Staphylococcus epidermidis CCSMS1601 as well as culture and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to staphylococcus epidermidis CCSMS1601 as well as a culture method and application of the staphylococcus epidermidis CCSMS1601. The staphylococcus epidermidis CCSMS1601 provided by the invention is preserved in the China General Microbiological Culture Collection Center on June 5, 2024, and the preservation number of the staphylococcus epidermidis CCSMS1601 is CGMCC (China General Microbiological Culture Collection Center) NO.30874. The staphylococcus epidermidis CCSMS1601 provided by the invention can be used for preparing the staphylococcus epidermidis. According to the present invention, the fermentation supernatant obtained by using the Staphylococcus epidermidis CCSMS1601 has the scalp flora regulation function so as to improve the dandruff, such that the scalp health is easily achieved; the strain CCSMS1601 disclosed by the invention has a good application prospect in the aspect of development of scalp care products for resisting inflammation, resisting oxidation and regulating scalp micro-ecology.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to Staphylococcus epidermidis CCSMS1601 and the culture and application thereof. Background Art

[0002] The scalp is a part of human skin and a unique ecosystem. The scalp microbial community includes a variety of microorganisms, such as bacteria, fungi and other microorganisms. The community composition and dynamic distribution of the scalp microecology are in an overall balance with the scalp surface tissue cells and the host's healthy immune status. The bacterial genera that reside on the scalp of healthy adults are Propionibacterium, Staphylococcus and Corynebacterium. The most important fungi on the scalp are Malassezia, which are less in number than bacteria, mainly Malassezia restricta and Malassezia globosa. Normal scalp microorganisms maintain the state of the scalp by consuming secretions, supporting metabolism, and improving the immune system. There is a complex interaction between these microorganisms, which play different roles in scalp health and the development of diseases.

[0003] Staphylococcus epidermidis is a coagulase-negative staphylococcus with spherical cells. It is a resident bacterium on the human scalp and plays a major role in maintaining the balance of scalp microecology and scalp self-purification. As an important component of the scalp flora, changes in its colonization ratio may cause an imbalance in the colonization of other flora, leading to the occurrence of scalp problems.

[0004] About 50% of the world's population suffers from dandruff problems. The main symptoms of dandruff include scalp itching, scaling, greasy hair, etc. In severe cases, it can also lead to inflammation and hair loss. The pathogenesis of dandruff is complex, including abnormal sebum secretion, fungal infection, scalp microecological disorder, stratum corneum metabolic disorder and other factors. At present, most studies have shown that compared with healthy people, the relative abundance of Propionibacterium acnes and Malassezia globosa in the dandruff area of ​​dandruff people decreases, while the relative abundance of Malassezia restricta increases.

[0005] At present, the research on the mechanism of dandruff and its prevention and treatment methods mainly focuses on Malassezia, and commonly used treatment drugs include shampoos containing antifungal ingredients, antibiotics, anti-inflammatory drugs, etc. However, long-term use of antifungal drugs will produce drug resistance and bring side effects such as dizziness and burning sensation. Therefore, antifungal treatment of dandruff alone may not be the only effective countermeasure. Comprehensive treatment is needed to restore the homeostasis between scalp microorganisms and protect the health of the scalp from the root. Summary of the invention

[0006] In order to solve the above problems, the purpose of the present invention is to provide a Staphylococcus epidermidis CCSMS1601 and its culture and application.

[0007] The present invention isolates a strain of Staphylococcus epidermidis CCSMS1601 from a healthy scalp, and the fermentation supernatant thereof has the advantages of promoting the proliferation of Propionibacterium acnes and Malassezia globosa, and inhibiting the proliferation of Malassezia restricta (it has the function of regulating the scalp flora, thereby improving dandruff and being beneficial to scalp health), and can be used in the development of anti-inflammatory and antioxidant scalp care and dandruff prevention and treatment related care products, and has good application prospects.

[0008] The purpose of the present invention can be achieved by the following technical solutions:

[0009] The first object of the present invention is to provide a Staphylococcus epidermidis (Staphylococcus epidermidis) CCSMS1601, which has been deposited in the General Microbiological Center of China Microorganism Culture Collection Administration on June 5, 2024, with a deposit number of CGMCC NO.30874.

[0010] In one embodiment of the present invention, the bacterial cells of Staphylococcus epidermidis CCSMS1601 are spherical and arranged in grape-like or singly shapes, with a diameter of 0.9 to 1.0 μm, without flagella, without spores, and Gram-positive.

[0011] In one embodiment of the present invention, Staphylococcus epidermidis CCSMS1601 was streaked and isolated on a TSA plate and aerobically cultured at 37°C for 16 hours, and the strain grew well. Its colonies were white and small round protrusions with a colony diameter of about 0.5 to 1 mm, with neat edges, smooth and moist surface, opaque, able to be drawn into threads, and without hemolysis zones.

[0012] The second object of the present invention is to provide a 16s rDNA sequence of Staphylococcus epidermidis CCSMS1601, as shown in SEQ ID NO.3.

[0013] A third object of the present invention is to provide a method for cultivating Staphylococcus epidermidis CCSMS1601, comprising the following steps:

[0014] Staphylococcus epidermidis CCSMS1601 was cultured in TSB liquid medium;

[0015] During the culture process, the pH of the TSB liquid culture medium was 4.0-9.0, the culture temperature was 30-40°C, and the culture time was 36 hours.

[0016] The fourth object of the present invention is to provide a fermentation method of Staphylococcus epidermidis CCSMS1601, comprising the following steps:

[0017] (S1) inoculating activated Staphylococcus epidermidis CCSMS1601 into TSB liquid culture medium to obtain seed solution;

[0018] (S2) inoculating the seed solution prepared in step (S1) into a fermentation medium to obtain a fermentation solution of Staphylococcus epidermidis CCSMS1601;

[0019] The fermentation liquid of Staphylococcus epidermidis CCSMS1601 is centrifuged to obtain a supernatant, which is a fermentation supernatant of Staphylococcus epidermidis CCSMS1601.

[0020] In one embodiment of the present invention, in step (S1), during the culturing process, the temperature is 30-40° C. and the time is 16-20 h.

[0021] In one embodiment of the present invention, in step (S2), during the fermentation process, the temperature is 30-40° C. and the time is 18-20 hours.

[0022] The fifth object of the present invention is to provide a fermentation supernatant of Staphylococcus epidermidis CCSMS1601, which is prepared by the above method;

[0023] The fermentation supernatant contained 456.13 μg / mL of acetic acid, 14.57 μg / mL of propionic acid, 3.62 μg / mL of isobutyric acid, 6.19 μg / mL of butyric acid, 8.84 μg / mL of isovaleric acid, and 14.66 μg / mL of valeric acid.

[0024] A sixth object of the present invention is to provide a use of a fermentation supernatant of Staphylococcus epidermidis CCSMS1601 in the preparation of a scalp care product.

[0025] In one embodiment of the present invention, the scalp care product is a scalp care product that promotes the proliferation of Propionibacterium acnes and Malassezia globosa and inhibits the proliferation of Malassezia restricta.

[0026] In one embodiment of the present invention, the scalp care product is an anti-inflammatory scalp care product;

[0027] Preferably, the anti-inflammatory scalp care product is a scalp care product that inhibits hyaluronidase (inhibition rate reaches 101.51%).

[0028] In one embodiment of the present invention, the scalp care product is an antioxidant scalp care product;

[0029] Preferably, the antioxidant scalp care product is a product that removes ABTS free radicals (IC 50 value is 1.82%) and DPPH free radical (IC 50 Value is 23.80%) scalp care products.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The Staphylococcus epidermidis CCSMS1601 provided by the present invention is rich in active ingredients such as short-chain fatty acids, has good anti-inflammatory effects, and its fermentation supernatant has an inhibition rate of 101.51% on hyaluronidase, the activities of T-SOD and GSH-PX in the fermentation supernatant are 27.32U / mL and 112.63U / mL, and the IC values ​​of scavenging ABTS free radicals and DPPH free radicals are 1.34 and 1.12, respectively. 50 The concentrations were 1.82 and 23.80%, showing good anti-inflammatory and antioxidant effects. Antibacterial tests showed that 25% (v / v) concentration of Staphylococcus epidermidis CCSMS1601 fermentation supernatant can promote the growth of Propionibacterium acnes CCSMS0630 and Malassezia globosa CCSMS1050, significantly inhibit the growth of Malassezia restricta CCSMS1060, regulate the microecological balance of the scalp, reduce the growth and reproduction of harmful bacteria, and help improve the health of dandruff scalp.

[0032] The Staphylococcus epidermidis CCSMS1601 provided by the present invention has good application prospects in the development of anti-inflammatory and antioxidant scalp care products, and fills the gap in the screening of the anti-inflammatory, antioxidant and scalp microecological efficacy of Staphylococcus epidermidis CCSMS1601. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The colony morphology (a) and bacterial cell photo (b) of Staphylococcus epidermidis CCSMS1601;

[0034] Figure 2 is the growth curve of Staphylococcus epidermidis CCSMS1601;

[0035] Figure 3 The results of the determination of the inhibition rate of hyaluronidase by the fermentation supernatant of Staphylococcus epidermidis CCSMS1601;

[0036] Figure 4 The scavenging ability of the fermentation supernatant of Staphylococcus epidermidis CCSMS1601 on ABTS free radicals;

[0037] Figure 5 The scavenging ability of the fermentation supernatant of Staphylococcus epidermidis CCSMS1601 on DPPH free radicals;

[0038] Figure 6 The results of the intervention of the fermentation supernatant of Staphylococcus epidermidis CCSMS1601 on Propionibacterium acnes CCSMS0630 at concentrations of 1.56%, 3.13%, 6.25%, 12.5% ​​and 25% respectively;

[0039] Figure 7 The results of the intervention of Staphylococcus epidermidis CCSMS1601 fermentation supernatant with concentrations of 1.56%, 3.13%, 6.25%, 12.5% ​​and 25% on Malassezia globosa CCSMS1050;

[0040] Figure 8These are the intervention results of the fermentation supernatant of Staphylococcus epidermidis CCSMS1601 with concentrations of 1.56%, 3.13%, 6.25%, 12.5% ​​and 25% on restrictive Malassezia CCSMS1060. DETAILED DESCRIPTION

[0041] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0042] In the following examples, unless otherwise specified, all reagents used are commercially available reagents, and all detection means and methods used are conventional detection means and methods in the art.

[0043] Experimental strains: Propionibacterium acnes CCSMS0630 (deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on January 18, 2024, with a deposit number of CGMCC NO.29674, and classified as Propionibacterium acnes CCSMS0630), Malassezia globosa CCSMS1050 (deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on August 26, 2024, with a deposit number of CGMCC NO.31746, and classified as Malassezia globose CCSMS1050), Malassezia restricta CCSMS1060 (deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on August 26, 2024, with a deposit number of CGMCC NO.31747, and classified as Malassezia restricta restricta)CCSMS1060) are all isolated from healthy human scalp skin in the laboratory.

[0044] Tryptic soy peptone agar blood culture medium (TSA plate): tryptic peptone 15g / L, soybean papain hydrolysate 5g / L, sodium chloride 5g / L, agar 15g / L, autoclave at 121℃ for 15min, pour into the plate.

[0045] TSA blood plate: 15 g / L trypticase, 5 g / L soybean papain hydrolysate, 5 g / L sodium chloride, 15 g / L agar, sterilize at 121℃ for 15 min, add 5% sterile defibrinated sheep blood when cooled to about 50℃ after sterilization, mix well and pour into plates.

[0046] TSB liquid medium: 15 g / L trypticase, 5 g / L soybean papain hydrolysate, sodium chloride, autoclave at 121°C for 15 min, set aside.

[0047] Malassezia culture medium (Dixon liquid medium) - 2693 modified mDixon: malt extract powder 36g / L, ox bile powder 20g / L, peptone 6g / L, glycerol 2g / L, oleic acid 2g / L, high pressure sterilization at 121℃ for 15min, set aside.

[0048] Preservation medium: 120 g skim milk powder, 880 mL distilled water, autoclave at 121°C for 15 min, set aside.

[0049] Example 1

[0050] This example provides the isolation, screening and purification of strain CCSMS1601.

[0051] (1) Recruit volunteers with healthy scalp who meet the following conditions:

[0052] 1) No topical antifungal drugs or corticosteroid preparations have been used on the scalp within two weeks;

[0053] 2) No oral antifungal drugs within two months;

[0054] 3) Not accompanied by atopic dermatitis, psoriasis or other scalp skin diseases;

[0055] 4) Age range: 18 to 30 years old, gender is not limited.

[0056] (2) Sample collection

[0057] Volunteers were asked not to wash their hair the night before sampling, and samples were taken at noon or afternoon the next day.

[0058] Moisten a sterile cotton swab made of polymer fiber material in a wetting solution (0.9% sodium chloride and 0.1% Tween-20), rub it back and forth on the selected area of ​​the scalp 50 times (the cotton swab should be wiped with a certain amount of force), place the sterile cotton swab in the sampling tube with sterile tweezers, and seal it with a sealing film; refrigerate the collected samples in an ice box, bring them back to the laboratory and place them in a 4°C refrigerator to isolate the strain as soon as possible.

[0059] (3) Sample pretreatment

[0060] In an ultra-clean workbench, use sterile scissors to cut off the sterile cotton swab head with the sample, place it in a 5 mL centrifuge tube (EP tube), draw 5 mL of sterile water into the centrifuge tube, mix thoroughly, and obtain the sample solution.

[0061] (4) Plate screening

[0062] Take 0.5 mL of the sample solution obtained in step (3) and add 4.5 mL of sterile water for gradient dilution. Select an appropriate dilution and apply 0.1 mL of the dilution on a TSA blood agar plate. Apply 2 plates for each dilution and place them in a 37°C incubator for aerobic culture for 24 h.

[0063] (5) Streaking, purification and preservation

[0064] According to the colony characteristics of coagulase-negative staphylococcus strains on blood agar plates, single colonies were picked and streaked onto TSA blood agar plates based on whether a hemolytic circle was produced, colony size, color, moisture, gloss, etc., and cultured in a 37°C constant temperature incubator for 16 hours. After multiple streaking and purification, the strain CCSMS1601 was obtained, placed in a preservation medium (using skim milk powder as a protective agent) and preserved in a preservation tube. After freeze-drying, it was stored in a -80°C deep freezer.

[0065] Example 2

[0066] This example provides the microbiological identification of strain CCSMS1601.

[0067] (1) Colony characteristics

[0068] The strain CCSMS1601 was streaked on TSA blood agar plates and cultured at 37°C for 16 h. The colony morphology of the strain on the agar plates was observed. Figure 1 As shown in a, from Figure 1 a It can be seen that the colonies of Staphylococcus epidermidis on TSA blood agar plates are white and small, forming round protrusions with neat edges. The colony diameter is about 0.5-1 mm, the surface is smooth, moist, opaque, and can be drawn into threads when picked, with no hemolysis zone;

[0069] (2) Bacterial characteristics

[0070] A small amount of strain CCSMS1601 was picked and smeared on a glass slide, stained with Gram stain, and the morphological characteristics of the bacteria were observed under a microscope. Figure 1 As shown in b, from Figure 1 b It can be seen that the Gram staining of the bacteria is positive, the bacteria are spherical, with a diameter of about 0.9 to 1.0 μm, arranged in grape shapes, and also arranged individually. They have no flagella, cannot move, and do not produce spores.

[0071] (3) Cultural characteristics

[0072] The strain CCSMS1601 was inoculated into TSB liquid culture medium and cultured at different temperatures. Studies have shown that the strain's minimum growth temperature is 15°C, the maximum growth temperature is 45°C, and the best growth temperature is 30-40°C. It was inoculated into TSB liquid culture medium with different pH values ​​and cultured at a constant temperature of 37°C. It was found that the highest growth pH was 9.0, the lowest growth pH was 4.0, and the optimal growth pH was 6.0.

[0073] (4) Genetic characteristics

[0074] 16S rDNA sequence analysis of strain CCSMS1601

[0075] DNA extraction method: Pick a single colony of the purified strain CCSMS1601 and inoculate it into 10 mL of TSB liquid culture medium. After culturing at 37°C for 16 h, centrifuge the bacterial solution (8000 r / min, 15 min) to collect the bacteria.

[0076] The genomic DNA was extracted using a genomic DNA extraction kit (Shanghai Bioengineering Co., Ltd.). Two synthetic universal primers (16s 27F (SEQ ID NO.1): 5'-GAGAGTTTGA TCCTGGCTCAG-3'; 16s1492R (SEQ ID NO.2): 5'-CGGCTACCTTGTTACGAC TT-3') were used for PCR amplification. The PCR product was recovered using a column PCR product purification kit (Shanghai Bioengineering Co., Ltd.), and after purification, it was sent to Meiji Bioengineering Co., Ltd. for sequencing. The 16S rDNA nucleotide sequence of the obtained strain CCSMS1601 is shown in SEQ ID NO.3, and the sequence length is 1384bp. It was sent to GenBank for Blast analysis. It was found that the strain with the highest homology to the strain CCSMS1601 was MT585523.1, with a homology of 100%.

[0077] Among them, the sequence shown in SEQ ID NO.3 is as follows:

[0078]

[0079] According to Goodfellow and O'Donnell, species with DNA G+C (mol%) ≤ 10% to 12% and 16S rRNA sequence homology ≥ 95% can be classified as one genus, and Embley and Stackebrangdt believe that species with 16S rRNA sequence homology ≥ 97% can be considered as one species. Therefore, it can be inferred that strain CCSMS1601 and Staphylococcus epidermidis belong to the same species.

[0080] Therefore, strain CCSMS1601 was identified as Staphylococcus epidermidis.

[0081] Based on the above-mentioned microbiological characteristics such as colony, bacterial morphology, culturing, physiological and biochemical identification and its genetic characteristics 16s rDNA, strain CCSMS1601 was identified as Staphylococcus epidermidis, and was deposited in the General Microbiology Center of China National Microbiological Culture Collection on June 5, 2024. Its preservation number is CGMCCNO.30874, and its classification name is Staphylococcus epidermidis (Staphylococcus epidermidis) CCSMS1601.

[0082] The activated Staphylococcus epidermidis CCSMS1601 was inoculated into TSB liquid culture medium at a rate of 2% (v / v), and cultured at 37°C with constant temperature shaking for 48 h. The OD value of the culture solution was measured at 600 nm every 4 h. 600 The growth curve of Staphylococcus epidermidis CCSMS1601 in TSB liquid medium was obtained by plotting the value against time. Figure 2 As shown, from Figure 2 It can be seen that Staphylococcus epidermidis CCSMS1601 grows rapidly in TSB broth liquid culture medium, enters the logarithmic growth phase in 8 hours, and reaches the stable phase in 36 hours.

[0083] Example 3

[0084] This example provides the preparation of the fermentation supernatant of Staphylococcus epidermidis CCSMS1601 (hereinafter referred to as "S. epidermidis CCSMS1601").

[0085] (1) Streak the S.epidermidis CCSMS1601 strain on a TSA plate, culture at 37°C for 18 h, and activate it twice; inoculate the activated S.epidermidis CCSMS1601 into TSB liquid culture medium, culture it on a shaker at 37°C for 18 h, and adjust the bacterial OD 600 In the range of 0.55 to 0.56, it is used as seed liquid.

[0086] (2) inoculating the seed solution prepared in step (1) into 150 mL of TSB liquid culture medium at an inoculum size of 1% (v / v), placing the culture medium in a 37° C. constant temperature shaking incubator, and culturing the culture medium at a speed of 160 r / min for 20 h to obtain a fermentation liquid of S. epidermidis CCSMS1601;

[0087] (3) The fermentation liquid prepared in step (2) was centrifuged at 9000 r / min for 10 min to obtain the centrifugal supernatant, which was the fermentation supernatant of S. epidermidis CCSMS1601.

[0088] Example 4

[0089] This example provides the determination of short-chain fatty acids in the fermentation supernatant of S. epidermidis CCSMS1601.

[0090] (1) Sample pretreatment

[0091] In a 10mL centrifuge tube, add 2mL of fermentation supernatant of S.epidermidis CCSMS1601, 0.4mL50% H2SO4 and 2mL of ether, mix well, shake on a shaker at 250r / min for 45min, then centrifuge at 4℃ and 10000r / min for 5min, take the upper organic phase, filter it through a 0.22μm organic filter membrane before GC injection, and keep the supernatant at -20℃ for later use.

[0092] (2) Gas chromatography conditions

[0093] GC was used to determine the short-chain fatty acids in the fermentation supernatant of S. epidermidis CCSMS1601. The instrument model was GC-2010plus; the chromatographic column was Agilent DB-WAX UI (60m×0.25μm×0.25mm); the injection port and detector temperature were set to 250℃; high-purity nitrogen was used as the carrier gas (flow rate 1.2mL / min), and the splitless detection mode was adopted; the heating conditions were: the sample volume was 1μL, and the temperature was increased from 75℃ to 180℃ at 10℃ / min and maintained for 1min, and then increased to 220℃ at 10℃ / min and maintained for 1min.

[0094] (3) Preparation of standard curve

[0095] Water-soluble fatty acid mixed standard solution (Northern Weiye Metrology Institute): glacial acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, a total of 6 components, the standard value of each is 1000μg / mL. Ultrapure water diluted mixed standard, respectively diluted to 5μg / mL, 10μg / mL, 20μg / mL, 50μg / mL, 100μg / mL, 200μg / mL, 500μg / mL, 800μg / mL, 1000μg / mL 9 concentration gradients, stored at -20℃ for use.

[0096] (4) Sample content determination

[0097] It was determined that the fermentation supernatant of S. epidermidis CCSMS1601 contained 6 short-chain fatty acids, and the specific contents are shown in Table 1 below.

[0098] Table 1 Results of short-chain fatty acid content in the fermentation supernatant of CCSMS1601

[0099]

[0100] Example 5

[0101] This example provides a determination of the hyaluronidase inhibition rate of the fermentation supernatant of S. epidermidis CCSMS1601.

[0102] (1) The reagents used are as follows:

[0103] CaCl2 solution: 2.5mmol / L;

[0104] NaOH solution: 0.4 mol / L;

[0105] Buffer: Solution A (0.2 mol / L acetic acid, 1.155 mL glacial acetic acid dissolved in 100 mL distilled water) 4.8 mL; Solution B (0.2 mol / L sodium acetate, 1.64 g CH3COONa dissolved in 100 mL distilled water) 45.2 mL, mixed and diluted to 100 mL to obtain an acetate buffer with a pH of 5.6;

[0106] Hyaluronidase solution: 500U / mL;

[0107] Sodium hyaluronate solution: 0.5 mg / mL;

[0108] Acetylacetone solution: 1.4 mL;

[0109] P-DAB colorimetric reagent-Ehrlihc reagent;

[0110] (2) Experimental procedures

[0111] 1) Take four test tubes and mark them as a, b, C, and D. Add 100 μL of 2.5 mmol / LCaCl2 solution and 500 μL of 600 U / mL hyaluronidase solution to test tubes a and c respectively, and treat at 37°C for 20 min. Add 100 μL of 2.5 mmol / LCaCl2 solution and 500 μL of acetate buffer to test tubes b and d, and treat at 37°C for 20 min.

[0112] 2) Add 500 μL of the fermentation supernatant of S. epidermidis CCSMS1601 to test tubes a and b, and add 500 μL of distilled water to test tubes c and d and treat at 37°C for 20 min;

[0113] 3) Add 500 μL of 0.5 mg / mL sodium hyaluronate solution to test tubes a, b, c, and d respectively, treat at 37°C for 30 min, and let stand at room temperature for 5 min;

[0114] 4) Add 500 μL of acetylacetone solution and 100 μL of NaOH solution to test tubes a, b, c, and d respectively, and place in a boiling water bath for 15 min and an ice bath for 5 min;

[0115] 5) Add 1 mL of P-DAB colorimetric reagent to test tubes a, b, c, and d respectively. After color development at room temperature for 20 min, take 200 μL from each test tube and place it in an ELISA plate to measure its absorbance at a wavelength of 530 nm.

[0116] The experiment was set up with 3 replicate wells and the average value was taken.

[0117] (3) Calculation of hyaluronidase inhibition rate

[0118]

[0119] Where: A: OD of test tube a 530 ;

[0120] B: OD of test tube b 530 ;

[0121] C: OD of test tube c 530 ;

[0122] D: OD of test tube d 530 .

[0123] The test results are as follows Figure 3 As shown, the hyaluronidase inhibition rate of the fermentation supernatant of Staphylococcus capitis CCSMS1601 was 101.51±4.85%, showing a good anti-inflammatory effect.

[0124] Example 6

[0125] This example provides T-SOD and GSH-PX enzyme assays in the fermentation supernatant of S. epidermidis CCSMS1601.

[0126] (I) Prepare the required working solution according to the instructions of the glutathione peroxidase (GSH-PX) test kit (Nanjing Jiancheng Bioengineering Institute) and conduct the following experiment

[0127] 1) Enzymatic reaction:

[0128] Table 2 Summary of enzymatic reactions

[0129]

[0130] Among them, the reagent 1 application solution was preheated at 37°C before use;

[0131] 2) Color reaction:

[0132] Table 3 Summary of color reaction

[0133]

[0134] Mix well, let stand at room temperature for 15 min, adjust to zero with distilled water at 412 nm, 1 cm optical path, and measure the OD value of each tube.

[0135] Calculation formula:

[0136]

[0137] 100 μL of the fermentation supernatant of S. epidermidis CCSMS1601 was taken and the GSH-PX activity was calculated to be 112.63±0.54 U / mL.

[0138] (2) According to the instructions of the total superoxide dismutase (T-SOD) assay kit (Nanjing Jiancheng Bioengineering Institute), the required working solution was prepared and the following experiment was performed:

[0139] Table 4 Reaction summary

[0140] Reagents Determination tube Control tube Reagent 1 Application Solution (mL) 1.0 1.0 Sample (mL) 0.1 0.0 Distilled water (mL) 0.0 0.05 Reagent 2 (mL) 0.1 0.1 Reagent 3 (mL) 0.1 0.1 Reagent 4 Application Solution (mL) 0.1 0.1

[0141] Mix thoroughly with a vortex mixer and place in a 37°C constant temperature water bath or air bath for 40 min.

[0142] Color developer (mL) 2 2

[0143] Mix well, leave at room temperature for 10 min, and compare colors at a wavelength of 550 nm, using a 1 cm optical path cuvette and adjusting to zero with distilled water.

[0144] Calculation formula:

[0145]

[0146] 100 μL of the fermentation supernatant of S. epidermidis CCSMS1601 was taken and the T-SOD activity was calculated to be 27.32±3.72 U / mL.

[0147] Example 7

[0148] This example provides an ABTS free radical scavenging assay of the fermentation supernatant of S. epidermidis CCSMS1601.

[0149] Mix equal volumes of 7 mmol / L ABTS and 2.6 mmol / L potassium persulfate solution, protect from light at room temperature for 14 h to obtain ABTS stock solution, and dilute it with anhydrous ethanol before use so that its absorbance at a wavelength of 734 nm is 0.70±0.02 to obtain ABTS working solution.

[0150] Control solution: Take a 10mL test tube, add 1.0mL ABTS working solution and 3.0mL anhydrous ethanol to the test tube to a final volume of 4.0mL, let it stand for 30min, and measure the absorbance at a wavelength of 734nm, which is recorded as a0;

[0151] Sample solution: dilute the fermentation supernatant of S.epidermidis CCSMS1601 by 20, 40, 60, 80, and 100 times, add 2.0 mL of anhydrous ethanol, and then add ABTS working solution to make the final volume 4.0 mL. Incubate in dark place for 30 minutes and measure the absorbance at a wavelength of 734 nm, which is recorded as a.

[0152] The experiment used the same mass concentration of V C As a positive control, each sample was repeated 3 times, and the ABTS free radical scavenging rate (%) = (a0-a) / a0×100%.

[0153] The experimental results are as follows Figure 4 As shown, through Figure 4 It can be found that the scavenging ability of S. epidermidis CCSMS1601 fermentation supernatant on ABTS free radicals increases with the increase of fermentation supernatant concentration. 50 The concentration was 1.82% (the original fermentation liquid concentration was 100%). The results showed that the fermentation supernatant of S. epidermidis CCSMS1601 had good antioxidant and free radical scavenging ability.

[0154] Example 8

[0155] This example provides a DPPH free radical scavenging test of the fermentation supernatant of S. epidermidis CCSMS1601.

[0156] Control group: 1 mL of 2 × 10 -4 mol / L DPPH solution, add water to make the final volume 4mL, keep it in dark for 30min at room temperature, and measure the absorbance at 517nm, which is recorded as a0;

[0157] Sample solution: Take 1 mL of DPPH solution and mix it with 1 mL of fermentation supernatant of S.epidermidis CCSMS1601 with concentrations of 5%, 2.5%, and 1.67%, respectively, and then add water to make the final volume 4 mL. After 30 minutes at room temperature away from light, measure the absorbance of the fermentation supernatant of different concentrations and record it as a.

[0158] The experiment used the same mass concentration of V C As a positive control, each sample was repeated 3 times, and the DPPH free radical scavenging rate (%) = (a0-a) / a0×100%.

[0159] The test results are as follows Figure 5 As shown, through Figure 5 It can be found that the scavenging ability of the fermentation supernatant of S. epidermidis CCSMS1601 on DPPH free radicals increases with the increase of concentration, and the IC 50 It is 23.80% (the original fermentation liquid concentration is 100%), indicating that the fermentation supernatant of S. epidermidis CCSMS1601 has good antioxidant capacity.

[0160] Example 9

[0161] This example provides the intervention of the fermentation supernatant of S. epidermidis CCSMS1601 on Propionibacterium acnes CCSMS0630.

[0162] (1) The concentration of the fermentation supernatant of S. epidermidis CCSMS1601 was selected by mixing the fermentation supernatant with sterile TSB liquid medium at a ratio of 1:1 using a half-dilution method to obtain fermentation supernatants with concentrations of 25, 12.5, 6.25, 3.125, and 1.562%;

[0163] (2) Preparation of bacterial suspension of Propionibacterium acnes CCSMS0630: Take out the freeze-dried tube of milk powder of Propionibacterium acnes strain stored at -80°C, streak TSA plates, and place them in a 37°C constant temperature incubator for anaerobically cultured for 54 h; after activation twice, pick a single colony of Propionibacterium acnes activated twice and inoculate it into a test tube containing 5 mL of TSB liquid culture medium, place it in a 37°C incubator, and culture it anaerobically for 54 h to adjust the bacterial OD 600 Around 0.3 to 0.4, keep aside.

[0164] (3) In the experimental group, 50 μL of Propionibacterium acnes CCSMS0630 bacterial suspension, 50 μL of S. epidermidis CCSMS1601 fermentation supernatant and 100 μL of TSB liquid culture medium were added to each well of the 96-well plate.

[0165] The blank group was added with 100 μL TSB liquid medium and 100 μL S.epidermidis CCSMS1601 fermentation supernatant.

[0166] Add 200 μL TSB liquid medium to the sterile control well.

[0167] The growth control wells were added with 150 μL TSB liquid medium and 50 μL Propionibacterium acnes CCSMS0630 bacterial suspension.

[0168] Four replicate wells were made in each group;

[0169] (4) The 96-well plate was incubated at 37°C for 48 h and the OD was measured using an ELISA reader. 600 The inhibition rate was calculated as follows: 1-(OD value of the experimental group-OD value of the experimental blank group) / (OD value of the growth control well-OD value of the sterile control well) × 100%. Figure 6 The results showed that the fermentation supernatant of S.epidermidis CCSMS1601 inhibited the growth of Propionibacterium acnes at low concentrations and promoted its growth at high concentrations. At a concentration of 25%, the growth rate of Propionibacterium acnes CCSMS0630 was 20.84±2.25%, which was the growth rate of Propionibacterium acnes in normal flora, and regulated the scalp microecology, which was helpful to improve scalp health.

[0170] Example 10

[0171] This example provides an intervention test of the fermentation supernatant of S. epidermidis CCSMS1601 on Malassezia globosa CCSMS1050.

[0172] (1) Selection of the concentration of the fermentation supernatant of S. epidermidis CCSMS1601: the fermentation supernatant of S. epidermidis CCSMS1601 was mixed with sterile Dixon medium at a ratio of 1:1 by a half-dilution method to obtain the fermentation supernatant of S. epidermidis CCSMS1601 with concentrations of 25, 12.5, 6.25, 3.13, and 1.56%;

[0173] (2) Preparation of Malassezia suspension: Take out the -80°C stored Malassezia spherica CCSMS1050 strain milk powder freeze-dried tube, streak the Dixon agar plate, streak it using the three-zone streak method, place it in a 32°C constant temperature incubator and culture it for 72 hours to make a first-generation activation plate, pick a single colony from the colonies grown on the first-generation plate, streak it on the second-generation plate, place it in a 32°C constant temperature incubator and culture it aerobically for 72 hours, and set it aside. Pick a single colony of the Malassezia that has been activated for two generations and inoculate it into a conical flask containing 100 mL of Dixon liquid culture medium, place it in a 32°C shaker, and shake and culture it at a speed of 150 r / min for 72 hours to adjust the bacterial OD 600 In the range of 0.7~0.8, keep in reserve.

[0174] (3) In the experimental group, 50 μL of Malassezia CCSMS1050 culture medium, 50 μL of S. epidermidis CCSMS1601 fermentation supernatant and 100 μL of Dixon liquid medium were added to each well of the 96-well plate.

[0175] The blank group was supplemented with 100 μL Dixon liquid medium and 100 μL fermentation supernatant of S. epidermidis CCSMS1601.

[0176] Add 200 μL Dixon liquid medium to the sterile control well.

[0177] The growth control wells were added with 150 μL Dixon liquid medium and 50 μL Malassezia CCSMS1050 bacterial solution, and 4 replicate wells were made for each group;

[0178] (4) The 96-well plate was incubated at 32°C for 72 h and the OD was measured using an ELISA reader. 600 The inhibition rate was calculated as follows: 1-(OD value of the experimental group-OD value of the experimental blank group) / (OD value of the growth control well-OD value of the sterile control well) × 100%. Figure 7The results showed that the fermentation supernatant of S.epidermidis CCSMS1601 inhibited the growth of Malassezia globosa CCSMS1050 at low concentrations and promoted its growth at high concentrations, and the proliferation rate increased with increasing concentration. At a concentration of 25%, the growth proliferation rate of Malassezia globosa CCSMS1050 was 22.42±4.48%, indicating that the fermentation supernatant of S.epidermidis CCSMS1601 at low concentrations can significantly promote the growth of Malassezia globosa, regulate the scalp microecology, and help improve scalp health.

[0179] Embodiment 11

[0180] This example provides an intervention test of the fermentation supernatant of S. epidermidis CCSMS1601 on Malassezia restricta CCSM1060.

[0181] (1) Selection of the concentration of the fermentation supernatant of S. epidermidis CCSMS1601: the fermentation supernatant was mixed with sterile Dixon medium at a ratio of 1:1 by a half-dilution method to obtain the fermentation supernatant of S. epidermidis CCSMS1601 with concentrations of 25, 12.5, 6.25, 3.13, and 1.56%;

[0182] (2) Preparation of Malassezia bacterial suspension: Take out the -80°C storage restricted Malassezia CCSM1060 strain milk powder freeze-dried tube, streak the Dixon agar plate, streak it using the three-zone streak method, place it in a 32°C constant temperature incubator and culture it for 72 hours to make a first-generation activation plate, pick a single colony from the colonies grown on the first-generation plate, streak it on the second-generation plate, place it in a 32°C constant temperature incubator and culture it aerobically for 72 hours, and set it aside. Pick a single colony of the Malassezia that has been activated for two generations and inoculate it into a conical flask containing 100 mL of Dixon liquid culture medium, place it in a 32°C shaker, and shake and culture it at a speed of 150r / min for 72 hours to adjust the bacterial OD 600 In the range of 0.7~0.8, keep in reserve.

[0183] (3) In the experimental group, 50 μL of Malassezia restricta CCSM1060 culture medium, 50 μL of S. epidermidis CCSMS1601 fermentation supernatant and 100 μL of Dixon liquid medium were added to each well of the 96-well plate.

[0184] The blank group was supplemented with 100 μL Dixon liquid medium and 100 μL fermentation supernatant of S. epidermidis CCSMS1601.

[0185] Add 200 μL Dixon liquid medium to the sterile control well.

[0186] The growth control wells were added with 150 μL Dixon liquid medium and 50 μL of Malassezia CCSM1060 bacterial solution.

[0187] Four replicate wells were made in each group;

[0188] (4) The 96-well plate was incubated at 32°C for 72 h and the OD was measured using an ELISA reader. 600 The inhibition rate was calculated as follows: 1-(OD value of the experimental group-OD value of the experimental blank group) / (OD value of the growth control well-OD value of the sterile control well) × 100%. Figure 8 The results showed that the fermentation supernatant of S. epidermidis CCSMS1601 had certain antifungal ability, and the inhibition rate of the fermentation supernatant of S. epidermidis CCSMS1601 on Malassezia restricta CCSM1060 was about 20%, and the growth inhibition rate of Malassezia restricta CCSM1060 was 23.29±2.04% at a concentration of 25%, indicating that the fermentation supernatant of S. epidermidis CCSMS1601 significantly inhibited the growth of Malassezia restricta, had potential in regulating the scalp microecology, and helped to improve scalp health.

[0189] In summary, the fermentation supernatant of S. epidermidis CCSMS1601 contains a variety of short-chain fatty acids, T-SOD enzyme and GSH-PX enzyme and other active ingredients, has good anti-inflammatory and antioxidant abilities, can significantly promote the growth of resident scalp fungi Propionibacterium acnes, and can promote the growth of Malassezia globosa, and has a certain inhibitory effect on the growth of Malassezia restricta. These characteristics indicate that the fermentation supernatant of S. epidermidis CCSMS1601 has good application prospects in the development of scalp care products for regulating scalp microecology, and the present invention lays a foundation for the exploration of functional skin bacteria.

[0190] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the explanation of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A Staphylococcus epidermidis CCSMS1601, characterized in that: It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 5, 2024, with the deposit number CGMCCNO.30874.

2. A 16s rDNA sequence of Staphylococcus epidermidis CCSMS1601, characterized in that: As shown in SEQ ID NO.

3.

3. A method for culturing Staphylococcus epidermidis CCSMS1601, characterized in that: The following steps are involved: Staphylococcus epidermidis CCSMS1601 was cultured in TSB liquid medium; During the culture process, the pH of the TSB liquid culture medium is 4.0-9.0, and the culture temperature is 30-40°C.

4. A fermentation method of Staphylococcus epidermidis CCSMS1601, characterized in that: The following steps are involved: (S1) inoculating activated Staphylococcus epidermidis CCSMS1601 into TSB liquid culture medium to obtain seed solution; (S2) inoculating the seed solution prepared in step (S1) into a fermentation medium to obtain a fermentation solution of Staphylococcus epidermidis CCSMS1601; The fermentation liquid of Staphylococcus epidermidis CCSMS1601 is centrifuged to obtain a supernatant, which is a fermentation supernatant of Staphylococcus epidermidis CCSMS1601.

5. The fermentation method of Staphylococcus epidermidis CCSMS1601 according to claim 4, characterized in that: In step (S1), during the culture process, the temperature is 30-40°C and the time is 16-20 hours.

6. The fermentation method of Staphylococcus epidermidis CCSMS1601 according to claim 4, characterized in that: In step (S2), during the fermentation process, the temperature is 30-40°C and the time is 18-20 hours.

7. A fermentation supernatant of Staphylococcus epidermidis CCSMS1601, characterized in that: It is prepared by the method according to any one of claims 4 to 6.

8. Use of the fermentation supernatant of Staphylococcus epidermidis CCSMS1601 according to claim 7 in the preparation of scalp care products.

9. The use according to claim 8, characterized in that: The scalp care product is an anti-inflammatory scalp care product.

10. The use according to claim 9, characterized in that: The scalp care product is an antioxidant scalp care product.

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