A preparation method of recombinant collagen and cosmetics for soothing skin

Abstract

The present invention belongs to the technical field of natural active collagen modification, and in particular, relates to a method for preparing small molecule recombinant collagen and its application. The present invention discloses a method for preparing recombinant collagen, which uses a degreasing solution, liposuction microspheres, a purification liquid and a cleaning liquid to remove impurities in animal skins, and in particular, uses liposuction microsphere technology to effectively remove bound lipid substances in animal skins, and then uses a composite enzyme solution and papain for two enzymatic hydrolysis, and the final collagen purity is greater than 95%, and the extraction rate is greater than 88%. And by grafting palmitic acid on small molecule collagen, not only the activity of collagen is improved, but also the small molecule collagen peptide has a lipophilic functional group, so that the small molecule collagen is more easily absorbed by the skin, can be used in cosmetics, can effectively remove free radicals, has the effect of delaying aging, and has the effect of locking water and moisturizing, and stabilizing the skin.

Description

Technical Field

[0001] The invention relates to the technical field of natural active collagen modification, and in particular to a preparation method and application of small molecule recombinant collagen. Background Art

[0002] Collagen extraction methods include acid extraction, alkaline extraction, enzyme extraction and neutral salt extraction. Acid extraction is beneficial to the natural structure of collagen and is the most commonly used method for collagen extraction. Dilute solutions of acidic solvents such as acetic acid, citric acid, hydrochloric acid and formic acid swell and dissolve collagen fibers, which can retain the triple helix structure of collagen to the greatest extent. Dilute acid solvents have better extraction effects on non-cross-linked or low-cross-linked collagen molecules in the tissues of young and middle-aged animals and are often used for the preliminary extraction of collagen. Enzyme extraction is usually used in combination with acid extraction to prepare non-immunogenic collagen by cleaving non-triple helical terminal peptides with pepsin. Neutral salt extraction uses the difference in the solubility of collagen in salt solutions of different concentrations to extract collagen. Commonly used reagents include sodium chloride, potassium chloride, sodium citrate and Tris-HCl.

[0003] However, no matter whether the collagen is prepared by acid extraction, alkali extraction, enzyme extraction or neutral salt extraction, there is a problem of impurity removal. Some proteins and enzymes are firmly bound to lipids or have more non-polar side chains in the molecules. They are insoluble in water, dilute salt, dilute acid or dilute alkali solutions and can be extracted with organic solvents. They have certain hydrophilicity and strong lipophilicity, but there will be different degrees of residues when adding organic solvents to remove lipid substances, and many of these organic solvents are toxic. The fat tissue in the raw materials must be fully removed. Once lipids are mixed in the collagen, they cannot be removed regardless of whether they are precipitated and dissolved by neutral or acid, and only a turbid collagen solution can be obtained in the end.

[0004] A Chinese patent with publication number CN102363798B discloses a method for preparing collagen sponge, which uses fresh pig skin as raw material, pre-treats, removes fat, and then treats it with acid enzymatic hydrolysis, and then uses ultrasonic treatment to further improve the extraction efficiency, and finally purifies it to obtain a high-purity collagen product. In the pig skin pre-treatment process in the patent, acetone or anhydrous ether is used as an organic solvent to remove lipids, which is prone to produce toxic residues.

[0005] A Chinese patent with publication number CN113563460B discloses a high-purity collagen extraction process, which includes an Achilles tendon pretreatment step and a collagen extraction and purification step. During the implementation process, the reagents in the pretreatment process, as well as the concentrations, mixing methods and addition sequences of various reagents are reasonably controlled, thereby significantly improving the removal efficiency of Achilles tendon fat during the pretreatment process, improving the purity and activity of collagen, and obtaining collagen with a complete triple helix structure. However, chemical reagents such as peracetic acid, sodium hydroxide, TritonX-114, sodium chloride and sodium carbonate, as well as Tween 80 have been used in the pretreatment process, which will cause excessive loss of collagen during the pretreatment process. In addition, peracetic acid and sodium hydroxide will cause the amino acids in the collagen to destroy tryptophan, serine and tyrosine in the collagen, and using too much salt solution may reduce the conformational stability of the collagen, which is not conducive to the extraction of natural structure collagen.

[0006] Therefore, how to prepare small molecule collagen without destroying the amino acid composition in collagen, maintaining the active ingredients of collagen, and improving the extraction purity of collagen is a difficult problem that needs to be solved urgently. Summary of the invention

[0007] In order to solve the above problems, the purpose of the present invention is to provide a method for preparing recombinant collagen to solve one or more technical problems existing in the prior art and at least provide a beneficial choice or create conditions.

[0008] In order to improve the purity of collagen, the present invention provides a method for preparing collagen from animal skin, which can effectively remove fat, calcium and foreign proteins in the animal skin. First, the animal skin is pretreated, specifically comprising the following steps:

[0009] S001, animal skin pretreatment: wash the animal skin with hot water at 50-60°C, remove the hair, scrape the fat layer close to the animal skin with a knife, soak the animal skin in a degreasing solution at 30-40°C for 10-15 minutes, drain, crush and pulp to obtain animal skin slurry for later use;

[0010] The degreasing solution is composed of the following parts by weight: 2 parts of anhydrous betaine, 4 parts of anhydrous ethanol, 2 parts of sorbitan monooleate, and 20 parts of water; the mass ratio of animal skin to degreasing solution is 1:5-8;

[0011] The degreasing solution can remove most of the fat in animal skins, but some lipids will remain in the skin tissue structure. The protein in animal skin tissue is soluble in water, dilute salt, dilute acid or dilute alkali solution, and betaine can react with fat to remove most of the free fat in animal skin tissue. However, there are still a few proteins bound to lipids in the skin tissue structure that are difficult to remove; in order to further remove the bound lipids without affecting the structure of collagen.

[0012] S002, adding lipoabsorbent microspheres to the animal skin slurry, stirring and reacting for 70 to 90 minutes, filtering the lipoabsorbent microspheres with a filter, and then soaking them in a purification solution for 10 to 20 minutes, and finally washing and soaking them in a cleaning solution for 10 to 20 minutes, centrifuging at a speed of 3500 rpm, washing with water, and drying to obtain a pretreated material for standby use;

[0013] The purification liquid is composed of the following components by mass: 2 parts of anhydrous betaine, 2 parts of sodium sulfate, and 10 parts of water; the cleaning liquid is composed of the following components by mass: 0.5 parts of citric acid, 2 parts of 1 mol / L lactic acid, and 50 parts of water;

[0014] The purification liquid made of anhydrous betaine, sodium sulfate and water can purify impurities on the one hand, and at the same time, the anhydrous betaine can stabilize the molecular structure of collagen, prevent the conformational stability of collagen from being reduced during processing, and improve the extraction rate of collagen.

[0015] The cleaning solution prepared with citric acid, lactic acid and water can, on the one hand, purify impurities; on the other hand, lactic acid and citric acid can react with calcium in animal skins to generate calcium lactate and calcium acetate, respectively, which can dissolve part of the calcium and achieve the purpose of removing calcium. Calcium will reduce the activity of protease and thus affect the extraction rate of collagen, and the presence of calcium will affect the purity of collagen.

[0016] S003, adding the pretreated material to deionized water, adjusting the pH value with citric acid, soaking and stirring, adding to a composite enzyme solution for soaking, and ultrasonic enzymolysis to obtain an enzymatic solution; the obtained enzymatic solution is centrifuged to collect the supernatant to obtain a crude collagen extract;

[0017] S004, hydrolyzing the crude collagen extract with papain again, and reacting it with beeswax dissolved in hot glycerol under alkaline conditions to obtain small molecule collagen peptides grafted with palmitic acid, which are recorded as recombinant collagen.

[0018] Furthermore, we provide a liposuction microsphere, which is prepared by starch modification, specifically:

[0019] S101, adding corn starch to deionized water, stirring evenly, slowly heating to 80-90° C., stirring for 20-40 minutes, the corn starch goes through the first stage and the second stage, and begins to gelatinize, and the stirring reaction is continued, and glycerol is added for 20-40 minutes, and the mixture is dried, crushed, and ground to obtain modified small molecule starch particles;

[0020] When the gelatinized starch is modified with glycerol, the gelatinized small-molecule starch particles can continue to expand and the mechanical strength becomes higher. The mass ratio of glycerol to corn starch is 1:5-8.

[0021] S102, dispersing the gelatinized starch granules in deionized water, adding sodium bicarbonate to adjust the pH value to 8.5-9, and then adding 3-chloro-2-hydroxypropyltrialkylammonium chloride to cause an etherification reaction, introducing quaternary ammonium substituent groups into the small molecule starch granules to generate cationic small molecule starch granules, and drying them for later use;

[0022] S103, dissolving distearoyloxyisopropylaluminate in ethyl acetate, then adding dropwise to the cationic small molecule starch particles at room temperature, stirring rapidly while adding dropwise, then heating in a constant temperature water bath at 100°C, reacting for 20 to 40 minutes, continuing stirring and cooling to 30 to 40°C, then adding octenylsuccinic anhydride diluted with glycerol, drying, grinding, and obtaining 40-50 um liposuction microspheres; the mass ratio of distearoyloxyisopropylaluminate, octenylsuccinic anhydride and starch is 1 to 2: 3 to 5: 8 to 12;

[0023] After the expanded starch small molecules are modified with distearyloxyisopropylaluminate and octenylsuccinic anhydride, the micropores on the starch particles increase and become larger, the surface of the starch particles becomes rough, the specific surface area increases, and the micropores on the microspheres also increase, so that the oil absorption increases, and the starch small molecule particles become hydrophobic and lipophilic liposorbent microspheres. Distearyloxyisopropylaluminate contains groups that can react with active hydrogen, so it can form a bond with the starch small molecule particles containing hydroxyl and carboxyl groups, improving the affinity and binding force between inorganic fillers and organic polymers. Octenylsuccinic anhydride strengthens the hydrophobic and lipophilic properties of the starch small molecule particles, so that the liposorbent microspheres can absorb and remove all the residual lipid proteins, free oils and bound lipid substances in the slurry.

[0024] Further, extracting collagen from the pretreated material obtained after the pretreatment, and preparing small molecule collagen peptide grafted with palmitic acid;

[0025] S201, adding the pretreated material to deionized water, adjusting the pH to 6 with citric acid, soaking and stirring for 30 to 60 minutes, adding to the composite enzyme solution and soaking, ultrasonically hydrolyzing for 10 to 12 hours at 2 to 5° C., with an ultrasonic power of 300 W, to obtain an enzymatic solution; the obtained enzymatic solution is centrifuged at 4° C. at a speed of 10,000 rpm for 20 minutes, and the supernatant is collected to obtain a crude collagen extract;

[0026] The complex enzyme solution includes pepsin, subtilisin and papain in a mass ratio of 10-12:2-4:3-5;

[0027] S202, add papain to the crude collagen extract, adjust the pH to 6-6.5 with citric acid, perform ultrasonic enzymolysis at 40-50°C for 2-3 hours, and then adjust the pH to 8.5-9.5 with sodium bicarbonate; add beeswax dissolved in hot glycerol while stirring, adjust the pH to 8.5-9.5 with sodium bicarbonate during the reaction, and react for 2-4 hours; collect the solution after the reaction, sterilize it, and purify it by nanofiltration for 2-5 hours; after purification, obtain a high concentration of small molecule collagen peptides grafted with palmitic acid, which are recorded as recombinant collagen.

[0028] The nanofiltration uses a Koch nanofiltration membrane, the Koch nanofiltration membrane has a molecular weight cutoff of 1-5KDa, a nanofiltration pressure of 1.0-1.5MPa, a mass ratio of glycerol to beeswax of 1:1, and a mass ratio of beeswax to collagen of 1:8-10;

[0029] Among them, recombinant collagen has good water solubility, retains the biological activity of natural active collagen, and contains multiple hydrophilic and lipophilic functional groups. Recombinant collagen is a small molecule collagen peptide, with palmitic acid grafted on its side chain, and is easily soluble in water. When used in cosmetics, it has the functions of locking in moisture and stabilizing the skin. Finally, a cosmetic is also provided, which includes a small molecule collagen peptide.

[0030] Since collagen is closely combined with a variety of proteoglycans, and its solubility is easily affected by pH and salt concentration, it is difficult to obtain collagen with high extraction rate and high purity by simple enzyme extraction. The purification process should be carried out at low temperature as much as possible. Too high temperature will denature the protein. In the traditional extraction method, the added pepsin cannot fully digest and dissolve the collagen; therefore, the mixed enzyme solution is used for extraction to promote the digestion and dissolution of collagen. After a period of enzymatic hydrolysis of the composite enzyme solution, collagen with a molecular weight greater than 10kDa can be obtained. Although collagen has some important biological activities, its large molecular weight is not conducive to skin absorption, which limits its biological activity. Collagen peptides refer to protein hydrolysis products mainly composed of small molecular peptides formed after the molecular chain of collagen disintegrates and breaks. Papain is used to hydrolyze large molecular collagen to obtain collagen peptides with a molecular weight of less than 5kDa. As the molecular weight of the peptide decreases, the ability to scavenge hydroxyl free radicals and superoxide anion free radicals and the reducing property are enhanced, and small molecule collagen peptides are easily absorbed by the skin. The collagen peptide molecule chain contains hydrophilic groups such as carboxyl and amino groups, which are easy to combine with water molecules. Beeswax contains a large amount of long-chain higher fatty acid esters, which have the effects of detoxification, muscle regeneration and pain relief. Grafting it on small molecule collagen not only changes the hydrophilic nature of small molecule collagen, greatly enhancing the oil solubility of small molecule collagen, but also has a synergistic effect with collagen, which increases the healing speed of the skin. This palmitic acid in beeswax has a shorter carbon chain than common stearic acid, and a smaller molecular weight after grafting, making it easier to penetrate into the dermis of the skin; and the double bond content in palmitic acid is more, which is easier to be absorbed by the skin and has the effect of stabilizing the skin. Collagen liquid can form a better water retention layer, provide all the moisture needed by the skin, and promote the synthesis of collagen in the skin. Collagen peptides contain a large number of hydrophilic groups such as amino, carboxyl, and hydroxyl groups. These groups can combine with water molecules to prevent water evaporation and are good moisturizers. Moreover, due to their small molecular weight, they have good compatibility with the skin and can enter the stratum corneum to maintain the normal water content of the stratum corneum, thus playing a deep moisturizing role. In addition to the hydrophilic groups, the grafted recombinant collagen is also grafted with a large number of lipophilic groups, which can firmly lock in moisture, effectively improve the structure of the skin's epidermis and dermis, increase the synthesis of collagen, and promote the formation of hyaluronic acid, thereby repairing the skin and maintaining skin elasticity.

[0031] Compared with the prior art, the beneficial effects of the present invention are:

[0032] 1. The present invention uses animal skin as raw material, pre-treats the animal skin with a degreasing solution, liposuction microspheres, a purification solution and a cleaning solution to remove impurities, especially effectively remove bound lipid substances in the animal skin, thereby improving the extraction rate and purity of collagen. The purity of the obtained collagen is at least 95%, and the extraction rate is at least 88%. The entire purification process of the present invention is simple, the process is stable, and the cost is low. It is a method for purifying collagen on a large scale.

[0033] 2. The present invention also provides a method for preparing recombinant collagen. After pre-treating animal skin, it is enzymolyzed twice with a composite enzyme solution and papain to obtain a small molecule collagen with a molecular weight of less than 3000Da. As the molecular weight of the peptide decreases, the ability to remove hydroxyl radicals and superoxide anion radicals and the reducing property are enhanced, and the small molecule collagen peptide is easily absorbed by the skin. And by grafting palmitic acid on the small molecule collagen peptide, the collagen peptide molecular chain contains hydrophilic groups such as carboxyl and amino groups, which are easy to combine with water molecules. Grafting it on the small molecule collagen not only changes the hydrophilic nature of the small molecule collagen, greatly enhancing the oil solubility of the small molecule collagen, but also has a synergistic effect with collagen, so that the healing speed of the skin is enhanced. It not only increases the activity of collagen, but also makes the small molecule collagen peptides have lipophilic functional groups, which effectively improves the structure of the skin epidermis and dermis, can increase the synthesis rate of collagen, and promote the formation of hyaluronic acid, thereby repairing the skin and maintaining skin elasticity. It has the effect of delaying aging, locking in moisture, and stabilizing the skin.

[0034] 3. The present invention has conducted a long-term study on the pretreatment technology of animal skins, and found that the pretreatment technology has a great influence on the performance of collagen. A method of liposuction microspheres is creatively proposed. Through the reasonable combination of degreasing solution and liposuction microspheres, and through reasonable proportioning, various reagents will not affect the triple helix structure of the obtained collagen. The present invention also provides a method for preparing liposuction microspheres, which are introduced into small molecule starch particles by quaternary ammonium substituent groups to generate small molecule starch particles with cations. Finally, after the expanded starch small molecules are modified with distearyloxyisopropylaluminate and octenylsuccinic anhydride, the micropores on the starch particles increase and enlarge, the starch particle surface becomes rough, the specific surface area increases, and the micropores on the microspheres also increase, so that the starch small molecule particles become hydrophobic and lipophilic liposuction microspheres. The liposuction microspheres are porous and have a large specific surface area, and have a good adsorption effect on oils and fats. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 , electron micrograph of the liposuction microspheres prepared in Example 1. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content described in the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0037] Example 1

[0038] The pretreatment of pigskin specifically includes the following steps:

[0039] Use 2 parts of anhydrous betaine, 4 parts of anhydrous ethanol, 2 parts of sorbitan monooleate, and 20 parts of water, and stir to obtain a degreasing solution.

[0040] Take 2 parts of pig skin, wash them with 60°C hot water, remove hair, scrape the fat layer of the pig skin with a knife, soak the pig skin in 10 parts of 40°C degreasing solution for 15 minutes, drain, crush and pulp to obtain animal skin slurry;

[0041] Further, liposuction microspheres are prepared, and the specific steps are as follows:

[0042] Add 20 parts of corn starch to deionized water, stir evenly, slowly heat to 90°C, stir for 40 minutes, the corn starch goes through the first stage and the second stage, starts to gelatinize, continue stirring and reacting, add 4 parts of glycerol and react for 40 minutes, dry, crush, grind, and obtain modified small molecule starch particles;

[0043] The gelatinized starch granules are dispersed in deionized water, sodium bicarbonate is added to adjust the pH value to 8.5, and then 2 parts of 3-chloro-2-hydroxypropyltrialkylammonium chloride are added to cause an etherification reaction, and quaternary ammonium substituent groups are introduced into the small molecule starch granules to generate cationic small molecule starch granules, which are dried and set aside; 1 part of distearoyloxyisopropylaluminate is dissolved in ethyl acetate, and then added dropwise to 8 parts of cationic small molecule starch granules at room temperature, stirring rapidly while adding dropwise, and then placed in a constant temperature water bath at 100°C for heating, reacting for 40 minutes, continuing to stir and cool to 40°C, and then adding 3 parts of octenylsuccinic anhydride diluted with glycerol, drying, and grinding to obtain 40-50um liposuction microspheres.

[0044] Take 2 parts of anhydrous betaine, 2 parts of sodium sulfate, and 10 parts of water to prepare a purification solution; take 0.5 parts of citric acid, 2 parts of 1 mol / L lactic acid, and 50 parts of water to prepare a cleaning solution.

[0045] Add 3 parts of liposorbent microspheres to 12 parts of animal skin slurry, stir and react for 90 minutes, filter the liposorbent microspheres with a filter, soak them in a purification solution for 20 minutes, and finally wash and soak them in a cleaning solution for 10 to 20 minutes, centrifuge at a speed of 3500 rpm, wash with water, and dry to obtain a pretreated material for standby use;

[0046] Finally, recombinant collagen was prepared by enzymatic hydrolysis, specifically:

[0047] Take 10 parts of pepsin, 4 parts of subtilisin and 5 parts of papain to obtain a composite enzyme solution;

[0048] Take 10 parts of the pretreated material, add it to deionized water, adjust the pH to 6 with citric acid, soak and stir for 60 minutes, add 0.03 parts of the complex enzyme solution and soak it, perform ultrasonic enzymolysis at 2°C for 12 hours, and the ultrasonic power is 300W to obtain an enzymatic solution; the obtained enzymatic solution is centrifuged at 10000rpm for 20 minutes at 4°C, and the supernatant is collected to obtain a crude collagen extract;

[0049] 0.03 parts of papain were added to 10 parts of crude collagen extract, the pH was adjusted to 6 with citric acid, ultrasonic enzymolysis was performed at 50°C for 3 hours, and then the pH was adjusted to 8.5 with sodium bicarbonate; 2 parts of beeswax dissolved in hot glycerol were added while stirring, and the pH was adjusted to 8.5 with sodium bicarbonate during the reaction, and the reaction was continued for 4 hours; after the reaction was completed, the solution was collected, sterilized, and purified by nanofiltration for 5 hours; after purification, a high concentration of small molecule collagen peptides grafted with palmitic acid were obtained, which were recorded as recombinant collagen.

[0050] Example 2

[0051] The pretreatment of pigskin specifically includes the following steps:

[0052] Use 2 parts of anhydrous betaine, 4 parts of anhydrous ethanol, 2 parts of sorbitan monooleate, and 20 parts of water, and stir to obtain a degreasing solution.

[0053] Take 2 parts of pig skin, wash them with 60°C hot water, remove hair, scrape the fat layer of the pig skin with a knife, soak the pig skin in 16 parts of 40°C degreasing solution for 15 minutes, drain, crush and pulp to obtain animal skin slurry;

[0054] Further, liposuction microspheres are prepared, and the specific steps are as follows:

[0055] 16 parts of corn starch were added to deionized water, stirred evenly, slowly heated to 80°C, and stirred for 40 minutes. After the first and second stages, the corn starch began to gelatinize. The reaction was continued by stirring. 2 parts of glycerol were added and reacted for 40 minutes. The mixture was dried, crushed, and ground to obtain modified small-molecule starch particles.

[0056] The gelatinized starch granules are dispersed in deionized water, sodium bicarbonate is added to adjust the pH value to 8.5, and then 2 parts of 3-chloro-2-hydroxypropyltrialkylammonium chloride are added to cause an etherification reaction, so as to introduce quaternary ammonium substituent groups into the small-molecule starch granules to generate small-molecule starch granules with cations, which are then dried for later use;

[0057] Dissolve 2 parts of distearoyloxyisopropylaluminate in ethyl acetate, then add dropwise to 12 parts of cationic small molecule starch granules at room temperature, stir rapidly while adding, then heat in a constant temperature water bath at 100°C, react for 40 minutes, continue stirring and cool to 40°C, then add 5 parts of octenylsuccinic anhydride diluted with glycerol, dry, grind, and obtain 40-50um liposuction microspheres.

[0058] Take 2 parts of anhydrous betaine, 2 parts of sodium sulfate, and 10 parts of water to prepare a purification solution; take 0.5 parts of citric acid, 2 parts of 1 mol / L lactic acid, and 50 parts of water to prepare a cleaning solution.

[0059] Add 4 parts of liposorbent microspheres to 20 parts of animal skin slurry, stir and react for 90 minutes, filter the liposorbent microspheres with a filter, soak them in a purification solution for 20 minutes, and finally wash and soak them in a cleaning solution for 20 minutes, centrifuge at a speed of 3500 rpm, wash with water, and dry to obtain a pretreated material for standby use;

[0060] Finally, recombinant collagen was prepared by enzymatic hydrolysis, specifically:

[0061] Take 12 parts of pepsin, 2 parts of subtilisin and 4 parts of papain to obtain a composite enzyme solution;

[0062] Take 10 parts of the pretreated material, add it to deionized water, adjust the pH to 6 with citric acid, soak and stir for 60 minutes, add 0.05 parts of the complex enzyme solution and soak it, perform ultrasonic enzymolysis at 2°C for 12 hours, and the ultrasonic power is 300W to obtain an enzymatic solution; the obtained enzymatic solution is centrifuged at 10000rpm for 20 minutes at 4°C, and the supernatant is collected to obtain a crude collagen extract;

[0063] 0.05 parts of papain were added to 8 parts of crude collagen extract, the pH was adjusted to 6 with citric acid, ultrasonic enzymolysis was performed at 50°C for 3 hours, and then the pH was adjusted to 8.5 with sodium bicarbonate; 2 parts of beeswax dissolved in hot glycerol were added while stirring, and the pH was adjusted to 8.5 with sodium bicarbonate during the reaction, and the reaction was continued for 4 hours; after the reaction was completed, the solution was collected, sterilized, and purified by nanofiltration for 5 hours; after purification, a high concentration of small molecule collagen peptides grafted with palmitic acid were obtained, which were recorded as recombinant collagen.

[0064] Example 3

[0065] The pretreatment of pigskin specifically includes the following steps:

[0066] Use 2 parts of anhydrous betaine, 4 parts of anhydrous ethanol, 2 parts of sorbitan monooleate, and 20 parts of water, and stir to obtain a degreasing solution.

[0067] Take 2 parts of pig skin, wash them with 60°C hot water, remove hair, scrape the fat layer of the pig skin with a knife, soak the pig skin in 12 parts of 40°C degreasing solution for 15 minutes, drain, crush and pulp to obtain animal skin slurry;

[0068] Further, liposuction microspheres are prepared, and the specific steps are as follows:

[0069] 16 parts of corn starch were added to deionized water, stirred evenly, slowly heated to 80°C, and stirred for 40 minutes. After the first and second stages, the corn starch began to gelatinize. The reaction was continued by stirring. 2 parts of glycerol were added and reacted for 40 minutes. The mixture was dried, crushed, and ground to obtain modified small-molecule starch particles.

[0070] The gelatinized starch granules are dispersed in deionized water, sodium bicarbonate is added to adjust the pH value to 8.5, and then 2 parts of 3-chloro-2-hydroxypropyltrialkylammonium chloride are added to cause an etherification reaction, so as to introduce quaternary ammonium substituent groups into the small-molecule starch granules to generate small-molecule starch granules with cations, which are then dried for later use;

[0071] Dissolve 2 parts of distearoyloxyisopropylaluminate in ethyl acetate, then add dropwise to 10 parts of cationic small molecule starch granules at room temperature, stir rapidly while adding, then heat in a constant temperature water bath at 100°C, react for 40 minutes, continue stirring and cool to 40°C, then add 4 parts of octenylsuccinic anhydride diluted with glycerol, dry, grind, and obtain 40-50um liposuction microspheres.

[0072] Take 2 parts of anhydrous betaine, 2 parts of sodium sulfate, and 10 parts of water to prepare a purification solution; take 0.5 parts of citric acid, 2 parts of 1 mol / L lactic acid, and 50 parts of water to prepare a cleaning solution.

[0073] Add 5 parts of liposorbent microspheres to 20 parts of animal skin slurry, stir and react for 90 minutes, filter the liposorbent microspheres with a filter, soak them in a purification solution for 20 minutes, and finally wash and soak them in a cleaning solution for 20 minutes, centrifuge at a speed of 3500 rpm, wash with water, and dry to obtain a pretreated material for standby use;

[0074] Finally, recombinant collagen was prepared by enzymatic hydrolysis, specifically:

[0075] Take 11 parts of pepsin, 3 parts of subtilisin and 3 parts of papain to obtain a composite enzyme solution;

[0076] Take 10 parts of the pretreated material, add it to deionized water, adjust the pH to 6 with citric acid, soak and stir for 60 minutes, add 0.04 parts of the complex enzyme solution and soak, perform ultrasonic enzymolysis at 2°C for 12 hours, the ultrasonic power is 300W, and obtain an enzymatic solution; the obtained enzymatic solution is centrifuged at 10000rpm for 20 minutes at 4°C, and the supernatant is collected to obtain a crude collagen extract;

[0077] 0.05 parts of papain were added to 8 parts of crude collagen extract, the pH was adjusted to 6 with citric acid, ultrasonic enzymolysis was performed at 50°C for 3 hours, and then the pH was adjusted to 8.5 with sodium bicarbonate; 2 parts of beeswax dissolved in hot glycerol were added while stirring, and the pH was adjusted to 8.5 with sodium bicarbonate during the reaction, and the reaction was continued for 4 hours; after the reaction was completed, the solution was collected, sterilized, and purified by nanofiltration for 5 hours; after purification, a high concentration of small molecule collagen peptides grafted with palmitic acid were obtained, which were recorded as recombinant collagen.

[0078] Comparative Example 1, the pretreated material obtained by soaking pig skin in a strong alkali soaking method commonly used at present is the same as that in Example 1;

[0079] Comparative Example 2: The pretreated material obtained by pretreating pigskin using the method in Example 1 with publication number CN113563460B; other aspects are the same as in Example 1;

[0080] Comparative Example 3, the palmitic acid in beeswax was not grafted, and the rest was the same as in Example 1;

[0081] Comparative Example 4, the pretreated material obtained by pretreating pig skin using the method in Example 1 with publication number CN113563460B; and no palmitic acid in beeswax was grafted, and the rest was the same as in Example 1;

[0082] Comparative Example 5, papain was not used for ultrasonic enzymatic hydrolysis at 40-50°C for 3 hours, and palmitic acid in beeswax was not grafted, and the molecular weight cut-off of the Koch nanofiltration membrane was 5-10KDa.

[0083] Others are the same as in Example 1;

[0084] Test data and performance analysis

[0085] 1.Performance test of liposuction microspheres

[0086] A pig skin with a fat content of 30% was used for a blank test. The concentration changes before and after grease removal were measured by HPLC, and the grease removal capabilities of the following methods were evaluated.

[0087] The total fat content of the pretreated material obtained by treating the pigskin slurry with the lipoabsorbent microspheres in Examples 1 to 3 was measured;

[0088] The pretreated pigskin in Comparative Example 1 was soaked in the strong alkali soaking method commonly used at present, and the total fat content was measured;

[0089] The pretreated pigskin in Comparative Example 2 was obtained by treating pigskin using the method in Example 1 with publication number CN113563460B;

[0090] Chromatographic column: Agilent ZORBAXSB-C18 column (5μm×4.6mm×250mm), mobile phase is methanol:0.02% (NH4)2HPO4=5:95, detection wavelength is 197nm, column temperature is 25℃, injection volume is 20μL, flow rate is 0.8mL / min, test results are shown in Table 1.

[0091] Table 1, fat removal rate

[0092]

[0093]

[0094] As can be seen from Table 1, the common alkali removal method of Comparative Example 1 can only remove the grease on the surface, and the grease in the pig skin structure is difficult to remove. The use of solutions such as peracetic acid, tributyl phosphate, Tween 80, sodium hydroxide, TritonX-114, sodium chloride and sodium carbonate in Comparative Example 2 can better remove the fat on the pig skin, and some free grease in the pig skin structure can also be well removed, but the grease bound to protein is difficult to remove. The use of liposuction microspheres, due to the oil absorption and charge of the liposuction microspheres, can also completely remove the bound grease in the slurry, and the removal effect is significantly improved, with a removal rate of more than 99%.

[0095] The particle size distribution of the liposorbent microspheres in water can be measured by a particle size analyzer. The particle size analyzer is used to analyze Examples 1-3 to obtain the corresponding particle size distribution of the liposorbent microspheres, as shown in Table 1;

[0096] The static nitrogen adsorption method is a common method for determining the specific surface area. In the JW-BK200C specific surface area and pore size analyzer, the lipoabsorbent microspheres were first vacuum degassed at 80°C for 8 hours, and then the lipoabsorbent microsphere samples of Examples 1-3 were tested at liquid nitrogen temperature to obtain the specific surface area (m 2 / g) and porosity (%); see Table 2

[0097] Table 2 Particle size distribution and specific surface area of ​​liposuction microspheres

[0098] Sample name Particle size distribution (μm) <![CDATA[Specific surface area (m 2 / g)]]> Porosity (%) Example 1 50～60 243.7 27.1 Example 2 50～60 240.5 26.5 Example 3 50～60 241.9 26.9

[0099] From Table 1, it can be seen that the particles of Examples 1-3 are relatively uniform, and the liposorbent microspheres have very good specific surface area and porosity, which are more conducive to the adsorption of oil from oily substances.

[0100] The morphology of the adsorbent material was observed using a field emission scanning electron microscope. Before the test, the lipoabsorbent microsphere sample in Example 1 was evenly dispersed on the conductive glue, and the scanning electron microscope parameters were adjusted, with the acceleration voltage set to 3.0 kV and the magnification to 1000 times; the morphology is shown in Figure 1 ,from Figure 1 It can be seen that the liposuction is a spherical microporous structure with small size and relatively uniform dispersion, and has a three-dimensional porous structure. This special morphology structure has a high specific surface area.

[0101] 2. Recombinant collagen extraction rate

[0102] The following performance test indicators were used to test the collagen of Examples 1-3 and Comparative Examples 1-5, respectively. The test results are shown in Table 3.

[0103] Extraction rate: The content of hydroxyproline in the hydrolyzed collagen samples and animal skins of Examples 1-3 and Comparative Examples 1-5 was tested, and the extraction rate of the hydrolyzed collagen was calculated.

[0104] Protein content: GB / T5009.5-2003 “Determination of protein in food” was used to determine the protein content in hydrolyzed collagen.

[0105] Molecular weight: The relative molecular mass of hydrolyzed collagen is determined using QB2732-2005 "Hydrolyzed Collagen", i.e. molecular weight

[0106] Ash content: GB / T5009.3-2003 “Determination of protein in food” was used to determine the ash content in hydrolyzed collagen.

[0107] Table 3 Performance test results

[0108]

[0109]

[0110] As can be seen from Table 3, after the animal skins of Examples 1-3 were treated with liposuction microspheres, the protein purity obtained was relatively high and the impurities were relatively few, indicating that the liposuction microspheres can fully react with the bound lipids in the animal skins, adsorb and remove them, thereby improving the extraction rate of collagen and the purity of the protein. However, as in Comparative Examples 1-2 and Comparative Example 4, the animal skins were treated with traditional acid-base solutions, which not only failed to completely remove lipid substances, but also destroyed the collagen structure in the animal skins, causing the loss of collagen and the increase of impurities, and the ash content was large. However, because Comparative Example 5 did not use papain for enzymatic hydrolysis again under 40-50°C, the collagen obtained was macromolecular collagen. It can also be concluded that papain is more active at 40-50°C than at low temperatures, so that the macromolecular collagen in the crude collagen extract is quickly hydrolyzed into small molecule collagen peptides.

[0111] 3. Free radical elimination rate

[0112] 1) DPPH-free radical scavenging experiment

[0113] The scavenging rates (%) of the collagens prepared in the above Examples 1-3 and Comparative Examples 1-5 and the blank control group were respectively detected as follows:

[0114] Experimental method: Use a 96-well plate, set up three replicate wells in each group, and the system is 200μL. Sample group: Take an appropriate amount of collagen sample, dissolve it in 100μL distilled water, make the final concentration of the sample to be tested in the system 100mg / mL, and add 100μL0.1mM DPPH solution to the reaction system; control group: Take 100μL distilled water, and then add 100μL0.1mM DPPH solution. After the reaction system is constructed, shake it in the dark for 10 minutes, and use an enzyme reader to test the absorbance at 520nm.

[0115] The calculation method of the clearance rate is: clearance rate (%) = [(A0-Ax) / A0] × 100%, A0 is the absorbance of the control group, and Ax is the absorbance of the sample group. The higher the clearance rate, the better the effect of collagen. The results of the DPPH free radical scavenging test are shown in Table 4.

[0116] 2) Hydroxyl free radical scavenging experiment

[0117] Experimental method: The collagen of sample group Examples 1-3 and Comparative Examples 1-5 was diluted to 1 / 100, and the negative control group was added in sequence to 0.1 parts of ferrous sulfate, 0.1 parts of ethanol, 0.1 parts of salicylic acid, 0.1 parts of sample or negative control, 1 part of plasma water, and 0.1 parts of hydrogen peroxide to a 1.5 mL centrifuge tube, tightly cover the tube, shake upside down, and keep warm in a 37°C water bath for 15 minutes. The solution was transferred to a 96-well plate with three replicate wells in each group. The absorbance at 510 nm was measured by an ELISA instrument. The hydroxyl radical scavenging rate was calculated by the following formula:

[0118] Hydroxyl radical scavenging rate % = {[A0-(Ax-Ax0)] / A0}×100%, wherein A0 is the absorbance of the negative control group, Ax is the absorbance of the sample group, and Ax0 is the absorbance of the sample background. The test results are shown in Table 4.

[0119] Table 4, experimental results of Examples 1-3 and Comparative Examples 1-5

[0120]

[0121]

[0122] As shown in Table 4, the recombinant collagen prepared in the present application has a strong scavenging ability for both hydroxyl radicals and DPPH radicals, indicating that the active ingredients in the recombinant collagen solution are good cosmetic antioxidant ingredients with anti-aging effects. As shown in Table 4, the addition of small molecule collagen peptides is more easily absorbed by the skin, making the scavenging ability for hydroxyl radicals and DPPH radicals significantly stronger than that of large molecule collagen. At the same time, the collagen grafted with palmitic acid in beeswax has a synergistic effect, which further enhances the scavenging rate of free radicals.

[0123] The above description is only used to illustrate the technical solution of the present invention rather than to limit it. Any equal modifications and changes made to the technical solution of the present invention by ordinary persons in the art, as long as they do not deviate from the overall concept of the present invention, are still within the scope of the present invention.

Claims

1. A method for preparing recombinant collagen, characterized in that: The method for preparing the recombinant collagen specifically comprises the following steps: S001, animal skin pretreatment: wash the animal skin with hot water at 50-60°C, remove the hair, scrape the fat layer of the animal skin with a knife, soak the animal skin in a degreasing solution at 30-40°C for 10-15 minutes, drain, crush and pulp to obtain animal skin slurry for later use; S002, adding lipoabsorbent microspheres to the animal skin slurry, stirring and reacting for 70 to 90 minutes, filtering the lipoabsorbent microspheres with a filter, and then soaking them in a purification solution for 10 to 20 minutes, and finally washing and soaking them in a cleaning solution for 10 to 20 minutes, centrifuging at a speed of 3500 rpm, washing with water, and drying to obtain a pretreated material for standby use; S003, adding the pretreated material to deionized water, adjusting the pH value with citric acid, soaking and stirring, adding the pretreated material to a composite enzyme solution, and performing ultrasonic enzymolysis to obtain an enzymatic solution; the obtained enzymatic solution is centrifuged to collect the supernatant to obtain a crude collagen extract; S004, hydrolyzing the crude collagen extract with papain again, and reacting it with beeswax dissolved in hot glycerol under alkaline conditions to obtain small molecule collagen peptides grafted with palmitic acid, which are recorded as recombinant collagen; The liposuction microspheres are prepared by modifying starch, specifically: S101, adding corn starch to deionized water, stirring evenly, slowly heating to 80-90° C., stirring for 20-40 minutes, the corn starch undergoes the first stage and the second stage, and begins to gelatinize, the stirring reaction is continued, glycerol is added for 20-40 minutes, drying, crushing, and grinding to obtain modified small molecule starch particles; S102, dispersing the gelatinized starch granules in deionized water, adding sodium bicarbonate to adjust the pH value to 8.5-9, and then adding 3-chloro-2-hydroxypropyltrialkylammonium chloride to cause an etherification reaction, introducing quaternary ammonium substituent groups into the small molecule starch granules to generate cationic small molecule starch granules, and drying them for later use; S103, dissolving distearoyloxyisopropylaluminate in ethyl acetate, and then adding dropwise to the cationic small molecule starch particles at room temperature, stirring rapidly while adding, and then heating in a constant temperature water bath at 100°C, reacting for 20 to 40 minutes, continuing to stir and cool to 30 to 40°C, and then adding octenylsuccinic anhydride diluted with glycerol, drying, and grinding to obtain 40-50um liposuction microspheres.

2. The method for preparing recombinant collagen according to claim 1, characterized in that: in, The degreasing solution is composed of the following components in parts by mass: 2 parts of anhydrous betaine, 4 parts of anhydrous ethanol, 2 parts of sorbitan monooleate, and 5 parts of water; the mass ratio of animal skin to the degreasing solution is 1:5-8; the purification solution is composed of the following components in parts by mass: 2 parts of anhydrous betaine, 2 parts of sodium sulfate, and 10 parts of water; the cleaning solution is composed of the following components in parts by mass: 0.5 parts of citric acid, 2 parts of 1 mol / L lactic acid, and 50 parts of water.

3. The method for preparing recombinant collagen according to claim 1, characterized in that: The pretreated material obtained after the pretreatment is subjected to collagen extraction, and a small molecule collagen peptide grafted with palmitic acid is prepared, specifically: S201, adding the pretreated material to deionized water, adjusting the pH to 6 with citric acid, soaking and stirring for 30 to 60 minutes, adding to the composite enzyme solution and soaking, ultrasonically hydrolyzing for 10 to 12 hours at 2 to 5° C., with an ultrasonic power of 300 W, to obtain an enzymatic solution; the obtained enzymatic solution is centrifuged at 4° C. at a speed of 10,000 rpm for 20 minutes, and the supernatant is collected to obtain a crude collagen extract; S202, add papain to the crude collagen extract, adjust the pH to 6-6.5 with citric acid, perform ultrasonic enzymolysis at 40-50°C for 2-3 hours, and then adjust the pH to 8.5-9.5 with sodium bicarbonate; add beeswax dissolved in hot glycerol while stirring, adjust the pH to 8.5-9.5 with sodium bicarbonate during the reaction, and react for 2-4 hours; collect the solution after the reaction, sterilize it, and purify it by nanofiltration for 2-5 hours; after purification, obtain a high concentration of small molecule collagen peptides grafted with palmitic acid, which are recorded as recombinant collagen.

4. The method for preparing recombinant collagen according to claim 3, characterized in that: Nanofiltration uses a Koch nanofiltration membrane with a molecular weight cutoff of 1 to 5 KDa, a nanofiltration pressure of 1.0 to 1.5 MPa, a mass ratio of glycerol to beeswax of 1:1, and a mass ratio of beeswax to collagen crude extract of 1:8 to 10.

5. The method for preparing recombinant collagen according to claim 3, characterized in that: The compound enzyme solution comprises pepsin, subtilisin and papain, and the mass ratio thereof is 10-12:2-4:3-5; and the addition amount of the compound enzyme solution is 1.0-2.0% of the mass of the animal skin.

6. The method for preparing recombinant collagen according to claim 1, characterized in that: The animal skin is one of cow skin, sheep skin, pig skin or chicken skin.

7. A small molecule collagen peptide, characterized in that: The small molecule collagen peptide is prepared according to the preparation method of recombinant collagen according to any one of claims 1 to 6, and is a small molecule collagen peptide, palmitic acid is grafted on its side chain, and is easily soluble in water.

8. The small molecule collagen peptide according to claim 7 is used in cosmetics to lock in moisture and stabilize the skin.

9. A cosmetic comprising a small molecule collagen peptide as claimed in claim 7.

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