Process for preparing hydrolyzed elastin for use as a cosmetic raw material and applications

Abstract

The application discloses a preparation process and application of hydrolyzed elastin used as a cosmetic raw material, and belongs to the technical field of cosmetic processing.The preparation process of the hydrolyzed elastin provided by the application discloses the traditional hot alkali method for removing grease and impurities, adopts low-concentration sodium bicarbonate and sucrose ester gradient ultrasonic degreasing, and removes impurities by dissolving in a weak alkali, so that the active structures of lock chain and isolock chain are retained, gradient enzymolysis is adopted by using microbial source elastase, the enzymolysis liquid is separated by circulation through a 1000Da ultrafiltration system after being subjected to multistage centrifugation and ceramic membrane filtration, large-molecule polypeptides are subjected to secondary enzymolysis by backflow, small-molecule active peptides with a molecular weight of less than 1000Da are enriched, and the total content of lock chain and isolock chain in the obtained product can reach 0.64g / 100g; the preparation process provided by the application is green and safe, cost is saved, and the process is beneficial to realizing industrialized production; and the prepared hydrolyzed elastin has excellent transdermal absorption and is suitable for being used as an anti-aging and skin repair cosmetic raw material.

Description

Technical Field

[0001] This invention relates to the field of cosmetic processing technology, and in particular to a process for preparing hydrolyzed elastin as a cosmetic raw material and its application. Background Technology

[0002] With rising living standards and improved material conditions, people's awareness of skincare has continued to upgrade, and cosmetics have become necessities in people's lives. Anti-aging and firming cosmetics, in particular, have become a core competitive area in the beauty industry. Consumers' demand for safe, non-irritating, highly active, and gentle natural bioactive ingredients continues to rise. Proteins are the most important building blocks of life, participating in metabolism, substance synthesis, information transmission, and disease monitoring. Using them as cosmetic ingredients is the safest and most effective. However, currently, collagen, keratin, and silk protein are added, with collagen being the primary addition, while elastin is almost non-existent. Many people believe that adding collagen to cosmetics can preserve youthful skin as they age, but in reality, elastin plays a more important role in maintaining and supporting skin elasticity.

[0003] The reduction of elastin is one of the main causes of skin sagging and aging. Although elastin only accounts for 2-5% of the total protein content in the skin, it is crucial for maintaining skin structure and function. Elastic fibers, like "springs," provide the skin with extensibility and resilience. Desmosin (Des) and isodesmosin (Ide) are unique components of elastin and also its characteristic amino acids, often used to evaluate the quality and purity of elastin peptides. They are covalently cross-linked by lysine residues to form a unique network structure, and changes in this network conformation give elastin sufficient elasticity. Adding animal tissue-derived elastin hydrolysates to cosmetics can delay skin aging, resist photochemical reactions, and promote cell proliferation. Elastin itself is insoluble in water, but hydrolyzed elastin after enzymatic and chemical treatment is water-soluble and has a small molecular weight, allowing it to cross the epidermal barrier and enter the superficial dermis to exert its effects. Elastin has the ability to resist aging and ultraviolet radiation, not only repairing already aged skin but also revitalizing aging skin. Because elastin is rich in hydrophobic amino acids, it is considered a good source of antioxidant peptides, which can protect the skin from oxidative stress damage by inhibiting oxidative stress and improving antioxidant capacity, thereby delaying skin aging and damage.

[0004] Elastin's high cross-linking and high content of hydrophobic amino acids make it insoluble in water and resistant to dilute acid / alkali hydrolysis, making the preparation of soluble elastin difficult. Currently, common methods for obtaining hydrolyzed elastin include enzymatic hydrolysis, acid hydrolysis, and organic alkali hydrolysis. However, acid and alkali hydrolysis methods have certain drawbacks. For example, they generate large amounts of waste liquid, causing environmental pollution; the hydrolysis intensity is high under high temperatures, making it difficult to control the degree of hydrolysis; and alkali hydrolysis leads to racemization of most amino acids. Enzymatic hydrolysis, on the other hand, has mild reaction conditions, fewer side reactions, and does not produce harmful substances. However, the enzyme currently used for enzymatic hydrolysis of elastin is mainly elastase, which has limited sources, high costs, and is difficult to commercialize.

[0005] Currently, the main raw materials for preparing hydrolyzed elastin are connective tissue from terrestrial animals and tissue from aquatic fish. However, raw materials such as bovine neck ligaments, animal cartilage, and porcine / bovine aorta have high procurement costs and require strong alkali pretreatment due to their dense structure, which can easily damage the active cross-linked structure of elastin. Most current preparation methods primarily use aquatic fish tissue. Skipjack tuna heart bulbs are a good raw material for elastin preparation. Because the bulbs need to repeatedly dilate and diastolic to buffer blood pressure, they have a high proportion of elastin, are rich in desmokinin and isodesmokinin, have fewer impurities, and are easier to prepare into small-molecule hydrolyzed elastin with excellent bioavailability. Summary of the Invention

[0006] The purpose of this invention is to provide a preparation process and application of hydrolyzed elastin as a cosmetic raw material, so as to provide a green and safe preparation process for highly active, small-molecule hydrolyzed elastin.

[0007] To achieve the above objectives, the present invention provides a process for preparing hydrolyzed elastin for use as a cosmetic ingredient, the method of which is as follows: S1. Take the heart arterial bulb tissue of bonito as raw material, crush it into pieces, wash it and soak it in physiological saline; S2. The arterial bulb fragments were soaked in a degreasing solution and then sonicated. After sonication, the soaking treatment was continued for a total of three cycles. The degreasing solution after sonication was allowed to stand in an ice bath. After the degreasing solution separated into layers, the aqueous phase was obtained. S3. The product is soaked in a mixed solution of sodium hydroxide and sodium carbonate under constant temperature and stirring conditions. The insoluble matter is crushed, and then the material is rinsed and allowed to stand to drain the free liquid. The product is then soaked a second time in a mixed solution of sodium hydroxide and sodium carbonate under constant temperature and stirring conditions. After the soaking is completed, the product is washed with purified water and dehydrated by vacuum filtration to obtain crude elastin. S4. Take crude elastin, add purified water to reconstitute it, then add Streptomyces griseus elastase, adjust the pH of the system, hydrolyze at a constant temperature, and then inactivate the enzyme at high temperature to complete the first enzymatic hydrolysis. Add Aspergillus oryzae neutral protease and Aspergillus oryzae aminopeptidase complex enzyme to the above system, adjust the pH of the system, hydrolyze at a constant temperature, and then inactivate the enzyme at high temperature to complete the second enzymatic hydrolysis. S5. After enzymatic hydrolysis, the liquid is centrifuged at low speed, and the supernatant is collected. Then, it is centrifuged at high speed to collect the clear crude hydrolysate. The crude hydrolysate is passed through a ceramic membrane for circulation filtration. The filtrate is sent to an ultrafiltration membrane system with a molecular weight cutoff of 1000 Da for circulation separation. Large molecular weight peptides with a molecular weight cutoff of >1000 Da are retained and enzymatically hydrolyzed twice more according to the above steps. Small molecular weight elastic peptide liquid with a molecular weight of <1000 Da is collected on the permeate side. S6. Connect the ultrafiltration permeate in series with three chromatography columns in the following order: cation exchange resin column → plant polyphenol adsorption resin column → amino chelate resin column. Pass the solution through the columns continuously at room temperature. S7. The purified hydrolyzed elastin solution is filtered through a filter membrane at room temperature under sterile conditions to obtain sterile peptide solution. The sterile peptide solution is then transferred to a vacuum low-temperature concentration tank. Low molecular weight sodium hyaluronate is added to the concentrate and stirred until homogeneous. S8. Low-temperature sterile spray drying is adopted, and after drying, the product is sieved to obtain hydrolyzed elastin powder.

[0008] Preferably, the defatting solution in S2 consists of a 0.8% sodium bicarbonate solution and a 0.5% food-grade sucrose ester in a volume ratio of 2:1, and the mass-volume ratio of arterial bulb fragments to defatting solution is 1g:8mL.

[0009] Preferably, in the first soaking of sodium hydroxide and sodium carbonate in S3, the mass concentration ratio of sodium hydroxide to sodium carbonate in the mixed solution is 1:40, and in the second soaking of sodium hydroxide and sodium carbonate in the mixed solution, the mass concentration ratio of sodium hydroxide to sodium carbonate is 1:20.

[0010] Preferably, in step S4, the amount of Streptomyces griseus elastase added is 0.8-1.2%, the pH value of the system after two enzymatic hydrolysis is 7.0-8.5, the constant temperature is 40-50℃, the hydrolysis time is 2-3h, the enzyme inactivation temperature is 85-95℃, and the enzyme inactivation treatment time is 20-40min.

[0011] Preferably, in step S4, the amount of Aspergillus oryzae neutral protease and Aspergillus oryzae aminopeptidase complex enzyme added is 1-2%, and the composition of Aspergillus oryzae neutral protease and Aspergillus oryzae aminopeptidase complex enzyme is 2:1 in mass ratio.

[0012] Preferably, in step S5, the low-speed centrifugation condition is centrifugation at 3000 r / min for 10 min, and the high-speed centrifugation condition is centrifugation at 8000 r / min for 15 min, and the ceramic membrane pore size is 80-120 nm.

[0013] Preferably, in step S6, the volume ratio of the three resin columns is 1:1.3:1.2; the flow rate of the feed solution is controlled at 3.5 BV / h, and the ambient temperature is 25-35℃; in step S7, the filter membrane used is 0.22μm, and the concentration conditions are that the concentration temperature does not exceed 60℃, the concentration is carried out to a peptide solid content of 30wt%, and the amount of low molecular weight sodium hyaluronate added is 0.7% of the peptide solid content; in step S8, the drying conditions are an inlet air temperature of 140℃ and an outlet air temperature of 70℃, and the dried product is passed through a 60-mesh sieve.

[0014] Preferably, in S1, the size of the fragmented cardiac arterial bulb is 5mm×5mm×5mm, and the cleaning conditions are: rinsing with running water for 10 minutes and standing in physiological saline for 2 hours; in S2, the soaking time is 15 minutes each time, the ultrasonic conditions are: power 350W, frequency 28kHz, ultrasonication for 10 minutes each time, the temperature under ice bath conditions is 4-5℃, and the standing time is 2-3 hours; in S3, the constant temperature during the two soakings is 40-50℃, the soaking time is 30-50 minutes, the material-liquid ratio is 1:5-1:6, the rinsing time is 3-5 minutes, the number of washings is 3-5 times, and the vacuum filtration dewatering filter cloth is 80 mesh.

[0015] Hydrolyzed elastin prepared using the above-described preparation process.

[0016] The above describes the application of the preparation process in the field of cosmetics manufacturing.

[0017] This invention utilizes a low-concentration weak alkali for multiple defatting and impurity removal processes, and introduces microbial elastase and aminopeptidase for segmented enzymatic hydrolysis, combined with a 1000 Da ultrafiltration system, to synergistically prepare hydrolyzed elastin with high activity and small molecular weight. The underlying principle is as follows: (1) Hydroxide (OH) under traditional strong alkaline conditions - It will attack the CN bonds at the cross-linking sites of the cyclic backbone of desmosin and isodesmosin, causing the ring to break and completely losing the repair and antioxidant activity of elastin. This application reduces the structural damage caused by the hot alkaline method through a mild weak alkaline process, and completely preserves the active structure of desmosin and isodesmosin, ensuring high-quality protein raw materials from enzymatic hydrolysis. (2) Elastin with complete cross-linked backbone has a regular and orderly molecular structure. Streptomyces gray elastase can specifically recognize the hydrophobic amino acid sequence of the cross-linked site of elastin, cut only the peptide bond of the macromolecular backbone, and disassemble it into medium-length peptides. Combined with neutral protease selectively cuts the hydrophobic peptide bond in the middle of the peptide chain, and aminopeptidase removes a small number of amino acids one by one from the N end of the peptide chain, the polarity and transdermal ability of the peptide are enhanced. The three enzymes work together to achieve precise control of the degree of hydrolysis. (3) When the large molecular peptides retained by ultrafiltration at 1000 Da are refluxed for secondary enzymatic hydrolysis, aminopeptidase, as an exonuclease, can work together with endonuclease to accelerate the conversion into small molecular peptides with a target of <1000 Da, reduce the proportion of large molecular peptides that are difficult to degrade in the system, and improve the protein recovery rate.

[0018] Therefore, the present invention provides a process for preparing hydrolyzed elastin as a cosmetic ingredient and its application, the specific technical effects of which are as follows: (1) The method for preparing hydrolyzed elastin as a cosmetic raw material provided by the present invention abandons the traditional hot alkaline method, fully preserves the active structure of elastin desmokinin and isodesmokinin, reduces the use of organic solvents, and realizes the green and environmentally friendly preparation process. (2) In this invention, a new microbial enzyme is introduced to perform two enzymatic hydrolysis processes. First, streptococcal elastase is added to cut the cross-linked backbone. After enzyme inactivation treatment, neutral protease and aminopeptidase are combined to modify the peptide chain. Endoenzymes and exoenzymes are combined to precisely control the degree of hydrolysis and reduce the generation of bitter free amino acids. (3) In this invention, a 1000Da ultrafiltration system is added for circulation separation, and large molecular peptides are retained and refluxed for secondary enzymatic hydrolysis to ensure that the enriched peptides are all small molecular peptides <1000Da. The hydrolyzed elastin prepared has stronger transdermal absorption capacity and is more suitable as a cosmetic raw material.

[0019] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0020] Figure 1 The graph shows the antioxidant properties of the prepared hydrolyzed elastin.

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0023] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0024] The instruments, equipment, and reagents used in the examples were all obtained through commercial means; the methods and steps not described in detail in the examples are all conventional techniques in the art; the degreasing solution used in the examples consists of sodium bicarbonate with a mass concentration of 0.8% and food-grade sucrose ester with a mass concentration of 0.5% (volume ratio of 2:1).

[0025] Example 1 Using skipjack tuna heart aortic bulb as raw material, it was crushed into blocks approximately 5mm × 5mm × 5mm in size (denoted as aortic bulb fragments). The fragments were washed with running water for 10 minutes and then soaked in physiological saline for 2 hours. After the time was up, the aortic bulb fragments were filtered out, drained, and mixed with defatting solution at a mass-to-volume ratio of 1g:8mL. The mixture was soaked for 15 minutes, then sonicated at 350W and 28kHz for 10 minutes. After sonication, the mixture was soaked for another 15 minutes, and then sonicated again at 350W and 28kHz for 10 minutes. This soaking-sonication process was repeated three times. The defatting solution after sonication was allowed to stand in an ice bath (approximately 4℃) for 2 hours. After the solution separated into layers, the aqueous phase was obtained. The obtained aqueous phase was soaked for 40 min in a mixed solution of sodium hydroxide and sodium carbonate (sodium hydroxide:sodium carbonate mass concentration ratio of 1:40, material-to-liquid ratio of 1:6) under constant temperature stirring at 40℃. The insoluble matter was then crushed using a homogenizer. The material was then rinsed with purified water for 3-5 min, and allowed to stand to drain the free liquid. A second soaking was then performed at 40℃ under constant temperature stirring in a mixed solution of sodium hydroxide and sodium carbonate (sodium hydroxide:sodium carbonate mass concentration ratio of 1:20, material-to-liquid ratio of 1:6) for 50 min. Afterward, the material was washed three times with purified water and then vacuum filtered through an 80-mesh filter cloth to obtain crude elastin.

[0026] Weigh 100g of crude elastin, reconstitute it with purified water, then add 1.0g of *Streptomyces griseus* elastase, adjust the pH to 7.5, and hydrolyze at 50℃ for 2 hours. After hydrolysis, inactivate the enzyme at 90℃ for 30 minutes to complete the first enzymatic hydrolysis. Add 1.5g of a complex enzyme of *Aspergillus oryzae* neutral protease and *Aspergillus oryzae* aminopeptidase (mass ratio 2:1, total addition 1.5% of substrate mass) to the above system, adjust the pH to 8.0, and hydrolyze at 50℃ for 3 hours. After hydrolysis, inactivate the enzyme at 90℃ for 30 minutes to complete the second enzymatic hydrolysis.

[0027] After enzymatic hydrolysis, the solution is centrifuged at 3000 rpm for 10 min, and the supernatant is collected. This supernatant is then centrifuged at 8000 rpm for 15 min, and the clarified crude hydrolysate is collected. The crude hydrolysate is then passed through a ceramic membrane with a pore size of 80-120 nm for circulating filtration. The filtrate is then transferred to an ultrafiltration membrane system with a molecular weight cutoff of 1000 Da for further separation. Large peptides with a molecular weight cutoff >1000 Da are retained and subjected to two more enzymatic hydrolysis cycles as described above. Small molecule elastic peptides with a molecular weight <1000 Da are collected from the permeate side.

[0028] The ultrafiltration permeate was sequentially packed into three chromatography columns in series. The column packing order was: cation exchange resin column → plant polyphenol adsorption resin column → amino chelating resin column, with a volume ratio of 1:1.3:1.2. The feed flow rate was controlled at 3.5 BV / h, and the column chromatography was performed continuously at room temperature. The purified hydrolyzed elastin solution was aseptically filtered through a 0.22 μm filter membrane at room temperature to obtain a sterile peptide solution. The sterile peptide solution was transferred to a vacuum low-temperature concentration tank, and the concentration temperature was controlled not to exceed 60℃. The concentration was carried out until the peptide solids content was 30 wt%. Low molecular weight sodium hyaluronate (0.7% of the peptide solids content) was added to the concentrate and stirred evenly. Low-temperature sterile spray drying was performed with an inlet air temperature of 140℃ and an outlet air temperature of 70℃. After drying, the solution was passed through a 60-mesh sieve to obtain 63.2 g of hydrolyzed elastin powder.

[0029] Comparative Example 1 Skipjack tuna heart arterial bulbs were used as raw material, crushed into blocks approximately 5mm × 5mm × 5mm in size, washed with running water for 10 minutes, and then soaked in physiological saline for 2 hours. The arterial bulb fragments were mixed with 0.1% sodium hydroxide solution at a mass-to-volume ratio of 1g:8mL and soaked at room temperature for 6 hours. After soaking, the mixture was washed with purified water until the pH value reached 7.0~7.5. The arterial bulb fragments were then mixed with 3 times the volume of purified water, and the pH value was adjusted to 9.0 with sodium hydroxide solution. The mixture was heated to 55℃, and the heated solution was allowed to stand in an ice bath (4℃) for 2 hours. After the defatted solution separated into layers, the aqueous phase was obtained by separation.

[0030] Weigh 100g of crude elastin, reconstitute it with purified water, then add 0.6g of alkaline protease, adjust the pH of the system to 7.5, and hydrolyze at a constant temperature of 50℃ for 2 hours. After hydrolysis, inactivate the enzyme at 90℃ for 30 minutes to complete the first enzymatic hydrolysis. Add 0.05g of flavor protease to the above system, adjust the pH of the system to 8.0, hydrolyze at a constant temperature of 50℃ for 3 hours, and inactivate the enzyme at 90℃ for 30 minutes to complete the second enzymatic hydrolysis.

[0031] After enzymatic hydrolysis, the solution is centrifuged at 3000 rpm for 10 min, and the supernatant is collected. This supernatant is then centrifuged at 8000 rpm for 15 min, and the clarified crude hydrolysate is collected. The crude hydrolysate is then passed through a ceramic membrane with a pore size of 80-120 nm for circulating filtration. The filtrate is then transferred to an ultrafiltration membrane system with a molecular weight cutoff of 1000 Da for further separation. Large peptides with a molecular weight cutoff >1000 Da are retained and subjected to two more enzymatic hydrolysis cycles as described above. Small molecule elastic peptides with a molecular weight <1000 Da are collected from the permeate side.

[0032] The ultrafiltration permeate was sequentially packed into three chromatography columns in series, with the column packing order as follows: cation exchange resin column → plant polyphenol adsorption resin column → amino chelating resin column, and the volume ratio of the three resin columns was 1:1.3:1.2. The feed flow rate was controlled at 3.5 BV / h, and the column chromatography was performed continuously at room temperature. The purified hydrolyzed elastin solution was then aseptically filtered through a 0.22 μm filter membrane at room temperature to obtain a sterile peptide solution. The sterile peptide solution was transferred to a vacuum low-temperature concentration tank, and the concentration temperature was controlled to not exceed 60℃. The solution was concentrated to a peptide solids content of 30 wt%. Low molecular weight sodium hyaluronate (0.7% of the peptide solids content) was added to the concentrate and stirred thoroughly. Low-temperature aseptic spray drying was performed with an inlet air temperature of 140℃ and an outlet air temperature of 70℃. After drying, the solution was passed through a 60-mesh sieve to obtain 55.4 g of hydrolyzed elastin powder.

[0033] Comparative Example 2 Skipjack tuna heart aortic bulb was used as raw material, crushed into pieces approximately 5mm × 5mm × 5mm in size, washed with running water for 10 minutes, and then soaked in physiological saline for 2 hours. A defatting solution was prepared, consisting of 0.8% sodium bicarbonate and 0.5% food-grade sucrose ester (volume ratio 2:1). The mixture was soaked in the solution at a mass-volume ratio of 1g:8mL for 15 minutes, followed by sonication at 350W and 28kHz for 10 minutes. After sonication, the mixture was soaked for another 15 minutes, and then sonicated for another 10 minutes under the same conditions, for a total of three cycles. The sonicated defatting solution was then allowed to stand in an ice bath (approximately 4℃) for 2 hours. After the solution separated into layers, the aqueous phase was obtained. The above product was soaked in a mixed solution of sodium hydroxide and sodium carbonate (sodium hydroxide:sodium carbonate mass concentration ratio of 1:40) (material-to-liquid ratio of 1:6) at a constant temperature of 40℃ with stirring for 40 min. The insoluble matter was then crushed using a homogenizer. The material was then rinsed with purified water for 3-5 min, and allowed to stand to drain the free liquid. A second soaking was then performed at 40℃ with stirring in a mixed solution of sodium hydroxide and sodium carbonate (sodium hydroxide:sodium carbonate mass concentration ratio of 1:20) (material-to-liquid ratio of 1:6) for 50 min. Afterward, the product was washed three times with purified water and then vacuum filtered through an 80-mesh filter cloth to obtain crude elastin.

[0034] Weigh 100g of crude elastin, reconstitute it with purified water, then add 0.6g of alkaline protease, adjust the pH of the system to 7.5, and hydrolyze at a constant temperature of 50℃ for 2 hours. After hydrolysis, inactivate the enzyme at 90℃ for 30 minutes to complete the first enzymatic hydrolysis. Add 0.05g of flavor protease to the above system, adjust the pH of the system to 8.0, hydrolyze at a constant temperature of 50℃ for 3 hours, and inactivate the enzyme at 90℃ for 30 minutes to complete the second enzymatic hydrolysis.

[0035] After enzymatic hydrolysis, the solution is centrifuged at 3000 rpm for 10 min, and the supernatant is collected. This supernatant is then centrifuged at 8000 rpm for 15 min, and the clarified crude hydrolysate is collected. The crude hydrolysate is then passed through a ceramic membrane with a pore size of 80-120 nm for circulating filtration. The filtrate is then transferred to an ultrafiltration membrane system with a molecular weight cutoff of 1000 Da for further separation. Large peptides with a molecular weight cutoff >1000 Da are retained and subjected to two more enzymatic hydrolysis cycles as described above. Small molecule elastic peptides with a molecular weight <1000 Da are collected from the permeate side.

[0036] The ultrafiltration permeate was sequentially packed into three chromatography columns in series. The column packing order was: cation exchange resin column → plant polyphenol adsorption resin column → amino chelating resin column, with a volume ratio of 1:1.3:1.2. The feed flow rate was controlled at 3.5 BV / h, and the column chromatography was performed continuously at room temperature. The purified hydrolyzed elastin solution was aseptically filtered through a 0.22 μm filter membrane at room temperature to obtain a sterile peptide solution. The sterile peptide solution was transferred to a vacuum low-temperature concentration tank, and the concentration temperature was controlled not to exceed 60℃. The concentration was carried out until the peptide solids content was 30 wt%. Low molecular weight sodium hyaluronate (0.7% of the peptide solids content) was added to the concentrate and stirred evenly. Low-temperature sterile spray drying was performed with an inlet air temperature of 140℃ and an outlet air temperature of 70℃. After drying, the solution was passed through a 60-mesh sieve to obtain 53.6 g of hydrolyzed elastin powder.

[0037] Example 1 Determination of desmokinin and isodesmokinin content in hydrolyzed elastin: Weigh 10 mg of the prepared hydrolyzed elastin sample into an ampoule, add 5 mL of 6M hydrochloric acid solution, seal the ampoule, and place it in an oven at 110℃ for 24 h for hydrolysis. After filtration, transfer the solution to a 50 mL volumetric flask and dilute to volume. Pipette 2 mL into a test tube and place it in a 60℃ oven until a small trace remains at the bottom. Add 2 mL of sample buffer and shake thoroughly to mix. Pipette 1 mL of the test sample through a 0.22 μm filter and analyze it using the instrument.

[0038] The contents of desmokine and isodesmokine in hydrolyzed elastin are shown in Table 1. The contents of desmokine and isodesmokine in the hydrolyzed elastin prepared in Example 1 are higher than those prepared in Comparative Example 1 and Comparative Example 2.

[0039] Table 1. Content of desmokinin and isodesmokinin in hydrolyzed elastin

[0040] Example 2 Determination of degree of hydrolysis and protein recovery: Total nitrogen was determined using the Kjeldahl method in GB 5009.5-2025 "National Food Safety Standard - Determination of Protein in Food". The degree of hydrolysis was determined using the formaldehyde titration method. Protein recovery was calculated using Formula I, and the degree of hydrolysis was calculated using Formula II.

[0041] Protein recovery rate (%) = total nitrogen mass of enzymatic hydrolysate / total nitrogen mass of raw material × 100 (Formula I).

[0042] Degree of hydrolysis (%) = Amino acid nitrogen content of enzymatic hydrolysate / Total nitrogen content of raw material × 100 (Formula II).

[0043] The results are shown in Table 2. The preparation method in Example 1 has a higher degree of hydrolysis and protein recovery rate compared with Comparative Example 1 and Comparative Example 2, which is beneficial to improving the utilization rate of resources.

[0044] Table 2. Degree of hydrolysis and protein recovery rate for different preparation methods

[0045] Example 3 Determination of the in vitro antioxidant activity of hydrolyzed elastin: (1) Determination of the free radical scavenging rate of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH).

[0046] Prepare a sample solution with a mass concentration of 8.0 mg / mL by distilling 40 mg of hydrolyzed elastin using distilled water. Take 3 mL of the sample solution in a test tube, add 3 mL of 0.1 mmol / L DPPH solution (prepared with 95% ethanol), shake to mix, and incubate in the dark for 30 min. Measure the absorbance at 517 nm, zeroing the tube with distilled water. Use 0.5 mg / mL ascorbic acid solution as a positive control. The DPPH free radical scavenging rate is calculated using the following formula: DPPH radical scavenging rate (%) = [A0 - (A1 - A2)] / A0 × 100%, Where A0 is the absorbance of distilled water + DPPH solution; A1 is the absorbance of sample + DPPH solution; and A2 is the absorbance of sample + 95% ethanol solution.

[0047] (2) Superoxide anion radical (O2) - Scavenging rate determination: Prepare a sample solution with a mass concentration of 8.0 mg / mL by distilling 20 mg of hydrolyzed elastin with distilled water. Take 1 mL of the sample solution in a test tube, add 4.5 mL of 50 mmol / L Tris-HCl buffer and 0.1 mL of 3 mmol / L pyrogallol solution, mix thoroughly, and incubate in a water bath at 25°C for 10 min. Stop the reaction by adding 1 mL of 8 mmol / L hydrochloric acid solution. Measure the absorbance at 320 nm, using distilled water as a blank control and 0.5 mg / mL ascorbic acid solution as a positive control. The superoxide anion radical scavenging rate is calculated using the following formula.

[0048] Superoxide anion radical scavenging rate (%) = [A3 - (A4 - A5)] / A3 × 100%, In the formula: A3 is the absorbance without sample solution and with pyrogallol solution; A4 is the absorbance with sample solution and pyrogallol solution; A5 is the absorbance with sample solution and without pyrogallol solution.

[0049] The results are as follows Figure 1As shown, the hydrolyzed elastin prepared in Example 1 has a higher DPPH free radical scavenging rate and superoxide anion free radical scavenging rate. The higher the scavenging rate, the stronger the ability of elastin to inhibit physiological reactive oxygen species and reduce cell damage, which means that Example 1 has better antioxidant activity.

[0050] Therefore, this invention discloses a preparation process and application of hydrolyzed elastin as a cosmetic ingredient. The hydrolyzed elastin preparation process provided by this invention abandons the traditional hot alkaline method for degreasing and impurity removal. Instead, it uses low-concentration sodium bicarbonate combined with sucrose ester gradient ultrasonic degreasing and weak alkaline dissolution to remove impurities, retaining the active structures of desmolysin and isodesmolysin. This method uses a novel microbial elastase gradient enzymatic hydrolysis. After multi-stage centrifugation and ceramic membrane filtration, the hydrolysate is circulated and separated through a 1000Da ultrafiltration system. Large molecular weight peptides are refluxed for secondary enzymatic hydrolysis to enrich small molecular weight active peptides with a molecular weight <1000Da. The total content of desmolysin and isodesmolysin in the obtained product can reach 0.64g / 100g. The preparation process provided by this invention is green, safe, cost-effective, and conducive to industrial production. The prepared hydrolyzed elastin has excellent transdermal absorption and is suitable for use as a raw material in anti-aging and repair cosmetics.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A process for preparing hydrolyzed elastin as a cosmetic ingredient, characterized in that, The method is as follows: S1. Take the heart arterial bulb of the skipjack tuna, break it into pieces, wash it, and soak it in physiological saline. S2. The fragments of the heart arterial bulb after soaking in S1 are soaked in a degreasing solution and then sonicated. After sonication, the soaking treatment is continued for a total of three soaking-sonication cycles. The degreasing solution after sonication is allowed to stand in an ice bath. After the degreasing solution separates into layers, the aqueous phase is obtained. S3. The aqueous phase obtained in S2 is soaked in a mixed solution of sodium hydroxide and sodium carbonate under constant temperature and stirring conditions. The insoluble matter is crushed, and then the material is rinsed and allowed to stand to drain the free liquid. The second soaking is carried out under constant temperature and stirring conditions in a mixed solution of sodium hydroxide and sodium carbonate. After the soaking is completed, it is washed with purified water, vacuum filtered and dehydrated to obtain crude elastin. S4. Take crude elastin, add purified water to reconstitute it, then add Streptomyces griseus elastase, adjust the pH of the system, hydrolyze at a constant temperature, and then inactivate the enzyme at high temperature to complete the first enzymatic hydrolysis. Add Aspergillus oryzae neutral protease and Aspergillus oryzae aminopeptidase complex enzyme to the above system, adjust the pH of the system, hydrolyze at a constant temperature, and then inactivate the enzyme at high temperature to complete the second enzymatic hydrolysis. S5. After enzymatic hydrolysis, the liquid is centrifuged at low speed, and the supernatant is collected. Then, it is centrifuged at high speed to collect the clear crude hydrolysate. The crude hydrolysate is passed through a ceramic membrane for circulation filtration. The filtrate is sent to an ultrafiltration membrane system with a molecular weight cutoff of 1000 Da for circulation separation. Large molecular weight peptides with a molecular weight cutoff of >1000 Da are retained and enzymatically hydrolyzed twice more according to the above steps. Small molecular weight elastic peptide liquid with a molecular weight of <1000 Da is collected on the permeate side. S6. Connect the ultrafiltration permeate in series with three chromatography columns in the following order: cation exchange resin column → plant polyphenol adsorption resin column → amino chelate resin column. Pass the solution through the columns continuously at room temperature. S7. The purified hydrolyzed elastin solution is filtered through a filter membrane at room temperature under sterile conditions to obtain sterile peptide solution. The sterile peptide solution is then transferred to a vacuum low-temperature concentration tank. Low molecular weight sodium hyaluronate is added to the concentrate and stirred until homogeneous. S8. Low-temperature sterile spray drying is adopted, and after drying, the product is sieved to obtain hydrolyzed elastin powder.

2. The process for preparing hydrolyzed elastin as a cosmetic raw material according to claim 1, characterized in that: The defatting solution in S2 consists of 0.8% sodium bicarbonate solution and 0.5% food-grade sucrose ester in a volume ratio of 2:1, and the mass-volume ratio of arterial bulb fragments to defatting solution is 1g:8mL.

3. The process for preparing hydrolyzed elastin as a cosmetic raw material according to claim 1, characterized in that: In the first soaking of sodium hydroxide and sodium carbonate in S3, the mass concentration ratio of sodium hydroxide to sodium carbonate is 1:40, and in the second soaking of sodium hydroxide and sodium carbonate, the mass concentration ratio of sodium hydroxide to sodium carbonate is 1:

20.

4. The process for preparing hydrolyzed elastin as a cosmetic raw material according to claim 1, characterized in that: The amount of Streptomyces griseus elastase added in S4 is 0.8-1.2%, the pH value of the two enzymatic hydrolysis systems is 7.0-8.5, the constant temperature is 40-50℃, the hydrolysis time is 2-3h, the enzyme inactivation temperature is 85-95℃, and the enzyme inactivation treatment time is 20-40min.

5. The process for preparing hydrolyzed elastin as a cosmetic raw material according to claim 1, characterized in that: In step S4, the amount of Aspergillus oryzae neutral protease and Aspergillus oryzae aminopeptidase complex enzyme added is 1-2%, and the composition of Aspergillus oryzae neutral protease and Aspergillus oryzae aminopeptidase complex enzyme is 2:1 in mass ratio.

6. The process for preparing hydrolyzed elastin as a cosmetic raw material according to claim 1, characterized in that: In S5, the low-speed centrifugation condition is 3000 r / min for 10 min, the high-speed centrifugation condition is 8000 r / min for 15 min, and the ceramic membrane pore size is 80-120 nm.

7. The process for preparing hydrolyzed elastin as a cosmetic raw material according to claim 1, characterized in that: In S6, the three resin columns are packed in a volume ratio of 1:1.3:1.2; the flow rate of the feed solution is controlled at 3.5 BV / h, and the ambient temperature is 25-35℃; in S7, the filter membrane used is 0.22μm, and the concentration conditions are that the concentration temperature does not exceed 60℃, and the concentration is carried out to a peptide solid content of 30wt%, with the addition of low molecular weight sodium hyaluronate accounting for 0.7% of the peptide solid content; in S8, the drying conditions are an inlet air temperature of 140℃ and an outlet air temperature of 70℃, and the dried product is passed through a 60-mesh sieve.

8. The process for preparing hydrolyzed elastin as a cosmetic raw material according to claim 1, characterized in that: In S1, the size of the broken skipjack tuna heart arterial bulb is 5mm×5mm×5mm. The cleaning conditions are: rinsing with running water for 10 minutes and standing in physiological saline for 2 hours. In S2, the soaking time is 15 minutes each time, the ultrasonic conditions are: power 350W, frequency 28kHz, ultrasonication for 10 minutes each time, ice bath temperature of 4-5℃, and standing time of 2-3 hours. In S3, the constant temperature during the two soakings is 40-50℃, the soaking time is 30-50 minutes, the material-liquid ratio is 1:5-1:6, the rinsing time is 3-5 minutes, the number of washings is 3-5 times, and the vacuum filtration dewatering filter cloth is 80 mesh.

9. A hydrolyzed elastin prepared by the preparation process according to any one of claims 1 to 8.

10. The application of the preparation process as described in any one of claims 1 to 8 in the field of cosmetic preparation.

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