as simple fatty liver, but without timely intervention, it can gradually develop into nonalcoholic steatohepatitis, liver fibrosis, and cirrhosis. Furthermore, MAFLD is mutually reinforcing with metabolic syndrome and type 2 diabetes, jointly promoting the development of arteriosclerotic heart disease, chronic kidney disease, liver decompensation, and malignant tumors such as hepatocellular carcinoma, placing a significant burden on public health. Therefore, in-depth research into the mechanisms of MAFLD occurrence and development and the search for effective interventions are crucial.

[0003] Probiotics are live microorganisms that, when administered in adequate amounts, have beneficial effects on the host's health and have been widely used for the prevention and treatment of microecological imbalances and metabolic-related diseases. Animal studies have shown that probiotics can regulate the expression of intestinal tight junction proteins claudin-1, ZO-1, occludin, and antimicrobial peptides Reg3a, Reg3b, and Reg3g, enhancing intestinal barrier function. Furthermore, by downregulating TLR-2 to reduce IL-17 secretion from hepatic γδT lymphocytes, they further inhibit macrophage polarization towards the pro-inflammatory M1 type, reducing IL-6 and TNF-α secretion, ultimately inhibiting the development of MAFLD. In clinical studies, probiotic treatment for MAFLD has shown positive effects in improving lipid metabolism, reducing insulin resistance, improving liver function, and alleviating liver inflammation and fibrosis. Although probiotics are considered an effective treatment for MAFLD, *Lactobacillus mucosa* (…) Lactobacillus mucosae, L. mucosae The specific mechanisms by which it works in treating MAFLD still require further investigation.

[0004] Extracellular vesicles (EVs) derived from probiotics are spherical vesicles with a lipid bilayer secreted by probiotics. They typically range in diameter from 20-400 nm and can carry various bioactive components (proteins, lipids, and nucleic acids). They can cross the mucus layer and migrate directly to other tissues or different cells of the host's immune system, thereby participating in biological functions such as bacterial stress response, biofilm formation, information exchange, and immune regulation. EVs produced by probiotics can activate host TLR2 signaling and enhance IgA antibody production through bacterial cell wall components, thereby strengthening the mucosal immune system, preventing infection, and maintaining intestinal flora balance. Furthermore, due to the close anatomical and functional connection between the intestine and liver—the "gut-liver axis"—the liver is also an active site for EV uptake. However… L. mucosae The specific molecular mechanisms by which EVs from this source improve MAFLD still require further investigation.

[0005] Based on this, this study aims to verify the results through animal and cell experiments. L. mucosae The role of EVs in MAFLD was investigated, and experimental techniques such as ELISA, immunohistochemistry, transcriptomics sequencing, high-throughput sequencing, qPCR, and Western blotting were used to explore new targets and ideas for the comprehensive treatment of MAFLD. Summary of the Invention

[0006] To address the core technical challenges of existing technologies, such as the lack of safe and effective targeted therapies for metabolic-related fatty liver disease (MAFLD), the risks of adverse liver reactions associated with current interventions, and the difficulties in gastrointestinal colonization, significant individual variability in efficacy, and limited clinical application of live probiotic preparations, this invention provides the application of *Lactobacillus mucosa* vesicles in the preparation of drugs for treating MAFLD. *Lactobacillus mucosa* HG003, isolated from fecal samples of healthy adults, exhibits significant antioxidant, anti-inflammatory, and lipid metabolism-regulating activities in vitro. Its secreted EVs are non-cytotoxic and non-immunogenic in vivo, and animal experiments have demonstrated good in vivo safety and liver-targeted enrichment characteristics.

[0007] This invention systematically validates the protective and therapeutic effects of *Lactobacillus mucosa* and its derived EVs in the development and progression of metabolic-associated fatty liver disease by integrating an animal model of metastatic fatty liver disease (MAFLD) with an in vitro experimental system of hepatocytes / macrophages. This study comprehensively utilizes multi-dimensional techniques, including enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC) staining, transcriptome sequencing, high-throughput sequencing of gut microbiota 16S rRNA, quantitative real-time PCR (qRT-PCR), and Western blotting, to deeply elucidate the key signaling pathways and molecular regulatory networks by which EVs regulate hepatic lipid synthesis and catabolism homeostasis, and inhibit hepatic oxidative stress and inflammatory cascade responses. It clarifies the core mechanism by which EVs improve liver pathological damage in MAFLD, ultimately providing safe and effective novel candidate drugs, core therapeutic targets, and scientific strategic support for the clinical intervention of metabolic-associated fatty liver disease.

[0008] To achieve the above objectives, the present invention provides a *Lactobacillus mucosa* strain named HG003 and classified as *Lactobacillus mucosa*. Lactobacillus mucosae The 16S rDNA gene sequence of *Lactobacillus mucosa* HG003, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.37136, is shown in SEQ ID NO: 1.

[0009] A second aspect of the present invention provides the use of extracellular vesicles derived from *Lactobacillus mucosa* as described in the present invention in the preparation of a drug for treating metabolic-related fatty liver disease.

[0010] Furthermore, the median particle size of the extracellular vesicles is 120-140 nm.

[0011] Furthermore, the extracellular vesicles are natural extracellular vesicles secreted into the fermentation broth by the *Lactobacillus mucosa* under conventional culture conditions (37°C, anaerobic environment, MRS medium), and the concentration of extracellular vesicle particles in the fermentation broth is ≥1×10⁻⁶. 11 per mL.

[0012] Furthermore, when the extracellular vesicles were applied to normal mice for verification of no significant toxicity, the results showed that there were no abnormal changes in the pathological morphology of the mouse liver tissue, and there were no statistically significant abnormalities in the core liver function indicators (alanine aminotransferase ALT, aspartate aminotransferase AST, and total bilirubin TBIL) (P>0.05).

[0013] Furthermore, when the drug is applied to MAFLD model mice, it is administered via gavage or intraperitoneal injection, and the concentration of extracellular vesicles in the drug is 1×10⁻⁶. 10 ~1×10 12 per mL.

[0014] Furthermore, after continuous treatment of MAFLD model mice with the aforementioned dose of drug for 4-6 weeks, compared with the model control group, the mice's body weight decreased by 6-8%, liver weight decreased by 22-26%, total liver cholesterol (TC) decreased by 21-23%, and liver triglycerides (TG) decreased by 20-25%.

[0015] Furthermore, the metabolic-related fatty liver disease includes non-alcoholic fatty liver disease and non-alcoholic steatohepatitis.

[0016] Furthermore, the metabolic-associated fatty liver disease includes liver fibrosis and / or cirrhosis caused by non-alcoholic fatty liver disease and / or non-alcoholic steatohepatitis.

[0017] A third aspect of the present invention provides a biological agent for treating metabolic-related fatty liver disease, said biological agent comprising the *Lactobacillus mucosa* or extracellular vesicles derived from *Lactobacillus mucosa* as described in the present invention.

[0018] Compared with the prior art, the present invention has at least the following beneficial effects:

[0019] 1. The targeting value of specific probiotics-extracellular vesicles in the treatment of the gut-hepatic axis was clarified: This study screened and isolated probiotic strains (Lactobacillus mucosa) with potential therapeutic effects on MAFLD. Lactobacillus mucosae HG003 was further clarified to be a key active ingredient for its therapeutic effect, namely extracellular vesicles. This provides direct evidence and material basis for developing a precision microecological treatment strategy based on extracellular vesicles derived from specific probiotics (rather than traditional live bacteria preparations), overcoming the limitations of low survival rate and unstable colonization in traditional probiotic therapies.

[0020] 2. A complete chain of evidence was constructed: The study combined 16S rRNA sequencing, transcriptomics, molecular biology and other multi-level technologies to systematically elucidate the systemic role of extracellular vesicles from Lactobacillus mucosa HG003 in improving hepatic steatosis, metabolic abnormalities, inflammation and oxidative stress by activating the hepatic AMPK signaling pathway at both the whole animal level and the cellular and molecular level.

[0021] 3. The study revealed a clear molecular pathway and action entity that can be translated into clinical intervention: The study not only confirmed the key role of the AMPK signaling pathway in mediating liver protection, but more importantly, it clarified the active components (EVs) and action pathways (gut-hepatic axis) of Lactobacillus mucosa HG003, providing a clear theoretical basis and intervention targets for developing precision treatment strategies based on vesicle drug delivery, vesicle component modification or AMPK pathway regulation. Attached Figure Description

[0022] Figure 1The following are evaluations of the probiotic properties of Lactobacillus mucosa HG003 in one embodiment of the present invention: (A) growth curve; (B) acid resistance test; (C) bile salt resistance test; (D) antioxidant activity assay; (E) cell adhesion test (scale bar = 50 μm); (F) hemolytic activity assay.

[0023] Figure 2 The following is an evaluation of the probiotic properties of *Lactobacillus mucosa* HG003 in one embodiment of the present invention: (A) Antibacterial activity assay; (BC) Antibiotic susceptibility test, n=3; wherein, c, culture medium control; m, bacterial suspension; PEN, penicillin; C, chloramphenicol; E, erythromycin; AMP, ampicillin; MY, lincomycin; CTP, ceftriaxone; TET, tetracycline; SXT, trimethoprim-sulfamethoxazole; GEN, gentamicin; CIP, ciprofloxacin.

[0024] Figure 3 This invention demonstrates that, in one embodiment, *Lactobacillus mucosa* HG003 intervention reduces hepatic lipid deposition in MAFLD mice: (A) Schematic diagram of animal experimental design; (B) Body weight and liver weight; (C) Serum TC and TG levels; (D) Blood glucose curve during GTT; (E) Blood glucose curve during ITT; (F) Macroscopic morphology of the liver; Data are expressed as mean ± standard deviation, and intergroup comparisons were performed using one-way ANOVA or Welch's ANOVA and Bonferroni or Dunnett's T3 post-hoc test; ns, no significant difference; *p<0.05, **p<0.01, ***p<0.001; Wherein, C, control group; M, MAFLD model group; MP, L. mucosae Treatment group; n=6 mice / group; TC, total cholesterol; TG, triglycerides.

[0025] Figure 4 This invention demonstrates that, in one embodiment, *Lactobacillus mucosa* HG003 intervention alleviates hepatic lipid deposition in MAFLD mice: (A) Representative photomicrographs of liver sections stained with HE (scale bar: 50 μm); (B) Serum ALT and AST levels; (C) Representative photomicrographs of liver sections stained with Oil Red O (scale bar: 50 μm); (D, E) Hepatic MDA, SOD, GSH, and CAT levels; (F) Serum IL-1β, IL-6, TNF-α, and LPS levels; Data are expressed as mean ± standard deviation, and intergroup comparisons were performed using one-way ANOVA or Welch's ANOVA and Bonferroni or Dunnett's T3 post-hoc test; ns, no significant difference; *p<0.05, **p<0.01, ***p<0.001; where C, control group; M, MAFLD model group; MP, L. mucosaeTreatment group; n=6 mice / group; ALT, alanine aminotransferase; AST, aspartate aminotransferase; MDA, malondialdehyde; SOD, superoxide dismutase; GSH, reduced glutathione; CAT, catalase; IL-1β, interleukin-1β; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; LPS, lipopolysaccharide.

[0026] Figure 5 This invention demonstrates that extracellular vesicles (EVs) derived from *Lactobacillus mucosa* HG003 alleviate FFA-induced lipid accumulation in HepG2 cells in one embodiment: (A) Schematic diagram of cell experiment design; (B) Representative image stained with Oil Red O (scale bar: 50 μm); (C) ALT and AST levels in cell supernatant and intracellular TC and TG contents; Data are expressed as mean ± standard deviation, and intergroup comparisons were performed using one-way ANOVA or Welch's ANOVA and Bonferroni or Dunnett's T3 post-hoc test; ns, no significant difference; *p<0.05, **p<0.01, ***p<0.001; Wherein, C, control group; M, MAFLD cell model group; MC, L. mucosae Cell genome; MS, L. mucosae Supernatant group.

[0027] Figure 6 This indicates that, in one embodiment of the present invention, extracellular vesicles (EVs) derived from *Lactobacillus mucosa* HG003 alleviate FFA-induced lipid accumulation in HepG2 cells: (A) L. mucosae Representative transmission electron microscopy (TEM) images (scale bar: 100 nm) and nanoparticle tracking analysis (NTA) of derived EVs; (B) L. mucosae Cellular uptake of derived EVs (scale bar: 10 μm); (C) Schematic diagram of cell experiment design; (D) Representative image of Oil Red O staining (scale bar: 50 μm); (E, F) ALT and AST levels in cell supernatant and intracellular TC and TG content; Data are expressed as mean ± standard deviation, and intergroup comparisons were performed using one-way ANOVA or Welch's ANOVA and Bonferroni or Dunnett's T3 post-hoc test; ns, no significant difference; *p<0.05, **p<0.01, ***p<0.001; Wherein, C, control group; M, MAFLD cell model group; ME, L. mucosaeDerived EVs group; MES, EVs with supernatant removed group; n=3 mice / group; EVs, extracellular vesicles; FFA, free fatty acids; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TC, total cholesterol; TG, triglycerides; TEM, transmission electron microscopy; NTA, nanoparticle tracking analysis.

[0028] Figure 7 This invention demonstrates that, in one embodiment, *Lactobacillus mucosa* HG003-derived EVs improve hepatic lipid deposition and glucose metabolism in MAFLD mice via the AMPK signaling pathway: (A) Schematic diagram of animal experimental design; (B) Mouse body weight and liver weight; (C) Serum TC, TG, HDL, LDL levels and liver TC and TG content; (D) Fasting blood glucose (FBG) and fasting insulin (FINS) levels; (E) Representative photomicrographs of liver sections stained with HE (scale bar: 50 μm); Data are expressed as mean ± standard deviation, and intergroup comparisons were performed using one-way ANOVA or Welch's ANOVA and Bonferroni or Dunnett's T3 post-hoc test; ns, no significant difference; *p<0.05, **p<0.01, ***p<0.001; where, C, control group; M, MAFLD model group; ME, L. mucosae Derivative EVs group; MEI, L. mucosae Derivative EVs and AMPK inhibitor (Compound C) groups; n=6 mice / group; EVs, extracellular vesicles; TC, total cholesterol; TG, triglycerides; HDL, high-density lipoprotein; LDL, low-density lipoprotein; FBG, fasting blood glucose; FINS, fasting insulin.

[0029] Figure 8 This invention demonstrates that, in one embodiment, *Lactobacillus mucosa* HG003-derived EVs improve hepatic lipid deposition and glucose metabolism in MAFLD mice via the AMPK signaling pathway: (A) blood glucose curves during GTT and ITT, and HOMA-IR and HOMA-IS indices; (B) serum ALT and AST levels; (C) representative photomicrographs of liver sections stained with Oil Red O (scale bar: 50 μm); data are expressed as mean ± standard deviation, and intergroup comparisons were performed using one-way ANOVA or Welch's ANOVA and Bonferroni or Dunnett's T3 post-hoc test; ns, no significant difference; *p<0.05, **p<0.01, ***p<0.001; where C, control group; M, MAFLD model group; ME, L. mucosae Derivative EVs group; MEI, L. mucosaeDerivative EVs and AMPK inhibitor (Compound C) groups; n=6 mice / group; EVs, extracellular vesicles; GTT, glucose tolerance test; ITT, insulin tolerance test; HOMA-IR, insulin resistance index assessed by homeostasis model; HOMA-IS, insulin sensitivity index assessed by homeostasis model; ALT, alanine aminotransferase; AST, aspartate aminotransferase.

[0030] Figure 9 This invention demonstrates that, in one embodiment of the present invention, *Lactobacillus mucosa* HG003-derived EVs reached mouse liver tissue without significant toxicity: (A) Representative images of in vitro fluorescence imaging of each organ in the PBS control group (CP group); (B) Representative images of in vitro fluorescence imaging of each organ in the extracellular vesicle treatment group (CE group); (C) Serum ALT, AST, CK-MB, CREA, BUN, and UA levels; Data are expressed as mean ± standard deviation or median (interquartile range), and comparisons between groups were performed using independent samples t-test or Mann-Whitney U test; ns, no significant difference; *p<0.05, **p<0.01, ***p<0.001; Wherein, the CP group was the PBS control group, and the CE group was the extracellular vesicle treatment group, with n=6 mice in each group; EVs: extracellular vesicles; TG: triglycerides; ALT: alanine aminotransferase; AST: aspartate aminotransferase; CK-MB: creatine kinase isoenzyme MB; CREA: creatinine; BUN: blood urea nitrogen; UA: uric acid.

[0031] Figure 10 This indicates that in one embodiment of the present invention, EVs derived from Lactobacillus mucosa HG003 reached mouse liver tissue and had no obvious toxicity: (A) HE-stained microscopic images of heart, liver, spleen, lung and kidney tissues in the CP group (scale bar: 50 μm); (B) Microscopic images of tissue sections of various organs in the CE group (scale bar: 50 μm); wherein, the CP group was the PBS control group, the CE group was the extracellular vesicle treatment group, and each group n=6 mice. Detailed Implementation

[0032] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] In the following embodiments:

[0035] EVs derived from *Lactobacillus mucosa* HG003 were obtained through the following methods: *Lactobacillus mucosa* ( L. mucosae After anaerobic culture in MRS liquid medium at 37°C until the stationary phase, the bacterial culture was collected and centrifuged at 10000×g for 30 minutes to remove bacterial cells. The supernatant was collected, filtered through a 0.22 μm filter membrane, and extracellular vesicles (EVs) were precipitated by ultracentrifugation (120000×g, 4°C, 90 minutes). The obtained EVs were finally resuspended in sterile PBS for later use. The morphology and structure of EVs were observed using transmission electron microscopy (HITACHI, Japan), and their particle size distribution and concentration were determined using nanoparticle tracking analysis technology (Particle Metrix, Germany).

[0036] Example 1: Isolation, purification and identification of Lactobacillus mucosa HG003.

[0037] 1.1 Separation and Purification

[0038] Place 1g of fresh, normal, healthy human feces into a 10mL centrifuge tube (containing 5mL of PBS solution). Then, centrifuge the sample (800rpm, 5min). After centrifugation, take 1mL of the supernatant and transfer it to a 1.5mL centrifuge tube for serial dilution with sterile PBS, with each dilution being 10-10. 1 ~10 9 Select appropriate concentration gradients (1-9) for plating (MRS medium). In this case, samples numbered 3, 5, 7, and 9 were selected, with 30 μL of each sample plated. The plates were then spread (the glass rods were repeatedly heated, and the plates were shaken before each sample addition). The plates were then incubated aerobically for 24-48 hours. Plates with 200-400 colonies were then inoculated into 5 mL of the corresponding liquid MRS medium in a sterile laminar flow hood. Based on the morphology, size, color, edge regularity, elevation, and growth rate of the colonies, 5-10 single colonies were selected and inoculated into the corresponding 5 mL liquid MRS medium for aerobic activation and incubation for 24-28 hours.

[0039] 1.2 Morphological identification

[0040] The purified bacterial strain was stained using Gram staining, and its morphology was observed under a 100x oil immersion microscope. The specific steps were as follows: The purified bacterial strain was inoculated into MRS solid medium and incubated at 37°C for 48 hours in an aerobic workstation. A small amount of a single colony of HG003 was taken, dipped into 2µL of physiological saline, and spread clockwise to form a colony of about 1cm. 2 The slide is uniformly thin and round. Place it at room temperature until the bacterial culture dries. Fix the slide by passing it through an alcohol lamp flame 1-2 times, being careful not to overheat it; the slide should be warm to the touch. Add crystal violet staining solution to the bacteria and stain for 1 minute. Wash with water, add iodine solution and stain for another 1 minute. Wash with water, add destaining solution, shake the slide, and destain for 30 seconds. Wash with water, absorb excess water, add safranin staining solution and stain for 1 minute. Wash with water, blot dry with filter paper, and examine under a 100x oil immersion microscope. Gram staining results show that the bacteria are Gram-positive and appear as short rods.

[0041] 1.3 Physiological and Biochemical Identification

[0042] (1) Growth kinetics: The growth kinetics of strain HG003 were characterized by a 48-hour growth curve plotted based on 600 nm absorbance measurement;

[0043] (2) Acid tolerance test: The strain was cultured in a 37°C incubator using the appropriate culture medium until its OD value reached 0.6, at which point the culture was terminated and the strain was retained for later use. 100 μL of the strain was diluted with PBS buffer. 1 10 3 10 5 Centrifuge at 6000g for 3 minutes and discard the supernatant; add PBS buffer with pH=2, 3, 5, 7, let stand for 4 hours, mix well, take 10μL and spread on a plate, incubate in a 37℃ incubator for 12-48 hours, count the viable bacteria, and record the experimental results.

[0044] (3) Bile salt tolerance test: The strain was cultured in a 37℃ incubator using the appropriate culture medium until its OD value reached 0.6, at which point the culture was terminated and the strain was retained for later use. 100 μL of the strain was diluted with PBS buffer. 1 10 3 10 5 Centrifuge at 6000g for 3 minutes and discard the supernatant; add culture medium containing 0.1%-0.3% (0, 0.1, 0.2, 0.3) ox bile salts and incubate at 37℃ for 12-48 hours. Mix well, take 10μL, spread on a plate, and incubate at 37℃ for 12-48 hours. Count the viable bacteria and record the experimental results.

[0045] (4) Cell adhesion experiment: The strain was cultured in a constant temperature incubator at 37℃ with the corresponding culture medium until the OD value was equal to 0.6, and the culture was terminated. The strain was retained for later use. The six-well cell culture plate was washed once with sterile PBS buffer, and a sterile coverslip was placed in it. 1 mL of the above bacterial culture medium was mixed with 1 mL of HT29 cell culture medium and added to the six-well plate. The plate was cultured in a cell culture incubator at 37℃. After 1-1.5 hours of culture, the six-well tissue cell culture plate was removed, the culture medium was aspirated, and the plate was washed repeatedly with PBS buffer 5 times. The plate was fixed with paraformaldehyde, stained with crystal violet, and observed and photographed under a microscope.

[0046] (5) Antibacterial experiment: The bacterial strains were inoculated into liquid culture medium and cultured in an anaerobic incubator at 37°C for 12-24 h. After centrifugation at 6000 rpm for 10 min, the supernatant was removed. Candida albicans, Pseudomonas aeruginosa, Listeria monocytogenes, Escherichia coli, Streptococcus pyogenes, and Staphylococcus aureus were spread on LB solid medium. Oxford cups were gently placed on the plate, and 250 μL of bacterial culture supernatant was aspirated into the Oxford cups (3 replicates per group). The plates were incubated at 37°C. The size of the inhibition zone was observed every 2 h, and the diameter of the inhibition zone was measured after 8 h.

[0047] (6) Hemolysis test: Streak HG003 strain on blood agar plates containing 5% defibrinated sheep blood and incubate at 37°C for 24-48 hours. Observe whether a transparent (β-hemolysis), grass-green (α-hemolysis), or unchanged (γ-hemolysis) hemolysis zone appears around the colony.

[0048] (7) Antibiotic resistance test: The standardized concentration of HG003 bacterial suspension was evenly spread on an agar plate, and commercial drug sensitivity test discs containing a specific concentration of antibiotics were attached. After 24 hours of incubation, the diameter of the inhibition zone was measured, and its sensitivity, intermediate or resistance was determined according to the CLSI standard.

[0049] (8) The results are as follows Figures 1-2 As shown:

[0050] Growth kinetics showed that the strain entered a rapid proliferation phase after about 8 hours of culture and reached a stable growth phase after about 20 hours. Figure 1 (A). Regarding tolerability, L. mucosae It showed good tolerance to different pH conditions (2.0, 3.0, 5.0, 7.0) and different concentrations of bile salts (0.0%, 0.1%, 0.2%, 0.3%), and plate count results showed that it maintained a high survival rate even after 4 hours of treatment. Figure 1 (Central BC). Regarding antioxidant capacity, L. mucosae p-2,2-Diphenyl-1-picrylhydrazine (DPPH), hydroxyl radical (OH) - ) and superoxide anion (O2) -The removal rates of Fe were 44.61%, 53.36%, and 74.63%, respectively; 2+ The chelating ability was 36.12%, and the total reducing power at 700 nm wavelength was 0.78. Figure 1 (D). Furthermore, this strain exhibits strong adhesion to HT-29 intestinal epithelial cells ( ). Figure 1 (E), and no hemolytic activity was observed ( Figure 1 (Middle F). L. mucosae It also has a significant inhibitory effect on a variety of common intestinal pathogens. Figure 2 (A), and exhibits some resistance to certain antibiotics ( Figure 2 (Chinese BC). In summary, L. mucosae It exhibits a variety of excellent probiotic properties in vitro, suggesting its potential as a candidate probiotic strain for MAFLD treatment.

[0051] 1.4 Molecular Identification

[0052] (1) Inoculate the isolated single bacteria into the corresponding 5 mL liquid culture medium;

[0053] (2) Preserve bacteria with 30% glycerol (one tube is sufficient for multiple samples), centrifuge (8000 rpm, 2 min), and discard the supernatant;

[0054] (3) Add 600 μL of lysis buffer (lysis buffer: 500 mM NaCl, 50 mM tris-HCl, pH 8.0, 50 mM EDTA, 4% SDS), 200 μL of Tris-saturated phenol and 0.3-0.4 g of glass beads to the precipitate, shake for 30 s and repeat 3 times until the cells are completely suspended, and centrifuge (8000 rpm, 1 min).

[0055] (4) Transfer the supernatant to a new 1.5 mL centrifuge tube, add 250 μL of 10 M ammonium acetate, place on ice for 10 min, and centrifuge (8000 rpm, 1 min).

[0056] (5) Take the supernatant above the organic layer onto the DNA adsorption column, erring on the side of less rather than more, and do not take the organic layer. Centrifuge (8000 rpm, 1 min).

[0057] (6) Wash once with 600 μL of 75% ethanol;

[0058] (7) Repeat (6);

[0059] (8) After spinning at 8000 rpm for 2 min, transfer the DNA adsorption column to a new EP tube and air dry for 30 min;

[0060] (9) Add 50µL of TE (pH=8.0) to the dried centrifuge tube, and send 25µL to Sangon Biotech (Shanghai) Co., Ltd. for sequencing;

[0061] The 16S rDNA sequence is as follows:

[0062]

[0063] 1.5 Results

[0064] Based on the 16S rDNA sequencing results, the bacterial strain was obtained by searching and comparing on NCBI. Specific bacteria were selected for secondary identification and then deposited into a bacterial library. The secondary sequencing results were submitted to the GenBank database of the National Center for Biotechnology Information (NCBI) for homology comparison using the basic local alignment search tool (BLAST). The results showed that HG003 had ≤99.8% homology with known *Lactobacillus mucosa* strains, confirming it as a novel strain.

[0065] 1.6 Preservation of strains

[0066] The purified HG003 strain was inoculated into MRS liquid medium and cultured for 24 h. Then, 30% glycerol was added and the culture was stored at -80℃. On December 22, 2025, it was deposited at the China General Microbiological Culture Collection Center (CGMCC), at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.37136 and classification name: Lactobacillus mucosae.

[0067] Example 2: Efficacy verification of Lactobacillus mucosa HG003 in treating MAFLD (mouse model verification)

[0068] The experimental protocol has been approved by the Laboratory Animal Ethics and Welfare Committee of Nanchang University (Approval No.: NCULAE-20241023001) and strictly followed the "Laboratory Animal Care and Use Guide" (8th Edition) published by the National Academy of Sciences Press in 2011.

[0069] 2.1 Laboratory Animals

[0070] Healthy male C57BL / 6J mice aged 6 - 8 weeks were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd. and were housed in the standard specific pathogen - free (SPF) animal facility (SYXK (Gan) 2021 - 0004) of the animal platform of the Biomedical Testing Center of Nanchang University. The housing environmental conditions were humidity 50% ± 10%, temperature 22°C ± 2°C, and a light - dark cycle of 12 hours / 12 hours. All mice had free access to food and water and were fed with standard feed. Before the experiment, all animals were acclimated for one week. The metabolic - associated fatty liver disease (MAFLD) model was constructed by feeding a high - fat diet (Xiaoshu Youtai, China, D12079B) continuously for 12 weeks (the high - fat diet contained 60% fat, 20% carbohydrates, and 20% protein).

[0071] 2.2 Experimental grouping

[0072] a. Control group (Group C = 6 mice): After being fed a normal common diet for 12 weeks, the mice were gavaged with 200 μL / d of sterile PBS solution for 4 weeks;

[0073] b. Model group (Group M = 6 mice): After being fed a high - fat diet for 12 weeks, the mice were gavaged with 200 μL / d of sterile PBS solution for 4 weeks;

[0074] c. Lactobacillus mucosae HG003 group (Group MP = 8 mice): After being fed a high - fat diet for 12 weeks, the mice were gavaged with 200 μL / d of PBS solution containing Lactobacillus mucosae HG003 (the strain concentration was 1×10 9 CFU / mL) for 4 weeks.

[0075] 2.3 Experimental procedures

[0076] (1)Observation of mice's food intake, water intake, and general conditions

[0077] Every day, observe and record the consumption of feed and water of mice in each group, and observe the general conditions of mice such as activity, hair luster, eating, defecation and urination, and mental state. Detect the symptoms of polydipsia and polyphagia and their remission in each group;

[0078] (2)Measurement of body weight, body length, and Lee's index

[0079] During the model establishment and intervention period, weigh and measure the body weight and body length of mice weekly and calculate Lee's index. Lee's index = body weight (g)^(1 / 3) × 10 / body length (cm);

[0080] (3)After the intervention, 6 mice in each group were sacrificed, and blood and liver tissue samples of the mice were taken for subsequent tests;

[0081] (4) Lipid metabolism and liver function-related indicators such as total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in serum and tissues were detected using a biochemical reagent kit (Nanjing Jiancheng, China). In addition, the activities of superoxide dismutase (SOD) and catalase (CAT), as well as the contents of glutathione (GSH) and malondialdehyde (MDA) in liver tissue were detected to comprehensively evaluate oxidative stress status. The levels of inflammatory factors and endotoxins in serum and tissue homogenates, including interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and lipopolysaccharide (LPS), were quantitatively analyzed using an ELISA kit (Jianglai Biotechnology, China). To evaluate systemic glucose homeostasis, fasting blood glucose (FBG) (Sinocare, China) and fasting insulin (FINS) (Jianglai Biotechnology, China) levels were measured, and the Homeostasis Model of Insulin Resistance Index (HOMA-IR) and Homeostasis Model of Insulin Sensitivity Index (HOMA-IS) were calculated accordingly. Furthermore, the body's glucose clearance capacity and insulin sensitivity were assessed using the intraperitoneal glucose tolerance test (IPGTT) and the intraperitoneal insulin tolerance test (IPITT). In cell experiments, the total cholesterol (TC) and triglyceride (TG) levels in cell pellets, as well as the activities of ALT and AST in cell culture supernatant, were measured. All assay procedures were strictly performed according to the corresponding kit instructions to ensure consistency of procedures and comparability of results.

[0082] (5) After liver tissue was fixed with 4% paraformaldehyde, embedded in paraffin and sectioned, the following staining was performed: HE staining (Servicebio, China, G1005) to observe tissue structure and inflammatory cell infiltration; Oil Red O staining (Servicebio, China, G1015) to detect the accumulation of neutral lipids in frozen sections.

[0083] (6) Statistical Analysis: All statistical analyses and data visualizations in this study were conducted based on independent biological replicates, and the specific sample sizes are indicated in the figure captions. Statistical analysis tools used included GraphPad Prism 9.0, SPSS 26.0, and R4.3.2. Normality of data was tested using the Shapiro-Wilk method. Normally distributed data were expressed as mean ± standard deviation. Intergroup comparisons were performed using unpaired t-tests or one-way ANOVA, with Bonferroni correction used for post-hoc multiple comparisons. If variances were unequal, Welch's t-test or Welch's ANOVA was used, with Dunnett's T3 correction used for post-hoc comparisons. Non-normally distributed data were expressed as median (interquartile range). Intergroup comparisons were performed using the Mann-Whitney U test or Kruskal-Wallis test, with Dunn's test used for post-hoc comparisons. Categorical data were expressed as frequency (percentage), with chi-square tests or Fisher's exact test used for intergroup comparisons. Correlation analysis was performed using the Spearman rank correlation test. All tests were two-tailed, and p < 0.05 was considered statistically significant.

[0084] 2.4 Experimental Results

[0085] like Figures 3-4 As shown, based on observations in previous preliminary experiments and in vitro studies... L. mucosae Given its probiotic properties, this invention further evaluated the therapeutic effect of this strain on MAFLD through in vivo experimental systems. Figure 3 (A). Compared with the MAFLD model group (M group) induced by a 60% high-fat diet, after... L. mucosae Mice treated by gavage for 4 weeks (MP group) showed significant reductions in both body weight and liver weight. Figure 3 (B, F). Meanwhile, the serum total cholesterol (TC) and triglyceride (TG) levels in the MP group ( Figure 3 Significant improvement was observed in C (in the middle jiao). Regarding glucose metabolism, the MP group mice showed superior glucose homeostasis, insulin resistance, and pancreatic β-cell function compared to the M group (in the middle jiao). Figure 3 (DE). HE staining of liver tissue showed that group M hepatocytes contained a large number of enlarged fat vacuoles, indicating severe steatohepatitis and tissue damage; while L. mucosae The intervention significantly alleviated the degree of hepatic steatosis. Figure 4 (A). Furthermore, Oil Red O staining of the liver further confirmed that lipid droplet deposition and fibrosis were significantly reduced in the MP group ( Figure 4 (C). Regarding inflammation and oxidative stress, L. mucosae The intervention significantly reduced serum levels of IL-1β, IL-6, TNF-α, and LPS. Figure 4It improved oxidative stress-related indicators in the liver, including malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), and glutathione (GSH). Figure 4 (DE). Furthermore, serum ALT and AST activities in the MP group were significantly lower than those in the M group, suggesting improved liver function. Figure 4 (B) In summary, L. mucosae It exhibited multifaceted ameliorative effects in the MAFLD mouse model, including regulating lipid metabolism, improving glucose homeostasis, reducing hepatic steatosis, inhibiting inflammation and oxidative stress, and promoting liver function recovery. These results collectively support its potential value as a probiotic strain for the treatment of MAFLD.

[0086] Example 3: Validation of the effect of EVs derived from Lactobacillus mucosa HG003 on acutely injured cells (HepG2 cell model validation).

[0087] 3.1 Cell Experiments

[0088] HepG2, AML12, and HT-29 cell lines were all purchased from the Cell Bank of the Chinese Academy of Sciences. All cells were cultured using Dulbecco modified Eagle medium (Servicebio, China, G4511) as the basal medium, supplemented with 10% heat-inactivated fetal bovine serum (Sempervivum, China, BC-SE-FBS07C) and 1% penicillin-streptomycin solution (Servicebio, China, G4003). For AML12 cells, 1% dexamethasone (OriLeaf, China, S17003) was added to the medium. All cells were cultured routinely in an incubator at 37°C, 5% CO2, and saturated humidity.

[0089] To establish a cell model of metabolic-associated fatty liver disease (MAFLD), HepG2 cells were treated with 0.75 mM free fatty acids (FFA) (Kunchuang Biotechnology, China, KC006) for 24 hours.

[0090] Preliminary experiment: To investigate the optimal concentrations of different components of *Lactobacillus mucosa* in improving MAFLD. In an FFA-induced HepG2 cell model, the same concentrations of [component name missing] were added... L. mucosae The supernatant, heat-inactivated bacterial cells, and their derived extracellular vesicles were cultured for another 24 hours.

[0091] 3.2 Grouping Methods and Handling Measures

[0092] (1) Group C: control group, PBS blank control.

[0093] (2) Group M: Model group, HepG2 cells were induced by FFA for 24 hours to form a model, and were given PBS Intervention for 24 hours.

[0094] (3) MC group: bacterial cell group, FFA induced HepG2 cells for 24h to form a model, and then... L. mucosae Bacterial intervention for 24 hours.

[0095] (4) MS group: supernatant group, FFA induced HepG2 cells for 24h to form a model, and then... L. mucosae Supernatant was used for intervention for 24 hours.

[0096] (5) ME group: vesicle group, FFA induced HepG2 cells for 24h to form a model, and vesicles were intervened for 24h.

[0097] 3.3 Experimental Procedure

[0098] (1) Observe the cell growth in each group;

[0099] (2) Compare the ALT and AST levels in each group;

[0100] Remove the well plate, aspirate the supernatant, wash the cells twice with PBS, add RIPA cell lysis buffer to lyse the cells for 30 min, then transfer the suspension to a 1.5 mL EP tube, centrifuge for 5 min, aspirate the supernatant, and use a biochemical kit to detect ALT and AST levels.

[0101] (3) Compare the TC and TG levels in each group;

[0102] Remove the well plate, aspirate the supernatant, wash the cells twice with PBS, add RIPA cell lysis buffer to lyse the cells for 30 min, then transfer the suspension to a 1.5 mL EP tube, centrifuge for 5 min, aspirate the supernatant, and use a biochemical kit to detect TC and TG levels.

[0103] (4) Cell Oil Red O staining

[0104] Oil Red O staining was used to assess intracellular lipid accumulation. Cells were washed with PBS, fixed with 4% paraformaldehyde, and then stained with Oil Red O working solution (Servicebio, China, G1015) at room temperature in the dark for 30 minutes. After differentiation with 60% isopropanol, cells were counterstained with hematoxylin and DAPI. After staining, slides were mounted and observed and images were acquired under an optical microscope (NiKon, Japan).

[0105] The q-PCR procedure was as follows: Total RNA was extracted from mouse liver tissue using TRIzol reagent (Life Technologies, USA), and its concentration and purity were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). One microgram of RNA was reverse transcribed into cDNA using a Hifair® AdvanceFast gDNA Removal RT Premix (Yeasen, China, 11151ES60). Real-time quantitative PCR was performed on the Applied Biosystems platform using a Hieff UNICON® SYBR Green Premix (Yeasen, China, 11185ES08). The PCR reaction conditions were as follows: initial denaturation at 95°C for 30 seconds; followed by 40 cycles, including denaturation at 95°C for 10 seconds and annealing extension at 60°C for 30 seconds. β-actin was used as an internal reference gene, and the relative expression level of the gene was calculated using the 2^–ΔΔCt method.

[0106] Western blot (WB) detection was performed as follows: Tissue and cell samples were lysed on ice using RIPA buffer (Solarbio, China) containing protease inhibitors, and the lysates were collected by centrifugation. Protein concentration was determined using the BCA method. Equal volumes of protein were separated by SDS-PAGE electrophoresis, transferred to a PVDF membrane (Millipore, USA), and blocked in 5% skim milk for 2 hours. The PVDF membrane was then incubated overnight with primary antibody at 4 °C, followed by incubation with HRP-labeled secondary antibody at room temperature for 2 hours. Protein bands were developed using a chemiluminescent substrate (Invitrogen, USA), and images were acquired using an automated gel imaging system (Tanon, China). ImageJ software was used for quantitative analysis of band intensity.

[0107] 3.4 Results

[0108] like Figures 5-6 As shown, for the investigation L. mucosae To identify the key active components responsible for the improvement, this study first treated HepG2 cells with 0.75 mM free fatty acids (FFA) for 24 hours to establish an in vitro MAFLD cell model. Subsequently, the bacterial culture was centrifuged to separate the supernatant and bacterial cell pellet. Based on determining the optimal intervention dose, the effects of different concentrations of the bacterial cell pellet (10...) on the supernatant and bacterial cell pellet were compared. 7 Effects of CFU / mL and bacterial supernatant (10%) on ALT, AST levels and intracellular TC and TG content in MAFLD cell supernatant. Figure 5 (A). The results show that, L. mucosae The supernatant significantly reduced ALT and AST levels and alleviated intracellular lipid accumulation, while bacterial cell precipitation did not produce a significant effect. Figure 5 (C). Oil Red O staining results further confirmed the role of the supernatant in reducing lipid deposition. Figure 5 (B). Recent studies have shown that extracellular vesicles (EVs) can serve as delivery carriers for bioactive molecules, exerting therapeutic effects in various disease models, and probiotic-derived EVs have demonstrated potential application value in metabolic diseases. Based on this, the present invention hypothesizes that... L. mucosae The source of EVs may play a key functional media role. To verify this hypothesis, this invention employs ultracentrifugation to extract EVs from... L. mucosae EVs were isolated from the culture and characterized using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). NTA results showed an EV particle concentration of 3.4 × 10⁻⁶. 11 particles / mL, median particle size is 129.1 nm ( Figure 6 (A); TEM images show that the extracted EVs exhibit a typical cup-shaped morphology ( Figure 6 (A). To further evaluate cellular uptake of EVs, this invention labeled EVs with PKH26 fluorescent dye and co-cultured them with HepG2 hepatocytes and HT-29 intestinal cells, respectively. Confocal microscopy showed that both cell types effectively internalized EVs. Figure 6 (Middle B). The effects of 50 μg / mL EVs and the supernatant after EV removal on MAFLD cells were then compared ( Figure 6 (C) It was found that EV treatment alone significantly improved ALT, AST, TC, TG levels and Oil Red O staining results, while the supernatant after EV removal showed no significant therapeutic effect. Figure 6 (middle DF). The above results indicate that L. mucosae The derived EVs are the key active ingredient that plays a role in improving liver lipids.

[0109] like Figures 7-8 As shown, in order to verify the central role of the AMPK pathway in animal models ( Figure 7 (A). The results showed that, after L. mucosae After 4 weeks of intervention with EVs via gavage, the body weight and liver weight of mice were significantly reduced. Figure 7 B), serum and liver lipid metabolism indicators ( Figure 7 (C) and liver function indicators ( Figure 8 Both the middle and lower limb glucose levels (B) showed significant improvement, and their glucose metabolism status was also better than that of the model group (B). Figure 7 D, Figure 8 (A). Liver HE staining ( Figure 7 (E) and Oil Red O staining ( Figure 8Further, in vivo studies (C) confirmed that EVs effectively alleviated hepatic steatosis. However, the combined use of the AMPK pathway inhibitor Compound C essentially blocked the aforementioned ameliorative effects of EVs. In summary, this study, through in vivo experiments, confirmed that... L. mucosae The EVs from this source mainly exert their multifaceted effects on MAFLD by activating the AMPK signaling pathway.

[0110] Example 4: L. mucosae Verification that the derived EVs showed no significant toxicity upon reaching mouse liver tissue (animal model validation).

[0111] To evaluate the in vivo biosafety, distribution characteristics, and potential application value of extracellular vesicles (EVs) derived from Lactobacillus mucosae, the present invention conducted the following systematic experiments:

[0112] ① Fluorescent labeling and tracing of extracellular vesicles

[0113] First, this invention uses the lipid-soluble fluorescent dye DiR to treat the purified... L. mucosae - EVs were labeled on a membrane. The labeled EVs were administered orally to mice fed a normal or high-fat diet via gavage, as described in Example 2. Twenty-four hours after gavage, whole-body imaging of the mice was performed using a small animal in vivo fluorescence imaging system to observe the dynamic distribution and enrichment of fluorescence signals in vivo in real time and non-invasively.

[0114] ②Analysis of in vivo distribution and bioavailability

[0115] In vivo imaging results showed ( Figure 9 Following oral administration of DiR-labeled EVs (absorbed by the intestines), the fluorescence signal generated by the EVs was significantly enriched in the liver and spleen regions, suggesting that the EVs can be absorbed through the intestines and effectively delivered to the liver via the portal venous system or the gut-hepatic axis. This distribution pattern provides key pharmacokinetic evidence for their direct action on liver target cells and improvement of hepatic steatosis.

[0116] ③Systemic toxicity assessment

[0117] To comprehensively evaluate the in vivo safety of EVs, this invention collects mouse serum after the EV intervention period ends and performs multiple biochemical tests.

[0118] Hepatotoxicity assessment: Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured.

[0119] Cardiotoxicity assessment: Detection of creatine kinase isoenzyme (CK-MB) levels, a marker of myocardial enzyme profile.

[0120] Nephrotoxicity assessment: Detect creatinine (CREA), uric acid (UA), and blood urea nitrogen (BUN) levels.

[0121] The results show that ( Figure 9 (C), compared with the control group, was given L. mucosae In mice treated with EVs, none of the above-mentioned serum biochemical indicators showed statistically significant increases and remained within the normal physiological range, preliminarily indicating that EV intervention did not cause significant damage to the liver, heart, and kidneys.

[0122] 4. Histopathological examination of vital organs

[0123] To further verify safety at the tissue morphology level, mice were euthanized at the experimental endpoint, and six vital organs—brain, heart, liver, spleen, lungs, and kidneys—were collected. These tissue samples were fixed, embedded in paraffin, sectioned, and then stained with hematoxylin and eosin (H&E).

[0124] Pathological observation was performed under a high-powered microscope. Figure 10 (AB). The results showed that in all groups of mice treated with EVs, the tissue structure of all examined organs was clear and intact, with no obvious inflammatory cell infiltration, necrosis, fibrosis or other pathological damage.

[0125] In summary, through an experimental strategy combining in vivo imaging, serum biochemistry, and histopathology, this invention confirms that orally administered... L. mucosae -EVs are efficiently absorbed from the intestine and specifically accumulate in the target organ, the liver. Simultaneously, in vivo intervention did not induce significant systemic toxicity or organ pathological damage. These results provide... L. mucosae -EVs, as a safe and well-targeted bioactive agent, provide important preclinical safety data to support the further development and clinical translation of metabolic-associated fatty liver disease (MAFLD).

[0126] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and variations that are directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. A strain of *Lactobacillus mucosa*, characterized in that, The strain was named HG003 and classified as Lactobacillus mucosa ( Lactobacillus mucosae It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.37136.

2. The use of the extracellular vesicles derived from *Lactobacillus mucosa* according to claim 1 in the preparation of a drug for treating metabolic-related fatty liver disease, characterized in that... The metabolic-related fatty liver disease mentioned above is simple fatty liver.

3. The application according to claim 2, characterized in that, The median particle size of the extracellular vesicles is 120-140 nm.

4. The application according to claim 2, characterized in that, The extracellular vesicles are natural extracellular vesicles secreted by the mucosal lactobacilli into a fermentation broth under conventional culture conditions, the concentration of extracellular vesicle particles in the fermentation broth being ≥ 1 x 10 11 particles / mL.

5. A biological agent for treating metabolic-related fatty liver disease, characterized in that, The biological agent comprises the live Lactobacillus mucosa of claim 1 or extracellular vesicles derived from Lactobacillus mucosa of claim 1.

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