Staphylococcus arlettae strain and application thereof

Abstract

This invention discloses an anti-inflammatory Staphylococcus arlettae strain and its applications. The strain, Staphylococcus arlettae CCSM0305, was isolated from healthy human tissue and deposited at the China Center for Type Culture Collection (CCTCC) on June 1, 2022, with accession number CCTCC No: M 2022778. This strain exhibits good inhibitory activity against hyaluronidase and can reduce the level of IL-6 produced by LPS-stimulated macrophages, showing promising application prospects in the development of anti-inflammatory and soothing microecological skincare products.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to an anti-inflammatory Staphylococcus alright strain and its application. Background Technology

[0002] As the organ with the largest surface area in the human body, the skin can resist exogenous pathogenic microorganisms. Its surface resident and transient microorganisms maintain a balance. Changes in the microbial composition will lead to an imbalance in skin surface homeostasis, resulting in alterations in skin structure and activation of the immune system, causing skin problems. The skin microecology is an ecosystem composed of microorganisms such as bacteria, fungi, and viruses, skin cells and their secretions, and the immune system. Imbalance in the skin microecology weakens or damages the skin's biological barrier, affecting normal physiological functions, even leading to infection and accelerated skin aging. Skin microecological imbalance is present in inflammatory skin problems such as atopic dermatitis, acne, and sensitive skin. Research has shown that adjusting the skin microecology is becoming a new approach to treating various skin problems. The skin microbiota, host skin, and environment constitute the skin micro-ecosystem, interacting and regulating each other to maintain a coordinated, physiological, and dynamic balance.

[0003] Skin microbiota, playing a crucial role in the skin's microecology, are essential for maintaining skin health. Healthy skin harbors a large number of potential beneficial bacteria. Numerous studies have shown that coagulase-negative staphylococci, such as Staphylococcus epidermidis, secrete antimicrobial peptides and break down sebum to produce short-chain fatty acids (SCFAs), nucleosides, and other metabolites. These bacteria possess various physiological functions, including antibacterial, anti-inflammatory, immunomodulatory, skin barrier enhancement, and anticancer effects, making them potential beneficial skin bacteria (Stacy A, Belkaid Y. Microbial guardians of skin health[J]. Science, 2019, 363(6424):227-228.). Given the vital role of skin microbiota in maintaining skin health, skin microbiota and their metabolites may be used in future skin health care. Previous studies have shown that microorganisms isolated from autologous skin and mixed with gels to create novel cosmetics have significantly improved skin hydration (Yuichi Nodakea, Saki Matsumotoa, b. Pilot study on novelskin care method by augmentation with Staphylococcus epidermidis, an autologous skin microbe-A blinded randomized clinical trial[J]. Journal of Dermatological Science 79, 2015, 119-126). The search for beneficial bacteria in the skin and their application in cosmetics holds great promise.

[0004] Currently, there is a lack of screening for skin bacteria and functional strains from healthy individuals in China, and there are no reports on the screening of Staphylococcus aureus. Isolating strains from healthy skin and screening and validating effective strains can lay the foundation for the development of beneficial skin bacteria and microecological skincare products. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention provides a coagulase-negative staphylococcus—Staphylococcus arlettae CCSM0305—and its applications. This strain exhibits good inhibitory activity against hyaluronidase and can reduce the level of IL-6 produced by LPS-stimulated macrophages, showing promising application prospects in the development of anti-inflammatory and soothing microecological skincare products.

[0006] The first objective of this invention is to provide a strain of Staphylococcus arlettae CCSM0305, which was isolated from healthy human skin and deposited on June 1, 2022, at the China Center for Type Culture Collection (depository address: Wuhan University, Wuhan, China), with accession number CCTCC No: M 2022778.

[0007] The colony characteristics of the above strains are as follows: from Figure 1 As can be seen from A, strain CCSM0305 has small, slightly yellowish colonies on TSA solid blood agar plates, forming round raised areas with neat edges. The colony diameter is approximately 1–1.2 mm, the surface is smooth, moist, and opaque, and it can be stretched into a string when picked up, without any hemolytic zone.

[0008] The bacterial cell characteristics of the above strains are as follows: from Figure 1 B shows that it is Gram-positive. The bacteria are spherical, about 0.9-1.0 μm in diameter, arranged in a grape-like pattern, or in a single arrangement. They are non-motile, lack flagella, and do not produce spores.

[0009] The second objective of this invention is to provide a fermentation method for Staphylococcus arlettae CCSM0305, characterized in that activated Staphylococcus arlettae CCSM0335 is inoculated into TSB liquid medium and cultured on a shaker at 30–40°C for 16–20 h, adjusting the OD of the bacterial cells. 600 The seed culture is prepared in the range of 0.9 to 1.1. The seed culture is inoculated into TSB liquid medium at a volume ratio of 1 to 3%, and cultured at 30 to 40°C with shaking for 10 to 15 hours to obtain the fermentation broth of Staphylococcus arlettae CCSM0305. The broth is then centrifuged, and the supernatant obtained is the fermentation supernatant.

[0010] A third objective of this invention is to provide the application of Staphylococcus arlettae CCSM0305 and the above-mentioned fermentation broth supernatant in skin anti-inflammatory applications.

[0011] The fourth objective of this invention is to provide the application of Staphylococcus arlettae CCSM0305 and the above-mentioned fermentation broth supernatant in combating skin damage caused by inflammatory factors and in preparing anti-inflammatory and soothing microecological skin care products.

[0012] The anti-inflammatory effects mentioned above include inhibiting hyaluronidase and reducing LPS-induced macrophage inflammatory factor IL-6.

[0013] Compared with existing technologies, this invention provides a strain of Staphylococcus arlettae CCSM0305 isolated from healthy human subjects. Experiments have shown that the fermentation supernatant of this strain inhibits hyaluronidase by 64%, and significantly reduces LPS-induced macrophage IL-6 expression by 17.9% compared to the positive control LPS. Therefore, this strain has promising applications in combating skin damage caused by inflammatory factors and in the development of anti-inflammatory and soothing microecological skincare products, filling a gap in the screening of Staphylococcus arlettae for anti-inflammatory efficacy. Attached Figure Description

[0014] Figure 1 The colony morphology and microscopic images of Staphylococcus arlettae CCSM0305 are shown in Figure A, which shows the colony morphology, and Figure B shows the microscopic image.

[0015] Figure 2 The growth curve of Staphylococcus arlettae CCSM0305;

[0016] Figure 3 The supernatant of Staphylococcus arlettae CCSM0305 fermentation was used to investigate the cytotoxicity of macrophages RAW264.7.

[0017] Figure 4 The effect of fermentation supernatant of Staphylococcus arlettae CCSM0305 on LPS-induced IL-6 expression in RAW264.7 macrophages. Detailed Implementation

[0018] The present invention is further illustrated below by way of embodiments, but these embodiments are not intended to limit the invention to their scope. Experimental methods not specifically described in the following embodiments are performed according to conventional methods and conditions, or as selected in the product manual. It should be understood that the specific embodiments described herein are for illustrative purposes only and do not constitute a limitation thereof.

[0019] Example 1: Isolation, screening and purification of strain CCSM0305

[0020] (1) Recruiting volunteers for perfect skin

[0021] Applicants must have healthy skin, be without makeup, have smooth skin with small pores, and be free of acne, pustules, inflammation, and peeling. Those with blemishes are also welcome to participate. Applicants must not have used any ointments in the past 3 months. Applicants must be between 18 and 30 years old, and gender is not limited.

[0022] (2) Sample collection

[0023] Volunteers are required to wash their face and perform basic skincare (applying toner, lotion, etc.) the night before sampling, but not to wash their face the next morning. Sampling is generally done at noon or in the afternoon. Select an area of ​​approximately 4cm x 4cm on the skin to be sampled. Moisten a sterile cotton swab made of polymer fiber in a wetting solution (containing 0.9% sodium chloride and 0.1% Tween-20) and rub it back and forth 50 times in the selected area (the swab should be applied with sufficient pressure). Place the sterile cotton swab into the sampling tube using sterile forceps and seal it with sealing film. Place the collected samples in an icebox for refrigeration, bring them back to the laboratory, and store them in a 4°C refrigerator for rapid bacterial isolation.

[0024] (3) Sample pretreatment

[0025] 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.

[0026] (4) Plate screening

[0027] Take 0.5 mL of the above sample solution 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 solid blood agar plates. Spread 2 plates for each dilution and incubate aerobically at 37°C for 24 h.

[0028] Tryptic soybean peptone agar blood medium (TSA solid blood plate): 15 g / L tryptic peptone, 5.0 g / L soybean peptone, 5.0 g / L sodium chloride, 15.0 g / L agar. Autoclave at 121°C for 15 min. When cooled to about 50°C, add 5% sterile defibrinated sheep blood, mix well, and pour into plates.

[0029] (5) Marking, purifying and preserving

[0030] Based on the colony characteristics of coagulase-negative staphylococcal strains on blood agar plates, including differences in colony size, color, moisture, and gloss (no hemolysis zone), single colonies were streaked onto TSA solid blood agar plates and incubated at 37°C for 16 hours. After repeated streaking and purification, the colonies were preserved in storage tubes using a culture medium containing skim milk powder as a cryoprotectant. After freeze-drying, the culture medium was stored at -85°C. The culture medium consisted of 120g skim milk powder and 880mL distilled water, autoclaved at 118°C for 15 minutes.

[0031] After the above screening, strain CCSM0305 was selected, and then the following microbiological identification was performed.

[0032] Example 2: Microbiological identification of strain CCSM0305

[0033] Microbiological identification was performed on strain CCSM0305 from Example 1.

[0034] (1) Colony characteristics:

[0035] Strawberry strain CCSM0305 was streaked onto TSA solid 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 from A, strain CCSM0305 has small, slightly yellowish colonies on TSA solid blood agar plates, forming round raised areas with neat edges. The colony diameter is approximately 1–1.2 mm, the surface is smooth, moist, and opaque, and it can be stretched into a string when picked up, without any hemolytic zone.

[0036] (2) Bacterial cell characteristics:

[0037] A small amount of strain CCSM0305 obtained in step (1) was picked and smeared on a glass slide, Gram stained, and the morphological characteristics of the bacteria were observed under a microscope. The results are shown in […]. Figure 1 As shown in B, from Figure 1 B shows that it is Gram-positive. The bacteria are spherical, about 0.9-1.0 μm in diameter, arranged in a grape-like pattern, or in a single arrangement. They are non-motile, lack flagella, and do not produce spores.

[0038] (3) Characteristics of training:

[0039] The strain CCSM0305 was inoculated into TSB liquid medium and cultured at different temperatures. The results showed that the minimum growth temperature of CCSM0305 was 15℃, the maximum growth temperature was 45℃, and the optimal growth temperature was 30-40℃. When the strain CCSM0305 was inoculated into TSB liquid medium with different pH values ​​and cultured at 37℃, the highest growth pH of the strain CCSM0305 was 9.0, the lowest growth pH was 4.0, and the optimal growth pH was 6.0.

[0040] Tryptic soybean broth (TSB): 17 g / L tryptic peptone, 3.0 g / L soybean papain digest, 5.0 g / L sodium chloride, 2.5 g / L potassium dihydrogen phosphate, 2.5 g / L glucose. Autoclave at 121°C for 15 min.

[0041] (4) Genetic characteristics (16S rDNA sequence analysis):

[0042] CCSM0305 genomic DNA extraction method: Select a purified CCSM0305 single colony and inoculate it into 10 mL of TSB liquid medium. After incubation at 37℃ for 14-16 h, centrifuge the bacterial solution (8000 r / min, 15 min) and collect the bacterial cells.

[0043]

[0044] The sample was sent to GenBank for BLAST analysis. Comparative analysis showed that strain CCSM0305 and Staphylococcus arlettae belong to the same species. Therefore, strain CCSM0305 was identified as Staphylococcus arlettae.

[0045] Based on the above-mentioned microbiological characteristics such as colony and cell morphology, culture, physiological and biochemical identification, as well as its genetic characteristics and 16S rDNA, strain CCSM0305 was identified as Staphylococcus arlettae. This strain was deposited at the China Center for Type Culture Collection (CCTCC) on June 1, 2022, with accession number CCTCC No: M 2022778.

[0046] Example 3: Plotting the growth curve of Staphylococcus arlettae CCSM0305

[0047] Activated Staphylococcus arlettae CCSM0305 was inoculated into TSB liquid medium at a 2% (v / v) inoculum and cultured at 37°C with shaking for 24 h. The OD value of the culture medium was measured at 600 nm every 2 h. 600 The growth curve of Staphylococcus arlettae CCSM0305 in TSB liquid medium was obtained by plotting values ​​against time. The results are as follows: Figure 2 As shown, from Figure 2 As can be seen, Staphylococcus arlettae CCSM0305 grows rapidly in TSB liquid medium, entering the logarithmic phase in about 2 hours and the stationary phase in about 12 hours.

[0048] Example 4: Determination of hyaluronidase inhibition rate in fermentation supernatant of Staphylococcus aureus CCSM0305

[0049] (1) Preparation of fermentation supernatant of Staphylococcus arlettae CCSM0305

[0050] Staphylococcus arlettae CCSM0305 strain was streaked onto TSA solid blood agar plates and incubated at 37°C for 16–20 h, activating the bacteria twice. The activated Staphylococcus arlettae CCSM0335 was then inoculated into TSB liquid medium and cultured at 37°C with shaking for 16–20 h, adjusting the bacterial OD value. 600 The seed culture was prepared in the range of 0.9–1.1. The seed culture was inoculated into 250 mL Erlenmeyer flasks of TSB liquid medium at a volume ratio of 2%, and cultured at 37 °C with shaking at 160 rpm for 12 h to obtain the fermentation broth of *Staphylococcus arlettae* CCSM0305. The fermentation broth was then centrifuged at 10000 rpm for 15 min, and the resulting supernatant was used as the fermentation supernatant.

[0051] Tryptic soybean broth (TSB): 17 g / L tryptic peptone, 3.0 g / L soybean papain digest, 5.0 g / L sodium chloride, 2.5 g / L potassium dihydrogen phosphate, 2.5 g / L glucose. Autoclave at 121°C for 15 min.

[0052] (2) The hyaluronidase inhibition rate in the metabolites of Staphylococcus arlettae CCSM0305 was determined by the hyaluronidase inhibition method. The steps are as follows:

[0053] ① Prepare a 12.5 mM calcium chloride solution, a 4000 U / mL hyaluronidase solution, and a 2 mg / mL sodium hyaluronate solution using 0.1 M acetic acid buffer solution;

[0054] ② Mix 1 mL of acetylacetone and 10 mL of 0.5 M sodium carbonate solution thoroughly and prepare fresh before use;

[0055] ③ Dissolve 1.5g of dimethylaminobenzaldehyde in 43.75mL of glacial acetic acid, mix well, and then add 6.25mL of 10M hydrochloric acid. Prepare fresh before use.

[0056] (3) Take the fermentation supernatant from step (1) as a sample and add the reagents from step (2) according to Table 1 to carry out the reaction.

[0057] Table 1: Experimental Design

[0058]

[0059]

[0060] The hyaluronidase inhibition rate of the fermentation supernatant of Staphylococcus arlettae CCSM0305 was calculated to be 64±14.65% according to the above formula.

[0061] Example 5: Test on the effect of Staphylococcus arlettae CCSM0305 fermentation supernatant on LPS-stimulated macrophage IL-6 production (1) Preparation of Staphylococcus arlettae CCSM0305 fermentation supernatant

[0062] Prepare the fermentation supernatant according to step (1) of Example 4, and then concentrate it to 1 / 4 of the original volume, which is the concentrated fermentation supernatant of Staphylococcus alliet.

[0063] (2) Cell viability test

[0064] RAW264.7 macrophages were cultured normally in high-glucose DMEM medium containing 10% fetal bovine serum under 5% CO2 and 37°C conditions. When the cells reached 80-90% confluence, they were digested with trypsin and cultured at 8 × 10⁻⁶ cells / day. 5 Cells were seeded at a density of cells / mL in 96-well plates. After 24 h of cell adhesion, 0.1%, 0.5%, 1%, 5%, and 10% CCSM0305 fermentation supernatant concentrate were added and cultured for 24 h, respectively. A control group was set up, and each experimental group had 3 parallel wells. After treatment, CCK-8 reagent was added and incubated for 1 h according to the manufacturer's instructions. The absorbance was measured at 450 nm, and the relative cell viability was calculated.

[0065] Relative cell viability % = (OD value of sample group / OD value of control group) × 100%

[0066] (3) IL-6 assay in LPS-induced RAW264.7 macrophages

[0067] RAW264.7 macrophages were cultured normally in high-glucose DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. When the cells reached 80-90% confluence, they were digested with trypsin and cultured at 8 × 10⁻⁶ cells / day. 5 Cells were seeded at a density of [number] cells / mL in 96-well plates. After 24 h of cell adhesion, concentrated LPS, LPS, and 5% CCSM0305 fermentation supernatant were added to the reaction wells. Dexamethasone (DEX) was used as a positive control, and a blank control group was set up. Each group had 3 parallel wells. After 24 h of culture, the supernatant was collected, and IL-6 inflammatory factor was detected using an ELISA kit.

[0068] from Figure 3It can be seen that, compared with the blank control group (NT), the addition of CCSM0305 fermentation supernatant concentrate at a concentration below 5% did not show cytotoxicity and significantly promoted the growth of RAW264.7 macrophages. Therefore, 5% CCSM0305 fermentation supernatant concentrate was selected for inflammatory factor experiments. The experimental results showed that ( Figure 4 Normal cells (NT) produce a small amount of the cytokine IL-6, while LPS treatment causes cells to secrete a large amount of the inflammatory cytokine IL-6. Adding concentrated CCSM0305 fermentation supernatant significantly reduced the amount of the inflammatory cytokine IL-6, with a reduction rate of 17.9%. Therefore, the CCSM0305 fermentation supernatant of *Staphylococcus albus* can significantly reduce the expression of the inflammatory cytokine IL-6 induced by LPS, demonstrating a good anti-inflammatory effect.

Claims

1. A strain of Staphylococcus alright ( Staphylococcus arlettae CCSM0305, its accession number is CCTCC No: M 2022778.

2. The Staphylococcus albus according to claim 1 ( Staphylococcus arlettae The fermentation method of CCSM0305 is characterized by, Fermentation was carried out using TSB liquid medium, and the fermentation broth was centrifuged to obtain the fermentation supernatant.

3. The Staphylococcus albus as described in claim 2 ( Staphylococcus arlettae The fermentation method of CCSM0305 is characterized by, The activated Staphylococcus aureus ( Staphylococcus arlettae CCSM0335 was inoculated into TSB liquid medium and cultured in a shaker at 30-40℃ for 16-20 h. The OD600 of the bacterial cells was adjusted to the range of 0.9-1.1 to serve as the seed culture. Inoculate the seed culture into TSB liquid medium at a volume ratio of 1-3%, and culture at 30-40℃ with shaking for 10-15 h to obtain Staphylococcus alright. Staphylococcus arlettae The fermentation broth of CCSM0305 was centrifuged to obtain the fermentation supernatant.

4. The fermentation supernatant prepared by the fermentation method of claim 2 or 3.

5. The Staphylococcus albus according to claim 1 ( Staphylococcus arlettae The application of the fermentation supernatant as described in CCSM0305 or claim 4 in the preparation of anti-inflammatory and soothing microecological preparations.

6. A microecological skincare product comprising the *Staphylococcus albus* as described in claim 1 (…). Staphylococcus arlettae CCSM0305 or the fermentation supernatant as described in claim 4.

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