CN116694474A - New exosome preparation method for skin care product and application thereof - Google Patents
New exosome preparation method for skin care product and application thereof Download PDFInfo
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- CN116694474A CN116694474A CN202310413120.3A CN202310413120A CN116694474A CN 116694474 A CN116694474 A CN 116694474A CN 202310413120 A CN202310413120 A CN 202310413120A CN 116694474 A CN116694474 A CN 116694474A
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Classifications
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P37/08—Antiallergic agents
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- 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
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
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Abstract
The invention relates to a novel method for preparing exocrine for skin care products, which comprises the following steps: culturing Chlorella by high-density heterotrophic fermentation, collecting Chlorella culture supernatant, and obtaining pure Chlorella exosomes by PEG precipitation method. Compared with the exosomes of animal origin, the original chlorella exosomes provided by the invention have the advantages of low culture cost, short culture period, large yield, high yield, easy extraction, better biocompatibility and low toxicity and side effects, and do not have the problem of foreign body immunogenicity and ethical problems. Experiments prove that the original chlorella exosomes have obvious proliferation promotion and MMP1 expression reduction on HaCaT and HDF, can obviously inhibit tyrosinase activity in melanocytes and reduce skin melanin production, and can be used for preparing cosmetics or skin care products for whitening, anti-inflammatory, antiallergic, skin tightening or skin tissue repair.
Description
Technical Field
The invention relates to the technical field of biological skin care products, in particular to a novel preparation method and application of exocrine substances which can be used for skin care products.
Background
Exosomes are the most essential parts of stem cells, and are referred to as small vesicles (30-150 nm) containing complex RNAs and proteins, which nowadays are specifically disc-shaped vesicles with diameters of 40-100 nm. Exosomes are extracellular vesicles secreted by cells, the main components of which are phospholipid bilayer and carry the relevant content. The extract of the exosomes can be extracted from various body fluids, such as human blood, urine, semen and saliva, or from various cell cultures.
Exosomes are mainly derived from multivesicular bodies formed by the invagination of intracellular lysosome particles, and are released into the extracellular matrix after fusion with the cell membrane via the outer membrane of the multivesicular bodies. After the first discovery in 1983, exosomes were considered as a way for cells to excrete waste, and today a large number of studies on their biological sources, their material constitution and transport, intercellular signaling and distribution in body fluids have found that exosomes have a wide variety of functions. The function of exosomes depends on the cell type from which they are derived, and they can be involved in a variety of aspects such as immune responses in the body, antigen presentation, cell migration, cell differentiation, tumor invasion, etc. For example, tumor-derived exosomes are involved in the exchange of genetic information between tumor cells and basal cells, resulting in the generation of a large number of new blood vessels that promote tumor growth and invasion. The exosomes derived from animal cells can be used in skin care products or medicines for resisting allergy and skin aging, can effectively reduce the number of skin wrinkles, and plays an anti-aging role.
However, the exosomes derived from animals are difficult to obtain, and have high cost, so that large-scale industrial application is difficult to realize. The main reasons include: (1) The exosomes from animal sources are mainly obtained by culturing umbilical cord stem cells and separating the umbilical cord stem cells from a culture solution, the stem cells are cultured by using a relatively expensive serum product, the culture scale is generally in the milliliter level, the culture period is longer, and the yield is low; (2) The exosomes of animal origin also have the problem of alloimmunogenicity, the safety of which cannot be guaranteed, and are affected by animal species and animal cell health conditions, and the stem cell sources need to be strictly controlled; (3) Due to lack of relevant laws and specifications, stem cell technology has many ethical problems in scientific research and application links, and it is difficult to find a balance between science and technology and ethics.
At present, little research is carried out on microalgae exosomes, the existing polymer precipitation method of the chlamydomonas reinhardtii exosomes cannot obtain pure exosome vesicles with complete structures, the biological activity of the extracted products is not verified experimentally, and the method is not suitable for being used in the field of beauty and skin care.
Disclosure of Invention
First, the technical problem to be solved
In view of the above-mentioned shortcomings and disadvantages of the prior art, the invention provides a new preparation method of exosomes for skin care products and application thereof, which solves the technical problems of difficult acquisition of exosomes of animal origin, high cost, difficult guarantee of safety and incapability of realizing large-scale industrialized application. Therefore, the essence of the invention is to provide a simple and easy extraction method for obtaining pure and complete microalgae exosomes, and the application rate of exosomes in the fields of skin care products/cosmetology and the like is improved.
(II) technical scheme
In a first aspect, the present invention provides a novel method for preparing an exosome useful for skin care products comprising: culturing Chlorella by high-density heterotrophic fermentation, collecting Chlorella culture supernatant, and obtaining pure Chlorella exosomes by PEG precipitation method.
According to a preferred embodiment of the present invention, the exosome acquisition method includes:
s1, culturing Chlorella
The culture of chlorella comprises shake flask culture and fermentation tank culture; the chlorella is original chlorella Auxenochlorella protothecoides 0710; the method for culturing the fermentation tank comprises the following steps: initial fermentation conditions: the temperature is 28+/-0.5 ℃, the pH=6.0-6.3, the dissolved oxygen is 100 percent, and the stirring speed is 200-400rpm;
during the culture process: feeding concentrated glucose and yeast extract in batches, controlling the glucose concentration in a fermentation tank to be 8-25g/L, and regulating the pH value to be 6.3-6.5 by NaOH; the dissolved oxygen is kept to be more than or equal to 20% by adjusting the stirring speed and ventilation;
s2, preparing exosome coarse body liquid
When the concentration of residual glucose in the fermentation tank is lower than 5g/L, precipitating algae cells by using a centrifugal force of 4500g-5000g for 1.5-3min, reserving supernatant, transferring to a new centrifuge tube, precipitating large fragments in the supernatant by using a centrifugal force of 4500g-5000g for 25-35min, and transferring the supernatant to a new container to obtain crude exosome body fluid;
s3, obtaining a pure exosome by a PEG precipitation method, which specifically comprises the following steps:
s31, filtering the exosome crude body liquid through a microfiltration membrane to obtain filtrate;
s32, preparing PEG and water into aqueous solution of PEG with concentration of 18-24wt%, wherein the weight average molecular weight of PEG is 2000-8000;
s33, uniformly mixing the aqueous solution of PEG and the filtrate according to the volume ratio of 1:1, and standing at low temperature for overnight storage for precipitation; taking out after 24 hours, and centrifuging for 50-80 minutes under the centrifugal force of 8000-12000 g to obtain a sediment product and a supernatant;
s34, re-suspending the sediment product of the centrifuge tube according to the proportion that 1.5-2mL of PBS is added into each 100mL of sediment product generated by the supernatant, so as to obtain exosomes.
According to a preferred embodiment of the present invention, in S2, the specific method is as follows: when the concentration of residual glucose in the fermentation tank is lower than 5g/L, centrifuging for 2min at 5000g to precipitate algae cells, transferring supernatant to a new centrifuge tube, and preserving at-80 ℃ in a refrigerator for standby; taking out the supernatant from the refrigerator at-80 ℃ one day before extraction, and thawing overnight in the refrigerator at 4 ℃; taking out completely thawed chlorella supernatant, centrifuging at 4deg.C and 5000g for 30min, discarding the lower precipitate, and transferring to a new container to obtain the crude extract of exosome.
According to a preferred embodiment of the present invention, the microfiltration membrane in S31 has a filtration pore of 0.22 μm;
the mass concentration of PBS in S32 is 19.3%, and the weight average molecular weight of PEG is 6000;
the centrifugation conditions in S33 were 4℃and 10000g of centrifugal force was applied for 1 hour.
According to a preferred embodiment of the present invention, in S1, the shake flask culture comprises:
s11, preparing a heterotrophic liquid culture medium:
preparing KH-containing liquid 2 PO 4 0.7-7g/L，K 2 HPO 4 0.3-3g/L，MgSO 4 .7H 2 O 0.3-2g/L，FeSO 4 .7H 2 0.003-0.02g/L of O, 1-20 g/L of vitamin B, 30-60g/L of glucose, 1-2.5g/L of yeast extract, 2-10g/L of glycine and 0.8-1.2mL/L of A5 trace element mother liquor, and sterilizing the aqueous solution at high temperature and high pressure to obtain the heterotrophic liquid culture medium;
the formula of the A5 microelement mother solution is as follows: h 3 BO 3 286mg/L,MnSO 4 ·7H 2 O 250mg/L,ZnSO 4 ·7H 2 O 22.2mg/L,CuSO 4 ·5H 2 O 7.9mg/L,Na 2 MoO 4 ·2H2O 2.1mg/L；
S12, shake flask culture
Absorbing a certain amount of cells of Chlorella primordialis Auxenochlorella protothecoides0710, adding into the heterotrophic liquid culture medium, shaking in the dark at 100-200rpm to 2-3×10 6 Individual algal cells/mL; continuously shaking culture in a dark constant-temperature shaking table at 28 ℃ and 180-250rpm until reaching a stationary growth phase; inoculating the shake-flask cultured algae liquid into a fermentation tank.
In addition, the CCK8 proliferation experiment proves that the exosome of the chlorella prototheca Auxenochlorella protothecoides0710 has the effects of promoting the expression of collagen fiber related genes in HDF, promoting the expression of the closely connected protein related genes in HaCaT and inhibiting the tyrosinase activity in melanocyte B16, and shows proliferation activity on human immortalized epidermal cells (HaCaT) and human skin fibroblasts (HDF) and inhibiting melanin production, so that the cosmetic or skin care product with the functions of whitening, anti-inflammatory, antiallergic, skin tightening or skin tissue repairing can be prepared.
(III) beneficial effects
Compared with the exosome of animal sources, the exosome of the original chlorella Auxenochlorella protothecoides0710 provided by the invention has the following advantages:
(1) The culture cost is low: unlike the expensive serum product for animal cell culture, the main culture medium components of the chlorella are glucose and inorganic salt during fermentation culture, the formula is simple, the price is low, and the culture cost in the production process is obviously reduced.
The culture period is shorter, the yield is large, and the yield is high: the method comprises the steps of carrying out fermentation culture on chlorella by using a fermentation tank, wherein the culture of the fermentation tank is generally 50L-10000L, a large amount of algae cells can be obtained rapidly, the culture density of the fermentation culture chlorella can reach 270g/L at most according to the prior art, and the cell culture density is far higher than that of animal cells; the culture period of the chlorella is as short as 5-10 days, which is far less than the time cost required by animal cell culture. Because the growth speed of the chlorella is high, the growth period is short, the culture of the chlorella can use light energy to fix carbon dioxide as a carbon source, or inorganic salt and saccharides with lower price are adopted, fermentation can reach several tons, the extraction of exosomes by using the microalgae is more beneficial to subsequent industrialized mass production, the maximum culture density of cells is estimated to be 100g/L according to the volume of a 50L fermentation tank and the culture time of about 10 days, and the cost of per 1mg of the chlorella exosomes obtained is less than 0.1 percent of the cost of the amniotic stem cell exosomes, and the cost is obviously different. Therefore, compared with the traditional animal umbilical cord stem cell culture method, the method for preparing the exosomes has quite optimistic industrial application prospect.
(2) Biological means are readily available to obtain more exosomes. The chlorella has better tolerance and stress resistance than animal cells, is easy to carry out genetic modification, pressure stress induction or stimulation and the like, so as to promote the chlorella to generate more exosomes, and achieves the purposes of improving the yield, reducing the cost and expanding the industrialized application on the premise that the obtained exosomes and animal cell-derived exosomes have equivalent skin care effects.
(3) The chlorella source exosomes are easy to extract, and the exosome vesicle structure with clear edges and complete structures can be obtained. The method adopts the PEG precipitation method to extract the chlorella exosomes, and extracts the chlorella exosomes through the steps of filtering, precipitating, centrifuging and the like, so that complicated instruments and equipment are not needed, and the process cost is low. The exosomes from animal cells are usually only sucrose or iodixanol density gradient centrifugation, and the exosomes from animal cells are very small in volume, only a few milliliters to tens milliliters, difficult to apply in industry and cannot be extracted in large scale from a few liters to tens of milliliters of cell culture fluid because the culture medium plasma serum contains more lipoproteins and PEG coprecipitation is easy to precipitate lipoproteins and other substances of human beings and has low purity.
(4) Unlike animal cell-derived exosomes, microalgae-derived exosomes are based on plant active substances, contain abundant contents, have the advantage of greenization, and thus have better biocompatibility and low toxicity. The exosome provided by the invention has no problem of alloimmunogenicity and high safety; there is no ethical problem.
(5) The invention uses the verified exosomes of the original chlorella Auxenochlorella protothecoides0710 to have proliferation promoting effect on HaCaT and HDF, and can improve the expression of the HDF collagen fiber related genes and HaCaT tight junction protein related genes. In addition, the chlorella exosomes can inhibit tyrosinase activity in melanocyte B16, reduce skin melanin production, and can be used for scar wound repair and whitening.
In conclusion, the preparation method of the chlorella exosome is simple, the preparation process is nontoxic and harmless, the process parameters are easy to control, the cost is low, exosome components are easy to be absorbed by cells, and the effects of whitening, resisting aging, resisting inflammation, moisturizing and tendering skin, resisting oxidation, repairing after sun, reducing spots, resisting inflammation, resisting allergy, relieving, tightening and the like are achieved by testing the effects of the chlorella exosome on skin care, so that the problems of whitening, skin wrinkles, preventing skin aging and the like are solved, the price of exosome skin care products is reduced, and people with beautiful appearance can consume the exosome skin care products.
Drawings
FIG. 1 is a transmission electron microscope image of chlorella exosomes.
FIG. 2 is a graph showing the effect of chlorella exosomes on proliferation of HDF cells.
FIG. 3 is a graph showing the effect of chlorella exosomes on HaCaT cell proliferation.
FIG. 4 shows a graph of HaCaT tight junction protein gene expression by Chlorella exosomes.
FIG. 5 is a graph showing the expression of HDF collagen fiber-related genes by chlorella exosomes.
FIG. 6 is a graph showing the effect of chlorella exosomes on tyrosinase activity in B16.
Detailed Description
The invention will be better explained by the following detailed description of the embodiments with reference to the drawings.
Example 1
The embodiment is a preparation method of chlorella exosomes, which comprises the following steps:
(1) Preparing chlorella heterotrophic culture solution containing KH 2 PO 4 5g/L，K 2 HPO 4 2g/L，MgSO 4 .7H 2 O 1.0g/L，Fe SO 4 .7H 2 O0.01 g/L, vitamin B1 g/L, glucose 50g/L, yeast extract 2g/L, glycine 6g/L, and A5 trace element mother liquor 1mL/L.
A5 microelement mother liquor formula is H3BO3 286mg/L, mnSO4.7H2O 250mg/L, znSO 4 ·7H 2 O 22.2mg/L,CuSO 4 ·5H 2 O 7.9mg/L,Na 2 MoO 4 ·2H 2 O2.1 mg/L. The heterotrophic original chlorella culture medium can be obtained after high-temperature high-pressure sterilization.
(2) A certain amount of cells of Chlorella primordialis Auxenochlorella protothecoides0710 are sucked, added into a certain volume of liquid culture medium, shake-cultured in the dark at 100-200rpm, and finally each milliliter of culture medium contains 2-3×106 algae cells. The cultures were placed in a dark thermostated shaker at 28℃and 180-250rpm, until a stationary growth phase was reached.
(3) Fermentation tank culture of chlorella
Fermenting and culturing the strain with 5L fermenter (GBJS-5L-AUTOBIO, zhenjiang eastern, china). The initial fermentation conditions are as follows: the temperature was 28.+ -. 0.5 ℃, the pH was 6.3, the oxygen solubility (pO 2) was 100%, and the stirring speed was 300rpm. Concentrated glucose and yeast extract were fed in batches and pH was adjusted to 6.5 with NaOH. In the fermentation process, the concentration of sucrose is controlled to be 8-25g/L by manual feeding, and other parameters are automatically controlled. The dissolved oxygen is maintained above 20% by adjusting stirring speed and ventilation.
(4) Preparation of crude exosome extract
When the residual glucose concentration was below 5g/L, algae were collected. Centrifuging the algae liquid with 5000g for 2min to precipitate algae cells, transferring supernatant to a new centrifuge tube, and storing in a refrigerator at-80deg.C for use.
Taking out the supernatant frozen into ice from the refrigerator at-80 ℃ in the day before extraction, and thawing overnight in the refrigerator at 4 ℃ for standby; taking out completely melted chlorella supernatant, and sub-packaging into a 100mL centrifuge tube (the liquid volume of the 100mL centrifuge tube is not more than 80 mL); centrifuging at 4deg.C for 30min with 5000g centrifugal force, and removing large pieces from supernatant; transferring the upper layer liquid into a new container after centrifugation, discarding the lower layer precipitate, and obtaining the exosome crude extract.
(5) PEG precipitation to obtain pure exosome component
Filtering the crude extract of exosome with 0.22um filter membrane to obtain filtrate. A PEG6000 solution (24 g PEG6000 for 100mL water) prepared in advance was mixed with the filtrate in a volume ratio of 1:1, mixing uniformly, starting to precipitate once, and standing in a refrigerator at 4 ℃ for overnight precipitation. After 24h, the suspension was removed and the floc was suspended in solution and centrifuged at 10000g for 1h at 4 ℃; the supernatant was discarded. The resulting product was resuspended in PBS at a rate of 100mL of supernatant plus 2mL, and pooled in a 1.5mL EP tube, and submitted for sample preparation (temporary failure to send samples frozen at-80 ℃) and the remainder frozen at-80 ℃ (exact volume indicated on EP tube).
(6) And (3) taking PBS heavy suspension, detecting the particle size of the chlorella exosomes by using a nano Coulter particle sizer, and calculating the content of the original supernatant particles to be 2.75E11pcs/mL according to the result, wherein the average particle size is 69nm.
(7) The extracted original chlorella exosome forms are observed by using a transmission electron microscope, so that exosome vesicle structures can be observed, the edges are clear, and the structures are complete (see figure 1).
Example 2
In this example, the biological activity of chlorella exosomes at the cell level was verified by the following experimental method:
(1) Effect of chlorella exosomes on HDF proliferation: HDF was inoculated into 96-well plates at 3000/well overnight for 5 columns of plates with DMEM (high sugar medium) containing 10% fbs (fetal bovine serum). After overnight adherence, the original medium was discarded, the first column was added with fresh medium as control and the second column was added with a concentration of 7.336 x 10 9 The third column of the chlorella exosomes with the concentration of 3.668 x 10 is added in the pcs/mL 10 The concentration of the chlorella exosomes added in the fourth column is 1.834 x 10 11 The concentration of the chlorella exosomes added in the fifth column is 9.17 x 10 11 The ccs/mL chlorella exosomes were detected 48h later for CCK 8. As is evident from fig. 2, the addition of chlorella exosomes significantly promoted the proliferation of HDF compared to the control group.
(2) Effect of chlorella exosomes on HaCaT proliferation: haCaT was inoculated at 5000/well into 96-well plates for overnight culture, 5 columns of plates, and DMEM (high sugar medium) containing 10% fbs (fetal bovine serum) was used as the medium. After overnight adherence, the original medium was discarded, the first column was added with fresh medium as control and the second column was added with a concentration of 5.5×10 9 The third column of the chlorella exosomes with the concentration of 5.5 x 10 is added 10 The concentration of the chlorella exosomes added in the fourth column is 1.1 x 10 11 The concentration of the chlorella exosomes of pcs/mL was 2.75 x 10 for the fifth column addition 11 The ccs/mL chlorella exosomes were detected 48h later for CCK 8. As is evident from FIG. 3, the addition of Chlorella exosomes significantly promoted the proliferation of HaCaT compared to the control group.
(3) Expression of HDF collagen fiber-related genes by chlorella exosomes: HDF was performed according to 10000/cm 2 Inoculated into 6-well plates overnight, plated 5-well, and culture medium was DMEM (high sugar medium) containing 10% fbs (fetal bovine serum). After overnight adherence, the original medium was discarded, fresh medium was added to the first well as a control and the second well was added at a concentration of 7.336 x 10 9 The PCs/mL chlorella exosomes were added to the third well at a concentration of 3.668 x 10 10 The concentration of the chlorella exosomes added to the fourth hole is 1.834 x 10 11 PCs/mL chlorella exosomes were added to the fifth well at a concentration of 9.17 x 10 11 And (3) performing RNA extraction, inversion and qpcr detection on the chlorella exosomes of pcs/mL after 48 hours. As is evident from FIG. 4, the addition of Chlorella exosomes significantly increased the expression of Claudin1, claudin5 and Claudin6 genes compared to the control group.
(4) Expression of closely related genes of the protein in HaCaT by Chlorella exosomes: haCaT was measured at 15000/cm 2 Inoculated into 6-well plates overnight, plated 5-well, and culture medium was DMEM (high sugar medium) containing 10% fbs (fetal bovine serum). After overnight adherence, the original medium was discarded, fresh medium was added to the first well as a control and the second well was added at a concentration of 5.5×10 9 The PCs/mL chlorella exosomes were added to a third well at a concentration of 5.5 x 10 10 Chlorella exocrine of pcs/mLThe body, fourth well, was added at a concentration of 1.1 x 10 11 The concentration of the chlorella exosomes of pcs/mL, the fifth hole is added and used at 2.75 x 10 11 And (3) performing RNA extraction, inversion and qpcr detection on the chlorella exosomes of pcs/mL after 48 hours. As is evident from fig. 5, compared with the control group, the addition of chlorella exosomes significantly increased the expression of COLA1, COLA2, FN1, ELN genes and decreased the expression of MMP1 (MMP 1 inhibits collagen synthesis), promoting collagen synthesis.
(5) Effect of chlorella exosomes on tyrosinase activity in melanoma cells B16: melanoma cell B16 was prepared at 15000/cm 2 Inoculated into 75 bottles overnight for culture, and five bottles of DMEM (high sugar medium) containing 10% FBS (fetal bovine serum) were used as the culture medium. After overnight adherence, the original medium was discarded, the first flask was added with fresh medium as control and the second flask was added at a concentration of 5.5×10 9 The third bottle is added with chlorella exosomes with the concentration of 5.5 x 10 per mL 10 The PCs/mL chlorella exosomes are added into a fourth bottle with the concentration of 1.1 x 10 11 The concentration of the chlorella exosomes of pcs/mL was 2.75 x 10 in fifth bottle 11 After 48 hours, digesting cells of the chlorella exosomes of pcs/mL, adding 1mL of extracting solution into 500 ten thousand melanocytes B16 in each bottle, performing ultrasonic crushing under the condition of 200W, opening 3s and closing 10s in each period, repeating for 30 times, centrifuging for 20 minutes at the temperature of 12000g at the temperature of 4 ℃, taking the supernatant, detecting by using a 475nm spectrophotometer, and converting to obtain tyrosinase activity data. As is evident from FIG. 6, the addition of Chlorella exosomes significantly reduced tyrosinase activity compared to the control group.
In conclusion, the chlorella exosomes provided by the invention have the characteristics of obviously promoting the proliferation of HDF and HaCaT, improving the expression of Claudin1, claudin5 and Claudin6 genes, improving the expression of COLA1, COLA2, FN1 and ELN genes, reducing the expression of MMP1 and inhibiting the tyrosinase activity in melanoma cells B16, and in addition, the experimental process also shows that the chlorella exosomes provided by the invention do not reduce the number and growth rate of living cells, which indicates that the exosomes have safety, so the chlorella exosomes can be used for preparing cosmetics or skin care products with the functions of whitening, anti-inflammatory, antiallergic, skin tightening or skin tissue repairing.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (9)
1. A novel method for preparing exosomes useful in skin care products, characterized in that it comprises: culturing Chlorella by high-density heterotrophic fermentation, collecting Chlorella culture supernatant, and obtaining pure Chlorella exosomes by PEG precipitation method.
2. A novel method for preparing exosomes useful in skin care products according to claim 1, characterized in that the exosome acquisition method comprises:
s1, culturing Chlorella
The culture of chlorella comprises shake flask culture and fermentation tank culture; the chlorella is original chlorella Auxenochlorella protothecoides 0710; the method for culturing the fermentation tank comprises the following steps: initial fermentation conditions: the temperature is 28+/-0.5 ℃, the pH=6.0-6.3, the dissolved oxygen is 100 percent, and the stirring speed is 200-400rpm;
during the culture process: feeding concentrated glucose and yeast extract in batches, controlling the glucose concentration in a fermentation tank to be 8-25g/L, and regulating the pH value to be 6.3-6.5 by NaOH; the dissolved oxygen is kept to be more than or equal to 20% by adjusting the stirring speed and ventilation;
s2, preparing exosome coarse body liquid
When the concentration of residual glucose in the fermentation tank is lower than 5g/L, precipitating algae cells by using a centrifugal force of 4500g-5000g for 1.5-3min, reserving supernatant, transferring to a new centrifuge tube, precipitating large fragments in the supernatant by using a centrifugal force of 4500g-5000g for 25-35min, and transferring the supernatant to a new container to obtain crude exosome body fluid;
s3, obtaining a pure exosome by a PEG precipitation method, which specifically comprises the following steps:
s31, filtering the exosome crude body liquid through a microfiltration membrane to obtain filtrate;
s32, preparing PEG and water into aqueous solution of PEG with concentration of 18-24wt%, wherein the weight average molecular weight of PEG is 2000-8000;
s33, uniformly mixing the aqueous solution of PEG and the filtrate according to the volume ratio of 1:1, and standing at low temperature for overnight storage for precipitation; taking out after 24 hours, and centrifuging for 50-80 minutes under the centrifugal force of 8000-12000 g to obtain a sediment product and a supernatant;
s34, re-suspending the sediment product of the centrifuge tube according to the proportion that 1.5-2mL of PBS is added into each 100mL of sediment product generated by the supernatant, so as to obtain exosomes.
3. The method for preparing the exosome for skin care products according to claim 2, wherein in S2, the specific method is as follows: when the concentration of residual glucose in the fermentation tank is lower than 5g/L, centrifuging for 2min at 5000g to precipitate algae cells, transferring supernatant to a new centrifuge tube, and preserving at-80 ℃ in a refrigerator for standby; taking out the supernatant from the refrigerator at-80 ℃ one day before extraction, and thawing overnight in the refrigerator at 4 ℃; taking out completely thawed chlorella supernatant, centrifuging at 4deg.C and 5000g for 30min, discarding the lower precipitate, and transferring to a new container to obtain the crude extract of exosome.
4. A novel method for preparing an exosome useful for skin care products according to claim 2 wherein the microfiltration membrane in S31 has a filtration pore of 0.22 μm;
the mass concentration of PBS in S32 is 19.3%, and the weight average molecular weight of PEG is 6000;
the centrifugation conditions in S33 were 4℃and 10000g of centrifugal force was applied for 1 hour.
5. A novel process for the preparation of exosomes useful in skin care products according to claim 2, characterized in that in S1 the shake flask culture comprises:
s11, preparing a heterotrophic liquid culture medium:
preparing KH-containing liquid 2 PO 4 0.7-7g/L，K 2 HPO 4 0.3-3g/L，MgSO 4 .7H 2 O 0.3-2g/L，FeSO 4 .7H 2 0.003-0.02g/L of O, 1-20 g/L of vitamin B, 30-60g/L of glucose, 1-2.5g/L of yeast extract, 2-10g/L of glycine and 0.8-1.2mL/L of A5 trace element mother liquor, and sterilizing the aqueous solution at high temperature and high pressure to obtain the heterotrophic liquid culture medium;
the formula of the A5 microelement mother solution is as follows: h 3 BO 3 286mg/L,MnSO 4 ·7H 2 O 250mg/L,ZnSO 4 ·7H 2 O 22.2mg/L,CuSO 4 ·5H 2 O 7.9mg/L,Na 2 MoO 4 ·2H2O 2.1mg/L；
S12, shake flask culture
Absorbing a certain amount of cells of Chlorella primordialis Auxenochlorella protothecoides0710, adding into the heterotrophic liquid culture medium, shaking in the dark at 100-200rpm to 2-3×10 6 Individual algal cells/mL; continuously shaking culture in a dark constant-temperature shaking table at 28 ℃ and 180-250rpm until reaching a stationary growth phase; inoculating the shake-flask cultured algae liquid into a fermentation tank.
6. Use of a novel exosome according to any one of claims 1 to 5 for the preparation of skin care products, prepared by a process for the preparation of exosomes.
7. Application of exosomes of Chlorella primordialis Auxenochlorella protothecoides0710 in preparing skin care product.
8. Use of an exosome of chlorella primordialis Auxenochlorella protothecoides0710 in the preparation of a cosmetic or skin care product for promoting expression of collagen fiber related genes in HDF, promoting expression of claudin related genes in HaCaT, and inhibiting tyrosinase activity in melanocyte B16.
9. The application of the exosomes of the chlorella primordialis Auxenochlorella protothecoides0710 in preparing cosmetics or skin care products for whitening, anti-inflammatory, antiallergic, skin tightening or skin tissue repair.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117243885A (en) * | 2023-11-15 | 2023-12-19 | 北京青藤谷禧干细胞科技研究院有限公司 | Stem cell exosome composition for improving skin and preparation method thereof |
| CN117243885B (en) * | 2023-11-15 | 2024-01-26 | 北京青藤谷禧干细胞科技研究院有限公司 | Stem cell exosome composition for improving skin and preparation method thereof |
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