Staphylococcus epidermidis and application thereof

Abstract

The invention relates to application of staphylococcus epidermidis XBYM001, XBYM002 and XBYM003 as well as fermentation liquor and an extract of the staphylococcus epidermidis XBYM001, XBYM002 and XBYM003 in preparation of a preparation for preventing or repairing skin aging. Wherein the staphylococcus epidermidis is preserved in the China Center for Type Culture Collection in Wuhan City, Hubei Province on March 27, 2024, the preservation number is XBYM001: CCTCC NO.M 2024572, the preservation number is CCTCC NO.M 2024572, and the preservation number is CCTCC NO.M 2024572. XBYM002: CCTCC (China Center For Type Culture Collection) NO.M 2024573; and XBYM003: CCTCC (China Center For Type Culture Collection) NO. M 2024574. By screening coagulase-negative staphylococcus epidermidis with good UVA / UVB radiation resistance and an anti-oxidation function, a potential microecological preparation skin care product capable of relieving cell oxidative damage caused by optical radiation so as to inhibit skin aging, stabilize skin barrier and maintain skin microecological balance is obtained.

Description

Technical Field

[0001] The invention relates to the field of bioengineering, in particular to Staphylococcus epidermidis and uses thereof. Background Art

[0002] The skin is the largest organ in the human body. It is also the first barrier between the human body and the external environment, playing a role in protection, temperature regulation, sensation, secretion, excretion and immunity.

[0003] Skin aging is highly correlated with ultraviolet radiation. Skin aging caused by ultraviolet radiation is also called photoaging. More than 80% of facial aging is caused by photoaging. On October 27, 2017, the World Health Organization's International Agency for Research on Cancer published a preliminary list of carcinogens for reference. Ultraviolet radiation (wavelength 100-400nm, including UVA, UVB and UVC) is on the list of Class I carcinogens. The culprit of photoaging is UVA and UVB in ultraviolet rays.

[0004] Different from the characteristics of skin aging caused by genetics, the activity of keratinocytes in the epidermis of photoaging skin decreases, the renewal rate slows down, and the epidermal barrier function weakens, leading to dry and peeling skin; the number of fibroblasts in the dermis decreases, the synthesis of collagen and elastin slows down, and the decomposition accelerates. In particular, UVA and UVB in sunlight can damage skin cells in many ways, such as directly damaging DNA, generating reactive oxygen species, inducing increased levels of MMPs, degrading extracellular matrix, and inducing inflammation, leading to a decline in skin function, rough and thickened skin, loose wrinkles, localized excessive pigmentation or capillary dilation, and even various benign or malignant tumors.

[0005] More and more studies have found that skin microecology has an important impact on skin health. During the aging process of the body, the skin microecology also undergoes dynamic changes.

[0006] The skin is inhabited by bacteria, archaea, fungi and viruses, and is a complex and dynamic ecosystem. These microorganisms are collectively referred to as the skin microbiome. The skin microecology plays a vital role in defending against invading pathogens, adjusting the human immune system and decomposing metabolites. Among them, Staphylococcus accounts for 16.8% of the total bacterial abundance on the skin, and Staphylococcus epidermidis accounts for 65%-90% of the staphylococci on the body surface, which is one of the main members of the microorganisms on the surface of normal human skin. Staphylococcus epidermidis is widely present in the stratum corneum of the skin and can be seen in the epidermal basement membrane in dry, moist and sebaceous gland areas. It belongs to the genus Staphylococcus of the Micrococaceae family of the Firmicutes phylum and is a relatively hydrophobic Gram-positive facultative anaerobic bacterium. The vast majority of S. epidermidis that do not produce toxic substances such as plasma coagulase and α-hemolysin are not pathogenic.

[0007] Keratinocytes in the human epidermis are the main cell population involved in resisting microbial pathogens. Keratinocytes actively participate in the innate immune response by producing cytokines, chemokines and antimicrobial peptides (AMPs). S. epidermidis is tightly bound to keratinocytes and can communicate with them through a variety of secretions. It is considered a commensal bacterium of the skin.

[0008] Studies have shown that S.epidermidis activates the unique innate immune signaling pathway in human keratinocytes through AMPs, and its cell wall component lipoteichoic acid helps the development and activation of the adaptive immune system, thereby regulating the skin immune process. By secreting sphingomyelinase, S.epidermidis can replenish ceramides on the skin, strengthen the tight junctions between keratinocytes, and thus maintain the homeostasis of the skin barrier.

[0009] Under normal physiological conditions, the symbiotic S. epidermidis inhibits the formation of Staphylococcus aureus biofilm by secreting the serine protease glutamine endopeptidase, and improves skin moisture by stimulating keratinocytes to produce antimicrobial peptides, maintaining a low pH value, and producing glycerol and organic acids through metabolism, thereby affecting the colonization of Staphylococcus aureus, controlling the overgrowth of pathogenic microorganisms, accelerating wound healing, and maintaining skin barrier function.

[0010] The imbalance of S.epidermidis abundance was found in a variety of skin diseases such as seborrheic dermatitis, acne and atopic dermatitis, which further indicates that S.epidermidis plays an important role in the skin as a skin commensal bacteria. In addition, a large number of studies have shown that coagulase-negative S.epidermidis can be used as a beneficial bacterium to exhibit good anti-inflammatory, antioxidant, immune regulation and other physiological functions.

[0011] The reduction of commensal S. epidermidis may reduce their benefits to the skin and contribute to skin aging. As an important player in the skin microbiome, maintaining the relative abundance and diversity of S. epidermidis may be a key factor in skin health. Summary of the invention

[0012] In response to the technical problems existing in the prior art, the present invention proposes the use of Staphylococcus epidermidis XBYM001, XBYM002, XBYM003 and their fermentation broth and extracts in the preparation of preparations for preventing or repairing skin aging; wherein, the Staphylococcus epidermidis was deposited in the China Type Culture Collection of Wuhan University, Wuhan City, Hubei Province on March 27, 2024, abbreviated as CCTCC, and the preservation numbers are: XBYM001: CCTCC NO.M2024572; XBYM002: CCTCC NO.M 2024573; and XBYM003: CCTCC NO.M 2024574.

[0013] In the application as described above, the skin aging is skin aging caused by radiation.

[0014] In the application as described above, the skin aging is photoaging of the skin caused by radiation.

[0015] A preparation for preventing or repairing skin aging, comprising Staphylococcus epidermidis, one or more of its fermentation broth and extracts thereof; wherein the Staphylococcus epidermidis is selected from one or more of XBYM001, XBYM002, and XBYM003, which were deposited in the China Center for Type Culture Collection in Wuhan, Hubei Province on March 27, 2024, abbreviated as CCTCC, and the deposit numbers are: XBYM001: CCTCC NO.M 2024572; XBYM002: CCTCC NO.M2024573; and XBYM003: CCTCC NO.M 2024574.

[0016] The preparation as described above is in the form of granules, powder, serum, suspension, viscous liquid or cream.

[0017] The preparation as described above, which is an external preparation for skin.

[0018] A method for preparing any of the above preparations, comprising: preparing Staphylococcus epidermidis fermentation broth, comprising: culturing Staphylococcus epidermidis in a liquid culture medium at 25-42 degrees Celsius for 18-24 hours; preferably the culture temperature is 37 degrees Celsius; centrifuging and collecting the supernatant, which is the fermentation broth.

[0019] The method as described above, wherein the concentration of Staphylococcus epidermidis cells is (0.3-8)×10 9 CFU / mL; preferably, the concentration of Staphylococcus epidermidis is about 1×10 9 CFU / mL.

[0020] A method for preparing any of the above preparations, comprising: preparing a Staphylococcus epidermidis extract, comprising: culturing Staphylococcus epidermidis in a liquid culture medium at 25-42 degrees Celsius for 18-24 hours; preferably the culturing temperature is 37 degrees Celsius; centrifuging and collecting the cells; and disrupting the cells to obtain a Staphylococcus epidermidis extract.

[0021] The bacterial cells were disrupted by ultrasonication as described above.

[0022] A skin care product comprising any one of the above preparations for preventing or repairing skin aging.

[0023] The present application obtains a potential microecological skin care product that can alleviate cell oxidative damage caused by light radiation, thereby inhibiting skin aging, stabilizing the skin barrier, and maintaining the balance of skin microecology by screening coagulase-negative Staphylococcus epidermidis with good resistance to UVA / UVB radiation and antioxidant function. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The preferred embodiments of the present invention will be further described in detail below with reference to the accompanying drawings, wherein:

[0025] Figure 1 This is a photo of a bacterial hemolysis experiment according to an embodiment of the present invention;

[0026] Figure 2 is a photograph of Gram staining results of bacteria after screening according to one embodiment of the present invention;

[0027] Figure 3 This is a PCR electrophoresis diagram of 16SrDNA detection of three strains after screening according to one embodiment of the present invention;

[0028] Figure 4A This is an analysis of sequencing results of 16SrDNA detection of XBYM001 strain according to an embodiment of the present invention;

[0029] Figure 4B This is an analysis of sequencing results of 16SrDNA detection of XBYM002 strain according to an embodiment of the present invention;

[0030] Figure 4C This is an analysis of the sequencing results of 16SrDNA detection of the XBYM003 strain according to an embodiment of the present invention;

[0031] Figure 5 is a bar graph of the total antioxidant capacity of three strains in different forms according to one embodiment of the present invention;

[0032] Figure 6 is a bar graph of the DPPH free radical scavenging ability of three strains in different forms according to one embodiment of the present invention;

[0033] Figure 7 is a bar graph of the hydroxyl radical scavenging abilities of three strains in different forms according to one embodiment of the present invention;

[0034] Figure 8 is a bar graph of catalase activity of three strains in different forms according to one embodiment of the present invention; and

[0035] Fig. 9 1 is a bar graph showing superoxide dismutase activities of three strains in different forms according to an embodiment of the present invention. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] In the following detailed description, reference may be made to the various specification drawings that are part of the present application and are used to illustrate specific embodiments of the present application. In the accompanying drawings, similar reference numerals describe substantially similar components in different figures. The various specific embodiments of the present application are described below in sufficient detail so that a person of ordinary skill in the art with relevant knowledge and skills in the art can implement the technical solutions of the present application. It should be understood that other embodiments may also be used or the embodiments of the present application may be changed.

[0038] The concept of microecological skin care is increasingly attracting the attention of many cosmetics companies. Therefore, some companies have begun to make bacteria and their lysates and fermentations into skin care products, and promise to "rebalance" the bacterial microecology of human skin in life, making the skin healthier and better able to resist damage to the skin barrier caused by environmental pollution, light, autoimmune disorders and other factors.

[0039] Therefore, based on the composition of the skin microbiome, this patent aims to develop a microecological skin care product that can effectively alleviate cell oxidative damage caused by light radiation, thereby inhibiting skin aging, stabilizing the skin barrier, and maintaining the balance of the skin's microecology by screening coagulase-negative Staphylococcus epidermidis with good resistance to UVA / UVB radiation and antioxidant function.

[0040] In this application, "skin aging" refers to the decline of skin tissue and the decline of the physiological function of the skin. The thickness of the skin changes significantly with age. As aging cells attach to the stratum corneum of the epidermis, the skin surface becomes hard and loses its luster. Subcutaneous fat decreases, and the elasticity and tension of the skin gradually lose due to changes in elastic fibers and collagen fibers, further leading to sagging skin and wrinkles. The functions of sebaceous glands and sweat glands decline, and the excretion of sweat and sebum decreases. The skin gradually loses its former luster and becomes dry. The blood circulation function is reduced to supplement the necessary nutrients for the skin. Among them, skin aging is highly correlated with ultraviolet radiation. Skin aging caused by ultraviolet radiation is also called photoaging. More than 80% of facial aging is caused by photoaging.

[0041] In this application, "fermentation liquid" refers to the liquid obtained after culturing bacteria in a liquid medium for a certain period of time. During the culturing process, the secretions of the bacteria are incorporated into the liquid medium to form a fermentation liquid.

[0042] In the present application, "cell-free extract" refers to an extract of bacterial cells. In some embodiments, the cell-free extract is obtained by disrupting the bacterial cells.

[0043] Example 1 Isolation of skin microorganisms

[0044] In the present application, the anti-UVA / UVB strains are isolated from the healthy face of a human body. Specifically, the method for isolating the strains includes skin swab sample collection, skin flora culture and isolation, and anti-UVA / UVB strain screening, as follows:

[0045] 1. Skin swab sample collection

[0046] Sample collection: Exclude subjects who have used antibiotics or steroids on their face in the previous seven days. In order to ensure sample concentration and prevent contamination, antibacterial toiletries and skin care products should not be used the night before sampling and on the day of sampling, and face washing is not allowed on the day of sampling. The sampler wears sterile gloves, presses a sterile cotton swab soaked in saline or sterile water on the sampling surface, applies steady pressure, and wipes the sampling surface steadily and slowly (sampling area 4cm×4cm, depending on actual conditions), and repeatedly scrapes three different parts of the subject (at least 15 times): the middle of the forehead, 1cm near the nose of the left cheek, and 1cm near the nose of the right cheek. When scraping, pay attention to slightly rotate the cotton swab to make full contact. Immediately after taking the sample, put the cotton swab back into the corresponding test tube and seal it.

[0047] 2. Culture and Isolation of Skin Flora

[0048] Immediately after the sample is collected, place the swab in a sampling tube for storage, suspend the skin sample in physiological saline or sterile water and stir it thoroughly, then discard the swab. Cover tightly, shake the sampling tube quickly for at least 30 seconds, perform appropriate gradient dilutions, spread on solid culture medium, and culture at 37°C for 24-48 hours. Pick a single colony on the plate and repeatedly streak it for isolation, purification and culture. Observe the colony morphology by Gram staining to select Gram-positive strains. Inoculate the selected strain into a liquid culture medium and culture it at 37°C for 24 hours. Activate and subculture three times to ensure that the strain is vigorous and obtain the initial screening strain.

[0049] 3. Screening of UVA / UVB-resistant strains

[0050] Prepare and activate the strains screened initially, dilute some of them appropriately, spread them on solid culture medium, and directly place them in a biochemical incubator at 37°C for 24-48h. After irradiating some of them under UVA / UVB, dilute them appropriately and spread them on solid culture medium, and place them in a biochemical incubator for 24-48h at 37°C. Observe the growth of each strain with or without UVA / UVB irradiation, and select strains with good growth as dominant strains to continue the experiment. Increase the irradiation time in a gradient manner, determine the tolerance of each dominant strain to the UVA / UVB radiation intensity, and select strains with better radiation resistance for biochemical identification and subsequent experiments.

[0051] In the present application, the liquid culture medium components involved include (g / L): peptone 10.0g, beef powder 8.0g, yeast powder 4.0g, glucose 20.0g, dipotassium hydrogen phosphate 2.0g, diammonium hydrogen citrate 2.0g, sodium acetate 5.0g, magnesium sulfate 0.2g, manganese sulfate 0.04g, Tween 80 1.0g; the solid culture medium components involved include: corresponding liquid culture medium + 2% agar. The above components are all purchased and are not used as a limitation of the present application.

[0052] UVA / UVB radiation dose: Select the radiation dose UVA as 10J / cm 2 and UVB is 40mJ / cm 2 , the radiation distance is 15cm. Use an ultraviolet radiation meter to measure the radiation intensity of UVA / UVB on the table inside the ultraviolet light box, according to the formula:

[0053] UVA / UVB radiation intensity = UVA / UVB radiation dose ÷ radiation time (seconds)

[0054] Calculate the corresponding radiation time of UVA and UVB respectively.

[0055] The results are shown in Table 1:

[0056] Table 1

[0057]

[0058] After multiple screenings, 9 strains were initially obtained for testing. The irradiation time was gradually increased to 45 min, and the irradiation dose reached 10 J / cm 2 At this time, the number of colonies of 5 strains can still reach 10 under this radiation intensity. 6 The above are strains 3-5 and 7-8, respectively, so these 5 test strains were selected for further physiological and biochemical identification.

[0059] Example 2 Physiological and biochemical identification of dominant strains

[0060] 1. Catalase test (catalase test)

[0061] The strains with better radiation resistance were screened and purified on the plate. Single colonies were picked and placed on a clean glass slide. 1-2 drops of 3% hydrogen peroxide solution were added to observe whether bubbles were generated. If bubbles were generated, it was catalase positive, and if there were no bubbles, it was negative. Among Gram-positive cocci, Staphylococci all produce catalase.

[0062] Result analysis: Single colonies of each strain were picked and added into 3% hydrogen peroxide solution. All five strains produced a large amount of bubbles. The test results are shown in Table 2, indicating that all five strains were catalase positive.

[0063] 2. Coagulase test

[0064] Coagulase-negative Staphylococci are normal bacteria that exist on the skin, oral cavity and intestines of healthy people and have always been considered to have no pathogenic effect. Therefore, coagulase is an important indicator for identifying whether Staphylococci are pathogenic. The selected dominant strains are inoculated into liquid culture medium at a 3% inoculation rate, cultured at 37°C for 24 hours, activated and subcultured 3 times to ensure the vitality of the bacteria. Add 0.5ml of sterile saline to each vial of freeze-dried plasma and shake it slightly until it is completely dissolved. Add 0.3ml of fresh culture of the dominant strain respectively, mix thoroughly, and culture in a 37°C incubator. Observe every half an hour for 6 hours. If coagulation (i.e., clots appear when tilted or inverted) or the coagulation volume is greater than half of the original volume, it is judged as a positive result, otherwise it is negative. Coagulase-negative strains are selected for subsequent identification experiments.

[0065] The results of the coagulase test are shown in Table 2. Result analysis: During the 6-hour experiment, the strains 5 and 8 were observed every half an hour. A small amount of flocculent precipitation appeared in the process, but no large-scale coagulation occurred; strains 3, 4 and 9 did not coagulate, so it was determined that the 5 tested strains were coagulase negative.

[0066] 3. Hemolysis Experiment

[0067] Staphylococcus epidermidis generally does not produce exotoxins (hemolytic toxins), so its invasiveness is weak and it is a non-pathogenic staphylococcus. However, it can also cause disease under certain conditions, causing suppurative infection and even sepsis. The dominant strains screened were streaked and inoculated on blood agar plates and cultured at 37°C for 24-48 hours. Take out and observe whether hemolytic rings are produced around each colony on the blood plate. The appearance of a 1-2mm grass-green ring around the colony indicates that the bacteria produce α-hemolysin; if the bacteria form a wide 2-4mm transparent hemolytic ring around the colony on the blood plate, the bacteria produce β-hemolysin; if many small gray-white colonies appear around the colony, the bacteria produce γ-hemolysin. According to the results, bacteria that do not produce hemolysin are selected as the final screened radiation-resistant dominant strains.

[0068] Result analysis:

[0069] Figure 1 The following are photos of hemolysis plates of the five strains. The hemolysis results are shown in Table 2. The culture results showed that the colonies of strains 3 and 5 grown on the blood plate had transparent hemolysis rings, indicating that both produced β-hemolysin and did not meet the screening conditions. There was no hemolysis ring around the colonies of strains 2, 8, and 9, which met the screening conditions and could be used as dominant strains for subsequent experiments.

[0070] Table 2

[0071]

[0072] Example 3 Colony morphology and identification of dominant strains

[0073] 1. Colony morphology of dominant strains

[0074] The dominant strains 4, 8, and 9 screened were inoculated on the plate and cultured at 37°C for 24-48 hours to observe the morphology of the single colonies of each strain. A small amount of single colonies were selected and smeared on a glass slide for Gram staining, and the morphological characteristics of each bacterial cell were observed under a microscope.

[0075] Result analysis: Figure 2 It can be seen that the three dominant strains are all Gram-positive under the microscope, and their bacterial morphology is spherical and arranged in a grape shape. On ordinary agar plates, they form raised, smooth, opaque, 1-2 mm diameter round colonies, which are white or lemon-colored. They were initially identified as Staphylococcus epidermidis, and further identified by 16SrDNA.

[0076] 2.16S rDNA identification and preservation

[0077] The bacterial 16SrDNA sequence sequencing method is used to identify the species of bacteria, including the steps of bacterial genomic DNA extraction, 16SrDNA specific primer PCR amplification, amplification product purification, DNA sequencing, sequence comparison, etc.

[0078] Result analysis:

[0079] Figure 3 This is a PCR electrophoresis diagram of 16SrDNA detection of three strains after screening according to one embodiment of the present invention; Figure 4A-4C This is an analysis of sequencing results of 16SrDNA detection of XBYM (001-003) strain according to an embodiment of the present invention.

[0080] according to Figure 3 and Figure 4A-4C The results showed that the three dominant strains screened from the skin were all identified as Staphylococcus, presumed to be Staphylococcus epidermidis, and were named respectively: strain 4 (Staphylococcus epidermidis XBYM001), strain 8 (Staphylococcus epidermidis XBYM002), and strain 9 (Staphylococcus epidermidis XBYM003).

[0081] The applicant deposited the above three strains in the China Center for Type Culture Collection (CCTCC) on March 27, 2024, and the deposit numbers are:

[0082] Staphylococcus epidermidis XBYM001: CCTCC NO.M 2024572;

[0083] Staphylococcus epidermidis XBYM002: CCTCC NO.M 2024573; and

[0084] Staphylococcus epidermidis XBYM003: CCTCC NO.M 2024574.

[0085] Example 4 Evaluation of the antioxidant capacity of three strains of Staphylococcus epidermidis

[0086] 1. Preparation of Staphylococcus epidermidis, its fermentation broth and its extract

[0087] Staphylococcus epidermidis was activated and cultured in liquid culture medium for 3 generations to keep the bacterial concentration at 1×10 9 CFU / mL) to ensure the activity, thereby obtaining the culture fermentation liquid. The activated bacterial culture liquid was centrifuged at 4000g, 4°C for 10 minutes, and the fermentation supernatant was collected, which was the Staphylococcus epidermidis fermentation liquid.

[0088] The bacterial cell pellet obtained above was resuspended in PBS, washed, and centrifuged at 4000 g, 4° C. for 10 min to remove the remaining culture medium, and the process was repeated three times to obtain a bacterial cell suspension.

[0089] A portion of the bacterial cell suspension was preserved, and the other portion was ultrasonically disrupted for 30 min at a power of 300 W, working for 5 s / interval of 5 s to obtain a cell-free extract.

[0090] The fermentation supernatant, bacterial cells, and cell-free extracts were prepared and used for subsequent experiments.

[0091] 2. Total Antioxidant Capacity (T-AOC)

[0092] This example uses the total antioxidant capacity (T-AOC) kit purchased from Nanjing Jiancheng Bioengineering Institute for determination. The determination principle is: ABTS is oxidized to green ABTS under the action of appropriate oxidants. ·+ , in the presence of antioxidants, ABTS ·+ The production of ABTS is inhibited and ABTS is measured at 405nm or 734nm ·+The total antioxidant capacity of the sample can be measured and calculated by the absorbance. Trolox is an analog of VE, with an antioxidant capacity similar to VE, and is used as a reference for the total antioxidant capacity of other antioxidants. For example, the total antioxidant capacity of Trolox is 1. Under the same concentration, the antioxidant capacity of other substances is expressed as a multiple of their antioxidant capacity compared to Trolox.

[0093]

[0094] Since the kit used is commercially available and the kit is not used to limit the scope of protection of the present application, the method for using the kit will not be described in detail here.

[0095] Figure 5 A bar graph showing the total antioxidant capacity of three strains in different forms according to an embodiment of the present invention is shown. Figure 5 As shown, the fermentation broths of the three strains XBYM (001-003) all had strong total antioxidant capacity, and the antioxidant capacity of the cell-free extracts was slightly weaker than that of the fermentation broth.

[0096] 3. Ability to scavenge DPPH free radicals

[0097] This example was measured according to the instructions of the DPPH free radical scavenging kit purchased from Nanjing Jiancheng Bioengineering Institute. DPPH (1,1-Diphenyl-2-picrylhydrazyl radical) is 1,1-diphenyl-2-picrylhydrazyl radical. It is widely used to quantitatively measure the antioxidant capacity of biological samples and foods.

[0098] This method is based on the fact that the DPPH free radical has a single electron, has a strong absorption at 517nm, and its alcohol solution is purple. When a free radical scavenger is present, its absorption gradually disappears due to pairing with its single electron. The lighter the color, the lower the A value, and the DPPH scavenging ability in the sample is quantitatively analyzed.

[0099] Since the kit used is commercially available and the kit is not used to limit the scope of protection of the present application, the method for using the kit will not be described in detail here.

[0100] Figure 6 3 strains of different forms of scavenging DPPH free radicals according to an embodiment of the present invention. Figure 7As shown in the figure, the fermentation broth, intact cells and cell-free extracts of XBYM (001-003) all have strong DPPH free radical scavenging ability. Among them, the scavenging ability of XBYM001 and XBYM003 fermentation broth is stronger, and XBYM001 can even exceed 21μg soluble vitamin E (Ttolox) / mL. The cell-free extract of XBYM002 has a higher DPPH free radical scavenging ability.

[0101] 4. Ability to remove hydroxyl radicals (OH.-)

[0102] This example is determined using a hydroxyl radical kit purchased from Nanjing Jiancheng Bioengineering Institute. The determination principle is: Fenton reaction is the most common chemical reaction to produce hydroxyl radicals, the amount of H2O2 is proportional to the amount of OH· produced by the Fenton reaction, and after an electron acceptor is given, a red substance is formed by color development with a Griess reagent, and its color is proportional to the amount of OH·.

[0103] Since the kit used is commercially available and the kit is not used to limit the scope of protection of the present application, the method for using the kit will not be described in detail here.

[0104] Figure 7 The hydroxyl radical scavenging ability bar graph of three strains in different forms according to one embodiment of the present invention is shown. Figure 7 As shown, XBYM (001-003) has a strong ability to inhibit hydroxyl free radicals, especially the fermentation broth of the three strains, the inhibition capacity can exceed 22U / mL.

[0105] 5. Catalase (CAT) activity

[0106] In this example, the CAT activity was determined by the ammonium molybdate method using a hydroxyl radical kit purchased from Nanjing Jiancheng Bioengineering Institute. The assay principle of the kit is that the reaction of catalase decomposing H2O2 can be quickly stopped by adding ammonium molybdate, and the remaining H2O2 reacts with ammonium molybdate to produce a light yellow complex, and the change in the complex is measured at 405nm to calculate the activity of CAT.

[0107] Among them, the CAT activity determination method of Staphylococcus epidermidis fermentation broth and cell-free extract can refer to the detection method of serum; the CAT activity determination method of bacterial cell suspension can refer to the detection method of tissue sample. The kit used is commercially available, and the kit is not used to limit the protection scope of this application, so the use of the kit is not repeated here.

[0108] Figure 8 The catalase activity bar graph of three strains in different forms according to one embodiment of the present invention is shown. Figure 8 As shown, the fermentation broth, intact cells and cell-free extracts of XBYM (001-003) all have strong catalase activity. Furthermore, the catalase activity of the intact cells of the strain is close to 10 U / mL.

[0109] 6. Superoxide dismutase (SOD) activity assay

[0110] In this example, the SOD activity was determined using a hydroxyl radical test kit purchased from Nanjing Jiancheng Bioengineering Institute. In this reaction system, the amount of enzyme corresponding to a 50% SOD inhibition rate is one SOD activity unit (U).

[0111] The measurement methods include:

[0112] 1. Obtain the sample to be tested. The sample to be tested is directly obtained fermentation broth, cell-free extract or collected bacterial cells.

[0113] 2. When the sample to be tested is bacterial cells, the bacterial cells are broken. The present application does not limit the breaking method, and methods such as grinding, ultrasonic breaking, repeated freezing and thawing, and chemical lysis can be used.

[0114] 3. Protein determination, the operation includes:

[0115]

[0116]

[0117] Among them, the enzyme diluent, enzyme working solution and substrate application solution are all prepared according to the reagents provided in the kit and the instructions. The SOD activity of the sample is calculated according to the following formula:

[0118] SOD 抑制率 =[(A 对照 -A 对照空白 )-(A 测定 -A 测定空白 )] / (A 对照 -A 对照空白 )X100%

[0119] SOD 活力 (U / mgprot)=SOD 抑制率 / (reaction system / dilution factor)(0.24ml / 0.02ml) / protein concentration of the sample to be tested (mgprot / ml)

[0120] Where A represents the reading of the ELISA reader.

[0121] After the determination, the SOD standard curve is obtained according to the calculation results. Fig. 9As shown, when the sample was fermentation broth, the SOD activity was the highest, and all three strains could reach above 20U / mL; while the SOD activity of the cell-free extract was almost zero.

[0122] The above embodiments are only used to illustrate the present invention, but not to limit the present invention. Ordinary technicians in the relevant technical field can make various changes and modifications without departing from the scope of the present invention. Therefore, all equivalent technical solutions should also fall within the scope of the present invention.

Claims

1. Use of Staphylococcus epidermidis XBYM001, XBYM002, XBYM003 and their fermentation broth and extracts in the preparation of preparations for preventing or repairing skin aging; wherein: The Staphylococcus epidermidis was deposited in the China Center for Type Culture Collection (CCTCC) in Wuhan, Hubei Province on March 27, 2024, with the deposit numbers: XBYM001: CCTCC NO.M 2024572; XBYM002: CCTCC NO.M 2024573; as well as XBYM003: CCTCC NO.M 2024574.

2. The use according to claim 1, wherein the skin aging is skin aging caused by radiation.

3. The use according to claim 2, wherein the skin aging is photoaging of the skin caused by radiation.

4. A preparation for preventing or repairing skin aging, comprising one or more of Staphylococcus epidermidis, its fermentation liquid and its extract; wherein: The Staphylococcus epidermidis is selected from one or more of XBYM001, XBYM002, and XBYM003, which were deposited in the China Center for Type Culture Collection in Wuhan, Hubei Province on March 27, 2024, referred to as CCTCC, and the deposit numbers are: XBYM001: CCTCC NO.M 2024572; XBYM002: CCTCC NO.M 2024573; as well as XBYM003: CCTCC NO.M 2024574.

5. The preparation according to claim 4, wherein the preparation is in the form of granules, powder, serum, suspension, mucus or cream. The preparation according to claim 4 , which is an external preparation for skin.

7. A method for preparing the preparation according to any one of claims 4 to 6, comprising: The method comprises preparing a Staphylococcus epidermidis fermentation broth, comprising: Cultivate Staphylococcus epidermidis in liquid culture medium at 25-42 degrees Celsius for 18-24 hours; Centrifuge and collect the supernatant, which is the fermentation broth.

8. The method according to claim 7, wherein: The concentration of Staphylococcus epidermidis is (0.3-8)×10 9 CFU / mL; preferably, the concentration of Staphylococcus epidermidis is about 1×10 9 CFU / mL.

9. A method for preparing the preparation according to any one of claims 4 to 6, comprising: A Staphylococcus epidermidis extract is prepared comprising: Cultivate Staphylococcus epidermidis in liquid culture medium at 25-42 degrees Celsius for 18-24 hours; Centrifuge and collect the cells; The bacterial cells were broken to obtain the Staphylococcus epidermidis extract.

10. A skin care product comprising the preparation for preventing or repairing skin aging according to any one of claims 4 to 6.

Images