Staphylococcus epidermidis CCSMS1601 and culture and application thereof

Abstract

The present application relates to the technical field of microorganisms, and particularly relates to a Staphylococcus epidermidis CCSMS1601 as well as culture and application thereof.The Staphylococcus epidermidis CCSMS1601 provided by the present application has been preserved in the China General Microbiological Culture Collection Center on June 5, 2024, and the preservation number is CGMCC NO.30874.The fermentation supernatant obtained by using the Staphylococcus epidermidis CCSMS1601 has the function of regulating scalp flora, and thus improves dandruff and is beneficial to scalp health;the strain CCSMS1601 has a good application prospect in the development of scalp care products with anti-inflammatory, antioxidant and scalp microecological regulation functions.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a Staphylococcus epidermidis CCSMS1601 and its cultivation and application. Background Technology

[0002] The scalp, an integral part of human skin, is a unique ecosystem. The scalp microbiome comprises various microorganisms, such as bacteria, fungi, and others. The composition and dynamic distribution of this microecological community maintain a holistic balance with the scalp's surface tissue cells and the host's healthy immune status. The most common bacterial genera residing on the scalp of healthy adults are Propionibacterium, Staphylococcus, and Corynebacterium. The most prevalent fungi on the scalp are Malassezia, which are fewer in number than bacteria, primarily including Malassezia restricta and Malassezia cocci. Normal scalp microbiota maintain scalp health through various means, including consuming secretions, supporting metabolism, and enhancing the immune system. These microorganisms interact in complex ways, playing different roles in scalp health and disease development.

[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 scalp's microecological balance and self-cleaning function. As an important component of the scalp flora, changes in its colonization ratio can lead to imbalances in other bacterial groups, resulting in scalp problems.

[0004] More than 50% of the world's population suffers from dandruff. The main symptoms of dandruff include itchy scalp, flaking, and oily hair; in severe cases, it can lead to inflammation and hair loss. The pathogenesis of dandruff is complex, involving multiple factors such as abnormal sebum secretion, fungal infection, scalp microecological dysbiosis, and stratum corneum metabolic disorders. Currently, most studies indicate that compared to healthy individuals, people with dandruff have a lower relative abundance of Propionibacterium acnes and Malassezia globosa, and a higher relative abundance of Malassezia restricta in the scalp areas.

[0005] Currently, research on the mechanisms and prevention of dandruff mainly focuses on Malassezia. Commonly used treatments include shampoos containing antifungal ingredients, antibiotics, and anti-inflammatory drugs. However, long-term use of antifungal drugs can lead to drug resistance and side effects such as dizziness and burning sensations. Therefore, simply treating dandruff with antifungal medications may not be the only effective strategy. A comprehensive approach is needed to restore the homeostasis of scalp microbiota and protect scalp health from the root cause. Summary of the Invention

[0006] To address the aforementioned problems, the present invention aims to provide a Staphylococcus epidermidis CCSMS1601 strain and its culture and application.

[0007] This invention isolates a strain of Staphylococcus epidermidis CCSMS1601 from healthy scalp. Its fermentation supernatant has the advantage of promoting the proliferation of Propionibacterium acnes and Malassezia spheroidae while inhibiting the proliferation of Malassezia restricta (it has the function of regulating scalp flora, thereby improving dandruff and benefiting scalp health). It can be used in the development of anti-inflammatory and antioxidant scalp care and dandruff prevention and treatment products, and has good application prospects.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] The first objective of this invention is to provide a Staphylococcus epidermidis CCSMS1601, which was deposited on June 5, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.30874.

[0010] In one embodiment of the present invention, the bacterial cells of Staphylococcus epidermidis CCSMS1601 are spherical and arranged in a grape-like or single manner, with a diameter of 0.9 to 1.0 μm, no flagella, do not produce spores, and are Gram-positive.

[0011] In one embodiment of the present invention, Staphylococcus epidermidis CCSMS1601 was streaked onto a TSA plate and cultured aerobically at 37°C for 16 hours. The strain grew well. Its colonies were small, white, round protrusions with a diameter of approximately 0.5–1 mm, with neat edges, a smooth and moist surface, and were opaque. They could be drawn into threads when picked up and did not exhibit hemolytic zones.

[0012] A second objective of this invention is to provide a 16S rDNA sequence of Staphylococcus epidermidis CCSMS1601, as shown in SEQ ID NO.3.

[0013] A third objective of this invention is to provide a method for culturing Staphylococcus epidermidis CCSMS1601, comprising the following steps:

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

[0015] During the cultivation process, the pH of the TSB liquid medium was 4.0–9.0, the cultivation temperature was 30–40℃, and the cultivation time was 36 h.

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

[0017] (S1) Activated Staphylococcus epidermidis CCSMS1601 was inoculated into TSB liquid medium and cultured to obtain seed culture.

[0018] (S2) The seed liquid prepared in step (S1) is inoculated into the fermentation medium to obtain the fermentation broth of Staphylococcus epidermidis CCSMS1601.

[0019] The fermentation broth of Staphylococcus epidermidis CCSMS1601 was centrifuged, and the resulting supernatant was the fermentation supernatant of Staphylococcus epidermidis CCSMS1601.

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

[0021] In one embodiment of the present invention, in step (S2), the fermentation process is carried out at a temperature of 30-40°C for 18-20 hours.

[0022] The fifth objective of this 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] The sixth objective of this invention is to provide the application of the fermentation supernatant of Staphylococcus epidermidis CCSMS1601 in the preparation of scalp care products.

[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 spheroidae while inhibiting 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 (with an inhibition rate of 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 designed to eliminate ABTS free radicals (IC). 50 The value was 1.82% and DPPH free radicals (IC). 50 Scalp care products with a value of 23.80%.

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

[0031] The *Staphylococcus epidermidis* CCSMS1601 strain provided by this invention is rich in short-chain fatty acids and other active ingredients, exhibiting good anti-inflammatory effects. Its fermentation supernatant showed an inhibition rate of 101.51% against hyaluronidase, and the activities of T-SOD and GSH-PX in the fermentation supernatant were 27.32 U / mL and 112.63 U / mL, respectively. The IC50 value for scavenging ABTS and DPPH free radicals was also significant. 50 Concentrations of 1.82% and 23.80% showed good anti-inflammatory and antioxidant effects. Antibacterial tests indicated that a 25% (v / v) concentration of Staphylococcus epidermidis CCSMS1601 fermentation supernatant promoted the growth of Propionibacterium acnes CCSMS0630 and Malassezia spheroides CCSMS1050, and significantly inhibited the growth of Malassezia restricta CCSMS1060. This can regulate the scalp's microecological balance, reduce the growth and reproduction of harmful bacteria, and help improve the health of dandruff-prone scalps.

[0032] The Staphylococcus epidermidis CCSMS1601 provided by this invention has good application prospects in the development of anti-inflammatory and antioxidant scalp care products, filling the gap in the screening of the anti-inflammatory, antioxidant and scalp microecological regulation effects of Staphylococcus epidermidis CCSMS1601. Attached Figure Description

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

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

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

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

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

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

[0039] Figure 7 The results of the intervention of Staphylococcus epidermidis CCSMS1601 fermentation supernatant at concentrations of 1.56%, 3.13%, 6.25%, 12.5%, and 25% on Malassezia CCSMS1050 are shown.

[0040] Figure 8The results show the effects of fermentation supernatant of Staphylococcus epidermidis CCSMS1601 at concentrations of 1.56%, 3.13%, 6.25%, 12.5%, and 25% on the restriction of Malassezia CCSMS1060. Detailed Implementation

[0041] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0042] Unless otherwise specified, all reagents used in the following embodiments are commercially available reagents, and all detection methods and techniques used are conventional detection methods and techniques in the art.

[0043] Experimental strains: *Propionibacterium acnes* CCSMS0630 (deposited at the China General Microbiological Culture Collection Center on January 18, 2024, with accession number CGMCC NO.29674, classified as *Propionibacterium acnes* CCSMS0630), *Malassezia globose* CCSMS1050 (deposited at the China General Microbiological Culture Collection Center on August 26, 2024, with accession number CGMCC NO.31746, classified as *Malassezia globose* CCSMS1050), and *Malassezia restrictiveis* CCSMS1060 (deposited at the China General Microbiological Culture Collection Center on August 26, 2024, with accession number CGMCC NO.31747, classified as *Malassezia restrictiveis*). (restricta)CCSMS1060) were all isolated from healthy human scalp skin in the laboratory.

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

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

[0046] Tryptic soybean liquid medium (TSB liquid medium): 15 g / L tryptic, 5 g / L soybean papain hydrolysate, sodium chloride, autoclave at 121°C for 15 min, ready for use.

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

[0048] Preservation medium: 120g skim milk powder, 880mL distilled water, autoclave at 121℃ for 15min, for later use.

[0049] Example 1

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

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

[0052] 1) No topical antifungal medications or corticosteroids have been used on the scalp within the past two weeks;

[0053] 2) Has not taken any oral antifungal medication within the past two months;

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

[0055] 4) Age 18-30, gender not limited.

[0056] (2) Sample collection

[0057] Volunteers were required to not wash their hair the night before sampling and to collect samples the following day at noon or in the afternoon.

[0058] Moisten a sterile cotton swab made of polymer fiber material in a wetting solution (0.9% sodium chloride and 0.1% Tween-20), and swab it back and forth 50 times in the selected area of ​​the scalp (the swab should be swiped with a certain amount of force). Place the sterile cotton swab in the sampling tube with sterile forceps and seal it with sealing film. Place the collected sample in an ice box for refrigeration, bring it back to the laboratory and place it in a 4°C refrigerator for isolation of the bacterial strain as soon as possible.

[0059] (3) Sample pretreatment

[0060] In a clean bench, use sterile scissors to cut off the sterile cotton swab tip containing the sample and place it in a 5mL centrifuge tube (EP tube). Add 5mL of sterile water to the centrifuge tube and mix thoroughly to 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 serial dilution. Select an appropriate dilution and spread 0.1 mL of the diluted solution onto TSA blood plates. Spread 2 plates for each dilution and place them in an incubator at 37°C for aerobic incubation for 24 h.

[0063] (5) Marking, purifying and preserving

[0064] Based on the colony characteristics of coagulase-negative staphylococcal strains on blood agar plates, and according to differences such as whether hemolysis zones are produced, colony size, color, moisture, and luster, single colonies were picked and streaked onto TSA blood agar plates and incubated at 37°C for 16 hours. After repeated streaking and purification, strain CCSMS1601 was obtained and stored in preservation medium (using skim milk powder as a protectant) in preservation tubes. After freeze-drying, it was stored in a -80°C cryogenic freezer.

[0065] Example 2

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

[0067] (1) Colony characteristics

[0068] Strawberry strain CCSMS1601 was streaked on TSA blood agar plates and incubated at 37°C for 16 hours. The colony morphology of the strain on the plates was then observed. Results are as follows: Figure 1 As shown in a, from Figure 1 As can be seen, Staphylococcus epidermidis colonies on TSA blood agar plates are white and small, forming round raised bumps with neat edges. The colony diameter is about 0.5-1 mm, the surface is smooth, moist, and opaque, and can be stretched into strings when picked up, without hemolytic zones.

[0069] (2) Bacterial characteristics

[0070] A small amount of bacterial cells from strain CCSMS1601 was picked and smeared onto a glass slide, Gram-stained, and the morphological characteristics of the cells were observed under a microscope. Results are shown below. Figure 1 As shown in b, from Figure 1 b shows that the bacterial cells are Gram-positive, and the cells are spherical with a diameter of about 0.9 to 1.0 μm, arranged in a grape-like pattern, or arranged singly. They are non-motile, do not produce spores, and lack flagella.

[0071] (3) Characteristics of Cultivation

[0072] The strain CCSMS1601 was inoculated into TSB liquid medium and cultured at different temperatures. The results showed that the minimum growth temperature of the strain was 15℃, the maximum growth temperature was 45℃, and the optimal growth temperature was 30-40℃. When inoculated into TSB liquid medium with different pH values ​​and cultured at 37℃, the highest growth pH was found to be 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: Select a single colony of purified strain CCSMS1601 and inoculate it into 10 mL of TSB liquid medium. After incubation at 37°C for 16 h, centrifuge the bacterial solution (8000 r / min, 15 min) to collect the bacterial cells.

[0076] Genomic DNA was extracted using a genomic DNA extraction kit (Sangon Biotech (Shanghai) Co., Ltd.). PCR amplification was performed using two synthetic universal primers (16S 27F (SEQ ID NO.1): 5'-GAGAGTTTGA TCCTGGCTCAG-3'; 16S 1492R (SEQ ID NO.2): 5'-CGGCTACCTTGTTACGAC TT-3'). PCR products were recovered using a column-based PCR product purification kit (Sangon Biotech (Shanghai) Co., Ltd.) and then sent to Meiji Biotechnology (Shanghai) Co., Ltd. for sequencing. The 16S rDNA nucleotide sequence of strain CCSMS1601 is shown in SEQ ID NO.3, with a sequence length of 1384 bp. Blast analysis was performed in GenBank. The strain with the highest homology to strain CCSMS1601 was MT585523.1, with 100% homology.

[0077] 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%–12% and 16S rRNA sequence homology ≥95% can be classified into the same genus. Furthermore, Embey and Stackebrangdt suggest that 16S rRNA sequence homology ≥97% can be considered a single 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 aforementioned microbiological characteristics, including colony and cell morphology, culture, physiological and biochemical identification, as well as its genetic characteristics (16S rDNA), strain CCSMS1601 was identified as Staphylococcus epidermidis and deposited at the China General Microbiological Culture Collection Center on June 5, 2024, with accession number CGMCCNO.30874 and classification name Staphylococcus epidermidis CCSMS1601.

[0082] Activated Staphylococcus epidermidis CCSMS1601 was inoculated into TSB liquid medium at a 2% (v / v) inoculum and cultured at 37°C with shaking for 48 h. The OD value of the culture medium 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 values ​​against time. The results are as follows: Figure 2 As shown, from Figure 2 As can be seen, Staphylococcus epidermidis CCSMS1601 grows rapidly in TSB broth liquid medium, entering the logarithmic growth phase after 8 hours and reaching the stationary phase after 36 hours.

[0083] Example 3

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

[0085] (1) The S. epidermidis CCSMS1601 strain was streaked onto TSA plates and incubated at 37℃ for 18 h to activate it twice; the activated S. epidermidis CCSMS1601 was inoculated into TSB liquid medium and cultured at 37℃ with shaking for 18 h to adjust the bacterial OD. 600 The concentration is in the range of 0.55 to 0.56, used as seed solution.

[0086] (2) The seed liquid prepared in step (1) was inoculated into 150 mL of TSB liquid medium at an inoculation rate of 1% (v / v), and placed in a constant temperature shaking incubator at 37℃. It was cultured at a speed of 160 r / min for 20 h to obtain the fermentation broth of S. epidermidis CCSMS1601.

[0087] (3) Centrifuge the fermentation broth prepared in step (2) at 9000 r / min for 10 min. The resulting supernatant is the fermentation supernatant of S. epidermidis CCSMS1601.

[0088] Example 4

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

[0090] (1) Sample pretreatment

[0091] Add 2 mL of fermentation supernatant of S. epidermidis CCSMS1601, 0.4 mL of 50% H2SO4 and 2 mL of diethyl ether to a 10 mL centrifuge tube and mix well. Mix at 250 r / min for 45 min on a shaker, then centrifuge at 10000 r / min for 5 min at 4 °C. Take the upper organic phase, filter it through a 0.22 μm organic filter membrane before GC injection, and retain the supernatant for storage at -20 °C for later use.

[0092] (2) Gas chromatography conditions

[0093] Short-chain fatty acids in the fermentation supernatant of *S. epidermidis* CCSMS1601 were determined by GC. The instrument used was a GC-2010plus; the column was an Agilent DB-WAX UI (60m × 0.25μm × 0.25mm); the injector and detector temperatures were set to 250℃; high-purity nitrogen was used as the carrier gas (flow rate 1.2mL / min), and splitless detection mode was employed; the temperature conditions were as follows: 1μL sample was loaded, and the temperature was increased from 75℃ to 180℃ at a rate of 10℃ / min and held for 1 min, then increased to 220℃ at a rate of 10℃ / min and held for 1 min.

[0094] (3) Preparation of standard curve

[0095] Water-soluble fatty acid mixed standard solution (Northern Weiye Metrology Research Institute): This solution comprises six components: glacial acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid, each with a standard value of 1000 μg / mL. The mixed standard is diluted with ultrapure water to obtain nine concentration gradients: 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 500 μg / mL, 800 μg / mL, and 1000 μg / mL. It is stored at -20℃ for later use.

[0096] (4) Sample content determination

[0097] The fermentation supernatant of S. epidermidis CCSMS1601 was determined to contain 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 fermentation supernatant of CCSMS1601

[0099]

[0100] Example 5

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

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

[0103] CaCl2 solution: 2.5 mmol / L;

[0104] NaOH solution: 0.4 mol / L;

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

[0106] Hyaluronidase solution: 500 U / mL;

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

[0108] Acetylacetone solution: 1.4 mL;

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

[0110] (2) Experimental steps

[0111] 1) Take four test tubes and label them a, b, C, and D. Add 100 μL of 2.5 mmol / L CaCl2 solution and 500 μL of 600 U / mL hyaluronidase solution to test tubes a and c, respectively, and treat them at 37°C for 20 min. Add 100 μL of 2.5 mmol / L CaCl2 solution and 500 μL of acetate buffer to test tubes b and d, and treat them at 37°C for 20 min.

[0112] 2) Add 500 μL of 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℃ 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℃ 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 incubate in boiling water for 15 min and in 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 into an ELISA plate and measure its absorbance at a wavelength of 530 nm.

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

[0117] (3) Calculate the hyaluronidase inhibition rate

[0118]

[0119] In the formula: 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%, indicating a good anti-inflammatory effect.

[0124] Example 6

[0125] This embodiment provides the determination of T-SOD and GSH-PX enzymes in the fermentation supernatant of S. epidermidis CCSMS1601.

[0126] (I) After preparing the required working solution according to the instructions of the glutathione peroxidase (GSH-PX) test kit (Nanjing Jiancheng Bioengineering Institute), the following experiment was conducted.

[0127] 1) Enzymatic reaction:

[0128] Table 2 Summary of Enzyme-Catalyzed Reactions

[0129]

[0130] Among them, the reagent 1 application solution should be preheated to 37°C before use;

[0131] 2) Color reaction:

[0132] Table 3 Summary of Colorimetric Reactions

[0133]

[0134] After mixing and standing at room temperature for 15 minutes, measure the OD value of each tube at 412 nm with a 1 cm optical path, using distilled water to zero the instrument.

[0135] Calculation formula:

[0136]

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

[0138] (2) After preparing the required working solution according to the instructions of the Total Superoxide Dismutase (T-SOD) Assay Kit (Nanjing Jiancheng Bioengineering Institute), the following experiments were conducted:

[0139] Table 4 Summary of Reactions

[0140] reagents Measurement 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 minutes.

[0142] Colorimetric reagent (mL) 2 2

[0143] Mix well, let stand at room temperature for 10 minutes, and measure the color at a wavelength of 550 nm using a 1 cm optical path cuvette and distilled water to zero the instrument.

[0144] Calculation formula:

[0145]

[0146] The fermentation supernatant of 100 μL 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 embodiment provides an ABTS free radical scavenging test of the fermentation supernatant of S. epidermidis CCSMS1601.

[0149] Mix equal volumes of 7 mmol / L ABTS with 2.6 mmol / L potassium persulfate solution and incubate at room temperature in the dark for 14 hours to obtain ABTS stock solution. Before use, dilute with anhydrous ethanol to obtain an absorbance of 0.70 ± 0.02 at a wavelength of 734 nm, thus obtaining ABTS working solution.

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

[0151] Sample solution: The fermentation supernatant of S. epidermidis CCSMS1601 was diluted 20, 40, 60, 80 and 100 times, 2.0 mL of anhydrous ethanol was added, and then ABTS working solution was added to make the final volume 4.0 mL. The mixture was allowed to stand in the dark for 30 min, and the absorbance was measured at a wavelength of 734 nm and recorded as a.

[0152] The experiment used V at the same mass concentration C As a positive control, each sample was repeated 3 times. 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 observed that the ABTS radical scavenging ability of the fermentation supernatant of *S. epidermidis* CCSMS1601 increases with increasing fermentation supernatant concentration, and the IC50 of the ABTS radical scavenging effect is [not specified]. 50 The concentration was 1.82% (the original fermentation broth concentration was 100%), indicating that the fermentation supernatant of S. epidermidis CCSMS1601 has a good ability to resist oxidation and scavenge free radicals.

[0154] Example 8

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

[0156] Control group: Take 1 mL of 2×10 -4 A mol / L DPPH solution was diluted with water to a final volume of 4 mL. After being kept at room temperature in the dark for 30 min, the absorbance was measured at 517 nm and recorded as a0.

[0157] Sample solution: Take 1 mL of DPPH solution and 1 mL of fermentation supernatant of S. epidermidis CCSMS1601 with concentrations of 5%, 2.5%, and 1.67%, respectively. Add water to make the final volume 4 mL. After incubation at room temperature in the dark for 30 min, measure the absorbance of fermentation supernatant of different concentrations and record it as a.

[0158] The experiment used V at the same mass concentration C As a positive control, each sample was repeated 3 times. 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 observed that the DPPH radical scavenging ability of the fermentation supernatant of *S. epidermidis* CCSMS1601 increases with increasing concentration, and the IC50 for DPPH radical scavenging... 50 The concentration was 23.80% (original fermentation broth concentration was 100%), indicating that the fermentation supernatant of S. epidermidis CCSMS1601 has good antioxidant capacity.

[0160] Example 9

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

[0162] (1) Selection of fermentation supernatant concentration of S. epidermidis CCSMS1601: The fermentation supernatant was mixed with sterile TSB liquid culture medium at a ratio of 1:1 using the 1:2 dilution method to obtain fermentation supernatant 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 Propionibacterium acnes strain milk powder stored at -80℃, streak it on a TSA plate, and place it in a constant temperature incubator at 37℃ for anaerobic culture for 54h; after activation twice, pick a single colony of Propionibacterium acnes activated for two generations and inoculate it into a test tube containing 5mL of TSB liquid medium, place it in a constant temperature incubator at 37℃ for anaerobic culture for 54h, and adjust the bacterial OD. 600 Keep it at around 0.3 to 0.4 for later use.

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

[0165] The experimental blank group was supplemented with 100 μL of TSB liquid medium and 100 μL of fermentation supernatant from 1.epidermidis CCSMS1601.

[0166] Add 200 μL of TSB liquid culture medium to the aseptic control well.

[0167] Add 150 μL of TSB liquid medium and 50 μL of Propionibacterium acnes CCSMS0630 bacterial suspension to the growth control well.

[0168] Make 4 duplicate holes in each group;

[0169] (4) Incubate the 96-well plate at 37°C for 48 hours, and measure the OD using a microplate reader. 600 The inhibition rate was calculated as follows: 1 - (OD value of experimental group - OD value of experimental blank group) / (OD value of growth control well - OD value of sterile control well) × 100%. The experimental results are as follows: Figure 6 The fermentation supernatant of *S. epidermidis* CCSMS1601 showed that low concentrations inhibited the growth of *Propionibacterium acnes*, while high concentrations promoted its growth. At a concentration of 25%, it inhibited the growth and proliferation rate of *Propionibacterium acnes* CCSMS0630 by 20.84 ± 2.25% compared to the normal flora of *Propionibacterium acnes*, thus regulating the scalp microecology and contributing to scalp health.

[0170] Example 10

[0171] This embodiment provides an intervention experiment of fermentation supernatant of S. epidermidis CCSMS1601 on spherical Malassezia CCSMS1050.

[0172] (1) Selection of fermentation supernatant concentration of S. epidermidis CCSMS1601: The fermentation supernatant of S. epidermidis CCSMS1601 was mixed with sterile Dixon medium at a ratio of 1:1 using the 1:2 dilution method to obtain fermentation supernatant of S. epidermidis CCSMS1601 with concentrations of 25, 12.5, 6.25, 3.13 and 1.56%;

[0173] (2) Preparation of Malassezia bacterial suspension: Take the -80℃ preserved spherical Malassezia CCSMS1050 strain milk powder freeze-dried tubes, streak them onto Dixon agar plates using the three-zone streak method, and incubate them at 32℃ for 72 hours to obtain the first-generation activated plates. Pick a single colony from the first-generation plates and streak it onto a second-generation plate, then incubate it at 32℃ for aerobic incubation for 72 hours. After two generations of activation, pick a single colony and inoculate it into an Erlenmeyer flask containing 100 mL of Dixon liquid medium. Place the flask in a shaker at 32℃ and shake at 150 rpm for 72 hours. Adjust the bacterial OD... 600 In the range of 0.7 to 0.8, keep it as a backup.

[0174] (3) In the experimental group, 50 μL of Malassezia CCSMS1050 bacterial culture, 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 experimental blank group was supplemented with 100 μL of Dixon liquid medium and 100 μL of fermentation supernatant from S. epidermidis CCSMS1601.

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

[0177] Add 150 μL of Dixon liquid medium and 50 μL of Malassezia CCSMS1050 bacterial suspension to each growth control well, and make 4 replicates for each group;

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

[0179] Example 11

[0180] This embodiment provides an intervention experiment of fermentation supernatant of S. epidermidis CCSMS1601 on restriction Malassezia CCSM1060.

[0181] (1) Selection of fermentation supernatant concentration of S. epidermidis CCSMS1601: The fermentation supernatant was mixed with sterile Dixon medium at a ratio of 1:1 using the 1:2 dilution method to obtain 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: Recycle -80℃ stored Malassezia CCSM1060 strain milk powder freeze-dried tubes, streak them onto Dixon agar plates using the three-zone streak method, and incubate at 32℃ for 72 hours to obtain first-generation activated plates. Single colonies from the first-generation plates are streaked onto second-generation plates and incubated aerobically at 32℃ for 72 hours. Single colonies from the two generations of activated Malassezia are then inoculated into Erlenmeyer flasks containing 100 mL of Dixon liquid medium and incubated at 32℃ with shaking at 150 rpm for 72 hours. Adjust the bacterial OD... 600 In the range of 0.7 to 0.8, keep it as a backup.

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

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

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

[0186] Add 150 μL of Dixon liquid medium and 50 μL of Malassezia CCSM1060 bacterial suspension to the growth control well.

[0187] Make 4 duplicate holes in each group;

[0188] (4) Incubate the 96-well plate at 32℃ for 72 h, and measure the OD using a microplate reader. 600 The inhibition rate was calculated as follows: 1 - (OD value of experimental group - OD value of experimental blank group) / (OD value of growth control well - OD value of sterile control well) × 100%. The experimental results are as follows: Figure 8 The fermentation supernatant of *S. epidermidis* CCSMS1601 showed that it has certain antifungal activity. The fermentation supernatant of *S. epidermidis* CCSMS1601 inhibited the growth of *Malassezia restrictorum* CCSM1060 by about 20%, and at a concentration of 25%, the growth inhibition rate of *Malassezia restrictorum* CCSM1060 was 23.29 ± 2.04%. This indicates that the fermentation supernatant of *S. epidermidis* CCSMS1601 significantly inhibits the growth of *Malassezia restrictorum* and has the potential to regulate scalp microecology and help improve scalp health.

[0189] In summary, the fermentation supernatant of *S. epidermidis* CCSMS1601 contains various short-chain fatty acids, T-SOD enzymes, and GSH-PX enzymes, exhibiting good anti-inflammatory and antioxidant capabilities. It can significantly promote the growth of resident scalp bacteria *Propionibacterium acnes*, while also promoting the growth of *Malassezia spheroidosa* and inhibiting the growth of *Malassezia spheroidosa*. These characteristics indicate that the fermentation supernatant of *S. epidermidis* CCSMS1601 has promising application prospects in the development of scalp care products that regulate the scalp microecology. This invention lays the foundation for the discovery of functional skin bacteria.

[0190] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the interpretation of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. A type of Staphylococcus epidermidis ( Staphylococcus epidermidis CCSMS1601, characterized in that, It was deposited at the China General Microbiological Culture Collection Center on June 5, 2024, with accession number CGMCCNO.30874.

2. A type of Staphylococcus epidermidis ( Staphylococcus epidermidis The cultivation method of CCSMS1601 is characterized by, Includes the following steps: Staphylococcus epidermidis ( Staphylococcus epidermidis CCSMS1601 was cultured in TSB liquid medium; During the cultivation process, the pH of the TSB liquid medium was 4.0~9.0, and the cultivation temperature was 30~40℃; Among them, the Staphylococcus epidermidis ( Staphylococcus epidermidis CCSMS1601 was deposited at the China General Microbiological Culture Collection Center on June 5, 2024, with accession number CGMCC NO.30874.

3. A type of Staphylococcus epidermidis ( Staphylococcus epidermidis The fermentation method of CCSMS1601 is characterized by, Includes the following steps: (S1) Activated Staphylococcus epidermidis ( Staphylococcus epidermidis CCSMS1601 was inoculated into TSB liquid medium and cultured to obtain seed culture; (S2) The seed culture prepared in step (S1) is inoculated into the fermentation medium to culture Staphylococcus epidermidis (S2). Staphylococcus epidermidis Fermentation broth of CCSMS1601; Among them, Staphylococcus epidermidis ( Staphylococcus epidermidis The supernatant obtained by centrifuging the fermentation broth of CCSMS1601 was Staphylococcus epidermidis (Staphylococcus epidermidis). Staphylococcus epidermidis Fermentation supernatant of CCSMS1601; The Staphylococcus epidermidis ( Staphylococcus epidermidis CCSMS1601 was deposited at the China General Microbiological Culture Collection Center on June 5, 2024, with accession number CGMCC NO.30874.

4. A Staphylococcus epidermidis strain according to claim 3 ( Staphylococcus epidermidis The fermentation method of CCSMS1601 is characterized by, In step (S1), the temperature is 30~40℃ and the time is 16~20h during the cultivation process.

5. A Staphylococcus epidermidis strain according to claim 3 ( Staphylococcus epidermidis The fermentation method of CCSMS1601 is characterized by, In step (S2), the fermentation process is carried out at a temperature of 30~40℃ for 18~20h.

6. A type of Staphylococcus epidermidis ( Staphylococcus epidermidis The fermentation supernatant of CCSMS1601 is characterized in that... It is prepared by any one of the methods described in claims 3 to 5.

7. A Staphylococcus epidermidis strain as described in claim 6 ( Staphylococcus epidermidis Application of fermentation supernatant of CCSMS1601 in the preparation of scalp care products.

8. The application according to claim 7, characterized in that, The scalp care product mentioned is an anti-inflammatory scalp care product.

9. The application according to claim 7, characterized in that, The scalp care product mentioned is an antioxidant scalp care product.

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