Witzia ciliaris and application thereof

Abstract

The invention discloses a strain of Weizmannia coagulans and application thereof, the classification name of the strain of Weizmannia coagulans L-B15 is Weizmannia coagulans L-B15, the strain of Weizmannia coagulans L-B15 is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation time is January 23, 2025, the preservation number is CGMCC No.33496, and the preservation number is CGMCC No.33496. The strain of Weizmannia coagulans L-B15 is named as Weizmannia coagulans L-B15. The preservation unit address of the bacillus coagulans L-B15 is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and application of the bacillus coagulans L-B15. The extracellular vesicles of the Witzia ciliaris L-B15 disclosed by the invention can be used as a unique active ingredient to be applied to a product for promoting wound healing.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms. More specifically, the present invention relates to a strain of Weissella coagulans and its applications. Background Art

[0002] Wound healing is an important physiological process for organisms to maintain tissue integrity and functional recovery. In clinical practice, the treatment of complex wounds such as chronic wounds, burns, and postoperative wounds still faces many challenges. Commonly used treatment methods in the prior art mainly include local antibiotic application, growth factor preparations, and physical dressings, but these methods have gradually revealed technical limitations in long-term practice.

[0003] Although traditional antibiotic treatment can effectively inhibit bacterial infections, with the widespread emergence of drug-resistant strains, its treatment effect has been significantly reduced. Clinical data show that the drug resistance rate of common wound infection bacteria such as Staphylococcus aureus to β-lactam antibiotics has exceeded 60%, forcing the use of higher doses or broad-spectrum antibiotics in treatment, which not only increases the risk of liver and kidney toxicity but also may disrupt the wound microecological balance. In addition, antibiotics cannot directly promote key healing processes such as epithelial cell migration and angiogenesis, resulting in limited effects in the treatment of non-infected wounds.

[0004] Although recombinant growth factor drugs (such as PDGF, EGF) can directly stimulate cell proliferation, their applications have obvious defects. These protein molecules are easily degraded by proteases in the wound exudate environment, with a half-life usually less than 6 hours, and frequent administration is required to maintain effective concentrations. More critically, the purity requirements for industrially produced growth factor preparations are high, and the production cost reaches thousands of dollars per milligram, severely restricting clinical popularization. Some studies have pointed out that the abnormal expression of certain growth factors in chronic wounds may even promote the formation of fibrotic scars.

[0005] Based on the above technical limitations, researchers have begun to focus on using extracellular vesicles (EVs) as a new type of therapeutic carrier and applying them in products for promoting wound healing. However, there is currently no report on the application of extracellular vesicles based on Weissella coagulans in promoting wound healing. Summary of the Invention

[0006] The purpose of the present invention is to provide a strain of Weissella coagulans and its applications to at least solve the above problems.

[0007] To achieve the purpose and other advantages of the present invention, a strain of Weissella coagulans L-B15 is provided, and its taxonomic name is: Weissella coagulans ( Weizmannia coagulans ), strain Weissella coagulans ( Weizmannia coagulans)L-B15 was deposited in the China General Microbiological Culture Collection Center (CGMCC) on January 23, 2025, with the deposit number CGMCC No. 33496, and the address of the deposit unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The present invention also provides an application of the above-mentioned Weizmannia coagulans in the preparation of a product for promoting wound healing.

[0009] Preferably, the only active ingredient of the product is the extracellular vesicles of Weizmannia 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, inoculate Weizmannia coagulans L-B15 into a liquid 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 perform anaerobic culture at 45-55 °C for 48-72 hours to obtain an initial fermentation broth; Step 2, centrifuge the initial fermentation broth at 8000-12000 g for 8-15 minutes to remove the bacteria, and collect the supernatant; filter the supernatant through a polyethersulfone membrane with a pore size of 0.45 μm and a polyvinylidene fluoride membrane with a pore size of 0.22 μm in sequence; ultracentrifuge the filtered supernatant at 100000-150000 g for 60-90 minutes at 4-8 °C, discard the supernatant, and obtain a primary vesicle precipitate; Step 3, resuspend the primary vesicle precipitate in a phosphate buffer solution with a pH of 7.9-8.5, add a calcium chloride solution with a final concentration of 5-10 mmol / L, and incubate with shaking in a water bath at 35-42 °C for 20-40 minutes; perform gradient sucrose density centrifugation on the incubated mixture, sequentially stack sucrose solutions with mass fractions of 15%, 30%, and 45%, and centrifuge at 80000-100000 g for 2-4 hours at 4-8 °C, and collect the vesicle fraction at the interface of the 30%-45% sucrose layer; Step 4, mix the collected vesicle fraction with a Tris-HCl buffer solution containing 0.05-0.2 mol / L trehalose, and extrude it repeatedly through a polycarbonate membrane with a pore size of 100-200 nm for 3-5 times; ultracentrifuge the extruded mixture at 100000-150000 g for 60-90 minutes at 4-8 °C, collect the precipitate and wash it twice with a Tris-HCl buffer solution with a pH of 7.4-7.6, and the precipitate is the extracellular vesicles.

[0011] Preferably, the method for preparing the 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 is continuously shaken at 200 - 400 rpm for 10 - 20 minutes under ice bath conditions, aliquoted into tubes with a volume of 0.5 - 1.5 mL / tube, and polypropylene cryotubes with a wall thickness of 1.2 - 1.8 mm are used. After aliquoting, it is pre-cooled to -40°C at a rate of 1 - 2°C per minute, and then transferred to -80°C for storage.

[0012] The present invention also provides a product for promoting wound healing, wherein the product uses the extracellular vesicles of the above-mentioned Weissella coagulans as the sole 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: First, for the first time, the extracellular vesicles of a specific strain of Weissella coagulans are applied in a product for promoting wound healing. Using natural metabolites of microorganisms to promote healing can reduce the toxic and side effects of chemically synthesized drugs or recombinant proteins, and components derived from microorganisms are more easily compatible with the host microenvironment. This application direction provides a new path for the development of non-antibiotic-dependent and microecology-friendly wound treatment strategies.

[0014] Second, by defining the sole active ingredient of the product as extracellular vesicles and clarifying its concentration range, the treatment targeting is significantly improved, and the risk of allergy or toxicity during clinical application is reduced. By controlling the vesicle concentration within a specific range, it can not only ensure the effective activation of cell migration but also avoid the excessive inflammatory response that may be caused by high doses.

[0015] Third, through the combination of a specific culture medium formula and a fractional purification process, the efficient preparation of high-purity extracellular vesicles is achieved. Anaerobic culture conditions and customized nutrient ratios can induce the strain to secrete specific functional molecules while inhibiting the generation of irrelevant metabolites. The combined technology of multi-stage membrane filtration and gradient centrifugation effectively removes culture medium residues and bacterial debris, significantly improving the vesicle purity. Calcium ion-assisted incubation and buffer extrusion treatment optimize the vesicle dispersion and membrane integrity, ensuring the activity of the vesicles. This preparation method improves the batch-to-batch consistency while ensuring the biological activity of the vesicles, providing a feasible path for industrial production.

[0016] Fourthly, by combining the cryoprotectant formulation with the programmed cooling process, the technical problem of long-term preservation of microbial vesicles is solved. The synergistic effect of trehalose and mannitol can form a vitreous matrix during freezing, reducing the mechanical damage of ice crystals to the vesicle membrane. The cryotube with a specific thickness and the controlled-rate precooling design reduce the risk of phase transition of the lipid bilayer caused by sudden temperature changes and maintain the integrity of the vesicle structure. This preservation method enables the vesicle preparation to be stably stored at -80°C for several months, with a significantly lower activity loss rate than the conventional freeze-drying method. At the same time, it avoids the damage to the preparation caused by repeated freezing and thawing, ensuring the reliability of the therapeutic effect during clinical use.

[0017] Other advantages, objectives, and features of the present invention will be partially reflected by the following description and partially understood by those skilled in the art through the research and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the colony morphology diagram of Weissella coagulans L-B15; Figure 2 is the Gram staining result of Weissella coagulans L-B15; Figure 3 is the phylogenetic tree of Weissella coagulans L-B15; Figure 4 is the transmission electron microscopy image of the extracellular vesicles of Weissella coagulans L-B15; Figure 5 is the scanning electron microscopy image of the process of extracellular vesicle secretion by Weissella coagulans L-B15; Figure 6 is the particle size diagram of the extracellular vesicles of Weissella coagulans L-B15; Figure 7 is the result diagram of the effect of extracellular vesicles of Weissella coagulans L-B15 at different concentrations on the viability of fibroblasts; Figure 8 is the result diagram of the effect of extracellular vesicles of Weissella coagulans L-B15 at different concentrations on the viability of damaged HFF; Figure 9 is the result of Edu staining of normal HFF cells treated with extracellular vesicles of Weissella coagulans L-B15 at different concentrations; Figure 10 is the result of Edu staining of damaged HFF cells treated with extracellular vesicles of Weissella coagulans L-B15 at different concentrations; Figure 11 is the result of the effect of extracellular vesicles of Weissella coagulans L-B15 at different concentrations on the migration ability of normal HFF cells; Figure 12 is the result of the effect of extracellular vesicles of Weissella coagulans L-B15 at different concentrations on the migration ability of damaged HFF cells; Figure 13 are fluorescence images of intracellular reactive oxygen species in cells of different treatment groups; Figure 14 are the Western Blot detection results of type III collagen in cells of different treatment groups; Figure 15 are the statistical results of type III collagen in cells of different treatment groups; Figure 16 are the results of the effect of extracellular vesicles of Weissella coagulans L-B15 on the scratch self-healing ability of fibroblasts; Figure 17 are the microscopic observation results of different treatment groups incubated for 0 h and 12 h; Figure 18 are the cell migration rates of different treatment groups cultured for 12 h.

[0019] Biomaterial preservation: Weissella coagulans L-B15, whose taxonomic name is: Weissella coagulans( Weizmannia coagulans ), strain Weissella coagulans(( Weizmannia coagulans )L-B15 was deposited in the China General Microbiological Culture Collection Center (CGMCC) on January 23, 2025, with the deposit number CGMCC No. 33496, and the address of the deposit unit is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Detailed implementation manners

[0020] The present invention will be further described in detail below in conjunction with the embodiments and the drawings, so that those skilled in the art can implement it according to the description in the specification.

[0021] It should be understood that the terms such as "having", "comprising" and "including" used herein do not exclude the presence or addition of one or more other elements or their combinations.

[0022] It should be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial sources unless otherwise specified.

[0023] Example 1: Screening and identification of Weissella coagulans.

[0024] Weizmannia coagulans L-B15 was isolated from milk powder. First, according to the high-temperature tolerance characteristic of Weizmannia coagulans, non-spore strains in the milk powder suspension were removed under the condition of 80 °C water bath. Then, the milk powder suspension was spread on MRS medium for cultivation, and strains with high-temperature tolerance characteristics were screened out. Then, strains with acid-producing characteristics were screened out from the strains with high-temperature tolerance characteristics, and the strains were spread on 513 solid medium for cultivation to identify acid-producing strains. Then, the strains with high-temperature tolerance and acid-producing characteristics screened out were expanded in cultivation, and the spot plate method was used to preliminarily screen strains with antibacterial ability, and strains with antibacterial activity in their crude protein extracts were screened out from them. The obtained strains were subjected to morphological observation and molecular biological identification, and finally 1 strain of Weizmannia coagulans was successfully obtained and named Weizmannia coagulans L-B15. The colony morphology of this strain is as Figure 1 shown. It grows well on MRS medium and forms circular colonies with a diameter of 2 mm - 3 mm in 36 h, which are white, shiny, with a moist surface and neat edges. The Gram staining result of this strain is shown in Figure 2 . Its morphology is rod-shaped, arranged singly or in pairs, and it is a Gram-positive bacterium. The phylogenetic tree of this strain is shown in Figure 3 . Strain L-B15 is located on the Weizmannia coagulans lineage, and the support degree of this lineage reaches 100%.

[0025] After morphological and molecular identification, the taxonomic naming of strain L-B15 is: Weizmannia coagulans ( Weizmannia coagulans ), and strain Weizmannia coagulans ( Weizmannia coagulans ) L-B15 was deposited in the China General Microbiological Culture Collection Center (CGMCC). The deposit time is: January 23, 2025, and the deposit number is: CGMCC No. 33496. The address of the deposit unit is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0026] Example 2: Preparation of extracellular vesicles of Weizmannia coagulans Inoculate the bacterial solution of Weissella coagulans L-B15 at a ratio of 2:100 into a liquid MRS medium, and continue culturing for 24 - 48 h to allow the bacteria to secrete extracellular vesicles (EVs) into the culture supernatant. After the culture is completed, collect the culture supernatant and perform pre-centrifugation, such as centrifuging at 10000 g or below for 10 - 60 min, and the pre-centrifugation can be repeated once or more (such as centrifuging at 5000 g for 30 min first, and then centrifuging the supernatant at 10000 g for 30 min) to remove insoluble small particles such as bacterial fragments. Collect the supernatant and filter it using a filtration device (0.22 μm filter membrane) to obtain the culture supernatant; subject the culture supernatant to ultracentrifugation at 100000 g or above for 60 - 100 min, such as centrifuging at 140000 g for 60 min, and the ultracentrifugation can be repeated once or more. Discard the supernatant, and the obtained precipitate is the extracellular vesicles of Weissella coagulans (H-EVs); resuspend it with PBS buffer to obtain a suspension of extracellular vesicles of Weissella coagulans. Before use, the suspension of extracellular vesicles of Weissella coagulans can be filtered through a 0.22 μm filter head to obtain a sterile suspension.

[0027] Perform morphological identification of the extracellular vesicles of Weissella coagulans (H-EVs) using a transmission electron microscope. It can be seen from the observation of the transmission electron microscope that H-EVs are round or oval vesicles, such as tray-shaped; under the electron microscope, a vesicle structure with a membrane structure of 50 nm - 100 nm can be clearly observed. The results are shown in Figure 4 ; observe the process of the secretion of extracellular vesicles by Weissella coagulans using a scanning electron microscope. The results show that on the surface of the bacterial cells, a protruding membrane structure can be observed, which is the extracellular vesicles secreting outward. The results are shown in Figure 5 . Measure the particle size of H-EVs in the suspension using a nanoparticle tracking analyzer. The results show that the particle size of H-EVs is mainly distributed between 50 - 200 nm, and it can be found that there are fewer miscellaneous peaks and a lower polydispersity index. The results are shown in Figure 6 .

[0028] Example 3: Preparation of extracellular vesicles of Weissella coagulans.

[0029] The bacterial solution of Weissella coagulans L-B15 was inoculated 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 at a ratio of 2:100, and cultured under anaerobic conditions at 50 °C for 72 hours to obtain an initial fermentation broth; subsequently, the fermentation broth was centrifuged at 10000 g for 10 minutes to remove the bacteria, the supernatant was collected and filtered successively through a 0.45 μm polyethersulfone membrane and a 0.22 μm polyvinylidene fluoride membrane, and the filtrate was ultracentrifuged at 120000 g for 75 minutes at 4 °C. After discarding the supernatant, a primary vesicle precipitate was obtained; the precipitate was resuspended in a phosphate buffer solution at pH 8.2, a calcium chloride solution with a final concentration of 7.5 mmol / L was added, and the mixture was incubated with shaking in a water bath at 37 °C for 30 minutes. Subsequently, gradient sucrose density centrifugation was performed (successively overlaying 15%, 30%, and 45% sucrose solutions, and slowly injecting from the bottom of the centrifuge tube in the order of 45% → 30% → 15% sucrose solution, with a volume ratio of 1:2:1 for each layer), and the vesicle fraction at the 30%-45% sucrose layer interface was collected after centrifugation at 80000 g for 3 hours at 4 °C; this fraction was mixed with a Tris-HCl buffer solution (pH 7.5) containing 0.1 mol / L trehalose and extruded through a 150 nm polycarbonate membrane 4 times. The extruded solution was ultracentrifuged at 120000 g for 70 minutes at 4 °C, and the precipitate was washed twice with a Tris-HCl buffer solution at pH 7.5 after discarding the supernatant; 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 for 15 minutes under ice bath conditions, aliquoted into 1.5 mL / tube polypropylene cryogenic storage tubes with a wall thickness of 1.5 mm, pre-cooled to -40 °C at a rate of 1.5 °C / minute, and then transferred to -80 °C for storage.

[0030] Experimental Example 1: Logarithmic-phase fibroblasts (HFF cells) were inoculated into a 96-well plate at 100 μL / well, and the cell concentration was 1.0×10 4 cells / mL. Different concentrations of H-EVs prepared in Example 2 (0 (blank control), 5×10 9 , 2.5×10 9 , 5×10 8 , 2.5×10 8 and 5×10 7 particles / mL) were added to the cell culture wells, and 6 replicate wells were set for each well. After culturing the cells for 24 h, the cell viability was detected by the CCK-8 assay, and the results are as Figure 7 shown. The results showed that H-EVs did not exhibit a toxic effect on the activity of HFF cells, and at low concentrations (10 7 ~10 8(particles / mL) can improve cell viability to a certain extent.

[0031] It is known that D-galactose can reduce cell activity in a concentration-dependent manner. In this experiment, HFF cells were induced with 50 mg / mL, causing the cell activity to decrease by approximately 50%. The protective effect of H-EVs on HFF cells was detected by the CCK-8 method, and the results are as Figure 8 shown. The addition of H-EVs can alleviate the decrease in cell viability caused by D-galactose, and there is a significant difference compared with the D-galactose treatment group (model group) under the treatment of low-concentration H-EVs (10 7 ~10 8 (particles / mL). Therefore, 5×10 8 , 2.5×10 8 and 5×10 7 particles / mL of H-EVs were used for subsequent studies.

[0032] Experimental Example 2: Cell Proliferation Experiment 1. Effect of H-EVs on the proliferation ability of normal HFF cells A proliferation experiment was conducted to analyze the proliferation ability of H-EVs-treated HFF cells. The specific experimental steps were as follows: HFF cells in the logarithmic growth phase were inoculated in a 6-well plate at 2 mL / well, with a cell concentration of 1.25×10 5 cells / mL. They were cultured at 37°C and 5% CO2 for 24 h until the cells adhered to the wall. Then they were randomly divided into four groups, and the specific treatment steps were as follows: Blank control group: Only medium was added; Experimental group 1 (5×10 8 (particles / mL H-EVs)): Medium containing 5×10 8 (particles / mL of H-EVs) was added; Experimental group 2 (2.5×10 8 (particles / mL H-EVs)): Medium containing 2.5×10 8 (particles / mL of H-EVs) was added; Experimental group 3 (5×10 7 (particles / mL H-EVs)): Medium containing 5×10 7 (particles / mL of H-EVs) was added.

[0033] Add 2 mL of EdU (10 µM) reagent (Beyotime, C0071S) to each well of the above-mentioned cell treatment groups and incubate for 2 h to label the cells. After washing three times with PBS, the cells are fixed in 4% paraformaldehyde solution (Dingguo Biotech, AR-0211) for 15 minutes, permeabilized with 0.3% Triton X-100 (GenStar, VA11410) for 15 minutes, and then incubated with the click reaction reagent in the dark at room temperature for 30 minutes. The click reaction means that the ethynyl group on EdU can undergo a covalent reaction with a fluorescently labeled small molecule azide probe (AlexaFluor 488 azide) catalyzed by monovalent copper ions to form a stable triazole ring, and the process of this reaction is very rapid. Through the click reaction, newly synthesized DNA will be labeled by the corresponding fluorescent probe, so that proliferating cells can be detected using an appropriate fluorescence detection device. Finally, the cell nuclei are counterstained with 1× Hoechst33342 (Hoechst 33342 fluorescent dye) reagent. The staining results are observed using a laser confocal microscope system ( Figure 9 ) It can be seen from Figure 9 that H-EVs can promote the proliferation of HFF cells, suggesting that it can promote the proliferation of cells at the wound site and thus promote wound healing during the wound healing process.

[0034] 2. Effect of H-EVs on the proliferative ability of damaged HFF cells Inoculate HFF cells in the logarithmic growth phase at 2 mL / well in a 6-well plate at a cell concentration of 1.25×105 cells / mL, and culture at 37 °C and 5% CO2 for 24 h until the cells adhere to the wall. Randomly divide them into 5 treatment groups, and the specific treatment steps are as follows: Blank control group: Only add the culture medium; Model group (D-galactose): Add 50 mg / mL of D-galactose to the culture medium and treat for 48 h; Experimental group 1 (D-galactose + 5×10 8 particles / mL H-EVs): Add 50 mg / mL of D-galactose and 5×10 8 particles / mL of H-EVs to the culture medium; The specific operation is to pretreat with D-galactose for 24 h, then add H-EVs, and then continue to culture in the culture medium containing D-galactose for another 24 h; Experimental group 2 (D-galactose + 2.5×10 8 particles / mL H-EVs): Add 50 mg / mL of D-galactose and 2.5×10 8H-EVs at [X] particles / mL; specifically, after pre-treating with D-galactose for 24 h, H-EVs were added, and then the cells were cultured for another 24 h in the medium containing D-galactose. Experimental group 3 (D-galactose + 5×10 7 particles / mL H-EVs): Add 50 mg / mL D-galactose and 5×10 7 particles / mL H-EVs to the medium; specifically, after pre-treating with D-galactose for 24 h, H-EVs were added, and then the cells were cultured for another 24 h in the medium containing D-galactose.

[0035] Add 2 mL of EdU (10 µM) reagent (Beyotime, C0071S) to each well of the cells in the above treatment groups and incubate for 2 h to label the cells. After washing three times with PBS, the cells were fixed in 4% paraformaldehyde solution (Dingguo Biotech, AR-0211) for 15 minutes, permeabilized with 0.3% Triton X-100 (GenStar, VA11410) for 15 minutes, and then incubated with the click reaction reagent in the dark at room temperature for 30 minutes. The click reaction means that the ethynyl group on EdU can undergo a covalent reaction with the fluorescently labeled small molecule azide probe (AlexaFluor 488 azide) catalyzed by monovalent copper ions to form a stable triazole ring, and the process of this reaction is very rapid. Through the click reaction, the newly synthesized DNA will be labeled by the corresponding fluorescent probe, so that the proliferating cells can be detected using an appropriate fluorescence detection device. Finally, the nuclei were counterstained with 1× Hoechst33342 (Hoechst 33342 fluorescent dye) reagent. The staining results were observed using a laser confocal microscope system ( Figure 10 ). It Figure 10 can be seen that for HFF cells treated with D-galactose, almost no cell proliferation was observed. However, after treatment with H-EVs, cell proliferation was observed again, indicating that H-EVs can alleviate the decline in cell proliferation ability induced by D-galactose. Therefore, H-EVs can promote wound healing by promoting cell proliferation at chronic non-healing wound sites.

[0036] Experimental Example 3: Cell migration experiment 1. Effect of H-EVs on the migration ability of normal HFF cells A migration experiment was performed to analyze the migration ability of HFF cells treated with H-EVs. The specific experimental steps were as follows: HFF cells in the logarithmic growth phase (1×10 5 ~5×10 5Cells / mL) were inoculated at 200 μL / well into the upper chamber of a Transwell chamber (containing a polycarbonate membrane), and a medium containing 15% FBS (fetal bovine serum) was added to the lower chamber. The cells were further cultured for 24 h at 37 °C and 5% CO2. This experiment was randomly divided into four groups, and the specific treatment steps were as follows: Blank control group: Only serum-free medium was added to the upper chamber; Experimental group 1 (5×10 8 particles / mL H-EVs): Serum-free medium containing 5×10 8 particles / mL of H-EVs was added to the upper chamber; Experimental group 2 (2.5×10 8 particles / mL H-EVs): Serum-free medium containing 2.5×10 8 particles / mL of H-EVs was added to the upper chamber; Experimental group 3 (5×10 7 particles / mL H-EVs): Serum-free medium containing 5×10 7 particles / mL of H-EVs was added to the upper chamber.

[0037] After the experiment was completed, the chambers were taken out, and the cells that had not migrated on the surface of the upper chamber membrane were gently wiped with a cotton swab; fixed with 4% paraformaldehyde for 15 minutes, washed with PBS; finally stained with 0.1% crystal violet for 20 minutes, and rinsed with PBS and air-dried. The number of migrated cells was observed under a microscope ( Figure 11 ). It can be seen from Figure 11 that H-EVs can promote the migration of HFF cells, suggesting that it can promote the migration of cells at the wound site to the damaged area and thus promote wound healing during the wound healing process.

[0038] 2. Effect of H-EVs on the migration ability of damaged cells HFF cells in the logarithmic growth phase (1×10 5 ~5×10 5 cells / mL, and the model group and experimental groups were all pretreated with 50 mg / mL of D-galactose for 24 h) were inoculated at 200 μL / well into the upper chamber of a Transwell chamber (containing a polycarbonate membrane), and a medium containing 15% FBS (fetal bovine serum) was added to the lower chamber. The cells were further cultured for 24 h at 37 °C and 5% CO2. This experiment was randomly divided into 5 groups, and the specific treatment steps were as follows: Blank control group: Only serum-free medium was added to the upper chamber; Model group: Only serum-free medium was added to the upper chamber; Experimental group 1 (5×10 8particles / mL H-EVs): Add serum-free medium containing 5×10 8 particles / mL of H-EVs to the upper chamber; Experimental group 2 (2.5×10 8 particles / mL H-EVs): Add serum-free medium containing 2.5×10 8 particles / mL of H-EVs to the upper chamber; Experimental group 3 (5×10 7 particles / mL H-EVs): Add serum-free medium containing 5×10 7 particles / mL of H-EVs to the upper chamber.

[0039] After the experiment is completed, take out the chamber, and gently wipe the cells that have not migrated on the surface of the upper chamber membrane with a cotton swab; fix with 4% paraformaldehyde for 15 minutes, wash with PBS; finally, stain with 0.1% crystal violet for 20 minutes, and rinse with PBS and air dry. Observe the number of migrated cells under a microscope ( Figure 12 ). It can be seen from Figure 12 that H-EVs can promote the migration of damaged HFF cells, suggesting that it can promote the migration of cells to the damaged site in chronic non-healing wounds, thereby promoting healing and repair.

[0040] Experimental example 4: Inoculate HFF cells in the logarithmic growth phase at 2 mL / well in a 6-well plate, with a cell concentration of 1.25×10 5 cells / mL, and culture at 37°C and 5% CO2 for 24 h until the cells adhere. Randomly divide them into 5 treatment groups, and the specific treatment steps are as follows: Blank control group: Only add medium; Model group (D-galactose): Add 50 mg / mL of D-galactose to the medium and treat for 48 h; Experimental group 1 (D-galactose + 5×10 8 particles / mL H-EVs): Add 50 mg / mL of D-galactose and 5×10 8 particles / mL of H-EVs to the medium; The specific operation is to pre-treat with D-galactose for 24 h, then add H-EVs, and then continue to culture in the medium containing D-galactose for another 24 h; Experimental group 2 (D-galactose + 2.5×10 8 particles / mL H-EVs): Add 50 mg / mL of D-galactose and 2.5×10 8H-EVs at [X] particles / mL; specifically, after pre-treating with D-galactose for 24 h, H-EVs were added and then the cells were cultured for another 24 h in the medium containing D-galactose. Experimental group 3 (D-galactose + 5×10 7 particles / mL H-EVs): Add 50 mg / mL D-galactose and 5×10 7 particles / mL H-EVs to the medium; specifically, after pre-treating with D-galactose for 24 h, H-EVs were added and then the cells were cultured for another 24 h in the medium containing D-galactose.

[0041] The following detections were performed on each of the above treatment groups respectively.

[0042] 1. ROS determination Add serum-free medium containing 10 μM DCFH-DA to each well of the above treatment groups for ROS detection. Then, the cells were observed under an inverted fluorescence microscope. The results showed that the treatment with different concentrations of H-EVs reduced the level of reactive oxygen species (ROS) induced by D-galactose, that is, H-EVs could significantly improve the ROS level at the damaged wound site, facilitating the transition of chronic wounds from the inflammatory phase to the proliferative phase and promoting healing. The results are as Figure 13 shown.

[0043] 2. Western blot (1) Total protein extraction: Take appropriate samples from each treatment group, add an equal volume of RIPA lysis buffer (RIPA:PMSF:phosphatase inhibitor = 100:1:1), pipette and mix well, and place on ice for 30 min, shaking on a vortex mixer for 30 s every 10 min. Centrifuge at 12,000 g for 10 min at 4 °C, and transfer the supernatant to a new ep tube to obtain the total protein product.

[0044] (2) Protein quantification: Quantify the extracted total protein according to the BCA kit instructions.

[0045] (3) Western blot: Prepare separating gels (lower layer) with different concentrations according to the protein molecular weight. When the separating gel is not yet solidified, quickly add the stacking gel (upper layer), and immediately insert the electrophoresis unit and let it stand for 25 - 40 min to fully solidify. Pull out the comb, fix the glass plate on the gel rack and place it in the electrophoresis tank, add electrophoresis buffer to submerge the gel, and load the samples evenly. According to the electrophoresis buffer instruction manual, end the electrophoresis when the marker has run far and the samples have migrated close to the bottom of the gel. Cut the gel, activate the PVDF membrane with methanol for 1 - 2 min in advance, soak the filter paper in the transfer buffer, and perform wet transfer at a constant current of 400 mA for 25 - 30 min (determine the transfer time according to the protein molecular weight): Place the transferred PVDF membrane on a shaker at room temperature in a rapid blocking solution for 5 - 15 min; After blocking, wash the membrane 5 times with the washing solution (PBST / TBST), 5 min each time, blot dry, and then place it in the corresponding primary antibody at 4 °C overnight; Take out the membrane incubated with the primary antibody, wash it 5 times with the washing solution (PBST / TBST), 5 min each time, blot dry, and then place it in the corresponding secondary antibody and incubate it on a shaker at room temperature for 45 min; After the incubation with the secondary antibody is completed, wash the membrane 5 times with the washing solution (PBST / TBST), 5 min each time, blot dry with absorbent paper, evenly spread the luminescent solution on the membrane, and use a gel imaging system and ImageJ software to perform image acquisition and gray-scale analysis. The results are as Figures 14 - 15 shown.

[0046] According to Figure 14 and Figure 15 it can be seen that compared with the control group, the level of type III collagen in the model group was significantly decreased. Treatment with 2.5×10 8 particles / mL H-EVs promoted the expression of type III collagen in D-galactose-induced cells. Therefore, H-EVs can promote wound healing by promoting the process of extracellular matrix remodeling.

[0047] Experimental Example 5: Cell scratch assay 1. Effect of H-EVs on the scratch self-healing ability of normal HFF cells A scratch assay was performed to analyze the migration ability of HFF cells treated with H-EVs. The specific steps were as follows: Seed HFF cells in the logarithmic growth phase into a 6-well plate at a concentration of 2.5×10 5 cells / well. After culturing for 24 h, use a 200 μL pipette tip to vertically create a cell scratch on the plate, and make the width of each scratch as consistent as possible. Then, wash each well twice with serum-free medium to remove cell debris, and randomly divide them into 4 treatment groups. The specific treatment steps are as follows: Blank control group: Only add serum-free medium; Experimental group 1 (5×10 8particles / mL H-EVs): Add serum-free medium containing 5×10 8 particles / mL of H-EVs; Experimental group 2 (2.5×10 8 particles / mL H-EVs): Add serum-free medium containing 2.5×10 8 particles / mL of H-EVs; Experimental group 3 (5×10 7 particles / mL H-EVs): Add serum-free medium containing 5×10 7 particles / mL of H-EVs.

[0048] After culturing for 24 h, take pictures of the same scratch position and observe the healing conditions among different groups. The results are as Figure 16 shown.

[0049] As Figure 16 can be seen, H-EVs can effectively promote the scratch self-healing ability of cells, suggesting that H-EVs can effectively promote the repair of damaged skin fibroblasts and thus promote wound healing.

[0050] 2. Effects of H-EVs on the scratch self-healing ability of damaged HFF cells Perform a scratch assay to analyze the migratory ability of HFF cells treated with H-EVs. The specific steps are as follows: Seed HFF cells in the logarithmic growth phase into a 6-well plate at a concentration of 2.5×10 5 cells / well. After culturing for 24 h, use a 200 μL pipette tip to generate a cell scratch perpendicular to the plate, and make the width of each scratch as consistent as possible. Then, wash the medium in each well twice with serum-free medium to remove cell debris, and randomly divide them into 5 treatment groups. The specific treatment steps are as follows: Blank control group: Only add medium containing 1% serum; Model group (D-galactose): Add medium containing 50 mg / mL D-galactose and 1% serum; Experimental group 1 (D-galactose + 5×10 8 particles / mL H-EVs): Add medium containing 50 mg / mL D-galactose and 5×10 8 particles / mL of H-EVs and 1% serum; Experimental group 2 (D-galactose + 2.5×10 8 particles / mL H-EVs): Add medium containing 50 mg / mL D-galactose and 2.5×10 8Medium containing 1% serum with H-EVs at [X] particles / mL; Experimental group 3 (D-galactose + 5×10 7 particles / mL H-EVs): Add medium containing 50 mg / mL D-galactose and 5×10 7 particles / mL of H-EVs with 1% serum.

[0051] After 12 h of treatment in each treatment group, an inverted fluorescence microscope was used to observe cell migration, and the scratch area was analyzed using ImageJ software. The cell migration rate was calculated according to the following formula, and the results of the control group were used for normalization. The results are as Figure 17 and Figure 18 shown.

[0052] Cell migration rate = (scratch area at 0 h - scratch area after culture) / scratch area at 0 h From Figure 17 and Figure 18 it can be seen that the migration rate of the model group induced by D-galactose was lower than that of the control group. After adding different concentrations of H-EVs, it could effectively promote the migration of damaged cells and help them play a role in promoting wound healing in chronic non-healing wounds.

[0053] Experimental example 6: Experiment on the promotion of skin wound healing in mice by extracellular vesicles 1. Experimental protocol 1.1 Experimental animals and grouping Healthy male C57BL / 6 mice (8 weeks old, weighing 20 - 25 g) were randomly divided into 3 groups (n = 8 / group): Control group: Topically apply normal saline; Example 2 group: Topically apply the extracellular vesicles prepared in Example 2 (2.5×10 8 particles / mL); Example 3 group: Topically apply the extracellular vesicles prepared in Example 3 (2.5×10 8 particles / mL). 1.2 Establishment of wound model After anesthesia, the back of the mice was shaved and disinfected, and a full-thickness skin defect wound was created using a 6 mm diameter biopsy punch. 1.3 Administration method Once a day, each group was topically applied with 100 μL of the corresponding solution (normal saline or vesicle suspension) for 14 consecutive days. 1.4 Observation indexes and detection methods Wound healing rate: The wound area was photographed daily, and the healing rate was calculated using ImageJ software.

[0054] Healing rate = [Initial area - Remaining area] / Initial area × 100% Histopathological analysis: Mice were sacrificed on the 7th and 14th days, and wound tissues were taken for: HE staining: To observe epidermal regeneration and granulation tissue thickness; Masson staining: To detect the amount of collagen deposition. Inflammatory factor detection: On the 3rd day, wound tissue homogenate was taken, and the levels of TNF-α and IL-6 were measured by ELISA. 2. Experimental results and analysis 2.1 Dynamic changes in wound healing rate The results are shown in the following table.

[0055] In the table, indicates p < 0.05 compared with the control group, indicates p < 0.01 compared with the control group; # indicates p < 0.05 compared with the Example 2 group; the same below.

[0056] As can be seen from the above table, the healing rate of the Example 3 group was significantly better than that of the Example 2 group and the control group at each time point (p < 0.05), indicating that the extracellular vesicles of the Example 3 group can accelerate wound closure, which may be related to the more complete structure and better activity of the extracellular vesicles in the Example 3 group.

[0057] 2.2 Histopathological analysis 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 the granulation tissue thickness of 300 ± 25 μm in the control group. Masson staining: On the 14th day, the amount of collagen deposition in the Example 3 group was 78.5 ± 4.2%, which was significantly higher than the amount of collagen deposition of 65.3 ± 3.8% in the Example 2 group and the amount of collagen deposition of 45.1 ± 3.5% in the control group. It can be seen that the granulation tissue thickness and the amount of collagen deposition in the Example 3 group are higher, indicating that its preparation method optimized the biological activity of the vesicles and promoted the reconstruction of the extracellular matrix.

[0058] 2.3 Inflammatory factor levels The results are shown in the following table.

[0059] As can be seen from the above table, 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 a more efficient anti-inflammatory effect.

[0060] 3. Conclusion The extracellular vesicles prepared in Example 3 were significantly improved in terms of purity, stability and biological activity through gradient purification, calcium ion incubation and cryoprotectant optimization. Compared with those in Example 2, the vesicles in Example 3 showed better performance in promoting epidermal regeneration, collagen deposition and inhibiting inflammatory response (p<0.05), which confirmed that the improvement of its preparation process played a key role in enhancing the curative effect of wound healing.

[0061] The number of devices and the processing scale described here are used to simplify the description of the present invention. Applications, modifications and variations of the application of Weissella coagulans and its applications of the present invention will be apparent to those skilled in the art.

[0062] Although the embodiments of the present invention have been disclosed as above, it is not limited to the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily achieved. 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 the examples shown and described herein.

Claims

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

2. Use of the Weissella coagulans according to claim 1 in the preparation of a product for promoting wound healing.

3. The use according to claim 2, characterized in that, The only active ingredient of the product is the extracellular vesicles of Weissella coagulans, and the effective concentration of the extracellular vesicles is 5×10 7 ~5×10 8 particles / ml.

4. The use according to claim 2, characterized in that, The extracellular vesicles are prepared by the following steps: Step 1: Inoculate Weizmannia coagulans L-B15 into a liquid 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 perform anaerobic culture at 45 - 55 °C for 48 - 72 hours to obtain an initial fermentation broth; Step 2: Centrifuge the initial fermentation broth at 8000 - 12000 g for 8 - 15 minutes to remove the bacteria, and collect the supernatant; filter the supernatant successively through a polyethersulfone membrane with a pore size of 0.45 μm and a polyvinylidene fluoride membrane with a pore size of 0.22 μm; ultracentrifuge the filtered supernatant at 100000 - 150000 g at 4 - 8 °C for 60 - 90 minutes, and discard the supernatant to obtain a primary vesicle precipitate; Step 3: Resuspend the primary vesicle precipitate in a phosphate buffer solution with a pH of 7.9 - 8.5, add a calcium chloride solution with a final concentration of 5 - 10 mmol / L, and incubate with shaking in a water bath at 35 - 42 °C for 20 - 40 minutes; perform gradient sucrose density centrifugation on the incubated mixture, successively stack sucrose solutions with mass fractions of 15%, 30%, and 45%, and centrifuge at 80000 - 100000 g at 4 - 8 °C for 2 - 4 hours, and collect the vesicle fraction at the interface of the 30% - 45% sucrose layer; Step 4: Mix the collected vesicle fraction with a Tris-HCl buffer solution containing 0.05 - 0.2 mol / L trehalose, and repeatedly extrude it 3 - 5 times through a polycarbonate membrane with a pore size of 100 - 200 nm; ultracentrifuge the extruded mixture at 100000 - 150000 g at 4 - 8 °C for 60 - 90 minutes, collect the precipitate and wash it twice with a Tris-HCl buffer solution with a pH of 7.4 - 7.6, and the precipitate is the extracellular vesicle.

5. The use according to claim 4, characterized in that, The method for preparing the extracellular vesicles further includes: 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 under ice bath conditions for 10 - 20 minutes, aliquoted into tubes with a volume of 0.5 - 1.5 mL / tube, and use polypropylene cryotubes with a wall thickness of 1.2 - 1.8 mm. After aliquoting, pre-cool it to -40 °C at a rate of 1 - 2 °C per minute, and then transfer it to -80 °C for storage.

6. A product for promoting wound healing, characterized in that, The product uses the extracellular vesicles of Weissella coagulans described in claim 1 as the only active ingredient, and the effective concentration of the extracellular vesicles is 5×10 7 ~5×10 8 particles / ml.

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