CN117243885A - Stem cell exosome composition for improving skin and preparation method thereof - Google Patents
Stem cell exosome composition for improving skin and preparation method thereof Download PDFInfo
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Abstract
The invention provides a stem cell exosome composition for improving skin and a preparation method thereof, belonging to the technical field of cosmetics. The pig adipose mesenchymal stem cells are subjected to in vitro culture and passage, centrifugation to obtain adipose stem cell exosomes, the rest culture solution is reserved, oat, nostoc sphaeroids, medlar and snail stock solution are mixed and subjected to enzymolysis to extract alcohol precipitation to obtain active polysaccharide, the rest of the active polysaccharide is mixed and fermented with the rest culture solution, zn and Cu are chelated, zymocyte is subjected to lysozyme enzymolysis to obtain a two-split yeast cell lysate, and the two-split yeast cell lysate, the fermentation product of the chelated Zn/Cu, acetyl dipeptide-1 cetyl ester, palmitoyl tripeptide-5 and glutathione are mixed and added into temperature-sensitive hydrogel containing the active polysaccharide to obtain the skin stem cell exosome composition.
Description
Technical Field
The invention relates to the technical field of cosmetics, in particular to a stem cell exosome composition for improving skin and a preparation method thereof.
Background
Skin aging is an important subject in the research of skin care and skin care cosmetics at present, and skin aging not only can seriously affect the beauty, but also can cause specific symptoms such as dryness, roughness, telangiectasia, elasticity reduction, irregular pigmentation, seborrheic keratosis and the like. According to the action mechanism, skin aging is mainly divided into two major types of endogenous aging and exogenous aging, wherein the endogenous aging is a physiological process influenced by genes, hormones and the like, and the exogenous aging mainly refers to aging of skin caused by exogenous physical and chemical stimulation, and ultraviolet rays are important factors for causing skin aging.
Ultraviolet rays can be divided into 3 wave bands according to the wavelength, namely long-wave ultraviolet UVA (320-400 nm), medium-wave ultraviolet UVB (275-320 nm) and short-wave ultraviolet UVC (230-275 nm), due to the blocking effect of the atmosphere, the UVC is almost fully absorbed by an ozone layer, ultraviolet rays which are incident on the surface of the earth are mainly UVA and UVB, and researches show that the damage of the UVB radiation with the same dose to the skin is 800-1000 times greater than the UVA, and the damage to the skin is mainly UVB. When skin is irradiated by excessive ultraviolet rays, skin tissue cells can generate oxidative stress to generate excessive Reactive Oxygen Species (ROS), the ROS can damage the integrity of cell membranes and an oxidation resistance system in cells, cell inflammation, apoptosis and tumor formation are further caused, meanwhile, excessive ultraviolet rays can degrade skin collagen, hyaluronic acid and elastin, so that skin elasticity becomes low, rough and wrinkles are formed, and if ultraviolet rays are irradiated for a long time, excessive sedimentation of melanin is possibly caused, and skin canceration can be induced by serious people.
Antioxidant stress is an important way to protect skin from ultraviolet injury, various antioxidant means are provided for researchers, such as the researchers in China currently propose that natural plant components such as raspberry (raspberry) extract, aloe extract, ginsenoside, methyl cardiolipin and the like are used for protecting the skin from the ultraviolet injury, and the researchers also propose that active peptides are used for protecting the skin from the ultraviolet injury, such as Chinese patent application CN112574282B, CN108703287B, CN107955062B, CN109415422B and the like, but the ultraviolet protection effect of the active peptides is still to be enhanced, and especially how to improve the efficacy of resisting the antioxidant stress, promote the repair process after injury, improve the collagen secretion of the skin and enable the skin to recover the elasticity and luster rapidly.
Exosomes (exosomes) are vesicle-like substances secreted by cells having a diameter of 40-150nm and a density in the range of 1.09-1.18g/ml with a bilayer phospholipid membrane structure. Exosomes such as neuronal cells, mesenchymal stem cells, fibroblasts, endothelial cells, megakaryocytes, etc. have been extracted from cell culture supernatants of various species, types, etc. Exosomes are also found in various body fluids of the human body, such as blood, saliva, urine, milk, etc. miRNA carried by exosomes can partially regulate the anti-aging effect of htNSC and play a corresponding role after htNSC enters cerebrospinal fluid.
Chinese patent No. 111821251B discloses a cosmetic composition and cosmetic, which comprises CD200 modified mesenchymal stem cell exosome as main ingredient, and can increase skin cell activity, promote skin cell metabolism, promote skin repair and delay skin aging after being applied to skin surface; chinese patent CN110540956B claims a simple method for preparing cytokines from placental mesenchymal stem cells, optimizes the preparation method of cytokines derived from placental mesenchymal stem cells, can prepare cytokines on a large scale, and promotes repair after skin injury and skin care. However, the stem cell product has complex components, the preparation process needs to be strictly controlled, and the stability of the skin care effect of the stem cell or the derivative thereof is poor, so that the application of the stem cell product in skin care cosmetics is limited to a certain extent.
Disclosure of Invention
The invention aims to provide a stem cell exosome composition for improving skin and a preparation method thereof, which have the effects of biocompatibility, low toxicity, low immunogenicity, good effect of absorbing barrier penetrating skin tissues by dermal cells, promoting keratinocytes, promoting migration, proliferation and secretion of collagen of fibroblasts, good effects of resisting oxidation, inflammation and skin photoaging, improving skin roughness, reducing melanin content, improving skin brightness, luster, elasticity, wrinkle resistance and the like, and have wide application prospects.
The technical scheme of the invention is realized as follows:
the invention provides a preparation method of a stem cell exosome composition for improving skin, which comprises the steps of carrying out in vitro culture and passage on pig adipose mesenchymal stem cells, centrifuging to obtain a adipose stem cell exosome, reserving the rest culture solution, mixing oat, nostoc sphaeroids kutz, medlar and snail stock solution, carrying out enzymolysis and extraction on alcohol precipitation to obtain active polysaccharide, mixing the rest culture solution with the rest culture solution, inoculating bifidobacterium longum and bifidobacterium breve, fermenting the obtained fermentation product to chelate Zn and Cu ions, carrying out enzymolysis on the fermentation product by lysozyme to obtain a bifidus yeast lysate, mixing the bifidus yeast lysate with the adipose stem cell exosome, the fermentation product to chelate Zn/Cu, acetyl dipeptide-1 cetyl ester, palmitoyl tripeptide-5 and glutathione, adding the mixture into a temperature sensitive hydrogel containing the active polysaccharide, and uniformly stirring and mixing to obtain the stem cell exosome composition for improving skin.
As a further improvement of the invention, the method comprises the following steps:
s1, extracting fat stem cell exosomes: re-suspending cells of the pig adipose-derived mesenchymal stem cells by using alpha-MEM complete culture solution, performing in-vitro culture, flushing the cells by using PBS (phosphate buffer solution) when the cell density is 70-80%, reserving the rest culture solution, inoculating the culture solution into a serum-free culture medium for culturing for 36-60h, collecting the cells, reserving the rest culture solution, and combining the rest culture solution with the previous culture solution to prepare the adipose-derived stem cell-free culture solution; washing cells with PBS solution, adding trypsin for digestion, centrifuging the culture medium at 270-320 Xg for 10-15min,1800-2200 Xg for 5-10min,8000-12000 Xg for 25-35min, and removing cells and cell fragments; continuing to centrifuge for 60-80min at 98000-102000 Xg, re-suspending the precipitate at the bottom, washing with PBS solution, and centrifuging for 60-80min at 98000-102000 Xg to remove protein polluted in exosomes, thereby obtaining fat stem cell exosomes;
S2, preparing active polysaccharide: cleaning herba Avenae Fatuae, nostoc sphaeroids kutz and fructus Lycii respectively, drying, pulverizing, adding snail stock solution, stirring, mixing, adding into water, adding complex enzyme, heating for enzymolysis, extracting, filtering, and collecting residue; adding ethanol into the filtrate for precipitation, filtering to obtain active polysaccharide, lyophilizing the filtrate, and mixing with the residue to obtain an extract mixture;
s3, activating zymophyte: inoculating bifidobacterium longum and bifidobacterium breve into a Gao's medium respectively, and performing activation culture to obtain bifidobacterium longum and bifidobacterium breve seed liquid;
s4, preparing a culture medium: uniformly mixing the fat stem cell-free culture solution in the step S1, the extraction mixture in the step S2 and water, and sterilizing to obtain a culture medium;
s5, fermenting: inoculating the seed solution of the bifidobacterium longum and the bifidobacterium breve prepared in the step S3 into the culture medium prepared in the step S4, fermenting and culturing under the anoxic condition, filtering, washing the solid with sterile water, collecting fermentation bacteria, and concentrating the obtained fermentation bacteria solution; lyophilizing the filtrate to obtain fermentation product;
s6, metal ion chelate: adding the fermentation product obtained in the step S5 into water, adding soluble copper salt and soluble zinc salt, stirring and mixing uniformly, dialyzing, and freeze-drying the dialyzate to obtain a Zn/Cu chelated fermentation product;
S7, preparing a yeast cell lysate: adding lysozyme into the fermentation broth prepared in the step S5 for enzymolysis, inactivating enzyme, centrifuging, collecting supernatant, and freeze-drying to obtain a two-split yeast lysate;
s8, preparing an additive: uniformly mixing acetyl dipeptide-1 cetyl ester, palmitoyl tripeptide-5 and glutathione to prepare an additive;
s9, preparing temperature-sensitive hydrogel: dissolving the active polysaccharide and sodium hyaluronate prepared in the step S2 in water to obtain a mixed sugar solution; dissolving hydroxypropyl methylcellulose in water, adding mixed sugar solution, stirring and mixing uniformly, adding glycerol, stirring and mixing uniformly, and obtaining temperature-sensitive hydrogel;
s10, preparation of a composition for improving the stem cell exosome of skin: uniformly mixing the fat stem cell exosome prepared in the step S1, the Zn/Cu chelated fermentation product prepared in the step S6, the yeast cell lysate prepared in the step S7 and the additive prepared in the step S8, adding the mixture into the temperature-sensitive hydrogel prepared in the step S9, and uniformly stirring and mixing the mixture to prepare the skin-improving stem cell exosome composition.
As a further improvement of the invention, the method of adding trypsin for digestion in the step S1 is to add 0.2-0.3wt% of trypsin for digestion for 3-5min at 36-38 ℃; in the step S2, the mass ratio of oat, nostoc sphaeroids kutz, medlar and snail stock solution is 5-7:10-15:3-5:7-10, the mass ratio of the compound enzyme is a mixture of cellulase and pectase, the mass ratio is 5-7:1-2, the temperature of heating enzymolysis extraction is 45-50 ℃ for 2-4h, the concentration of ethanol added to the system ethanol is 70-80wt%, and the precipitation time is 3-5h.
As a further improvement of the invention, the condition of the activation culture in the step S3 is 36-38 ℃,70-100r/min, the activation time is 24-36h, and the bacterial seed liquid contains 10 percent of bacteria 8 -10 9 cfu/mL; the mass ratio of the fat stem cell-free culture solution to the extraction mixture to the water in the step S4 is 20-25:7-10:100.
As a further improvement of the invention, the seed liquid of the bifidobacterium longum and the bifidobacterium breve in the step S5 has the inoculation amount of 2-4v/v percent and 1-3v/v percent respectively, the condition of the fermentation culture is 36-38 ℃, the fermentation culture time is 70-100r/min, the fermentation culture time is 48-72h, and the bacterial content of the fermentation bacterial liquid is 10 10 -10 11 cfu/mL; the mass ratio of the fermentation product, the soluble copper salt and the soluble zinc salt in the step S6 is 15-20:0.7-1.2:0.5-1, wherein the soluble copper salt is selected from at least one of copper chloride, copper sulfate and copper nitrate, the soluble zinc salt is selected from at least one of zinc chloride, zinc sulfate and zinc nitrate, the aperture of the dialysis bag for dialysis is 1-1.5kDa, and the dialysis time is 3-5h.
As a further improvement of the invention, in the step S7, the mass ratio of the fermentation broth to the lysozyme is 1000:3-5, the enzymolysis temperature is 40-45 ℃ and the time is 2-4 hours, and the centrifugation condition is 5000-7000r/min for 10-15min; the mass ratio of the acetyl dipeptide-1 cetyl ester, the palmitoyl tripeptide-5 and the glutathione in the step S8 is 3-5:1-3:2-4.
As a further improvement of the invention, the mass ratio of the active polysaccharide, the sodium hyaluronate, the hydroxypropyl methylcellulose and the glycerol in the step S9 is 5-7:7-10:6-8:2-4; in the step S10, the mass ratio of the fat stem cell exosomes to the fermentation products of chelating Zn/Cu to the yeast cell lysate to the additives to the temperature-sensitive hydrogel is 1-3:3-5:2-4:0.5-1:15-20.
As a further improvement of the invention, the method specifically comprises the following steps:
s1, extracting fat stem cell exosomes: re-suspending cells of the pig adipose-derived mesenchymal stem cells by using alpha-MEM complete culture solution, performing in-vitro culture, flushing the cells by using PBS (phosphate buffer solution) when the cell density is 70-80%, reserving the rest culture solution, inoculating the culture solution into a serum-free culture medium for culturing for 36-60h, collecting the cells, reserving the rest culture solution, and combining the rest culture solution with the previous culture solution to prepare the adipose-derived stem cell-free culture solution; then washing the cells with PBS solution, adding into 0.2-0.3wt% trypsin solution, wherein the cell density is 10 8 -10 9 Digesting at 36-38deg.C for 3-5min, centrifuging at 2-4deg.C for 10-15min, centrifuging at 270-320 Xg for 5-10min, centrifuging at 1800-2200 Xg for 25-35min, centrifuging at 8000-12000 Xg, and removing cells and cell fragments; continuing to centrifuge for 60-80min at 98000-102000 Xg, re-suspending the precipitate at the bottom, washing with PBS solution, and centrifuging for 60-80min at 98000-102000 Xg to remove protein polluted in exosomes, thereby obtaining fat stem cell exosomes;
S2, preparing active polysaccharide: respectively cleaning 5-7 parts by weight of oat, 10-15 parts by weight of nostoc sphaeroids kutz and 3-5 parts by weight of medlar, drying, crushing, adding 7-10 parts by weight of snail stock solution, stirring and mixing uniformly, adding 200 parts by weight of water, adding 0.5-1 part by weight of complex enzyme, heating to 45-50 ℃, carrying out enzymolysis and extraction for 2-4 hours, filtering, and reserving filter residues; adding ethanol into the filtrate until the concentration of the system ethanol is 70-80wt%, precipitating for 3-5h, filtering to obtain active polysaccharide, lyophilizing the filtrate, and mixing with the residue to obtain an extract mixture;
the compound enzyme is a mixture of cellulase and pectase, and the mass ratio is 5-7:1-2;
s3, activating zymophyte: inoculating Bifidobacterium longum and Bifidobacterium breve into Gao's medium at 36-38deg.C, 70-100r/min, and performing active culture for 24-36 hr to obtain seed solution of Bifidobacterium longum and Bifidobacterium breve with bacterial content of 10 8 -10 9 cfu/mL；
S4, preparing a culture medium: uniformly mixing 20-25 parts by weight of the fat stem cell-free culture solution in the step S1, 7-10 parts by weight of the extraction mixture in the step S2 and 100 parts by weight of deionized water, and sterilizing to obtain a culture medium;
s5, fermenting: inoculating the seed liquid of Bifidobacterium longum and Bifidobacterium breve obtained in the step S3 into the culture medium obtained in the step S4, wherein the inoculum sizes of the seed liquid of Bifidobacterium longum and Bifidobacterium breve are respectively 2-4v/v% and 1-3v/v%, under the anoxic condition, the temperature is 36-38 ℃ and the speed is 70-100r/min, the fermentation culture time is 48-72h, filtering, washing the solid with sterile water, collecting the fermentation bacteria, concentrating the obtained fermentation bacteria liquid, and the bacterial content is 10 10 -10 11 cfu/mL; lyophilizing the filtrate to obtain fermentation product;
s6, metal ion chelate: adding 15-20 parts by weight of the fermentation product obtained in the step S5 into 200 parts by weight of water, adding 0.7-1 part by weight of soluble copper salt and 0.5-1 part by weight of soluble zinc salt, stirring and mixing uniformly, dialyzing for 3-5 hours by using a dialysis bag with the aperture of 1-1.5kDa, and freeze-drying the dialysate to obtain a Zn/Cu chelated fermentation product;
s7, preparing a yeast cell lysate: adding 3-5 parts by weight of lysozyme into 1000 parts by weight of the fermentation broth prepared in the step S5, carrying out enzymolysis for 2-4 hours at 40-45 ℃, inactivating enzyme, centrifuging for 10-15 minutes at 5000-7000r/min, collecting supernatant, and freeze-drying to obtain a two-split yeast lysate;
s8, preparing an additive: uniformly mixing 3-5 parts by weight of acetyl dipeptide-1 cetyl ester, 1-3 parts by weight of palmitoyl tripeptide-5 and 2-4 parts by weight of glutathione to prepare an additive;
s9, preparing temperature-sensitive hydrogel: dissolving 5-7 parts by weight of the active polysaccharide prepared in the step S2 and 7-10 parts by weight of sodium hyaluronate in 200 parts by weight of water to obtain a mixed sugar solution; dissolving 6-8 parts by weight of hydroxypropyl methylcellulose in 100 parts by weight of water, adding a mixed sugar solution, stirring and mixing uniformly, adding 2-4 parts by weight of glycerol, stirring and mixing uniformly, and preparing the temperature-sensitive hydrogel;
S10, preparation of a composition for improving the stem cell exosome of skin: uniformly mixing 1-3 parts by weight of the fat stem cell exosome prepared in the step S1, 3-5 parts by weight of the fermentation product of chelated Zn/Cu prepared in the step S6, 2-4 parts by weight of the yeast cell lysate prepared in the step S7 and 0.5-1 part by weight of the additive prepared in the step S8, adding the mixture into 15-20 parts by weight of the temperature-sensitive hydrogel prepared in the step S9, and uniformly stirring and mixing to prepare the stem cell exosome composition for improving skin.
The invention further provides a stem cell exosome composition for improving skin prepared by the preparation method.
The invention further provides application of the stem cell exosome composition for improving skin in preparing products for resisting skin aging, promoting skin elasticity recovery, whitening, removing wrinkles and preserving moisture.
The invention has the following beneficial effects:
the adipose-derived stem cell exosome carries rich protein, RNA, DNA and lipid molecules, has biocompatibility, low toxicity and low immunogenicity, can penetrate through physiological barriers of organism tissues, participates in substance exchange and information communication among cells, can promote migration, proliferation and collagen secretion of keratinocytes and fibroblasts, can remarkably improve skin roughness, reduce melanin content, improve skin brightness, luster, elasticity, wrinkle resistance and other effects, and has obvious effects of resisting skin aging, wound repair and scar repair. Meanwhile, the culture solution in the in-vitro culture process of the pig adipose-derived mesenchymal stem cells is collected, and the obtained adipose-derived stem cell-free culture solution contains abundant active substances such as cytokines (including vascular endothelial growth factor, hepatocyte growth factor, basic fibroblast growth factor and the like), exovesicles, exosomes, DNA, RNA and the like which are secreted by the adipose-derived stem cells, so that the growth of the skin fibroblasts can be promoted, the proliferation and collagen synthesis capacities of the skin fibroblasts can be improved, and wrinkles, fine lines and pigmentation can be reduced, the recovery of skin elasticity can be promoted, and meanwhile, the promotion effect on proliferation and migration of the fibroblasts can be promoted, and wound healing and elimination of skin scars can be promoted. On the other hand, the fat stem cell-free culture solution also contains rich nutritional factors, and can promote the rapid proliferation of zymophyte.
According to the invention, oat, nostoc sphaeroids kutz, medlar and snail stock solution are subjected to mixed enzymolysis, wherein the oat, nostoc sphaeroids kutz and medlar all contain rich polysaccharide and protein substances, the snail stock solution mainly comprises allantoin, glycollic acid, collagen, glucuronic acid, trehalose and the like, the rupture of plant cell walls can be promoted through enzymolysis, the dissolution of active substances such as polysaccharide, protein and the like in cells can be promoted, and further, the active polysaccharide containing oat polysaccharide, nostoc sphaeroids kutz polysaccharide, medlar polysaccharide and snail stock solution polysaccharide is obtained through adding ethanol for precipitation; meanwhile, the enzymolysis product and the filter material contain rich nutrient substances, and the nutrient substances are mixed with the cell-free culture solution of the adipose-derived stem cells, so that the rapid proliferation of zymophyte can be further promoted. After inoculating the fermentation bacteria of the bifidobacterium genus for fermentation, the obtained fermentation product is a fermentation product of the saccharomyces cerevisiae, contains rich oat polypeptide, nostoc polypeptide, phycobiliprotein derived from nostoc sphaeroids, medlar polypeptide, collagen peptide in snail stock solution and the like, and has good effects of resisting oxidation, absorbing moisture and preserving moisture, chelating metal ions, resisting inflammation, stimulating cells to produce collagen, promoting wound proliferation repair, resisting stimulation, easing pain and anesthesia, removing necrotic tissue and promoting granulation and the like.
The prepared fermentation product is chelated with Cu ions and Zn ions, a small amount of Cu ions can promote skin cutin formation, effectively promote skin cell proliferation, activate the aminopeptidase in the synthesis process of collagen, resist inflammation, prevent sunburn and delay aging, stabilize and protect cell membranes, promote cell growth and keep skin elasticity. The Zn ions in a small amount can strengthen the resistance of the skin, reduce the invasion of external bacteria, play a role in preventing skin infection, promote the synthesis of collagen in a human body, promote the healing of wounds, facilitate the recovery of the skin, participate in the metabolism of the skin, accelerate the repair of the skin and facilitate the metabolism of the skin. The synergistic effect of the two further improves the effects of the composition on resisting oxidation, inflammation, sun protection, aging resistance, healing promotion and wrinkle resistance of the skin, and has synergistic effect.
In addition, the concentrated bacterial liquid is subjected to enzymolysis by lysozyme to obtain the yeast cell lysate, and a large amount of active substances are generated by bifidobacteria in the fermentation process, and are released in cells by enzymolysis by lysozyme, so that the concentrated bacterial liquid comprises various amino acids, proteins and various molecular mediums, has the effects of regulating and balancing skin and regulating immune functions, has good effects of resisting wrinkles, tightening, repairing, sun-screening, resisting oxidation and resisting aging, can reduce the damage caused by external stimulus such as ultraviolet rays and promote the repair of damaged DNA.
Acetyl dipeptide-1 cetyl esters have a similar activity to alpha-MSH and can act by modulating NF- κB to interfere with the occurrence of inflammatory responses, by reducing skin inflammation and improving skin barrier. Palmitoyl tripeptide-5 can inhibit melanin production by inhibiting tyrosinase activity, down-regulating MITF and tyrosinase mRNA levels, and significantly reduce uv-induced precipitation of skin pigments. The glutathione can remove excessive free radicals and some peroxides in skin tissues, remarkably prevent lipid peroxidation of mitochondria in cells, has the effects of protecting skin and resisting cell aging, and has the synergistic effect by adding the three.
The temperature-sensitive hydrogel is a solution state at room temperature and can form gel at a certain temperature, the raw materials of the temperature-sensitive hydrogel comprise active polysaccharide, sodium hyaluronate, hydroxypropyl methylcellulose and glycerin, wherein the hydroxypropyl methylcellulose is a cellulose-derived temperature-sensitive hydrophilic polymer, the temperature-sensitive hydrophilic polymer has no toxicity, low cost and high swelling property and surface activity, the sodium hyaluronate is a polysaccharide compound with moisturizing and skin moisture absorption promoting functions, meanwhile, the polysaccharide component contained in the active polysaccharide can participate in forming the temperature-sensitive hydrogel, and simultaneously, the active polysaccharide can play a good role in moisturizing, antioxidation, anti-inflammatory, anti-wrinkle and other effects, and meanwhile, the active polysaccharide can play a good role in promoting transdermal absorption, and can form the hydrogel at the skin surface temperature by reasonably mixing the active polysaccharide, and can fully load substances including a dicyanoyeast cell lysate, an fat stem cell exosome, a chelated Zn/fermentation product, acetyl dipeptide-1 cetyl palmitate, tripeptide-5 and glutathione, so that the active polysaccharide can be fully coated on the skin surface, and the skin surface can be well released and the skin can be well, and the skin adhesion can be well released, and the skin is well absorbed at the skin surface temperature.
The stem cell exosome composition for improving skin prepared by the invention has the effects of biocompatibility, low toxicity and low immunogenicity, and good effect of being absorbed by cells in dermis layer through a barrier of skin tissue, can promote keratinocytes, promote migration, proliferation and secretion of collagen of fibroblasts, has good effects of resisting oxidation, inflammation and skin photoaging, improving skin roughness, reducing melanin content, improving skin brightness, luster, elasticity, wrinkle resistance and the like, and has wide application prospect.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.
FIG. 1 is a TEM image of the fat stem cell exosomes prepared in example 1.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Porcine adipose mesenchymal stem cells purchased from Shanghai's ancestor biotechnology Co., ltd; snail stock solution with content >90% purchased from Guangzhou Heng-Yongshi Biotechnology Co., ltd; cellulase, 1.1 ten thousand U/g, pectase, 2.5 ten thousand U/g, trypsin and 20 ten thousand U/g; lysozyme, 200 ten thousand U/g, purchased from Xia Cheng (Beijing) Biotechnology development Co., ltd; sodium hyaluronate with molecular weight of 80-120 Da and purity of >99% is purchased from Siamiwanfant biotechnology Co., ltd; bifidobacterium longum, 100 hundred million cfu/g, bifidobacterium breve, 100 hundred million cfu/g, purchased from Zhongke Jiayi bioengineering technologies, inc.
Example 1
The embodiment provides a preparation method of a stem cell exosome composition for improving skin, which specifically comprises the following steps:
s1, extracting fat stem cell exosomes: resuspension of cells with alpha-MEM complete culture solution, in vitro culture, washing with PBS solution when cell density is 70%, inoculating into serum-free culture medium, and culturing to cell density of 2×10 7 After culturing for 36h, collecting cells, reserving the rest culture solution, and combining the rest culture solution with the previous culture solution to obtain a cell-free culture solution of the adipose-derived stem cells; then the cells were rinsed with PBS and added to 0.2wt% trypsin solution, the cell density in the solution being 3X 10 8 Digesting at 36 ℃ for 3min per mL, centrifuging the culture medium at 2 ℃ for 10min at 270 Xg, 5min at 1800 Xg and 25min at 8000 Xg in sequence, and removing cells and cell fragments; centrifugation was continued at 98000 Xg for 60min, and the bottom pellet was resuspended and taken up with PBS solution flushing, finally removing the polluted protein in the exosome by centrifugation at 98000 Xg for 60min, and finally obtaining the fat stem cell exosome, wherein fig. 1 is a TEM image of the fat stem cell exosome, and the exosome is mainly spherical, has complete structure and has a diameter of mainly 30-110 nm;
s2, preparing active polysaccharide: respectively cleaning 5 parts by weight of oat, 10 parts by weight of nostoc sphaeroids kutz and 3 parts by weight of medlar, drying, crushing, adding 7 parts by weight of snail stock solution, stirring and mixing for 20min, adding 200 parts by weight of water, adding 0.5 part by weight of complex enzyme, heating to 45 ℃, carrying out enzymolysis and extraction for 2h, filtering, and reserving filter residues; adding ethanol into the filtrate until the concentration of the ethanol in the system is 70wt%, precipitating for 3h, filtering to obtain active polysaccharide, freeze-drying the filtrate, and mixing with the filter residue under stirring for 10min to obtain an extraction mixture;
the compound enzyme is a mixture of cellulase and pectase, and the mass ratio is 5:1;
s3, activating zymophyte: inoculating Bifidobacterium longum and Bifidobacterium breve respectively into Gao's culture medium, and performing active culture at 36deg.C and 70r/min for 24 hr under anoxic condition to obtain seed solution of Bifidobacterium longum and Bifidobacterium breve with bacterial content of 2×10 8 cfu/mL；
S4, preparing a culture medium: stirring and mixing 20 parts by weight of the fat stem cell-free culture solution in the step S1, 7 parts by weight of the extraction mixture in the step S2 and 100 parts by weight of deionized water for 10min, and sterilizing to obtain a culture medium;
s5, fermenting: inoculating the seed liquid of Bifidobacterium longum and Bifidobacterium breve obtained in the step S3 into the culture medium obtained in the step S4, wherein the inoculum sizes of the seed liquid of Bifidobacterium longum and Bifidobacterium breve are respectively 2v/v% and 1v/v%, fermenting and culturing at 36 ℃ under the anoxic condition for 48 hours at 70r/min, filtering, washing the solid with sterile water, collecting fermentation bacteria, concentrating the obtained fermentation bacteria liquid, and the bacterial content is 4 multiplied by 10 10 cfu/mL; lyophilizing the filtrate to obtain fermentation product;
s6, metal ion chelate: adding 15 parts by weight of the fermentation product obtained in the step S5 into 200 parts by weight of water, adding 0.7 part by weight of copper chloride and 0.5 part by weight of zinc chloride, stirring and mixing for reaction for 30min, dialyzing for 3h by using a dialysis bag with the aperture of 1kDa, and freeze-drying the dialysate to obtain a Zn/Cu chelated fermentation product;
s7, preparing a yeast cell lysate: adding 3 parts by weight of lysozyme into 1000 parts by weight of the fermentation broth prepared in the step S5, carrying out enzymolysis for 2 hours at 40 ℃, inactivating the lysozyme, centrifuging for 10 minutes at 5000r/min, collecting supernatant, and freeze-drying to prepare a two-split yeast lysate;
S8, preparing an additive: mixing 3 parts by weight of acetyl dipeptide-1 cetyl ester, 1 part by weight of palmitoyl tripeptide-5 and 2 parts by weight of glutathione for 15min under stirring to obtain an additive;
s9, preparing temperature-sensitive hydrogel: dissolving 5 parts by weight of the active polysaccharide prepared in the step S2 and 7 parts by weight of sodium hyaluronate in 200 parts by weight of water to obtain a mixed sugar solution; dissolving 6 parts by weight of hydroxypropyl methylcellulose in 100 parts by weight of water, adding a mixed sugar solution, stirring and mixing for 15min, adding 2 parts by weight of glycerol, and stirring and mixing for 15min to obtain a temperature-sensitive hydrogel;
s10, preparation of a composition for improving the stem cell exosome of skin: and (3) stirring and mixing 1 part by weight of the fat stem cell exosome prepared in the step (S1), 3 parts by weight of the fermentation product chelated with Zn/Cu prepared in the step (S6), 2 parts by weight of the yeast cell lysate prepared in the step (S7) and 0.5 part by weight of the additive prepared in the step (S8) for 15 minutes, adding the mixture into 15 parts by weight of the temperature-sensitive hydrogel prepared in the step (S9), and stirring and mixing for 20 minutes to prepare the skin-improving stem cell exosome composition.
Example 2
The embodiment provides a preparation method of a stem cell exosome composition for improving skin, which specifically comprises the following steps:
s1, extracting fat stem cell exosomes: resuspending cells of pig fat mesenchymal stem cells with alpha-MEM complete culture solution, culturing in vitro, washing cells with PBS solution when cell density is 80%, inoculating the rest culture solution into serum-free culture medium, and culturing to cell density of 4X10% 7 After culturing for 60 hours, collecting cells, reserving the rest culture solution, and combining the rest culture solution with the previous culture solution to prepare a cell-free culture solution of the adipose-derived stem cells; the cells were then rinsed with PBS and 0.3wt% trypsin was addedIn the solution, the cell density in the solution is 10 9 Digesting at 38deg.C for 5min, centrifuging at 4deg.C for 15min, centrifuging at 220X1 for 10min, centrifuging at 12000 Xg for 35min, and removing cells and cell debris; continuing to centrifuge for 80min at 102000 Xg, re-suspending the sediment at the bottom, flushing with PBS solution, and centrifuging for 80min at 102000 Xg to remove the polluted protein in the exosomes, thus finally obtaining the fat stem cell exosomes;
s2, preparing active polysaccharide: respectively cleaning, drying, crushing and adding 7 parts by weight of oat, 15 parts by weight of nostoc sphaeroids kutz and 5 parts by weight of medlar, adding 10 parts by weight of snail stock solution, stirring and mixing for 20min, adding 200 parts by weight of water, adding 1 part by weight of complex enzyme, heating to 50 ℃, carrying out enzymolysis and extraction for 4h, filtering, and reserving filter residues; adding ethanol into the filtrate until the concentration of the system ethanol is 80wt%, precipitating for 5h, filtering to obtain active polysaccharide, lyophilizing the filtrate, and mixing with the residue under stirring for 10min to obtain an extract mixture;
The compound enzyme is a mixture of cellulase and pectase, and the mass ratio is 7:2;
s3, activating zymophyte: inoculating Bifidobacterium longum and Bifidobacterium breve respectively into Gao's culture medium, and performing activation culture at 38deg.C and 100r/min for 36 hr under anoxic condition to obtain seed solution of Bifidobacterium longum and Bifidobacterium breve with bacterial content of 10 9 cfu/mL；
S4, preparing a culture medium: mixing 25 parts by weight of the adipose-derived stem cell-free culture solution in the step S1, 10 parts by weight of the extraction mixture in the step S2 and 100 parts by weight of deionized water for 10 minutes under stirring, and sterilizing to obtain a culture medium;
s5, fermenting: inoculating the seed liquid of Bifidobacterium longum and Bifidobacterium breve obtained in the step S3 into the culture medium obtained in the step S4, wherein the inoculum sizes of the seed liquid of Bifidobacterium longum and Bifidobacterium breve are respectively 4v/v% and 3v/v%, fermenting and culturing at 38 ℃ and 100r/min for 72h under the anoxic condition, filtering, washing the solid with sterile water, collecting fermentation bacteria, concentrating the obtained fermentation bacteria liquid, and the bacterial content is 10 11 cfu/mL; lyophilizing the filtrate to obtain fermentation product;
s6, metal ion chelate: adding 20 parts by weight of the fermentation product obtained in the step S5 into 200 parts by weight of water, adding 1 part by weight of copper sulfate and 1 part by weight of zinc sulfate, stirring and mixing for reaction for 30min, dialyzing for 5h by using a dialysis bag with the aperture of 1.5kDa, and freeze-drying the dialysate to obtain a Zn/Cu chelated fermentation product;
S7, preparing a yeast cell lysate: adding 5 parts by weight of lysozyme into 1000 parts by weight of the fermentation broth prepared in the step S5, carrying out enzymolysis for 4 hours at 45 ℃, inactivating the enzyme, centrifuging for 15 minutes at 7000r/min, collecting supernatant, and freeze-drying to prepare a two-split yeast lysate;
s8, preparing an additive: mixing 5 parts by weight of acetyl dipeptide-1 cetyl ester, 3 parts by weight of palmitoyl tripeptide-5 and 4 parts by weight of glutathione for 15min under stirring to obtain an additive;
s9, preparing temperature-sensitive hydrogel: dissolving 7 parts by weight of the active polysaccharide prepared in the step S2 and 10 parts by weight of sodium hyaluronate in 200 parts by weight of water to obtain a mixed sugar solution; dissolving 8 parts by weight of hydroxypropyl methylcellulose in 100 parts by weight of water, adding a mixed sugar solution, stirring and mixing for 15min, adding 4 parts by weight of glycerol, and stirring and mixing for 15min to obtain a temperature-sensitive hydrogel;
s10, preparation of a composition for improving the stem cell exosome of skin: 3 parts by weight of the fat stem cell exosome prepared in the step S1, 5 parts by weight of the fermentation product chelated with Zn/Cu prepared in the step S6, 4 parts by weight of the yeast cell lysate prepared in the step S7 and 1 part by weight of the additive prepared in the step S8 are stirred and mixed for 15min, and then 20 parts by weight of the additive is added into the temperature-sensitive hydrogel prepared in the step S9, and the mixture is stirred and mixed for 20min, so that the stem cell exosome composition for improving skin is prepared.
Example 3
The embodiment provides a preparation method of a stem cell exosome composition for improving skin, which specifically comprises the following steps:
s1, extracting fat stem cell exosomes: resuspending cells of pig fat mesenchymal stem cells with alpha-MEM complete culture solution, culturing in vitro, washing cells with PBS solution when cell density is 75%, inoculating the rest culture solution into serum-free culture medium, and culturing to cell density of 3×10 7 After culturing for 48h, the cells are collected and the remaining culture solution is used for the cultureCombining the previous culture solutions to prepare a cell-free culture solution of the adipose-derived stem cells; then the cells were rinsed with PBS and added to 0.25wt% trypsin solution, the cell density in the solution being 7X 10 8 Digesting at 37 ℃ for 4min per mL, centrifuging at 3 ℃ the culture medium at 300 Xg for 12min, at 2000 Xg for 7min and at 10000 Xg for 30min in sequence, and removing cells and cell fragments; continuing to centrifuge for 70min at 100000 Xg, re-suspending the sediment at the bottom, flushing with PBS solution, and centrifuging for 70min at 100000 Xg to remove the polluted protein in the exosomes, thus finally obtaining the fat stem cell exosomes;
s2, preparing active polysaccharide: cleaning 6 parts by weight of oat, 12 parts by weight of nostoc sphaeroids kutz and 4 parts by weight of medlar respectively, drying, crushing, adding 8.5 parts by weight of snail stock solution, stirring and mixing for 20min, adding 200 parts by weight of water, adding 0.7 part by weight of complex enzyme, heating to 47 ℃, carrying out enzymolysis and extraction for 3h, filtering, and reserving filter residues; adding ethanol into the filtrate until the concentration of the system ethanol is 75wt%, precipitating for 4h, filtering to obtain active polysaccharide, lyophilizing the filtrate, mixing with the residue under stirring for 10min to obtain an extract mixture;
The compound enzyme is a mixture of cellulase and pectase, and the mass ratio is 6:1.5;
s3, activating zymophyte: inoculating Bifidobacterium longum and Bifidobacterium breve respectively into Gao's culture medium, and performing activation culture at 37deg.C and 85r/min for 30 hr under anoxic condition to obtain seed solution of Bifidobacterium longum and Bifidobacterium breve with bacterial content of 5×10 8 cfu/mL；
S4, preparing a culture medium: mixing 22 parts by weight of the adipose-derived stem cell-free culture solution in the step S1, 8.5 parts by weight of the extraction mixture in the step S2 and 100 parts by weight of deionized water for 10min under stirring, and sterilizing to obtain a culture medium;
s5, fermenting: inoculating the seed liquid of Bifidobacterium longum and Bifidobacterium breve obtained in the step S3 into the culture medium obtained in the step S4, wherein the inoculum sizes of the seed liquid of Bifidobacterium longum and Bifidobacterium breve are respectively 3v/v% and 2v/v%, fermenting and culturing for 56h at 37 ℃ under the anoxic condition and 85r/min, filtering, washing the solid with sterile water, collecting fermentation bacteria, concentrating the obtained fermentation bacteria liquid, and the bacterial content is 6 multiplied by 10 10 cfu/mL; lyophilizing the filtrate to obtain fermentation product;
s6, metal ion chelate: adding 17 parts by weight of the fermentation product obtained in the step S5 into 200 parts by weight of water, adding 0.85 part by weight of copper nitrate and 0.7 part by weight of zinc nitrate, stirring and mixing for reaction for 30min, dialyzing for 4h by using a dialysis bag with the aperture of 1.2kDa, and freeze-drying the dialyzate to obtain a Zn/Cu chelated fermentation product;
S7, preparing a yeast cell lysate: adding 4 parts by weight of lysozyme into 1000 parts by weight of the fermentation broth prepared in the step S5, carrying out enzymolysis for 3 hours at 42 ℃, inactivating the lysozyme, centrifuging for 12 minutes at 6000r/min, collecting supernatant, and freeze-drying to prepare a two-split yeast lysate;
s8, preparing an additive: mixing 4 parts by weight of acetyl dipeptide-1 cetyl ester, 2 parts by weight of palmitoyl tripeptide-5 and 3 parts by weight of glutathione for 15min under stirring to obtain an additive;
s9, preparing temperature-sensitive hydrogel: dissolving 6 parts by weight of the active polysaccharide prepared in the step S2 and 8.5 parts by weight of sodium hyaluronate in 200 parts by weight of water to obtain a mixed sugar solution; dissolving 7 parts by weight of hydroxypropyl methylcellulose in 100 parts by weight of water, adding a mixed sugar solution, stirring and mixing for 15min, adding 3 parts by weight of glycerol, and stirring and mixing for 15min to obtain a temperature-sensitive hydrogel;
s10, preparation of a composition for improving the stem cell exosome of skin: mixing 2 parts by weight of the fat stem cell exosome prepared in the step S1, 4 parts by weight of the fermentation product chelated with Zn/Cu prepared in the step S6, 3 parts by weight of the yeast cell lysate prepared in the step S7 and 0.7 part by weight of the additive prepared in the step S8 for 15min, adding 17 parts by weight of the temperature-sensitive hydrogel prepared in the step S9, and stirring and mixing for 20min to prepare the skin-improving stem cell exosome composition.
Example 4
The difference compared to example 3 is that the complex enzyme is a single cellulase.
Example 5
The difference compared to example 3 is that the complex enzyme is a single pectase.
Comparative example 1
In comparison with example 3, the difference is that no complex enzyme was added in step S2.
The method comprises the following steps:
s2, preparing active polysaccharide: cleaning 6 parts by weight of oat, 12 parts by weight of nostoc sphaeroids kutz and 4 parts by weight of medlar respectively, drying, crushing, adding 8.5 parts by weight of snail stock solution, stirring and mixing for 20min, adding 200 parts by weight of water, heating to 47 ℃, extracting for 3h, filtering, and reserving filter residues; adding ethanol into the filtrate until the concentration of the ethanol in the system is 75wt%, precipitating for 4h, filtering to obtain active polysaccharide, lyophilizing the filtrate, and mixing with the residue under stirring for 10min to obtain extract mixture.
Comparative example 2
The difference from example 3 is that no snail stock solution was added in step S2.
The method comprises the following steps:
s2, preparing active polysaccharide: cleaning 6 parts by weight of oat, 12 parts by weight of nostoc sphaeroids kutz and 4 parts by weight of medlar respectively, drying, crushing, stirring and mixing for 20min, adding into 200 parts by weight of water, adding 0.7 part by weight of complex enzyme, heating to 47 ℃, carrying out enzymolysis and extraction for 3h, filtering, and reserving filter residues; adding ethanol into the filtrate until the concentration of the system ethanol is 75wt%, precipitating for 4h, filtering to obtain active polysaccharide, lyophilizing the filtrate, mixing with the residue under stirring for 10min to obtain an extract mixture;
The compound enzyme is a mixture of cellulase and pectase, and the mass ratio is 6:1.5.
Comparative example 3
In comparison with example 3, the difference is that no adipose-derived stem cell-free culture solution was added in step S4.
The method comprises the following steps:
s4, preparing a culture medium: 30.5 parts by weight of the extraction mixture in the step S2 and 100 parts by weight of deionized water are stirred and mixed for 10 minutes, and sterilized to obtain a culture medium.
Comparative example 4
The difference compared to example 3 is that no extraction mixture is added in step S4.
The method comprises the following steps:
s4, preparing a culture medium: and (2) stirring and mixing 30.5 parts by weight of the adipose-derived stem cell-free culture solution in the step (S1) with 100 parts by weight of deionized water for 10min, and sterilizing to obtain the culture medium.
Comparative example 5
The difference from example 3 is that the seed solution of bifidobacterium longum strain was not inoculated in step S5.
The method comprises the following steps:
s5, fermenting: inoculating the seed liquid of the bifidobacterium breve prepared in the step S3 into the culture medium prepared in the step S4, wherein the inoculum size of the seed liquid of the bifidobacterium breve is 5v/v%, the fermentation culture time is 56h at 37 ℃ and 85r/min under the anoxic condition, filtering, washing the solid with sterile water, collecting the fermentation bacteria, and concentrating the obtained fermentation bacteria liquid with the bacterial content of 6 multiplied by 10 10 cfu/mL; and freeze-drying the filtrate to obtain a fermentation product.
Comparative example 6
The difference from example 3 is that the seed solution of bifidobacterium breve was not inoculated in step S5.
The method comprises the following steps:
s5, fermenting: inoculating the seed liquid of the bifidobacterium breve prepared in the step S3 into the culture medium prepared in the step S4, wherein the inoculum size of the seed liquid of the bifidobacterium breve is 5v/v%, the fermentation culture time is 56h at 37 ℃ and 85r/min under the anoxic condition, filtering, washing the solid with sterile water, collecting the fermentation bacteria, and concentrating the obtained fermentation bacteria liquid with the bacterial content of 6 multiplied by 10 10 cfu/mL; and freeze-drying the filtrate to obtain a fermentation product.
Comparative example 7
In comparison with example 3, the difference is that steps S3 to S6 are not performed.
The method comprises the following steps:
s1, extracting fat stem cell exosomes: resuspending cells of pig fat mesenchymal stem cells with alpha-MEM complete culture solution, culturing in vitro, washing cells with PBS solution when cell density is 75%, inoculating the rest culture solution into serum-free culture medium, and culturing to cell density of 3×10 7 After culturing for 48 hours, collecting cells, reserving the rest culture solution, and combining the rest culture solution with the previous culture solution to prepare a cell-free culture solution of the adipose-derived stem cells; then washed with PBS solution Cells were added to a 0.25wt% trypsin solution having a cell density of 7X 10 8 Digesting at 37 ℃ for 4min per mL, centrifuging at 3 ℃ the culture medium at 300 Xg for 12min, at 2000 Xg for 7min and at 10000 Xg for 30min in sequence, and removing cells and cell fragments; continuing to centrifuge for 70min at 100000 Xg, re-suspending the sediment at the bottom, flushing with PBS solution, and centrifuging for 70min at 100000 Xg to remove the polluted protein in the exosomes, thus finally obtaining the fat stem cell exosomes;
s2, preparing active polysaccharide: cleaning 6 parts by weight of oat, 12 parts by weight of nostoc sphaeroids kutz and 4 parts by weight of medlar respectively, drying, crushing, adding 8.5 parts by weight of snail stock solution, stirring and mixing for 20min, adding 200 parts by weight of water, adding 0.7 part by weight of complex enzyme, heating to 47 ℃, carrying out enzymolysis and extraction for 3h, filtering, and reserving filter residues; adding ethanol into the filtrate until the concentration of the system ethanol is 75wt%, precipitating for 4h, filtering to obtain active polysaccharide, lyophilizing the filtrate, mixing with the residue under stirring for 10min to obtain an extract mixture;
the compound enzyme is a mixture of cellulase and pectase, and the mass ratio is 6:1.5;
s3, preparing an additive: mixing 4 parts by weight of acetyl dipeptide-1 cetyl ester, 2 parts by weight of palmitoyl tripeptide-5 and 3 parts by weight of glutathione for 15min under stirring to obtain an additive;
S4, preparing temperature-sensitive hydrogel: dissolving 6 parts by weight of the active polysaccharide prepared in the step S2 and 8.5 parts by weight of sodium hyaluronate in 200 parts by weight of water to obtain a mixed sugar solution; dissolving 7 parts by weight of hydroxypropyl methylcellulose in 100 parts by weight of water, adding a mixed sugar solution, stirring and mixing for 15min, adding 3 parts by weight of glycerol, and stirring and mixing for 15min to obtain a temperature-sensitive hydrogel;
s5 preparation of a skin-improving stem cell exosome composition: and (3) stirring and mixing 2 parts by weight of the adipose-derived stem cell exosome prepared in the step (S1) and 0.7 part by weight of the additive prepared in the step (S4) for 15min, adding 17 parts by weight of the temperature-sensitive hydrogel prepared in the step (S4), and stirring and mixing for 20min to prepare the skin-improving stem cell exosome composition.
Comparative example 8
The difference from example 3 is that copper nitrate is not added in step S6.
The method comprises the following steps:
s6, metal ion chelate: and (3) adding 17 parts by weight of the fermentation product obtained in the step (S5) into 200 parts by weight of water, adding 1.55 parts by weight of copper nitrate, stirring and mixing for reaction for 30min, dialyzing for 4h by using a dialysis bag with the aperture of 1.2kDa, and freeze-drying the dialysate to obtain the Zn-chelated fermentation product.
Comparative example 9
The difference from example 3 is that zinc nitrate is not added in step S6.
The method comprises the following steps:
s6, metal ion chelate: and (3) adding 17 parts by weight of the fermentation product obtained in the step (S5) into 200 parts by weight of water, adding 1.55 parts by weight of copper nitrate, stirring and mixing for reaction for 30min, dialyzing for 4h by using a dialysis bag with the aperture of 1.2kDa, and freeze-drying the dialysate to obtain the Cu-chelated fermentation product.
Comparative example 10
In comparison with example 3, the difference is that step S6 is not performed.
The method comprises the following steps:
s1, extracting fat stem cell exosomes: resuspending cells of pig fat mesenchymal stem cells with alpha-MEM complete culture solution, culturing in vitro, washing cells with PBS solution when cell density is 75%, inoculating the rest culture solution into serum-free culture medium, and culturing to cell density of 3×10 7 After culturing for 48 hours, collecting cells, reserving the rest culture solution, and combining the rest culture solution with the previous culture solution to prepare a cell-free culture solution of the adipose-derived stem cells; then the cells were rinsed with PBS and added to 0.25wt% trypsin solution, the cell density in the solution being 7X 10 8 Digesting at 37 ℃ for 4min per mL, centrifuging at 3 ℃ the culture medium at 300 Xg for 12min, at 2000 Xg for 7min and at 10000 Xg for 30min in sequence, and removing cells and cell fragments; continuing to centrifuge for 70min at 100000 Xg, re-suspending the sediment at the bottom, flushing with PBS solution, and centrifuging for 70min at 100000 Xg to remove the polluted protein in the exosomes, thus finally obtaining the fat stem cell exosomes;
S2, preparing active polysaccharide: cleaning 6 parts by weight of oat, 12 parts by weight of nostoc sphaeroids kutz and 4 parts by weight of medlar respectively, drying, crushing, adding 8.5 parts by weight of snail stock solution, stirring and mixing for 20min, adding 200 parts by weight of water, adding 0.7 part by weight of complex enzyme, heating to 47 ℃, carrying out enzymolysis and extraction for 3h, filtering, and reserving filter residues; adding ethanol into the filtrate until the concentration of the system ethanol is 75wt%, precipitating for 4h, filtering to obtain active polysaccharide, lyophilizing the filtrate, mixing with the residue under stirring for 10min to obtain an extract mixture;
the compound enzyme is a mixture of cellulase and pectase, and the mass ratio is 6:1.5;
s3, activating zymophyte: inoculating Bifidobacterium longum and Bifidobacterium breve respectively into Gao's culture medium, and performing activation culture at 37deg.C and 85r/min for 30 hr under anoxic condition to obtain seed solution of Bifidobacterium longum and Bifidobacterium breve with bacterial content of 5×10 8 cfu/mL；
S4, preparing a culture medium: mixing 22 parts by weight of the adipose-derived stem cell-free culture solution in the step S1, 8.5 parts by weight of the extraction mixture in the step S2 and 100 parts by weight of deionized water for 10min under stirring, and sterilizing to obtain a culture medium;
s5, fermenting: inoculating the seed liquid of Bifidobacterium longum and Bifidobacterium breve obtained in the step S3 into the culture medium obtained in the step S4, wherein the inoculum sizes of the seed liquid of Bifidobacterium longum and Bifidobacterium breve are respectively 3v/v% and 2v/v%, fermenting and culturing for 56h at 37 ℃ under the anoxic condition and 85r/min, filtering, washing the solid with sterile water, collecting fermentation bacteria, concentrating the obtained fermentation bacteria liquid, and the bacterial content is 6 multiplied by 10 10 cfu/mL; lyophilizing the filtrate to obtain fermentation product;
s6, preparing a yeast cell lysate: adding 4 parts by weight of lysozyme into 1000 parts by weight of the fermentation broth prepared in the step S5, carrying out enzymolysis for 3 hours at 42 ℃, inactivating the lysozyme, centrifuging for 12 minutes at 6000r/min, collecting supernatant, and freeze-drying to prepare a two-split yeast lysate;
s7, preparing an additive: mixing 4 parts by weight of acetyl dipeptide-1 cetyl ester, 2 parts by weight of palmitoyl tripeptide-5 and 3 parts by weight of glutathione for 15min under stirring to obtain an additive;
s8, preparing temperature-sensitive hydrogel: dissolving 6 parts by weight of the active polysaccharide prepared in the step S2 and 8.5 parts by weight of sodium hyaluronate in 200 parts by weight of water to obtain a mixed sugar solution; dissolving 7 parts by weight of hydroxypropyl methylcellulose in 100 parts by weight of water, adding a mixed sugar solution, stirring and mixing for 15min, adding 3 parts by weight of glycerol, and stirring and mixing for 15min to obtain a temperature-sensitive hydrogel;
s9, preparation of a composition for improving the stem cell exosome of skin: and (3) stirring and mixing 2 parts by weight of the fat stem cell exosome prepared in the step (S1), 4 parts by weight of the fermentation product prepared in the step (S5), 3 parts by weight of the yeast cell lysate prepared in the step (S6) and 0.7 part by weight of the additive prepared in the step (S7) for 15min, adding 17 parts by weight of the temperature-sensitive hydrogel prepared in the step (S8), and stirring and mixing for 20min to prepare the skin-improving stem cell exosome composition.
Comparative example 11
The difference compared to example 3 is that no active polysaccharide is added in step S9.
The method comprises the following steps:
s9, preparing temperature-sensitive hydrogel: dissolving 14.5 parts by weight of sodium hyaluronate in 200 parts by weight of water to obtain a mixed sugar solution; and (3) dissolving 7 parts by weight of hydroxypropyl methylcellulose in 100 parts by weight of water, adding the mixed sugar solution, stirring and mixing for 15min, adding 3 parts by weight of glycerol, and stirring and mixing for 15min to obtain the temperature-sensitive hydrogel.
Comparative example 12
In comparison with example 3, the difference is that no adipose stem cell exosomes were added in step S10.
The method comprises the following steps:
s10, preparation of a composition for improving the stem cell exosome of skin: 4 parts by weight of the Zn/Cu chelated fermentation product prepared in the step S6, 3 parts by weight of the yeast cell lysate prepared in the step S7 and 0.7 part by weight of the additive prepared in the step S8 are stirred and mixed for 15min, 17 parts by weight of the temperature-sensitive hydrogel prepared in the step S9 is added, and stirring and mixing are carried out for 20min, so that the skin-improving stem cell exosome composition is prepared.
Comparative example 13
The difference compared with example 3 is that the fermentation product chelating Zn/Cu is not added in step S10.
The method comprises the following steps:
s10, preparation of a composition for improving the stem cell exosome of skin: and (3) mixing 2 parts by weight of the fat stem cell exosome prepared in the step (S1), 3 parts by weight of the bipartite yeast lysate prepared in the step (S7) and 0.7 part by weight of the additive prepared in the step (S8) for 15 minutes under stirring, adding 17 parts by weight of the temperature-sensitive hydrogel prepared in the step (S9), and stirring and mixing for 20 minutes to prepare the stem cell exosome composition for improving skin.
Comparative example 14
In comparison with example 3, the difference is that no yeast lysate was added in step S10.
The method comprises the following steps:
s10, preparation of a composition for improving the stem cell exosome of skin: and (3) mixing 2 parts by weight of the fat stem cell exosome prepared in the step (S1), 4 parts by weight of the Zn/Cu chelated fermentation product prepared in the step (S6) and 0.7 part by weight of the additive prepared in the step (S8) for 15 minutes under stirring, adding 17 parts by weight of the temperature-sensitive hydrogel prepared in the step (S9), and stirring and mixing for 20 minutes to prepare the skin-improving stem cell exosome composition.
Comparative example 15
The difference from example 3 is that no additive is added in step S10.
The method comprises the following steps:
s10, preparation of a composition for improving the stem cell exosome of skin: and (3) stirring and mixing 2 parts by weight of the fat stem cell exosome prepared in the step (S1), 4 parts by weight of the fermentation product chelated with Zn/Cu prepared in the step (S6) and 3 parts by weight of the yeast cell lysate prepared in the step (S7) for 15min, adding 17 parts by weight of the temperature-sensitive hydrogel prepared in the step (S9), and stirring and mixing for 20min to prepare the skin-improving stem cell exosome composition.
Test example 1
The temperature-sensitive hydrogels prepared in examples 1 to 3 of the present invention and comparative example 11 were tested.
Placing the obtained temperature-sensitive hydrogel in a constant temperature water bath, taking out test tubes every 10min, inverting to see whether the hydrogel flows, if so, continuing heating at 0.5deg.C, and if not, defining the temperature as T of hydrogel gel . Determining T gel After that, at 37 DEG CThe gel time of each set of hydrogels was measured and recorded as the point in time at which each set of samples did not flow. The results are shown in Table 1.
TABLE 1
As shown in the table above, the gel temperature of the temperature-sensitive hydrogel prepared in examples 1 to 3 of the present invention is between 35.5 and 36.6 ℃ and is close to the skin temperature of human body, and can be converted into a non-flowing hydrogel after being heated to 37 ℃. In comparative example 11, no active polysaccharide was added, and the gelation temperature was high, which was not conducive to gel formation on the skin surface and promoted the absorption of active ingredients by the skin.
Test example 2 skin healing promotion experiment
Balb/c mice were randomly divided into control groups (PBS solution), examples 1-5, and comparative examples 1-15 (the skin-improving stem cell exosome compositions prepared in the respective groups were formulated as 10mg/mL solutions), 6 each. The mice were anesthetized with 1% sodium pentobarbital, back dehaired, physiological saline flushed, and iodine sterilized, and a wound model was prepared by cutting off the whole skin with a diameter of about 1cm on the dehaired back. 100. Mu.L of Escherichia coli (ATCC 25922) (2X 10) 6 cfu/mL) was inoculated onto the skin wound. After 1d infection, 0.5mL of the solution of the control group and each experimental group is uniformly coated on the back wound surface, the coating is carried out 1 time every 3 days, the wound surface healing area is observed and recorded on the 7 th day after the wound, the wound surface healing rate is calculated, the observation is continued, the complete healing time is recorded, and the result is shown in Table 2.
Wound healing rate = (initial wound area-wound healing area)/initial wound area×100%
TABLE 2
Annotation:for comparison with the control group, P<0.05。
From the above table, the skin-improving stem cell exosome compositions prepared in examples 1 to 3 of the present invention have a good effect of promoting skin wound healing.
Test example 3 antioxidant test
Human immortalized epidermal cells HaCaT cells were seeded in 96-well plates (1X 10) 4 Well), to a cell confluence of about 80% -90%. The medium in each well was aspirated and removed, the drugs of examples 1 to 5 or comparative examples 1 to 15 (medium of the skin-improving stem cell exosome composition prepared at a final concentration of 0.05 wt%), the positive group (medium of vitamin C at a final concentration of 0.05%) or the control group (medium without any drug added) were added to each well, the medium was aspirated and removed after incubation in an incubator for 24 hours, 100. Mu.L of DMEM medium containing DCFH-DA fluorescent probes was added, the incubator was incubated for 25 minutes, washed with PBS buffer, and then PBS buffer was added, and the mixture was subjected to irradiation under UVA (dose 5.0J/cm) 2 ). And detecting the fluorescence intensity by a fluorescence enzyme-labeled instrument immediately after the irradiation is finished. The ROS levels for each group were calculated as 100% for the control group. The results are shown in Table 3.
ROD level = experimental/control fluorescence x 100%
TABLE 3 Table 3
Annotation:for comparison with the control group, P<0.05。
As is clear from the above table, the skin-improving stem cell exosome compositions prepared in examples 1 to 3 of the present invention have excellent antioxidant properties.
Test example 4 promotion of human skin fibroblast proliferation
After human skin fibroblasts were cultured in vitro for 24 hours, the skin-improving stem cell exosome compositions prepared in examples 1 to 5 or comparative examples 1 to 15 were added at a final concentration of 10mg/L, the blank group was not added with the composition, and after further culturing for 48 hours, CCK8 reagent was added for incubation for 2 hours, and absorbance at a wavelength of 450nm was detected using a microplate reader, and relative cell viability was calculated with respect to 100% of the control group. The results are shown in Table 4.
Relative cell viability (%) = experimental OD/blank OD x 100%
TABLE 4 Table 4
From the above table, it is clear that the skin-improving stem cell exosome compositions prepared in examples 1 to 3 of the present invention can significantly promote proliferation of human skin fibroblasts.
Test example 5
Healthy women aged 33-50 years were selected as subjects with macroscopic fine lines or wrinkles at the canthus, no moles or acne on the face, and randomized into groups of examples 1-5 and comparative examples 1-15, 5 persons each.
Discharge conditions: pregnant or lactating women; a person with sensitive facial skin; the face has severely disturbed epidermal features such as scratches, birthmarks, pimples, acne marks, etc.; patients who received dermatological doctor treatment within 3 months.
The testing method comprises the following steps: and (5) half-face testing. Subjects were uniformly coated with 0.5mL of the skin-improving stem cell exosome compositions prepared in the examples 1-5 and comparative examples 1-15 on the left half face between 8 and 8 half a day in the morning, and the right half face was continuously tested with an equal amount of clear water for 4 weeks without using other skin care products during the test. Testing was performed before the start of the experiment and after the end of the experiment. Prior to testing, the subjects were cleaned with clear water, rubbed off with water, allowed to sit still in a constant temperature and humidity environment (25.+ -. 1 ℃ C., 70.+ -. 10% relative humidity) for 30 minutes, and then assayed. The results are shown in Table 5.
Skin stratum corneum moisture content:
the moisture content of the skin horny layer of the left and right cheeks was measured by a moisture tester, and the difference between the two was calculated, and the average value of 5 measurements was taken. The difference is positive, and the larger the difference, the better the moisture retention improvement of the skin.
Skin elasticity:
skin elasticity tester was used to measure the skin at the tail of the left and right eyes. The difference between the two is calculated, and the values of the skin elasticity parameter R5 (the elastic amount of the rebound portion/the elastic amount of the stretch portion) and the tightening parameter F4 (the area formed by the maximum stretch amount and time) are selected as the comparison indices. The average of 5 measurements was taken.
The difference value of the skin elasticity parameter R5 is a positive number, and the larger the difference value is, the better the skin elasticity is;
the tightening parameter F4 is negative in difference and the greater the absolute value of the difference, the more tightening the skin.
Skin gloss:
the skin glossiness of the skin of the left and right cheeks was measured using a skin glossiness tester, the difference between the two was calculated, and the average value of 5 measurements was taken. The difference is positive and the greater the difference, the more pronounced the improvement in skin gloss.
TABLE 5
From the above table, the skin care composition with the effects of relieving and resisting aging prepared in the embodiments 1-3 can obviously improve the moisture content of skin horny layer, improve skin elasticity, tighten skin, lighten fine wrinkles, improve skin glossiness and have good effects of resisting aging and relieving.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. A preparation method of a stem cell exosome composition for improving skin is characterized by culturing and passaging pig adipose mesenchymal stem cells in vitro, centrifuging to obtain adipose stem cell exosome, reserving the rest culture solution, mixing oat, nostoc sphaeroids kutz, medlar and snail stock solution, performing enzymolysis and extraction on alcohol precipitation to obtain active polysaccharide, mixing the rest culture solution with the rest culture solution, inoculating bifidobacterium longum and bifidobacterium breve for fermentation, chelating Zn and Cu ions with the obtained fermentation product, performing enzymolysis on the fermentation product by lysozyme to obtain a bifidus yeast lysate, mixing the bifidus yeast exosome with the adipose stem cell exosome, the fermentation product chelating Zn/Cu, acetyl dipeptide-1 cetyl ester, palmitoyl tripeptide-5 and glutathione, adding the mixture into a temperature sensitive hydrogel containing the active polysaccharide, and uniformly stirring and mixing to obtain the stem cell exosome composition for improving skin.
2. The method of manufacturing according to claim 1, comprising the steps of:
s1, extracting fat stem cell exosomes: re-suspending cells of the pig adipose-derived mesenchymal stem cells by using alpha-MEM complete culture solution, performing in-vitro culture, flushing the cells by using PBS (phosphate buffer solution) when the cell density is 70-80%, reserving the rest culture solution, inoculating the culture solution into a serum-free culture medium for culturing for 36-60h, collecting the cells, reserving the rest culture solution, and combining the rest culture solution with the previous culture solution to prepare the adipose-derived stem cell-free culture solution; washing cells with PBS solution, adding trypsin for digestion, centrifuging the culture medium at 270-320 Xg for 10-15min,1800-2200 Xg for 5-10min,8000-12000 Xg for 25-35min, and removing cells and cell fragments; continuing to centrifuge for 60-80min at 98000-102000 Xg, re-suspending the precipitate at the bottom, washing with PBS solution, and centrifuging for 60-80min at 98000-102000 Xg to remove protein polluted in exosomes, thereby obtaining fat stem cell exosomes;
s2, preparing active polysaccharide: cleaning herba Avenae Fatuae, nostoc sphaeroids kutz and fructus Lycii respectively, drying, pulverizing, adding snail stock solution, stirring, mixing, adding into water, adding complex enzyme, heating for enzymolysis, extracting, filtering, and collecting residue; adding ethanol into the filtrate for precipitation, filtering to obtain active polysaccharide, lyophilizing the filtrate, and mixing with the residue to obtain an extract mixture;
S3, activating zymophyte: inoculating bifidobacterium longum and bifidobacterium breve into a Gao's medium respectively, and performing activation culture to obtain bifidobacterium longum and bifidobacterium breve seed liquid;
s4, preparing a culture medium: uniformly mixing the fat stem cell-free culture solution in the step S1, the extraction mixture in the step S2 and water, and sterilizing to obtain a culture medium;
s5, fermenting: inoculating the seed solution of the bifidobacterium longum and the bifidobacterium breve prepared in the step S3 into the culture medium prepared in the step S4, fermenting and culturing under the anoxic condition, filtering, washing the solid with sterile water, collecting fermentation bacteria, and concentrating the obtained fermentation bacteria solution; lyophilizing the filtrate to obtain fermentation product;
s6, metal ion chelate: adding the fermentation product obtained in the step S5 into water, adding soluble copper salt and soluble zinc salt, stirring and mixing uniformly, dialyzing, and freeze-drying the dialyzate to obtain a Zn/Cu chelated fermentation product;
s7, preparing a yeast cell lysate: adding lysozyme into the fermentation broth prepared in the step S5 for enzymolysis, inactivating enzyme, centrifuging, collecting supernatant, and freeze-drying to obtain a two-split yeast lysate;
s8, preparing an additive: uniformly mixing acetyl dipeptide-1 cetyl ester, palmitoyl tripeptide-5 and glutathione to prepare an additive;
S9, preparing temperature-sensitive hydrogel: dissolving the active polysaccharide and sodium hyaluronate prepared in the step S2 in water to obtain a mixed sugar solution; dissolving hydroxypropyl methylcellulose in water, adding mixed sugar solution, stirring and mixing uniformly, adding glycerol, stirring and mixing uniformly, and obtaining temperature-sensitive hydrogel;
s10, preparation of a composition for improving the stem cell exosome of skin: uniformly mixing the fat stem cell exosome prepared in the step S1, the Zn/Cu chelated fermentation product prepared in the step S6, the yeast cell lysate prepared in the step S7 and the additive prepared in the step S8, adding the mixture into the temperature-sensitive hydrogel prepared in the step S9, and uniformly stirring and mixing the mixture to prepare the skin-improving stem cell exosome composition.
3. The preparation method according to claim 2, wherein the method of adding trypsin for digestion in step S1 is adding 0.2-0.3wt% trypsin for digestion at 36-38 ℃ for 3-5min; in the step S2, the mass ratio of oat, nostoc sphaeroids kutz, medlar and snail stock solution is 5-7:10-15:3-5:7-10, the mass ratio of the compound enzyme is a mixture of cellulase and pectase, the mass ratio is 5-7:1-2, the temperature of heating enzymolysis extraction is 45-50 ℃ for 2-4h, the concentration of ethanol added to the system ethanol is 70-80wt%, and the precipitation time is 3-5h.
4. The method according to claim 2, wherein the conditions for the activation culture in step S3 are 36-38deg.C, 70-100r/min, the activation time is 24-36h, and the bacterial seed solution has a bacterial content of 10 8 -10 9 cfu/mL; the mass ratio of the fat stem cell-free culture solution to the extraction mixture to the water in the step S4 is 20-25:7-10:100.
5. The method according to claim 2, wherein the seed solution of Bifidobacterium longum and Bifidobacterium breve in step S5 has inoculum sizes of 2-4v/v% and 1-3v/v%, respectively, the conditions of fermentation culture are 36-38deg.C, 70-100r/min, the fermentation culture time is 48-72h, and the bacterial content of the fermentation broth is 10 10 -10 11 cfu/mL; the mass ratio of the fermentation product to the soluble copper salt to the soluble zinc salt in the step S6 is 15-20:0.7-1.2:0.5-1, the soluble copper salt is at least one of copper chloride, copper sulfate and copper nitrate, the soluble zinc salt is at least one of zinc chloride, zinc sulfate and zinc nitrate, the aperture of a dialysis bag for dialysis is 1-1.5kDa, and the dialysis time is 3-5h.
6. The preparation method according to claim 2, wherein in the step S7, the mass ratio of the fermentation broth to the lysozyme is 1000:3-5, the enzymolysis temperature is 40-45 ℃, the enzymolysis time is 2-4h, and the centrifugation condition is 5000-7000r/min for 10-15min; the mass ratio of the acetyl dipeptide-1 cetyl ester, the palmitoyl tripeptide-5 and the glutathione in the step S8 is 3-5:1-3:2-4.
7. The preparation method according to claim 2, wherein the mass ratio of the active polysaccharide, sodium hyaluronate, hydroxypropyl methylcellulose, and glycerin in step S9 is 5-7:7-10:6-8:2-4; in the step S10, the mass ratio of the fat stem cell exosomes to the fermentation products of chelating Zn/Cu to the yeast cell lysate to the additives to the temperature-sensitive hydrogel is 1-3:3-5:2-4:0.5-1:15-20.
8. The preparation method according to claim 2, characterized by comprising the following steps:
s1, extracting fat stem cell exosomes: re-suspending cells of the pig adipose-derived mesenchymal stem cells by using alpha-MEM complete culture solution, performing in-vitro culture, flushing the cells by using PBS (phosphate buffer solution) when the cell density is 70-80%, reserving the rest culture solution, inoculating the culture solution into a serum-free culture medium for culturing for 36-60h, collecting the cells, reserving the rest culture solution, and combining the rest culture solution with the previous culture solution to prepare the adipose-derived stem cell-free culture solution; then washing the cells with PBS solution, adding into 0.2-0.3wt% trypsin solution, wherein the cell density is 10 8 -10 9 Digesting at 36-38deg.C for 3-5min, centrifuging at 2-4deg.C for 10-15min, centrifuging at 270-320 Xg for 5-10min, centrifuging at 1800-2200 Xg for 25-35min, centrifuging at 8000-12000 Xg, and removing cells and cell fragments; continuing to centrifuge for 60-80min at 98000-102000 Xg, re-suspending the precipitate at the bottom, washing with PBS solution, and centrifuging for 60-80min at 98000-102000 Xg to remove protein polluted in exosomes, thereby obtaining fat stem cell exosomes;
S2, preparing active polysaccharide: respectively cleaning 5-7 parts by weight of oat, 10-15 parts by weight of nostoc sphaeroids kutz and 3-5 parts by weight of medlar, drying, crushing, adding 7-10 parts by weight of snail stock solution, stirring and mixing uniformly, adding 200 parts by weight of water, adding 0.5-1 part by weight of complex enzyme, heating to 45-50 ℃, carrying out enzymolysis and extraction for 2-4 hours, filtering, and reserving filter residues; adding ethanol into the filtrate until the concentration of the system ethanol is 70-80wt%, precipitating for 3-5h, filtering to obtain active polysaccharide, lyophilizing the filtrate, and mixing with the residue to obtain an extract mixture;
the compound enzyme is a mixture of cellulase and pectase, and the mass ratio is 5-7:1-2;
s3, activating zymophyte: inoculating Bifidobacterium longum and Bifidobacterium breve into Gao's medium at 36-38deg.C, 70-100r/min, and performing active culture for 24-36 hr to obtain seed solution of Bifidobacterium longum and Bifidobacterium breve with bacterial content of 10 8 -10 9 cfu/mL；
S4, preparing a culture medium: uniformly mixing 20-25 parts by weight of the fat stem cell-free culture solution in the step S1, 7-10 parts by weight of the extraction mixture in the step S2 and 100 parts by weight of deionized water, and sterilizing to obtain a culture medium;
s5, fermenting: inoculating the seed liquid of Bifidobacterium longum and Bifidobacterium breve obtained in the step S3 into the culture medium obtained in the step S4, wherein the inoculum sizes of the seed liquid of Bifidobacterium longum and Bifidobacterium breve are respectively 2-4v/v% and 1-3v/v%, under the anoxic condition, the temperature is 36-38 ℃ and the speed is 70-100r/min, the fermentation culture time is 48-72h, filtering, washing the solid with sterile water, collecting the fermentation bacteria, concentrating the obtained fermentation bacteria liquid, and the bacterial content is 10 10 -10 11 cfu/mL; lyophilizing the filtrate to obtain fermentation product;
s6, metal ion chelate: adding 15-20 parts by weight of the fermentation product obtained in the step S5 into 200 parts by weight of water, adding 0.7-1 part by weight of soluble copper salt and 0.5-1 part by weight of soluble zinc salt, stirring and mixing uniformly, dialyzing for 3-5 hours by using a dialysis bag with the aperture of 1-1.5kDa, and freeze-drying the dialysate to obtain a Zn/Cu chelated fermentation product;
s7, preparing a yeast cell lysate: adding 3-5 parts by weight of lysozyme into 1000 parts by weight of the fermentation broth prepared in the step S5, carrying out enzymolysis for 2-4 hours at 40-45 ℃, inactivating enzyme, centrifuging for 10-15 minutes at 5000-7000r/min, collecting supernatant, and freeze-drying to obtain a two-split yeast lysate;
s8, preparing an additive: uniformly mixing 3-5 parts by weight of acetyl dipeptide-1 cetyl ester, 1-3 parts by weight of palmitoyl tripeptide-5 and 2-4 parts by weight of glutathione to prepare an additive;
s9, preparing temperature-sensitive hydrogel: dissolving 5-7 parts by weight of the active polysaccharide prepared in the step S2 and 7-10 parts by weight of sodium hyaluronate in 200 parts by weight of water to obtain a mixed sugar solution; dissolving 6-8 parts by weight of hydroxypropyl methylcellulose in 100 parts by weight of water, adding a mixed sugar solution, stirring and mixing uniformly, adding 2-4 parts by weight of glycerol, stirring and mixing uniformly, and preparing the temperature-sensitive hydrogel;
S10, preparation of a composition for improving the stem cell exosome of skin: uniformly mixing 1-3 parts by weight of the fat stem cell exosome prepared in the step S1, 3-5 parts by weight of the fermentation product of chelated Zn/Cu prepared in the step S6, 2-4 parts by weight of the yeast cell lysate prepared in the step S7 and 0.5-1 part by weight of the additive prepared in the step S8, adding the mixture into 15-20 parts by weight of the temperature-sensitive hydrogel prepared in the step S9, and uniformly stirring and mixing to prepare the stem cell exosome composition for improving skin.
9. A skin improving stem cell exosome composition prepared by the method of any one of claims 1-8.
10. Use of the skin improving stem cell exosome composition according to claim 9 for preparing a product for skin aging resistance, skin elasticity restoration promotion, whitening, wrinkle removal, and moisture preservation.
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Citations (37)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB474822A (en) * | 1935-02-01 | 1937-11-08 | Carl Wilhelm Horter | Process for the production of specifically active enzyme preparations |
| BE691881A (en) * | 1965-12-28 | 1967-06-28 | ||
| SU1659480A1 (en) * | 1989-06-20 | 1991-06-30 | Научно-Исследовательский Конструкторско-Технологический Институт Биологически Активных Веществ | Strain of bacterium klebsiella pneumoniae - a producer of restrictase kpn 378 1 |
| WO2012174978A1 (en) * | 2011-06-20 | 2012-12-27 | 天津大学 | Strain improvement and process optimization in two-step mixed fermentation for production of vitamin c |
| CN105483059A (en) * | 2016-01-18 | 2016-04-13 | 王军 | Method for cultivating bifidobacteria through inulin |
| CN108721200A (en) * | 2018-06-07 | 2018-11-02 | 武汉赛云博生物科技有限公司 | A kind of preparation method and application of the excretion body cosmetic formulation in human mesenchymal stem cell source |
| CN110452842A (en) * | 2019-08-01 | 2019-11-15 | 诺佰克（武汉）生物科技有限公司 | Bifidobacterium lactis nbk-W13 and its application |
| CN110548001A (en) * | 2019-09-06 | 2019-12-10 | 沈阳细胞治疗工程技术研发中心有限公司 | Repair anti-aging skin care product containing umbilical cord mesenchymal stem cell exosomes |
| CN111139200A (en) * | 2020-01-14 | 2020-05-12 | 广州栋方生物科技股份有限公司 | Bifidobacterium lysate and preparation method thereof |
| CN111184679A (en) * | 2020-01-18 | 2020-05-22 | 西安九州再生医学集团有限公司 | Striae gravidarum repair preparation containing stem cell exosomes and preparation method and application thereof |
| CN111407716A (en) * | 2019-07-08 | 2020-07-14 | 广东先康达生物科技有限公司 | Umbilical cord mesenchymal stem cell exosome mask |
| CN111643442A (en) * | 2020-01-06 | 2020-09-11 | 热休(厦门)细胞生物科技有限公司 | Exosome beauty liquid and preparation method thereof |
| CN111803408A (en) * | 2020-08-02 | 2020-10-23 | 广州君研生物科技有限公司 | Dischizosaccharomyces cerevisiae fermentation product filtrate and application thereof in skin care products |
| CN111821252A (en) * | 2020-08-12 | 2020-10-27 | 辽宁盛京干细胞科技有限公司 | Anti-wrinkle essence containing adipose-derived stem cell extract and preparation method thereof |
| CN112675123A (en) * | 2021-01-21 | 2021-04-20 | 上海迪馥化妆品有限公司 | Active composition of three white ingredients, preparation method and application thereof |
| CN113262195A (en) * | 2021-06-02 | 2021-08-17 | 上海南滨江细胞生物科技有限公司 | Face cream containing stem cell exosomes and preparation method thereof |
| CN113662906A (en) * | 2021-08-19 | 2021-11-19 | 蓝海细胞(北京)生物科技有限公司 | Preparation method of stem cell exosome anti-aging essence |
| CN113730361A (en) * | 2021-10-19 | 2021-12-03 | 陕西科美致尚生物科技有限公司 | Exosome preparation with needle-free injection effect suitable for mucous membrane and preparation method thereof |
| CN113750030A (en) * | 2021-10-11 | 2021-12-07 | 李俊 | Anti-aging essence containing exosomes and preparation method thereof |
| CN114480549A (en) * | 2022-03-24 | 2022-05-13 | 世联生物工程无锡有限公司 | Bioactive peptide prepared from snail, and preparation method and application thereof |
| CN114591902A (en) * | 2022-03-30 | 2022-06-07 | 深圳市茵冠生物科技有限公司 | Culture method and application of mesenchymal stem cells |
| CN114703247A (en) * | 2022-06-07 | 2022-07-05 | 北京幸福能量健康科技有限公司 | High-absorptivity composite protein composition and preparation method and application thereof |
| CN114767728A (en) * | 2022-04-25 | 2022-07-22 | 扬州市职业大学(扬州开放大学) | Composition for preventing and treating Parkinson's disease and preparation method thereof |
| CN115074285A (en) * | 2022-07-01 | 2022-09-20 | 中科华启(北京)生物技术研究院有限公司 | Bifidobacterium enzymolysis lysate and preparation process and application thereof |
| CN115282065A (en) * | 2022-08-11 | 2022-11-04 | 顾帅 | Lyophilized powder containing mesenchymal stem cell exosomes and preparation method and application thereof |
| CN115349639A (en) * | 2022-10-17 | 2022-11-18 | 天津市宝恒生物科技有限公司 | Probiotic slow-release system for improving immunity and preparation method and application thereof |
| CN115404252A (en) * | 2022-08-31 | 2022-11-29 | 北京北清博育信息技术研究有限公司 | Auricularia auricula polysaccharide and application and preparation method thereof |
| KR102499346B1 (en) * | 2022-01-24 | 2023-02-14 | (주)제이비 바이오 | Natural complex fermented liquid and solid fermented ultrafine powder using it to increase active ingredients and improve absorption in the body |
| CN115944665A (en) * | 2022-10-17 | 2023-04-11 | 天津市宝恒生物科技有限公司 | Probiotic agent for improving intestinal flora balance and preparation method and application thereof |
| CN115969957A (en) * | 2022-12-21 | 2023-04-18 | 浙江佰穗莱生命健康科技有限公司 | Compound probiotic preparation for inflammatory bowel diseases and preparation method thereof |
| CN116035205A (en) * | 2023-03-14 | 2023-05-02 | 广东华南医药技术有限责任公司 | Probiotic composition for regulating metabolism and preparation method thereof |
| CN116426456A (en) * | 2023-03-08 | 2023-07-14 | 广州元基细胞生物科技有限公司 | Method for preparing lysate of saccharomyces cerevisiae product by utilizing plant exosome fermentation and application thereof |
| CN116459203A (en) * | 2023-05-24 | 2023-07-21 | 杭州拾光欣雅生物技术有限公司 | Preparation method and application of bifidobacterium/brown rice fermentation product lysate |
| CN116694474A (en) * | 2023-04-10 | 2023-09-05 | 深圳元育生物科技有限公司 | New exosome preparation method for skin care product and application thereof |
| CN116726139A (en) * | 2023-06-06 | 2023-09-12 | 南京峦创生命科技有限公司 | Composition containing palmitoyl carnitine and preparation method and application thereof |
| WO2023177208A1 (en) * | 2022-03-18 | 2023-09-21 | 주식회사 엠디헬스케어 | Method for preparing lactic acid bacterium-derived extracellular vesicles with improved yield |
| CN117025452A (en) * | 2023-07-21 | 2023-11-10 | 华南理工大学 | High-density culture method of bifidobacterium longum subspecies infantis B2-01 |
-
2023
- 2023-11-15 CN CN202311516539.8A patent/CN117243885B/en active Active
Patent Citations (37)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB474822A (en) * | 1935-02-01 | 1937-11-08 | Carl Wilhelm Horter | Process for the production of specifically active enzyme preparations |
| BE691881A (en) * | 1965-12-28 | 1967-06-28 | ||
| SU1659480A1 (en) * | 1989-06-20 | 1991-06-30 | Научно-Исследовательский Конструкторско-Технологический Институт Биологически Активных Веществ | Strain of bacterium klebsiella pneumoniae - a producer of restrictase kpn 378 1 |
| WO2012174978A1 (en) * | 2011-06-20 | 2012-12-27 | 天津大学 | Strain improvement and process optimization in two-step mixed fermentation for production of vitamin c |
| CN105483059A (en) * | 2016-01-18 | 2016-04-13 | 王军 | Method for cultivating bifidobacteria through inulin |
| CN108721200A (en) * | 2018-06-07 | 2018-11-02 | 武汉赛云博生物科技有限公司 | A kind of preparation method and application of the excretion body cosmetic formulation in human mesenchymal stem cell source |
| CN111407716A (en) * | 2019-07-08 | 2020-07-14 | 广东先康达生物科技有限公司 | Umbilical cord mesenchymal stem cell exosome mask |
| CN110452842A (en) * | 2019-08-01 | 2019-11-15 | 诺佰克（武汉）生物科技有限公司 | Bifidobacterium lactis nbk-W13 and its application |
| CN110548001A (en) * | 2019-09-06 | 2019-12-10 | 沈阳细胞治疗工程技术研发中心有限公司 | Repair anti-aging skin care product containing umbilical cord mesenchymal stem cell exosomes |
| CN111643442A (en) * | 2020-01-06 | 2020-09-11 | 热休(厦门)细胞生物科技有限公司 | Exosome beauty liquid and preparation method thereof |
| CN111139200A (en) * | 2020-01-14 | 2020-05-12 | 广州栋方生物科技股份有限公司 | Bifidobacterium lysate and preparation method thereof |
| CN111184679A (en) * | 2020-01-18 | 2020-05-22 | 西安九州再生医学集团有限公司 | Striae gravidarum repair preparation containing stem cell exosomes and preparation method and application thereof |
| CN111803408A (en) * | 2020-08-02 | 2020-10-23 | 广州君研生物科技有限公司 | Dischizosaccharomyces cerevisiae fermentation product filtrate and application thereof in skin care products |
| CN111821252A (en) * | 2020-08-12 | 2020-10-27 | 辽宁盛京干细胞科技有限公司 | Anti-wrinkle essence containing adipose-derived stem cell extract and preparation method thereof |
| CN112675123A (en) * | 2021-01-21 | 2021-04-20 | 上海迪馥化妆品有限公司 | Active composition of three white ingredients, preparation method and application thereof |
| CN113262195A (en) * | 2021-06-02 | 2021-08-17 | 上海南滨江细胞生物科技有限公司 | Face cream containing stem cell exosomes and preparation method thereof |
| CN113662906A (en) * | 2021-08-19 | 2021-11-19 | 蓝海细胞(北京)生物科技有限公司 | Preparation method of stem cell exosome anti-aging essence |
| CN113750030A (en) * | 2021-10-11 | 2021-12-07 | 李俊 | Anti-aging essence containing exosomes and preparation method thereof |
| CN113730361A (en) * | 2021-10-19 | 2021-12-03 | 陕西科美致尚生物科技有限公司 | Exosome preparation with needle-free injection effect suitable for mucous membrane and preparation method thereof |
| KR102499346B1 (en) * | 2022-01-24 | 2023-02-14 | (주)제이비 바이오 | Natural complex fermented liquid and solid fermented ultrafine powder using it to increase active ingredients and improve absorption in the body |
| WO2023177208A1 (en) * | 2022-03-18 | 2023-09-21 | 주식회사 엠디헬스케어 | Method for preparing lactic acid bacterium-derived extracellular vesicles with improved yield |
| CN114480549A (en) * | 2022-03-24 | 2022-05-13 | 世联生物工程无锡有限公司 | Bioactive peptide prepared from snail, and preparation method and application thereof |
| CN114591902A (en) * | 2022-03-30 | 2022-06-07 | 深圳市茵冠生物科技有限公司 | Culture method and application of mesenchymal stem cells |
| CN114767728A (en) * | 2022-04-25 | 2022-07-22 | 扬州市职业大学(扬州开放大学) | Composition for preventing and treating Parkinson's disease and preparation method thereof |
| CN114703247A (en) * | 2022-06-07 | 2022-07-05 | 北京幸福能量健康科技有限公司 | High-absorptivity composite protein composition and preparation method and application thereof |
| CN115074285A (en) * | 2022-07-01 | 2022-09-20 | 中科华启(北京)生物技术研究院有限公司 | Bifidobacterium enzymolysis lysate and preparation process and application thereof |
| CN115282065A (en) * | 2022-08-11 | 2022-11-04 | 顾帅 | Lyophilized powder containing mesenchymal stem cell exosomes and preparation method and application thereof |
| CN115404252A (en) * | 2022-08-31 | 2022-11-29 | 北京北清博育信息技术研究有限公司 | Auricularia auricula polysaccharide and application and preparation method thereof |
| CN115944665A (en) * | 2022-10-17 | 2023-04-11 | 天津市宝恒生物科技有限公司 | Probiotic agent for improving intestinal flora balance and preparation method and application thereof |
| CN115349639A (en) * | 2022-10-17 | 2022-11-18 | 天津市宝恒生物科技有限公司 | Probiotic slow-release system for improving immunity and preparation method and application thereof |
| CN115969957A (en) * | 2022-12-21 | 2023-04-18 | 浙江佰穗莱生命健康科技有限公司 | Compound probiotic preparation for inflammatory bowel diseases and preparation method thereof |
| CN116426456A (en) * | 2023-03-08 | 2023-07-14 | 广州元基细胞生物科技有限公司 | Method for preparing lysate of saccharomyces cerevisiae product by utilizing plant exosome fermentation and application thereof |
| CN116035205A (en) * | 2023-03-14 | 2023-05-02 | 广东华南医药技术有限责任公司 | Probiotic composition for regulating metabolism and preparation method thereof |
| CN116694474A (en) * | 2023-04-10 | 2023-09-05 | 深圳元育生物科技有限公司 | New exosome preparation method for skin care product and application thereof |
| CN116459203A (en) * | 2023-05-24 | 2023-07-21 | 杭州拾光欣雅生物技术有限公司 | Preparation method and application of bifidobacterium/brown rice fermentation product lysate |
| CN116726139A (en) * | 2023-06-06 | 2023-09-12 | 南京峦创生命科技有限公司 | Composition containing palmitoyl carnitine and preparation method and application thereof |
| CN117025452A (en) * | 2023-07-21 | 2023-11-10 | 华南理工大学 | High-density culture method of bifidobacterium longum subspecies infantis B2-01 |
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