CN117645968A - Application of prebiotics in promoting growth of staphylococcus epidermidis CCSM0322 - Google Patents
Application of prebiotics in promoting growth of staphylococcus epidermidis CCSM0322 Download PDFInfo
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- CN117645968A CN117645968A CN202311653025.7A CN202311653025A CN117645968A CN 117645968 A CN117645968 A CN 117645968A CN 202311653025 A CN202311653025 A CN 202311653025A CN 117645968 A CN117645968 A CN 117645968A
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- oligosaccharide
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- staphylococcus epidermidis
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- 210000002374 sebum Anatomy 0.000 description 1
- 235000000053 special nutrition Nutrition 0.000 description 1
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- 239000012086 standard solution Substances 0.000 description 1
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- 231100000820 toxicity test Toxicity 0.000 description 1
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Abstract
The invention discloses an application of a prebiotic for promoting the growth of staphylococcus epidermidis CCSM0322, wherein the prebiotic is preferably fructo-oligosaccharide. The addition of fructo-oligosaccharide remarkably improves the growth of staphylococcus epidermidis CCSM0322 thallus and generates more short-chain fatty acids such as acetic acid, isovaleric acid and the like. The fermentation supernatant obtained by fermenting staphylococcus epidermidis CCSM0322 by a culture medium containing fructo-oligosaccharide can obviously inhibit staphylococcus aureus biofilm formation, reduce the generation of ROS (reactive oxygen species) of keratinocytes induced by UVB (ultraviolet B), has good antibacterial and antioxidant effects, and has wide application prospects in preparing antioxidant and allergy-relieving skin care products.
Description
Technical Field
The invention relates to an application of prebiotics in promoting the growth of staphylococcus epidermidis CCSM0322, and belongs to the technical field of microorganisms.
Background
Staphylococcus epidermidis (Staphylococcus epidermidis) is a resident dominant bacterium on human skin and plays an important role in maintaining skin health. A number of studies have shown that Staphylococcus epidermidis has a variety of physiological functions (Stacy A, belkaid Y. Microbiol guardians of skin health [ J ]. Science,2019,363 (6424):227-228.). Staphylococcus epidermidis metabolism produces antibacterial peptides, short Chain Fatty Acids (SCFAs), etc. to combat infections, suppress inflammatory factors and suppress pathogenic bacteria. The cell wall component lipoteichoic acid of staphylococcus epidermidis can reduce inflammation caused by propionibacterium acnes. The 6-N-hydroxyaminopurine (6-HAP) produced by the metabolism of staphylococcus epidermidis inhibits the activity of DNA polymerase and reduces the incidence of UVB induced skin tumors in mice. Staphylococcus epidermidis can also promote wound healing. Studies have demonstrated that Staphylococcus epidermidis has multiple functions, but not all Staphylococcus epidermidis has the above functions, with strain variability. The probiotics are to verify the probiotics function on the level of the strain, and the efficacy of the strain is clear. The selection of good strains is a time-consuming and laborious task, and we will appreciate that more and more functional staphylococcus epidermidis is being mined. At present, although skin probiotics in the true sense have not been scientifically defined and validated, a great deal of research has shown that: staphylococcus epidermidis is a potential probiotic on the skin, also known as "next generation probiotic" (NGP, next Generation Probiotics).
Prebiotics (pre-biotics) are compounds which cannot be hydrolyzed by human digestive enzymes, can be decomposed and utilized by intestinal flora after reaching the intestinal tract of a human body, promote the proliferation of probiotics in the intestinal tract and generate beneficial metabolites. Currently recognized prebiotics are fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), isomalto-oligosaccharides (IMO), inulin (Inulin), part of polysaccharides and part of dietary fibers, etc. The prebiotic concept is derived from food nutrition science and is based on intestinal probiotics such as lactobacillus, bifidobacterium etc. based on carbohydrates. At present, the prebiotic components such as trehalose, hyaluronic acid, beta-glucan, GOS, FOS, inulin and the like are added into the formulation of the cosmetic, so that the cosmetic has the effects of moisturizing, resisting aging, balancing skin microecology, improving skin state and the like. We see the accepted prebiotics in the cosmetic industry, just borrowing the concept of intestinal prebiotics, and at present, no deep research on the growth, metabolism and skin beneficial effect of skin bacteria is seen to verify whether the prebiotics are the prebiotics of skin bacteria.
The growth environment of the skin bacteria is different from that of lactic acid bacteria, and the skin bacteria form special nutrition requirements and metabolic pathways in long-term coexistence with a host, so that the skin can utilize sebum secreted by the skin, cells which are exfoliated from the epidermis and the like as food to synthesize nutrient substances and metabolic products which are required by the skin. The prebiotic component of intestinal bacteria is likely not the best "food" for skin bacteria. Therefore, it is imperative to search for the prebiotics of the dermatophytes by studying the appropriate nutritional conditions of the dermatophytes, analyzing the metabolites and mechanisms that exert efficacy and maintain skin health.
The applicant has developed a staphylococcus epidermidis CCSM0322 (issued patent number: ZH 202210967956.3) which has been preserved in China Center for Type Culture Collection (CCTCC) for 1 month 1 of 2022, and has a preservation number of CCTCC No. M2022774. Experiments prove that: the staphylococcus epidermidis CCSM0322 strain is a skin potential probiotic with good anti-inflammatory and whitening effects. It can be seen that CCSM0322 strain is a strain with development potential. The growth of the strain is independent of the nutritional requirements, and different nutritional requirements and culture conditions have obvious differences in the production of metabolites of the strain. The difference in nutrition of the strain causes the difference in metabolites of the strain, thereby affecting the efficacy on the skin. In contrast, the prior art lacks prebiotic screening studies of staphylococcus epidermidis.
Disclosure of Invention
Aiming at the problems, the invention comprehensively examines the influence of 4 common oligosaccharides (fructo-oligosaccharide, galacto-oligosaccharide, isomaltooligosaccharide and inulin) on staphylococcus epidermidis CCSM0322 from the effects of thallus proliferation, fermentation supernatant pH value, short-chain fatty acid yield, inhibition on staphylococcus aureus biomembrane and UVB-induced ROS and inflammatory factors, screens the prebiotics of staphylococcus epidermidis CCSM0322 and lays a foundation for the development of skin probiotics in the future.
The invention firstly provides application of a prebiotic in promoting the growth of staphylococcus epidermidis CCSM0322 and generating more short-chain fatty acids such as acetic acid, isovaleric acid and the like by metabolism, wherein the prebiotic is any one of oligosaccharides such as fructo-oligosaccharide, galacto-oligosaccharide, isomaltooligosaccharide, inulin and the like, and preferably the prebiotic is fructo-oligosaccharide. Wherein the concentration of the prebiotics is 0.1-3%.
The invention also provides an application of fermentation supernatant obtained by fermenting staphylococcus epidermidis CCSM0322 with a culture medium containing prebiotics in preparing skin care products for inhibiting formation of staphylococcus aureus biomembrane.
The invention also provides an application of fermentation supernatant obtained by fermenting staphylococcus epidermidis CCSM0322 with a culture medium containing prebiotics in preparing skin care products for reducing ROS of keratinocytes induced by UVB.
The invention also provides an application of fermentation supernatant obtained by fermenting staphylococcus epidermidis CCSM0322 with a culture medium containing prebiotics in preparing an antioxidant, allergy-relieving and skin-care product.
The prebiotic is preferably fructooligosaccharides.
The preparation method of the fermentation supernatant comprises the following steps: inoculating CCSM0322 seed solution into sugar-free TSB culture medium containing 0.1-3% prebiotics (fructo-oligosaccharide, galacto-oligosaccharide, isomaltooligosaccharide and inulin) according to the volume ratio of 2-3%, shake culturing at 35-40deg.C for 8-20 hr, centrifuging, and collecting supernatant to obtain fermentation supernatant of CCSM0322 fermented by each prebiotic.
The invention has the technical effects that: the invention provides a prebiotic for promoting the growth and application of staphylococcus epidermidis, and the prebiotic is preferably fructo-oligosaccharide. The addition of fructo-oligosaccharide remarkably improves the growth of staphylococcus epidermidis CCSM0322 thallus and generates more short-chain fatty acids such as acetic acid, isovaleric acid and the like. The TSB fermentation supernatant of the strain is obtained by adding fructo-oligosaccharide for fermentation, so that the formation of staphylococcus aureus biological films is obviously inhibited, the generation of ROS (reactive oxygen species) of keratinocytes induced by UVB is reduced, the strain has good antibacterial and antioxidant effects, and the strain has wide application prospect in preparing antioxidant and allergy-relieving skin care products. The prebiotics obtained by screening of the invention promote the growth of the strain CCSM0322 and improve the probiotic characteristics of the strain. The invention lays a foundation for the development of functional skin bacteria.
Drawings
FIG. 1 is a graph showing the growth of CCSM0322 of the epidermal grape ball with different concentrations of oligosaccharide added in example 1 of the present invention; wherein A, B, C, D is the graph of adding fructo-oligosaccharide, galacto-oligosaccharide, isomalto-oligosaccharide and inulin with different concentrations;
FIG. 2 is a graph showing the growth of CCSM0322 of example 2 of the present invention with 2% of different oligosaccharides added to the epidermal grape bulb;
FIG. 3 is a graph showing the pH of the epidermal grape bulb CCSM0322 growth with the addition of 2% different oligosaccharides in example 2 of the present invention;
FIG. 4 is a graph showing the effect of different oligosaccharides on the production of short chain fatty acids acetic acid (a) and isovaleric acid (b) by Staphylococcus epidermidis CCSM0322 in example 2 of the present invention;
FIG. 5 is a graph showing the adhesion of the fermentation supernatants of different oligosaccharide fermented epidermal grape balls CCSM0322 to a golden yellow grape ball biofilm in example 3 of the present invention;
FIG. 6 is a graph showing cytotoxicity of various oligosaccharide fermented epidermic grape globus CCSM0322 fermented supernatants against primary keratinocytes in example 4 of the present invention.
Detailed Description
In order to make the technical means, the creation features, the achievement of the purpose and the effect of the present invention easy to understand, the present invention is specifically described below with reference to the embodiments and the drawings.
The materials and strain media used in the following examples are as follows:
fructooligosaccharides (bowling organism share limited), galactooligosaccharides (bowling organism share limited), isomaltooligosaccharides (bowling organism share limited) and inulin (purity > 90%, adamas life).
Sugar-free TSB medium: tryptone 17.0g/L; 3.0g/L soybean peptone; k (K) 2 HPO 4 2.5g/L; sodium chloride 5.0g/L; adding fructo-oligosaccharide, galacto-oligosaccharide, isomaltooligosaccharide and inulin with different concentrations, and sterilizing at 121deg.C for 15 min.
TSB broth: tryptone 17.0g/L; 3.0g/L soybean peptone; sodium chloride 5.0g/L; k (K) 2 HPO 4 2.5g/L; glucose 2.5g/L; sterilizing at 121deg.C for 15 min.
TSA medium: 15.0g/L of tryptone; 5.0g/L of soybean papain hydrolysate; sodium chloride 5.0g/L; 15.0g/L of agar; sterilizing at 121deg.C for 15 min.
The preparation method of the seed liquid comprises the following steps:
1) Strain activation
Taking out a staphylococcus epidermidis CCSM0322 streak TSA solid plate preserved at-80 ℃, and placing the solid plate in a 37 ℃ constant temperature incubator for aerobic culture for 16-20h; activating for 2 times for standby.
2) Seed liquid preparation
Inoculating Staphylococcus epidermidis CCSM0322 into TSB liquid culture medium, shake culturing at 37deg.C for 16-20 hr, and measuring OD of fermentation broth 600 Adjust to the proper concentration range and adjust the OD 600 To 0.55-0.56 as seed solution.
Example 1: effect of different oligosaccharides on growth of Staphylococcus epidermidis CCSM0322
Fructo-oligosaccharide, galacto-oligosaccharide, isomalto-oligosaccharide and inulin are added into sugarless TSB culture medium to prepare oligosaccharide culture medium with serial concentration (0.018%, 0.036%, 0.072%, 0.15% and 0.3%), and sterilizing at 121deg.C for 15 min. Inoculating CCSM0322 seed solution into each fermentation tube according to 2.5% inoculum size, shake culturing at 37deg.C with 160r/min shaker for 16-18 hr, and measuring OD of each tube fermentation liquid 600 Values, 6 replicates per sample, were averaged, and blank (sugarless TSB medium), control 1 (sugarless TSB medium+Staphylococcus epidermidis CCSM 0322), and control 2 (sugarless TSB medium+oligosaccharide) were set. The strain was grown at various concentrations of fructo-oligosaccharide, galacto-oligosaccharide, isomalto-oligosaccharide and inulin, the results are shown in FIG. 1, and the EC was calculated 50 Values.
EC 50 Are commonly used to evaluate the activation or promotion of cells by drugs and the like, EC 50 Lower values indicate better results. As can be seen from fig. 1: the addition of 4 kinds of oligosaccharides with different concentrations can well promote the growth of staphylococcus epidermidis CCSM0322, and the EC that fructo-oligosaccharide, galacto-oligosaccharide, isomaltooligosaccharide and inulin promote the growth of CCSM0322 is obtained through calculation 50 Values 0.1319%,30.16%,0.2008%,0.1396%, respectively. The effect of promoting the growth of the strain is fructo-oligosaccharide>Inulin>Oligomeric isomaltose>Galactooligosaccharides, of which fructooligosaccharides promote the most favorable cells of staphylococcus epidermidis CCSM 0322.
Example 2: effect of 2% oligosaccharide addition on the growth curve of Staphylococcus epidermidis CCSM0322
Fructo-oligosaccharide, galacto-oligosaccharide, isomaltooligosaccharide and inulin are respectively added into sugarless TSB culture medium to prepare oligosaccharide culture medium with 2% additive concentration, and 2% glucose is added as control group, and autoclaved at 121deg.C for 15min for use. Inoculating CCSM0322 seed solution into each fermentation tube according to 2.5% inoculum size, shake culturing at 37deg.C with 160r/min shaker, selecting 8 time points of 0, 4, 8, 12, 16, 20, 24, 28 hr, sampling every 4 hr, sampling at each time point, and measuring cell growth OD 600 pH of supernatantShort chain fatty acid content.
Wherein the determination of short chain fatty acids:
1) The sample processing method comprises the following steps: centrifuging the fermentation broth at 10000r/min for 15min to collect supernatant, and adding 0.4mL50% H into 10mL centrifuge tube 2 SO 4 Mixing with 2mL diethyl ether, shaking at 250r/min for 45min, centrifuging at 4deg.C and 10000r/min for 5min, collecting upper organic phase, filtering with 0.22 μm organic filter membrane before GC loading analysis, and preserving at-20deg.C;
2) Gas chromatography conditions: using a Shimadzu GC instrument (GC-2010 plus); chromatographic column: agilent DB-WAX UI (60 m 0.25 μm 0.25 mm); the sample inlet and detector temperatures were set at 250 ℃; high-purity nitrogen is used as carrier gas (flow rate is 12.1 mL/min), and a non-split detection mode is adopted; heating conditions: the loading was 1. Mu.L, which was maintained at 10℃/min to 180℃for 1min from 75℃and at 10℃/min to 220℃for 1min. The mixed standard substance is adopted: water-soluble fatty acid mixed standard solution (north bureau of metric research institute): glacial acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid total 6 fractions, each 1000 μg/mL, diluted with ultrapure water to a gradient of 5, 10, 20, 50, 100, 200, 500 and 800 μg/mL.
The growth curve of the strain CCSM0322 added with 2% of oligosaccharide is shown in figure 2, and compared with a control group (sugar-free TSB culture medium), the growth of the bacterial cells is obviously promoted by adding the oligosaccharide, the logarithmic growth phase is shortened, the specific growth speed is improved, and the bacterial cell concentration is obviously increased as shown in figure 2. Compared with 4 kinds of oligosaccharides, the growth promoting effect is fructo-oligosaccharide > inulin > isomaltooligosaccharide > galacto-oligosaccharide when observed at a time point of 16h of culture. At the same time, the pH change at each time point was observed, as shown in FIG. 3, and the addition of oligosaccharide significantly promoted the acid production by the bacterial growth and the pH decrease of the fermentation broth compared with the control group (sugar-free TSB medium). The fructo-oligosaccharide, the isomaltooligosaccharide, the galacto-oligosaccharide and the glucose have the same trend, the pH of the fermentation liquid is in a descending trend along with the increase of the culture time, the time point of the rapid pH value descending is 8-12h, and the pH value is slowly changed. Wherein the pH value of the fermentation liquor of the inulin gradually rises after the fermentation liquor is cultured for 12 hours, and the fermentation liquor shows different trends.
We monitored the short chain fatty acid changes at various time points simultaneously, see figure 4, and found that after oligosaccharide addition, 2 short chain fatty acids, mainly acetic acid and isovaleric acid, were produced. Wherein the fructo-oligosaccharide, the isomaltooligosaccharide, the galacto-oligosaccharide and the glucose have substantially uniform behaviors. With increasing culture time, the acetogenic and isovaleric acid content increased significantly, and the acetic acid content was greater than the isovaleric acid content. Wherein after inulin is cultured for 20 hours, the acetic acid content is in a decreasing trend, and the isovaleric acid content is gradually increased, so that different trends are shown. Meanwhile, the total amount of acetic acid and isovaleric acid at each time point is calculated, and the time point of 12 hours is taken as an example, fructo-oligosaccharide > isomaltooligosaccharide > galacto-oligosaccharide > inulin.
As can be seen from fig. 4, the addition of each oligosaccharide significantly promoted the production of 2 short chain fatty acids such as acetic acid and isovaleric acid by staphylococcus epidermidis CCSM0322 in TSB medium, unlike the use of oligosaccharides for the main production of acetic acid, propionic acid and butyric acid by intestinal flora. Previous studies have also demonstrated that the variety of short chain fatty acids produced by the addition of different carbon sources or precursor substances is also different, e.g., staphylococcus epidermidis strain ATCC 12228 ferments glycerol to produce short chain fatty acids such as acetic acid, butyric acid, succinic acid, etc., further demonstrating that butyric acid activates the short chain fatty acid FFAR2 receptor to exert its anti-inflammatory effect (Keshari S, balasubramaniam A, myagmardoloonjin B, et al, butyl acid from probiotic staphylococcus epidermidis in the skin microbiome down-regulates the ultraviolet-induced pro-inch insulator IL-6cytokine via short-chain fatty acid receptor [ J ]. International journal of molecular sciences,2019,20 (18): 4477.).
Research has shown that staphylococcus epidermidis is used as symbiotic bacteria for healthy skin, and the metabolism of staphylococcus epidermidis generates short chain fatty acid, which is favorable for inhibiting the proliferation of pathogenic bacteria, reducing infection and having anti-inflammatory effect. The addition of each oligosaccharide promotes the growth of CCSM0322 thallus and the production of short chain fatty acid, wherein the fructooligosaccharide has the best effect.
Example 3: effect of addition of different oligosaccharide fermentation supernatants on Staphylococcus aureus biofilm
1) Activation and culture of indicator bacteria
Will be-80Scribing a TSA solid plate by a staphylococcus aureus CCSM0424 (preservation number: CCTCC No. M2022074) preserved at the temperature, and placing the solid plate in a constant temperature incubator at the temperature of 37 ℃ for aerobic culture for 16-20h; activating for 2 times for standby. Inoculating CCSM0424 of Staphylococcus aureus into TSB liquid culture medium, shake culturing at 37deg.C for 16-20 hr, and determining OD of fermentation broth 600 Adjust to the proper concentration range and adjust the OD 600 To 0.55-0.56 as seed solution.
2) Preparation of different oligosaccharide fermentation supernatants
Inoculating CCSM0322 seed solution into sugar-free TSB culture medium containing 2% oligosaccharide (fructo-oligosaccharide, galacto-oligosaccharide, isomaltooligosaccharide and inulin) according to 2.5% inoculum size, shake culturing at 37deg.C at 160r/min for 8 hr, centrifuging at 10000r/min for 15min, and collecting supernatant to obtain fermentation supernatant of CCSM0322 fermented by different oligosaccharides.
3) Semi-quantitative determination of biological film by crystal violet
Experimental grouping: group a (blank): 200. Mu.L of blank TSB medium; group B (control): 50. Mu.L of bacterial suspension + 50. Mu.L of physiological saline + 100. Mu.L of TSB medium; group C (experimental group): 50. Mu.L of bacterial suspension + 50. Mu.L of each strain was fermented at different culture times with supernatant + 100. Mu.L of TSB medium.
Adding fermentation supernatant of corresponding oligosaccharide into each well of 96-well cell culture plate according to groups, arranging 4 multiple wells in each group, respectively culturing in a constant temperature incubator at 37deg.C for 4, 8, 12, 16, 20, 24, and 28 hr, taking out 96-well plate, sucking out residual culture medium, adding 200 μl of 0.9% physiological saline into each well, sucking out 0.9% physiological saline to remove non-adhered bacteria, washing the well plate three times, adding 0.01% crystal violet solution into completely dried well plate, dyeing for 15min, washing with sterile deionized water twice, washing out excessive crystal violet solution, oven drying, releasing fixed crystal violet solution with 95% ethanol, and measuring OD 595 Absorbance values.
Staphylococcus aureus CCSM0424 is a laboratory isolated strain from skin and has been determined to have the ability to produce a biofilm by congo red experiments and semi-quantitative staining of crystal violet, so the CCSM0424 strain was used as the strain for the study of biofilms. The graph of adhesion of the fermentation supernatant of each oligosaccharide fermentation epidermal grape ball CCSM0322 to the staphylococcus aureus biofilm was measured, as shown in FIG. 5, the addition of the oligosaccharide CCSM0322 fermentation supernatant can reduce the biofilm formation capacity of the staphylococcus aureus, wherein the CCSM0322 fermentation supernatant obtained by adding fructo-oligosaccharide and glucose can obviously inhibit the generation of the CCSM0424 strain biofilm, and the formation amount of the biofilm is obviously reduced compared with the control group when the fermentation supernatant is kept at a low film-producing level in the whole growth process of the staphylococcus aureus. The inhibition effect of adding fructo-oligosaccharide is obviously better than that of other 3 kinds of oligosaccharides from the aspect of inhibiting the formation of staphylococcus aureus biofilm by the 4 kinds of oligosaccharides.
Example 4: preparation of fermentation supernatant of different Staphylococcus epidermidis CCSM0322 to reduce ROS content of keratinocytes caused by UVB by adding fructooligosaccharide fermentation supernatant
The staphylococcus epidermidis CCSM0322 strain is streaked on a TSA plate, cultured for 16-20 hours at the constant temperature of 37 ℃ and activated for 2 times. The CCSM0322 seed solution was inoculated into a sugar-free TSB (labeled 287C), 2% fructo-oligosaccharide (labeled 287G) and 2% glucose (labeled 287P) sugar-free TSB medium according to an inoculum size of 2.5%, shake-cultured at 37℃with a shaker at 160r/min for 16 hours, and the supernatant was collected by centrifugation at 10000r/min at 4℃at the end of the culture to obtain a fermentation supernatant of CCSM0322 fermented with sugar-free TSB, fructo-oligosaccharide and glucose.
(2) Cell culture
Human primary skin keratinocytes (NEKs) were isolated from normal skin tissue using Promocell-specific complete medium (Keratinocyte Growth Medium, C-20011) at 37℃with 5% CO 2 Conventional culture under conditions, after cells grew to near confluence, passaging was done by trypsinization, 1 passage per 5 d.
(3) Cell viability assay
Taking NEKs cells in optimal growth state, performing conventional treatment, and adjusting the cell suspension density to 8×10 4 -1×10 5 mu.L of cell suspension per well was inoculated into 96-well plates at 37℃and 5% CO 2 Culturing in an incubator. Adding 0.1%, 0.5%, 1%, 2.5%, 5% and 10% sugar-free TSB, 2% fructo-oligosaccharide and 2% glucose, respectively, and culturing the CCSM0322 fermentation supernatant for 24 hr, and setting control group, each experimentThe group was provided with 3 parallel holes. After 24 hours of incubation, 10. Mu.LCCK-8 reagent (Japanese homozygote CK-04) was added to each well and incubated for 2 hours in a conventional manner. The absorbance at 450nm was measured using an enzyme-labeled instrument, and the reference wavelength was 600nm or more.
(4) ROS detection
NEKs cells in optimal growth state were taken at 2X 10 4 The individual/well cell suspensions were seeded in 96-well plates. NEKs cells were exposed to 312nm wavelength radiation at a dose of 10mJ/cm 2 Under UVB radiation (Spectroline Model EB-160C ultraviolet lamp, USA). After UVB irradiation, cells were washed with PBS, and CCSM0322 fermentation supernatant containing 10% sugarless TSB, 2% fructooligosaccharides and 2% glucose was added, fresh medium was added and incubated for 24h. After incubation for 24h, 5 μlros fluorescent chromogenic reagent (CellRox, thermo) was added to each well and washed with PBS after conventional incubation for 1 h. The fluorescence value was measured using an enzyme-labeled instrument, the excitation light wavelength was 485nm, and the emission light was 520nm.
Toxicity experiments of sugar-free TSB, 2% fructo-oligosaccharide and 2% glucose CCSM0322 fermentation supernatants on primary keratinocytes as shown in FIG. 6, it can be seen from the graph that sugar-free TSB (labeled 287C), 2% fructo-oligosaccharide (labeled 287G) and 2% glucose (labeled 287P) CCSM0322 fermentation supernatants were added in an amount ranging from 0.1% to 10% concentration, and showed no significant cytotoxicity, wherein sugar-free TSB (labeled 287C) and 2% fructo-oligosaccharide (labeled 287G) CCSM0322 fermentation supernatants were able to promote growth of primary skin keratinocytes in humans, as compared to the control group. Here we selected 10% CCSM0322 fermentation supernatant for UVB-induced inflammation model experiments, the experimental results are shown in table 1.
TABLE 1 ROS of different fermentation supernatants to UVB-induced primary keratinocytes
The experimental results show (Table 1) that different added fermentation supernatants have a difference in the effect of UVB-induced reactive oxygen species ROS produced by primary keratinocytes. After UVB irradiation (UVB group), cells produced a large amount of reactive oxygen, and CCSM0322 fermentation supernatant with 10% fructo-oligosaccharides added showed a scavenging rate of 10.5% for reactive oxygen radical ROS, indicating the ability to scavenge free radicals. The sugarless TSB group has weaker protective effect on UVB-induced damage, while the glucose group also has protective effect on the damage, but has no obvious effect of fructo-oligosaccharide group. It can be seen that the addition of fructooligosaccharides promotes metabolism of CCSM0322 strain to produce beneficial substances, protecting the primary keratinocytes from damage caused by UVB-induced reactive oxygen species.
In summary, the invention provides a prebiotic of staphylococcus epidermidis, which can obviously promote the growth of staphylococcus epidermidis and improve the beneficial effects of bacterial strain fermentation supernatant on promoting skin health. The prebiotics are preferably fructo-oligosaccharides, and the addition of the fructo-oligosaccharides obviously improves the growth of staphylococcus epidermidis CCSM0322 thalli and generates more short-chain fatty acids such as acetic acid, isovaleric acid and the like through metabolism. The TSB fermentation supernatant of the strain is obtained by adding fructo-oligosaccharide for fermentation, so that the formation of staphylococcus aureus biological membranes is obviously inhibited, the generation of ROS (reactive oxygen species) of keratinocytes induced by UVB is reduced, and the strain has good antibacterial and antioxidant effects. The prebiotics obtained by screening have the advantages of promoting the growth of the strain CCSM0322 and improving the probiotic characteristics of the strain. The invention lays a foundation for the development of functional skin bacteria.
Claims (6)
1. Use of a prebiotic for promoting the growth of staphylococcus epidermidis CCSM0322, metabolizing to produce more acetic acid and isovaleric acid, said prebiotic being any one of fructo-oligosaccharides, galacto-oligosaccharides, isomaltooligosaccharides and inulin.
2. The use according to claim 1, wherein the prebiotic is fructo-oligosaccharide.
3. An application of fermentation supernatant obtained by fermenting staphylococcus epidermidis CCSM0322 with a culture medium containing prebiotics in preparing skin care product for inhibiting formation of staphylococcus aureus biofilm; the prebiotic is fructo-oligosaccharide.
4. Use of a fermentation supernatant obtained by fermenting staphylococcus epidermidis CCSM0322 with a prebiotic-containing medium for the preparation of a skin care product for reducing UVB-induced ROS of keratinocytes; the prebiotic is fructo-oligosaccharide.
5. An application of fermentation supernatant obtained by fermenting staphylococcus epidermidis CCSM0322 with a culture medium containing prebiotics in preparing antioxidant, allergy-relieving and skin-caring products; the prebiotic is fructo-oligosaccharide.
6. The use according to any one of claims 3 to 5, wherein the fermentation supernatant is prepared by a process comprising: inoculating CCSM0322 seed solution into sugar-free TSB culture medium containing 0.1-3% fructo-oligosaccharide according to 2-3% inoculum size, shake culturing at 35-40deg.C for 8-20 hr, centrifuging, and collecting supernatant.
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| US20210069088A1 (en) * | 2018-03-08 | 2021-03-11 | Plexus Worldwide, LLC. | Compositions and methods for skin renewal |
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| US20210069088A1 (en) * | 2018-03-08 | 2021-03-11 | Plexus Worldwide, LLC. | Compositions and methods for skin renewal |
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