CN116855559A - Preparation method and application of snow cover algae oligosaccharide for regulating skin microecology - Google Patents
Preparation method and application of snow cover algae oligosaccharide for regulating skin microecology Download PDFInfo
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/005—Antimicrobial preparations
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/10—Washing or bathing preparations
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/12—Disaccharides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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- Wood Science & Technology (AREA)
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- Animal Behavior & Ethology (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Dermatology (AREA)
- General Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
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- Birds (AREA)
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Abstract
The invention belongs to the technical field of cosmetics, and provides a preparation method and application of a snow cover algae oligosaccharide for regulating and controlling skin microecology. The results of the examples show that the snow cover algae oligosaccharide can promote beneficial bacteria staphylococcus epidermidis and bifidobacterium on the skin surface, inhibit harmful bacteria staphylococcus aureus, propionibacterium acnes, malassezia and the like on the skin surface, has the function of regulating and controlling the balance of skin flora, and can also repair and strengthen skin barriers and relieve skin inflammation. The invention also discloses the application of the snow cover algae oligosaccharide in preparing the functional cosmetics with the functions of regulating skin microecology, repairing skin barrier and relieving skin inflammation, the application range of the snow cover algae oligosaccharide is enlarged, and the commercial utilization value of the snow cover algae oligosaccharide is improved.
Description
Technical Field
The invention belongs to the technical field of cosmetics, and particularly relates to a preparation method and application of a snow cover algae oligosaccharide for regulating skin microecology.
Background
The snow cover algae (Chlamydomo nasnivalis) belongs to the class of Chlorophyta (Chlorophyta), chlorophyceae (Chlorophyceae), volvocales (Volvocales), and is a special single-cell low-class plant group which can be grown in the annual snow-accumulating areas such as mountains, polar regions and the like throughout the year. The snow cover algae is rich in high-quality protein, linolenic acid, polysaccharide, nucleotide, polypeptide, vitamin, trace elements and other substances, and has antiviral, anticancer, antioxidant, antiinflammatory and other effects.
The oligosaccharides are compounds polymerized by 2-10 glycosidic bonds, are widely existing in natural plants, and have the advantages of improving the immunity of organisms, regulating the microecological environment of intestinal tracts, being low in relative molecular weight, good in water solubility, easy to absorb moisture, high in biological activity and the like, and also have the characteristics of pure nature, no pollution and the like, so that the oligosaccharide substances are widely applied to industries such as foods, medical treatment, health care products and the like. The content of saccharide in the snow cover algae cell in the green stage is high, and the snow cover algae oligosaccharide can be obtained by separating the snow cover algae from the snow cover algae by a biological fermentation technology.
Disclosure of Invention
The invention aims to provide the snow cover algae oligosaccharide for regulating and controlling the skin microecology and the preparation method thereof, which have the functions of regulating and controlling the skin flora, repairing the skin barrier and relieving the skin inflammation, and can be widely applied to cosmetic products.
In order to achieve the above object, the present invention is achieved by the following technical solutions.
In a first aspect, the invention provides a preparation method of a snow cover algae oligosaccharide for regulating skin microecology, wherein the snow cover algae oligosaccharide is obtained from polar algae snow cover algae by a biological fermentation technology, and the preparation method comprises the following specific steps:
s1, activating and culturing strains: taking out the preserved bacillus subtilis from the inclined plane, and transferring the bacillus subtilis into a triangular flask for seed shake flask culture (LB culture medium);
s2, fermentation: when the fungus suspension OD 600 The bacterial suspension is inoculated into a fermentation culture medium containing 15-30wt% of snow cover algae powder and 2-3wt% of glucose according to the inoculum size of 10-20wt% of volume fraction when the value is 3-5. Stationary culture at 30deg.C, when fermentation broth OD 600 Stopping fermentation at 120-140, centrifuging the fermentation broth at 8000-10000r/min for 10-15min, and collecting thallus and fermentation supernatant respectively;
s3, cell wall breaking: homogenizing and breaking the wall of the fermentation thalli to obtain a thalli wall-broken extracting solution;
s4, extracting and refining: mixing the fermentation supernatant in the step S2 with the cell wall-broken extracting solution in the step S3, evaporating to obtain a dried product, dissolving the dried product with petroleum ether, filtering, dissolving filter residues with 70-90% ethanol, filtering and evaporating to obtain the snow cover algae oligosaccharide.
Preferably, the bacillus subtilis (bacillus subtilis) in the step S1 is preserved in the China general microbiological culture Collection center with a preservation number of CGMCC No.26359 and a preservation date of 2022, 12 months and 30 days.
Preferably, the seed shake flask culture in the step S1 has an inoculum size of 1-2%, and is cultured at 37+ -2deg.C and 220-240rpm for 15-20 hr.
Preferably, the pressure of the homogeneous wall breaking in the step S3 is 80-100Mpa.
Preferably, the purity of the snow cover algae oligosaccharide in the step S4 is 52-68%.
In a second aspect, the present invention provides a snow cover algae oligosaccharide for regulating skin microecology, which is prepared by adopting the preparation method of the snow cover algae oligosaccharide.
The snow cover algae oligosaccharide has the functions of regulating the balance of skin flora, repairing skin barrier function and relieving skin inflammation, and has the functions of regulating the flora to inhibit the growth and proliferation of harmful bacteria and promote the growth and proliferation of probiotics; the harmful bacteria include but are not limited to propionibacterium acnes, malassezia and the like besides staphylococcus aureus; the probiotics include, but are not limited to, bifidobacteria and the like in addition to surface staphylococci.
The effect of the skin barrier repair is in particular to promote proliferation and migration of skin epidermis-related cells.
The effect of relieving skin inflammation is specifically relieving skin inflammation caused by chemical stimulus such as surfactant; inhibiting the expression of inflammation promoting factors TNF-alpha, IL-1 beta and IL-8mRNA, and promoting the expression of inflammation inhibiting factors IL-13; skin inflammation is alleviated by a route that promotes autophagy.
In a third aspect, the invention provides an application of the snow cover algae oligosaccharide for regulating skin microecology in the aspect of functional cosmetics with the functions of regulating skin microecology, repairing skin barrier and relieving skin inflammation.
Preferably, the functional cosmetic includes, but is not limited to, a cleanser, a toner, a lotion, an emulsion, a mask, a cream, an essence, and the like.
The beneficial effects of the invention are as follows:
the snow cover algae oligosaccharide is obtained through a biological fermentation technology, is green in source and high in human safety.
The snow cover algae oligosaccharide disclosed by the invention can obviously inhibit the proliferation of skin harmful bacteria, promote the growth and proliferation of probiotics, regulate skin microecology and be beneficial to the establishment and regulation of skin steady state.
The snow cover algae oligosaccharide disclosed by the invention can repair and consolidate skin barriers, relieve skin inflammation, effectively improve skin state and maintain skin health.
Drawings
FIG. 1 shows the regulation of characteristic species by Xueyeriaceae oligosaccharide;
FIG. 2 shows the relative repair rate of the action of the snow cover algae oligosaccharides on the tail of the zebra fish;
FIG. 3 is a graph showing the soothing effect of Xueclothing algae oligosaccharides on the inflammation model of zebra fish;
FIG. 4 shows the effect of Xuefukania oligosaccharides on inflammatory mRNA expression levels;
FIG. 5 shows the effect of Xuefua oligosaccharides on key proteins of the autophagy-related pathway.
Detailed description of the preferred embodiments
In order to more clearly illustrate the present invention, the present invention will be further described with reference to preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and that this invention is not limited to the details given herein.
Example 1
Activating and culturing strains: and taking out the preserved bacillus subtilis CGMCC No.26359 from the inclined plane, transferring the bacillus subtilis CGMCC No.26359 into a triangular flask, and culturing the bacillus subtilis with the inoculum size of 1 percent and 220rpm for 15 hours by shaking the seed in the triangular flask. .
Fermentation: when the fungus suspension OD 600 The bacterial suspension is inoculated into a fermentation medium containing 15wt% of snow cover algae powder and 2wt% of glucose according to the inoculation amount of 10% of volume fraction at the value of 3. Stationary culture at 28deg.C, when fermentation broth OD 600 Stopping fermentation at 120, centrifuging the fermentation liquor at 8000r/min for 10min, and collecting thallus and fermentation supernatant respectively.
Breaking cell walls: homogenizing and breaking the wall of the fermentation thalli under the pressure of 80MPa to obtain a thalli wall-breaking extracting solution.
Extracting and refining: mixing the fermentation supernatant with the cell wall-broken extract, evaporating to obtain a dry product, dissolving the dry product with petroleum ether, filtering, dissolving the filter residue with 70% ethanol, and filtering and evaporating to obtain the snow cover algae oligosaccharide with the purity of 52%.
Example 2
Activating and culturing strains: and taking out the preserved bacillus subtilis CGMCC No.26359 from the inclined plane, transferring the bacillus subtilis CGMCC No.26359 into a triangular flask, and culturing the bacillus subtilis with seed shaking (LB culture medium) in an inoculum size of 1.5 percent at 37 ℃ for 17.5 hours at 230 rpm. .
Fermentation: when the fungus suspension OD 600 The bacterial suspension is inoculated into a fermentation medium containing 22wt% of snow cover algae powder and 2.5wt% of glucose according to the inoculation amount of 15% of volume fraction at the time of value 4. Stationary culture at 30deg.C, when fermentation broth OD 600 Fermentation was stopped at 130, and the fermentation broth was centrifuged at 9000r/min for 12min, and the cell and fermentation supernatant were collected, respectively.
Breaking cell walls: homogenizing and breaking the wall of the fermentation thalli under the pressure of 90MPa to obtain a thalli wall-breaking extracting solution.
Extracting and refining: mixing the fermentation supernatant with the cell wall-broken extract, evaporating to obtain a dry product, dissolving the dry product with petroleum ether, filtering, dissolving the filter residue with 80% ethanol, and filtering and evaporating to obtain the snow cover algae oligosaccharide with the purity of 61%.
Example 3
Activating and culturing strains: and taking out the preserved bacillus subtilis CGMCC No.26359 from the inclined plane, transferring the bacillus subtilis CGMCC No.26359 into a triangular flask, and culturing the bacillus subtilis with the inoculum size of 2 percent and at 39 ℃ and 240rpm for 20 hours by shaking the seeds in the flask. .
Fermentation: when the fungus suspension OD 600 The bacterial suspension is inoculated into a fermentation medium containing 30wt% of snow cover algae powder and 3wt% of glucose according to the inoculation amount of 20% of volume fraction at the time of value of 5. Stationary culture at 32deg.C, when fermentation broth OD 600 Stopping fermentation at 140, centrifuging the fermentation broth at 10000r/min for 15min, and collecting thallus and fermentation supernatant respectively.
Breaking cell walls: homogenizing and breaking the wall of the fermentation thalli under the pressure of 100MPa to obtain a thalli wall-breaking extracting solution.
Extracting and refining: mixing the fermentation supernatant with the cell wall-broken extract, evaporating to obtain a dry product, dissolving the dry product with petroleum ether, filtering, dissolving the filter residue with 90% ethanol, and filtering and evaporating to obtain the snow cover algae oligosaccharide with the purity of 68%.
Example 4
Skin micro-ecological efficacy control test of the snow cover algae oligosaccharide obtained in example 2
1. In vitro bacteriostasis experiment
Strain activation: respectively inoculating characteristic strains (Staphylococcus epidermidis, lactobacillus, staphylococcus aureus, propionibacterium acnes, malachite, and Candida) into corresponding nutrient agar culture medium for activation, culturing in a 37 deg.C constant temperature incubator for 24 hr, respectively picking 1 ring of activated strains, placing into 9mL of sterile water, shaking, and making into a series of bacterial suspensions with concentration of about 10 7 CFU/mL, ready for use.
Measuring a bacteriostasis zone: processing qualitative filter paper into 9mm round filter paper sheet, placing into a dry plate, sterilizing at 121deg.C for 20min, and soaking into different concentration of Celastracene oligosaccharide water solution (1, 2, 10, 20 mg/mL) to make it fully absorbed for use; pouring the sterilized nutrient agar culture medium into a plate after melting, cooling and solidifying, respectively adding 0.4mL of bacterial suspension into the plate by using a liquid taking device, uniformly coating the plate by using a sterile coater, clamping the soaked filter paper sheets in the bacteria-containing plate by using sterile forceps, and taking the filter paper sheets with each concentration at a certain distance as a reference; each plate is pasted with 5 pieces, and each strain is repeated for 3 times; then each plate is inverted in a constant temperature incubator at 37 ℃ for 48 hours; taking out, and measuring the diameter of the inhibition zone.
2. In vitro growth-promoting experiments
Strain activation: inoculating staphylococcus on the surface of a probiotic bacterial strain into an MRS solid culture medium, culturing for 16 hours at 37 ℃, selecting single bacterial colony, inoculating into an MRS liquid culture medium, culturing for 18 hours at 37 ℃ for activation, and continuously carrying out passage for 2 times to obtain experimental bacterial liquid; the cells were centrifuged at 6000rpm for 8min, and washed twice with 0.9% physiological saline to obtain cells for subsequent experiments.
Strain growth assay: investigating the probiotics by the change of absorbance values of thalliIn vitro proliferation effect, quantitative concentrated bacterial solutions (final concentration about 5×10 were inoculated 6 CFU/mL) in the split-packed MRS liquid culture medium and MRS modified culture medium, anaerobic culturing at 37 ℃ for 12 hours, sampling and measuring absorbance value; the absorbance measurement wavelength is 600nm, and the sterile culture medium is used as a blank control; three replicates were run for each group.
3. Experimental results
As shown in fig. 1, the snow cover algae oligosaccharide can obviously inhibit harmful bacteria staphylococcus aureus and propionibacterium acnes, has a certain inhibition effect on fungi malassezia and candida, has a remarkable promotion effect on beneficial bacteria bifidobacteria, and has a certain promotion effect on staphylococcus epidermidis.
The result shows that the snow cover algae oligosaccharide can regulate and control skin flora and balance skin microecology by inhibiting harmful bacteria and promoting growth beneficial bacteria.
Example 5
Skin barrier repair efficacy test on the snow Chlamydomonas oligosaccharide obtained in example 2
1. Zebra fish tail break repair
Preparation: 2dpf wild type zebra fish larvae.
Tail breaking: the tail fin part of the juvenile fish is cut off by a surgical knife under a microscope, and the spine of the juvenile fish cannot be cut. The recording was photographed using ImageView.
Administration: the young fish after tail cutting is added into 2ml of aqueous solution containing samples with different concentrations for conventional culture. After 24h, the same parameters were used to record under a microscope with ImageView.
Calculating a relative repair rate:
2. experimental results
According to fig. 2, in the experiment of repairing the tail of zebra fish, the snow cover algae oligosaccharide can significantly promote the repairing effect, and the relationship of dose-effect is shown.
The results show that the snow cover algae oligosaccharides have repairing and maintaining effects on skin barriers.
Example 6
The snow cover algae oligosaccharide obtained in example 2 was subjected to an efficacy test for relieving inflammation
1. Zebra fish skin inflammation model
Selecting 2dpf transgenic neutrophil green fluorescent strain zebra fish in six hole plates, setting a blank control group, a model group and high, medium and low concentration snow chlamydomonas oligosaccharide groups at 20 tails of each hole, adding 60 mug/mL SLS, and establishing a zebra fish skin infiltration inflammation relieving model, wherein compared with the blank control group, the quantity of neutrophil on the surface of the zebra fish skin of the model control group is obviously increased, so that the zebra fish relaxation model is successfully established. After the high, medium and low concentration of the snow cover algae oligosaccharides are treated for 24 hours, the snow cover algae oligosaccharides are photographed under a fluorescence microscope, and the inflammation relieving efficacy of the snow cover algae oligosaccharides is evaluated according to the statistical analysis result of the number of neutrophils on the surface of the skin.
2. Detecting expression level of inflammatory factors TNF-alpha, IL-1 beta, IL-8 and IL-13mRNA in cell inflammation model by qPCR technology
Cell culture: haCaT cells (purchased from chinese cell resource pool) were selected. DMEM culture medium is selected, and 10% of serum plus 1% of diabody is added in the preparation of the culture medium. Selecting cell culture bottles for culture, adding 5-6 mL of culture medium into each bottle, and carrying out passage every 2-3 days. When the cells grow to 80% -90% of the bottom surface of the culture flask, digesting with 0.25% of pancreatin, homogenizing the cells, and diluting to 10% 6 And each mL. The cell suspension was then transferred to 60mm dishes, 3mL of cell suspension was added to each dish, and the mixture was allowed to stand after homogenization. After the cells are totally settled, the cells are placed at 37 ℃ and 5 percent CO 2 Culturing in incubator for 24 hr, and adding medicine.
Experimental grouping: the experiment is divided into six groups, namely a blank control group, a model group and four sample groups, wherein the concentration of the snow chlamydomonas oligosaccharide in the sample groups is 1,2, 10 and 20mg/mL respectively. SLS was added as an inflammation inducer to both the model and sample groups.
RNA extraction: RNA extraction was performed using Trizol method. First, the 24h petri dish was removed, the medium was removed, and then washed twice with 4 ℃ pre-chilled PBS. 1mL Trizol was added to each dish, adherent cells were blown down, and then transferredThe mixture was allowed to stand in a 1.5mL centrifuge tube without enzyme for 3min. Then 380 mu L of chloroform is added, vortex mixing is carried out, and standing still is carried out for 3min. Setting a program of 4 ℃/12000g/15min by using a refrigerated centrifuge, and carefully transferring the upper liquid after completion>500 μl) into a new enzyme-free 1.5mL centrifuge tube. Adding isopropanol according to the volume of the transfer liquid at a ratio of 1:1, reversing and uniformly mixing, and then placing into a refrigerator at the temperature of minus 20 ℃ for standing for 10-15 min. And then centrifuged at 4 ℃ C./12000 g/10min, and the supernatant is discarded. 1mL of 75% ethanol (DEPC formulation) was added, the pellet was washed, post-centrifuged, 4℃12000g/5min, and the liquid was removed as dry as possible. Finally, the precipitated RNA was dried, and after the RNA was slightly dried, 20. Mu.L of DEPC water was added to dissolve the RNA. Finally, electrophoresis and OD are carried out 260 /OD 280 And (3) detecting to determine whether the RNA can be used for subsequent experiments.
Reverse transcription: the synthesis of the first strand of the cDNA was performed using a reverse transcription kit (purchased from Wohanovular). 20. Mu.L of a reaction system was prepared as follows:
| temperature (temperature) | Time |
| 25℃ | 5min |
| 42℃ | 30min |
| 85℃ | 5sec |
qPCR detection: quantitative mRNA detection was performed using qPCR detection kit (purchased from wuhansaiville), with the addition of primers. 20. Mu.L of a reaction system was prepared as follows:
based on the obtained CT value, the calculation was performed by using a 2-DeltaCT method with beta-Actain as an internal reference.
3. Detection of key proteins of cell autophagy-related pathway AMPK/mTOR by Western Blot Western blotting
Cell culture and experimental grouping were as above.
Western blotting detection: after 48h, HACAT cells were collected, washed 2 times with PBS (1000 r.min-1, 5 min), and lysed using the cell lysate kit, respectively. After the protein is quantified, 50 mug of protein is respectively added into a loading buffer solution, and the protein is denatured for 10min at 95 ℃. After 8% polyacrylamide-SDS gel electrophoresis, electrotransfer to nitrocellulose membrane, 5% skimmed milk powder is closed, and then corresponding primary antibody and secondary antibody are sequentially added, and incubated for 2h at room temperature, and washed with TBST buffer solution for 5 times and 10min each time. After adding the chemiluminescent reagent, the mixture was put into a cassette for tabletting, and then developed, fixed and imaged in sequence, and the grey value of the bands was analyzed by using Image J software.
3. Experimental results
According to fig. 3, in the zebra fish skin inflammation model, compared with a blank group, the migration number of neutrophils in the model group is remarkably increased, and the migration number of neutrophils in the group added with the snow cover algae oligosaccharides all shows a decreasing trend, and the higher the concentration of the snow cover algae oligosaccharides, the more obvious the decreasing effect, and the snow cover algae oligosaccharides have remarkable relieving effect on the SLS induced zebra fish skin inflammation.
According to FIG. 4, compared with the model group, the mRNA transcription level of the genes IL-1 beta, IL-8 and TNF-alpha of the snow cover algae oligosaccharide pro-inflammatory factors has obvious inhibition effect and has certain promotion effect on the mRNA transcription level of the genes IL-13 of the inflammation inhibitor.
According to FIG. 5, the Xuefuano oligosaccharide promotes the LC3-B, P-AMPK factor of the APMK/mTOR pathway associated with autophagy, and inhibits the p-mTOR factor, the former promotes autophagy, and the latter is the opposite.
The above results indicate that the snow cover algae oligosaccharides can relieve skin inflammation by acting on transcription of inflammatory factors and promoting autophagy.
Claims (7)
1. The preparation method of the snow cover algae oligosaccharide for regulating skin microecology is characterized in that the snow cover algae oligosaccharide is obtained from polar algae snow cover algae by a biological fermentation technology, and the preparation method comprises the following specific steps:
s1, activating and culturing strains: taking out the preserved bacillus subtilis from the inclined plane, and transferring the bacillus subtilis into a triangular flask for seed shake flask culture (LB culture medium);
s2, fermentation: when the fungus suspension OD 600 The bacterial suspension is inoculated into a fermentation culture medium containing 15-30wt% of snow cover algae powder and 2-3wt% of glucose according to the inoculum size of 10-20wt% of volume fraction when the value is 3-5. Stationary culture at 30+ -2deg.C, and fermenting to obtain fermentation liquid OD 600 Stopping fermentation at 120-140, centrifuging the fermentation broth at 8000-10000r/min for 10-15min, and collecting thallus and fermentation supernatant respectively;
s3, cell wall breaking: homogenizing and breaking the wall of the fermentation thalli to obtain a thalli wall-broken extracting solution;
s4, extracting and refining: mixing the fermentation supernatant in the step S2 with the cell wall-broken extracting solution in the step S3, evaporating to obtain a dried product, dissolving the dried product with petroleum ether, filtering, dissolving filter residues with 70-90% ethanol, filtering and evaporating to obtain the snow cover algae oligosaccharide;
the bacillus subtilis in the step S1 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.26359 and the preservation date of 2022, 12 months and 30 days.
2. The method for preparing the snow cover algae oligosaccharide for regulating skin microecology according to claim 1, wherein the seed shake flask culture in the step S1 is 1-2% in inoculum size, and the seed shake flask culture is carried out at 37±2 ℃ for 15-20h at 220-240 rpm.
3. The method for preparing the snow cover algae oligosaccharide for regulating skin microecology according to claim 1, wherein the pressure of homogenizing wall breaking in the step S3 is 80-100Mpa.
4. The method for preparing the snow cover algae oligosaccharide for regulating skin microecology according to claim 1, wherein the purity of the snow cover algae oligosaccharide in the step S4 is 52-68%.
5. The snow cover algae oligosaccharide for regulating skin microecology is characterized in that the snow cover algae oligosaccharide is prepared by the preparation method of the snow cover algae oligosaccharide according to any one of claims 1-4.
6. Use of the snow cover algae oligosaccharide for regulating skin microecology according to claims 1-5, characterized in that the snow cover algae oligosaccharide is used in functional cosmetics with skin microecology regulation, skin barrier repair and skin inflammation relief.
7. The use of a snow cover algae oligosaccharide for regulating skin microecology according to claim 6, wherein the functional cosmetic comprises a face cleansing cream, a toner, a lotion, an emulsion, a mask, a cream and an essence.
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