Staphylococcus epidermidis NCU-07, probiotic postbiotics and application of staphylococcus epidermidis NCU-07 and probiotic postbiotics

Abstract

The invention discloses staphylococcus epidermidis NCU-07, a probiotic metagen and application, and relates to the technical field of biological medicine. The staphylococcus epidermidis NCU-07 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on January 18, 2023, and the preservation number is CGMCC NO.26493. The staphylococcus epidermidis NCU-07 is preserved in the China General Microbiological Culture Collection The bacterial strain is prepared into the probiotic metagen, and the application of the probiotic metagen, sulfadiazine silver cream and a compound coptis chinensis oil liniment in a medicine composition for treating skin burn wounds is provided in a combined mode of the probiotic metagen, the sulfadiazine silver cream and the compound coptis chinensis oil liniment. The probiotic metagen disclosed by the invention is beneficial to promoting the healing of the II-degree burn wound of a patient, can enhance the treatment effect when being combined with the sulfadiazine silver cream and the compound coptis oil liniment, relieves the itching symptom of the II-degree burn wound of the patient, has no obvious influence on the pain symptom of the wound, increases the diversity of skin florae, and can be used for treating the II-degree burn wound of the patient. Colonization and proliferation of pathogenic microorganisms can be inhibited, and proliferation of skin symbiotic bacteria is promoted.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a strain of Staphylococcus epidermidis NCU-07, probiotic postbiotics, and their applications. Background Art

[0002] Burn is a common type of trauma worldwide. Currently, the treatment of second-degree burn wounds in hospitalized patients mainly involves removing necrotic tissues, protecting the wound with external dressings, and using antibiotics to prevent infection. In burn outpatient clinics, patients are mainly given simple dressing changes and bandaging, and then they need to come to the outpatient clinic for dressing changes every day. In addition, although silver sulfadiazine is one of the gold standard drugs for treating burn wounds and is widely used clinically, with the in-depth clinical research, it has been found that silver ions (Ag + ) in SSD have antibacterial effects, but at high concentrations, they may be toxic to host cells, inhibit the proliferation of keratinocytes and fibroblasts, and angiogenesis, thereby delaying epithelialization and granulation tissue formation. Therefore, in view of the side effects of drugs such as silver sulfadiazine and better treatment for outpatient burn patients, there is an urgent need to explore and develop optimized treatment strategies for second-degree skin burn wounds. Summary of the Invention

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

[0004] For skin burns, the present invention screened a strain of Staphylococcus epidermidis NCU-07, and prepared Staphylococcus epidermidis NCU-07 into probiotic postbiotics. The present invention also proposes to apply Staphylococcus epidermidis NCU-07 or probiotic postbiotics in drugs for treating skin burn wounds, and combine probiotic postbiotics with traditional drugs to evaluate the potential efficacy of probiotic postbiotics in assisting silver sulfadiazine in treating second-degree burn wounds.

[0005] The technical solution of the present invention is as follows: In the first aspect of the present invention, a strain of Staphylococcus epidermidis NCU-07 is provided, and the Staphylococcus epidermidis ( Staphylococcus epidermidis ) NCU-07 was deposited on January 18, 2023 at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, deposit number: CGMCC NO.26493, deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] Optionally, the Staphylococcus epidermidis NCU-07 was screened from the wounds of patients with rosacea.

[0007] In the second aspect of the present invention, probiotic postbiotics prepared by using the Staphylococcus epidermidis NCU-07 are provided.

[0008] The third aspect of the present invention provides a method for preparing the probiotic postbiotic as described above, comprising the following steps: S1. Prepare a bacterial solution of Staphylococcus epidermidis NCU-07, and transfer the bacterial solution to a solid medium for aerobic culture; S2. Pick a single colony from the solid medium and inoculate it into a liquid medium for secondary culture; S3. Aspirate the bacterial solution from the bacterium solution of the secondary culture into a liquid medium for continuous culture until Staphylococcus epidermidis NCU-07 grows to the logarithmic growth phase; then centrifuge the culture solution at low temperature, discard the bottom precipitate, collect the supernatant, filter to remove the bacteria and impurities, collect the supernatant, sterilize it to obtain the probiotic postbiotic.

[0009] Optionally, the conditions for aerobic culture in S1 include: aerobic culture at 37 °C for 24 h - 48 h; The conditions for secondary culture in S2 include: aerobic culture at 37 °C for 24 h - 48 h; S3 includes the following specific steps: Aspirate the bacterial solution from the bacterium solution of the secondary culture into a liquid medium for continuous culture until Staphylococcus epidermidis NCU-07 grows for 12 h - 16 h to the logarithmic growth phase; then centrifuge the culture solution in a refrigerated high-speed centrifuge at 4 °C and 8000 r / min for 10 min, discard the bottom precipitate, collect the supernatant, filter to remove the bacteria and impurities with a 0.22 μm microporous membrane, collect the supernatant, and sterilize it at 121 °C for 30 min to obtain the probiotic postbiotic.

[0010] The fourth aspect of the present invention provides a biological preparation, the active ingredient of which includes the Staphylococcus epidermidis NCU-07 as described above or the probiotic postbiotic.

[0011] The fifth aspect of the present invention provides an auxiliary drug for the treatment of skin burn wounds, and the auxiliary drug includes the Staphylococcus epidermidis NCU-07 as described above or the probiotic postbiotic.

[0012] The sixth aspect of the present invention provides the application of the Staphylococcus epidermidis NCU-07 as described above or the probiotic postbiotic in the preparation of drugs for treating skin burn wounds.

[0013] The seventh aspect of the present invention provides the application of the Staphylococcus epidermidis NCU-07 as described above or the probiotic postbiotic in combination with silver sulfadiazine cream and compound Coptis chinensis oil paint in the drug combination for treating skin burn wounds.

[0014] The skin burn wound is a skin second-degree burn wound (between superficial second-degree and deep second-degree).

[0015] The combined usage method is as follows: apply the probiotic postbiogen and silver sulfadiazine cream to the skin burn wound surface, and then cover it with a gauze containing the compound Coptis chinensis oil preparation.

[0016] The present invention has at least one of the following beneficial effects: The present invention screened out a strain of Staphylococcus epidermidis NCU-07 and prepared the strain into a probiotic postbiogen. Through experiments, it was found that the probiotic postbiogen helps to promote the healing of patients' second-degree burn wounds, and can enhance the therapeutic effect when used in combination with silver sulfadiazine cream and compound Coptis chinensis oil preparation. The probiotic postbiogen can also relieve the itching symptoms of patients' second-degree burn wounds, but has no obvious effect on the wound pain symptoms. The probiotic postbiogen increases the diversity of skin flora, can inhibit the colonization and proliferation of pathogenic microorganisms, and promote the proliferation of skin symbiotic bacteria. Description of the Drawings

[0017] Figure 1 For the comparison of the wound healing conditions of two groups of patients, in the figure, A is the comparison of the healing times of the two groups, B is the comparison of the number of patients with wound healing at D8 in the two groups, C is the comparison of the number of patients with wound healing at D15 in the two groups, D is the wound healing condition of the patients in the experimental group, and E is the wound healing condition of the patients in the control group.

[0018] Figure 2 For the comparison of the wound pain scores of two groups of patients before and after treatment. Among them, A, B, and C in the figure are the comparisons of the wound pain scores of two groups of patients before and after treatment, and D in the figure is the visual analogue scale (VAS).

[0019] Figure 3 For the comparison of the wound itching scores of two groups of patients before and after treatment. Among them, A and B in the figure are the comparisons of the wound pain scores of two groups of patients before and after treatment, and C in the figure is the visual analogue scale (VAS).

[0020] Figure 4 For the Venn diagram showing the ASVs of each group of samples. A1: before treatment in the control group; A2: after treatment in the control group; B1: before treatment in the experimental group; B2: after treatment in the experimental group.

[0021] Figure 5 For the rarefaction curve of skin flora samples. A1: before treatment in the control group; A2: after treatment in the control group; B1: before treatment in the experimental group; B2: after treatment in the experimental group.

[0022] Figure 6 For the abundance rank curve of skin flora samples. A1: before treatment in the control group; A2: after treatment in the control group; B1: before treatment in the experimental group; B2: after treatment in the experimental group.

[0023] Figure 7Comparison of α-diversity of four groups of skin microbiota samples. A1: Before treatment in the control group; A2: After treatment in the control group; B1: Before treatment in the experimental group; B2: After treatment in the experimental group.

[0024] Figure 8 PCoA plot based on Jaccard distance matrix. A1: Before treatment in the control group; A2: After treatment in the control group; B1: Before treatment in the experimental group; B2: After treatment in the experimental group.

[0025] Figure 9 PCoA plot based on weighted UniFrac distance matrix. A1: Before treatment in the control group; A2: After treatment in the control group; B1: Before treatment in the experimental group; B2: After treatment in the experimental group.

[0026] Figure 10 Composition diagram of the top 10 phyla of species. The abscissa in the figure is the name of each group, and the ordinate is the relative abundance. A1: Before treatment in the control group; A2: After treatment in the control group; B1: Before treatment in the experimental group; B2: After treatment in the experimental group.

[0027] Figure 11 Comparison diagram of species at the phylum level. A1: Before treatment in the control group; A2: After treatment in the control group; B1: Before treatment in the experimental group; B2: After treatment in the experimental group.

[0028] Figure 12 Composition diagram of the top 20 genera of species. The abscissa in the figure is the name of each group, and the ordinate is the relative abundance. A1: Before treatment in the control group; A2: After treatment in the control group; B1: Before treatment in the experimental group; B2: After treatment in the experimental group.

[0029] Figure 13 Comparison diagram of species at the genus level. * indicates P < 0.05. A1: Before treatment in the control group; A2: After treatment in the control group; B1: Before treatment in the experimental group; B2: After treatment in the experimental group. Detailed implementation mode

[0030] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention clearer, the present 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 only used to explain the present invention and are not used to limit the present invention.

[0031] Example 1 Isolation and identification of Staphylococcus epidermidis NCU-07 Staphylococcus epidermidis NCU-07 in this example was isolated from the wound surface of a rosacea patient. The specific isolation method is as follows: I. Isolation experiment process 1. Sample collection and processing Source: Wound surface of rosacea patients.

[0032] Sampling method: Use a sterile swab to dip into normal saline and then wipe the target area.

[0033] Enrichment culture: Inoculate the sample into Brain Heart Infusion Broth (BHI broth) containing 7.5% NaCl and culture at 37°C for 18 - 24 hours to enrich salt-tolerant Staphylococcus.

[0034] Serial dilution: Serial dilute the sample 10 0 ~10 7 times, select an appropriate concentration gradient for plating. For new samples, 10 1 times, 10 3 times, 10 4 times, 10 5 times can be selected, and plate 30 μL; for samples after cryopreservation, 10 1 times, 10 2 times, 10 3 times, 10 4 times can be plated with 30 μL. Prepare the selected solid medium for liquid culture.

[0035] 2. Isolation on solid medium Medium selection: Ordinary nutrient agar plate (LB, main components: beef extract, yeast extract, peptone, sodium chloride, agar powder, distilled water).

[0036] Coating / streaking method: Take the enrichment broth and streak inoculate it on the plate, culture at 37°C for 24 - 48 hours.

[0037] Colony observation: Staphylococcus epidermidis forms white / grayish-white, smooth, moist, neatly edged, round convex colonies on agar.

[0038] II. Screening and purification 1. Primary screening (morphological screening) Gram staining: Pick a suspicious colony, smear it, perform Gram staining and then examine it under the microscope to confirm whether it is Gram-positive cocci (purple, arranged in grape-like clusters) according to the shape of the bacteria, etc.

[0039] Catalase test: Drop 3% H2O2, those that produce bubbles are positive (all Staphylococcus are catalase positive, which can be distinguished from Streptococcus).

[0040] 2. Purification culture Isolation of single colony: Pick a single colony, repeatedly streak inoculate it on a blood agar plate, and culture at 37°C for 24 hours to ensure obtaining a pure culture.

[0041] Preservation: The purified strain can be inoculated on an agar slant or in a glycerol preservation tube (long-term preservation at -80°C).

[0042] III. Identification process 1. Biochemical identification Coagulase test: Staphylococcus epidermidis is coagulase-negative (to distinguish from Staphylococcus aureus).

[0043] Method: Mix the colony with rabbit plasma and incubate at 37°C for 4 hours. No coagulation is negative.

[0044] Oxidase test: Negative (to exclude Gram-negative bacteria such as Pseudomonas).

[0045] 2. Molecular biology identification DNA extraction a. Preserve the bacteria with 30% glycerol (500 μL glycerol + 500 μL bacterial solution).

[0046] b. Centrifuge the bacterial solution (8000 rpm, 2 min) and discard the supernatant.

[0047] c. Add 600 μL of lysis solution (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, 3 times, until the bacteria are completely suspended, and centrifuge (8000 rpm, 1 min).

[0048] 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).

[0049] e. Take the supernatant above the organic layer onto the DNA adsorption column, do not take the organic layer, and centrifuge (8000 rpm, 1 min).

[0050] f. Wash once with 600 μL of 75% ethanol.

[0051] g. Repeat (f).

[0052] h. Spin dry at 8000 rpm for 2 min, then transfer the DNA adsorption column to a new ep tube and air dry for 30 min.

[0053] i. Add 50 μL of TE (pH = 8.0) to the dried centrifuge tube, and send 25 μL for high-throughput sequencing.

[0054] According to the sequencing result sequence, search and compare on NCBI to obtain the strain result. Select special bacteria for secondary identification and then preserve them in the strain bank.

[0055] After identification, the isolated strain is Staphylococcus epidermidis ( Staphylococcus epidermidis), and it was numbered NCU-07. After determination, the 16S rDNA sequence of NCU-07 is as shown in SEQ ID NO. 1.

[0056] SEQ ID NO. 1:

[0057] Example 2 Preparation of Probiotic Postbiotics The Staphylococcus epidermidis NCU-07 in Example 1 was prepared into probiotic postbiotics, and the specific method is as follows: (1) Cultivation of the strain: The Staphylococcus epidermidis NCU-07 was formulated into a bacterial solution. 200 μL of the bacterial solution was transferred to a solid medium (TBS + 2% agar), spread evenly with a glass rod, and aerobically cultured at 37 °C for 48 h.

[0058] (2) Subculture of the strain: A single colony was picked from the solid medium and inoculated into a TBS liquid medium (5 mL), and cultured in a constant temperature shaker at 37 °C for 48 h to obtain a second-generation bacterial solution.

[0059] (3) Measuring the OD value: The cultured Staphylococcus epidermidis was transferred to a fresh TBS liquid medium for inoculation, and the inoculation amount was 2%. The mixture was filled into 10 test tubes, and the 10 test tubes were placed in a 37 °C incubator for cultivation. One tube of Staphylococcus epidermidis (3 replicates, 3 mL per well) was taken out 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), and the absorbance of the bacterial solution at a wavelength of 600 nm was detected with a spectrophotometer to plot the growth curve.

[0060] (4) Plate colony counting: The cultured Staphylococcus epidermidis was transferred to a fresh TBS liquid medium for inoculation, and the inoculation amount was 2%. The mixture was filled into 10 test tubes, and the 10 test tubes were placed in a 37 °C incubator for cultivation. One tube of Staphylococcus epidermidis (2 plates were coated for each gradient at each time point) was taken out at regular intervals (0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, 36 h, 48 h). 100 μL of the bacterial solution was pipetted into a sterile centrifuge tube, 900 μL of sterile triple-distilled water was added to the centrifuge tube, and mixed thoroughly to dilute it to 10 -1 bacterial solution. 100 μL of 10 -1 bacterial solution was taken, 900 μL of sterile triple-distilled water was added, and diluted to 10 -2 bacterial solution. Dilute it step by step in this way to 1×10 -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 times. Take the bacterial solution concentrations of 1×10 -5 times, 1×10 -6 times and 1×10-7 Coat it in multiples. Use a pipette to transfer 100 µL of the bacterial solution to the central position of a sterile solid medium each time, and use a sterile spreader to spread it, gently expanding the bacterial solution outward along the concentric circle direction first to make it evenly distributed. Let it stand at room temperature for 10 min to allow the bacterial solution to soak into the medium. After coating, place the petri dish in an incubator at 37°C and incubate it upside down for 24 h.

[0061] (5) Prepare the supernatant: Pipette 200 μL of the bacterial solution from the second-generation bacterial solution in step (2) into 6 mL of LTBS liquid medium and continue culturing until the bacteria grow to the logarithmic growth phase (12 h - 16 h). Then centrifuge the bacterial solution in a refrigerated high-speed centrifuge at 4°C and 8000 r / min for 10 min, discard the bottom precipitate, collect the supernatant, filter it through a 0.22-μm microporous membrane to remove bacteria and other impurities, collect the supernatant, sterilize it at 121°C for 30 min, and aseptically dispense and cool it, then store it at -80°C for later use to obtain probiotic postbiotics.

[0062] Example 3 Adjuvant efficacy of probiotic postbiotics combined with silver sulfadiazine cream and compound Coptis oil paint in the treatment of second-degree skin burns (between superficial second-degree and deep second-degree) 1. Research objects and data (1) Determine the research objects: From October 2023 to December 2024, patient recruitment was carried out in the Burn Department of the First Affiliated Hospital of Nanchang University. Before participating in the trial, all patients signed a written informed consent form. Finally, 80 burn patients hospitalized in the Burn Department of the First Affiliated Hospital of Nanchang University were determined as the research objects, regardless of gender.

[0063] (2) Sample and data sources: In this study, general clinical data were all sourced from the electronic medical record system of the First Affiliated Hospital of Nanchang University. The wound healing situation was recorded in a spreadsheet after being evaluated by the researchers, and the pain and itching scores were evaluated using a visual analogue scale (VAS) and recorded in a spreadsheet. Before treatment, on the 8th day of treatment, or at the end of treatment, microbial samples from the enrolled wound surfaces of patients were collected using a skin sterile swab, stored in a -80°C refrigerator, and 16S rRNA high-throughput sequencing was completed by Personalbio Technology Co., Ltd.

[0064] (3) Ethical note: This study has been approved by the hospital's ethics committee [Ethical number: IIT(2023)Clinical Ethics Review No. 151], and the enrolled research objects are informed about the content of this study and have signed the informed consent form. This trial has been registered in the Medical Research Registration and Filing Information System, with the registration number: MR-36-23-031854.

[0065] (4) Inclusion criteria: 1) Aged 18 to 65 years old, regardless of gender; 2) According to the latest domestic "Burn Surgery" diagnostic criteria, patients clinically diagnosed with second-degree burns, with the causative factors being flame, hot water, steam, metal, etc., but excluding chemical burns and electric shock injuries, the burn time is within 48 hours, and the total burn area does not exceed 30% of the total body surface area at most; 3) Those 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 does not exceed 2% of the total body surface area at most; 4) The 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.

[0066] 2. Research protocol: Patients were randomly grouped into a control group and an experimental group through Excel. 41 burn patients were assigned to the silver sulfadiazine and compound Coptis oil application group (Group A, control group), and 39 burn patients were assigned to the probiotic combined with silver sulfadiazine and compound Coptis oil application group (Group B, experimental group). During the study, 1 participant in Group A withdrew from the intervention due to automatic discharge, and 3 participants withdrew from the intervention for their own reasons. 4 participants in Group B withdrew from the intervention for their own reasons. Finally, 72 participants completed the intervention study and were subsequently included in the final analysis. Among them, 37 burn patients were included in the control group (Group A), 31 were male (83.80%), 6 were female (16.20%), aged 19 to 59 years old, with an average age of 45 years old (30.50 years old, 55.00 years old). The enrollment site of 20 people was the upper limb (54.10%), the enrollment site of 9 people was the lower limb (24.30%), and the enrollment site of 8 people was the trunk (21.60%). In addition, there were 16 cases of flame burns (43.20%), 15 cases of scalds (40.50%), and 6 cases of electric spark burns (16.20%). The total burn area was 2% - 29% TBSA, with an average of 10% (5.00%, 10.00%) TBSA. The experimental group (Group B) included a total of 35 burn patients, 25 were male (71.40%), 10 were female (28.60%), aged 20 to 62 years old, with an average age of 38 years old (26.00 years old, 53.00 years old). The enrollment site of 17 people was the upper limb (48.60%), the enrollment site of 7 people was the lower limb (20.00%), and the enrollment site of 11 people was the trunk (31.40%). In addition, there were 12 cases of flame burns (34.30%), 15 cases of scalds (42.90%), and 8 cases of electric spark burns (22.90%). The total burn area was 2% - 29% TBSA, with an average of 10% (6.00%, 10.00%) TBSA. Comparison of the demographic characteristics and clinical features of the two groups of research objects showed no statistical difference (P>0.05). In addition, there was no statistical significance in the vital signs, white blood cells, platelets, glucose, etc. between the two groups of patients at the time of enrollment (P>0.05). Therefore, the two groups of patients were homogeneous. See Table 1 for details.

[0067] Table 1 Comparison of baseline characteristics and clinical data between the experimental group and the control group Note 1: "-" indicates that there is no such statistic, and the test statistics in the table are all the absolute values of the test statistics.

[0068] Note 2: Since the data of average age, burn area, T, R, P, SBP, DBP, white blood cells, platelets, glucose, total bilirubin, PT, and APTT in Table 1 do not conform to the normal distribution, the median (interquartile range) M (P25, P75) is used to represent them, where M is the median, P25 indicates that 25% of the data is less than or equal to this value, and P75 indicates that 75% of the data is less than or equal to this value.

[0069] Note 3: TBSA represents the total body surface area.

[0070] To evaluate whether the postbiotics of Staphylococcus epidermidis can assist in promoting the healing of second-degree burn wounds, the sulfadiazine silver cream plus compound Coptis chinensis oil preparation (Group A, n = 37) and probiotic postbiotics plus sulfadiazine silver cream plus compound Coptis chinensis oil preparation (Group B, n = 35) were used on the enrolled wounds of the two groups of patients. The wound healing time of the two groups of burn patients and the number of healed patients at D8 and D15 or at the time of wound healing were monitored, and the results are as follows: 1. Comparison of wound healing time between the two groups of patients The data of the wound healing time (days) of the two groups of patients were tested to be non-normally distributed. The median (interquartile range) was used to statistically describe the data, and the Mann-Whitney test was used, with P = 0.002 (P < 0.05). The results showed that compared with the control group (Group A), after adding probiotic postbiotics, the overall wound healing time of burn patients was shortened, and the difference in healing time between the two groups was statistically significant (P < 0.05). See Table 2 and Figure 1 A, D, and E in

[0071] Table 2 Comparison of wound healing time between the two groups of patients (days) Note: The test statistics in the table are all the absolute values of the test statistics.

[0072] 2. Comparison of the number of healed patients at D8 between the two groups of patients The difference in the number of healed patients between the two groups at D8 was compared and analyzed. After chi-square test, P = 0.002 (P < 0.05). The results indicated that there was a statistical difference in the wound healing situation between the two groups of patients at D8 (P < 0.05). See Table 3 and Figure 1 B in

[0073] Table 3 Comparison of the number of healed patients at D8 between the two groups of patients (cases) Note: The test statistics in the table are all the absolute values of the test statistics.

[0074] 3. Comparison of the number of healed patients at D15 between the two groups of patients Comparative analysis of the difference in the number of healed patients between the two groups at D15. After chi-square test, P => 0.99 (P > 0.05). The results suggest that there is no statistical difference in the wound healing situation between the two groups at D15 (P > 0.05), indicating that regardless of whether probiotic postbiotics are added or not, the wounds of both groups of patients can heal within 15 days. See Table 4 and Figure 1 C in

[0075] Table 4 Comparison of the number of patients with wound healing at D15 in the two groups (cases) Note: The test statistics in the table are all the absolute values of the test statistics.

[0076] 4. Analysis of the remission of main symptoms before and after treatment After that, in order to compare the differences in wound pain and itching between the two groups of patients during the wound healing process, the wound pain indexes of the patients were scored at D0, D4 days and the end of treatment (EOT), and the wound itching indexes of the patients were scored at D0 and the end of treatment (VAS visual analogue scale).

[0077] (1) Comparison of wound pain scores of the two groups of patients before and after treatment The pain degree of the patients was scored by VAS ( Figure 2 D in P ). The data of wound pain scores of the two groups of patients before and after treatment were tested to be non-normally distributed. The median (interquartile range) was used to statistically describe the data, and the Mann-Whitney test was adopted. The results showed that there were no significant differences in the pain scores of the two groups of patients, regardless of whether probiotic postbiotics were added or not, at the three stages of D0, D4 and the end of treatment (EOT) ( Figure 2 > 0.05). See Table 5 and

[0078] Table 5 Comparison of wound pain scores of the two groups of patients before and after treatment Note: The test statistics in the table are all the absolute values of the test statistics.

[0079] (2)Comparison of wound itching scores of the two groups of patients before and after treatment The pain degree of the patients was scored by VAS. The data of wound itching scores of the two groups of patients before and after treatment were tested to be non-normally distributed. The median (interquartile range) was used to statistically describe the data, and the Mann-Whitney test was adopted. The results showed that the group with probiotic postbiotics added (experimental group) could significantly relieve the wound itching symptoms during treatment (P < 0.05). See Table 6 and Figure 3 。

[0080] Table 6 Comparison of wound itching scores of the two groups of patients before and after treatment Note: The test statistics in the table are all absolute values of the test statistics.

[0081] 5. 16S rRNA High-throughput Sequencing During the corresponding time, swab samples of the patients' wound surfaces were collected. The samples were immediately stored in a -80°C refrigerator after collection and then transported to Personalbio Technology Co., Ltd. in dry ice for batch sequencing. Professional personnel from Personalbio Technology Co., Ltd. processed the samples for 16S rDNA amplicon sequencing. The steps included: pretreatment of the samples, extraction and quantification of total microbial DNA, PCR amplification of the bacterial 16S rRNA gene, quantification and pooling of PCR products, library construction, and on-machine 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 machine was the Illumina NovaSeq 6000. After obtaining the off-machine data, DADA2 was used to process the raw data to obtain high-quality amplicon sequence variants (ASVs), and then they were compared with the database and species annotated. Based on the annotation results of ASV and species, the Alpha diversity and species composition of the microbial community were analyzed.

[0082] The analysis of the sequencing results is as follows: Research has shown that the colonization and growth of pathogenic microorganisms can damage neonatal epithelial cells and the extracellular matrix by secreting toxins and enzymes, or inhibit the healing process by forming biofilms. Therefore, microbial community samples from the second-degree burn wound sites of the patients were collected and sequenced. Before treatment and on the 8th day of treatment or at the end of treatment, 99 skin microbial samples were collected from the lesion sites, including 24 in group A1, 25 in group A2, 24 in group B1, and 26 in group B2. Among them, A1: before treatment in the control group; A2: after treatment in the control group; B1: before treatment in the experimental group; B2: after treatment in the experimental group. Subsequently, these samples were subjected to 16S rRNA sequencing analysis to investigate the changes in the skin microbial community of the burn wounds.

[0083] (1) Basic Analysis of Sequencing Data As a visualization tool, the Venn diagram can clearly represent the relationships between sets, count the number of members in each set respectively, and then intuitively show the presence or absence between groups and the number of species shared between groups. The Venn diagrams drawn according to different groupings are as follows ( Figure 4) The results showed that there were 145 shared amplicon sequence variants (ASVs) among the four groups of microbial samples. The total number of ASVs in the four groups was B2 > B1 > A1 > A2. The number of shared ASVs between group A1 and group A2 was 54, and the number of shared ASVs between group B1 and group B2 was 197.

[0084] (2)α-diversity analysis 1) Rarefaction curves: The sequence numbers of a total of 99 samples from the four groups were evaluated using rarefaction curves, as Figure 5 . The impact of sequencing depth on the observed sample diversity was reflected by the flatness of the curve. When the curve tended to flatten, it indicated that the sequencing data had sufficiently covered the biodiversity information present in the samples. The results showed that these rarefaction curves presented a gradually flattening trend, indicating that the sequence numbers of each group of samples were sufficient and met the sufficient conditions for microbial diversity analysis.

[0085] 2) Abundance rank curves Abundance rank curves can describe the richness and evenness of species within a sample. The evenness of community composition was characterized by the flatness of the curve shape. There was a direct correlation between the flatness of the curve and the evenness of community composition: when the curve tended to change smoothly, the abundance differences between classifications were smaller, and the evenness of community composition was higher; conversely, an increase in the steepness of the curve reflected a lower homogenization characteristic of species distribution. The more diverse the species in the sample, the wider the width. As Figure 6 shown, each group of samples showed a large curve span in the horizontal direction, especially after adding probiotic postbiotics (group B2), indicating that the species within the community were relatively rich.

[0086] 3) α-diversity indices α-diversity is used to evaluate the species composition characteristics in a local homogeneous habitat. The main indicators include richness, diversity, and evenness, etc. In this study, richness and diversity were characterized by the species richness estimator index, species richness index, and Shannon index, respectively. The values of the species richness estimator index and species richness index were positively correlated with the richness of the community, and the value of the Shannon index was positively correlated with the diversity of the community. Moreover, the higher the sequencing coverage index, the smaller the proportion of species not detected in the sample. As shown in Table 7 below, the sequencing coverage indices of each group of samples were all above 99%, indicating that the detection of sequences in the samples was relatively sufficient and most species had been covered. Then, the species richness estimator, species richness index, and Shannon index in each group of samples were analyzed and plotted Figure 7 for illustration.

[0087] Table 7 Sequencing coverage index values of the four groups of samples According to the result prompts, as shown in Table 8, the estimated species richness index and the species richness index of the control group on the eighth day of treatment (A2) were significantly lower than those before treatment (A1), with statistical differences (P < 0.05). The Shannon index decreased slightly, but there was no statistical difference (P > 0.05). After treatment in the experimental group (B2), as shown in Table 9, the estimated species richness, Shannon, and species richness indices were significantly increased compared with those before treatment, with statistical differences (P < 0.05). When comparing the estimated species richness, Shannon, and species richness indices of the experimental group and the control group after treatment, as shown in Table 10, there were statistical differences (P < 0.05). The above results indicate that the combined treatment with probiotic postbiotics increased the Alpha diversity of the patients' wounds.

[0088] Table 8 Comparison of skin flora α-diversity indices before and after treatment in the control group Note: The test statistics in the table are all the absolute values of the test statistics.

[0089] Table 9 Comparison of skin flora α-diversity indices before and after treatment in the experimental group Note: The test statistics in the table are all the absolute values of the test statistics.

[0090] Table 10 Comparison of skin flora α-diversity indices after treatment between the control group and the experimental group Note: The test statistics in the table are all the absolute values of the test statistics.

[0091] 4) β-diversity analysis The Beta diversity index focuses on the comparison of diversity between different habitats, that is, the differences between samples. Principal Coordinate Analysis (PCoA, also known as Classical Multidimensional Scaling CMDS) is a distance matrix-based dimensionality reduction technique used to visualize data points in a low-dimensional space while preserving their original distance structure as much as possible.

[0092] This study mainly performed principal coordinate analysis through weighted UniFrac distance and Jaccard distance. The results showed that, as Figure 8 、 Figure 9 , after treatment, there was a tendency for the samples of the group without added probiotic postbiotics to gradually separate from those of the group with added probiotic postbiotics, and most samples did not overlap, indicating that there were significant differences in the skin wound flora between the groups.

[0093] 5) Analysis of skin flora composition To analyze the microbial composition of the skin wounds of two groups of patients before and after treatment, the relative abundance distribution maps of the microbiota were drawn at the phylum and genus taxonomic levels, and then the species with relatively high abundance or related to burns were compared for differences. 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 of groups A1, A2, B1, and B2, respectively ( Figure 10 ). Table 11 lists the proportions of the top four phyla in each group. Further analysis showed that after treatment in the control group, the abundances of Actinobacteria and Proteobacteria increased, while the abundance of Firmicutes increased after treatment in the experimental group, as shown in Figure 11 , but these were not statistically significant (P>0.05).

[0094] Table 11 Abundances of species at the phylum level At the genus level, we selected representative skin microbiota closely related to the benefits and infections of 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 inhibit pathogenic bacteria normally, and some specific strains also have probiotic functions. The representative bacteria in the other two genera are Streptococcus pyogenes and Pseudomonas aeruginosa, which are common in burn infections. Further analysis found that, as shown in Figure 12 , Figure 13 , after treatment of burn patients, the abundance of Staphylococcus in the postbiogenic group supplemented with Staphylococcus epidermidis (group B2) increased significantly compared with that before treatment, with statistical significance (P<0.05). Compared with Propionibacterium, the abundance increased slightly after treatment in the experimental group and decreased slightly after treatment in the control group, without statistical significance (P>0.05). In the pathogenic genera, although the abundances of Streptococcus and Pseudomonas had no statistical differences between the two groups, the abundances increased in the control group and decreased in the experimental group. This indicates that probiotic postbiotics can promote the colonization and proliferation of skin commensal bacteria and reduce the abundance of pathogenic bacteria.

[0095] Table 12 Abundances of species at the genus level In summary, based on the above experiments, it can be concluded that: 1. Compared with the control group (Group A), the addition of postbiotics of Staphylococcus epidermidis significantly shortened the overall wound healing time of burn patients (P<0.05). The healing time of the control group was 9 (8.00, 10.00) days, and that of the experimental group was 7 (7.00, 8.00) days. At D8, 13 (28.60%) people in the control group had their wounds healed, while 25 (71.40%) people in the experimental group had their wounds healed. There was a statistically significant difference in the number of people with wound healing between the two groups at D8 (P<0.05).

[0096] 2. During the treatment of the two groups of patients, the itching score of the wound during treatment in the group with the addition of postbiotics of Staphylococcus epidermidis (experimental group) was significantly lower than that of the control group (P<0.05). Before the intervention, on the fourth day after the intervention, and at the end of the treatment, there was no significant difference in the pain score of the enrolled wounds between the two groups of patients (P>0.05).

[0097] 3. Basic information of skin microbiota sample sequencing: A total of 99 samples were collected and sequenced, including 24 in Group A1 (before treatment in the control group), 25 in Group A2 (after treatment in the control group), 24 in Group B1 (before treatment in the experimental group), and 26 in Group B2 (after treatment in the experimental group). The total number of ASVs in the four groups was B2>B1>A1>A2.

[0098] 4. In the experimental group after the intervention, the species richness estimate, Shannon, and species richness index in α-diversity were significantly increased compared with those before the treatment, with statistical differences (P<0.05). On the contrary, the species richness estimate index and species richness index in the control group were significantly decreased compared with those before the intervention (P<0.05). In the β-diversity analysis, principal coordinate analysis using weighted UniFrac distance and Jaccard distance showed that after the treatment, there was a tendency for the samples of the group without adding probiotic postbiotics to gradually separate from the samples of the group with the addition of postbiotics of Staphylococcus epidermidis, and most of them did not overlap, indicating differences in the skin wound microbiota between groups.

[0099] 5. By comparing the abundances at the phylum level and genus level of species between groups, after the intervention, the relative abundance of Firmicutes at the phylum level in the wound microbiota of the experimental group increased compared with that of the control group. At the genus level, after the treatment, the abundance of Staphylococcus in the experimental group increased significantly compared with that before the treatment, with statistical differences (P<0.05). In Propionibacterium, the abundance in the experimental group increased slightly after the treatment, while the abundance in the control group decreased slightly after the treatment. In the pathogenic genera, the abundances of Streptococcus and Pseudomonas decreased slightly in the experimental group.

[0100] Therefore, the following conclusions can be drawn from the above experimental results: 1. Postbiotics of Staphylococcus epidermidis helps to promote the healing of second-degree burn wounds in patients and can enhance the therapeutic effect when combined with silver sulfadiazine cream and compound Coptis chinensis oil paint.

[0101] 2. Postbiotics of Staphylococcus epidermidis can relieve the itching symptoms of patients with second-degree burn wounds, but have no obvious effect on the pain symptoms of the wounds.

[0102] 3. Postbiotics of Staphylococcus epidermidis increase the diversity of skin flora, can inhibit the colonization and proliferation of pathogenic microorganisms, and promote the proliferation of skin commensal bacteria.

[0103] As mentioned above, only the preferred specific embodiments of the present invention are described, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and should be covered within the protection scope of the present invention.

Claims

1. A strain of Staphylococcus epidermidis NCU-07, characterized in that, The Staphylococcus epidermidis ( Staphylococcus epidermidis ) NCU-07 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 18, 2023, with the deposit number: CGMCC NO. 26493, and the deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Staphylococcus epidermidis NCU-07 according to claim 1, characterized in that The Staphylococcus epidermidis NCU-07 was screened from the wound surface of patients with rosacea.

3. A probiotic postbiotic prepared from the Staphylococcus epidermidis NCU-07 according to any one of claims 1 to 2.

4. The preparation method of the probiotic postbiotics according to claim 3, wherein, It includes the following steps: S1. Prepare the Staphylococcus epidermidis NCU-07 into a bacterial solution, and transfer the bacterial solution to a solid medium for aerobic culture; S2. Pick a single colony on the solid medium and inoculate it into a liquid medium for secondary culture; S3. Absorb the bacterial solution from the second-generation cultured bacterial solution into the liquid medium and continue to culture it until the Staphylococcus epidermidis NCU-07 grows to the logarithmic growth phase; then centrifuge the culture solution at low temperature, discard the bottom precipitate, collect the supernatant, filter to remove the bacteria and impurities, collect the supernatant again, sterilize it to obtain the probiotic postbiotic.

5. According to the preparation method described in claim 4, wherein The conditions for aerobic culture in S1 include: aerobic culture at 37°C for 24 h - 48 h; The conditions for secondary culture in S2 include: aerobic culture at 37°C for 24 h - 48 h; S3 includes the following specific steps: Absorb the bacterial solution from the second-generation cultured bacterial solution into the liquid medium and continue to culture it for 12 h - 16 h until the Staphylococcus epidermidis NCU-07 grows to the logarithmic growth phase; then centrifuge the culture solution in a refrigerated high-speed centrifuge at 4°C and 8000 r / min for 10 min, discard the bottom precipitate, collect the supernatant, filter to remove the bacteria and impurities with a 0.22 μm microporous membrane, collect the supernatant again, sterilize it at 121°C for 30 min to obtain the probiotic postbiotic.

6. A biological preparation, characterized in that, Its active ingredient includes the Staphylococcus epidermidis NCU-07 according to any one of claims 1 to 2 or the probiotic postbiotic according to claim 3.

7. An auxiliary drug for treating skin burn wounds, characterized in that, The adjuvant drug includes the Staphylococcus epidermidis NCU-07 according to any one of claims 1 to 2 or the probiotic postbiotic according to claim 3.

8. Use of the Staphylococcus epidermidis NCU-07 according to any one of claims 1 to 2 or the probiotic postbiotic according to claim 3 in the preparation of a drug for treating skin burn wounds.

9. Use of the Staphylococcus epidermidis NCU-07 according to any one of claims 1 to 2 or the probiotic postbiotic according to claim 3 in combination with silver sulfadiazine cream and compound Coptis chinensis oil paint in the application of a drug combination for treating skin burn wounds.

10. The application according to claim 9, wherein The skin burn wound is a second-degree skin burn wound; The method of combined use is as follows: Apply the probiotic postbiotic and silver sulfadiazine cream to the skin burn wound, and then cover it with a gauze containing the compound Coptis chinensis oil paint.

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