Lactobacillus strain and its use in the preparation of an exopolysaccharide dressing for promoting skin wound repair

Abstract

The application discloses a lactic acid bacterial strain and application of the lactic acid bacterial strain in preparation of an exopolysaccharide dressing for promoting skin wound repair, the strain is named as Lactiplantibacillus plantarum, and is preserved in the China Center for Type Culture Collection on August 31, 2023, with a preservation number of CCTCC M 20231553 and a preservation address of Wuhan University, Wuhan, China. Research results show that the EPS secreted by the Lactiplantibacillus plantarum T1 not only has good antioxidant activity, but also shows good antibacterial effect on exogenous pathogenic bacteria Staphylococcus aureus, can effectively promote the proliferation and repair of epidermal cells HaCaT, has reliable biological safety, effectively promotes wound healing, and has wide application prospect in development of medical dressing products.

Description

Technical Field

[0001] This invention belongs to the field of food functional active ingredient processing technology. Specifically, it relates to a strain of lactic acid bacteria and its application in the preparation of a lactic acid bacteria extracellular polysaccharide dressing that promotes skin wound repair. Background Technology

[0002] The skin is the body's first line of defense against external physical, chemical, and biological hazards, playing a vital role in the body's health and homeostasis. Therefore, maintaining skin integrity is crucial for human health. Currently, the treatment of wound inflammation and ulceration caused by lesions has become a hot research topic, involving the overuse of antibiotics and the challenge of chronic wounds. Studies have shown that infection is one of the most significant external factors affecting wound healing, especially microbial infections such as Staphylococcus aureus. These pathogenic bacteria can significantly slow down wound healing and may even lead to tissue necrosis. Therefore, there is an urgent need for a safe dressing with inherent antibacterial properties that can effectively inhibit bacterial growth and promote wound repair without relying on drug intervention.

[0003] Lactic acid bacteria polysaccharides are complex carbohydrate polymers composed of monosaccharides such as glucose and galactose linked by glycosidic bonds, and are widely distributed in various lactic acid bacteria resources. Their unique molecular structure endows them with a variety of biological activities. Although extracellular polysaccharides from lactic acid bacteria have attracted widespread attention in clinical practice due to their remarkable biological activities, there are no reported cases of their safe and reliable application in wound healing research. Therefore, developing functional wound healing dressings using green, safe, and reliable lactic acid bacteria polysaccharide resources would not only help deepen our understanding of their role in promoting wound healing but also provide a safe and reliable treatment option for wound repair. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of lactic acid bacteria and its application in the production of lactic acid bacteria extracellular polysaccharides that can promote wound healing.

[0005] Another objective of this invention is to provide a lactic acid bacteria extracellular polysaccharide and its applications. The preparation method of this lactic acid bacteria extracellular polysaccharide is simple and efficient, exhibits strong biocompatibility, good antibacterial properties, and can prevent bacterial infection of wounds and promote skin wound healing.

[0006] Another objective of this invention is to provide a lactic acid bacteria extracellular polysaccharide dressing that promotes skin wound repair. This lactic acid bacteria extracellular polysaccharide dressing is a safe and reliable lactic acid bacteria-derived extracellular polysaccharide dressing with high biocompatibility and can be used for skin wound repair.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] In a first aspect, the present invention seeks protection for a strain of lactic acid bacteria that produces extracellular polysaccharides, classified as *Lactiplantibacillus plantarum*, which was deposited on August 31, 2023, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20231553, located at Wuhan University, Wuhan, China.

[0009] Secondly, the present invention seeks protection for the use of the above-mentioned lactic acid bacteria strains in the production of lactic acid bacteria extracellular polysaccharides that can promote wound healing.

[0010] Thirdly, the present invention seeks protection for the use of the extracellular polysaccharides of lactic acid bacteria secreted by the above-mentioned lactic acid bacteria strains in the following (1) or (2) or (3):

[0011] (1) Promote wound healing or prepare products that promote wound healing;

[0012] (2) Preparation of drug carriers;

[0013] (3) Prepare dressing.

[0014] Furthermore, the lactic acid bacteria extracellular polysaccharides mentioned above are obtained by fermentation culture of the aforementioned lactic acid bacteria strain. Even further, the lactic acid bacteria extracellular polysaccharides are prepared by fermentation culture of the aforementioned lactic acid bacteria strain, followed by separation and purification of the extracellular polysaccharides in the fermentation broth.

[0015] Furthermore, the separation and purification process is as follows:

[0016] (1) Centrifuge the fermentation broth and collect the supernatant; treat the supernatant with 4% (v / v) trichloroacetic acid solution, centrifuge to remove protein; then add anhydrous ethanol for treatment and centrifuge to obtain crude polysaccharide precipitate;

[0017] (2) The crude polysaccharide precipitate was dissolved in deionized water and then transferred into a dialysis bag with a molecular weight cutoff of 8,000-14,000 Da for water dialysis. The dialysis solution was freeze-dried to obtain crude extracellular polysaccharide.

[0018] (3) The crude extracellular polysaccharide was further purified using a DEAE-52 anion exchange column. A gradient elution was performed using deionized water, 0.1M and 0.3M NaCl solutions at a flow rate of 1 mL / min to collect the purified polysaccharide fraction. The sugar content was determined by the phenol-sulfuric acid method. Finally, the main fraction was dialyzed with pure water and freeze-dried to obtain the purified lactic acid bacteria extracellular polysaccharide. The main fraction is the component obtained after purification with deionized water.

[0019] Fourthly, the present invention claims protection for a dressing that promotes the repair of skin wounds, the dressing containing lactic acid bacteria extracellular polysaccharides secreted by the said lactic acid bacteria strain.

[0020] This invention screened a lactic acid bacteria strain that produces extracellular polysaccharides. This strain was isolated from kefir grains in Aksu, Xinjiang. 16S rDNA sequencing analysis identified it as *Lactiplantibacillus plantarum*, and it was named *Lactiplantibacillus plantarum*. During the research, technicians first extracted, isolated, and purified the extracellular polysaccharides from the lactic acid bacteria. The purified extracellular polysaccharides from the lactic acid bacteria were then studied as follows:

[0021] (1) Evaluation of in vitro antioxidant activity;

[0022] (2) Evaluation of in vitro biofilm inhibitory activity of pathogenic bacteria;

[0023] (3) Evaluation of bioactivity in HaCaT cell compatibility and safety;

[0024] (4) Comprehensive evaluation of its effect on wound repair in mice as a wound dressing.

[0025] The results show that this invention isolated and purified an extracellular polysaccharide (EPS) from a lactic acid bacteria strain that produces high levels of EPS, with a molecular weight of 1.41 × 10⁻⁶. 6 Da, composed of galactose and glucose in a molar ratio of 1.21:1.00, is an extracellular polysaccharide from lactic acid bacteria. It exhibits excellent in vitro antioxidant activity, effectively scavenging reactive free radicals such as ABTS, DPPH, and hydroxyl radicals. Simultaneously, this extracellular polysaccharide effectively inhibits biofilm formation in pathogenic Staphylococcus aureus, thus promoting wound healing. HacaT cell experiments demonstrated that the extracellular polysaccharide exhibits good biocompatibility and effectively promotes cell proliferation. Cell scratch assays showed that the extracellular polysaccharide effectively promotes cell repair. Further investigation using wound repair experiments in male ICR mice revealed that, compared to the control group, the extracellular polysaccharide group effectively accelerated the repair of superficial wounds in mice within 14 days.

[0026] Compared with the prior art, the advantages and effects of the present invention are as follows:

[0027] 1) The polysaccharide prepared by this invention uses naturally sourced lactic acid bacteria as the parent material, has good biocompatibility and is renewable, inexpensive and green.

[0028] 2) The lactic acid bacteria polysaccharide prepared by this invention has excellent antioxidant capacity and is suitable for various types of wounds.

[0029] 3) The lactic acid bacteria polysaccharide prepared by this invention has good biofilm inhibition activity against pathogenic bacteria.

[0030] The foregoing summary of certain objectives of the invention is not exhaustive and does not represent all objectives of the invention. Other objectives and advantages of the invention will become clearer from the following description, which, together with the accompanying drawings, illustrations, and embodiments, and specific details of the invention, forms part of this specification. This includes exemplary embodiments of the invention, illustrating various objectives and features of the invention. Attached Figure Description

[0031] Figure 1 This refers to the extraction and purification steps of lactic acid bacteria extracellular polysaccharides in Example 1.

[0032] Figure 2 This is a high-performance liquid chromatography gel permeation chromatogram of lactic acid bacteria extracellular polysaccharides.

[0033] Figure 3 This is a chromatogram showing the monosaccharide composition of extracellular polysaccharides from lactic acid bacteria.

[0034] Figure 4 The in vitro antioxidant properties of lactic acid bacteria extracellular polysaccharides.

[0035] Figure 5 The extracellular polysaccharide of lactic acid bacteria exhibits inhibitory activity against Staphylococcus aureus biofilm.

[0036] Figure 6 To assess the biosafety of lactic acid bacteria extracellular polysaccharides to HaCaT cells, the PBS group served as a negative control.

[0037] Figure 7 This image shows the effect of lactic acid bacteria extracellular polysaccharides on scratch repair in HaCaT cells.

[0038] Figure 8 The value represents the migration rate of HaCaT cells affected by lactic acid bacteria extracellular polysaccharides.

[0039] Figure 9 To measure the in vitro hemolysis rate of lactic acid bacteria extracellular polysaccharides, deionized water was used as a positive control and PBS as a negative control.

[0040] Figure 10 This is a photograph of the effect of lactic acid bacteria extracellular polysaccharide on the repair of skin damage in mice.

[0041] Figure 11 The effect of lactic acid bacteria extracellular polysaccharide on the repair rate of skin damage in mice. Detailed Implementation

[0042] The method of the present invention will be described below through specific embodiments, but the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0043] Example 1: Screening of lactic acid bacteria strains

[0044] This invention screened a strain of lactic acid bacteria that produces extracellular polysaccharides. This strain was isolated from kefir grains in Aksu, Xinjiang. The strain was identified as *Lactiplantibacillus plantarum* T1 by 16S sequencing analysis and was deposited at the China Center for Type Culture Collection (CCTCC) on August 31, 2023, with accession number CCTCC M 20231553, located at Wuhan University, Wuhan, China.

[0045] (1) Screening method:

[0046] Kefir grains were sourced from dairy products made by residents in Aksu, Xinjiang. After sampling in sterile containers, the samples were sent back to the laboratory under low-temperature conditions for the isolation and purification of lactic acid bacteria. First, MRS liquid and solid culture media were prepared for the growth and plating of lactic acid bacteria colonies. Then, a piece of kefir grain was selected in a laminar flow hood and immersed in MRS liquid culture medium, followed by thorough vortexing. After 24 hours of incubation, the culture medium containing the bacterial solution was serially diluted with 0.85% (g / 100ml) sterile physiological saline, and then the colonies were plated. After culturing at 37℃ for 30 hours, the colony morphology on the solid plates was observed. Single colonies with differences in size, color, and fibrillation were selected for streak purification and labeled. The streak purification process was repeated 3-4 times. Single colonies were then selected for Gram staining and microscopic examination to ensure that the selected strains were Gram-positive and catalase-negative, and were non-spore-forming. The purified strains were selected through the above operations, and then activated by liquid culture. The cultured single bacteria were then sent to a biotechnology company for 16S rDNA sequencing. The sequencing results were compared with the bioinformatics data in the GenBank database to determine that strain T1 was *Lactobacillus plantarum*.

[0047] Example 2: Extraction and physicochemical composition analysis of extracellular polysaccharides from lactic acid bacteria

[0048] (1) Extraction of extracellular polysaccharides from lactic acid bacteria

[0049] A glycerol-preserved lactic acid bacteria strain (Lactiplantibacillus plantarum T1) was inoculated into MRS liquid medium and activated twice at 30°C for 20 h each time. Subsequently, it was transferred to a large flask of MRS medium at a ratio of 4% (v / v) and incubated at 30°C for 28 h. The fermented medium was centrifuged at 12000 rpm for 15 min to obtain a supernatant containing extracellular polysaccharides (EPS). The supernatant was then treated with 4% (v / v) trichloroacetic acid (TCA) solution, allowed to stand at 4°C for 6 h, and centrifuged to remove proteins. Then, three volumes of anhydrous ethanol were added, and the mixture was allowed to stand overnight at 4°C to precipitate crude polysaccharides. The crude extracellular polysaccharide precipitate was then obtained by centrifugation and dissolved in deionized water. The precipitate was transferred to a dialysis bag (MD34, 8000-14000 Da) and dialyzed against running water for 72 h. The dialysate was freeze-dried to obtain a dried crude extracellular polysaccharide sample. The extracellular polysaccharides were further purified using a DEAE-52 anion exchange column (2.6 × 30 cm). Gradient elution was performed using deionized water, 0.1 M, and 0.3 M NaCl solutions at a flow rate of 1 mL / min. Twenty tubes (10 mL each) were collected under each elution condition. The sugar content in each tube was determined by the phenol-sulfuric acid method. The fractions obtained primarily through deionized water purification were collected, dialyzed against pure water, and freeze-dried to obtain purified lactic acid bacteria extracellular polysaccharides for further research (see [link to study]). Figure 1 ).

[0050] (2) Physicochemical composition analysis of polysaccharides

[0051] High-performance liquid chromatography-gel permeation chromatography (HPGPC) was used to determine the homogeneity and relative molecular mass of polysaccharides. The selected columns were an Ultrahydrogel™ guard column (6 mm × 40 mm) and an Ultrahydrogel™ linear gel column (7.8 mm × 300 mm). The mobile phase was 0.02% (w / v, g / 100 mL) sodium azide, the flow rate was 0.6 mL / min, and the detectors were a differential detection detector (RID) and a UV detector, with the detection wavelength set at 280 nm. The column temperature was 35 °C, the injection volume was 20 μL, and the data acquisition time was 30 min.

[0052] The monosaccharide composition of polysaccharides was determined using high-performance anion exchange chromatography with a pulsed amperometric detector (HPAEC-PAD). First, 5 mg of the polysaccharide sample was weighed into a glass tube, and 0.5 mL of 12 M H₂SO₄ was added. A stir bar was added to the glass tube, and the mixture was magnetically stirred for 30 min in an ice bath. After the reaction, deionized water was added to dilute the H₂SO₄ concentration to 2 M, and then the mixture was transferred to a 120 °C oil bath for hydrolysis for 2 h. The hydrolysate was cooled and diluted 100-fold, then filtered through a 0.22 μm pinhole filter before analysis. Different monosaccharide standards and their standard curves were established using the same procedure, mainly including fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, and glucuronic acid. Chromatographic conditions were performed using a Dionex ICS-5000 ion-exchange chromatography system, equipped with a pulsed amperometric detector, a CarboPac™ PA20 guard column, and a CarboPac™ PA20 analytical column (4 mm × 250 mm). Mobile phase A was ultrapure water, mobile phase B was 250 mmol / L NaOH solution, and mobile phase C was 1 M NaOAc solution. Separation was achieved using gradient elution. The flow rate was set at 0.5 mL / min, and the elution program is detailed in Table 1.

[0053] Table 1. Mobile phase elution program for Dionex chromatography system

[0054]

[0055] The results showed that the purified extracellular polysaccharide had a single and symmetrical chromatographic peak, indicating the homogeneity of the polysaccharide component. The molecular weight (Mw) of the polysaccharide was determined to be 1.41 × 10⁻⁶. 6 Da is composed of galactose and glucose in a molar ratio of 1.21:1.00. Figure 2 , Figure 3 ).

[0056] Example 3: Verification of the effect of lactic acid bacteria extracellular polysaccharides

[0057] 1. Evaluation of the in vitro antioxidant activity of polysaccharide dressings

[0058] 1.1. DPPH free radical scavenging ability

[0059] Weigh the lactic acid bacteria extracellular polysaccharide sample obtained in Example 2, and prepare lactic acid bacteria extracellular polysaccharide sample solutions of different concentrations (0.5, 1.0, 2.0, 4.0 mg / mL) using deionized water. Then, measure 1.0 mL of the lactic acid bacteria extracellular polysaccharide sample solution and mix it with 2.0 mL of deionized water and 0.2 mL of ethanol-2,2-biphenyl-1-picrylhydrazine (DPPH) solution (0.4 mM). Then, let it stand for 30 min in the dark at room temperature, and measure the absorbance at 517 nm. The experiment was repeated in triplicate.

[0060] DPPH free radical scavenging activity (%) = [1-(A 样品 -A0) / A 空白 ]×100

[0061] Where A0 represents the sample in relation to A 样品 Under the same conditions, water was used instead of DPPH radical solution for absorbance, and deionized water and vitamin C were used as blank and positive controls, respectively.

[0062] 1.2. ABTS+ free radical scavenging ability

[0063] Weigh the lactic acid bacteria extracellular polysaccharide sample obtained in Example 2, and prepare lactic acid bacteria extracellular polysaccharide solutions of different concentrations (0.5, 1.0, 2.0, 4.0 mg / mL) using deionized water. Mix the polysaccharide sample solutions of different concentrations with 2,2-adiazon-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS)+ solution at a volume ratio of 1:1. The blank group is distilled water, and the control group is distilled water instead of ABTS+ working solution. Place at room temperature in the dark for 6 min, and measure its absorbance at a wavelength of 734 nm. The experiment is repeated in triplicate.

[0064] ABTS+ free radical scavenging capacity (%) = [A 空白 -(A 样品 -A 对照 ) / A 空白 ]×100%

[0065] Where A 空白 To add ABTS+ solution and distilled water; A 样品 To add ABTS+ solution to the sample solution; A 对照 The sample solution and distilled water were used, with vitamin C as a positive control.

[0066] 1.3. Hydroxyl radical scavenging ability

[0067] 0.75 mM FeSO4 (1 mL), 0.75 mM 1,10-phenanthroline (1 mL), 0.15 M sodium phosphate buffer (pH 7.4, 1.5 mL), and H2O2 (0.01%, v / v, 1 mL) were mixed separately. Then, 1.0 mL of different concentrations of lactic acid bacteria extracellular polysaccharide solutions (0.5, 1.0, 2.0, 4.0 mg / mL) prepared with deionized water were added to the mixture, and the mixture was reacted at 37 °C for 30 min. The absorbance was measured at 536 nm, and the experiment was repeated in triplicate.

[0068] Hydroxyl radical scavenging activity (%) = [(A 样品 -A 空白 ) / (A0-A空白 )]×100

[0069] Where A0 represents the absorbance of deionized water in the measurement system, excluding H2O2 and the sample. VC and deionized water are used as blank and positive controls, respectively.

[0070] The results showed that as the concentration of extracellular polysaccharides from lactic acid bacteria increased within the range of 0.5 to 4 mg / mL, the polysaccharide concentration had an effect on DPPH and ABTS. + The scavenging activity of EPS against hydroxyl radicals was dose-dependent. At a concentration of 4.0 mg / mL, the free radical scavenging activities of EPS against the three radicals were 62.37%, 59.64%, and 72.49%, respectively (see [link to article]). Figure 4 ).

[0071] 2. In vitro antibacterial properties analysis of polysaccharide dressings

[0072] The in vitro antibacterial properties of lactic acid bacteria extracellular polysaccharides against Staphylococcus aureus were evaluated by measuring changes in biofilm formation caused by these polysaccharides. First, 10 μL of bacterial suspension (10 8 CFU mL -1 Add 200 μL of LB medium containing different concentrations of the lactic acid bacteria extracellular polysaccharide obtained in Example 2. Then incubate at 37°C for 18-24 hours. After washing 2-3 times with PBS, dry in an oven for 10 min, then stain with crystal violet for 15-30 s, wash with PBS until colorless, add 100 μL of glacial acetic acid-ethanol decolorizing solution, and measure the absorbance of the suspension at 595 nm using a microplate reader to evaluate the antibacterial properties of the polysaccharide against pathogenic bacterial biofilms. The inhibition rate can be calculated using the following formula:

[0073] Biomembrane inhibition rate (%) = [(OD 595,空白 -OD 595,多糖 ) / OD 595,空白 )]×100

[0074] Conclusion: Bacterial infection exacerbates exudate formation, thus delaying wound healing. The EPS at a concentration of 4 mg / mL showed a 54.35% inhibition rate against Staphylococcus aureus biofilm, indicating that this polysaccharide, as a wound dressing, can effectively reduce Staphylococcus aureus biofilm formation, thereby better promoting wound repair (see...). Figure 5 ).

[0075] 3. Safety evaluation of polysaccharide dressings on HaCaT cells

[0076] The cells were placed in DMEM medium containing 10% fetal bovine serum and cultured in a cell culture incubator at 37°C. The control group was the one without polysaccharide. The polysaccharide obtained in Example 2 was added to the culture medium after sterilization, and then filtered through a 0.22 μm filter to obtain a sterile polysaccharide solution. Subsequently, cells in the logarithmic growth phase were diluted to 2 × 10⁻⁶ cells / mL. 4 The samples were inoculated into 96-well plates and incubated for another 24 hours in an incubator. The absorbance (OD) of the solution at 450 nm was measured using a CCK-8 assay kit.

[0077] Conclusion: Cell compatibility is an important factor in the safety of polysaccharide samples. This EPS sample exhibited good biocompatibility with HaCaT cells and promoted cell proliferation at concentrations ranging from 0 to 400 μg / mL, indicating that the lactic acid bacteria polysaccharide obtained in Example 2 has good biocompatibility. Figure 6 ).

[0078] 4. Cellular scratch repair assessment

[0079] HaCaT cells were used to assess scratch repair capacity in order to evaluate the repair activity of polysaccharide samples. First, cells were prepared at a concentration of 5 × 10⁻⁶. 3 HaCaT cells were seeded in 6-well cell culture plates at a seed volume of 1 ml and cultured in a cell culture incubator. When the cell density reached 90%, horizontal scratches were made in the wells using a ruler and a 200 μL micropipette tip, and the culture medium in the wells was aspirated. After washing twice with PBS buffer, culture medium containing different concentrations of polysaccharide samples was added to each well. The culture medium without the sample served as a blank control. The scratch area of ​​HaCaT cells was measured at 0, 12, and 24 hours. All experiments were performed in triplicate.

[0080] Conclusion: Compared with the control group, the EPS group significantly promoted cell migration. At a concentration of 400 μg / mL, the cell migration rate reached 35.54% after 24 h, significantly higher than the 4.53% in the control group. This indicates that the extracellular polysaccharide EPS from lactic acid bacteria can effectively promote the proliferation and differentiation of epidermal cells (see...). Figure 7 and Figure 8 ).

[0081] 5. Biocompatibility assessment of polysaccharide dressings

[0082] First, fresh whole blood was collected from ICR mice. 20 mL of PBS buffer was mixed with 10 mL of whole blood, and the mixture was centrifuged at 5000 rpm for 5 min to collect red blood cells. The collected red blood cells were then mixed with twice the volume of PBS buffer and centrifuged again to remove impurities from the blood. The dried polysaccharide powder obtained in Example 2 was weighed and dispersed in PBS buffer to prepare a suspension with a concentration range of 10-40 mg / mL. Then, different concentrations of polysaccharide solutions were mixed with red blood cells at a volume ratio of 1:1. After incubation at 25°C for 2 h, the samples were centrifuged at 5000 rpm for 5 min, and the absorbance of the supernatant was measured at 540 nm. PBS buffer and deionized water were used as negative and positive controls, respectively. The percentage of hemolysis was calculated using the following method:

[0083] Hemolysis rate (%) = (OD) 540样品 -OD 540 PBS ) / (OD 540水 -OD 540 PBS )×100

[0084] Conclusion: The biocompatibility of the sample is crucial for its application in the biomedical field. Within a concentration range of 10-40 mg / mL, the hemolysis rate of this EPS was below the international standard of 5%, indicating a certain degree of biosafety (see...). Figure 9 ).

[0085] 6. Analysis of wound repair in male ICR mice

[0086] Male ICR mice (33-35g) were anesthetized via intraperitoneal injection of chloral hydrate (10%, w / v, g / 100ml). A 6mm diameter incision was made in the smooth back of the mouse using a punch. The polysaccharide solution obtained in Example 2 was used for treatment for 14 days at concentrations of 20 and 40 mg / mL. A PBS-treated group served as a blank control, and 3M wound bandages served as a positive control. Each group contained 12 animals. Wound healing was photographed on days 0, 3, 7, and 14.

[0087] Conclusion: Three days after wound treatment in mice, there was no significant difference in wound repair among the groups. After day 7, the wound repair rate of the EPS polysaccharide-treated group was significantly higher than that of other groups, and the high-concentration polysaccharide group was significantly better than the low-concentration polysaccharide group. On day 14, EPS at 40 mg / mL achieved a wound repair rate of 98.67% in mice, significantly better than the control group. This indicates that the extracellular polysaccharide EPS secreted by *Lactobacillus plantarum* can effectively promote the repair of skin wounds (see...). Figure 10 and Figure 11 ).

[0088] 7. Discussion:

[0089] This invention utilizes a lactic acid bacteria strain isolated from kefir grains in Aksu, Xinjiang, for fermentation, sugar extraction, and purification to obtain homogeneous, high-purity extracellular polysaccharides (EPS). Its excellent antioxidant activity was evaluated through in vitro free radical scavenging. Its potential antibacterial activity was explored through in vitro biofilm inhibition experiments against Staphylococcus aureus. Its biocompatibility with HaCaT cells was assessed for biosafety. The repair activity of the polysaccharide as a dressing was investigated using a mouse wound model. These results indicate that the EPS secreted by *Lactobacillus plantarum* T1 not only possesses good antioxidant activity but also inhibits biofilm formation by *Staphylococcus aureus*. Furthermore, EPS effectively promotes the proliferation and repair of HaCaT epidermal cells, demonstrating reliable biosafety. Mouse experiments showed that, compared to the control group, this EPS, as a polysaccharide dressing, effectively promoted wound repair in mice. In summary, the extracellular polysaccharide derived from this lactic acid bacteria strain is not only simple to prepare as a wound dressing, but also has excellent biocompatibility. It exhibits good antibacterial effects against exogenous pathogenic bacteria Staphylococcus aureus and effectively promotes wound healing. Therefore, its development into medical dressing products has broad application value and prospects.

[0090] sequence list

[0091] Lactiplantibacillus plantarum T1 strain 16S rDNA

[0092] TTGATCACTTAGCGGCTGGTTCCTAAAAGGTTACCCCACCGACTTTGGGTGTTACAAA

[0093] CTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCAT

[0094] GCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATC

[0095] CGAACTGAGAATGGCTTTAAGAGATTAGCTTACTCTCGCGAGTTCGCAACTCGTTGTA

[0096] CCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCA

[0097] TCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACCAGAGTGCCCAACTTAATGCT

[0098] GGCAACTGATAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACA

[0099] CGAGCTGACGACAACCATGCACCACCTGTATCCATGTCCCCGAAGGGAACGTCTAAT

[0100] CTCTTAGATTTGCATAGTATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTA

[0101] AACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTG

[0102] CGGCCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGA

[0103] AACCCTCCAACACTTAGCATTCATCGTTTACGGTATGGACTACCAGGGTATCTAATCCT

[0104] GTTTGCTACCCATACTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCG

[0105] CCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTC

[0106] CTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTTCTTCGGTTGAGCCGAAGGCT

[0107] TTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAA

[0108] CGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGG

[0109] TTAAATACCGTCAATACCTGAACAGTTACTCTCAGATATGTTCTTCTTTAACAACAGAG

[0110] TTTTACGAGCCGAAACCCCTTCTTCACTCACGCGCGGTGGCTCATCAGACTTTCGTTC

[0111] CATGTGAGATCCCCTACTGCTGCCTCCGGTAAGAGTTTGGTCCTGTTCTCAGTCCTAA

[0112] TGTGGTCGAATGACCTCCTCAGGATCGACTTACGGTATTA。

Claims

1. The application of extracellular polysaccharides secreted by Lactiplantibacillus plantarum strain T1 (accession number CCTCC M 20231553) in the preparation of products promoting skin wound repair, characterized in that... The extracellular polysaccharide has a molecular weight of 1.41 × 10⁶ Da, is composed of galactose and glucose in a molar ratio of 1.21:1.00, and has the activity of inhibiting Staphylococcus aureus biofilm formation and promoting HaCaT cell proliferation.

2. The application according to claim 1, characterized in that, The extracellular polysaccharide was obtained by fermentation culture of strain T1.

3. The application according to claim 2, characterized in that, The extracellular polysaccharide was prepared by fermenting the strain T1 and then separating and purifying the extracellular polysaccharide in the fermentation broth.

4. The application according to claim 3, characterized in that, The separation and purification process is as follows: (1) Centrifuge the fermentation broth and collect the supernatant; treat the supernatant with 4% trichloroacetic acid solution, centrifuge to remove protein; then add anhydrous ethanol for treatment and centrifuge to obtain crude polysaccharide precipitate; (2) The crude polysaccharide precipitate was dissolved in deionized water and then transferred to a dialysis bag with a molecular weight cutoff of 8,000-14,000 Da for water dialysis. The dialysis solution was freeze-dried to obtain crude extracellular polysaccharide. (3) The crude extracellular polysaccharide was further purified using a DEAE-52 anion exchange column. The purified polysaccharide fraction was collected by gradient elution using deionized water, 0.1 M and 0.3 M NaCl solutions at a flow rate of 1 mL / min. The sugar content was detected by the phenol-sulfuric acid method. Finally, the polysaccharide fraction was dialyzed with pure water and freeze-dried to obtain the purified lactic acid bacteria extracellular polysaccharide.

5. A dressing that promotes skin wound healing, characterized in that, The dressing contains extracellular polysaccharides secreted by Lactiplantibacillus plantarum strain T1 with accession number CCTCC M20231553. The extracellular polysaccharides have a molecular weight of 1.41 × 10⁶ Da and are composed of galactose and glucose in a molar ratio of 1.21:1.

00. The extracellular polysaccharides have the activity of inhibiting Staphylococcus aureus biofilm formation and promoting HaCaT cell proliferation.

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