Silk fibroin essence liquid, preparation method and application of silk fibroin essence liquid in cosmetics

Abstract

The invention relates to the technical field of cosmetic product preparation, in particular to silk fibroin essence liquid, a preparation method and application of the silk fibroin essence liquid in cosmetics. Wherein in the preparation of the refined silk fibroin liquid, silk is degummed to obtain macromolecular silk fibroin, the macromolecular silk fibroin is dissolved in a mixed solution to obtain the silk fibroin liquid, and uniform dispersion is realized. Through dialysis desalination, concentration and enzymolysis, the structural properties of macromolecular silk fibroin are changed, and a silk fibroin enzymatic hydrolysate is separated to obtain a silk fibroin enzymatic hydrolysate which serves as an important macromolecular product to become a fermentation substrate. After fermentation, silk fibroin fermentation liquor is obtained, and components beneficial to skin care are generated. And mixing the small molecular silk fibroin peptide enzymatic hydrolysate with the silk fibroin fermentation liquor to obtain refined liquor. The silk fibroin enzymatic hydrolysate and fermentation liquor derived from macromolecular silk fibroin have good effects of resisting wrinkles, tightening, repairing and the like.

Description

Technical Field

[0001] The invention relates to the technical field of cosmetic product preparation, and in particular to a silk fibroin extract, a preparation method and application thereof in cosmetics. Background Art

[0002] As people's living standards improve, the requirements for the efficacy and quality of cosmetics are increasing. The market demand for functional skin care products such as anti-wrinkle, firming, and repair continues to expand, and consumers are eager to find products that can truly improve skin problems and enhance skin health. Silk fibroin has received widespread attention in the cosmetics field due to its unique physical and chemical properties and biological activity.

[0003] Silk fibroin is the main component of silk. It is abundant in source and has good biocompatibility, moisture retention and degradability. Studies have shown that the various amino acids and peptides in silk fibroin can provide nutrition to the skin, promote cell metabolism and collagen synthesis, and have a nourishing and repairing effect on the skin. However, natural silk fibroin has some limitations, and the direct application in cosmetics is not effective.

[0004] Although traditional silk fibroin processing methods can improve its performance to a certain extent, they still have many shortcomings. The products obtained by the early simple hydrolysis technology have complex components, and it is difficult to accurately control their molecular weight and active ingredients, resulting in unstable product quality and insignificant skin care effects. At the same time, microbial contamination problems often occur during the production process, affecting the safety and stability of the product.

[0005] In addition, most of the existing silk protein-related products on the market have a single function and cannot meet consumers' demand for multiple skin care effects. Pure moisturizing products cannot effectively solve the problem of anti-wrinkle and firming skin, and products that focus on anti-wrinkle are weak in repairing the skin barrier. Summary of the invention

[0006] 1. Technical issues to be resolved The technical problem to be solved by the present invention is that the existing silk fibroin moisturizing products on the market cannot effectively solve the problem of anti-wrinkle and firming of the skin, and the products focusing on anti-wrinkle are relatively weak in repairing the skin barrier.

[0007] (II) Technical solution To this end, the first aspect of the present invention mentions a method for preparing a silk fibroin extract, comprising: Step S100: placing the silk in a sodium carbonate solution of a preset concentration to undergo a degumming treatment to obtain macromolecular silk fibroin; Step S200: preparing a mixed solution according to the molar ratio of lithium bromide:methanol:water of 2:(3-5):(5-7), placing the macromolecular silk fibroin in the mixed solution and dissolving it to obtain a silk fibroin solution; Step S300: After desalting the silk fibroin solution, the silk fibroin solution is concentrated by polyethylene glycol; Step S400: enzymatically hydrolyzing the concentrated silk fibroin solution to obtain a silk fibroin enzymatic hydrolyzate; Step S500: separating the silk fibroin enzymatic hydrolysate to obtain a first silk fibroin peptide enzymatic hydrolysate and a second silk fibroin protein enzymatic hydrolysate with different molecular weights; Step S600: mixing activated Staphylococcus epidermidis and Lactobacillus plantarum into the first silk fibroin protein hydrolysate according to a preset ratio for fermentation, and collecting the silk fibroin protein fermentation liquid after the preset fermentation time; Step S700: Mix the second silk fibroin peptide hydrolyzate and the silk fibroin fermentation solution in a mass ratio of 1:(0.5-2) to obtain a silk fibroin extract.

[0008] Furthermore, the concentration of the sodium carbonate solution is 5 g / L-20 g / L, and the material-liquid ratio of silk to sodium carbonate solution is (1:20)-(1:30) by mass percentage.

[0009] Furthermore, in terms of mass percentage, the mass ratio of the macromolecular silk fibroin to the mixed solution is (1:40-80).

[0010] Further, step S300 includes: putting the dissolved silk fibroin solution into a dialysis bag, placing it in a large beaker filled with deionized water for dialysis and desalination, replacing the dialysis external fluid every 2-3 hours, and dialysis for 12-24 hours until no lithium bromide ions are detected in the dialysis external fluid. After the dialysis is completed, the silk fibroin solution and polyethylene glycol are transferred to a centrifuge tube in a mass ratio of 1: (1-5) for concentration.

[0011] Further, step S400 includes: adjusting the pH of the concentrated silk fibroin solution to 6.0-7.0, mixing the mass percentages of papain: silk fibroin at 1:100-1:50 and flavor protease: silk fibroin at 1:100-1:50, performing a composite hydrolysis reaction for 4-8 hours, and obtaining an enzymatic solution after the reaction is completed.

[0012] Furthermore, step S400 includes: heating the enzymatic hydrolysate at 90-100° C. for 10-15 minutes to obtain a silk fibroin enzymatic hydrolysate.

[0013] Further, step S600 includes: mixing activated Staphylococcus epidermidis and Lactobacillus plantarum in a ratio of (1:1-3) to form a mixed strain, placing a fermentation container containing the mixed strain and silk fibroin enzymatic hydrolysate in a sterile environment at a temperature of 30-35°C for fermentation.

[0014] Further, step S600 includes: after the mixed strain and the silk fibroin protein hydrolysate are co-fermented for 6-30 hours, centrifuged at 3000-5000 rpm for 10-15 minutes to precipitate the bacteria, and the supernatant is collected to obtain the silk fibroin fermentation liquid.

[0015] The second aspect of the present application mentions a silk fibroin extract, which is prepared by the preparation method of a silk fibroin extract mentioned in the first aspect above.

[0016] The third aspect of the present application mentions the application of a silk fibroin extract in cosmetics.

[0017] (III) Beneficial effects The present invention discloses a silk fibroin extract, a preparation method and application thereof in cosmetics, wherein the method comprises: placing silk in a sodium carbonate solution of a preset concentration for degumming to obtain macromolecular silk fibroin; preparing a mixed solution according to a molar ratio of lithium bromide:methanol:water of 2:(3-5):(5-7), placing the macromolecular silk fibroin in the mixed solution for dissolution to obtain a silk fibroin liquid; dialyzing the silk fibroin liquid for desalination, and concentrating the silk fibroin liquid with polyethylene glycol; adding papain and flavor protease to the concentrated silk fibroin liquid for The method comprises the steps of: performing enzymatic hydrolysis to obtain a silk fibroin enzymatic hydrolysate; separating the silk fibroin enzymatic hydrolysate by an ultrafiltration concentration tube to obtain a small molecule silk fibroin protein peptide enzymatic hydrolysate of less than 10 kd and a silk fibroin protein enzyme enzymatic hydrolysate of greater than 10 kd, respectively; mixing activated Staphylococcus epidermidis and Lactobacillus plantarum into the silk fibroin protein enzyme enzymatic hydrolysate of greater than 10 kd in a preset ratio for fermentation, and collecting the silk fibroin protein fermentation liquid after a preset fermentation time; mixing the small molecule silk fibroin protein peptide enzymatic hydrolysate of less than 10 kd and the silk fibroin protein fermentation liquid in a mass ratio of 1:(0.5-2) to obtain a silk fibroin protein extract.

[0018] The silk protein extract prepared by this method has significant effects in anti-wrinkle, firming and repair. In terms of anti-wrinkle and firming, the larger peptides and proteins in the silk protein hydrolysate greater than 10kd obtained by ultrafiltration separation can form a protective film on the skin surface, lock in moisture, increase skin luster and elasticity, and reduce wrinkles; during the fermentation process, the polysaccharides and other metabolites produced by the activation of Staphylococcus epidermidis and Lactobacillus plantarum can further enhance the skin's moisturizing ability, make the skin hydrated and plump, improve skin sagging, and enhance firmness. In terms of repair efficacy, the small molecule silk protein peptide hydrolysate less than 10kd can quickly penetrate the skin's stratum corneum, penetrate deep into the bottom layer of the skin, provide nutrition and repair signals to the skin, and promote cell repair and regeneration; enzymes and other components in the fermentation broth participate in intracellular biochemical reactions, accelerate the renewal of aging cells, and vitamins provide antioxidant protection, resist free radical damage, and repair damaged skin in all directions, thereby showing good anti-wrinkle, firming and repair effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to make the contents of the present invention more clearly understood, the present invention is further described in detail below based on the specifics of the present invention and in conjunction with the accompanying drawings.

[0020] Figure 1 A flow chart of a method for preparing a silk fibroin extract solution mentioned in the present invention; Figure 2 A comparison chart of the expression of collagen I in cells by the products prepared in the embodiments of the present invention; Figure 3 This is a comparison chart of the migration rates of keratinocytes of the products prepared in the embodiments mentioned in the present invention. DETAILED DESCRIPTION

[0021] The present invention will be further described below in conjunction with the accompanying drawings and details so that those skilled in the art can better understand the present invention and implement it, but the examples are not intended to limit the present invention.

[0022] refer to Figure 1 The first aspect of the present invention relates to a method for preparing a silk fibroin extract, comprising: Step S100: placing the silk in a sodium carbonate solution of a preset concentration for degumming to obtain macromolecular silk fibroin.

[0023] It should be noted that the surface of silk contains impurities such as sericin, which will affect the subsequent extraction and performance of silk fibroin. Degumming with sodium carbonate solution can selectively remove sericin and separate the macromolecular silk fibroin in silk. Controlling the concentration of sodium carbonate solution at 5g / L-20g / L and the material-liquid ratio of silk to sodium carbonate solution at (1:20)-(1:30) can effectively degumming while avoiding damage to the structure of silk fibroin caused by excessive treatment, ensuring the integrity and quality of macromolecular silk fibroin, and laying the foundation for the subsequent preparation of high-quality silk fibroin extract.

[0024] Step S200: preparing a mixed solution according to the molar ratio of lithium bromide:methanol:water of 2:(3-5):(5-7), dissolving the macromolecular silk fibroin in the mixed solution, and obtaining a silk fibroin solution.

[0025] It should be noted that the mixed solution can provide a suitable dissolving environment and has good dissolving properties for macromolecular silk fibroin. It is ensured that the polarity, ionic strength and other properties of the mixed solution are conducive to the destruction of the interaction between silk fibroin molecules, so that it is fully dissolved to form a uniform silk fibroin solution. The mass ratio of macromolecular silk fibroin to the mixed solution is controlled to (1:40-80), which ensures that the concentration of silk fibroin in the solution is moderate, which is not only conducive to subsequent operations, but also allows the silk fibroin to be fully dissolved without oversaturation or precipitation, providing a stable solution system for subsequent processing.

[0026] Step S300: After the silk fibroin solution is dialyzed to remove salt, the silk fibroin solution is concentrated by polyethylene glycol.

[0027] It should be noted that during the dissolution process, lithium bromide ions are introduced into the silk fibroin solution, which will affect the subsequent enzymatic hydrolysis reaction and the properties of silk fibroin. By dialysis desalination, the dialysis external fluid is replaced every 2-3 hours, and the dialysis is performed for 12-24 hours, which can effectively remove lithium bromide ions in the silk fibroin solution, obtain a pure silk fibroin solution, improve the purity of silk fibroin, and reduce the interference of impurities on subsequent reactions.

[0028] Transferring the silk fibroin solution and polyethylene glycol in a mass ratio of 1: (1-5) to a centrifuge tube for concentration can remove water from the solution and increase the concentration of silk fibroin. The appropriate mass ratio can effectively achieve the purpose of concentration without affecting the structure and properties of silk fibroin, so that the silk fibroin solution reaches an appropriate concentration, which is beneficial to the subsequent enzymatic hydrolysis reaction and improves the reaction efficiency.

[0029] Step S400: adding papain and flavor protease to the concentrated silk fibroin solution for enzymolysis to obtain a silk fibroin enzymolysis solution.

[0030] In the present application, the pH of the concentrated silk fibroin solution is adjusted to 6.0-7.0, which is conducive to efficient enzymatic hydrolysis of the silk fibroin, decomposing the macromolecular silk fibroin into smaller peptide segments, and improving the availability and biological activity of the silk fibroin.

[0031] In the present application, the mass percentage of papain: silk protein is 1:100-1:50, and the mass percentage of flavor protease: silk protein is 1:100-1:50, and the two enzymes act synergistically to perform a composite hydrolysis reaction on the silk protein. Different enzymes act on different sites of the silk protein, making the enzymatic reaction more sufficient and comprehensive, producing diversified peptide segments, enriching the composition of the silk protein enzymatic hydrolyzate, and helping to improve the functional diversity of the silk protein extract.

[0032] In the present application, the composite hydrolysis reaction is carried out for 4-8 hours to ensure that the enzymatic reaction reaches an appropriate degree, neither excessive enzymatic hydrolysis resulting in the peptide being too short and losing certain functions, nor insufficient enzymatic hydrolysis resulting in excessive residues of macromolecular silk protein. Through enzymatic hydrolysis, a mixture of peptides of moderate size and having different functions can be obtained, providing rich raw materials for the subsequent preparation of silk protein extracts with multiple functions.

[0033] In this application, the enzymatic solution is heated at 90-100°C for 10-15 minutes, which can inactivate the enzyme, terminate the enzymatic reaction, and prevent the enzyme from continuing to act and causing excessive hydrolysis of the peptide segment. At the same time, high temperature treatment has a bactericidal effect, reduces the number of microorganisms in the enzymatic solution, reduces the risk of subsequent microbial contamination, and ensures the quality stability of the silk fibroin enzymatic solution. In addition, appropriate high temperature treatment will cause some fine-tuning of the molecular structure of silk fibroin, prompting the spatial structure of some peptide segments to change, exposing more potential active sites, thereby enhancing the biological activity and functional properties of the silk fibroin extract.

[0034] Step S500: Separate the silk fibroin hydrolysate through an ultrafiltration concentration tube to obtain a first silk fibroin protein peptide hydrolysate and a second silk fibroin protein hydrolysate with different molecular weights; wherein the first silk fibroin protein peptide hydrolysate is a silk fibroin protein peptide hydrolysate with a molecular weight greater than 10 kd, and the second silk fibroin protein peptide hydrolysate is a small molecule silk fibroin protein peptide hydrolysate with a molecular weight less than 10 kd.

[0035] It should be noted that the ultrafiltration concentration tube separates the silk fibroin hydrolysate based on the difference in molecular weight, and can effectively classify the silk fibroin peptides after enzymatic hydrolysis according to the molecular weight to obtain small molecule silk fibroin peptide hydrolysate less than 10kd and silk fibroin protein hydrolysate greater than 10kd. Due to its small molecular weight, small molecule silk fibroin peptides are easier to penetrate the stratum corneum of the skin, can be absorbed and utilized by the skin more quickly, and provide immediate nourishment and repair effects to the skin; while the larger peptides and proteins in the silk fibroin protein hydrolysate greater than 10kd have different functions, forming a protective film on the surface of the skin, which helps to lock in moisture and increase the skin's luster and elasticity. Through separation, a variety of raw material options are provided for the subsequent preparation of silk fibroin protein extracts according to different needs and functions, and products with specific functions can be more accurately formulated to meet different skin types and skin care needs.

[0036] Step S600: Mixing activated Staphylococcus epidermidis and Lactobacillus plantarum into a silk fibroin protein hydrolysate with a pre-set ratio for fermentation, and collecting the silk fibroin protein fermentation liquid after a pre-set fermentation time.

[0037] Activated Staphylococcus epidermidis and Lactobacillus plantarum were purchased from Kunshan Laber Instrument Equipment Co., Ltd., among which the preservation number of Lactobacillus plantarum is CICC21804, and the preservation number of activated Staphylococcus epidermidis is CICC10294.

[0038] It should be noted that the activated Staphylococcus epidermidis and Lactobacillus plantarum are mixed at a ratio of 1:1-1:3, and the two strains have different metabolic characteristics and functions. Staphylococcus epidermidis and Lactobacillus plantarum use the nutrients in the silk protein hydrolysate to grow and reproduce, and secrete a variety of metabolites. These metabolites include organic acids, enzymes, vitamins, polysaccharides, etc., which enrich the components of the silk protein fermentation liquid.

[0039] It should be noted that the fermentation container containing the mixed strain and silk fibroin enzymatic hydrolysate is placed in a sterile environment at a temperature of 30-35°C for fermentation to ensure the activity of the strain and the normal metabolic activity. The sterile environment can prevent contamination by foreign bacteria, ensure the stability and repeatability of the fermentation process, and ensure the quality and safety of the fermentation product.

[0040] It should be noted that the mixed strain and the silk fibroin protein hydrolysate are fermented together for 6-30 hours, so that the strain has enough time to fully metabolize the silk fibroin protein hydrolysate. The fermentation reaction proceeds gradually, the structure and properties of the silk fibroin protein change, and more biologically active substances are produced. If the fermentation time is too short, insufficient metabolites are generated, and the fermentation effect is not obvious; if the fermentation time is too long, it will lead to excessive fermentation, produce bad odors or affect the stability of the product.

[0041] It should be noted that after the fermentation is completed, centrifugation is performed at 3000-5000 rpm for 10-15 minutes to precipitate the bacteria, and the supernatant is taken to obtain the silk fibroin fermentation liquid. The bacterial cells in the fermentation liquid can be effectively removed to obtain a clarified silk fibroin fermentation liquid, thereby improving the purity and stability of the fermentation liquid, and facilitating subsequent mixing with a small molecule silk fibroin peptide enzymatic hydrolyzate and application in cosmetics.

[0042] Step S700: mixing the small molecule silk fibroin peptide hydrolysate less than 10 kd and the silk fibroin fermentation liquid in a preset ratio to obtain a silk fibroin extract.

[0043] The small molecule silk protein peptide hydrolysate less than 10kd and the silk protein fermentation liquid are mixed in a preset ratio to achieve the complementary advantages of different functional components. The high permeability and easy absorption of the small molecule silk protein peptide hydrolysate, combined with the rich metabolites in the silk protein fermentation liquid, the fermented and modified silk protein and other ingredients, make the silk protein extract have more comprehensive skin care effects.

[0044] At the same time, this method of mixing according to preset proportions ensures the stability and consistency of product quality, which is conducive to large-scale production and market promotion.

[0045] It should be noted that small molecule peptides can quickly penetrate into the bottom layer of the skin, providing nutrition and repair signals to the skin, and the organic acids in the fermentation liquid can adjust the pH value of the skin surface, making it closer to the natural pH environment of the skin, enhancing the barrier function of the skin, and improving the skin's resistance; enzyme substances participate in the biochemical reactions in skin cells, promote cell metabolism, and accelerate the renewal of aging cells; vitamins can provide antioxidant protection for the skin, resist free radical damage to the skin, and delay skin aging; polysaccharides help to form a moisturizing film on the skin surface, further enhancing the skin's moisturizing ability and keeping the skin hydrated. The silk protein extract mentioned in this application can not only protect the skin from multiple levels and meet the needs of different skin problems, but also use the synergistic effect between different ingredients to exert a more powerful effect than a single ingredient.

[0046] The second aspect of the present application mentions a silk fibroin extract, which is prepared by the preparation method of the silk fibroin extract mentioned in the first aspect above. It has all the beneficial effects mentioned in the first aspect above, which will not be repeated here.

[0047] The third aspect of the present application mentions the application of a silk fibroin extract in cosmetics.

[0048] In order to better understand the technical solution, the technical solution of the present invention is described in detail below by way of embodiments.

[0049] Example Step S100: degumming to obtain macromolecular silk fibroin; Prepare a sodium carbonate solution with a concentration of 10g / L. Weigh 10g of silk and measure 250g of sodium carbonate solution according to the mass percentage of silk to sodium carbonate solution of 1:25. Put the silk into the sodium carbonate solution, place it in a constant temperature water bath, heat and stir at 90-100°C for 30-60 minutes, and stir continuously during the degumming to make the cocoon degummed evenly. After degumming, centrifuge at a speed of 3000-5000rpm for 5-10 minutes, collect the silk fibroin, and wash it repeatedly with deionized water to remove the residual sodium carbonate until the pH of the washing solution is close to neutral to obtain macromolecular silk fibroin.

[0050] Step S200: preparing a mixed solution and dissolving the macromolecular silk fibroin; The mixed solution was prepared according to the molar ratio of lithium bromide: methanol: water of 2: 4: 6. 5 g of the macromolecular silk fibroin after degumming was weighed, and 300 g of the mixed solution was measured according to the mass percentage of the macromolecular silk fibroin to the mixed solution of 1: 60. The macromolecular silk fibroin was added to the mixed solution, and stirred evenly to fully dissolve it to obtain a silk fibroin solution.

[0051] Step S300: dialysis to remove salt and concentrate the silk fibroin solution; The dissolved silk fibroin solution was placed in a dialysis bag and placed in a large beaker filled with deionized water for dialysis and desalination. The dialysis external solution was replaced every 2 hours for 18 hours until no lithium bromide ions were detected in the dialysis external solution. After the dialysis was completed, the silk fibroin solution and polyethylene glycol were transferred to a centrifuge tube at a mass ratio of 1:3 for concentration to obtain a concentrated silk fibroin solution, wherein the average molecular weight of the polyethylene glycol was 20,000.

[0052] Step S400: obtaining a silk fibroin enzymatic hydrolyzate by enzymatic reaction; The concentrated silk fibroin solution was adjusted to pH 6.5. The mixture was mixed according to the mass percentage of papain: silk fibroin of 1:80 and the mass percentage of flavor protease: silk fibroin of 1:80. That is, appropriate amounts of papain and flavor protease were added to the silk fibroin solution and the composite hydrolysis reaction was carried out for 6 hours. After the reaction was completed, the enzymatic hydrolyzate was heated at 95°C for 12 minutes to obtain a silk fibroin enzymatic hydrolyzate.

[0053] Step S500: separating the silk fibroin enzymatic hydrolysate; The silk fibroin enzymatic hydrolysate was separated by ultrafiltration concentration tube to obtain small molecule silk fibroin peptide enzymatic hydrolysate less than 10 kd and silk fibroin protein enzymatic hydrolysate greater than 10 kd.

[0054] Step S600: fermenting to prepare silk fibroin fermentation liquid; After the Staphylococcus epidermidis and Lactobacillus plantarum are activated, they are mixed and cultured in a ratio of 1:2. The mixed cultured strains are added to a silk protein hydrolysate greater than 10 kd, and the fermentation container containing the mixed strains and the silk protein hydrolysate is placed in a sterile environment at a temperature of 32° C. for fermentation for 18 hours. After the fermentation is completed, centrifugation is performed at 4000 rpm for 12 minutes to precipitate the bacteria, and the supernatant is taken to obtain a silk protein fermentation liquid.

[0055] Step S700: mixing to obtain a silk fibroin extract; The enzymatic hydrolyzate of small molecule silk protein peptides less than 10 kd and the silk protein fermentation liquid were mixed in a mass ratio of 1:1 and stirred evenly to obtain the silk protein extract. Result analysis; The Pierce quantitative colorimetric peptide detection kit was used, and based on its usage method, the polypeptide concentration of the fermentation broth obtained by adding Staphylococcus epidermidis and Lactobacillus plantarum to the mixed fermentation of silk fibroin was determined: 1% concentration of the above concentrated protein solution was added to the fermentation medium. As can be seen from Table 1, the polypeptide concentration in the fermentation broth obtained after mixed fermentation changes with time, and the polypeptide concentration reaches a maximum of 257μg / ml after 24 hours of fermentation. When the fermentation time continues to extend, the polypeptide concentration shows a downward trend. Therefore, the time for mixed fermentation is selected as 24h, and the fermentation broth at this time is used to determine the skin care effect.

[0056] Table 1 6h 12h 18h 24h 30h Peptide concentration 56μg / ml 113μg / ml 185μg / ml 257μg / ml 204μg / ml Skin care efficacy determination; The determination was carried out with reference to the type 1 collagen kit and its usage method of Nanjing Jiancheng Bioengineering Institute. The protein solution of less than 10 kd and the fermentation broth after fermentation were mixed in a certain ratio. The following ratios were selected in this study: the protein solution of less than 10 kd and the fermentation broth after fermentation were mixed in a ratio of 1:1 and recorded as S1, the protein solution of less than 10 kd and the fermentation broth after fermentation were mixed in a ratio of 1:2 and recorded as S2, and the protein solution of less than 10 kd and the fermentation broth after fermentation were mixed in a ratio of 2:1 and recorded as S3.

[0057] The effect of the samples in the embodiment on the expression of collagen I in cells was studied by establishing an in vitro fibroblast model. Figure 2 It can be seen that the expression level of collagen I in the fibroblasts of the S2 sample group was the highest at 50.1 ng / ml, which was significantly different from that of the blank control group. This shows that the sample obtained by mixing the protein solution less than 10 kd and the fermented liquid in this ratio has significant anti-wrinkle and firming effects.

[0058] By establishing an in vitro keratinocyte model, the effects of samples on cell scratch test were studied. Figure 3 It can be seen that the migration rate of keratinocytes by the S2 sample reached 1.9, which was significantly different from that of the blank control group, indicating that the sample obtained by mixing the protein solution less than 10kd and the fermented liquid in this ratio had a significant repair effect.

[0059] Obviously, the above is only an example for clear explanation and is not a limitation of the implementation method. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from this are still within the protection scope of the invention.

[0060] In the description of the present invention, it should be understood that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0061] In the present invention, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium; it can be the internal connection of two elements or the interaction relationship between two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0062] In the present invention, unless otherwise clearly specified and limited, when a first feature is “on” or “below” a second feature, it may be that the first and second features are in direct contact, or that the first and second features are in indirect contact through an intermediate medium. Moreover, when a first feature is “above”, “above” or “above” a second feature, it may be that the first feature is directly above or obliquely above the second feature, or it may simply mean that the first feature is higher in level than the second feature. When a first feature is “below”, “below” or “below” a second feature, it may be that the first feature is directly below or obliquely below the second feature, or it may simply mean that the first feature is lower in level than the second feature.

[0063] In the description of this specification, the description of the terms "one", "some", "", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the or example are included in at least one or examples of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same or examples. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different or examples and the features of the different or examples described in this specification without contradiction.

[0064] Although the present invention has been shown and described above, it is to be understood that the above is exemplary and is not to be construed as limiting the present invention. A person skilled in the art may make changes, modifications, substitutions and variations to the above within the scope of the present invention.

Claims

1. A method for preparing a silk fibroin extract, characterized in that: include: Step S100: placing the silk in a sodium carbonate solution of a preset concentration to undergo a degumming treatment to obtain a macromolecular silk fibroin; Step S200: preparing a mixed solution according to the molar ratio of lithium bromide:methanol:water of 2:(3-5):(5-7), placing the macromolecular silk fibroin in the mixed solution and dissolving it to obtain a silk fibroin solution; Step S300: After desalting the silk fibroin solution, the silk fibroin solution is concentrated by polyethylene glycol; Step S400: enzymatically hydrolyzing the concentrated silk fibroin solution to obtain a silk fibroin enzymatic hydrolyzate; Step S500: separating the silk fibroin enzymatic hydrolysate to obtain a first silk fibroin peptide enzymatic hydrolysate and a second silk fibroin protein enzymatic hydrolysate with different molecular weights; Step S600: mixing activated Staphylococcus epidermidis and Lactobacillus plantarum into the first silk fibroin protein hydrolysate according to a preset ratio for fermentation, and collecting the silk fibroin protein fermentation liquid after the preset fermentation time; Step S700: Mix the second silk fibroin peptide hydrolysate and the silk fibroin fermentation solution in a mass ratio of 1:(0.5-2) to obtain a silk fibroin extract.

2. The method for preparing a silk fibroin extract according to claim 1, characterized in that ； The concentration of the sodium carbonate solution is 5g / L-20g / L, and the material-liquid ratio of silk to the sodium carbonate solution is (1:20)-(1:30) in terms of mass percentage.

3. The method for preparing a silk fibroin extract according to claim 1, characterized in that ； In terms of mass percentage, the mass ratio of the macromolecular silk fibroin to the mixed solution is (1:40-80).

4. The method for preparing a silk fibroin extract according to claim 1, characterized in that ； Step S300 includes: putting the dissolved silk fibroin solution into a dialysis bag, placing it in a large beaker filled with deionized water for dialysis and desalination, replacing the dialysis external fluid every 2-3 hours, and dialysis for 12-24 hours until no lithium bromide ions are detected in the dialysis external fluid. After the dialysis is completed, the silk fibroin solution and polyethylene glycol are transferred to a centrifuge tube in a mass ratio of 1: (1-5) for concentration.

5. The method for preparing a silk fibroin extract according to claim 1, characterized in that ； Step S400 includes: adjusting the pH of the concentrated silk fibroin solution to 6.0-7.0, mixing papain: silk fibroin at a mass percentage of 1:100-1:50 and flavor protease: silk fibroin at a mass percentage of 1:100-1:50, performing a composite hydrolysis reaction for 4-8 hours, and obtaining an enzymatic solution after the reaction is completed.

6. The method for preparing a silk fibroin extract according to claim 5, characterized in that ； Step S400 includes: heating the enzymatic hydrolyzate at 90-100° C. for 10-15 minutes to obtain a silk fibroin enzymatic hydrolyzate.

7. The method for preparing a silk fibroin extract according to claim 1, characterized in that ； Step S600 includes: mixing activated Staphylococcus epidermidis and Lactobacillus plantarum in a ratio of (1:1-3) to form a mixed strain, placing a fermentation container containing the mixed strain and silk fibroin enzymatic hydrolysate in a sterile environment at a temperature of 30-35° C. for fermentation.

8. The method for preparing a silk fibroin extract according to claim 7, characterized in that ； Step S600 includes: after the mixed strain and the silk fibroin protein hydrolysate are co-fermented for 6-30 hours, centrifuged at 3000-5000 rpm for 10-15 minutes to precipitate the bacteria, and taking the supernatant to obtain the silk fibroin fermentation liquid.

9. A silk fibroin extract, prepared by the method for preparing a silk fibroin extract as claimed in any one of claims 1 to 8.

10. Use of the silk fibroin extract according to claim 9 in cosmetics.

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