Lactobacillus mucilaginosus HG002 and application thereof

Abstract

The invention discloses lactobacillus mucus HG002 and application thereof, and belongs to the field of microbial medicine. The lactobacillus mucilaginosus HG002 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC NO.36670, and the 16S rDNA gene sequence of the lactobacillus mucilaginosus HG002 is as shown in SEQ ID NO: 1. The strain is separated from excrement of healthy adults, the relieving effect of ALF is remarkably improved through the brand-new fermentation lactobacillus mucus HG002 strain and derivative EVs thereof in combination with a synergistic effect mechanism and optimized product design, many defects in the prior art are overcome, and wide application prospects and industrialization value are achieved.

Description

Technical Field

[0001] This invention relates to the field of microbial medicine, specifically to a fermenting Lactobacillus mucinus HG002 and its applications. Background Technology

[0002] Acute liver failure (ALF) is an acute impairment of liver function that occurs without underlying chronic liver disease. It is primarily characterized by severe coagulation disorders and jaundice, with grade II or higher hepatic encephalopathy developing within two weeks. ALF can be caused by viral infection, drug-induced injury, toxic injury, traumatic events, or other unknown causes. Liver transplantation and artificial liver therapy are currently the two main clinical treatments for ALF. However, the scarcity of donor livers, the high incidence of postoperative complications, and the exorbitant cost limit the widespread clinical application of liver transplantation, while the efficacy of artificial liver therapy is also relatively limited. Therefore, the search for new treatment methods is of great significance.

[0003] Due to the close anatomical relationship between the liver and intestines, changes in liver disease often lead to changes in the gut microbiota. Animal studies have shown that *Lactobacillus fermentum* (…) Limosilactobacillus fermentum , L. fermentum Non-alcoholic fatty liver disease can be improved by regulating metabolism. L. fermentum HF06 can improve liver damage secondary to ulcerative colitis through its anti-inflammatory effects. Furthermore, in in vitro cell experiments... L. fermentum MG4237 and MG4294 can prevent ethanol-induced liver damage in HepG2 cells.

[0004] Extracellular vesicles (EVs) derived from probiotics are spherical vesicles with a lipid bilayer, typically ranging from 20 to 400 nm in diameter, secreted by probiotics. They can carry various bioactive components (proteins, lipids, and nucleic acids) across the mucus layer to other tissues or different cells of the host's immune system, thereby participating in biological functions such as microbial stress response, biofilm formation, information exchange, and immune regulation. Probiotic-derived EVs can activate host TLR2 signaling and enhance IgA antibody production through microbial cell wall components, thus strengthening the mucosal immune system, preventing infection, and maintaining gut microbiota balance. Furthermore, due to the close anatomical and functional connection between the gut and liver—the "gut-liver axis"—the liver is also an active site for EV uptake.

[0005] However, the specific mechanisms of action, protective effects, and underlying molecular mechanisms of fermenting Lactobacillus mucinus and its derived EVs in a mouse model of lipopolysaccharide (LPS) / galactosamine (D-Gal)-induced acute liver failure remain unclear. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a fermenting *Lactobacillus mucinus* HG002 strain and its applications. The strain was isolated from the feces of healthy adults. This invention utilizes a novel fermenting *Lactobacillus mucinus* strain. Limosilactobacillus fermentum The HG002 strain and its derived EVs, combined with a synergistic mechanism and optimized product design, significantly improve the alleviating effect of ALF, overcome many shortcomings of existing technologies, and have broad application prospects and industrialization value.

[0007] In this invention, previous studies have found that patients with acute-on-chronic liver failure (ACLF) have an imbalance in their gut microbiota, with an overall increase in opportunistic pathogens and an overall decrease in beneficial bacteria, specifically Escherichia coli (E. coli). Escherichia ) and Streptococcus spp. Streptococcus The relative abundance of Collins spp. increased, and Collins spp. ( Collinsella The relative abundance of *Lactobacillus mucinus* HG002 and its derived EVs was reduced. This study aims to verify the role of *Lactobacillus mucinus* HG002 and its derived EVs in ALF through animal and cell experiments. ELISA, flow cytometry, immunohistochemistry, transcriptomics sequencing, qPCR, and Western blotting will be used to explore the specific molecular mechanisms by which EVs exert hepatoprotective effects in ALF, providing new targets and strategies for the comprehensive treatment of ALF.

[0008] To achieve the above objectives, the present invention provides a fermenting *Lactobacillus mucinus* strain named HG002, which is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC NO.36670. The 16S rDNA gene sequence of *Lactobacillus mucinus* HG002 is shown in SEQ ID NO: 1.

[0009] Furthermore, the strain has the following characteristics: physiological and biochemical characteristics: Gram-positive, non-spore-forming, rod-shaped; tolerant to acidic environments of pH 2.0-7.0, and tolerant to 0.0%-0.3% bile salts.

[0010] Furthermore, the strain possesses the following characteristics: EV production capacity: after 40 hours of aerobic culture, the EV yield in the fermentation broth is ≥5.0 × 10⁻⁶. 10 The number of EVs per mL is 100-200 nm.

[0011] Furthermore, the culture conditions for the strain are as follows: MRS medium, aerobic culture at 37°C for 14-24 hours, with the pH maintained at 5.5-6.5 during the culture process.

[0012] A second aspect of the present invention provides EVs derived from *Lactobacillus fermentatus* HG002 described in this invention, wherein the EVs are obtained by the following method: *Lactobacillus fermentatus* HG002 strain is cultured aerobically at 37°C for 40 h in MRS medium to a concentration ≥1×10⁻⁶. 9 The culture suspension of CFU / mL was centrifuged at 2000 ×g for 10 min, 5000 ×g for 20 min, and then at 10000 ×g for 30 min to remove bacterial cells. The supernatant was filtered through a 0.22 μm filter membrane and ultraconcentrated at 150000 ×g for 70 min. The precipitate containing EVs was washed with PBS, ultracentrifuged, resuspended in PBS, and stored at -80℃ for later use.

[0013] Furthermore, the EVs have a particle size of 100-200 nm, a Zeta potential of -10 to -30 mV, and transmission electron microscopy shows that they have a typical crescent-shaped structure and do not contain intact bacterial cells or bacterial cell fragments.

[0014] A third aspect of the present invention provides the use of the fermented Lactobacillus mucinus HG002 and / or the EVs described herein in the preparation of a product for alleviating acute liver failure, wherein the product alleviates ALF by regulating the balance of intestinal flora, inhibiting the liver inflammatory response (reducing TNF-α and IL-6 expression), and reducing hepatocyte apoptosis.

[0015] Furthermore, the application includes: using Lactobacillus fermentum HG002 as an effective dose to intervene in a mouse model of acute liver failure, with a dose of 1×10⁻⁶. 9 -1×10 11 CFU / kg body weight / day

[0016] Furthermore, the application includes: an effective dose of EVs for intervention in a mouse model of acute liver failure is 200-300 μL / mouse / day, and the EVs concentration is 5.1 × 10⁻⁶. 10 per mL.

[0017] Furthermore, compared to the control group of mice with acute liver failure who were not treated, the mice with acute liver failure were treated with the fermented Lactobacillus mucinus HG002, and the mice with acute liver failure were treated with the EVs ...

[0018] Further, compared with the acute liver failure model control group of the mice without administration, the IL-6, IL-1β and TNF-α levels in the liver tissue of the mice after intervention of the fermented M. mucusolvens HG002 are reduced by 40-50%; the IL-6, IL-1β and TNF-α levels in the liver tissue of the mice after intervention of the EVs are reduced by 42-60%.

[0019] Further, the acute liver failure is caused by drug-induced liver injury, viral liver injury or alcoholic liver injury.

[0020] The fourth aspect of the present application provides a product for relieving acute liver failure, and the active ingredient of the product is selected from the fermented M. mucusolvens HG002 and / or the EVs of the present application; wherein the viable cell count of the fermented M. mucusolvens is ≥1×10 9 CFU / mL, and the concentration of the EVs is ≥5×10 10 CFU / mL.

[0021] Compared with the prior art, the present application has at least the following beneficial effects: (1) The HG002 strain of the present application is a newly isolated and identified M. mucusolvens strain, and the 16S rDNA gene sequence (as shown in SEQ ID NO. 1) has a homology of ≤99.8% with the known M. mucusolvens strain, and has unique physiological and biochemical characteristics; at the same time, the strain has excellent acid and bile salt resistance, can successfully pass through the human gastrointestinal barrier and colonize, and solves the problem of low colonization efficiency of ordinary probiotics; more importantly, the strain has a significant EVs production capacity, which provides a stable strain source for subsequent large-scale preparation and application of EVs, and has an essential advantage over the existing probiotic strains that do not produce EVs or have weak EVs production capacity.

[0022] (2) The EVs isolated and purified from the culture solution of the HG002 strain have clear characteristic parameters, high purity and no complete bacterial body pollution; the EVs have significant liver targeting, can be effectively taken up by liver cells, solve the defects of traditional probiotics that need to indirectly regulate liver function through the intestinal tract and have low efficiency, and at the same time reduce the administration dose and improve the use safety, which has a significant advantage over the existing probiotics.

[0023] (3) The present application innovatively proposes that the EVs secreted by the strain HG002 can be used for relieving ALF; the mechanism can play a role from two dimensions of "regulating intestinal flora balance" and "directly targeting and inhibiting liver inflammation and reducing liver cell apoptosis", forming a double protection mechanism, and providing a new technical path for the precise treatment of ALF.

[0024] (4) The strain HG002, EVs and their products of the present invention can effectively alleviate ALF caused by a variety of etiologies and are applicable to a wide range of people. The strains used are derived from the feces of healthy human beings. EVs are natural secretions of strain HG002 and have good biocompatibility. They have no obvious toxic side effects and are safer than traditional chemical hepatoprotective drugs (if they have no obvious side effects). At the same time, the technical solution of the present invention is simple to operate and highly reproducible (the coefficient of variation is ≤5% after 3 experiments), which is convenient for clinical promotion and application. It can effectively reduce the treatment cost of ALF and has important clinical significance and social value. Attached Figure Description

[0025] Figure 1 The following are the evaluation results of the probiotic properties of *Lactobacillus fermentans* in one embodiment of the present invention: (A) growth curve; (B) antioxidant properties evaluation; (C) hemolysis test; (D) acid resistance evaluation; (E) bile salt resistance evaluation; (F) adhesion test.

[0026] Figure 2 The following are evaluations of the probiotic properties of *Lactobacillus fermentans* in one embodiment of the present invention: (A) antibacterial test; (B) drug sensitivity test. Notes: PEN: penicillin; C: chloramphenicol; E: erythromycin; AMP: ampicillin; MY: lincomycin; CTP: ceftriaxone; TET: tetracycline; SXT: trimethoprim-sulfamethoxazole; GEN: gentamicin; CIP: ciprofloxacin.

[0027] Figure 3 This invention demonstrates that fermentation of *Lactobacillus mucinus* alleviates liver injury in mice with acute liver failure in one embodiment: (A) Schematic diagram of experimental design; (B) Serum ALT and AST levels; (C) Serum IL-6, IL-1β, and TNF-α levels; (D) Liver IL-6, IL-1β, and TNF-α levels; (E) Representative images of liver tissue H&E staining (scale bar: 100 μm); (F) Relative expression levels of oxidative stress markers (MDA, SOD, GSH) in liver tissue. Data are expressed as mean ± standard deviation and were statistically analyzed using one-way ANOVA (Dunnett's post-hoc test). ns: No significant difference; * p <0.05,** p <0.01, *** p <0.001. Wherein, C: control group; M: acute liver failure model group; P: fermentation *Lactobacillus mucinus* intervention group (n=6 per group). Note: L. fermentum : Fermenting Lactobacillus mucinus; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; IL-6: Interleukin-6; IL-1β: Interleukin-1β; TNF-α: Tumor necrosis factor-α; MDA: Malondialdehyde; SOD: Superoxide dismutase; GSH: Glutathione.

[0028] Figure 4 Figure 10 shows that the extracellular vesicles of fermented L. muceris have a significant protective effect on LPS / D-GALN-induced HepG2 cell damage in an embodiment of the present application: (A) cell viability detection after 1 pg / mL LPS and 5 mg / mL D-GALN treatment for 12, 24, 36 and 48 hours; (B) schematic diagram of cell experiment design; (C) cell viability detection after 24 hours of pre-treatment with fermented L. muceris culture supernatant or bacterial body precipitate; (D) ALT and AST levels in cell culture supernatant; (E) nanoparticle tracking analysis to detect the particle size distribution of extracellular vesicles of fermented L. muceris; (F) representative image of extracellular vesicles of fermented L. muceris under transmission electron microscope (scale bar: 100 nm); (G) PKH26-labeled extracellular vesicles of fermented L. muceris are internalized by HepG2 cells (scale bar: 10 pm); (H) effect of different concentrations of extracellular vesicles of fermented L. muceris on cell viability; (I) ALT and AST levels in cell culture supernatant. Data are expressed as mean ± standard deviation, and statistical analysis was performed by one-way ANOVA (Dunnett's post-hoc test). ns: no significant difference; p <0.05,** p <0.01,*** p <0.001. Wherein, C: control group; M: liver injury model group; S: fermented L. muceris supernatant group; T: fermented L. muceris bacterial body group; EVs: extracellular vesicles of fermented L. muceris group; S(-EV): extracellular vesicle-removed supernatant group (n=3-6). Note: L. fermentum : fermented L. muceris; EVs: extracellular vesicles; LPS: lipopolysaccharide; D-GalN: D-galactosamine; ALT: glutamic-pyruvic transaminase; AST: glutamic-oxalacetic transaminase.

[0029] Figure 5 Figure 11 shows that EVs reduce liver damage in acute liver failure mice in an embodiment of the present application: (A) schematic diagram of experimental design; (B) serum ALT and AST levels; (C) serum IL-6, IL-1β and TNF-α levels; (D) liver IL-6, IL-1β and TNF-α levels. Data are expressed as mean ± standard deviation, and statistical analysis was performed by one-way ANOVA (Dunnett's post-hoc test). ns: no significant difference; p <0.05,** p <0.01,*** p<0.001. Wherein, C: control group; M: acute liver failure model group; EVs: EVs intervention group (n=6 in each group); EVs+ANI: EVs combined with ANI intervention group. Note: EVs: extracellular vesicles; ALT: glutamic-pyruvic transaminase; AST: glutamic-oxalacetic transaminase; IL-6: interleukin 6; IL-1β: interleukin 1β; TNF-α: tumor necrosis factor-α.

[0030] Figure 6 Figure 2 shows the HE staining of mouse liver tissue in an embodiment of the present application, which shows that EVs can reduce liver tissue damage in acute liver failure mice. DETAILED DESCRIPTION

[0031] The endpoints of the ranges and any values disclosed herein are not limited to the precise values recited as the exact dimensions are not critical to the present application. Any numerical value, however, can be expressed as a range to include any and all expressed, implied or potentially interposed values, subject to more limiting ranges if so specified. For values which are not binary or discrete, the endpoints of the ranges are included in the disclosure as well as intermediate values which are not explicitly listed.

[0032] According to one aspect of the present application, a fermented Lactobacillus muiyi is provided, the strain of which is named HG002, and is preserved in the China General Microbiological Culture Collection Center, with the preservation number of CGMCC NO. 36670. The 16S rDNA gene sequence of the fermented Lactobacillus muiyi HG002 is shown in SEQ ID NO: 1.

[0033] In some embodiments, the strain has the following characteristics: physiological and biochemical characteristics: gram-positive, non-spore, rod-shaped; resistant to pH 2.0-7.0 acidic environment, resistant to 0.0%-0.3% bile salt.

[0034] In some embodiments, the strain has the following characteristics: EVs production capacity: after 40h of aerobic culture, the EVs production in the fermentation broth is ≥5.0×10 10 nm.

[0035] In some embodiments, the culture conditions of the strain are as follows: MRS medium, 37°C aerobic culture for 14-24h, and the pH is maintained at 5.5-6.5 during the culture process.

[0036] According to a second aspect of the present application, an EVs derived from the fermented Lactobacillus muiyi HG002 is provided, and the EVs are obtained by the following method: the fermented Lactobacillus muiyi HG002 strain is cultured in MRS medium at 37°C for 40h, and the concentration is ≥1×10 9The culture suspension of CFU / mL was centrifuged at 2000 x g for 10 minutes, at 5000 x g for 20 minutes, and then at 10000 x g for 30 minutes to remove the bacterial bodies, and the supernatant was filtered through a 0.22 μm filter membrane, ultra-concentrated at 150000 x g for 70 minutes, and the precipitate containing EVs was washed with PBS, ultra-centrifuged, resuspended in PBS, and stored at -80°C for later use.

[0037] In some embodiments, the particle size of the EVs is 100-200 nm, the Zeta potential is -10 to -30 mV, and the transmission electron microscopy shows a typical crescent structure without complete bacterial bodies and bacterial body fragments.

[0038] According to a third aspect of the present application, the application provides use of the fermented Lactobacillus muiis HG002 and / or the EVs in the preparation of a product for alleviating acute liver failure, which alleviates ALF by regulating the balance of intestinal flora, inhibiting the inflammatory response of the liver (reducing the expression of TNF-α and IL-6), and reducing hepatocyte apoptosis.

[0039] In some embodiments, the use includes: the effective dose of the fermented Lactobacillus muiis HG002 for intervention in a mouse acute liver failure model is 1 x 10 9 -1 x 10 11 CFU / kg body weight / day.

[0040] In some embodiments, the use includes: the effective dose of the EVs for intervention in a mouse acute liver failure model is 200-300 μL per mouse per day, and the concentration of the EVs is 5.1 x 10 10 / mL.

[0041] In some embodiments, compared with a control group of a mouse acute liver failure model without administration, after intervention of the mouse acute liver failure model with the fermented Lactobacillus muiis HG002, the serum ALT and AST levels of the mouse are reduced by 31%-52%; after intervention of the mouse acute liver failure model with the EVs, the serum ALT and AST levels of the mouse are reduced by 39%-56%.

[0042] In some embodiments, compared with a control group of a mouse acute liver failure model without administration, after intervention of the mouse acute liver failure model with the fermented Lactobacillus muiis HG002, the IL-6, IL-1β and TNF-α levels in the liver tissue are reduced by 40-50%; after intervention of the mouse acute liver failure model with the EVs, the IL-6, IL-1β and TNF-α levels in the liver tissue are reduced by 42-60%.

[0043] In some embodiments, the acute liver failure is induced by drug-induced liver injury, viral liver injury, or alcoholic liver injury.

[0044] According to a fourth aspect of the present application, there is provided a product for alleviating acute liver failure, the active ingredient of the product being selected from the fermentable Lactobacillus mucus HG002 and / or the EVs described in the present application; wherein the viable cell count of the fermentable Lactobacillus mucus is ≥ 1 x 10 9 CFU / mL, and the concentration of the EVs is ≥ 5 x 10 10 CFU / mL.

[0045] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative labor fall within the protection scope of the present application.

[0046] In the following embodiments: The reagents used include: phosphate buffered saline (Selleck, China, G4202), crystal violet staining solution (Selleck, China, G1014), lipopolysaccharide (Sigma, USA, L8880), D-galactosamine (Sigma, USA, G1639), anisomycin (Selleck, China; S7409), agar powder (Solebo, China, G4003), MRS medium (Hibio, China, HB0384-5), LB medium (Hibio, China, HB0128), DMEM medium (Selleck, China, G4511), fetal bovine serum (Sunbeam, China, BC-SE-FBS07C), penicillin-streptomycin solution (Selleck, China, G4003), PKH26 red fluorescent dye (Yumai Bio, China, UR52302), ELISA kit (Jianglai Bio, China; IL-6, JLW20268; IL-1β, JLW18442; TNF-α, JLW10484), biochemical kit (Nanjing Jianshen Biological Engineering Institute, China; GSH, A006-2-1; MDA, A003-1-2; SOD, A001-3-2).

[0047] The instruments used include: optical microscope (NiKon, Japan), laser confocal microscope (Perkin Elmer & Olympus, Germany), transmission electron microscope (Hitachi HT7700, Japan), and nanoparticle tracking analysis system (Particle Metrix PMX-120, Germany), fully automatic biochemical analyzer (Rayto Chemray 800, China), pipette (Selleck, China, SPIP).

[0048] Example 1: Isolation, purification and identification of fermenting Lactobacillus mucinus HG002.

[0049] 1.1 Separation and Purification 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 For each sample, a suitable concentration gradient (1-9) was selected for plating (MRS medium). Samples numbered 3, 5, 7, and 9 were used, with 30 μL of each sample plated. The plates were repeatedly heated with glass rods, and the plates were shaken before each sample addition. The plates were then incubated in an aerobic incubator 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.

[0050] 1.2 Morphological identification 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 HG002 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.

[0051] 1.3 Physiological and Biochemical Identification (1) Growth kinetics: The growth kinetics of strain HG002 were characterized by 24-hour growth curves plotted based on 600nm absorbance measurements; (2) Acid resistance test: HG002 cells in the logarithmic phase were washed with PBS and resuspended in acidic buffer solutions of pH 2.0, 3.0, 5.0 and 7.0. They were incubated at 37°C for 4 hours and viable cells were counted by plate method. (3) Bile salt tolerance test: HG002 cells in the logarithmic phase were washed with PBS and resuspended in bovine bile salt solutions of different concentrations (0.1%, 0.2%, 0.3%). They were incubated at 37°C for 4 hours and viable cells were counted by plate method. (4) Cell adhesion experiment: Human colon cancer HT-29 cells were used to form a monolayer on a culture plate. Logarithmic bacteria were resuspended in PBS and co-incubated with the cells for 1-2 hours. Afterwards, the cells were thoroughly washed with sterile PBS to remove unadhered bacteria. The cells were observed and photographed under an optical microscope. (5) Antibacterial test: The bacterial suspensions of common intestinal pathogens (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, β-hemolytic streptococci, Shigella flexneri, Listeria monocytogenes) were spread on agar plates, Oxford cups were placed on the surface of the plates, HG002 culture supernatant was added to the Oxford cups, and the plates were incubated at 37°C for 24 hours. The diameter of the inhibition zone was measured to evaluate the antibacterial activity. (6) Hemolysis test: Streak HG002 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 no change (γ hemolysis) hemolysis zone appears around the colony. (7) Antibiotic resistance test: The standardized concentration of HG002 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. (8) The results are as follows Figures 1-2 As shown: The growth curve shows that this strain enters a rapid proliferation phase after 4 hours and reaches a stationary phase after 14 hours. Figure 1 (A); This invention also found that it possesses significant antioxidant capabilities, with a DPPH free radical scavenging rate of 48.51%, a hydroxyl radical (•OH) scavenging rate of 61.99%, and a superoxide anion (•O2) scavenging rate of 61.99%. - The scavenging rate was 60.3%, the iron ion chelation capacity reached 45.33%, and the total reducing power was 0.68 (OD700). Figure 1 (B); This strain also tolerates acidic environments of pH 2.0–7.0 and bile salt concentrations of 0.0%–0.3%, and plate counting showed that it maintained a high survival rate even after 4 hours of culture. Figure 1 (D, E) Safety and functional experiments showed that this strain had no hemolytic activity. Figure 1 (C), exhibits strong adhesion to HT-29 intestinal epithelial cells ( Figure 1 (F) has inhibitory effects on a variety of pathogenic bacteria, including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, β-hemolytic streptococci, Shigella flexneri, and Listeria monocytogenes. Figure 2(A). Drug sensitivity tests showed that it was sensitive to antibiotics such as erythromycin, penicillin, ciprofloxacin, ampicillin, lincomycin, ceftriaxone, gentamicin, trimethoprim-sulfamethoxazole, chloramphenicol, and tetracycline. Figure 2 (B)

[0052] In conclusion, this study confirms that *Lactobacillus fermentatus* possesses both significant hepatoprotective effects and outstanding probiotic properties, making it a promising candidate probiotic strain for development.

[0053] 1.4 Molecular Identification (1) Inoculate the isolated single bacteria into the corresponding 5 mL liquid culture medium; (2) Preserve bacteria with 30% glycerol (one tube is sufficient for multiple samples), centrifuge (8000 rpm, 2 min), and discard the supernatant; (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). (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). (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). (6) Wash once with 600 μL of 75% ethanol; (7) Repeat (6); (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; (9) Add 50µL of TE (pH=8.0) to the dried centrifuge tube, and send 25µL to Sangon Biotech (Shanghai) Co., Ltd. for sequencing; The 16S rDNA sequence is as follows:

[0054] 1.5Results According to the sequence of 16S rDNA sequencing results, the strain results were obtained by searching and matching on NCBI. After secondary identification of special bacteria, they were preserved in the germ bank. The secondary sequencing results were submitted to the Gen Bank database of the national center for biotechnology information (NCBI) for basic local alignment search tool (BLAST) homology comparison, and the results showed that the homology of HG002 with known L. fermentum strains was ≤99.8%, proving that it was a new strain.

[0055] 1.6Strain preservation The purified HG002 strain was inoculated in MRS liquid medium, cultured for 24 h, 30% glycerol was added, and stored at -80°C; on November 18, 2025, it was preserved in the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Beichen West Road, Chaoyang District, Beijing, with the preservation number CGMCC NO. 36670.

[0056] Example 2: Verification of the effect of L. fermentum HG002 and EVs in relieving ALF (mouse model verification) The experimental plan has been approved by the Nanchang University Experimental Animal Ethics and Welfare Committee (approval number: NCULAE-202502211001) and strictly followed the Guide for the Care and Use of Laboratory Animals (8th edition) published by the National Academy of Sciences Press in 2011.

[0057] 2.1Experimental animals A total of 24 6-8-week-old C57BL / 6J male mice were purchased from Beijing Sperbio Biotechnology Co., Ltd. The mice were acclimated for 1 week in a specific pathogen-free (SPF) level experimental animal center. They were kept under a 12-hour light and 12-hour dark cycle, with a temperature maintained at 22°C±3°C and a relative humidity of 50%±15%. There were 4-5 mice per cage, which could freely access food and water. The mice were given a 7-day acclimation period before the experiment, and the temperature, humidity, noise, and other conditions in the animal room were controlled within the specified range and cleaned daily.

[0058] 2.2Experimental grouping a. Blank group (group C = 6): After 7 days of normal feeding, the mice were given 200 μL of sterile PBS liquid per mouse by gavage, which lasted for 7 days; b. Model group (M group = 6): 200 μL / each of sterile PBS was administered intragastrically every day from day 1 to day 7, and 10 mL / kg of LPS / D-Gal mixture was injected intraperitoneally on day 8 to establish the ALF model; c. Fermented L. m. HG002 group (P group = 6): 200 μL / each of sterile PBS was administered intragastrically every day from day 1 to day 7, and 10 mL / kg of LPS / D-Gal mixture was injected intraperitoneally on day 8 to establish the ALF model; L. fermentum 1 x 10 9 CFU / mL) of L. m. HG002 bacterial solution was administered intragastrically every day from day 1 to day 7, and 10 mL / kg of LPS / D-Gal mixture was injected intraperitoneally on day 8 to establish the ALF model; d. EVs group (EVs group = 6): 250 μL / each of EVs (5.1 x 10 10 CFU / mL) was administered intragastrically every day from day 1 to day 7, and 10 mL / kg of LPS / D-Gal mixture was injected intraperitoneally on day 8 to establish the ALF model.

[0059] 2.3 Experimental procedure (1) Preparation of LPS / D-Gal mixed solution: 1 μg / mL LPS + 80 mg / mL D-Gal; (2) L. fermentum (3) Preparation of L. m. HG002 bacterial solution and EVs: L. m. HG002 was transplanted into MRS medium and cultured aerobically at 37°C for 40 h, with the pH maintained at 5.5-6.5 during the culture process; part of the culture suspension was centrifuged at 2000 x g for 10 min, at 5000 x g for 20 min, and then at 10000 x g for 30 min to remove the bacterial bodies; the supernatant was filtered through a 0.22 μm filter membrane, and the EVs-containing precipitate was washed with PBS, ultracentrifuged, resuspended in PBS, and stored at -80°C for later use; (3) Six mice were sacrificed in each group 5 hours after modeling on day 8, and blood and liver tissue samples were taken from the mice for subsequent detection; (4) The serum ALT and AST levels were detected using a full-automatic biochemical analyzer, and the serum and liver tissue IL-1β, IL-6, and TNF-α levels were detected using an ELISA kit; (5) Liver tissue photographing (morphological changes): after washing twice with sterile PBS, the liver tissue was fixed with 4% paraformaldehyde for histopathological HE staining (pathological changes) and TUNEL staining (cell apoptosis); (6) Intestinal flora detection: intestinal content microbial DNA was extracted, and 16S rDNA high-throughput sequencing was used to analyze the flora diversity (Alpha diversity index) and the relative abundance of beneficial bacteria (Bifidobacterium and lactic acid bacteria); (7) Liver tissue pathology score: The liver tissue was fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, stained with HE, and observed under a microscope to observe the damage of hepatocytes. The score was based on "the degree of hepatocyte necrosis and the degree of inflammatory cell infiltration" (0-4 points, the higher the score, the more serious the damage). (8) Statistical analysis: One-way ANOVA was performed using SPSS 26.0 software. p <0.05 indicates a statistically significant difference.

[0060] 2.4 Experimental Results like Figure 3 As shown, based on the observed in vitro probiotic properties of fermenting *Lactobacillus mucinus* HG002, this invention further evaluates its therapeutic effect through in vivo experiments. Figure 3 (A). Compared with the acute liver failure model group (M group), the serum ALT and AST levels of mice treated with *Lactobacillus mucinus* HG002 via gavage (P group) were significantly reduced. Figure 3 (Middle B). Given that cytokine storm is a key pathological mechanism driving the development of acute liver failure, this invention examined the expression levels of pro-inflammatory cytokines. The results showed that fermentation with *Lactobacillus mucinus* significantly downregulated the expression of IL-6, IL-1β, and TNF-α in the serum and liver tissue of mice with acute liver failure. Figure 3 Based on the crucial role of oxidative stress in the pathogenesis of acute liver failure and the importance of the liver's antioxidant system, this invention simultaneously assessed the oxidative stress status of liver tissue. Results showed that the activities of antioxidant enzymes GSH and SOD were significantly upregulated in group P, while the content of lipid peroxidation product MDA was significantly decreased. Figure 3 (F). Histological analysis further demonstrated that pretreatment with fermented *Lactobacillus mucinus* effectively improved liver inflammation and necrosis (F). Figure 3 (E).

[0061] In summary, the results of this study indicate that fermented Lactobacillus mucinus can alleviate LPS / D-GalN-induced acute liver failure.

[0062] Example 3: Validation of the effect of fermented Lactobacillus mucinus HG002 and EVs on acutely injured cells (HepG2 cell model validation).

[0063] 3.1 First round of cell experiments – evaluating the effective components of probiotics 3.1.1 Preprocessing Acute injury cell specific modeling method: take HepG2 cells with DMEM / F12 medium (10% FBS + high glucose DMEM base medium + 1% double antibody) for conventional culture for 12h, and the culture conditions in the incubator are set as 37℃, 5% CO2, and the culture solution is changed every 2-3 days. When the cell fusion reaches 80%, the subculture can be carried out. Take the logarithmic growth phase of HepG2 cells, use LPS (100 ng / mL) and D-Gal (44 μg / mL) to culture for 24h to construct an acute injury cell model.

[0064] Supernatant preparation process: ferment Lactobacillus muciaparius HG002 to the platform phase, centrifuge (8000 rpm, 5 min) and take the supernatant for subsequent experiments.

[0065] 3.1.2 Grouping method and treatment measures (1) C group: blank control group, HepG2 cells were treated with PBS solution (1% medium volume) for 28h; (2) M group: LPS / D-Gal group, HepG2 cells were pretreated with PBS solution (1% medium volume) for 4h, and then LPS (100 ng / mL) and D-Gal (44 μg / mL) were added to culture HepG2 cells for 24h; (3) T group: LPS / D-Gal+cell group, the cells (10 9 CFU / mL L. fermentum HG002 bacterial solution coating liquid) (1% medium volume) pretreated HepG2 cells for 4h, and then LPS (100 ng / mL) and D-Gal (44 μg / mL) were added to culture HepG2 cells for 24h; (4) S group: LPS / D-Gal+supernatant group, supernatant (1% medium volume) pretreated HepG2 cells for 4h, and then LPS (100 ng / mL) and D-Gal (44 μg / mL) were added to culture HepG2 cells for 24h.

[0066] 3.1.3 Detection index: (1) Observe the growth of cells in each group; (2) Detect the growth and apoptosis index of cells: CCK8 and flow cytometry detect cell apoptosis and reactive oxygen species; (3) Compare the levels of ALT and AST in each group; Take out the well plate, absorb the supernatant, wash the cells with PBS for 2 times, add RIPA cell lysis solution to lyse the cells for 30min, then transfer the suspension to a 1.5mL EP tube, centrifuge for 5min, absorb the supernatant, and use biochemical kit to detect the levels of ALT and AST.

[0067] 3.2 Second round of cell experiment-evaluation of the effectiveness of EVs 3.2.1 Grouping Methods and Handling Measures (1) Group C: Blank control group, treated with 200 μL PBS solution; (2) M group: LPS / D-Gal group, treated with 200μL PBS for 4h, then added LPS (100ng / mL) and D-Gal (44μg / mL) to culture HepG2 cells for 24h; (3) EVs group: LPS / D-Gal+EVs group, given 200μL EVs (5.1×10⁻⁶). 10 HepG2 cells were pretreated with cells / mL for 4 hours, followed by the same procedure. (4) S(-EVs) group: LPS / D-Gal+ supernatant for removing EVs group: HepG2 cells were pretreated with 200 μL of supernatant for removing EVs for 4 h, and the same procedure was followed.

[0068] 3.2.2 Detection indicators: Same as 3.1.3.

[0069] 3.3 Results like Figure 4 As shown, the CCK-8 experiment confirmed the successful construction of this damage model. Figure 4 (A). Subsequently, the present invention separates the bacterial culture into a supernatant and a bacterial precipitate by centrifugation ( ). Figure 4 (B) This invention found that pretreatment of the supernatant of fermented *Lactobacillus mucilaginosus* significantly improved cell viability and reduced ALT and AST levels, while cell precipitation showed no significant effect. Figure 4 (C, D)

[0070] Given that extracellular vesicles (EVs) can deliver a variety of bioactive substances to exert their functions, and that increasing evidence suggests that probiotic-derived EVs (p-EVs) have therapeutic potential in disease models such as influenza A virus infection and lung cancer, this invention hypothesizes that EVs derived from *Lactobacillus fermentans* (p-EVs)... L. fermentum EVs are the key active mediators for their beneficial effects. To verify this hypothesis, this invention first used ultracentrifugation to separate EVs produced by fermenting *Lactobacillus mucilaginosus*, and then characterized them by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). NTA showed a particle concentration of 5.1 × 10⁻⁶. 10 The number of particles per mL was 157.0 nm, with an average particle size of 157.0 nm. Figure 4 (E). TEM showed that the separated EVs exhibited a typical cup-shaped morphology (…). Figure 4 (Middle F). To further assess the uptake of EVs by hepatocytes, EVs were labeled with PKH26 fluorescent dye and co-incubated with HepG2 cells. Confocal microscopy analysis showed that EVs could be efficiently internalized by HepG2 cells ( Figure 4 (G).

[0071] To determine the optimal dosage of EVs for exerting therapeutic effect, the present application pretreated acute injured HepG2 cells with different concentrations of EVs, and the results showed that 200 μg / mL EVs could significantly enhance cell viability. But further increasing the concentration of EVs did not produce additional improvement effect Figure 4 in the middle H). Then the present application compared the effects of 200 μg / mL EVs and EVs-removed probiotic culture supernatant (S(-EV) group) on injured cells. The present application found that EVs pretreatment could significantly reduce the levels of ALT and AST in culture supernatant, while EVs-removed supernatant had no significant effect Figure 4 in the middle I). These findings prove that EVs are the key mediators for fermented M. viscous to exert liver protection function.

[0072] Example 4: Verification of the effect of EVs in alleviating ALF (mouse model verification).

[0073] 4.1 Experimental animals A total of 18 C57BL / 6J male mice aged 6-8 weeks were purchased from Beijing Sibeifu Biotechnology Co., Ltd. The mice were acclimated for 1 week in a specific pathogen-free (SPF) level experimental animal center. The light and dark cycle was maintained at 12 hours of light and 12 hours of darkness, the temperature was maintained at 22°C ± 3°C, the relative humidity was 50% ± 15%, 4-5 mice per cage, free access to food and water, the mice were given a 7-day acclimation period before the experiment, and the temperature, humidity, noise, etc. in the animal room were controlled within the specified range, and the cleaning was done on a daily basis.

[0074] 4.2 Experimental grouping a. Blank group (C group = 6): After 7 days of normal feeding, the mice were given 200 μL / each of sterile PBS liquid by gavage, which lasted for 7 days; b. Model group (M group = 6): On days 1 to 7, 200 μL / each of sterile PBS liquid was given by gavage every day, and on day 8, 10 mL / kg of LPS / D-Gal mixture was injected intraperitoneally to establish the model, and the ALF model was established 5 hours later; c. EVs group (EVs group = 6): On days 1 to 7, 250 μL / each of EVs (5.1 x 10 10 individual / mL) was given by gavage every day, and on day 8, the ALF model was established by the above method; d. EVs+pathway agonist group (EVs+ANI group = 6): On days 1 to 7, 250 μL / each of EVs (5.1 x 10 10On day 8, an ALF model was established using the above method. Two hours before establishing the ALF model, 20 mg / kg of anethole was injected intraperitoneally to activate the JNK / p38 MAPK signaling pathway.

[0075] 4.3 Experimental Procedure Same as 2.3.

[0076] 4.4 Experimental Results like Figure 5 As shown, this invention further evaluated the therapeutic effect of EVs on ALF mice through in vivo experiments. Figure 5 (A) and used JNK / p38 MAPK signaling pathway agonists to verify the importance of the MAPK signaling pathway in the hepatoprotective effect mediated by EVs. Compared with the acute liver failure model group (M group), the serum ALT and AST levels of mice treated with EVs by gavage (EVs group) were significantly reduced. Figure 5 In addition, EV treatment downregulated the expression of IL-6, IL-1β, and TNF-α in mouse serum and liver tissue. Figure 5 Medium CD).

[0077] like Figure 6 As shown, histological analysis further confirmed that EV pretreatment effectively improved liver inflammation and necrosis. All of these effects were reversed by ANI.

[0078] In summary, the results of this study indicate that fermented Lactobacillus mucin-derived EVs can alleviate LPS / D-GalN-induced acute liver failure.

[0079] 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 fermenting *Lactobacillus mucinus*, characterized in that, The strain was named HG002 and deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.36670. The 16S rDNA gene sequence of Lactobacillus fermentans HG002 is shown in SEQ ID NO:

1.

2. The fermented *Lactobacillus mucinus* according to claim 1, characterized in that, The strain has the following characteristics: Physiological and biochemical characteristics: Gram-positive, non-spore-forming, rod-shaped; tolerant of acidic environments (pH 2.0-7.0), and tolerates 0.0%-0.3% bile salts; and / or EV production capacity: After 40 hours of aerobic culture, the EV yield in the fermentation broth is ≥5.0 × 10⁻⁶. 10 The number of EVs per mL is 100-200 nm.

3. The fermented *Lactobacillus mucinus* according to claim 1, characterized in that, The culture conditions for the strain were as follows: MRS medium, aerobic culture at 37°C for 14-24 hours, with the pH maintained at 5.5-6.5 during the culture process.

4. An EV derived from *Lactobacillus fermentans* HG002 as described in claim 1, characterized in that, The EVs were obtained by the following method: Lactobacillus fermentum strain HG002 was cultured aerobically at 37°C for 40 h in MRS medium at a concentration ≥1×10⁻⁶. 9 The culture suspension of CFU / mL was centrifuged at 2000 ×g for 10 min, 5000 ×g for 20 min, and then at 10000 ×g for 30 min to remove bacterial cells. The supernatant was filtered through a 0.22 μm filter membrane and ultraconcentrated at 150000 ×g for 70 min. The precipitate containing EVs was washed with PBS, ultracentrifuged, resuspended in PBS, and stored at -80℃ for later use.

5. The EVs according to claim 4, characterized in that, The EVs have a particle size of 100-200 nm, a zeta potential of -10 to -30 mV, and transmission electron microscopy shows that they have a typical crescent-shaped structure and do not contain intact bacterial cells or bacterial fragments.

6. The use of the fermented *Lactobacillus mucinus* HG002 of claim 1 and / or the EVs of claim 4 in the preparation of a product for alleviating acute liver failure, characterized in that... The product alleviates ALF by regulating the balance of intestinal flora, inhibiting liver inflammation, and reducing hepatocyte apoptosis.

7. The application according to claim 6, characterized in that, The applications include: The effective dose of *Lactobacillus fermentum* HG002 in the intervention of a mouse model of acute liver failure was 1×10⁻⁶. 9 -1×10 11 CFU / kg body weight / day; and / or The effective dose for intervention in a mouse model of acute liver failure using EVs is 200-300 μL / mouse / day, with an EV concentration of 5.1 × 10⁻⁶. 10 per mL.

8. The application according to claim 7, characterized in that, Compared to the control group of mice with acute liver failure who did not receive the drug, intervention with the aforementioned *Lactobacillus mucinus* HG002 reduced serum ALT and AST levels by 31%-52% in the mouse acute liver failure model; intervention with the aforementioned EVs reduced serum ALT and AST levels by 39%-56%; and / or Compared to the control group of mice with acute liver failure who did not receive the drug, the levels of IL-6, IL-1β, and TNF-α in the liver tissue decreased by 40-50% after intervention with the aforementioned fermented Lactobacillus mucinus HG002; and the levels of IL-6, IL-1β, and TNF-α in the liver tissue decreased by 42-60% after intervention with the aforementioned EVs in the mouse acute liver failure model.

9. The application according to claim 6, characterized in that, The acute liver failure was caused by drug-induced liver injury, viral liver injury, or alcoholic liver injury.

10. A product for relieving acute liver failure, characterized in that, The active ingredient of the product is selected from the *Lactobacillus fermentans* HG002 of claim 1 and / or the *EVs* of claim 4; wherein the viable count of the *Lactobacillus fermentans* is ≥1×10⁻⁶. 9 CFU / mL, wherein the EVs concentration is ≥5×10 10 per mL.

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