Staphylococcus epidermidis ncu-07, probiotic metabolite and application thereof

Abstract

The application discloses a staphylococcus epidermidis NCU-07, probiotic postbiotic and application, relates to the field of biological medicine technology. The staphylococcus epidermidis NCU-07 is preserved in the China General Microbiological Culture Collection Center on January 18, 2023, and the preservation number is CGMCC NO.26493. The strain is prepared into probiotic postbiotic, and the application of the probiotic postbiotic in combination with silver sulfadiazine cream and compound coptis oil ointment in a drug combination for treating skin burn wounds is provided. The probiotic postbiotic disclosed by the application is helpful to promote the healing of the Ⅱ degree burn wound of a patient, can enhance the therapeutic effect when being used in combination with silver sulfadiazine cream and compound coptis oil ointment, relieve the itching symptoms of the Ⅱ degree burn wound of the patient, has no obvious influence on the pain symptoms of the wound, increases the diversity of skin flora, can inhibit the colonization and proliferation of pathogenic microorganisms, and promote the proliferation of skin symbiotic bacteria.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a strain of Staphylococcus epidermidis NCU-07, probiotic postbiotics, and their applications. Background Technology

[0002] Burns are a common type of trauma worldwide. Currently, the management of second-degree burn wounds treated in hospitals mainly involves debridement of necrotic tissue, application of dressings to protect the wound, and antibiotic treatment to prevent infection. In burn outpatient clinics, patients primarily receive simple dressing changes and bandaging, requiring daily visits for further dressing changes. Furthermore, although silver sulfadiazine is one of the gold standard drugs for treating burn wounds and is widely used clinically, further clinical research has revealed that silver ions (Ag) in SSDs... + While it possesses antibacterial properties, high concentrations may be toxic to host cells, inhibiting the proliferation of keratinocytes and fibroblasts, as well as angiogenesis, thereby delaying epithelialization and granulation tissue formation. Therefore, given the side effects of drugs like silver sulfadiazine and the need for better treatment of outpatient burn patients, there is an urgent need to explore and develop optimized treatment strategies for second-degree skin burns. Summary of the Invention

[0003] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a strain of Staphylococcus epidermidis NCU-07, a probiotic postbiotic and its application.

[0004] For skin burns, this invention screened and obtained a strain of Staphylococcus epidermidis NCU-07, and prepared Staphylococcus epidermidis NCU-07 into a probiotic postbiotic. This invention also proposes a method of applying Staphylococcus epidermidis NCU-07 or the probiotic postbiotic in the treatment of skin burn wounds, combining the probiotic postbiotic with traditional drugs, to evaluate the potential efficacy of the probiotic postbiotic as an adjunct to silver sulfadiazine in the treatment of second-degree burn wounds.

[0005] The technical solution of the present invention is as follows:

[0006] The first aspect of the present invention provides a strain of Staphylococcus epidermidis NCU-07, wherein the Staphylococcus epidermidis ( Staphylococcus epidermidis NCU-07 was deposited on January 18, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.26493 and address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0007] Optionally, the Staphylococcus epidermidis NCU-07 is obtained by screening from lesions of patients with rosacea.

[0008] A second aspect of the present invention provides a probiotic postbiotic prepared using the aforementioned Staphylococcus epidermidis NCU-07.

[0009] A third aspect of the present invention provides a method for preparing the probiotic postbiotic, comprising the following steps:

[0010] S1. Prepare a bacterial suspension of Staphylococcus epidermidis NCU-07, and transfer the bacterial suspension to a solid culture medium for aerobic culture;

[0011] S2. Pick a single colony from the solid culture medium and inoculate it into the liquid culture medium for second-generation culture.

[0012] S3. Take the bacterial culture from the second-generation culture and transfer it to a liquid culture medium for further culture, allowing Staphylococcus epidermidis NCU-07 to grow to the logarithmic growth phase; then centrifuge the culture at low temperature, discard the bottom precipitate, collect the supernatant, filter to remove bacteria and impurities, collect the supernatant, sterilize, and obtain probiotic post-biotics.

[0013] Optionally, the conditions for aerobic culture in S1 include: aerobic culture at 37°C for 24-48 hours;

[0014] The conditions for second-generation culture in S2 include: aerobic culture at 37℃ for 24-48 hours;

[0015] S3 includes the following specific steps: The bacterial culture from the second-generation culture is transferred to a liquid culture medium for further cultivation, allowing *Staphylococcus epidermidis* NCU-07 to grow for 12-16 hours until the logarithmic growth phase. The culture is then centrifuged in a refrigerated high-speed centrifuge at 4°C and 8000 rpm for 10 minutes. The bottom precipitate is discarded, and the supernatant is collected. The supernatant is filtered through a 0.22 μm microporous membrane to remove bacterial cells and impurities. The supernatant is then collected and sterilized at 121°C for 30 minutes to obtain the probiotic post-biotic.

[0016] A fourth aspect of the present invention provides a biological agent whose active ingredient includes the aforementioned Staphylococcus epidermidis NCU-07 or the aforementioned probiotic postbiotic.

[0017] A fifth aspect of the present invention provides an adjuvant drug for the treatment of skin burn wounds, the adjuvant drug comprising the aforementioned Staphylococcus epidermidis NCU-07 or the aforementioned probiotic postbiotic.

[0018] The sixth aspect of the present invention provides the use of the aforementioned Staphylococcus epidermidis NCU-07 or the aforementioned probiotic postbiotic in the preparation of a medicament for treating skin burn wounds.

[0019] The application of the Staphylococcus epidermidis NCU-07 or the probiotic postbiotic described in the seventh aspect of the present invention in combination with silver sulfadiazine cream and compound Coptis chinensis oil in the treatment of skin burn wounds.

[0020] The skin burn wound is a second-degree skin burn wound (between superficial second degree and deep second degree).

[0021] The combined method of use is as follows: apply the probiotic postbiotic and silver sulfadiazine cream to the burn wound on the skin, and then cover it with gauze containing the compound berberine oil ointment.

[0022] This invention has at least one of the following beneficial effects:

[0023] This invention screened and obtained a strain of Staphylococcus epidermidis NCU-07, and prepared this bacterium into a probiotic postbiotic. Experiments showed that this probiotic postbiotic helps promote the healing of second-degree burn wounds and can enhance the therapeutic effect when used in combination with silver sulfadiazine cream and compound Coptis chinensis oil ointment. The probiotic postbiotic can also relieve itching symptoms of second-degree burn wounds, but has no significant effect on wound pain. The probiotic postbiotic increases the diversity of the skin flora, can inhibit the colonization and proliferation of pathogenic microorganisms, and promote the proliferation of symbiotic skin bacteria. Attached Figure Description

[0024] Figure 1 To compare the wound healing of the two groups of patients, A in the figure represents the comparison of healing time between the two groups, B in the figure represents the comparison of the number of patients whose wounds have healed at D8 in the two groups, C in the figure represents the comparison of the number of patients whose wounds have healed at D15 in the two groups, D in the figure represents the wound healing of the experimental group, and E in the figure represents the wound healing of the control group.

[0025] Figure 2 The figures show a comparison of wound pain scores before and after treatment in two groups of patients. Figures A, B, and C show the comparison of wound pain scores before and after treatment in two groups of patients. Figure D shows the visual analog scale (VAS).

[0026] Figure 3 The comparison of wound itching scores before and after treatment in two groups of patients is shown. In the figure, A and B represent the comparison of wound pain scores before and after treatment in the two groups of patients, and C represents the visual analog scale (VAS).

[0027] Figure 4 The Venn diagram shows the ASVs of each group. A1: Control group before treatment; A2: Control group after treatment; B1: Experimental group before treatment; B2: Experimental group after treatment.

[0028] Figure 5 The curves show the sparse distribution of skin microbiota. A1: Control group before treatment; A2: Control group after treatment; B1: Experimental group before treatment; B2: Experimental group after treatment.

[0029] Figure 6This is a curve showing the abundance of skin microbiota samples. A1: Control group before treatment; A2: Control group after treatment; B1: Experimental group before treatment; B2: Experimental group after treatment.

[0030] Figure 7 This study compares the alpha diversity of skin microbiota samples from four groups. A1: Control group before treatment; A2: Control group after treatment; B1: Experimental group before treatment; B2: Experimental group after treatment.

[0031] Figure 8 This is the PCoA graph based on the Jaccard distance matrix. A1: Control group before treatment; A2: Control group after treatment; B1: Experimental group before treatment; B2: Experimental group after treatment.

[0032] Figure 9 This is a PCoA graph based on the weighted unified fast distance matrix. A1: Control group before treatment; A2: Control group after treatment; B1: Experimental group before treatment; B2: Experimental group after treatment.

[0033] Figure 10 This is a diagram showing the top 10 phyla of species. The horizontal axis represents the group name, and the vertical axis represents the relative abundance. A1: Control group before treatment; A2: Control group after treatment; B1: Experimental group before treatment; B2: Experimental group after treatment.

[0034] Figure 11 This is a comparison diagram at the phylum level. A1: Control group before treatment; A2: Control group after treatment; B1: Experimental group before treatment; B2: Experimental group after treatment.

[0035] Figure 12 This is a graph showing the top 20 genera of species. The horizontal axis represents the group name, and the vertical axis represents the relative abundance. A1: Control group before treatment; A2: Control group after treatment; B1: Experimental group before treatment; B2: Experimental group after treatment.

[0036] Figure 13 This is a comparison plot at the species / genus level. * indicates P < 0.05. A1: Control group before treatment; A2: Control group after treatment; B1: Experimental group before treatment; B2: Experimental group after treatment. Detailed Implementation

[0037] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0038] Example 1: Isolation and identification of Staphylococcus epidermidis NCU-07

[0039] In this embodiment, Staphylococcus epidermidis NCU-07 was isolated from the wound of a patient with rosacea. The specific isolation method is as follows:

[0040] I. Separation Experiment Procedure

[0041] 1. Sample Collection and Processing

[0042] Source: Rosacea patient's wound.

[0043] Sampling method: Use a sterile swab dipped in physiological saline to wipe the target area.

[0044] Enrichment culture: The sample was inoculated into brain heart broth containing 7.5% NaCl (BHI broth) and incubated at 37°C for 18-24 hours to enrich salt-tolerant Staphylococcus.

[0045] Gradual dilution: Dilute the sample serially by 10. 0 ~10 7 Choose an appropriate concentration gradient for plating. New samples can be selected at 10... 1 times, 10 3 times, 10 4 times, 10 5 Double the volume, coat 30 μL; after freezing, the sample can be stored for 10 days. 1 times, 10 2 times, 10 3 times, 10 4 Spread 30 μL. Prepare the selected solid culture medium for liquid culture.

[0046] 2. Isolation of solid culture media

[0047] Culture medium selection: ordinary nutrient agar plates (LB, main components are: beef extract, yeast extract, peptone, sodium chloride, agar powder, distilled water).

[0048] Stamping / spreading method: Stamp the enrichment broth onto a plate and incubate at 37°C for 24-48 hours.

[0049] Colony observation: Staphylococcus epidermidis forms white / grayish-white, smooth, moist, round, raised colonies with neat edges on agar.

[0050] II. Screening and Purification

[0051] 1. Initial screening (morphological screening)

[0052] Gram staining: Pick up suspicious colony smears, Gram stain them and examine them under a microscope. Confirm whether they are Gram-positive cocci (purple, arranged in grape-like clusters) based on the shape of the bacteria.

[0053] Catalase test: Add 3% H2O2. If bubbles are produced, the result is positive (all Staphylococcus spp. are catalase positive, which can distinguish them from Streptococcus).

[0054] 2. Purification culture

[0055] Single colony isolation: Pick a single colony, streak it repeatedly onto a blood agar plate, and incubate at 37°C for 24 hours to ensure a pure culture is obtained.

[0056] Preservation: The purified strain can be inoculated onto agar slants or glycerol preservation tubes (for long-term storage at -80℃).

[0057] III. Identification Process

[0058] 1. Biochemical identification

[0059] Coagulase test: Staphylococcus epidermidis is coagulase negative (to distinguish it from Staphylococcus aureus).

[0060] Method: The bacterial colonies were mixed with rabbit plasma and incubated at 37°C for 4 hours. No coagulation was considered negative.

[0061] Oxidase test: negative (excluding Gram-negative bacteria such as Pseudomonas).

[0062] 2. Molecular biological identification

[0063] DNA extraction

[0064] a. Use 30% glycerol to preserve bacteria (500uL of glycerol + 500uL of bacterial solution).

[0065] b. Centrifuge the bacterial culture (8000 rpm, 2 min) and discard the supernatant.

[0066] c. Add 600 μL of lysis buffer (lysis buffer: 500 mM NaCl, 50 mM tris-HCl, pH 8.0, 50 mM EDTA, 4% SDS), 200 μL of Tris-saturated phenol, and 0.3 g–0.4 g of glass beads to the precipitate. Shake for 30 s, repeat 3 times, until the cells are completely resuspended. Centrifuge (8000 rpm, 1 min).

[0067] d. Transfer the supernatant to a new 1.5 mL centrifuge tube, add 250 μL of 10 M ammonium acetate, place on ice for 10 min, and centrifuge (8000 rpm, 1 min).

[0068] e. Transfer the supernatant above the organic layer onto the DNA adsorption column, being careful not to collect the organic layer, and centrifuge (8000 rpm, 1 min).

[0069] f. Wash once with 600 μL of 75% ethanol.

[0070] g. Repeat (f).

[0071] After spinning at 8000 rpm for 2 minutes, transfer the DNA adsorption column to a new EP tube and air dry for 30 minutes.

[0072] i. Add 50 µL of TE (pH=8.0) to the dried centrifuge tube, and send 25 µL for high-throughput sequencing.

[0073] Based on the sequencing results, the bacterial strains were obtained by searching and comparing them on NCBI. Specific strains were selected for secondary identification and then preserved in a bacterial bank.

[0074] The strain isolated after screening was identified as Staphylococcus epidermidis (Staphylococcus epidermidis). Staphylococcus epidermidis It was designated NCU-07. The 16S rDNA sequence of NCU-07 was determined to be as shown in SEQ ID NO. 1.

[0075] SEQ ID NO. 1:

[0076]

[0077] Example 2 Preparation of probiotic postbiotics

[0078] The Staphylococcus epidermidis NCU-07 from Example 1 was prepared as a probiotic postbiotic, and the specific method is as follows:

[0079] (1) Culture of bacterial strain: Staphylococcus epidermidis NCU-07 was prepared into a bacterial suspension. 200 μL of the bacterial suspension was transferred to a solid culture medium (TBS + 2% agar), spread evenly with a glass rod, and cultured at 37°C for 48 h with aerobic conditions.

[0080] (2) Expanding the culture of the strain: pick a single colony from the solid culture medium and inoculate it into TBS liquid culture medium (5 mL). Incubate at 37°C for 48 h in a constant temperature shaker to obtain the second generation of bacterial culture.

[0081] (3) OD value measurement: The cultured Staphylococcus epidermidis was transferred to fresh TBS liquid medium with an inoculation amount of 2%, and mixed into 10 test tubes. The 10 test tubes were placed in an incubator at 37℃. At regular intervals (0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 24 h, 36 h, 48 h), one tube of Staphylococcus epidermidis (3 replicates, 3 mL per well) was taken out and the absorbance of the bacterial solution at 600 nm wavelength was measured with a spectrophotometer. The growth curve was plotted.

[0082] (4) Plate colony counting: The cultured Staphylococcus epidermidis was transferred to fresh TBS liquid medium at an inoculation rate of 2%, and mixed into 10 test tubes. The 10 test tubes were placed in an incubator at 37°C. At regular intervals (0h, 2h, 4h, 6h, 8h, 10h, 12h, 24h, 36h, 48h), one tube of Staphylococcus epidermidis was taken out (two plates were made for each time point and each gradient). 100 µL of bacterial culture was transferred to a sterile centrifuge tube, and 900 µL of sterile triple-distilled water was added to the centrifuge tube. The mixture was thoroughly mixed and diluted to 10:1. -1 Bacterial culture, transfer 100 µL using a pipette. -1 Dilute the bacterial culture with 900 µL of sterile triple-distilled water to a concentration of 10. -2 The bacterial suspension was diluted stepwise to 1×10⁻⁶ using this method. -1 times, 1×10 -2 times, 1×10 -3 times, 1×10 -4 times, 1×10 -5 times, 1×10 -6 times, 1×10 -7 The bacterial culture concentration was 1×10⁻⁶. -5times, 1×10 -6 Multiplied by 1×10 -7 Apply the bacterial solution using a pipette. Transfer 100 µL of bacterial solution to the center of a sterile solid culture medium each time, and spread it using a sterile spreader, gently expanding outwards in concentric circles to ensure even distribution. Let it stand at room temperature for 10 minutes to allow the bacterial solution to immerse in the medium. After spreading, place the petri dish in an inverted 37°C incubator for 24 hours.

[0083] (5) Preparation of supernatant: Take 200 μL of bacterial culture from the second-generation bacterial culture in step (2) and transfer it to a liquid medium containing 6 m L LTBS for further culture until the bacteria reach the logarithmic growth phase (12h-16h). Then centrifuge the bacterial culture in a refrigerated high-speed centrifuge at 4℃ and 8000 r / min for 10 min, discard the bottom precipitate, collect the supernatant, filter it with a 0.22 μm microporous membrane to remove bacterial cells and other impurities, collect the supernatant, sterilize it at 121℃ for 30 min, aseptically aliquot and cool it, and store it at -80℃ for later use to obtain probiotic post-biotics.

[0084] Example 3: Adjunctive therapeutic effect of probiotic post-biotic combined with silver sulfadiazine cream and compound coptis oil ointment in the treatment of second-degree burns (superficial to deep second degree).

[0085] 1. Research subjects and data

[0086] (1) Determining the study subjects: From October 2023 to December 2024, patient recruitment was conducted in the Department of Burns at the First Affiliated Hospital of Nanchang University. All patients signed a written informed consent form before participating in the trial. Ultimately, 80 burn patients hospitalized in the Department of Burns at the First Affiliated Hospital of Nanchang University were selected as the study subjects, regardless of gender.

[0087] (2) Sample and data sources: General clinical data in this study were obtained from the electronic medical record system of the First Affiliated Hospital of Nanchang University. Wound healing status was assessed by researchers and recorded in spreadsheets. Pain and pruritus scores were assessed using the Visual Analogue Scale (VAS) and recorded in spreadsheets. Microbial samples from the wounds of enrolled patients were collected by sterile skin swabs before treatment, on day 8 of treatment, or at the end of treatment. The samples were stored at -80°C and 16S rRNA high-throughput sequencing was performed by Paiseno Biotechnology Co., Ltd.

[0088] (3) Ethical Statement: This study has been approved by the hospital's ethics committee [ethics number: IIT (2023) Linlun Shen No. 151]. All subjects included in the study were informed of the content of this study and have signed informed consent forms. This trial has been registered and filed in the Medical Research Registration and Filing Information System, filing number: MR-36-23-031854.

[0089] (4) Inclusion criteria:

[0090] 1) Age 18-65 years, gender not limited; 2) Patients clinically diagnosed with second-degree burns according to the latest domestic "Burn Surgery" diagnostic criteria, with the causative factors being flame, hot water, steam, metal, etc., but excluding chemical burns and electrical burns, the burn time being within 48 hours, and the total burn area not exceeding 30% of the total surface area; 3) Patients with relatively independent burn wounds on the trunk and limbs that can be used as target wounds, and the burn area of ​​the target wound not exceeding 2% of the total surface area; 4) Burn depth is second-degree burns (between superficial second-degree and deep second-degree); 5) Able to comply with the research procedures in the protocol; 6) Understand and voluntarily sign the informed consent form approved by the ethics committee.

[0091] 2. Research Plan:

[0092] Patients were randomly assigned to control and experimental groups using Excel. 41 burn patients were assigned to the silver sulfadiazine and compound coptis oil treatment group (Group A, control group), and 39 burn patients were assigned to the probiotic combined with silver sulfadiazine and compound coptis oil treatment group (Group B, experimental group). During the study, one participant in Group A withdrew due to voluntary discharge, and three participants withdrew for personal reasons. Four participants in Group B withdrew for personal reasons. Ultimately, 72 participants completed the intervention and were included in the final analysis. The control group (Group A) included 37 burn patients, 31 males (83.80%) and 6 females (16.20%), aged 19-59 years, with a mean age of 45 years (30.50 years, 55.00 years). Of the 20 patients, 54.10% had upper limb burns, 9 had lower limb burns (24.30%), and 8 had trunk burns (21.60%). In addition, 16 patients (43.20%) had flame burns, 15 had scald burns (40.50%), and 6 had electrical spark burns (16.20%). The total burn area ranged from 2% to 29% TBSA, with a mean of 10% (5.00%, 10.00%) TBSA. The experimental group (Group B) included 35 burn patients: 25 males (71.40%) and 10 females (28.60%), aged 20-62 years, with a mean age of 38 years (26.00 years, 53.00 years). Of the 17 participants, 48.60% had upper limb burns, 7 had lower limb burns (20.00%), and 11 had trunk burns (31.40%). In addition, 12 cases (34.30%) were flame burns, 15 cases (42.90%) were hot liquid burns, and 8 cases (22.90%) were electrical spark burns. The total burn area ranged from 2% to 29% TBSA, with an average of 10% (6.00%, 10.00%) TBSA. There were no statistically significant differences in demographic and clinical characteristics between the two groups (P>0.05). Furthermore, there were no statistically significant differences in vital signs, white blood cell count, platelet count, and glucose levels between the two groups at enrollment (P>0.05). Therefore, the two groups were homogeneous. See Table 1 for details.

[0093] Table 1. Comparison of baseline characteristics and clinical data between the experimental and control groups.

[0094]

[0095] Note 1: "-" indicates that there is no such statistic. All test statistics in the table are absolute values ​​of the test statistics.

[0096] Note 2: The average age, burn area, T, R, P, SBP, DBP, white blood cells, platelets, glucose, total bilirubin PT, and APTT data in Table 1 are represented by the median (interquartile range) M (P25, P75) because they do not conform to a normal distribution. M is the median, P25 indicates that 25% of the data are less than or equal to this value, and P75 indicates that 75% of the data are less than or equal to this value.

[0097] Note 3: TBSA represents the total body surface area.

[0098] To evaluate whether Staphylococcus epidermidis postbiotics can aid in the healing of second-degree burn wounds, two groups of patients were treated with either silver sulfadiazine cream plus compound coptis oil ointment (Group A, n=37) or probiotic postbiotics plus silver sulfadiazine cream plus compound coptis oil ointment (Group B, n=35). The wound healing time and the number of patients who had healed at days 8 and 15, or at the time of wound healing, were monitored in both groups. The results are as follows:

[0099] 1. Comparison of wound healing time between the two groups of patients

[0100] The wound healing time (days) data of the two groups of patients were found to be non-normally distributed. The median (interquartile range) was used for statistical description, and the Mann-Whitney test was employed. P=0.002 (P<0.05). The results showed that, compared with the control group (Group A), the overall wound healing time of burn patients was shortened after the addition of probiotics (Shengyuan). The difference in healing time between the two groups was statistically significant (P<0.05). See Table 2 for details. Figure 1 A, D, and E in the diagram.

[0101] Table 2 Comparison of wound healing time between the two groups of patients (days)

[0102]

[0103] Note: All test statistics in the table are absolute values ​​of the test statistics.

[0104] 2. Comparison of the number of patients whose wounds healed at day 8 between the two groups.

[0105] A comparative analysis of the difference in the number of patients who had healed between the two groups at day 8 yielded a p-value of P=0.002 (P<0.05) using a chi-square test, indicating a statistically significant difference in wound healing between the two groups at day 8 (P<0.05). See Table 3 for details. Figure 1 B in the middle.

[0106] Table 3 Comparison of the number of patients whose wounds healed at day 8 between the two groups (cases)

[0107]

[0108] Note: All test statistics in the table are absolute values ​​of the test statistics.

[0109] 3. Comparison of the number of patients whose wounds healed at day 15 between the two groups.

[0110] A comparative analysis of the difference in the number of patients who had healed between the two groups at day 15 showed a p-value of >0.99 (P>0.05) using a chi-square test. This indicates that there was no statistically significant difference in wound healing between the two groups at day 15 (P>0.05), suggesting that regardless of the addition of probiotics or postbiotics, wounds in both groups healed within 15 days. See Table 4 for details. Figure 1 C in the middle.

[0111] Table 4 Comparison of the number of patients whose wounds healed at day 15 between the two groups (cases)

[0112]

[0113] Note: All test statistics in the table are absolute values ​​of the test statistics.

[0114] 4. Analysis of the relief of main symptoms before and after treatment

[0115] To compare the differences in wound pain and itching between the two groups of patients during the wound healing process, wound pain was scored on days 0, 4, and at the end of treatment (EOT), and wound itching was scored on days 0 and at the end of treatment (VAS visual analog scale).

[0116] (1) Comparison of wound pain scores before and after treatment between the two groups of patients

[0117] The patient's pain level was scored using the VAS (Visual Analogue Scale). Figure 2 (D) The wound pain scores of the two groups of patients before and after treatment were found to be non-normally distributed. The median (interquartile range) was used for statistical description, and the Mann-Whitney test was employed. Results showed that, regardless of whether probiotics or post-probiotics were added, there were no significant differences in pain scores between the two groups at D0, D4, and the end of treatment (EOT). P >0.05). See Table 5 for details. Figure 2 .

[0118] Table 5 Comparison of wound pain scores before and after treatment in the two groups of patients.

[0119]

[0120] Note: All test statistics in the table are absolute values ​​of the test statistics.

[0121] (2) Comparison of wound itching scores before and after treatment between the two groups of patients

[0122] Pain levels were assessed using the VAS (Visual Analogue Scale). The wound itching scores before and after treatment in both groups were found to be non-normally distributed. The median (interquartile range) was used for statistical description, and the Mann-Whitney test was employed. Results showed that the probiotic-added group (experimental group) significantly alleviated wound itching symptoms during treatment (P<0.05). See Table 6 for details. Figure 3 .

[0123] Table 6 Comparison of wound itching scores before and after treatment in the two groups of patients.

[0124]

[0125] Note: All test statistics in the table are absolute values ​​of the test statistics.

[0126] 5. 16S rRNA high-throughput sequencing

[0127] Wound swab samples were collected from patients within the designated timeframe. Immediately after collection, the samples were stored at -80°C and transported to Paisenuo Biotechnology Co., Ltd. via dry ice for batch sequencing. 16S rDNA amplicon sequencing was performed by professionals from Paisenuo Biotechnology Co., Ltd., including sample pretreatment, extraction and quantification of total microbiome DNA, PCR amplification of bacterial 16S rRNA genes, PCR product quantification and pooling, library construction, and sequencing. The sequencing region was the bacterial 16S rRNA V3V4(a) region. The upstream primer was 338F: CTCCTACGGGAGGCAGCA (SEQ ID NO. 2), and the downstream primer was 806R: GGACTACHVGGGTWTCTAAT (SEQ ID NO. 3). The sequencing instrument was an Illumina NovaSeq6000 sequencer. After obtaining the data from the anatomical machine, DADA2 was used to process the raw data to obtain high-quality amplicon sequence variants (ASVs). These were then compared with the database and annotated by species. Based on the annotation results of ASVs and species, the alpha diversity and species composition of the bacterial community were analyzed.

[0128] The sequencing results are analyzed as follows:

[0129] Studies have shown that the colonization and growth of pathogenic microorganisms can destroy newly formed epithelial cells and extracellular matrix by secreting toxins and enzymes, or inhibit the healing process by forming biofilms. Therefore, microbial samples from patients with second-degree burn wounds were collected and sequenced. Ninety-nine skin microbial samples were collected from the lesion site before treatment and on day 8 or at the end of treatment, including 24 samples from group A1, 25 samples from group A2, 24 samples from group B1, and 26 samples from group B2. A1: control group before treatment; A2: control group after treatment; B1: experimental group before treatment; B2: experimental group after treatment. Subsequently, 16S rRNA sequencing analysis was performed on these samples to investigate changes in the skin microbiome of the burn wound.

[0130] (1) Basic analysis of sequencing data

[0131] Venn diagrams, as a visualization tool, can clearly represent the relationships between sets, count the number of members in each set, and thus intuitively show the presence or absence of species between groups, as well as the number of species shared between groups. Venn diagrams drawn according to different groupings are shown below ( Figure 4 The results showed that 145 shared taxa units (ASVs) existed in the four groups of microbial samples, with the total number of ASVs in the order B2>B1>A1>A2. Groups A1 and A2 had a total of 54 ASVs, while groups B1 and B2 had a total of 197 ASVs.

[0132] (2) α-diversity analysis

[0133] 1) Sparse curves: The number of sequences in four groups of a total of 99 samples was evaluated using sparse curves, such as... Figure 5 The impact of sequencing depth on the diversity of observed samples is reflected by the flatness of the curve. When the curve tends to flatten, it indicates that the sequencing data has sufficiently covered the biodiversity information present in the samples. The results show that these sparse curves exhibit a gradually flattening trend, indicating that the number of sequences in each group of samples is sufficient, meeting the adequate conditions for conducting microbial diversity analysis.

[0134] 2) Abundance grade curve

[0135] Abundance ranking curves can describe the richness and evenness of species within a sample. The evenness of community composition is characterized by the smoothness of the curve's shape, and there is a direct correlation between curve smoothness and evenness: when the curve tends to be smooth, the abundance differences between taxa are smaller, and the evenness of community composition is higher; conversely, an increased curve steepness reflects a lower degree of homogeneity in species distribution. The greater the species diversity and breadth in a sample, the better. Figure 6As shown, each group of samples exhibits a large curve span in the horizontal direction, especially after the addition of probiotics and biogener (Group B2), indicating that the community is relatively rich in species.

[0136] 3) α Diversity Index

[0137] Alpha diversity is used to assess the species composition characteristics of locally homogeneous habitats. Key indicators include richness, diversity, and evenness. In this study, richness and diversity were characterized by the species richness estimation index, species richness index, and Shannon index, respectively. The species richness estimation index and species richness index values ​​were positively correlated with community richness, while the Shannon index value was positively correlated with community diversity. A higher sequencing coverage index indicates a lower proportion of undetected species in the sample. As shown in Table 7, the sequencing coverage index for each group of samples was above 99%, indicating that the sequences in the samples were sufficiently detected, and most species were covered. Subsequently, the species richness estimation, species richness index, and Shannon index for each group of samples were analyzed, and plotted... Figure 7 illustrate.

[0138] Table 7 Sequencing coverage index values ​​of four groups of samples

[0139]

[0140] According to the results, as shown in Table 8, on day 8 of treatment in the control group (A2), the estimated species richness index and species richness index were significantly lower than before treatment (A1), with statistically significant differences (P<0.05). The Shannon index was slightly lower, but not statistically significant (P>0.05). In contrast, after treatment in the experimental group (B2), as shown in Table 9, the estimated species richness, Shannon index, and species richness index were significantly higher than before treatment, with statistically significant differences (P<0.05). Furthermore, the estimated species richness, Shannon index, and species richness index after treatment in the experimental group and the control group were statistically different (P<0.05) compared to the control group. These results indicate that combined probiotic and biogenic treatment increased the alpha diversity of the patient's wound.

[0141] Table 8 Comparison of skin microbiota α diversity index before and after treatment in the control group

[0142]

[0143] Note: All test statistics in the table are absolute values ​​of the test statistics.

[0144] Table 9 Comparison of skin microbiota α diversity index before and after treatment in the experimental group

[0145]

[0146] Note: All test statistics in the table are absolute values ​​of the test statistics.

[0147] Table 10 Comparison of skin microbiota α diversity index between control group and experimental group after treatment.

[0148]

[0149] Note: All test statistics in the table are absolute values ​​of the test statistics.

[0150] 4) Beta diversity analysis

[0151] The Beta diversity index focuses on comparing diversity across different habitats, i.e., the differences between samples. Principal coordinate analysis (PCoA, also known as classic multidimensional scaling CMDS) is a dimensionality reduction technique based on a distance matrix, used to visualize data points in a low-dimensional space while preserving their original distance structure as much as possible.

[0152] This study primarily uses weighted unified fast distance and Jaccard distance for principal coordinate analysis. The results show that, Figure 8 , Figure 9 After treatment, the samples from the probiotic group without added probiotics and the probiotic group with added probiotics showed a gradual separation trend, with most samples not overlapping, indicating that there were significant differences in the skin wound flora between the groups.

[0153] 5) Analysis of the composition of skin microbiota

[0154] To analyze the microbial community composition before and after skin wound treatment in the two groups of patients, relative abundance distribution maps of the microbial communities were plotted at the phylum and genus levels. Differences were then compared among species with high relative abundance or those associated with burns. At the phylum level, the most common populations in these four treatment groups were Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes, accounting for 95.23%, 96.96%, 94.49%, and 98.23% of the total sequencing results in groups A1, A2, B1, and B2, respectively. Figure 10 Table 11 lists the percentage of the top four phyla in each group. Further analysis shows that after treatment, the abundance of Actinobacteria and Proteobacteria increased in the control group, while the abundance of Firmicutes increased in the experimental group. Figure 11 However, none of these differences were statistically significant (P>0.05).

[0155] Table 11 Abundance at the phylum level

[0156]

[0157] At the genus level, we selected representative skin microbiota closely related to both beneficial and infectious burn wounds for further analysis. Table 12 lists the specific proportions of Staphylococcus, Propionibacterium, Streptococcus, and Pseudomonas in each group. Studies have shown that Staphylococcus epidermidis can promote wound healing, while Propionibacterium has a dual nature; it can normally inhibit pathogens, and certain strains also have beneficial functions. Representative bacteria from the other two genera are Streptococcus pyogenes and Pseudomonas aeruginosa, which are common in burn infections. Further analysis revealed that, as... Figure 12 , Figure 13 In burn patients, after treatment, the abundance of Staphylococcus spp. in the group receiving Staphylococcus epidermidis postbiotic supplementation (Group B2) significantly increased compared to before treatment, with a statistically significant difference (P<0.05). Among Propionibacterium spp., the abundance in the experimental group slightly increased after treatment, while the abundance in the control group slightly decreased, with no statistically significant difference (P>0.05). While there was no statistically significant difference in the abundance of Streptococcus and Pseudomonas spp. between the two groups, the abundance increased in the control group and decreased in the experimental group. This indicates that probiotic postbiotics can promote the colonization and proliferation of dermal symbiotic bacteria and reduce the abundance of pathogenic bacteria.

[0158] Table 12 Species-Generation Level Abundance

[0159]

[0160] In summary, based on the above experiments, we can conclude that:

[0161] 1. Compared with the control group (Group A), the addition of Staphylococcus epidermidis significantly shortened the overall wound healing time in burn patients (P<0.05). The healing time in the control group was 9 (8.00, 10.00) days, while the healing time in the experimental group was 7 (7.00, 8.00) days. At day 8, 13 (28.60%) patients in the control group had wound healing, while 25 (71.40%) patients in the experimental group had wound healing. There was a statistically significant difference in the number of patients with wound healing between the two groups at day 8 (P<0.05).

[0162] 2. During treatment, the wound itching score in the Shengyuan group (experimental group) was significantly lower than that in the control group after the addition of Staphylococcus epidermidis (P<0.05). There were no significant differences in wound pain scores between the two groups before intervention, on the fourth day after intervention, and at the end of treatment (P>0.05).

[0163] 3. Basic data on skin microbiota sequencing: 99 samples were collected and sequenced, including 24 samples from group A1 (control group before treatment), 25 samples from group A2 (control group after treatment), 24 samples from group B1 (experimental group before treatment), and 26 samples from group B2 (experimental group after treatment). The total number of ASVs in the four groups was B2>B1>A1>A2.

[0164] 4. In the experimental group, after intervention, the estimated species richness, Shannon index, and species richness index of α-diversity significantly increased compared to before treatment, with statistically significant differences (P<0.05). Conversely, in the control group, the estimated species richness index and species richness index significantly decreased after intervention compared to before intervention (P<0.05). In β-diversity analysis, principal coordinate analysis using weighted unified fast distance and Jaccard distance showed that after treatment, the prebiotic group without probiotics and the prebiotic group with Staphylococcus epidermidis showed a gradual trend of separation, with most samples not overlapping, indicating differences in the skin wound microbiota between the groups.

[0165] 5. Through comparisons of abundance at the phylum and genus levels between groups, after intervention, the relative abundance of Firmicutes in the wound microbiota of the experimental group increased compared to the control group. At the genus level, the abundance of Staphylococcus in the experimental group significantly increased after treatment compared to before treatment, with a statistically significant difference (P<0.05). Among Propionibacterium, the abundance slightly increased in the experimental group after treatment, while it slightly decreased in the control group. Among pathogenic genera, the abundance of Streptococcus and Pseudomonas decreased slightly in the experimental group.

[0166] Therefore, the following conclusions can be drawn from the above experimental results:

[0167] 1. Staphylococcus epidermidis postbiotics help promote the healing of second-degree burn wounds and can enhance the therapeutic effect when used in combination with silver sulfadiazine cream and compound berberine oil ointment.

[0168] 2. Staphylococcus epidermidis metabiotic can relieve itching symptoms of second-degree burn wounds, but has no significant effect on wound pain symptoms.

[0169] 3. Staphylococcus epidermidis postbiotics increase the diversity of skin flora, can inhibit the colonization and proliferation of pathogenic microorganisms, and promote the proliferation of skin symbiotic bacteria.

[0170] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A medicament for treating a skin burn wound, characterized by, The drugs include Staphylococcus epidermidis (S. epidermidis) Staphylococcus epidermidis ) NCU-07, and sulfadiazine silver cream and compound coptis oil paint; The Staphylococcus epidermidis NCU-07 is preserved in the China General Microbiological Culture Collection Center on January 18, 2023, and the preservation number is CGMCC NO. 26493, and the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing; The Staphylococcus epidermidis NCU-07 is screened from the wound surface of a patient with rosacea; The skin burn wound is a skin Ⅱ degree burn wound; The preparation method of the Staphylococcus epidermidis NCU-07 probiotic includes the following steps: S1, Staphylococcus epidermidis NCU 07 Prepare into bacterial liquid, transfer the bacterial liquid to solid culture medium for aerobic culture; S2, picking a single colony on the solid culture medium and inoculating it into a liquid culture medium for secondary culture; S3, the bacteria in the second generation of culture were collected and cultured in liquid medium to obtain Staphylococcus epidermidis NCU-03 postbiotic. 07 grown to logarithmic phase; the culture solution was centrifuged at low temperature, the bottom precipitate was discarded, the supernatant was collected, filtered to remove bacteria and impurities, the supernatant was collected again, sterilized, and Staphylococcus epidermidis NCU-07 postbiotic was obtained.

2. Use of Staphylococcus epidermidis NCU-07 probiotic in the preparation of a medicament for the treatment of skin burn wounds, characterized in that, The drug for treating the skin burn wound includes the Staphylococcus epidermidis NCU-07 probiotic, sulfadiazine silver cream and compound coptis oil; The Staphylococcus epidermidis NCU-07 is preserved in the China General Microbiological Culture Collection Center on January 18, 2023, and the preservation number is CGMCC NO. 26493, and the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing; The Staphylococcus epidermidis NCU-07 is screened from the wound surface of a patient with rosacea; The skin burn wound is a skin Ⅱ degree burn wound; The preparation method of the Staphylococcus epidermidis NCU-07 probiotic includes the following steps: S1, Staphylococcus epidermidis NCU 07 prepare into a bacterial solution, and transfer the bacterial solution to a solid culture medium for aerobic culture; S2, picking a single colony on the solid culture medium and inoculating it into a liquid culture medium for secondary culture; S3, the bacteria in the second generation of culture were collected and cultured in liquid medium to obtain the S. epidermidis NCU-03 postbiotic. 07, the bacteria were grown to logarithmic phase, and the culture was centrifuged at low temperature. The precipitate was discarded, and the supernatant was collected, filtered to remove bacteria and impurities, and collected again. The supernatant was sterilized to obtain the S. epidermidis NCU-07 postbiotic.

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