A strain of Weizmannella coagulans and its application

Abstract

The present invention discloses a strain of Weizmannella coagulans and its application. The classification of Weizmannella coagulans L-B15 is named as Weizmannella coagulans ( Weizmannia coagulans ), strain Weizmannella coagulans ( Weizmannia coagulans Weizmannella coagulans L-B15 was deposited with the General Microbiology Center of the China General Culture Collection Administration (CGMCC) on January 23, 2025, with the deposit number CGMCC No. 33496. The depository address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The Weizmannella coagulans L-B15 and its extracellular vesicles can be used as the sole active ingredient in products that promote wound healing.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and more particularly to a strain of Weizmannella coagulans and its application. Background Art

[0002] Wound healing is a vital physiological process for maintaining tissue integrity and restoring function. In clinical practice, the management of complex wounds, such as chronic wounds, burns, and postoperative wounds, remains challenging. Commonly used treatments include topical antibiotics, growth factor preparations, and physical dressings. However, these approaches have gradually demonstrated their limitations in long-term practice.

[0003] While traditional antibiotic therapy can effectively suppress bacterial infections, its effectiveness has been significantly reduced with the widespread emergence of drug-resistant strains. Clinical data show that the resistance rate of common wound-infecting bacteria, such as Staphylococcus aureus, to β-lactam antibiotics has exceeded 60%, forcing the use of higher doses or broad-spectrum antibiotics during treatment. This not only increases the risk of hepatotoxicity and renal toxicity but also may disrupt the wound microecological balance. Furthermore, antibiotics are unable to directly promote key healing processes, such as epithelial cell migration and angiogenesis, limiting their effectiveness in treating non-infected wounds.

[0004] While recombinant growth factor drugs (such as PDGF and EGF) can directly stimulate cell proliferation, their application has significant drawbacks. These proteins are easily degraded by proteases in wound exudate, with a half-life typically less than six hours, requiring frequent dosing to maintain effective concentrations. Furthermore, industrially produced growth factor preparations require high purity levels, resulting in production costs reaching thousands of dollars per milligram, severely hindering their widespread clinical adoption. Studies have shown that abnormal expression of certain growth factors in chronic wounds may actually promote fibrotic scar formation.

[0005] Based on the above technical limitations, researchers have begun to focus on using extracellular vesicles (EVs) as new therapeutic carriers and applying them in products that promote wound healing. However, the application of extracellular vesicles based on Weizmannella coagulans in promoting wound healing has not yet been reported. Summary of the Invention

[0006] The object of the present invention is to provide a strain of Weizmannella coagulans and its application, so as to at least solve the above problems.

[0007] In order to achieve the purpose and other advantages of the present invention, a strain of Weizmannella coagulans L-B15 is provided, which is classified and named as Weizmannella coagulans ( Weizmannia coagulans ), strain Weizmannella coagulans ( Weizmannia coagulans)L-B15 was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) on January 23, 2025. The deposit number is CGMCC No.33496. The address of the deposit unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The present invention also provides a use of the Weizmannella coagulans in preparing a product for promoting wound healing.

[0009] Preferably, the only active ingredient of the product is the extracellular vesicles of Weizmannella coagulans, and the effective concentration of the extracellular vesicles is 5×10 7 ~5×10 8 particles / ml.

[0010] Preferably, the extracellular vesicles are prepared by the following steps: step 1, inoculating Weizmannella coagulans L-B15 into a liquid culture medium containing 0.5-1.5 g / L yeast extract, 2-4 g / L peptone, 0.3-0.7 g / L sodium chloride and 5-15 g / L glucose, and anaerobically culturing at 45-55°C for 48-72 hours to obtain an initial fermentation broth; step 2, centrifuging the initial fermentation broth at 8000-12000g for 8-15 minutes to remove the bacteria, and collecting the supernatant; filtering the supernatant through a 0.45 μm pore size polyethersulfone membrane and a 0.22 μm pore size polyvinylidene fluoride membrane in sequence; ultracentrifuging the filtered supernatant at 100000-150000g for 60-90 minutes at 4-8°C, and discarding the supernatant to obtain a primary vesicle precipitate; step 3, resuspending the primary vesicle precipitate in a 5% PH value; To a phosphate buffer solution with a final concentration of 7.9-8.5, add a calcium chloride solution with a final concentration of 5-10 m mol / L, and incubate in a water bath at 35-42°C for 20-40 minutes; the incubated mixture is subjected to gradient sucrose density centrifugation, and sucrose solutions with mass fractions of 15%, 30% and 45% are stacked in sequence, and centrifuged at 80,000-100,000g at 4-8°C for 2-4 hours to collect the vesicle components located at the interface of the 30%-45% sucrose layer; step 4, the collected vesicle components are mixed with Tris-HCl buffer containing 0.05-0.2 mol / L trehalose, and repeatedly extruded 3-5 times through a polycarbonate membrane with a pore size of 100-200 nm; the extruded mixture is centrifuged at 100,000-150,000g at 4-8°C. The mixture was ultracentrifuged at speed g for 60-90 minutes, and the precipitate was collected and washed twice with Tris-HCl buffer at pH 7.4-7.6. The precipitate was the extracellular vesicles.

[0011] Preferably, the method for preparing extracellular vesicles further comprises: step five, mixing the washed precipitate with a cryoprotectant containing 0.05-0.2 mol / L trehalose and 0.05-0.1 mol / L mannitol, the mixing step being continuously shaken at 200-400 rpm for 10-20 minutes under ice bath conditions, the aliquoting volume being 0.5-1.5 mL / tube, and using polypropylene cryovials with a wall thickness of 1.2-1.8 mm, after aliquoting, precooling to -40°C at a rate of 1-2°C per minute, and then transferring to -80°C for storage.

[0012] The present invention also provides a product for promoting wound healing, wherein the product contains the extracellular vesicles of Weizmannella coagulans as the only active ingredient, and the effective concentration of the extracellular vesicles is 5×10 7 ~5×10 8 particles / ml.

[0013] The present invention has at least the following beneficial effects:

[0014] First, for the first time, extracellular vesicles from a specific strain of Weizmannella coagulans have been used in a wound healing product. This approach, leveraging natural microbial metabolites to promote healing, can reduce the toxic side effects of synthetic drugs or recombinant proteins, and microbial-derived ingredients are more compatible with the host microenvironment. This application provides a new path for developing antibiotic-independent, microbiome-friendly wound treatment strategies.

[0015] Second, by limiting the product's sole active ingredient to extracellular vesicles and defining their concentration range, therapeutic targeting is significantly improved, reducing the risk of allergic reactions or toxicity during clinical use. By controlling vesicle concentration within a specific range, effective activation of cell migration is ensured while avoiding the excessive inflammatory response that high doses may trigger.

[0016] Third, by combining a specific culture medium formulation with a graded purification process, efficient preparation of high-purity extracellular vesicles was achieved. Anaerobic culture conditions and customized nutrient ratios can induce the strain to secrete specific functional molecules while inhibiting the production of irrelevant metabolites. The combination of multi-stage membrane filtration and gradient centrifugation effectively removes culture medium residues and bacterial debris, significantly improving the purity of the vesicles. Calcium ion-assisted incubation and buffer extrusion treatment optimize the dispersion and membrane integrity of the vesicles, ensuring the activity of the vesicles. This preparation method improves batch-to-batch consistency while ensuring the biological activity of the vesicles, providing a feasible path for industrial production.

[0017] Fourth, by combining the cryoprotectant formula with a programmed cooling process, the technical difficulties in the long-term preservation of microbial vesicles have been solved. The synergistic effect of trehalose and mannitol can form a glassy matrix during the freezing process, reducing the mechanical damage of ice crystals to the vesicle membrane. The specific thickness of the cryotube and the controlled rate pre-cooling design reduce the risk of lipid bilayer phase transition caused by sudden temperature changes and maintain the integrity of the vesicle structure. This preservation method enables the vesicle preparation to be stably stored for several months at -80°C, with an activity loss rate significantly lower than that of conventional freeze-drying methods. At the same time, it avoids the damage to the preparation caused by repeated freezing and thawing, ensuring the reliability of the efficacy during clinical use.

[0018] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a colony morphology diagram of Weizmannella coagulans L-B15;

[0020] Figure 2 This is the Gram stain result of Weizmannella coagulans L-B15;

[0021] Figure 3 is the phylogenetic tree of Weizmannella coagulans L-B15;

[0022] Figure 4 is a transmission electron micrograph of the extracellular vesicles of Weizmannella coagulans L-B15;

[0023] Figure 5 This is a scanning electron micrograph of the process of extracellular vesicle secretion by Weizmannella coagulans L-B15;

[0024] Figure 6 is a graph showing the particle size of extracellular vesicles of Weizmannella flocculata L-B15;

[0025] Figure 7 This figure shows the effect of different concentrations of extracellular vesicles of Weizmannella L-B15 on the viability of fibroblasts;

[0026] Figure 8 This figure shows the effect of different concentrations of extracellular vesicles of Weizmannella L-B15 on the activity of damaged HFFs;

[0027] Figure 9 This is the result of Edu staining of normal HFF cells treated with extracellular vesicles of Weizmannella L-B15 at different concentrations;

[0028] Figure 10 The results of Edu staining of HFF cells damaged by treatment with extracellular vesicles of Weizmannella L-B15 at different concentrations;

[0029] Figure 11 This is the result of the effect of different concentrations of extracellular vesicles coagulating Weizmannella L-B15 on the migration ability of normal HFF cells;

[0030] Figure 12 This is the result of the effect of different concentrations of extracellular vesicles of Weizmannella L-B15 on the migration ability of damaged HFF cells;

[0031] Figure 13 are the fluorescence images of intracellular reactive oxygen species in different treatment groups;

[0032] Figure 14 is the Western Blot detection result of type III collagen in cells of different treatment groups;

[0033] Figure 15 is the statistical result of type III collagen in cells of different treatment groups;

[0034] Figure 16 This is the result of the effect of extracellular vesicles of Weizmannella L-B15 on the self-healing ability of fibroblast scratches;

[0035] Figure 17 are the microscopic observation results of different treatment groups at 0 h and 12 h of incubation;

[0036] Figure 18 is the cell migration rate of different treatment groups after 12 h of culture.

[0037] Deposit of biological materials:

[0038] Weizmannella coagulans L-B15, whose classification is named: Weizmannella coagulans ( Weizmannia coagulans ), strain Weizmannella coagulans (( Weizmannia coagulans )L-B15 was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) on January 23, 2025. The deposit number is CGMCC No.33496. The address of the deposit unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0039] The present invention will be further described in detail below with reference to the embodiments and drawings so that those skilled in the art can implement the invention with reference to the description.

[0040] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0041] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0042] Example 1: Screening and identification of Weizmannella coagulans.

[0043] Weizmannella coagulans L-B15 was isolated from milk powder. First, based on the high temperature resistance of Weizmannella coagulans, non-spore strains were removed from the milk powder suspension under the condition of an 80°C water bath, and then the milk powder suspension was spread on MRS culture medium for culture and screening of strains with high temperature resistance; then, strains with acid production characteristics were screened out from the strains with high temperature resistance, and the strains were spread on 513 solid culture medium for culture and identification of acid-producing strains; the strains screened out with high temperature resistance and acid production characteristics were expanded and cultured, and the spot plate method was used to preliminarily screen strains with antibacterial ability, and strains whose crude protein extracts had antibacterial activity were screened out. The obtained strains were subjected to morphological observation and molecular biological identification, and finally a strain of Weizmannella coagulans was successfully obtained and named Weizmannella coagulans L-B15. The colony morphology of this strain is as follows Figure 1 As shown, it grows well on MRS medium and forms round colonies with a diameter of 2mm-3mm in 36 hours. They are white and shiny, with a moist surface and neat edges. The Gram staining results of this strain are shown in Figure 2 , rod-shaped, arranged singly or in pairs, and is a Gram-positive bacterium. The phylogenetic tree of this strain is shown in Figure 3 , strain L-B15 was located on the Weizmannia coagulans lineage, and the support for this lineage reached 100%.

[0044] After morphological and molecular identification, the strain L-B15 was classified as Weizmannella coagulans ( Weizmannia coagulans ), strain Weizmannella coagulans ( Weizmannia coagulans )L-B15 was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) on January 23, 2025. The deposit number is CGMCC No.33496. The address of the deposit unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0045] Example 2: Preparation of Weizmannella coagulans extracellular vesicles

[0046] The bacterial suspension of Weizmannella coagulans L-B15 was inoculated into liquid MRS culture medium at a ratio of 2:100 and cultured for 24-48 hours to allow the bacteria to secrete extracellular vesicles (EVs) into the culture supernatant. After the culture was completed, the culture supernatant was collected for pre-centrifugation, such as centrifugation at 10,000 g or less for 10-60 minutes. The pre-centrifugation could be repeated once or more (such as centrifugation at 5,000 g for 30 minutes, and then the supernatant was centrifuged at 10,000 g for 30 minutes) to remove insoluble small particles such as bacterial fragments, collect the supernatant, and filter it using a suction filtration device (0.22 μm filter membrane) to obtain the culture supernatant; the culture supernatant was ultracentrifuged at 100,000 g or above for 60-100 minutes, such as at 140,000 g. Centrifuge for 60 minutes. Repeat ultracentrifugation once or more. Discard the supernatant. The resulting pellet is the coagulated Weizmannella extracellular vesicles (H-EVs). Resuspend in PBS buffer to obtain a coagulated Weizmannella extracellular vesicle suspension. Filter the coagulated Weizmannella extracellular vesicle suspension through a 0.22 μm filter before use to obtain a sterile suspension.

[0047] Transmission electron microscopy was used to identify the morphology of Weizmannella coagulans extracellular vesicles (H-EVs). Transmission electron microscopy showed that H-EVs were round or oval vesicles, like trays; under the electron microscope, 50nm-100nm membrane-bound vesicle structures were clearly observed. Figure 4 The process of Weizmann's bacteria secreting extracellular vesicles was observed using a scanning electron microscope. The results showed that on the surface of the bacteria, a bulging membrane structure could be observed, indicating that extracellular vesicles were being secreted outward. Figure 5 The particle size of H-EVs in the suspension was determined using a nanoparticle tracking analyzer. The results showed that the particle size of H-EVs was mainly distributed between 50-200 nm, with fewer impurity peaks and a lower polydispersity index. Figure 6 .

[0048] Example 3: Preparation of coagulated Weizmannella extracellular vesicles.

[0049] A culture of Weizmannella coagulans L-B15 was inoculated at a ratio of 2:100 into a liquid medium containing 1.0 g / L yeast extract, 3.0 g / L peptone, 0.5 g / L sodium chloride, and 10 g / L glucose. The culture was anaerobically cultured at 50°C for 72 h to obtain an initial fermentation broth. The fermentation broth was then centrifuged at 10,000 g for 10 min to remove the bacteria. The supernatant was collected and filtered through a 0.45 μm polyethersulfone membrane and then a 0.22 μm polyvinylidene fluoride membrane. The filtrate was ultracentrifuged at 120,000 g for 75 min at 4°C. The supernatant was discarded to obtain a primary vesicle pellet. The pellet was resuspended in phosphate buffer (pH 8.2), and calcium chloride solution (final concentration 7.5 mmol / L) was added. The pellet was incubated in a 37°C water bath with shaking for 30 min. The pellet was then subjected to sucrose gradient density centrifugation (15%, 30%, and 45% sucrose solutions were stacked sequentially, and the following sequence was slowly injected from the bottom of the centrifuge tube upwards: 45% → 30% → The vesicle fraction at the interface of the 30%-45% sucrose layer was collected after centrifugation at 80,000 g for 3 hours at 4°C. This fraction was mixed with Tris-HCl buffer (pH 7.5) containing 0.1 mol / L trehalose and repeatedly extruded through a 150 nm polycarbonate membrane four times. The extrudate was ultracentrifuged at 120,000 g for 70 minutes at 4°C. The supernatant was discarded, and the pellet was washed twice with Tris-HCl buffer (pH 7.5). Finally, the washed vesicles were mixed with a cryoprotectant containing 0.1 mol / L trehalose and 0.075 mol / L mannitol, shaken at 300 rpm in an ice bath for 15 minutes, and aliquoted into 1.5 mL / tube (1.5 mm wall thickness) polypropylene cryovials. The tubes were precooled to -40°C at a rate of 1.5°C / min and then transferred to -80°C for storage.

[0050] Experimental Example 1:

[0051] Fibroblasts (HFF cells) in the logarithmic growth phase were seeded in 96-well plates at a concentration of 1.0 × 10 4 cells / mL, and H-EVs prepared in Example 2 at different concentrations (0 (blank control), 5×10 9 , 2.5×10 9 , 5×10 8 , 2.5×10 8 and 5×10 7 Particles / mL (particles / mL), each well was set up with 6 replicates. After 24 hours of cell culture, cell viability was detected by CCK-8 assay. The results are shown in Figure 2. Figure 7 The results showed that H-EVs had no toxic effect on HFF cell activity and at low concentrations (10 7 ~10 8particles / mL) can improve cell viability to a certain extent.

[0052] It is known that D-galactose can reduce cell viability in a concentration-dependent manner. In this experiment, HFF cells were induced with 50 mg / mL, which reduced cell viability by about 50%. The protective effect of H-EVs on HFF cells was detected by CCK-8 assay. The results are as follows: Figure 8 As shown in the results, the addition of H-EVs can alleviate the decrease in cell viability caused by D-galactose, and at low concentrations of H-EVs (10 7 ~10 8 particles / mL) showed significant difference compared with the D-galactose treatment group (model group). 8 , 2.5×10 8 and 5×10 7 Particles / mL of H-EVs were used for subsequent studies.

[0053] Experimental Example 2:

[0054] Cell proliferation assay

[0055] 1. Effect of H-EVs on the proliferation of normal HFF cells

[0056] Proliferation experiments were performed to analyze the proliferation capacity of HFF cells treated with H-EVs. The specific experimental steps were as follows: HFF cells in the logarithmic growth phase were seeded in 6-well plates at a concentration of 2 mL / well and a cell density of 1.25×10 5 Cells were cultured at 37°C and 5% CO2 for 24 h until the cells adhered to the wall. The cells were randomly divided into four groups. The specific treatment steps were as follows:

[0057] Blank control group: only culture medium was added;

[0058] Experimental group 1 (5×10 8 particles / mL H-EVs): add 5×10 8 particles / mL of culture medium for H-EVs;

[0059] Experimental group 2 (2.5×10 8 particles / mL H-EVs): add 2.5×10 8 particles / mL of culture medium for H-EVs;

[0060] Experimental group 3 (5×10 7 particles / mL H-EVs): add 5×10 7 particles / mL of H-EVs culture medium.

[0061] To label cells in each treatment group, 2 mL of EdU (10 µM) reagent (Beyotime, C0071S) was added to each well and incubated for 2 hours. After washing three times with PBS, cells were fixed in 4% paraformaldehyde (Dingguo Biotechnology, AR-0211) for 15 minutes, permeabilized with 0.3% Triton X-100 (GenStar, VA11410) for 15 minutes, and then incubated with click reaction reagent for 30 minutes at room temperature in the dark. The click reaction involves the covalent reaction of the acetylene group on EdU with a fluorescently labeled small molecule azide probe (AlexaFluor 488 azide) catalyzed by monovalent copper ions, forming a stable triazole ring. This reaction is very rapid. Through the click reaction, newly synthesized DNA is labeled with the corresponding fluorescent probe, allowing detection of proliferating cells using appropriate fluorescence detection equipment. Finally, cell nuclei were counterstained with 1× Hoechst 33342 reagent. The staining results were observed using a laser confocal microscope system ( Figure 9 ).Depend on Figure 9 It can be seen that H-EVs can promote the proliferation of HFF cells, suggesting that it can promote the proliferation of cells in the wound and thus promote healing during the wound healing process.

[0062] 2. Effect of H-EVs on the proliferation of damaged HFF cells

[0063] HFF cells in the logarithmic growth phase were seeded in 6-well plates at a density of 1.25 × 105 cells / mL at 2 mL / well. The cells were cultured at 37°C and 5% CO2 for 24 h until the cells adhered to the plate. The cells were then randomly divided into five treatment groups. The specific treatment steps were as follows:

[0064] Blank control group: only culture medium was added;

[0065] Model group (D-galactose): 50 mg / mL D-galactose was added to the culture medium and treated for 48 h;

[0066] Experimental group 1 (D-galactose + 5 × 10 8 particles / mL H-EVs): 50 mg / mL D-galactose and 5×10 8 particles / mL of H-EVs; the specific operation was to pre-treat with D-galactose for 24 hours, then add H-EVs, and then continue to culture in the medium containing D-galactose for another 24 hours;

[0067] Experimental group 2 (D-galactose + 2.5 × 10 8particles / mL H-EVs): 50 mg / mL D-galactose and 2.5×10 8 particles / mL of H-EVs; the specific operation was to pre-treat with D-galactose for 24 hours, then add H-EVs, and then continue to culture in the medium containing D-galactose for another 24 hours;

[0068] Experimental group 3 (D-galactose + 5 × 10 7 particles / mL H-EVs): 50 mg / mL D-galactose and 5×10 7 particles / mL of H-EVs; the specific operation was to pre-treat with D-galactose for 24 hours, then add H-EVs, and then continue to culture in the medium containing D-galactose for another 24 hours.

[0069] To label cells in each treatment group, 2 mL of EdU (10 µM) reagent (Beyotime, C0071S) was added to each well and incubated for 2 hours. After washing three times with PBS, cells were fixed in 4% paraformaldehyde (Dingguo Biotechnology, AR-0211) for 15 minutes, permeabilized with 0.3% Triton X-100 (GenStar, VA11410) for 15 minutes, and then incubated with click reaction reagent for 30 minutes at room temperature in the dark. The click reaction involves the covalent reaction of the acetylene group on EdU with a fluorescently labeled small molecule azide probe (AlexaFluor 488 azide) catalyzed by monovalent copper ions, forming a stable triazole ring. This reaction is very rapid. Through the click reaction, newly synthesized DNA is labeled with the corresponding fluorescent probe, allowing detection of proliferating cells using appropriate fluorescence detection equipment. Finally, cell nuclei were counterstained with 1× Hoechst 33342 reagent. The staining results were observed using a laser confocal microscope system ( Figure 10 ).Depend on Figure 10 As can be seen, HFF cells treated with D-galactose showed little cell proliferation. However, after treatment with H-EVs, cell proliferation was observed again, indicating that H-EVs can alleviate the D-galactose-induced decrease in cell proliferation. Therefore, H-EVs may promote wound healing by promoting cell proliferation in chronic, difficult-to-heal wounds.

[0070] Experimental Example 3:

[0071] Cell migration assay

[0072] 1. Effect of H-EVs on the migration ability of normal HFF cells

[0073] Migration experiments were performed to analyze the migration ability of HFF cells treated with H-EVs. The specific experimental steps were as follows: HFF cells (1×10 5 ~5×10 5 Cells / mL) were seeded at 200 μL / well into the upper chamber of a Transwell chamber (containing a polycarbonate membrane). Culture medium containing 15% FBS (fetal bovine serum) was added to the lower chamber and cultured for 24 hours at 37°C and 5% CO2. The experiment was randomly divided into four groups, and the specific treatment steps were as follows:

[0074] Blank control group: serum-free culture medium was added to the upper chamber only;

[0075] Experimental group 1 (5×10 8 particles / mL H-EVs): 5×10 8 particles / mL of serum-free culture medium for H-EVs;

[0076] Experimental group 2 (2.5×10 8 particles / mL H-EVs): 2.5×10 8 particles / mL of serum-free culture medium for H-EVs;

[0077] Experimental group 3 (5×10 7 particles / mL H-EVs): 5×10 7 particles / mL of serum-free culture medium for H-EVs.

[0078] After the experiment, the chamber was removed and the non-migrated cells on the upper chamber membrane were gently wiped with a cotton swab; fixed with 4% paraformaldehyde for 15 minutes and washed with PBS; finally, stained with 0.1% crystal violet for 20 minutes, rinsed with PBS, and dried. The number of migrated cells was observed under a microscope ( Figure 11 ).Depend on Figure 11 It can be seen that H-EVs can promote the migration of HFF cells, suggesting that it can promote the migration of cells at the wound to the damaged site during the wound healing process and thus promote healing.

[0079] 2. Effect of H-EVs on the migration ability of damaged cells

[0080] HFF cells (1×10 5 ~5×10 5Cells / mL (both the model and experimental groups were pre-treated with 50 mg / mL D-galactose for 24 hours) were seeded at 200 μL / well in the upper chamber of a Transwell chamber (containing a polycarbonate membrane). Culture medium containing 15% FBS (fetal bovine serum) was added to the lower chamber and cultured for an additional 24 hours at 37°C and 5% CO2. The experiment was randomly divided into five groups, and the specific treatment steps were as follows:

[0081] Blank control group: serum-free culture medium was added to the upper chamber only;

[0082] Model group: only serum-free culture medium was added to the upper chamber;

[0083] Experimental group 1 (5×10 8 particles / mL H-EVs): 5×10 8 particles / mL of serum-free culture medium for H-EVs;

[0084] Experimental group 2 (2.5×10 8 particles / mL H-EVs): 2.5×10 8 particles / mL of serum-free culture medium for H-EVs;

[0085] Experimental group 3 (5×10 7 particles / mL H-EVs): 5×10 7 particles / mL of serum-free culture medium for H-EVs.

[0086] After the experiment, the chamber was removed and the non-migrated cells on the upper chamber membrane were gently wiped with a cotton swab; fixed with 4% paraformaldehyde for 15 minutes and washed with PBS; finally, stained with 0.1% crystal violet for 20 minutes, rinsed with PBS, and dried. The number of migrated cells was observed under a microscope ( Figure 12 ).Depend on Figure 12 It can be seen that H-EVs can promote the migration of damaged HFF cells, suggesting that they can promote cell migration to the damaged site in chronic difficult-to-heal wounds and thus promote healing and repair.

[0087] Experimental Example 4:

[0088] HFF cells in the logarithmic growth phase were seeded in 6-well plates at 2 mL / well, with a cell concentration of 1.25 × 10 5 Cells were cultured at 37°C, 5% CO2 for 24 h until the cells adhered to the wall and randomly divided into 5 treatment groups. The specific treatment steps are as follows:

[0089] Blank control group: only culture medium was added;

[0090] Model group (D-galactose): 50 mg / mL D-galactose was added to the culture medium and treated for 48 h;

[0091] Experimental group 1 (D-galactose + 5 × 10 8 particles / mL H-EVs): 50 mg / mL D-galactose and 5×10 8 particles / mL of H-EVs; the specific operation was to pre-treat with D-galactose for 24 hours, then add H-EVs, and then continue to culture in the medium containing D-galactose for another 24 hours;

[0092] Experimental group 2 (D-galactose + 2.5 × 10 8 particles / mL H-EVs): 50 mg / mL D-galactose and 2.5×10 8 particles / mL of H-EVs; the specific operation was to pre-treat with D-galactose for 24 hours, then add H-EVs, and then continue to culture in the medium containing D-galactose for another 24 hours;

[0093] Experimental group 3 (D-galactose + 5 × 10 7 particles / mL H-EVs): 50 mg / mL D-galactose and 5×10 7 particles / mL of H-EVs; the specific operation was to pre-treat with D-galactose for 24 hours, then add H-EVs, and then continue to culture in the medium containing D-galactose for another 24 hours.

[0094] Each of the above treatment groups was subjected to the following tests.

[0095] 1. ROS determination

[0096] Serum-free culture medium containing 10 μM DCFH-DA was added to each well of the above treatment groups for ROS detection. The cells were then observed using an inverted fluorescence microscope. The results showed that treatment with different concentrations of H-EVs reduced the level of reactive oxygen species (ROS) induced by D-galactose, indicating that H-EVs can significantly improve the level of reactive oxygen species in the wound, facilitate the transition of chronic wounds from the inflammatory phase to the proliferative phase, and promote healing. Figure 13 shown.

[0097] 2. Western Blot

[0098] (1) Total protein extraction: Take an appropriate amount of sample from each treatment group, add an equal volume of RIPA lysis buffer (RIPA: PMSF: phosphatase inhibitor = 100:1:1), pipette to mix, and place on ice for 30 min. Oscillate on a vortex mixer for 30 s every 10 min. Centrifuge at 12,000 g for 10 min at 4°C. Transfer the supernatant to a new EP tube to obtain the total protein product.

[0099] (2) Protein quantification: Quantify the extracted total protein according to the instructions of the BCA kit.

[0100] (3) Protein blotting: Prepare separation gel (lower layer) of different concentrations according to the molecular weight of the protein. When the separation gel has not solidified, quickly add the concentrated gel (upper layer) and quickly insert it into the electrophoresis chamber. Let it stand for 25 to 40 minutes to fully solidify. Pull out the comb, fix the glass plate on the gel making rack and put it into the electrophoresis tank. Add electrophoresis buffer to immerse the gel and load the sample in equal parts. According to the instructions of the electrophoresis buffer, wait for the maker to run and the sample to migrate to the bottom of the gel before the electrophoresis is stopped. Cut the gel, activate the PVDF membrane with methanol in advance for 1 to 2 minutes, soak the filter paper in the transfer solution, and wet transfer at a constant current of 400mA for 25 to 30 minutes (determine the transfer time according to the protein molecular weight): Place the transferred PVDF membrane in a fast blocking solution and shake it at room temperature for 5 to 15 minutes; after blocking, wash the membrane with PBS (PBS). The membrane was washed with PBST / TBST for 5 times, each time for 5 minutes, and after absorbing the water, it was placed in the corresponding protein primary antibody at 4°C overnight; the membrane incubated with the primary antibody was removed and washed with PBST / TBST for 5 times, each time for 5 minutes, and after absorbing the water, it was placed in the corresponding secondary antibody and incubated on a shaker at room temperature for 45 minutes; after the secondary antibody incubation was completed, the membrane was washed with PBST / TBST for 5 times, each time for 5 minutes, and after absorbing the water with absorbent paper, the membrane was evenly covered with luminescent liquid, and the gel imaging system was used for imaging and ImageJ software for image acquisition and grayscale analysis. The results are shown in Figure 2. Figures 14 and 15 shown.

[0101] according to Figure 14 and Figure 15 It can be seen that the level of type III collagen in the model group was significantly reduced compared with the control group. 8 Particles / mL H-EVs treatment promoted the expression of type III collagen in D-galactose-induced cells. Therefore, H-EVs can promote wound healing by promoting the remodeling of the extracellular matrix.

[0102] Experimental Example 5:

[0103] Cell wound scratch assay

[0104] 1. Effect of H-EVs on the scratch self-healing ability of normal HFF cells

[0105] The scratch test was performed to analyze the migration ability of HFF cells treated with H-EVs. The specific steps were as follows: HFF cells in the logarithmic growth phase were cultured at 2.5×10 5 Cells were seeded into 6-well plates at a concentration of 10 cells / well. After 24 hours of culture, cells were scratched perpendicular to the plate using a 200 μL pipette tip, with each scratch being as uniform in width as possible. Each well was then washed twice with serum-free medium to remove cell debris and randomly assigned to four treatment groups. The specific treatment steps were as follows:

[0106] Blank control group: only serum-free culture medium was added;

[0107] Experimental group 1 (5×10 8 particles / mL H-EVs): add 5×10 8 particles / mL of serum-free culture medium for H-EVs;

[0108] Experimental group 2 (2.5×10 8 particles / mL H-EVs): add 2.5×10 8 particles / mL of serum-free culture medium for H-EVs;

[0109] Experimental group 3 (5×10 7 particles / mL H-EVs): add 5×10 7 particles / mL of serum-free culture medium for H-EVs.

[0110] After 24 hours of culture, the same scratch position was photographed to observe the healing conditions of different groups. Figure 16 shown.

[0111] Depend on Figure 16 It can be seen that H-EVs can effectively promote the self-healing ability of cells after scratches, suggesting that H-EVs can effectively promote the repair of skin fibroblasts after damage, thereby promoting wound healing.

[0112] 2. Effect of H-EVs on the self-healing ability of damaged HFF cells

[0113] The scratch test was performed to analyze the migration ability of HFF cells treated with H-EVs. The specific steps were as follows: HFF cells in the logarithmic growth phase were cultured at 2.5×10 5 Cells were seeded into 6-well plates at a concentration of 10 cells / well. After 24 hours of culture, cells were scratched perpendicular to the plate using a 200 μL pipette tip, with each scratch being as uniform in width as possible. Each well was then washed twice with serum-free medium to remove cell debris and randomly assigned to five treatment groups. The specific treatment steps were as follows:

[0114] Blank control group: only culture medium containing 1% serum was added;

[0115] Model group (D-galactose): medium containing 50 mg / mL D-galactose and 1% serum was added;

[0116] Experimental group 1 (D-galactose + 5 × 10 8 particles / mL H-EVs): 50 mg / mL D-galactose and 5 × 10 8 particles / mL of H-EVs in culture medium containing 1% serum;

[0117] Experimental group 2 (D-galactose + 2.5 × 10 8 particles / mL H-EVs): 50 mg / mL D-galactose and 2.5×10 8 particles / mL of H-EVs in culture medium containing 1% serum;

[0118] Experimental group 3 (D-galactose + 5 × 10 7 particles / mL H-EVs): 50 mg / mL D-galactose and 5 × 10 7 particles / mL of H-EVs in culture medium containing 1% serum.

[0119] After 12 h of treatment in each treatment group, cell migration was observed using an inverted fluorescence microscope, and the scratch area was analyzed using ImageJ software. The cell migration rate was calculated according to the following formula and normalized with the results of the control group. The results are shown in Figure 2. Figure 17 and Figure 18 shown.

[0120] Cell migration rate = (scratch area at 0 h - scratch area after culture) / scratch area at 0 h

[0121] from Figure 17 and Figure 18 It can be seen that the migration rate of the D-galactose-induced model group is lower than that of the control group. After adding different concentrations of H-EVs, it can effectively promote the migration of damaged cells and help them play a role in promoting healing in chronic difficult-to-heal wounds.

[0122] Experimental Example 6:

[0123] Extracellular vesicles promote mouse skin wound healing experiment

[0124] 1. Experimental plan

[0125] 1.1 Experimental animals and groups

[0126] Healthy male C57BL / 6 mice (8 weeks old, weighing 20-25 g) were randomly divided into 3 groups (n=8 / group):

[0127] Control group: local application of normal saline;

[0128] Example 2 group: The extracellular vesicles prepared in Example 2 (2.5×10 8 particles / mL);

[0129] Example 3 group: Topically applied the extracellular vesicles prepared in Example 3 (2.5×10 8 particles / mL).

[0130] 1.2 Wound model establishment

[0131] After the mice were anesthetized, their backs were shaved and disinfected, and full-thickness skin incisions were made using a 6 mm diameter biopsy punch.

[0132] 1.3 Administration

[0133] Each group was smeared with 100 μL of the corresponding solution (normal saline or vesicle suspension) once a day for 14 consecutive days.

[0134] 1.4 Observation indicators and detection methods

[0135] Wound healing rate: Wound area was recorded daily and the healing rate was calculated using ImageJ software.

[0136] Healing rate = [initial area - remaining area] / initial area × 100%

[0137] Histopathological analysis: Mice were sacrificed on the 7th and 14th days, and wound tissues were taken for:

[0138] HE staining: observe epidermal regeneration and granulation tissue thickness;

[0139] Masson staining: detect the amount of collagen deposition.

[0140] Inflammatory factor detection: Wound tissue homogenate was obtained on the third day, and TNF-α and IL-6 levels were determined by ELISA.

[0141] 2. Experimental results and analysis

[0142] 2.1 Dynamic changes in wound healing rate

[0143] See the table below for the results.

[0144]

[0145] In the table, Compared with the control group, p < 0.05, = indicates p<0.01 compared with the control group; # indicates p<0.05 compared with the Example 2 group; the same below.

[0146] As can be seen from the above table, the healing rate of Example 3 group was significantly better than that of Example 2 group and the control group at each time point (p<0.05), indicating that the extracellular vesicles of Example 3 group can accelerate wound closure, which may be related to the more complete structure and better activity of the extracellular vesicles of Example 3 group.

[0147] 2.2 Histopathological analysis

[0148] HE staining: On the 7th day, the epidermis of the Example 3 group was completely regenerated, and the thickness of the granulation tissue was 520±45 μm, which was significantly higher than the granulation tissue thickness of 420±38 μm in the Example 2 group and 300±25 μm in the control group.

[0149] Masson staining: On the 14th day, the collagen deposition amount in the Example 3 group was 78.5±4.2%, which was significantly higher than the collagen deposition amount in the Example 2 group (65.3±3.8%) and the collagen deposition amount in the control group (45.1±3.5%).

[0150] It can be seen that the granulation tissue thickness and collagen deposition amount of Example 3 group are higher, indicating that its preparation method optimizes the biological activity of vesicles and promotes the reconstruction of extracellular matrix.

[0151] 2.3 Inflammatory factor levels

[0152] See the table below for the results.

[0153]

[0154] As can be seen from the table above, the levels of TNF-α and IL-6 in the Example 3 group were the lowest, suggesting that its vesicles can improve the healing microenvironment through more efficient anti-inflammatory effects.

[0155] 3. Conclusion

[0156] The extracellular vesicles prepared in Example 3 were purified by fractionation, incubated with calcium ions, and optimized with cryoprotectants, significantly improving their purity, stability, and bioactivity. Compared to Example 2, the vesicles in Example 3 performed significantly better in promoting epidermal regeneration, collagen deposition, and inhibiting inflammatory responses (p < 0.05), demonstrating that improved preparation technology plays a key role in enhancing wound healing efficacy.

[0157] The number of equipment and process scales described herein are intended to simplify the description of the invention. Applications, modifications, and variations of the Weizmannella coagulans and its uses will be readily apparent to those skilled in the art.

[0158] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. Weizmannella coagulans L-B15, characterized in that Its classification name is: Weizmannella coagulans Weizmannia coagulans ), strain Weizmannella coagulans ( Weizmannia coagulans )L-B15 was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) on January 23, 2025. The deposit number is CGMCC No.33496. The address of the deposit unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The use of Weizmannella coagulans in the preparation of a product for promoting wound healing according to claim 1, characterized in that: The only active ingredient of the product is the extracellular vesicles of the coagulant Weizmannella, and the effective concentration of the extracellular vesicles is 5×10 7 ~5×10 8 particles / ml; The extracellular vesicles are prepared by the following steps: Step 1, inoculating Weizmannella coagulans L-B15 into a liquid culture medium containing 0.5-1.5 g / L yeast extract, 2-4 g / L peptone, 0.3-0.7 g / L sodium chloride and 5-15 g / L glucose, and anaerobically culturing at 45-55° C. for 48-72 hours to obtain an initial fermentation broth; Step 2: Centrifuge the initial fermentation broth at 8000-12000g for 8-15 minutes to remove the bacteria and collect the supernatant; filter the supernatant sequentially through a 0.45 μm pore size polyethersulfone membrane and a 0.22 μm pore size polyvinylidene fluoride membrane; ultracentrifuge the filtered supernatant at 100,000-150,000g for 60-90 minutes at 4-8°C, and discard the supernatant to obtain a primary vesicle precipitate; Step 3: Resuspend the primary vesicle pellet in phosphate buffer (pH 7.9-8.5), add calcium chloride solution with a final concentration of 5-10 mmol / L, and incubate in a 35-42°C water bath with shaking for 20-40 minutes; subject the incubated mixture to gradient sucrose density centrifugation, stacking sucrose solutions with mass fractions of 15%, 30%, and 45% in sequence, and centrifuge at 80,000-100,000 g at 4-8°C for 2-4 hours to collect the vesicle components at the interface of the 30%-45% sucrose layer; Step 4: The collected vesicle fraction is mixed with Tris-HCl buffer containing 0.05-0.2 mol / L trehalose, and repeatedly extruded through a polycarbonate membrane with a pore size of 100-200 nm for 3-5 times; the extruded mixture is ultracentrifuged at 100,000-150,000 g for 60-90 minutes at 4-8°C, and the precipitate is collected and washed twice with Tris-HCl buffer with a pH of 7.4-7.

6. The precipitate is the extracellular vesicle; Step 5: Mix the washed precipitate with a cryoprotectant containing 0.05-0.2 mol / L trehalose and 0.05-0.1 mol / L mannitol. The mixing step is continuously shaken at 200-400 rpm for 10-20 minutes under ice bath conditions. The aliquot volume is 0.5-1.5 mL / tube, and polypropylene cryovials with a wall thickness of 1.2-1.8 mm are used. After aliquoting, precool to -40°C at a rate of 1-2°C per minute, and then transferred to -80°C for storage.

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