Functional peptide fragment and silk fibroin peptide having Anti-wrinkle effect, preparation method therefor, and application thereof
Patent Information
- Application Number
- PCT/CN2026/103371
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2025-04-14
- Filing Date
- 2026-06-12
- Publication Date
- 2026-10-22
Smart Images
Figure CN2026103371_22102026_PF_FP_ABST
Abstract
Description
A functional peptide with anti-wrinkle effects, silk fibroin peptide, its preparation method and application
[0001] This application claims priority to Chinese Patent Application No. 202510468052.X, filed on April 14, 2025, entitled "A functional peptide with anti-wrinkle effect, silk fibroin peptide and its preparation method and application", the entire contents of which are incorporated herein by reference. Technical Field
[0002] This application relates to biotechnology, and more particularly to a functional peptide with anti-wrinkle effects, a silk fibroin peptide, its preparation method and application. Background Technology
[0003] Skin aging and wrinkle formation is a complex and multifactorial natural process. This process involves changes in skin structure, decline in cell function, and the influence of the external environment, among other aspects.
[0004] First, from a skin structure perspective, as we age, the dermis gradually thins, and the production of collagen and elastin fibers decreases, leading to a loss of skin elasticity and firmness. The atrophy of the subcutaneous fat layer also contributes to skin laxity and wrinkle formation. Simultaneously, with skin aging, the secretion of moisture and oil decreases, making the skin more prone to dryness and fine lines. Second, declining cell function is also a significant cause of skin aging. The metabolic capacity of skin cells weakens with age, with fewer new cells and more aging cells, resulting in decreased skin repair capabilities and increased susceptibility to damage from the external environment. External environmental factors, such as ultraviolet radiation, air pollution, and unhealthy lifestyle habits, also accelerate skin aging and wrinkle formation. Ultraviolet radiation can damage collagen and elastin fibers in the skin, leading to sagging and wrinkles; harmful substances in air pollution also damage the skin, further accelerating aging.
[0005] Therefore, enhancing the health of the dermis, improving skin cell function, and strengthening the skin's ability to repair damage from the external environment are key to delaying skin aging and resisting wrinkle formation. Summary of the Invention
[0006] This application provides a functional peptide and silk fibroin peptide with anti-wrinkle effects, which can upregulate the relative expression levels of type I collagen (col1a1a, col1a1b) and elastin (elna, elnb) genes in the skin to achieve anti-wrinkle effects.
[0007] This application provides a method for preparing the aforementioned silk fibroin peptides. The silk fibroin peptides can be obtained through the selection of enzyme preparations, enzyme treatment processes, and corresponding separation and purification processes. This preparation method is simple and rapid, and convenient for large-scale production.
[0008] This application also provides the application of the above-mentioned functional peptides, and / or the above-mentioned silk fibroin peptides, and / or the silk fibroin peptides prepared by the above-mentioned preparation method in the preparation of anti-wrinkle related products.
[0009] This application provides a functional peptide with anti-wrinkle effects, wherein the functional peptide includes peptide GA, peptide GAY and peptide AA.
[0010] This application provides a silk fibroin peptide with anti-wrinkle effects, wherein the silk fibroin peptide includes the aforementioned functional peptide segments.
[0011] The silk fibroin peptides described above, wherein, based on the mass of the silk fibroin peptides, the mass content of peptide GA is ≥0.07%, the mass content of peptide GAY is ≥0.01%, and the mass content of peptide AA is ≥0.02%.
[0012] The silk fibroin peptides described above, wherein the moisture content is ≤2g / 100g and the ash content is ≤30g / 100g; and / or,
[0013] The protein content in silk fibroin peptides is ≥70g / 100g; and / or,
[0014] The content of acid-soluble protein in silk fibroin peptides is ≥70g / 100g; and / or,
[0015] The free amino acid content in silk fibroin peptides is ≤20g / 100g; and / or,
[0016] Silk fibroin peptides contain ≥50g / 100g peptides; and / or,
[0017] The mass content of peptides with a molecular weight of less than 1000u in silk fibroin peptides is ≥80%.
[0018] The silk fibroin peptides mentioned above are obtained by using silkworm cocoon protein as raw material, and then undergoing enzymatic hydrolysis and separation purification.
[0019] The enzymatic hydrolysis process includes enzymatic hydrolysis using at least one of neutral protease and carboxypeptidase A, and at least includes neutral protease.
[0020] This application provides a method for preparing the above-mentioned silk fibroin peptide, comprising the following steps:
[0021] Silk fibroin peptides were obtained by enzymatic hydrolysis and separation and purification of silk cocoon protein as raw material.
[0022] The enzymatic hydrolysis process includes enzymatic hydrolysis using at least one of neutral protease and carboxypeptidase A, and at least includes neutral protease.
[0023] In the preparation method described above, the enzymatic hydrolysis treatment is carried out at a temperature of 45-55℃ for 4.0-6.0 h, and the pH value is 6.5-7.5; and / or,
[0024] The enzyme activity of neutral proteases is 140,000-160,000 U / g; and / or,
[0025] Based on the amount of silkworm cocoon protein per gram, the amount of neutral protease added is 0.2 wt%-2 wt%; and / or,
[0026] The enzyme activity of carboxypeptidase A is 4000-6000 U / g; and / or,
[0027] Based on each gram of silkworm cocoon protein, the amount of carboxypeptidase A added is 0.2wt%-2wt%.
[0028] The preparation method described above, wherein silkworm cocoon protein can be obtained through the following steps:
[0029] Silkworm cocoons and water were treated at 110-130℃ and 0.05-0.17MPa for 0.5-1.5h, and the insoluble matter was obtained by filtration. The insoluble matter, water and sodium hydroxide were then treated at 110-130℃ and 0.05-0.17MPa for 1.0-2.0h to obtain silkworm cocoon protein.
[0030] In the preparation method described above, the separation and purification process includes at least one of diatomaceous earth adsorption treatment, cation exchange resin treatment, anion exchange resin treatment, and macroporous resin treatment.
[0031] The preparation method described above, wherein the above-mentioned functional peptides, and / or the above-mentioned silk fibroin peptides, and / or the silk fibroin peptides prepared by the above-mentioned preparation method are used in the preparation of anti-wrinkle related products.
[0032] This application provides a functional peptide with anti-wrinkle effects, including peptide GA, peptide GAY, and peptide AA. This functional peptide can increase skin elasticity by upregulating the relative expression levels of type I collagen (col1a1a, col1a1b) and elastin (elna, elnb) genes, thereby achieving an anti-wrinkle effect. Attached Figure Description
[0033] Figure 1 is a quantitative chromatogram of the Gly-Ala (GA) peptide fragment of silk fibroin peptide in Example 3;
[0034] Figure 2 is a quantitative chromatogram of the Gly-Ala-Tyr (GAY) peptide fragment of silk fibroin peptide in Example 3;
[0035] Figure 3 is a quantitative chromatogram of the Ala-Ala (AA) peptide fragment of silk fibroin peptide in Example 3;
[0036] Figure 4 shows the relative expression levels of the col1a1a gene in Example 4.
[0037] Figure 5 shows the relative expression levels of the col1a1b gene in Example 4.
[0038] Figure 6 shows the relative expression levels of the elna gene in Example 4;
[0039] Figure 7 shows the relative expression levels of the elnb gene in Example 4;
[0040] Figure 8 shows the ion mass spectrum of the peptide Gly-Ala (GA) in Example 6;
[0041] Figure 9 shows the ion mass spectrum of the peptide Gly-Ala-Tyr (GAY) in Example 6;
[0042] Figure 10 shows the ion mass spectrum of the peptide Ala-Ala (AA) product in Example 6;
[0043] Figure 11 shows the standard curve of the standard peptide Gly-Ala (GA) in Example 6;
[0044] Figure 12 shows the quantitative chromatogram of the standard peptide Gly-Ala (GA) in Example 6;
[0045] Figure 13 is a quantitative chromatogram of the Gly-Ala (GA) peptide fragment in silk fibroin peptide No. 1 in Example 6;
[0046] Figure 14 is a quantitative chromatogram of Gly-Ala (GA) peptide in silk fibroin peptide No. 2 in Example 6;
[0047] Figure 15 is a quantitative chromatogram of the Gly-Ala (GA) peptide fragment in silk fibroin peptide No. 3 in Example 6;
[0048] Figure 16 is a quantitative chromatogram of Gly-Ala (GA) peptide in silk fibroin peptide No. 4 in Example 6;
[0049] Figure 17 is a quantitative chromatogram of the Gly-Ala (GA) peptide fragment in silk fibroin peptide No. 5 in Example 6;
[0050] Figure 18 shows the standard curve of the standard peptide Gly-Ala-Tyr (GAY) in Example 6;
[0051] Figure 19 shows the quantitative chromatogram of the standard peptide Gly-Ala-Tyr (GAY) in Example 6;
[0052] Figure 20 is a quantitative chromatogram of the Gly-Ala-Tyr (GAY) peptide fragment in silk fibroin peptide No. 1 in Example 6;
[0053] Figure 21 is a quantitative chromatogram of the Gly-Ala-Tyr (GAY) peptide fragment in silk fibroin peptide No. 2 in Example 6;
[0054] Figure 22 is a quantitative chromatogram of the Gly-Ala-Tyr (GAY) peptide fragment in silk fibroin peptide No. 3 in Example 6;
[0055] Figure 23 is a quantitative chromatogram of the Gly-Ala-Tyr (GAY) peptide fragment in silk fibroin peptide No. 4 in Example 6;
[0056] Figure 24 is a quantitative chromatogram of the Gly-Ala-Tyr (GAY) peptide fragment in silk fibroin peptide No. 5 in Example 6;
[0057] Figure 25 shows the standard curve of the standard peptide Ala-Ala (AA) in Example 6;
[0058] Figure 26 shows the quantitative chromatogram of the standard peptide Ala-Ala (AA) in Example 6;
[0059] Figure 27 is a quantitative chromatogram of peptide Ala-Ala(AA) in silk fibroin peptide No. 1 in Example 6;
[0060] Figure 28 is a quantitative chromatogram of peptide Ala-Ala(AA) in silk fibroin peptide No. 2 in Example 6;
[0061] Figure 29 is a quantitative chromatogram of peptide Ala-Ala(AA) in silk fibroin peptide No. 3 in Example 6;
[0062] Figure 30 is a quantitative chromatogram of peptide Ala-Ala(AA) in silk fibroin peptide No. 4 in Example 6;
[0063] Figure 31 is a quantitative chromatogram of peptide Ala-Ala (AA) in silk fibroin peptide No. 5 in Example 6. Detailed Implementation
[0064] To enable those skilled in the art to better understand the solutions of this application, a further detailed description of this application is provided below. The specific embodiments listed below are merely descriptions of the principles and features of this application; the examples are only for explaining this application and are not intended to limit its scope. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this application.
[0065] The dermal connective tissue of the skin is composed of cellular components and the extracellular matrix (ECM). Collagen and elastin are important components of the ECM, accounting for 70% and 2-5% of the skin's dry matter, respectively. Collagen is the skin's main stress-resistant substance, maintaining its resilience. Secreted by fibroblasts, it cross-links to form a fibrous network of collagen fibers. Type I collagen is a triple-helix molecule composed of two α1 chains and one α2 chain, encoded by the COL1A1 and COL1A2 genes, respectively. The zebrafish genes col1a1a and col1a1b are homologous to the human type I collagen gene COL1A1. Elastin, like collagen, is synthesized by fibroblasts and exists in the ECM as elastic fibers. These elastic fibers wrap around collagen fibers, possessing high extensibility and resilience, giving the skin strong elasticity and recoil force, responsible for restoring deformed skin to its normal shape. The zebrafish genes elna and elnb are homologous to the human elastin gene ELN. Existing research has shown that the reason for loose and inelastic skin is that collagen and elastin in the dermis break down under the hydrolysis of matrix metalloproteinases, and are loosely and disorderly deposited in the ECM of the skin, which cannot provide sufficient mechanical support for the skin.
[0066] Therefore, to address the problem of loose and inelastic skin, this application provides, in its first aspect, a functional peptide with anti-wrinkle effects, comprising peptide GA, peptide GAY, and peptide AA. Peptide GA is a dipeptide composed of glycine and alanine (Gly-Ala, GA), peptide GAY is a tripeptide composed of glycine, alanine, and tyrosine (Gly-Ala-Tyr, GAY), and peptide AA is a dipeptide composed of alanine and alanine (Ala-Ala, AA).
[0067] Experimental results have shown that the aforementioned functional peptides can increase skin elasticity by upregulating the relative expression levels of type I collagen (col1a1a, col1a1b) and elastin (elna, elnb) genes, thereby achieving an anti-wrinkle effect.
[0068] A second aspect of this application provides a silk fibroin peptide with anti-wrinkle effects, wherein the silk fibroin peptide includes the aforementioned functional peptide segments.
[0069] Silk fibroin peptides are hydrolyzed products of silkworm cocoon protein. They are small-molecule active peptides obtained through bioprocessing techniques such as protein extraction, protein denaturation, enzymatic hydrolysis, and purification of silkworm cocoon protein. They retain the excellent properties of silkworm cocoon protein while also possessing many new applications, thus demonstrating promising development prospects. Experimental verification has shown that, because silk fibroin peptides include the aforementioned functional peptide segments, they can also increase skin elasticity by upregulating the relative expression levels of type I collagen (col1a1a, col1a1b) and elastin (elna, elnb) genes, thereby achieving an anti-wrinkle effect.
[0070] In the above technical solution, based on the quality of silk fibroin peptides, the mass content of peptide GA is ≥0.07%, the mass content of peptide GAY is ≥0.01%, and the mass content of peptide AA is ≥0.02%.
[0071] This application regulates the functional peptides and their content in the composition of silk fibroin peptides to meet the above-mentioned range, thereby promoting the upregulation of the relative expression levels of skin type I collagen (col1a1a, col1a1b) and elastin (elna, elnb) genes by silk fibroin peptides, thereby increasing skin elasticity and ultimately achieving the effect of anti-wrinkle.
[0072] Furthermore, this application found that improving the preparation process of silk fibroin peptides can increase the content of functional peptides in the silk fibroin peptides, thereby achieving better anti-wrinkle effects. Specifically, based on the quality of the silk fibroin peptides, the mass content of peptide GA is ≥1.08%, the mass content of peptide GAY is ≥0.18%, and the mass content of peptide AA is ≥0.02%.
[0073] In the above technical solutions, the silk fibroin peptides have a moisture content ≤2g / 100g and an ash content ≤30g / 100g, indicating that the silk fibroin peptides have fewer impurities and higher quality; the silk fibroin peptides have a protein content ≥70g / 100g, indicating that the silk fibroin peptides are rich in protein; the silk fibroin peptides have an acid-soluble protein content ≥70g / 100g, because large molecular weight proteins are easily precipitated when exposed to acid, so the content of acid-soluble protein to some extent means the content of lower molecular weight proteins, indicating that the silk fibroin peptides have a lower molecular weight; the silk fibroin peptides have a free amino acid content ≤20g / 100g, which can reflect the degree of hydrolysis and composition of the silk fibroin peptides; the silk fibroin peptides have a peptide content ≥50g / 100g, indicating that the silk fibroin peptides have a rich peptide content; the silk fibroin peptides have a peptide mass content of less than 1000u ≥80%, indicating that the silk fibroin peptides have a high degree of hydrolysis, a small molecular weight, and are easily absorbed and utilized by the body. It can be understood that satisfying at least one of the above solutions can enhance the anti-wrinkle effect of silk fibroin peptides.
[0074] In the above technical solution, silk fibroin peptides are obtained by using silkworm cocoon protein as raw material, and then undergoing enzymatic hydrolysis and separation and purification treatment; wherein, the enzymatic hydrolysis treatment includes using at least one of neutral protease and carboxypeptidase A, and at least includes neutral protease for enzymatic hydrolysis.
[0075] Silkworm cocoon protein is a natural high-molecular-weight protein with unique biocompatibility, excellent mechanical properties, and bioactivity, making it widely applicable in various fields. Preparing silkworm cocoon protein into silk fibroin peptides transforms the large-molecule silkworm cocoon protein, which cannot be directly utilized by organisms, into small-molecule silk fibroin peptides that can be absorbed and utilized, have well-defined structural characteristics, and possess bioactivity. This not only improves bioactivity but also endows silkworm cocoon protein with higher added value and a wider range of applications, thus possessing greater economic value. Furthermore, the silk fibroin peptides provided in this application are obtained through enzymatic hydrolysis and separation and purification processes, resulting in relatively low cost, a simple preparation method, and higher safety, facilitating subsequent large-scale production.
[0076] The silkworm cocoon protein used in this application only needs to meet the requirement that the protein content (dry basis) is not less than 95%. It is understood that the silkworm cocoon protein used in this application can be in solid or liquid form. In one embodiment of this application, the silkworm cocoon protein used is a liquid silkworm cocoon protein extract.
[0077] This application utilizes at least one of neutral protease and carboxypeptidase A, with a minimum of neutral protease, to enzymatically hydrolyze silkworm cocoon protein. Neutral protease is an endopeptidase, applicable to various protein hydrolysis processes, capable of hydrolyzing large protein molecules into amino acids and other products under specific temperature and pH conditions. Carboxypeptidase is an enzyme that catalyzes the hydrolysis of amino acids containing carboxyl terms in polypeptide chains; its enzyme activity is zinc-dependent. Carboxypeptidase A hydrolyzes carboxyl terms formed by aromatic and neutral aliphatic amino acids, such as tyrosine, phenylalanine, and alanine. It is understood that this application may use only neutral protease or both neutral protease and carboxypeptidase A to enzymatically hydrolyze silkworm cocoon protein. By regulating the enzymatic hydrolysis and purification processes of silkworm cocoon protein, this application ensures that the final silk fibroin peptides meet the predetermined functional peptide composition.
[0078] A third aspect of this application provides a method for preparing the above-mentioned silk fibroin peptide, comprising the following steps:
[0079] Silk fibroin peptides were obtained by enzymatic hydrolysis and separation and purification of silk cocoon protein as raw material.
[0080] The enzymatic hydrolysis process includes enzymatic hydrolysis using at least one of neutral protease and carboxypeptidase A, and at least includes neutral protease.
[0081] Extensive research has been conducted to explore how to ensure the enzymatic hydrolysis and purification of silkworm cocoon proteins contain the desired peptides GA, GAY, and AA. This research has demonstrated that the selection of enzymes and the enzyme treatment process significantly impact the results. During the research, it was discovered that enzymatic hydrolysis and purification of silkworm cocoon proteins, with specific enzyme types, can yield silk fibroin peptides containing GA, GAY, and AA peptides. This preparation method is simple, rapid, and suitable for large-scale production.
[0082] In the above technical solutions, controlling the temperature, time, and pH of the enzymatic hydrolysis treatment helps to promote the enzymatic hydrolysis reaction. Specifically, the temperature for enzymatic hydrolysis can be 45-55℃, the time can be 4.0-6.0h, and the pH can be 6.5-7.5. When the enzymatic hydrolysis treatment includes using neutral protease, the temperature can be 45-55℃, the time can be 4.5-5.5h, and the pH can be 7.0, which helps to better exert the enzymatic hydrolysis effect of neutral protease and promote the full release of functional peptides. When the enzymatic hydrolysis treatment includes sequentially using neutral protease and carboxypeptidase A for the first and second enzymatic hydrolysis, the temperature for the first enzymatic hydrolysis is 45-55℃, the time is 2.5-3.5h, and the pH is 7.0; the temperature for the second enzymatic hydrolysis is 45-55℃, the time is 1.5-2.5h, and the pH is 7.0; this helps to further release functional peptides based on the release of functional peptides by neutral protease. In practice, the pH value can be adjusted to the optimal pH environment for neutral protease hydrolysis, i.e., pH 7.0, to obtain a hydrolysate suitable for the first hydrolysis by neutral protease. The pH value can be adjusted using sodium hydroxide (NaOH) or hydrochloric acid (HCl). Then, at the suitable hydrolysis temperature for neutral protease, i.e., 45-55℃, neutral protease is added to the hydrolysate, and hydrolysis is carried out for 2.5-3.5 hours. This allows the neutral protease to fully act on the protein in the hydrolysate, opening up the higher-order structures of the protein to expose more cleavage sites. Next, carboxypeptidase A, which can hydrolyze the C-terminus of aromatic and neutral aliphatic amino acids, is added, and hydrolysis is carried out for 1.5-2.5 hours to obtain a large number of peptides with C-terminal alanine or tyrosine residues.
[0083] Regulating the enzyme activity and dosage of neutral protease or carboxypeptidase A can also help promote enzymatic hydrolysis, thereby promoting the release of functional peptides. Specifically, the enzyme activity of neutral protease is 140,000-160,000 U; the dosage of neutral protease is 0.2 wt%-2 wt% per gram of cocoon protein; the enzyme activity of carboxypeptidase A is 4,000-6,000 U; the dosage of carboxypeptidase A is 0.2 wt%-2 wt% per gram of cocoon protein.
[0084] It is understandable that if at least one of the following factors—temperature, time, pH, activity of neutral protease, amount of neutral protease added, activity of carboxypeptidase A, and amount of carboxypeptidase A added—meets the above range, the release of functional peptides can be promoted, thereby enhancing the anti-wrinkle effect of silk fibroin peptides.
[0085] In addition, after the enzymatic hydrolysis is completed, enzyme inactivation can be performed to cause the enzyme preparation to lose its catalytic activity. This application does not limit the method of enzyme inactivation; conventional enzyme inactivation methods in the art can be used, such as heating to 95°C and holding for 20 minutes.
[0086] After enzyme inactivation, centrifugation can be performed, and the supernatant can be collected for subsequent operations. Specifically, the centrifugation speed can be 2500-3500 rpm / min, and the centrifugation time can be 5-15 min.
[0087] In the above technical solution, silkworm cocoon protein can be obtained through the following steps:
[0088] Silkworm cocoons and water were treated at 110-130℃ and 0.05-0.17MPa for 0.5-1.5h, and the insoluble matter was obtained by filtration. The insoluble matter, water and sodium hydroxide were then treated at 110-130℃ and 0.05-0.17MPa for 1.0-2.0h to obtain silkworm cocoon protein.
[0089] In one specific embodiment, pupa-free, shelled cocoons can be used. After washing with clean water and drying, 20 times the weight of the pupa-free, shelled cocoons of water are added, and the mixture is pressure-treated at 121°C and 0.11 MPa for 1 hour. After filtration, insoluble matter is obtained. Then, 20 times the weight of the pupa-free, shelled cocoons of water and 30% the weight of the pupa-free, shelled cocoons of sodium hydroxide are added to the insoluble matter, and the mixture is pressure-treated at 121°C and 0.11 MPa for 1.5 hours. After cooling to 50°C, a cocoon protein pretreatment solution is obtained, which is cocoon protein with a protein content of 75% (dry basis).
[0090] The above method can effectively enrich silkworm cocoon protein, laying a good foundation for obtaining the target silk fibroin peptide in the future.
[0091] In the above technical solutions, the separation and purification process includes at least one of diatomaceous earth adsorption treatment, cation exchange resin treatment, anion exchange resin treatment, and macroporous resin treatment.
[0092] Diatomaceous earth adsorption treatment refers to mixing diatomaceous earth with the solution to be separated and purified, and then filtering to remove the diatomaceous earth to obtain the separated and purified solution. Specifically, diatomaceous earth and the solution to be separated and purified can be mixed at a mass ratio of 1:(18-22).
[0093] Cation exchange resin treatment refers to passing the solution to be separated and purified through a cation exchange resin column. Collection of the effluent begins when the UV detection value reaches 150-250 mAu, and collection stops when the UV detection value falls below 150-250 mAu. The collected effluent is the separated and purified solution. Specifically, the solution to be separated and purified can be passed through a 500 mL pre-equilibrated cation exchange resin column at a linear flow rate of 10 mL / min. Collection of the effluent begins when the UV detection value reaches 200 mAu. After all the separated and purified solution has been treated, pure water is used instead of the solution to be separated and purified and passed through the cation exchange resin column. Collection of the effluent stops when the UV detection value falls below 200 mAu.
[0094] Anion exchange resin treatment refers to passing the solution to be separated and purified through an anion exchange resin column. Collection of the eluent begins when the UV detection value reaches 150-250 mAu, and collection stops when the UV detection value falls below 150-250 mAu. The collected eluent is the separated and purified solution. Specifically, the solution to be separated and purified can be passed through a 100 mL pre-equilibrated anion exchange resin column at a linear flow rate of 10 mL / min. Collection of the eluent begins when the UV detection value reaches 200 mAu. After all the separated and purified solution has been treated, pure water is used instead of the solution to be separated and purified and passed through the anion exchange resin column. Collection of the eluent stops when the UV detection value falls below 200 mAu.
[0095] Macroporous resin treatment refers to passing the solution to be separated and purified through a macroporous resin column. Collection of the eluent begins when the UV detection value reaches 150-250 mAu, and collection stops when the UV detection value falls below 150-250 mAu. The collected eluent is the separated and purified solution. Specifically, the solution to be separated and purified can be passed through a 400 mL pre-equilibrated macroporous resin column at a linear flow rate of 10 mL / min. Collection of the eluent begins when the UV detection value reaches 200 mAu. After all the separated and purified solution has been treated, pure water is used instead of the solution to be separated and purified and passed through the macroporous resin column. Collection of the eluent stops when the UV detection value falls below 200 mAu.
[0096] Using the above separation and purification methods can further enrich the functional peptides in silk fibroin, enabling silk fibroin to exert a better anti-wrinkle effect.
[0097] It is understood that this application does not limit the order of the above-mentioned enzymatic hydrolysis and separation and purification processes. Enzymatic hydrolysis can be performed first, followed by separation and purification, or separation and purification can be performed first, followed by enzymatic hydrolysis.
[0098] In addition to enzymatic hydrolysis and separation purification, the process includes: filtration using a ceramic membrane, followed by rotary evaporation concentration, diatomaceous earth adsorption, and spray drying to obtain silk fibroin peptides. These steps further enrich the functional peptides.
[0099] Specifically, the solution after enzymatic hydrolysis and separation purification can be filtered through a 500-600nm ceramic membrane to obtain a filtrate. The filtrate is then concentrated by rotary evaporation using a rotary evaporator at a temperature of 70-90℃ and a vacuum of -0.1MPa. After removing 1600-1800mL of water from the filtrate, the remaining permeate is obtained. The remaining permeate is then mixed with 5-15g of diatomaceous earth and filtered to remove the diatomaceous earth, resulting in a clear liquid. Finally, the inlet air temperature of the spray drying tower is set to 130-150℃ and the outlet air temperature is set to 80-100℃, and the clear liquid is spray-dried to obtain silk fibroin peptides.
[0100] In one specific embodiment, the silkworm cocoon protein can be first enzymatically hydrolyzed to obtain an enzymatic hydrolysate. The hydrolysate is then adsorbed with diatomaceous earth and filtered through a ceramic membrane to obtain a filtrate. The filtrate is then concentrated by rotary evaporation to obtain a permeate. The permeate is then adsorbed with diatomaceous earth again to obtain a clear liquid. Finally, the clear liquid is spray-dried to obtain silk fibroin peptides.
[0101] In one specific embodiment, silkworm cocoon protein can be first treated with diatomaceous earth adsorption, then with cation exchange resin, and then with enzymatic hydrolysis to obtain an enzymatic hydrolysate. The hydrolysate is then filtered through a ceramic membrane to obtain a filtrate. The filtrate is then concentrated by rotary evaporation to obtain a permeate. The permeate is then treated with diatomaceous earth adsorption again to obtain a clear liquid. Finally, the clear liquid is spray-dried to obtain silk fibroin peptides.
[0102] In one specific embodiment, silkworm cocoon protein can be first treated with diatomaceous earth adsorption, cation exchange resin, and anion exchange resin, and then enzymatically hydrolyzed to obtain an enzymatic hydrolysate. The hydrolysate is then filtered through a ceramic membrane to obtain a filtrate. The filtrate is then concentrated by rotary evaporation to obtain a permeate. The permeate is then treated with diatomaceous earth adsorption again to obtain a clear liquid. Finally, the clear liquid is spray-dried to obtain silk fibroin peptides.
[0103] In one specific embodiment, silkworm cocoon protein can be first treated with diatomaceous earth adsorption and macroporous resin, and then enzymatically hydrolyzed to obtain an enzymatic hydrolysate. The hydrolysate is then filtered through a ceramic membrane to obtain a filtrate. The filtrate is then concentrated by rotary evaporation to obtain a permeate. The permeate is then treated with diatomaceous earth adsorption again to obtain a clear liquid. Finally, the clear liquid is spray-dried to obtain silk fibroin peptides.
[0104] In one specific embodiment, silkworm cocoon protein can be first enzymatically hydrolyzed to obtain an enzymatic hydrolysate, then subjected to diatomaceous earth adsorption treatment, cation exchange resin treatment, and then enzymatically hydrolyzed again to obtain an enzymatic hydrolysate. The enzymatic hydrolysate is then filtered through a ceramic membrane to obtain a filtrate. The filtrate is then concentrated by rotary evaporation to obtain a permeate. The permeate is then subjected to diatomaceous earth adsorption treatment again to obtain a clear liquid. Finally, the clear liquid is spray-dried to obtain silk fibroin peptides.
[0105] In one specific embodiment, silkworm cocoon protein can be first subjected to diatomaceous earth adsorption treatment, cation exchange resin treatment, and enzymatic hydrolysis to obtain an enzymatic hydrolysate. Then, it can be subjected to diatomaceous earth adsorption treatment, cation exchange resin treatment, and enzymatic hydrolysis again to obtain an enzymatic hydrolysate. The enzymatic hydrolysate is then filtered through a ceramic membrane to obtain a filtrate. The filtrate is then concentrated by rotary evaporation to obtain a permeate. The permeate is then subjected to diatomaceous earth adsorption treatment again to obtain a clear liquid. Finally, the clear liquid is spray-dried to obtain silk fibroin peptides.
[0106] The fourth aspect of this application provides the application of the above-mentioned functional peptide, the above-mentioned silk fibroin peptide, and the silk fibroin peptide prepared by the above-mentioned preparation method in the preparation of anti-wrinkle related products.
[0107] It is understood that the aforementioned related products include, but are not limited to, food, health products, skincare products, cosmetics, and pharmaceuticals. Extensive research data demonstrates that the functional peptides in this application, including peptides GA, GAY, and AA, possess significant anti-wrinkle effects. These peptides can be applied not only to conventional food, health products, skincare products, and cosmetics—such as anti-wrinkle foods, anti-wrinkle products, anti-wrinkle skincare products, and anti-wrinkle cosmetics—but also to the preparation of anti-wrinkle drugs, thereby broadening the application scope of silk fibroin peptides and providing new raw materials for anti-wrinkle related products.
[0108] The technical solution of this application will be further explained and illustrated below with reference to specific embodiments.
[0109] The materials and reagents used in the following examples are shown in Table 1, and the instruments and equipment used are shown in Table 2. For other experimental methods not specifically specified, they are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, all reagents used are commercially available or readily available from public sources.
[0110] Table 1
[0111] Table 2
[0112] Example 1: Detection of hydrolyzed amino acids in silkworm cocoon protein
[0113] Referring to the national standard GB 5009.124-2016, "Determination of Amino Acids in Food," the hydrolyzed amino acids of silkworm cocoon protein were tested, and the results are shown in Table 3. Based on the hydrolyzed amino acid composition of silkworm cocoon protein, glycine, alanine, and tyrosine are the three most abundant amino acids. Furthermore, based on the description of the amino acid structure of silkworm cocoon protein in the NCBI database, it can be inferred that silk fibroin peptides should contain peptide structures composed of glycine, alanine, and tyrosine, and possess biological activity. Therefore, to release the biologically active peptide structures, this application employs enzymatic hydrolysis and purification to obtain silk fibroin peptides from silkworm cocoon protein.
[0114] Table 3
[0115] Example 2: Preparation of silk fibroin peptides
[0116] (1) Preprocessing:
[0117] Weigh 100g of unpeupted, shelled cocoons (silkworms), wash with pure water, discard the washing water, and then add 2000mL of pure water at a mass ratio of 1:20. Maintain pressure at 121℃ and 0.11MPa for 1 hour. Filter to remove the filtrate, retaining the insoluble substances. Weigh pure water at a mass ratio of 1:20 (insoluble substances to pure water) and add it to the insoluble substances. Then add 30g of sodium hydroxide, which is 30% of the mass of the unpeupted, shelled cocoons (silkworms). Maintain pressure at 121℃ and 0.11MPa for 1.5 hours. After cooling to 50℃, obtain the silkworm cocoon protein pretreatment solution.
[0118] (2) Preparation:
[0119] The pretreated silkworm cocoon protein solution was adjusted to pH 7 with hydrochloric acid and then enzymatically hydrolyzed: 0.15g of neutral protease was added, and enzymatic hydrolysis was carried out at 50℃ for 5h. After hydrolysis, the solution was heated to 95℃ at a rate of 5℃ / min and kept at this temperature for 20min to inactivate the protease. The solution was then centrifuged at 3000r / min for 10min to obtain the supernatant. The supernatant was coarsely filtered through diatomaceous earth, followed by filtration through a ceramic membrane (550nm) to obtain the filtrate. The filtrate was concentrated by rotary evaporation using a rotary evaporator at 80℃ and a vacuum of -0.1MPa. After removing 1700mL of water from the filtrate, the remaining permeate was obtained. This permeate was mixed with 10g of diatomaceous earth and filtered through the diatomaceous earth to obtain the clear liquid. The inlet air temperature of the spray drying tower was set to 140℃ and the outlet air temperature to 90℃. The clear liquid was then spray-dried to obtain silk fibroin peptides.
[0120] Example 3: Quantitative analysis of silk fibroin peptides
[0121] (1) Yield of silk fibroin peptides:
[0122] The yield of silk fibroin peptides was calculated using the following formula: W1 / W2, where W1 is the mass of silk fibroin peptides and W2 is the mass of silkworm cocoons without pupae and shells (silkworms). The calculated yield of silk fibroin peptides was 46.28%.
[0123] (2) Detection of molecular weight distribution of silk fibroin peptides:
[0124] Molecular weight distribution was determined using high-performance gel filtration chromatography. Five peptide standards—acetyl-acetyl-acetyl (molecular weight 189), acetyl-acetyl-tyrosine-arginine (molecular weight 451), bacitracin (molecular weight 1450), aprotinin (molecular weight 6500), and cytochrome C (molecular weight 12500)—were prepared into 0.1% (m / v) solutions to obtain peptide standard solutions. 100 mg of the silk fibroin peptide sample obtained in Example 2 was weighed and dissolved in the mobile phase (prepared by a volume ratio of acetonitrile, water, and trifluoroacetic acid of 45:55:0.1), vortexed, and diluted to 10 mL to obtain the sample solution to be tested. The peptide standard solution and the sample solution to be tested were filtered through a 0.2 μm PTFE filter membrane and then injected into the system using high-performance liquid chromatography (HPLC) with gel filtration. The injection volume was 10 μL, the flow rate was 0.5 mL / min, the detection wavelength was 220 nm, the column temperature was 30 °C, and the detection was performed using a UV detector. Data processing was conducted using GPC software to obtain the chromatographic data of the peptide standard solution and to construct a relative molecular weight calibration curve, as well as the chromatographic data of the sample. The chromatographic data of the sample were substituted into the relative molecular weight calibration curve equation to calculate the peptide molecular weight and its distribution range. The relative percentage of peptides in different molecular weight ranges was calculated using the peak area normalization method. The analysis revealed that the proportion of hydrolysates with a relative molecular mass less than 1000 u in the silk fibroin peptide was approximately 80.15%.
[0125] (3) Detection of moisture and ash content of silk fibroin peptides:
[0126] The moisture content of the silk fibroin peptide obtained in Example 2 was determined to be 1.03 g / 100 g using the national standard method GB 5009.3-2016; the ash content of the silk fibroin peptide obtained in Example 2 was determined to be 27.85 g / 100 g using the national standard method GB 5009.4-2016.
[0127] (4) Detection of protein, peptide content, and free amino acid content of silk fibroin peptides:
[0128] Using the national standard method GB 5009.5-2016, the total nitrogen (protein) content (dry basis) of the silk fibroin peptide obtained in Example 2 was determined to be 70.98 g / 100 g; referring to the national standard method GB 22729-2008, the acid-soluble protein content (dry basis) of the silk fibroin peptide obtained in Example 2 was determined to be 70.65 g / 100 g, its free amino acid content was 19.39 g / 100 g, and its peptide content (dry basis) was 51.26 g / 100 g.
[0129] (5) Characteristic structural identification of silk fibroin peptides:
[0130] The characteristic structure of silk fibroin peptides was identified using a system coupled with a triple quadrupole mass spectrometer manufactured by Shimadzu Corporation of Japan, specifically including: LC-30AD×2 infusion pump, SIL-30AC autosampler, CTO-20AC column oven, CBM-20A system controller, LCMS-8060 triple quadrupole mass spectrometer, and LabSolutions Ver.5.91 chromatography workstation.
[0131] Weigh 2.0 mg of the silk fibroin peptide sample obtained in Example 2, dissolve it in ultrapure water, vortex mix, and bring the volume to 100 mL to obtain a silk fibroin peptide solution. Filter the silk fibroin peptide solution through a 0.2 μm PTFE filter membrane to obtain the sample to be tested. Connect an Inertsil ODS-3 column to a liquid chromatograph, and inject 10 μL of the sample to be tested. In positive ion mode, set the mass-to-charge ratio scan range to 100-600, and perform a Q3-Scan scan to collect peak mass-to-charge ratio information to obtain precursor ion information of the peptide. The liquid chromatography conditions are as follows: mobile phase A is pure water (containing 0.1% (v / v) formic acid), mobile phase B is 100% acetonitrile (containing 0.1% (v / v) formic acid), and the flow rate is 0.2 mL / min. The gradient elution program specifically includes: first gradient elution time of 0-15 min, mobile phase B content of 0%-40%; second gradient elution time of 15-20 min, mobile phase B content of 40%-80%; third gradient elution time of 20-25 min, mobile phase B content of 80%; and fourth gradient elution time of 25.1-35 min, mobile phase B content of 0%. The column oven temperature is 40℃. The mass spectrometry conditions are as follows: ionization mode is electrospray ionization (ESI), ion spray voltage is +4.5 kV, nebulizer gas (nitrogen) flow rate is 3.0 L / min, heating gas (nitrogen) flow rate is 10 L / min, drying gas (nitrogen) flow rate is 10 L / min, desolvation tube temperature is 250℃, heating module temperature is 400℃, and ion source temperature is 300℃.
[0132] High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) was used to perform precursor ion (Q3) scanning on the sample to obtain the total ion chromatogram of the main peptides in silk fibroin peptides. Subsequently, product ion (PIS) scanning was performed on the sample to fragment the peak precursor ions and collect fragment ion information of the peptides to obtain product ion information. The injection volume was set to 10 μL, the precursor ions were input based on the Q3 scan analysis results, and the collision energies were set to -25V and -35V. The HPLC and mass spectrometry conditions were the same as those for the Q3 scan analysis. Based on the product ion information generated after collisions, the NCBI database, and the target characteristic amino acid residues, three peptide structures were finally identified: Gly-Ala (GA), Gly-Ala-Tyr (GAY), and Ala-Ala (AA), as detailed in Table 4.
[0133] Table 4
[0134] (6) Quantification of functional peptides in silk fibroin:
[0135] Accurately weigh 2.0 mg of standard peptides Gly-Ala (GA), Gly-Ala-Tyr (GAY), and Ala-Ala (AA), respectively, dissolve them in ultrapure water, vortex to mix, and bring the volume to 100 mL to obtain a standard stock solution with a concentration of 20 μg / mL. The above standard stock solution was serially diluted with ultrapure water to 1.95 ng / mL, 3.91 ng / mL, 7.81 ng / mL, 15.63 ng / mL, 31.25 ng / mL, 62.5 ng / mL, 125 ng / mL, 250 ng / mL, and 500 ng / mL, and filtered through a 0.22 μm aqueous polyethersulfone membrane to obtain a series of standard working solutions with varying concentrations. The silk fibroin peptide obtained in Example 2 was prepared into a stock solution with a concentration of 10.0 mg / mL using ultrapure water, then diluted 1000 times and filtered through a 0.22 μm aqueous polyethersulfone filter membrane to obtain a sample solution with a concentration of 20 μg / mL.
[0136] Inject 10 μL of the above-mentioned standard working solution and the sample solution to be tested, respectively, and perform quantitative analysis in positive ion mode. The quantitative chromatograms and standard curves corresponding to the standard peptides Gly-Ala (GA), Gly-Ala-Tyr (GAY), and Ala-Ala (AA), as well as the quantitative chromatograms corresponding to the peptides Gly-Ala (GA), Gly-Ala-Tyr (GAY), and Ala-Ala (AA) in the sample to be tested, can be seen in Figures 1, 2, and 3. Figure 1 is the quantitative chromatogram of the Gly-Ala (GA) peptide in silk fibroin, Figure 2 is the quantitative chromatogram of the Gly-Ala-Tyr (GAY) peptide in silk fibroin, and Figure 3 is the quantitative chromatogram of the Ala-Ala (AA) peptide in silk fibroin. The liquid chromatography conditions were as follows: the column was an Inertsil ODS-3 (particle size 5 μm, inner diameter 2.1 mm, column length 250 mm); mobile phase A was pure water (containing 0.1% (v / v) formic acid), mobile phase B was 100% acetonitrile (containing 0.1% (v / v) formic acid), and the flow rate was 0.2 mL / min. The gradient elution program specifically included: the first gradient elution time was 0 min–15 min, with mobile phase B accounting for 0%–40%; the second gradient elution time was 15 min–20 min, with mobile phase B accounting for 40%–80%; the third gradient elution time was 20 min–25 min, with mobile phase B accounting for 80%; and the fourth gradient elution time was 25.1 min–35 min, with mobile phase B accounting for 0%. The column oven temperature was 40℃. The mass spectrometry conditions were as follows: ionization mode was electrospray ionization (ESI); ion spray voltage was +4.5 kV, nebulizing gas (nitrogen) flow rate was 3.0 L / min, heating gas (nitrogen) flow rate was 10 L / min, drying gas (nitrogen) flow rate was 10 L / min, desolvation tube temperature was 250 °C, heating module temperature was 400 °C, and ion source temperature was 300 °C; scanning mode was multiple reaction monitoring (MRM), residence time was 100 ms, and delay time was 3 ms.
[0137] The content of Gly-Ala (GA), Gly-Ala-Tyr (GAY), and Ala-Ala (AA) in the silk fibroin peptide was calculated to be 0.07% based on the standard curve.
[0138] Example 4: Efficacy Evaluation of Silk Fibroin Peptides
[0139] The dermal connective tissue of the skin is composed of cellular components and the extracellular matrix (ECM). Key components of the ECM include collagen and elastin, accounting for 70% and 2-5% of the skin's dry matter, respectively. Collagen is the skin's main stress-resistant substance, maintaining its resilience. Secreted by fibroblasts, it cross-links to form a fibrous network of collagen fibers. Type I collagen is a triple-helix molecule composed of two α1 chains and one α2 chain, encoded by the COL1A1 and COL1A2 genes, respectively. The zebrafish genes col1a1a and col1a1b are homologous to the human type I collagen gene COL1A1. Elastin, like collagen, is synthesized by fibroblasts and exists in the ECM as elastic fibers. These fibers wrap around collagen fibers, possessing high extensibility and resilience, giving the skin strong elasticity and recoil, responsible for restoring deformed skin to its normal shape. The zebrafish genes elna and elnb are homologous to the human elastin gene ELN. The reason for loose and inelastic skin is that collagen and elastin in the dermis break down under the hydrolysis of matrix metalloproteinases, resulting in loose and disordered deposition in the ECM of the skin, which cannot provide sufficient mechanical support for the skin. In this embodiment, the efficacy of silk fibroin peptides in increasing skin elasticity is evaluated by detecting the relative expression levels of type I collagen (col1a1a, col1a1b) and elastin (elna, elnb) genes.
[0140] Wild-type AB strain zebrafish, 4 days post-fertilization (4dpf), were randomly selected and placed in 6-well plates, with 30 zebrafish per well. The normal control group was treated with purified water; the experimental groups were treated with purified water containing silk fibroin peptides obtained in Example 2, prepared to final concentrations of 125 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL, and 2000 μg / mL, respectively; the positive control groups included Gly-Ala (GA), Gly-Ala-Tyr (GAY), and Ala-Ala (AA) groups, where the corresponding peptides were prepared with purified water to final concentrations of 125 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL, and 2000 μg / mL, respectively, for zebrafish treatment. Each well contained 3 mL of solution. After treatment at 28°C for 24 h, the maximum detectable concentration (MTC) of the above treatments in zebrafish was determined. The results are shown in Table 5. Under the conditions of this example, the MTC of silk fibroin peptide treatment, Gly-Ala (GA) treatment, and Ala-Ala (AA) treatment was 2000 μg / mL, while the MTC of Gly-Ala-Tyr (GAY) treatment was 250 μg / mL.
[0141] Wild-type AB strain zebrafish (4 dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish per well. The normal control group, experimental group, and positive control group were all treated with MTC concentration. Three parallel experiments were conducted, with 3 mL per well. After treatment at 28℃ for 24 h, total RNA was extracted from the zebrafish using the RNA-Quick Purification Kit. The concentration and purity (A260 / A280 ratio) of the total zebrafish RNA were measured using a UV-Vis spectrophotometer. The results are shown in Table 6. The A260 / A280 ratios were all between 1.8 and 2.2, indicating that the extracted total zebrafish RNA was of good quality and suitable for subsequent q-PCR experiments. 2.00 μg of total zebrafish RNA was used to synthesize 20.0 μL of cDNA according to the FastKing cDNA first-strand synthesis kit instructions. The expression of the col1a1a, col1a1b, elna, and elnb genes was detected by q-PCR using the primers shown in Table 7. Using β-actin as an internal control for gene expression, the relative RNA expression levels of the col1a1a, col1a1b, elna, and elnb genes were calculated. The results are shown in Table 8 and Figures 4, 5, 6, and 7. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software. p < 0.05 indicated statistical significance. Compared with the normal control group, *p < 0.05, **p < 0.01, and ***p < 0.001. Compared with the silk fibroin peptide treatment, the Gly-Ala (GA) treatment, Gly-Ala-Tyr (GAY) treatment, and Ala-Ala (AA) treatment showed &p < 0.05, &&p < 0.01, and &&&p < 0.001. To distinguish them from *, & is used here to indicate differences.
[0142] Under the conditions of this embodiment, as shown in Table 8 and Figures 4, 5, 6 and 7, silk fibroin peptide treatment, Gly-Ala (GA) treatment, Gly-Ala-Tyr (GAY) treatment and Ala-Ala (AA) treatment all have anti-wrinkle effects. Specifically, Gly-Ala (GA) treatment, Gly-Ala-Tyr (GAY) treatment and Ala-Ala (AA) treatment upregulate the relative expression levels of col1a1a, col1a1b, elna and elnb genes, while silk fibroin peptide treatment upregulates the relative expression levels of col1a1a, elna and elnb genes. This indicates that the silk fibroin peptides obtained in this application, as well as the peptides Gly-Ala (GA), Gly-Ala-Tyr (GAY), and Ala-Ala (AA), all have anti-wrinkle effects. Among them, the peptides Gly-Ala (GA), Gly-Ala-Tyr (GAY), and Ala-Ala (AA) can achieve anti-wrinkle effects by upregulating the relative expression levels of the col1a1a, col1a1b, elna, and elnb genes. The silk fibroin peptides obtained in this application achieve anti-wrinkle effects by upregulating the relative expression levels of the col1a1a, elna, and elnb genes.
[0143] Table 5
[0144] Table 6
[0145] Table 7
[0146] Table 8
[0147] Example 5: Optimization of the preparation method of silk fibroin peptides
[0148] Based on the experimental results of Example 3, it was found that increasing the content of Gly-Ala (GA), Gly-Ala-Tyr (GAY), and Ala-Ala (AA) peptides in silk fibroin peptides can improve their anti-wrinkle efficacy. Therefore, silk fibroin peptides can be purified by optimizing enzymatic hydrolysis and separation / purification processes to obtain high-content Gly-Ala (GA), Gly-Ala-Tyr (GAY), and Ala-Ala (AA) peptides. Enzymatic hydrolysis can be performed using carboxylpeptidases capable of hydrolyzing the carboxyl terminus of aromatic and neutral aliphatic amino acids to obtain peptides with a C-terminus of alanine (Ala, A) or tyrosine (Tyr, Y). Separation / purification can be performed using ion exchange resins or macroporous adsorption resins to adsorb and remove non-target peptides to increase the content of the target peptides. Through the above optimization strategies, this example prepared five types of silk fibroin peptides using different preparation methods, as follows:
[0149] (1) No. 1 Silk Peptide
[0150] The pretreatment steps were the same as in Example 2, yielding a silkworm cocoon protein pretreated solution. The silkworm cocoon protein pretreated solution was coarsely filtered using diatomaceous earth, and the coarse filtrate was purified using a cation exchange resin (manufactured by Shanghai Xun'er Chemical Technology Co., Ltd., model XRK539A). Specifically, the coarse filtrate was passed through a 500 mL pre-equilibrated cation exchange resin column at a linear flow rate of 10 mL / min. Effluent collection began when the UV detection value reached 200 mAu. After all the coarse filtrate was treated, pure water was used instead of the coarse filtrate and passed through the cation exchange resin column. Effluent collection was stopped when the UV detection value fell below 200 mAu. Add 0.15 g of neutral protease with an activity of 150,000 U / g to the collected effluent (pH 7 at this point), and hydrolyze at 50°C for 3 h to obtain the first hydrolysate. Add 0.15 g of carboxypeptidase A with an activity of 5,000 U / g to the first hydrolysate, and hydrolyze at 50°C for 2 h to obtain the second hydrolysate. After the second hydrolysis, heat the second hydrolysate to 95°C and incubate for 20 min to inactivate the protease, then centrifuge at 3000 r / min for 10 min to obtain the supernatant. Filter the supernatant through a ceramic membrane (550 nm) to obtain the filtrate. Concentrate the filtrate by rotary evaporation using a rotary evaporator at 80°C and a vacuum of -0.1 MPa. After removing 1700 mL of water from the filtrate, the remaining permeate is obtained. Mix this permeate with 10 g of diatomaceous earth, filter through the diatomaceous earth, and obtain the clear liquid. The inlet air temperature of the spray drying tower was set to 140℃ and the outlet air temperature was set to 90℃. The clear liquid was spray dried to obtain No. 1 silk fibroin peptide.
[0151] (2) No. 2 Silk Peptide
[0152] The pretreatment steps were the same as in Example 2, resulting in a silkworm cocoon protein pretreated solution. Diatomaceous earth was used to coarsely filter the silkworm cocoon protein pretreated solution, and the coarse filtrate was then purified first with a cation exchange resin and then with anion exchange resin. Specifically, the coarse filtrate was passed through a 400 mL pre-equilibrated cation exchange resin column at a linear flow rate of 10 mL / min. When the UV detection value reached 200 mAu, the effluent was collected. After all the coarse filtrate was treated, pure water was used instead of the coarse filtrate and passed through the cation exchange resin column. When the UV detection value was below 200 mAu, the collection of the effluent was stopped. The effluent purified by cation exchange resin was passed through a 100 mL pre-equilibrated anion exchange resin column (manufactured by Tianjin Xinyue Huamei Environmental Protection Technology Co., Ltd., model 201×7FC) at a linear flow rate of 10 mL / min. Effluent collection began when the UV detection value reached 200 mAu. After all the effluent purified by cation exchange resin was processed, pure water was used instead of the cation exchange resin purified effluent and passed through the anion exchange resin column. Effluent collection was stopped when the UV detection value fell below 200 mAu. 0.15 g of neutral protease with an enzyme activity of 150,000 U / g was added to the collected effluent (at pH 7), and enzymatic hydrolysis was carried out at 50℃ for 3 h to obtain the first enzymatic hydrolysate. 0.15 g of carboxypeptidase A with an enzyme activity of 5,000 U / g was added to the first enzymatic hydrolysate, and enzymatic hydrolysis was carried out at 50℃ for 2 h to obtain the second enzymatic hydrolysate. After the second enzymatic hydrolysis, the hydrolysate was heated to 95℃ and incubated for 20 min to inactivate the protease. It was then centrifuged at 3000 r / min for 10 min to obtain the supernatant. The supernatant was filtered through a ceramic membrane (550 nm) to obtain the filtrate. The filtrate was concentrated by rotary evaporation using a rotary evaporator at 80℃ and a vacuum of -0.1 MPa. After removing 1700 mL of water from the filtrate, the remaining permeate was obtained. This permeate was mixed with 10 g of diatomaceous earth and filtered through the diatomaceous earth to obtain the clear liquid. The inlet air temperature of the spray drying tower was set to 140℃ and the outlet air temperature to 90℃. The clear liquid was then spray-dried to obtain silk fibroin peptide No. 2.
[0153] (3) No. 3 Silk Peptide
[0154] The pretreatment steps were the same as in Example 2, resulting in a silkworm cocoon protein pretreated solution. The silkworm cocoon protein pretreated solution was coarsely filtered using diatomaceous earth, and the coarse filtrate was then purified using macroporous resin. Specifically, the coarse filtrate was passed through a 400 mL pre-equilibrated macroporous resin column (manufactured by Tianjin Xinyue Huamei Environmental Protection Technology Co., Ltd., model D101) at a linear flow rate of 10 mL / min. Effluent collection began when the UV detection value reached 200 mAu. After all the coarse filtrate was treated, pure water was used instead of coarse filtrate and passed through the macroporous resin column. Effluent collection was stopped when the UV detection value fell below 200 mAu. The pH of the collected effluent was adjusted to 7 using hydrochloric acid. Then, 0.15 g of neutral protease with an activity of 150,000 U / g was added, and the mixture was hydrolyzed at 50°C for 3 hours to obtain the first hydrolysate. Next, 0.15 g of carboxypeptidase A with an activity of 5,000 U / g was added to the first hydrolysate, and the mixture was hydrolyzed at 50°C for 2 hours to obtain the second hydrolysate. After the second hydrolysis, the second hydrolysate was heated to 95°C and incubated for 20 minutes to inactivate the protease. Then, it was centrifuged at 3000 rpm for 10 minutes to obtain the supernatant. The supernatant was filtered through a ceramic membrane (550 nm) to obtain the filtrate. The filtrate was concentrated by rotary evaporation using a rotary evaporator at 80°C and a vacuum of -0.1 MPa. After removing 1700 mL of water from the filtrate, the remaining permeate was obtained. This permeate was mixed with 10 g of diatomaceous earth and filtered through the diatomaceous earth to obtain the clear liquid. The inlet air temperature of the spray drying tower was set to 140℃ and the outlet air temperature was set to 90℃. The clear liquid was spray dried to obtain No. 3 silk fibroin peptide.
[0155] (4) No. 4 Silk Peptide
[0156] The pretreatment steps were the same as in Example 2, yielding a silkworm cocoon protein pretreatment solution. The pH of the pretreatment solution was adjusted to 7 using hydrochloric acid, and then 0.15 g of neutral protease with an enzyme activity of 150,000 U / g was added. The solution was hydrolyzed at 50°C for 3 hours to obtain the first hydrolysate. Then, 0.15 g of carboxypeptidase A with an enzyme activity of 5,000 U / g was added to the first hydrolysate, and the solution was hydrolyzed at 50°C for 2 hours to obtain the second hydrolysate. After the second hydrolysis, the second hydrolysate was heated to 95°C and incubated for 20 minutes to inactivate the protease. Subsequently, it was centrifuged at 3000 r / min for 10 minutes to obtain the supernatant. The supernatant was coarsely filtered using diatomaceous earth, and the coarse filtrate was then purified using cation exchange resin. Specifically, the coarse filtrate was passed through a 500 mL pre-equilibrated cation exchange resin column at a linear flow rate of 10 mL / min. Effluent collection began when the UV detection value reached 200 mAu. After all the coarse filtrate was treated, pure water was used instead of the coarse filtrate and passed through the cation exchange resin column. Effluent collection was stopped when the UV detection value fell below 200 mAu. The effluent was then filtered through a ceramic membrane (550 nm) to obtain the filtrate. The filtrate was concentrated by rotary evaporation using a rotary evaporator at 80℃ and a vacuum of -0.1 MPa. After removing 1700 mL of water from the filtrate, the remaining permeate was obtained. This permeate was mixed with 10 g of diatomaceous earth and filtered through the diatomaceous earth to obtain the clarified liquid. The inlet air temperature of the spray drying tower was set to 140℃ and the outlet air temperature to 90℃. The clarified liquid was then spray-dried to obtain silk fibroin peptide No. 4.
[0157] (5) No. 5 Silk Peptide
[0158] The pretreatment steps were the same as in Example 2, yielding a silkworm cocoon protein pretreated solution. The pretreated solution was coarsely filtered using diatomaceous earth, and then purified using a cation exchange resin (manufactured by Shanghai Xun'er Chemical Technology Co., Ltd., model XRK539A). Specifically, the coarse filtrate was passed through a 500mL pre-equilibrated cation exchange resin column at a linear flow rate of 10mL / min. Effluent collection began when the UV detection value reached 200mAu. After all the coarse filtrate was treated, pure water was used instead, flowing through the cation exchange resin column until the UV detection value fell below 200mAu. 0.3g of a neutral protease with an enzyme activity of 150,000 U / g was added to the collected effluent (at pH 7), and the mixture was enzymatically hydrolyzed at 50℃ for 5 hours to obtain the hydrolysate. After enzymatic hydrolysis, the hydrolysate was heated to 95℃ and incubated for 20 min to inactivate the protease. Then, it was centrifuged at 3000 r / min for 10 min to obtain the supernatant. The supernatant was filtered through a ceramic membrane (550 nm) to obtain the filtrate. The filtrate was concentrated by rotary evaporation using a rotary evaporator at 80℃ and a vacuum of -0.1 MPa. After removing 1700 mL of water from the filtrate, the remaining permeate was obtained. This permeate was mixed with 10 g of diatomaceous earth and filtered through the diatomaceous earth to obtain the clear liquid. The inlet air temperature of the spray drying tower was set to 140℃ and the outlet air temperature to 90℃. The clear liquid was then spray-dried to obtain silk fibroin peptide No. 5.
[0159] Example 6: Quantitative analysis of five silk fibroin peptides
[0160] (1) Physicochemical parameters of silk fibroin peptides:
[0161] Following the method of Example 2, the molecular weight distribution, moisture and ash content, protein, peptide content, free amino acid content, and yield of silk fibroin peptides 1-5 obtained in Example 4 were detected. The results are shown in Table 9.
[0162] Table 9
[0163] (2) Identification of functional peptides in silk fibroin:
[0164] Weigh 2.0 mg of silk fibroin peptide samples 1-5 obtained in Example 4, dissolve them in ultrapure water, vortex mix, and bring the volume to 100 mL to obtain a silk fibroin peptide solution. Filter the above silk fibroin peptide solution through a 0.2 μm PTFE filter membrane to obtain the sample to be tested. In positive ion mode, with collision energies set to -25 V and -35 V, perform product ion scanning (PIS) on the sample to be tested to obtain product ion mass spectra, as shown in Figures 8, 9, and 10. Figure 8 shows the product ion mass spectrum of peptide Gly-Ala (GA), Figure 9 shows the product ion mass spectrum of peptide Gly-Ala-Tyr (GAY), and Figure 10 shows the product ion mass spectrum of peptide Ala-Ala (AA). In Figures 8, 9, and 10, A represents the product ion mass spectrum obtained with a collision energy of -35 V, and B represents the product ion mass spectrum obtained with a collision energy of -25 V. Based on the information of the product ions generated after the collision and the NCBI and UniProt databases, three peptide structures were finally identified: Gly-Ala (GA), Gly-Ala-Tyr (GAY), and Ala-Ala (AA), as detailed in Table 10.
[0165] Table 10
[0166] (3) Quantification of functional peptides in silk fibroin:
[0167] Accurately weigh 2.0 mg of standard peptides Gly-Ala (GA), Gly-Ala-Tyr (GAY), and Ala-Ala (AA), respectively, dissolve them in ultrapure water, vortex to mix, and bring the volume to 100 mL to obtain a standard stock solution with a concentration of 20 μg / mL. The above standard stock solution was serially diluted with ultrapure water to 1.95 ng / mL, 3.91 ng / mL, 7.81 ng / mL, 15.63 ng / mL, 31.25 ng / mL, 62.5 ng / mL, 125 ng / mL, 250 ng / mL, and 500 ng / mL, and filtered through a 0.22 μm aqueous polyethersulfone membrane to obtain a series of standard working solutions with varying concentrations. The silk fibroin peptides 1-5 obtained in Example 4 were prepared into stock solutions with a concentration of 10.0 mg / mL using ultrapure water, then diluted 1000 times and filtered through a 0.22 μm aqueous polyethersulfone filter membrane to obtain a sample solution with a concentration of 20 μg / mL.
[0168] Inject 10 μL of the above-mentioned standard working solution and the sample solution to be tested, respectively, and perform quantitative analysis in positive ion mode. This yields quantitative chromatograms and standard curves for the standard peptides Gly-Ala (GA), Gly-Ala-Tyr (GAY), and Ala-Ala (AA), as well as quantitative chromatograms for the peptides Gly-Ala (GA), Gly-Ala-Tyr (GAY), and Ala-Ala (AA) in the sample to be tested. See Figures 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, and 31. Figure 11 shows the standard curve for the standard peptide Gly-Ala (GA), and Figure 12 shows the standard curve for the standard peptide Gly-Ala (GA). Figures 13, 14, 15, 16, and 17 show the quantitative chromatograms of Gly-Ala (GA) peptides from silk fibroin peptides 1-5, respectively. Figure 18 shows the standard curve of the standard peptide Gly-Ala-Tyr (GAY), and Figure 19 shows the quantitative chromatogram of the standard peptide Gly-Ala-Tyr (GAY). Figures 20, 21, 22, 23, and 24 show the quantitative chromatograms of Gly-Ala-Tyr (GAY) peptides from silk fibroin peptides 1-5, respectively. Figure 25 shows the standard curve of the standard peptide Ala-Ala (AA), and Figure 26 shows the quantitative chromatogram of the standard peptide Ala-Ala (AA). Figures 27, 28, 29, 30, and 31 show the quantitative chromatograms of Ala-Ala (AA) peptides from silk fibroin peptides 1-5, respectively. The contents of Gly-Ala (GA), Gly-Ala-Tyr (GAY), and Ala-Ala (AA) peptides in silk fibroin peptides, calculated according to the standard curve, are shown in Table 11. As shown in Table 11, the contents of Gly-Ala (GA), Gly-Ala-Tyr (GAY), and Ala-Ala (AA) peptides in silk fibroin peptide No. 1 are the highest, at 4.40%, 0.31%, and 0.11%, respectively.
[0169] The liquid chromatography conditions were as follows: the column was an Inertsil ODS-3 (particle size 5 μm, inner diameter 2.1 mm, column length 250 mm); mobile phase A was pure water (containing 0.1% (v / v) formic acid), mobile phase B was 100% acetonitrile (containing 0.1% (v / v) formic acid), and the flow rate was 0.2 mL / min. The gradient elution program specifically included: the first gradient elution time was 0 min–15 min, with mobile phase B accounting for 0%–40%; the second gradient elution time was 15 min–20 min, with mobile phase B accounting for 40%–80%; the third gradient elution time was 20 min–25 min, with mobile phase B accounting for 80%; and the fourth gradient elution time was 25.1 min–35 min, with mobile phase B accounting for 0%. The column oven temperature was 40℃. The mass spectrometry conditions were as follows: ionization mode was electrospray ionization (ESI); ion spray voltage was +4.5 kV, nebulizing gas (nitrogen) flow rate was 3.0 L / min, heating gas (nitrogen) flow rate was 10 L / min, drying gas (nitrogen) flow rate was 10 L / min, desolvation tube temperature was 250 °C, heating module temperature was 400 °C, and ion source temperature was 300 °C; scanning mode was multiple reaction monitoring (MRM), residence time was 100 ms, and delay time was 3 ms; the MRM parameters were optimized and can be found in Table 12, where * indicates quantitative ions.
[0170] Table 11
[0171] Table 12
[0172] In summary, the protein content of No. 1 silk fibroin peptide is 83.39%, the peptide content is 64.76%, the yield is 46.67%, and the proportion of molecular weight below 1000u is as high as 94.43%. The contents of its characteristic functional peptides Gly-Ala (GA), Gly-Ala-Tyr (GAY), and Ala-Ala (AA) can reach 4.40%, 0.31%, and 0.11%, respectively. Therefore, No. 1 silk fibroin peptide is the silk fibroin peptide with the best anti-wrinkle effect, and the preparation method of No. 1 silk fibroin peptide is the best preparation method for preparing silk fibroin peptides with anti-wrinkle effect. Meanwhile, the contents of Gly-Ala (GA) in silk fibroin peptides No. 1-5 are all greater than 0.07%, the contents of Gly-Ala-Tyr (GAY) are all greater than 0.01%, and the contents of Ala-Ala (AA) are all greater than or equal to 0.02%. This means that the contents of the functional peptides GA, GAY, and AA in silk fibroin peptides No. 1-5 are higher than those in the silk fibroin peptide of Example 2. This indicates that silk fibroin peptides No. 1-5 all have good anti-wrinkle potential, and correspondingly, their preparation method can produce silk fibroin peptides with anti-wrinkle effects.
[0173] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and not to limit them; although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or make equivalent substitutions for some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.
Claims
A functional peptide with anti-wrinkle effects, wherein, The functional peptides include peptide GA, peptide GAY, and peptide AA. A silk fibroin peptide with anti-wrinkle effects, wherein, The silk fibroin peptide includes the functional peptide segment described in claim 1. According to claim 2, the silk fibroin peptide, wherein, Based on the mass of the silk fibroin peptide, the mass content of peptide GA is ≥0.07%, the mass content of peptide GAY is ≥0.01%, and the mass content of peptide AA is ≥0.02%. According to claim 2 or 3, the silk fibroin peptide, wherein, The moisture content of the silk fibroin peptide is ≤2%. g / 100g, ash content ≤30g / 100g; and / or, The protein content of the silk fibroin peptide is ≥70g / 100g; and / or, The content of acid-soluble protein in the silk fibroin peptide is ≥70g / 100g; and / or, The free amino acid content in the silk fibroin peptide is ≤20g / 100g; and / or, The silk fibroin peptides contain ≥50g / 100g peptide content; and / or, The silk fibroin peptide contains ≥80% peptides with a molecular weight of less than 1000u. The silk fibroin peptide according to any one of claims 2-4, wherein, The silk fibroin peptide is obtained by enzymatic hydrolysis and separation and purification of silkworm cocoon protein as raw material. The enzymatic hydrolysis process includes enzymatic hydrolysis using at least one of neutral protease and carboxypeptidase A, and at least includes neutral protease. A method for preparing silk fibroin peptide according to any one of claims 2-5, wherein, Includes the following steps: The silk fibroin peptide was obtained by enzymatic hydrolysis and separation and purification of silk cocoon protein as raw material. The enzymatic hydrolysis process includes enzymatic hydrolysis using at least one of neutral protease and carboxypeptidase A, and at least includes neutral protease. According to the preparation method of claim 6, wherein, The enzymatic hydrolysis treatment is performed at a temperature of 45-55℃ for 4.0-6.0 hours, with a pH value of 6.5-7.5; and / or, The neutral protease has an enzyme activity of 140,000-160,000 U / g; and / or, Based on each gram of the silkworm cocoon protein, the amount of neutral protease added is 0.2 wt%-2 wt%; and / or, The carboxypeptidase A has an enzyme activity of 4000-6000 U / g; and / or, The amount of carboxypeptidase A added per gram of the silkworm cocoon protein is 0.2 wt% to 2 wt%. The preparation method according to claim 6 or 7, wherein, The silkworm cocoon protein can be obtained through the following steps: Silkworm cocoons and water are treated at 110-130℃ and 0.05-0.17MPa for 0.5-1.5 hours, and the insoluble matter is obtained by filtration. The insoluble matter, water and sodium hydroxide are then treated at 110-130℃ and 0.05-0.17MPa for 1.0-2.0 hours to obtain the silkworm cocoon protein. The preparation method according to any one of claims 6-8, wherein, The separation and purification process includes at least one of diatomaceous earth adsorption treatment, cation exchange resin treatment, anion exchange resin treatment, and macroporous resin treatment. The application of the functional peptide of claim 1, and / or the silk fibroin peptide of any one of claims 2-5, and / or the silk fibroin peptide prepared by the preparation method of any one of claims 6-9 in the preparation of anti-wrinkle related products.