Preparation method and application of anti-aging and anti-oxidation composite peptide

Abstract

The application provides a preparation method and application of an anti-aging and anti-oxidation composite peptide, and belongs to the technical field of biology. The composite peptide is extracted from mud snails and white flowers of horse chestnut, and comprises the following steps: live body electric stimulation induction of the mud snails, biomimetic mineralization wall breaking of the mud snails, composite directional enzymolysis of the mud snails and the white flowers of horse chestnut, crosslinking and purification. The obtained composite peptide can not only effectively remove free radicals and reduce the damage of oxidative stress to cells, but also has the effects of promoting collagen synthesis, stimulating cell regeneration, improving skin elasticity, slowing down wrinkle formation and improving skin gloss. The composite peptide can be widely applied to cosmetics and medicines as an effective component for delaying aging and protecting cell health.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a preparation method and application of an anti-aging and anti-oxidation composite peptide. BACKGROUND

[0002] Aging is a gradual loss of function and adaptability in the life cycle of an organism, and is a natural and complex physiological phenomenon. From the cellular perspective, aging is often accompanied by apoptosis, functional decline, and DNA damage, which leads to the weakening of tissue and organ function and affects the overall health of the body. At the same time, aging also involves changes in hormone levels, a decline in immune system function, and an increase in oxidative stress. In the aging process, a variety of factors play a role. Genetic factors play an important role, as certain genes can affect the rate and manner of aging in individuals. Environmental factors such as ultraviolet light, pollutants, and diet can affect the health of cells and tissues and accelerate aging. Unhealthy lifestyle habits, such as unhealthy diet, lack of exercise, smoking, etc., can also cause premature aging. In addition, psychological factors such as mental stress and depression can also affect the aging process.

[0003] The free radical theory, as one of the representative theories of aging, believes that aging is caused by the attack of free radicals on cells and excessive oxidation. In the normal metabolic process of cells, free radicals are generated, which have strong reactivity and can cause oxidation of various substances in cells, damaging biological membranes, and cross-linking proteins, nucleic acids and other macromolecules, affecting their normal function. With age, the body's defense ability against free radicals gradually weakens, and the accumulation of free radicals can trigger a series of degenerative changes related to aging. Numerous studies have shown that the key to maintaining health and combating aging lies in antioxidant, i.e. removing excess free radicals in the body.

[0004] Bioactive peptides are peptide compounds that are beneficial to the life activities of the organism or have physiological effects, with a relative molecular mass of less than 6000 Da, and have a variety of biological functions such as antioxidant, antihypertensive, antibacterial, antithrombotic, immunomodulatory, etc. Antioxidant peptides, as a kind of bioactive peptides, not only have the function of removing free radicals in the body, but also have obvious protective effects on mitochondrial damage caused by ultraviolet light and lipid peroxidation induced by free radicals. Its mechanism of action also includes providing hydrogen to antioxidant enzymes, chelating metal ions, etc. In recent years, antioxidant peptides have shown great potential in oxidative stress-related diseases and have received widespread attention. In the application field, collagen peptides, as the enzymatic products of collagen, are absorbed in the form of small peptides and free amino acids, with the characteristics of low molecular weight, easy absorption, low viscosity, etc., and also have antioxidant and anti-aging effects, and have been widely used in the fields of medicine, food, daily chemical industry, etc. However, there are many types of anti-aging and antioxidant peptides, with varying effects, and the efficacy needs to be continuously improved. SUMMARY

[0005] The present application aims to provide a new type of composite peptide extracted from mud snails and white clover, which is obtained by the following steps: living body electric stimulation induction of mud snails, biomimetic mineralization of the shell of the mud snails, composite directional enzymolysis of the mud snails and white clover, and cross-linking and purification.

[0006] The present application aims to provide a new type of composite peptide extracted from mud snails and white clover, which is obtained by the following steps: living body electric stimulation induction of mud snails, biomimetic mineralization of the shell of the mud snails, composite directional enzymolysis of the mud snails and white clover, and cross-linking and purification.

[0007] The preparation method of the composite peptide comprises the following steps:

[0008] S1 living body electric stimulation induction: the mud snails and physiological saline are placed in an electric stimulation reaction container in a mass ratio of 1: (4-5) for electric stimulation treatment;

[0009] S2 biomimetic mineralization of the shell: the mud snails subjected to the living body electric stimulation induction are added to a calcium chloride aqueous solution together with physiological saline, CO2 is introduced to gradually increase the pH of the system to 7.4-8.0, and in this process, calcium carbonate microcrystals are gradually formed; under the osmotic pressure, the generated calcium carbonate microcrystals directionally pierce the cell membrane of the mud snails to release the active substances in the cells, and the broken shell is obtained;

[0010] S3 composite directional enzymolysis: the broken shell is added with white clover in a mass ratio of (3-4):(1-1.5), and then added with thermophilic protease for enzymolysis, and then added with bacillus subtilis protease for continued enzymolysis; the enzymolysis is continued after cooling, and then the enzyme is inactivated to obtain an enzymolysis solution;

[0011] S4 cross-linking and purification: the enzymolysis solution is added with transglutaminase for reaction, and then the enzyme is inactivated to complete the residue cross-linking reaction; centrifugation is performed, and the supernatant is subjected to ultrafiltration to collect the product with a molecular weight of 2 kDa-5 kDa, and then the product is freeze-dried to obtain the anti-aging and anti-oxidation composite peptide.

[0012] The preparation method of the composite peptide comprises the following steps:

[0013] S1 living body electric stimulation induction: the mud snails and physiological saline are placed in an electric stimulation reaction container in a mass ratio of 1: (4-5) for electric stimulation treatment;

[0014] S2 biomimetic mineralization wall breaking: the mud eel treated by electric stimulation is added to calcium chloride aqueous solution together with physiological saline, CO2 is introduced, and the pH value of the system is gradually increased to 7.4-8.0, in the process, calcium carbonate microcrystals are gradually formed; under the action of osmotic pressure, the generated calcium carbonate microcrystals directionally pierce the cell membrane of the mud eel cells, and the intracellular active substances are completely released, and a wall broken product is obtained;

[0015] S3 composite directional enzyme hydrolysis: the wall broken product is added with white flowers of fresh knapweed in a mass ratio of (3-4):(1-1.5), then thermolysin is added, and the enzyme hydrolysis is carried out at 60-65℃ for 10-15min, the temperature is lowered to 48-52℃, then protease is added, and the enzyme hydrolysis is continued for 30-40min; the temperature is lowered to 35-38℃, and the enzyme hydrolysis is continued for 60-70min, and the enzyme is inactivated, and an enzyme hydrolysate is obtained; through the series of enzyme hydrolysis processes, the preferential cutting of the alpha-helix structure is realized, and small molecule active peptide segments are obtained;

[0016] S4 cross-linking and purification: transglutaminase is added to the enzyme hydrolysate, and the reaction is carried out at 37-45℃ for 4-6h, the enzyme is inactivated, and the residue cross-linking reaction is completed; then centrifugation is carried out at a speed of 7000-8000r / min for 15-20min, the supernatant is taken, ultrafiltration is carried out by using an ultrafiltration membrane, the component product with a molecular weight of 2-5kDa is collected, a freeze-drying protective agent is added, and freeze-drying is carried out, and an anti-aging and anti-oxidation composite peptide is obtained.

[0017] In S1 of the above preparation method, the mud eel is a live mud eel after shelling; the pulse electric field parameters of the electric stimulation treatment are: intensity 14-18kV / cm, pulse width 10-15μs, and treatment time 30-60min.

[0018] In S2 of the above preparation method, the mass concentration of the calcium chloride aqueous solution is 2.0%-3.0%; the amount of the calcium chloride aqueous solution is 2-3 times the volume of the physiological saline.

[0019] In S2 of the above preparation method, the whole process of the biomimetic mineralization wall breaking is carried out in a constant temperature environment of 36-38℃, and the duration is 2-3h, and the microcrystals and the cells are contacted by shaking every 15-20min during the process.

[0020] In S3 of the above preparation method, the white flowers of fresh knapweed are used as the white flowers of knapweed; the white flowers of knapweed are pre-ground and crushed.

[0021] In S3 of the above preparation method, the addition amount of the thermolysin is 2%-5% of the mass of the mud eel; the addition amount of the protease is 2%-5% of the mass of the mud eel.

[0022] In S4 of the above preparation method, the transglutaminase is added in an amount of 0.2% to 0.5% of the mass of the mud snail.

[0023] In S3 and S4 of the above preparation method, the enzyme inactivation temperature is 80 to 90 DEG C, and the enzyme inactivation time is 10 to 20 min.

[0024] In S4 of the above preparation method, the freeze-drying protective agent is added in an amount of 4% to 6% of the mass of the component product; the freeze-drying protective agent contains 18 to 22 g / 100 mL trehalose, 6 to 8 g / 100 mL dextran, and 8 to 10 g / 100 mL mannitol, and the solvent is sterile water.

[0025] The application of the anti-aging and anti-oxidation composite peptide in medicine and cosmetics.

[0026] The preparation method and application of the anti-aging and anti-oxidation composite peptide have the following beneficial effects:

[0027] I. The stress metabolic pathway of the mud snail is activated by in-vivo electric stimulation, the content of GSH in the cell is increased, and the synthesis of thiol active peptides is promoted, thereby laying a foundation for the subsequent extraction of active peptides with antioxidant capacity. The mud snail is electrically stimulated by setting specific pulse electric field parameters. This external stimulation can trigger the stress response in the mud snail, change the metabolic pathway in the cell, and promote the generation of GSH and other substances. GSH is an important antioxidant and can also participate in the synthesis of thiol active peptides.

[0028] II. The cell wall of the mud snail is broken under mild conditions, and the active substances in the cell are completely released. The formation and action process of the calcium carbonate microcrystals have little effect on the structure and function of the active substances in the cell. The pH value of the system is gradually increased by using the CO2 gradient release method, so that the calcium ions in the calcium chloride aqueous solution combine with the carbonate ions to form calcium carbonate microcrystals. Under the action of osmotic pressure, the microcrystals directionally pierce the cell membrane of the mud snail, thereby achieving the purpose of breaking the cell wall and maintaining the integrity of the active substances in the cell.

[0029] III. Through the synergistic action of multiple enzymes at different temperatures, the protein in the mud snail and the white flower of the horse chestnut is directionally hydrolyzed, the alpha-helix structure is preferentially cut, and small molecule active peptide segments with specific structure and function are obtained, which is conducive to improving the antioxidant and anti-aging activity of the composite peptide. Different enzymes have optimal activity at specific temperatures. The action of thermophilic protease and subtilisin in succession can cut the protein with specificity according to different structures. The alpha-helix structure is preferentially cut at a higher temperature by using thermophilic protease, then the temperature is lowered to allow subtilisin to continue to act, and the protein is further hydrolyzed, so that small molecule active peptide segments are obtained.

[0030] IV. Transglutaminase can catalyze the cross-linking reaction between the gamma-carboxamide group of glutamine residues in the peptide molecule and the epsilon-amino group of lysine residues, thereby changing the structure and functional properties of the peptide, improving the free radical scavenging ability, changing the spatial conformation of the peptide segment, enhancing its binding ability with cell surface receptors, activating more signal pathways in cells, thereby promoting collagen synthesis and cell regeneration. The purification steps such as ultrafiltration and lyophilization can obtain high-purity anti-aging antioxidant composite peptides with good storage properties.

[0031] In summary, the composite peptide is extracted from mud snails and white flowers of horse chestnut, including live body electric stimulation induction of mud snails, biomimetic mineralization of mud snails, composite directional enzymolysis of mud snails and white flowers of horse chestnut, and cross-linking and purification. The composite peptide obtained not only can effectively scavenge free radicals and reduce the damage of oxidative stress to cells, but also has the effects of promoting collagen synthesis, stimulating cell regeneration, improving skin elasticity, slowing down wrinkle formation, and improving skin gloss. The composite peptide can be widely used in cosmetics and drugs as an effective ingredient for delaying aging and protecting cell health. In addition, it can also be used for treating diseases related to oxidative damage, providing a new way to improve human health level. DETAILED DESCRIPTION

[0032] The application will be further described in combination with specific implementation examples, but the application is not limited to these examples.

[0033] Example 1

[0034] An anti-aging antioxidant composite peptide, the composite peptide is extracted from mud snails and white flowers of horse chestnut, including live body electric stimulation induction of mud snails, biomimetic mineralization of mud snails, composite directional enzymolysis of mud snails and white flowers of horse chestnut, and cross-linking and purification.

[0035] The preparation method of the above-mentioned anti-aging antioxidant composite peptide comprises the following steps:

[0036] S1 live body electric stimulation induction: select the live body of mud snails after shelling, put the mud snails and physiological saline into an electric stimulation reaction container according to a mass ratio of 1:4.5, set the pulse electric field parameters: intensity 15 kV / cm, pulse width 12 μs, and perform electric stimulation treatment for 50 min; activate the stress metabolic pathway of mud snails to increase the content of GSH (glutathione) in cells and promote the synthesis of thiol active peptides;

[0037] S2 biomimetic mineralization wall breaking: prepare a calcium chloride aqueous solution with a mass concentration of 2.5%; add the electrically stimulated mud snails and the physiological saline into the calcium chloride aqueous solution, the amount of the calcium chloride aqueous solution is 2.5 times the volume of the physiological saline, and CO2 is introduced to gradually increase the pH value of the system to 7.6, the whole process of biomimetic mineralization wall breaking is carried out in a constant temperature environment at 37℃, and the duration is 2.5h, and the system is shaken every 15min to promote the contact between the microcrystals and the cells; in this process, calcium carbonate microcrystals are gradually formed; under the osmotic pressure, the generated calcium carbonate microcrystals directionally pierce the cell membranes of the mud snail cells, and the active substances in the cells are completely released, and the wall broken material is obtained;

[0038] S3 composite directional enzymolysis: grinding and crushing the fresh white flowers of the horse-tail grass; adding the white flowers of the horse-tail grass into the wall broken material according to a mass ratio of 3.5:1.2 of the mud snails to the white flowers of the horse-tail grass, and then adding 3.5% of the protease of thermophilic bacteria by the mass of the mud snails, and carrying out enzymolysis for 12min at 62℃; cooling to 50℃, adding 3.5% of the protease of bacillus subtilis by the mass of the mud snails, and continuing to carry out enzymolysis for 35min; cooling to 37℃, and continuing to carry out enzymolysis for 60min, and then inactivating the enzyme at 85℃ for 15min to obtain an enzymolysis liquid; through the series of enzymolysis processes, the preferential cutting of the alpha-helix structure is realized, and small molecule active peptide segments are obtained;

[0039] S4 cross-linking and purification: adding 0.3% of the transglutaminase by the mass of the mud snails into the enzymolysis liquid, and reacting at 38℃ for 5h, and then inactivating the enzyme at 85℃ for 15min to complete the residue cross-linking reaction; then centrifuging at a speed of 7500r / min for 18min, taking the supernatant, and using an ultrafiltration membrane to perform ultrafiltration, collecting the component product with a molecular weight of 2kDa-5kDa, adding 5% of the freeze-drying protective agent by the mass of the component product, and freeze-drying to obtain the anti-aging and anti-oxidation composite peptide.

[0040] The freeze-drying protective agent contains trehalose 20g / 100mL, dextran 7g / 100mL, mannitol 9g / 100mL, and the rest solvent is sterile water.

[0041] Example 2

[0042] An anti-aging and anti-oxidation composite peptide is extracted from mud snails and white flowers of horse-tail grass, and includes the living body electric stimulation induction of the mud snails, the biomimetic mineralization wall breaking of the mud snails, the composite directional enzymolysis of the mud snails and the white flowers of the horse-tail grass, and the cross-linking and purification.

[0043] The preparation method of the above anti-aging and anti-oxidation composite peptide includes the following steps:

[0044] S1 living body electric stimulation induction: select the shelled mud snail, put the mud snail and physiological saline into the electric stimulation reaction container according to the mass ratio of 1:4, the pulse electric field parameters are set as follows: intensity 14kV / cm, pulse width 10μs, and electric stimulation treatment is performed for 30min; the stress metabolic pathway of the mud snail is activated, the content of GSH (glutathione) in the cell is increased, and the synthesis of thiol active peptide is promoted;

[0045] S2 biomimetic mineralization wall breaking: a calcium chloride aqueous solution with a mass concentration of 2.0% is prepared; the mud snail subjected to electric stimulation treatment is added into the calcium chloride aqueous solution together with physiological saline, the amount of the calcium chloride aqueous solution is 2 times the volume of the physiological saline, CO2 is introduced to gradually increase the pH value of the system to 7.4, the whole process of biomimetic mineralization wall breaking is carried out in a constant temperature environment at 36℃, and the duration is 2h, the system is shaken every 18min to promote the contact between the microcrystals and the cells, and in this process, calcium carbonate microcrystals are gradually formed; under the osmotic pressure, the generated calcium carbonate microcrystals directionally pierce the cell membrane of the mud snail to completely release the active substances in the cell, and the wall broken material is obtained;

[0046] S3 composite directional enzymolysis: fresh white flowers of horse-tail grass are ground and crushed; the wall broken material is added with the white flowers of horse-tail grass according to the mass ratio of mud snail to white flowers of horse-tail grass 3:1, then 2% of thermophilic protease by the mass of the mud snail is added, the enzymolysis is carried out at 60℃ for 10min, the temperature is reduced to 48℃, 2% of subtilisin by the mass of the mud snail is added, and the enzymolysis is continuously carried out for 30min; the temperature is reduced to 35℃, the enzymolysis is continuously carried out for 60min, and the enzyme is inactivated at 80℃ for 10min, and the enzymolysis liquid is obtained; through the series of enzymolysis processes, the preferential cutting of the alpha-helix structure is realized, and small molecule active peptide segments are obtained;

[0047] S4 cross-linking and purification: 0.2% of transglutaminase by the mass of the mud snail is added into the enzymolysis liquid, reaction is carried out at 37℃ for 4h, the enzyme is inactivated at 80℃ for 10min, and the residue cross-linking reaction is completed; then the supernatant is obtained by centrifugation at a speed of 7000r / min for 15min, ultrafiltration is carried out on the supernatant by using an ultrafiltration membrane, the component product with a molecular weight of 2kDa-5kDa is collected, 4% of freeze-drying protective agent by the mass of the component product is added, freeze-drying is carried out, and the anti-aging and anti-oxidation composite peptide is obtained.

[0048] The freeze-drying protective agent contains trehalose 18g / 100mL, dextran 6g / 100mL, mannitol 8g / 100m, and the rest solvent is sterile water.

[0049] Example 3

[0050] An anti-aging and anti-oxidation composite peptide is extracted from mud snails and white flowers of horse-tail grass, and the composite peptide is obtained through the living body electric stimulation induction of the mud snail, the biomimetic mineralization wall breaking of the mud snail, the composite directional enzymolysis of the mud snail and the white flowers of horse-tail grass, and the cross-linking and purification.

[0051] The preparation method of the anti-aging and anti-oxidation composite peptide comprises the following steps:

[0052] S1 living body electric stimulation induction: select the mud snail after shelling, put the mud snail and physiological saline into an electric stimulation reaction container according to a mass ratio of 1:4, set the pulse electric field parameters: intensity 18 kV / cm, pulse width 10 μs, and perform electric stimulation treatment for 60 min; activate the stress metabolic pathway of the mud snail, so as to improve the content of GSH (glutathione) in the cell and promote the synthesis of thiol active peptide;

[0053] S2 biomimetic mineralization wall breaking: prepare a calcium chloride aqueous solution with a mass concentration of 2.0%; put the mud snail subjected to electric stimulation treatment and the physiological saline into the calcium chloride aqueous solution, the amount of the calcium chloride aqueous solution is 3 times the volume of the physiological saline, CO2 is introduced to gradually increase the pH value of the system to 7.5, the whole process of biomimetic mineralization wall breaking is performed in a constant temperature environment at 38℃, and the duration is 2 h, the system is shaken every 20 min to promote the contact between the microcrystals and the cells, and in this process, calcium carbonate microcrystals are gradually formed; under the osmotic pressure, the generated calcium carbonate microcrystals directionally pierce the cell membrane of the mud snail cell, and the active substances in the cell are completely released, so that the wall broken material is obtained;

[0054] S3 composite directional enzyme hydrolysis: grind and crush the white flowers of fresh horse-tail grass; according to a mass ratio of 3:1.5 of the mud snail to the white flowers of horse-tail grass, the wall broken material is added with the white flowers of horse-tail grass, then 2% of the mass of the mud snail of thermophilic protease is added, the enzyme hydrolysis is performed at 65℃ for 10 min, the temperature is reduced to 52℃, 2% of the mass of the mud snail of subtilisin is added, and the enzyme hydrolysis is continuously performed for 40 min; the temperature is reduced to 35℃, the enzyme hydrolysis is continuously performed for 70 min, and the enzyme is inactivated at 80℃ for 20 min, so that the enzyme hydrolysate is obtained; through the series of enzyme hydrolysis processes, the preferential cutting of the alpha-helix structure is realized, and the small molecule active peptide segment is obtained;

[0055] S4 cross-linking and purification: 0.2% of the mass of the mud snail of transglutaminase is added to the enzyme hydrolysate, reaction is performed at 42℃ for 4 h, the enzyme is inactivated at 90℃ for 10 min, and the residue cross-linking reaction is completed; then the enzyme hydrolysate is centrifuged at a speed of 8000 r / min for 15 min, the supernatant is taken, ultrafiltration is performed on the supernatant by using an ultrafiltration membrane, the component product with a molecular weight of 2 kDa-5 kDa is collected, 6% of the mass of the component product of freeze-drying protective agent is added, freeze-drying is performed, and the anti-aging and anti-oxidation composite peptide is obtained.

[0056] The freeze-drying protective agent contains trehalose 18 g / 100 mL, dextran 8 g / 100 mL, mannitol 8 g / 100 mL, and the rest solvent is sterile water.

[0057] Example 4

[0058] The application discloses an anti-aging and anti-oxidation composite peptide, which is extracted from a freshwater mussel and a white flower of a horse chestnut, and is prepared through the following steps: living body electric stimulation induction of the mussel, biomimetic mineralization wall breaking of the mussel, composite directional enzymolysis of the mussel and the white flower of the horse chestnut, and cross-linking and purification.

[0059] The application further discloses a preparation method of the anti-aging and anti-oxidation composite peptide.

[0060] S1 living body electric stimulation induction: the shelled living body mussel is put into an electric stimulation reaction container together with physiological saline at a mass ratio of 1:5, pulse electric field parameters are set as follows: intensity 18 kV / cm, pulse width 15 mu s, and electric stimulation treatment is performed for 60 min; the stress metabolic pathway of the mussel is activated, the content of GSH (glutathione) in the cell is increased, and the synthesis of thiol active peptides is promoted.

[0061] S2 biomimetic mineralization wall breaking: a calcium chloride aqueous solution with a mass concentration of 3.0% is prepared; the electric stimulation treated mussel is added into the calcium chloride aqueous solution together with the physiological saline, the amount of the calcium chloride aqueous solution is 3 times the volume of the physiological saline, CO2 is introduced, the pH value of the system is gradually increased to 8.0, the whole process of the biomimetic mineralization wall breaking is performed in a constant temperature environment at 38 DEG C, and the duration is 3 h; the system is shaken every 20 min to promote the contact between the microcrystals and the cells; in the process, calcium carbonate microcrystals are gradually formed; under the action of the osmotic pressure, the formed calcium carbonate microcrystals directionally pierce the cell membrane of the mussel cells, and the active substances in the cells are completely released, so that the wall broken product is obtained.

[0062] S3 composite directional enzymolysis: the white flower of the horse chestnut is ground and crushed; the wall broken product is added with the white flower of the horse chestnut at a mass ratio of 4:1.5, then 5% of thermophilic protease by mass of the mussel is added, and enzymolysis is performed at 65 DEG C for 15 min; the temperature is reduced to 52 DEG C, 5% of subtilisin by mass of the mussel is added, and enzymolysis is continuously performed for 40 min; the temperature is reduced to 38 DEG C, and enzymolysis is continuously performed for 70 min; the temperature is reduced to 90 DEG C, and the enzyme is inactivated for 20 min, so that the enzymolysis liquid is obtained; through the series of enzymolysis processes, the preferential cutting of the alpha-helix structure is realized, and small molecule active peptide segments are obtained.

[0063] S4 cross-linking and purification: 0.5% of transglutaminase by mass of the mussel is added into the enzymolysis liquid, reaction is performed at 45 DEG C for 6 h, the temperature is reduced to 90 DEG C, and the enzyme is inactivated for 20 min, so that the residue cross-linking reaction is completed; then, centrifugation is performed at a rotating speed of 8000 r / min for 20 min, the supernatant is taken, ultrafiltration is performed on the supernatant by using an ultrafiltration membrane, the component product with a molecular weight of 2 kDa-5 kDa is collected, 6% of a freeze-drying protective agent by mass of the component product is added, and freeze-drying is performed, so that the anti-aging and anti-oxidation composite peptide is obtained.

[0064] The freeze-drying protective agent contains trehalose 22 g / 100 mL, dextran 8 g / 100 mL, mannitol 10 g / 100 mL, and the rest is sterile water.

[0065] Example 5

[0066] The anti-aging and anti-oxidation composite peptide is extracted from a freshwater mussel and a white flower of a horse chestnut, and comprises the following steps: living body electric stimulation induction of the mussel, biomimetic mineralization wall breaking of the mussel, composite directional enzymolysis of the mussel and the white flower of the horse chestnut, crosslinking, and purification.

[0067] The preparation method of the anti-aging and anti-oxidation composite peptide comprises the following steps:

[0068] S1 living body electric stimulation induction: select a shelled living mussel, put the mussel and physiological saline into an electric stimulation reaction container according to a mass ratio of 1:5, set the pulse electric field parameters as follows: intensity 14 kV / cm, pulse width 15 μs, and perform electric stimulation treatment for 30 min; activate the stress metabolic pathway of the mussel, so as to improve the content of GSH (glutathione) in the cell and promote the synthesis of thiol active peptides;

[0069] S2 biomimetic mineralization wall breaking: prepare a calcium chloride aqueous solution with a mass concentration of 3.0%; put the mussel subjected to the electric stimulation treatment and the physiological saline into the calcium chloride aqueous solution, the amount of the calcium chloride aqueous solution is 2 times the volume of the physiological saline, introduce CO2 to gradually increase the pH value of the system to 8.0, the whole process of the biomimetic mineralization wall breaking is performed in a constant temperature environment at 36℃, and the duration is 3 h, the system is shaken every 15 min to promote the contact between the microcrystals and the cells, and in this process, calcium carbonate microcrystals are gradually formed; under the osmotic pressure, the generated calcium carbonate microcrystals directionally pierce the cell membrane of the mussel to completely release the active substances in the cell, and wall broken substances are obtained;

[0070] S3 composite directional enzymolysis: grind and crush a fresh white flower of a horse chestnut; according to a mass ratio of 4:1 of the mussel to the white flower of the horse chestnut, add the white flower of the horse chestnut to the wall broken substances, then add 5% of thermophilic protease based on the mass of the mussel, perform enzymolysis at 60℃ for 15 min, cool to 48℃, add 5% of subtilisin based on the mass of the mussel, continue to perform enzymolysis for 30 min, cool to 38℃, continue to perform enzymolysis for 60 min, and then perform enzyme inactivation at 90℃ for 10 min to obtain an enzymolysis liquid; through the series of enzymolysis processes, preferential cutting of an alpha-helix structure is realized, and small molecule active peptide segments are obtained;

[0071] S4 cross-linking and purification: 0.5% transglutaminase of the mud snail is added to the enzymatic solution, and the reaction is carried out at 40℃ for 6h, and the enzyme is inactivated at 80℃ for 20min to complete the residue cross-linking reaction; then centrifugation is carried out at 7000r / min for 20min, the supernatant is taken, ultrafiltration is carried out by using an ultrafiltration membrane, the component product with a molecular weight of 2kDa-5kDa is collected, 4% freeze-drying protective agent of the component product is added, and freeze-drying is carried out to obtain the anti-aging and anti-oxidation composite peptide.

[0072] In the formula, the freeze-drying protective agent contains trehalose 22g / 100mL, dextran 6g / 100mL, mannitol 10g / 100mL, and the rest is sterile water.

[0073] In each of the above examples, the thermolysin is derived from Shanghai Hongshun Biological Technology Co., Ltd., and the enzyme activity is 50,000U / g; the subtilisin is derived from Xi'an Xinlu Biological Technology Co., Ltd., and the enzyme activity is 20,000U / g; and the transglutaminase is derived from Guangzhou Baode Biological Technology Co., Ltd., and the enzyme activity is 100,000U / g.

[0074] The anti-aging and anti-oxidation composite peptide prepared in each of the above examples is applied in drugs and cosmetics.

[0075] Comparative Example 1

[0076] In the preparation method, S1 is omitted, and the in-vivo electric stimulation induction is not performed; and other methods and parameters are the same as those in Example 1.

[0077] Comparative Example 2

[0078] In the preparation method, S2 is omitted, and the biomimetic mineralization wall breaking is not performed, and direct crushing treatment is performed; and other methods and parameters are the same as those in Example 1.

[0079] Comparative Example 3

[0080] In the preparation method, the white flowers of the caraxer are not added in S3; and other methods and parameters are the same as those in Example 1.

[0081] Comparative Example 4

[0082] In the preparation method, the thermolysin is not used for enzymolysis in S3; and other methods and parameters are the same as those in Example 1.

[0083] Comparative Example 5

[0084] In the preparation method, the subtilisin is not added in S3; and other methods and parameters are the same as those in Example 1.

[0085] Comparative Example 6

[0086] In the preparation method, the variable-temperature enzymolysis is not performed in S3, and all enzymolysis temperatures are at 37℃; and other methods and parameters are the same as those in Example 1.

[0087] Comparative Example 7

[0088] In S4 of the preparation method, no transglutaminase cross-linking reaction was performed, and direct centrifugation was performed; other methods and parameters were the same as in Example 1.

[0089] The above-mentioned each complex peptide product was detected.

[0090] I. DPPH free radical scavenging rate:

[0091] A 0.1 mmol / L DPPH ethanol solution was prepared. Complex peptide solutions of different concentrations were prepared: the complex peptides obtained in each example and comparative example were prepared into a solution with a concentration of 0.5 mg / mL using deionized water.

[0092] 1 mL of the complex peptide solution was taken, 3 mL of the DPPH ethanol solution was added, mixed, and reacted in the dark for 30 min. 1 mL of deionized water was added instead of the complex peptide solution to add 3 mL of the DPPH ethanol solution as a blank control, and ascorbic acid solutions with the same concentration gradient were used as a positive control. After the reaction was completed, the absorbance of each reaction system was measured at a wavelength of 517 nm using a UV-visible spectrophotometer, and the scavenging rate was calculated: scavenging rate (%) = [1 - (Ai - Aj) / A0] x 100%, wherein A0 is the absorbance of the blank control, Ai is the absorbance after the addition of the complex peptide solution, and Aj is the absorbance of the complex peptide solution itself. The results are shown in Table 1 below.

[0093] Table 1 DPPH free radical scavenging rate detection results

[0094] Sample DPPH radical scavenging rate (%) Sample DPPH radical scavenging rate (%) Example 1 85.36 Comparative Example 1 70.42 Example 2 82.95 Comparative Example 2 75.38 Example 3 83.47 Comparative Example 3 78.71 Example 4 86.23 Comparative Example 4 73.06 Example 5 84.19 Comparative Example 5 74.25 Comparative Example 6 76.39 Comparative Example 7 80.28

[0095] From the above results, it can be seen that the complex peptides of Comparative Examples 1 to 5 have high DPPH free radical scavenging effects.

[0096] The stress metabolic pathway in the cells of the mud snail is not activated, the content of GSH (glutathione) in the cells cannot be improved, and the synthesis of thiol active peptides is reduced without the induction of in vivo electrical stimulation in Comparative Example 1. Thiol active peptides have antioxidant capacity, and a decrease in the content of thiol active peptides directly leads to a decrease in the overall antioxidant capacity of the complex peptides. Therefore, in the detection of the three free radical scavenging rates, the data are lower than those of each of the examples. In Comparative Example 2, the cell membrane of the mud snail is not directionally pierced by the calcium carbonate microcrystals under the action of osmotic pressure without biomimetic mineralization and wall breaking, and the active substances in the cells cannot be completely released. The active substances in the cells are an important source of the antioxidant capacity of the complex peptides, and the insufficient release of the active substances reduces the active peptides that can participate in the antioxidant reaction, thereby reducing the antioxidant effect of the complex peptides, and the three free radical scavenging rates are also reduced. In S3 of Comparative Example 3, the trichosanthes kirilowii is not added, and the active ingredients in the trichosanthes kirilowii are lacking. In the process of complex directional enzymolysis, the ingredients in the trichosanthes kirilowii and the mud snail interact to produce small molecular peptide segments with higher antioxidant activity. Without the trichosanthes kirilowii, these high-efficiency antioxidant small molecular peptide segments cannot be formed, thereby affecting the antioxidant performance of the complex peptides, and the free radical scavenging rate data are lower than those of the examples. In S3 of Comparative Example 4, the protease is not used for enzymolysis, and the preferential cutting of the a-helix structure cannot be achieved. The effective cutting of the a-helix structure is crucial for obtaining small molecular active peptide segments, and without the action of the protease, the enzymolysis process is incomplete, the number and quality of the obtained small molecular active peptide segments are reduced, the antioxidant capacity of the complex peptides is reduced, and the three free radical scavenging rates are also reduced. In S3 of Comparative Example 5, the subtilisin is not added, and the substrate cannot be completely subjected to enzymolysis. The subtilisin plays an important role in the entire enzymolysis process, and cooperates with the protease to better cut the protein structure and obtain ideal small molecular active peptide segments. Without the subtilisin, the antioxidant capacity of the enzymolysis product is insufficient, and the free radical scavenging rate data are not ideal. In S3 of Comparative Example 6, the variable-temperature enzymolysis is not performed, and all the enzymolysis temperatures are 37℃. Different enzymes have optimal activity at different temperatures, and the variable-temperature enzymolysis is to adapt to the optimal activity temperatures of the protease and the subtilisin to achieve better enzymolysis effect. The enzymes cannot fully exert their activity at a fixed enzymolysis temperature of 37℃, the enzymolysis effect is not good, the number and quality of the obtained small molecular active peptide segments are not as good as those of the examples, the antioxidant performance is affected, and the free radical scavenging rate is reduced. In S4 of Comparative Example 7, the transglutaminase cross-linking reaction is not performed. The transglutaminase can catalyze the cross-linking reaction between the γ-carboxamide group of the glutamine residue and the ε-amino group of the lysine residue in the peptide molecule, thereby changing the structure and functional characteristics of the peptide. Without the transglutaminase cross-linking reaction, the structure characteristics are changed, the reaction capacity with free radicals is affected, the antioxidant function of the complex peptides cannot be fully exerted, and the free radical scavenging rate data are lower than those of the examples.

[0097] II. Collagen synthesis promotion and cell regeneration stimulation detection:

[0098] 1. Collagen synthesis promotion detection:

[0099] The cryopreserved human skin fibroblasts were taken out from the liquid nitrogen tank and quickly placed in a 37°C water bath for rapid thawing. The thawed cells were transferred to a centrifuge tube containing 5 mL of complete medium (DMEM medium containing 10% fetal bovine serum, 1% double-antibiotic, i.e., penicillin-streptomycin mixture), centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. An appropriate amount of complete medium was added to resuspend the cells, which were inoculated into a T25 cell culture bottle and incubated in a cell incubator at 37°C and 5% CO2. When the cell confluence reached more than 80%, the old culture medium was discarded, the cells were gently washed twice with PBS buffer, 1 mL of 0.25% trypsin digestion solution was added, and the cells were incubated at 37°C for 2 min. When the cells were observed under a microscope to be rounded and the intercellular space was enlarged, 2 mL of complete medium was added to terminate the digestion, and the cells were blown into a single cell suspension for subculture at a ratio of 1:3.

[0100] The well-grown cells were inoculated into a 96-well cell culture plate at a density of 5 x 10 4 cells per well, 200 μL of complete medium was added to each well, and the cells were incubated for 24 h to adhere to the wall. Each example and comparative example group (100 μg / mL of complex peptide was added to each well, and 200 μL was added). The culture plate was returned to the cell incubator for continued incubation for 48 h. After the incubation, the cell culture supernatant in each well was carefully aspirated and transferred to a new centrifuge tube. According to the instructions of the collagen ELISA detection kit, 100 μL of sample (i.e., the collected cell culture supernatant) was added to the corresponding well, followed by the addition of 100 μL of enzyme-labeled antibody working solution, gentle mixing, sealing with a sealing film, incubation at 37°C for 1.5 h. After the incubation, the plate was washed 5 times with a plate washer, each time for 30 s, and then dried. 100 μL of substrate color developing solution was added to each well, and the reaction was carried out at 37°C for 20 min in the dark. When the standard sample well was obviously colored, 50 μL of stop solution was added to each well to terminate the reaction, and the absorbance of each well was measured at a wavelength of 450 nm on an enzyme-labeled instrument. The collagen content in the sample was calculated. The detection results are shown in Table 2.

[0101] 2. Cell regeneration stimulation detection:

[0102] The cell culture was the same as the cell recovery and subculture steps in the collagen synthesis promotion detection. Human skin fibroblasts were inoculated into a 96-well cell culture plate at a density of 3 x 10 3 cells per well, 100 μL of complete medium was added to each well, and the cells were incubated for 24 h to adhere to the wall.

[0103] Each of the examples and comparative examples group (add culture medium containing the concentration of 100 μg / mL of composite peptide, add 100 μL). The culture plate was placed in the cell culture box and cultured for 24 h, 48 h, 72 h, respectively. 2 h before the end of each time point, 10 μL of CCK-8 solution was added to each well, mixed gently to avoid bubbles, and incubated. After incubation, the absorbance of each well was measured at 450 nm wavelength on a microplate reader, and the data was recorded. The test results are shown in Table 2.

[0104] Table 2 Test results of promoting collagen synthesis and stimulating cell regeneration ability

[0105] Sample Collagen synthesis amount (ng / mL, 100 μg / mL concentration) Cell proliferation absorbance (48 h, 100 μg / mL concentration) Example 1 120 1.25 Example 2 115 1.21 Example 3 118 1.23 Example 4 122 1.27 Example 5 121 1.26 Comparative Example 1 81 0.90 Comparative Example 2 93 0.95 Comparative Example 3 95 1.04 Comparative Example 4 86 0.92 Comparative Example 5 88 0.93 Comparative Example 6 92 0.98 Comparative Example 7 103 1.05

[0106] From the above results, it can be seen that the composite peptide of each example has good effect of promoting collagen synthesis and stimulating cell regeneration ability.

[0107] In Comparative Example 1, the stress metabolic pathway in the cell of the mud snail was not activated due to the omission of in vivo electric stimulation induction. Under normal circumstances, in vivo electric stimulation induction can increase the content of GSH (glutathione) in the cell and promote the synthesis of thiol active peptides. These active substances can regulate the oxidation-reduction state in the cell, activate the expression of genes related to collagen synthesis, such as COL1A1, and also activate the signal pathways related to cell proliferation, such as the ERK / MAPK signal pathway. In Comparative Example 1, due to the lack of these activation processes, the expression level of related genes is low, the activity of key enzymes for collagen synthesis in the cell, such as prolyl hydroxylase, is also low, resulting in a decrease in the amount of collagen synthesis. In terms of cell proliferation, the ERK / MAPK signal pathway and other signal pathways are not effectively activated, the cell cycle is blocked, and the number of cells in the proliferation phase is reduced, so the absorbance of cell proliferation is low.

[0108] In Comparative Example 2, the cell membrane of the mud snail cell cannot be directly pierced by the calcium carbonate microcrystals under the action of osmotic pressure without the process of biomimetic mineralization and smashing, and the active substances in the cell cannot be completely released. The active substances in the cell are the key components for the composite peptide to promote collagen synthesis and cell regeneration, and the release of active substances is insufficient, which reduces the amount of active peptides, growth factors and other substances that can participate in the reaction. These substances are essential for maintaining normal cell metabolism, promoting collagen synthesis and stimulating cell proliferation. For example, some active peptides can bind to receptors on the cell surface, activate downstream signal transduction, and promote collagen synthesis. In Comparative Example 2, due to the lack of active substances, the cell surface receptors cannot be fully activated, the signal transduction is blocked, and the amount of collagen synthesis and the ability of cell proliferation are affected, resulting in lower test data than the examples.

[0109] The S3 of Comparative Example 3 does not add the white flowers of the axle grass, and lacks the active ingredients in the white flowers of the axle grass. The white flowers of the axle grass contain various bioactive ingredients, which synergize with the ingredients in the mud snail during the complex directional enzymolysis process, and can produce small molecule peptide segments with higher bioactivity. These small molecule peptide segments can promote collagen synthesis and cell regeneration through multiple pathways, such as regulating transcription factors in cells, promoting transcription and translation of collagen genes, and also stimulating cells to secrete more growth factors, such as transforming growth factor beta (TGF-β), which can further promote collagen synthesis and cell proliferation. In Comparative Example 3, due to the lack of active ingredients in the white flowers of the axle grass, these efficient synergies cannot be formed, resulting in a weakened effect of the complex peptide in stimulating cell collagen synthesis and proliferation, and the data is not as good as the examples.

[0110] Comparative Examples 4 and 5: In S3, neither protease nor subtilisin is used, which cannot effectively cut the raw materials to obtain small molecule active peptide segments that promote cell function. Protease and subtilisin play a key role in the complex directional enzymolysis process, they can specifically cut the specific structure of the protein, protease preferentially cuts the alpha-helix structure, and subtilisin further acts in the subsequent enzymolysis process to degrade the protein into small molecule peptides. These small molecule peptides have specific amino acid sequences and structures, which can bind to receptors on the cell surface, activate intracellular signaling pathways, and promote collagen synthesis and cell proliferation. In Comparative Example 4, the lack of protease makes it impossible to effectively cut the alpha-helix structure, and the number and quality of small molecule peptides obtained are reduced, which cannot fully activate the intracellular signaling pathways. In Comparative Example 5, the lack of subtilisin makes the enzymolysis process incomplete, and it is also impossible to obtain small molecule peptides with optimal activity, thereby leading to a decrease in the collagen synthesis and regeneration capacity of cells, and the detection data is low.

[0111] In S3 of Comparative Example 6, no temperature-variable enzymolysis is performed, and all enzymolysis temperatures are at 37°C. Different enzymes have optimal activity at different temperatures, and the optimal temperature of protease is relatively high, and the optimal temperature of subtilisin is relatively low. Temperature-variable enzymolysis is to adapt to the optimal activity temperature of the two enzymes at different stages to achieve better enzymolysis effect. At 37°C, both enzymes cannot work at their optimal activity state, the enzymolysis efficiency is reduced, and the number and quality of small molecule active peptides obtained are insufficient. These small molecule active peptides are crucial for promoting cell function, and the decrease in their number and quality makes them unable to fully play a role in promoting cell synthesis and proliferation, so the amount of collagen synthesis and the cell proliferation capacity are lower than the examples.

[0112] The transglutaminase cross-linking reaction is not performed in S4 of Comparative Example 7. Transglutaminase can catalyze the cross-linking reaction between the γ-carboxamide group of the glutamine residue and the ε-amino group of the lysine residue in the peptide molecule, thereby changing the structural and functional properties of the peptide, changing the spatial conformation of the peptide segment, enhancing its binding ability to the cell surface receptor, activating more signal pathways in the cell, and thereby promoting collagen synthesis and cell regeneration. In Comparative Example 7, the transglutaminase cross-linking reaction is not performed, the stability and activity of the peptide segment are reduced, the binding ability to the cell surface receptor and the activation ability to the signal pathway in the cell are weakened, and the effect of promoting collagen synthesis and cell regeneration is not as good as that of the examples.

Claims

1. An anti-aging antioxidant composite peptide, characterized in that, The composite peptide is extracted from mud snails and white flowers of horse chestnut, comprising the following steps: living body electric stimulation induction of mud snails, biomimetic mineralization wall breaking, then composite directional enzymolysis with white flowers of horse chestnut, and crosslinking and purification. The preparation method of the composite peptide comprises the following steps: S1 living body electric stimulation induction: the mud snails and physiological saline are put into an electric stimulation reaction container for electric stimulation treatment at a mass ratio of 1: (4-5); The pulse electric field parameters of the electric stimulation treatment are: intensity 14 kV / cm-18 kV / cm, pulse width 10 μs-15 μs, and treatment time 30 min-60 min; S2 biomimetic mineralization wall breaking: the mud snails and physiological saline subjected to living body electric stimulation induction are added into a calcium chloride aqueous solution, CO2 is introduced to gradually increase the pH of the system to 7.4-8.0, and in this process, calcium carbonate microcrystals are gradually formed; under the action of osmotic pressure, the generated calcium carbonate microcrystals directionally pierce the cell membrane of the mud snails to release the active substances in the cells, and the wall broken product is obtained; S3 composite directional enzymolysis: the wall broken product is added with white flowers of horse chestnut at a mass ratio of (3-4):(1-1.5), and then thermophilic proteinase is added for enzymolysis at 60°C-65°C, the temperature is lowered to 48°C-52°C, and then subtilisin is added for continuous enzymolysis; the temperature is lowered to 35°C-38°C, and continuous enzymolysis is carried out, and then the enzyme is inactivated to obtain an enzymolysis liquid; S4 crosslinking and purification: transglutaminase is added to the enzymolysis liquid for reaction, the enzyme is inactivated, the residue crosslinking reaction is completed, centrifugation is carried out, the supernatant is subjected to ultrafiltration, the product with a molecular weight of 2 kDa-5 kDa is collected, and then freeze-drying is carried out to obtain the anti-aging and anti-oxidation composite peptide.

2. The method for preparing an anti-aging and antioxidant complex peptide according to claim 1, characterized in that, In S1, the mud snails are living body mud snails after shelling.

3. The method for preparing an anti-aging and antioxidant complex peptide according to claim 1, characterized in that, In S2, the mass concentration of the calcium chloride aqueous solution is 2.0%-3.0%, and the amount of the calcium chloride aqueous solution is 2-3 times the volume of the physiological saline.

4. The method for preparing an anti-aging and antioxidant complex peptide according to claim 1, characterized in that, In S2, the whole process of the biomimetic mineralization wall breaking is carried out in a constant temperature environment at 36°C-38°C for 2-3 h, and the microcrystals and cells are contacted by shaking every 15-20 min.

5. The method for preparing an anti-aging and antioxidant complex peptide according to claim 1, characterized in that, In S3, the white flowers of horse chestnut are white flowers of fresh horse chestnut, and the white flowers of horse chestnut are pre-ground and crushed.

6. The method for preparing an anti-aging and antioxidant complex peptide according to claim 1, characterized in that, In S3, the addition amount of the thermophilic proteinase is 2%-5% of the mass of the mud snails, and the addition amount of the subtilisin is 2%-5% of the mass of the mud snails.

7. The method for preparing an anti-aging and antioxidant complex peptide according to claim 1, characterized in that, In S4, the addition amount of the transglutaminase is 0.2%-0.5% of the mass of the mud snails.

8. The method for preparing an anti-aging and antioxidant complex peptide according to claim 1, characterized in that, In S3 and S4, the temperature for inactivating the enzyme is 80°C-90°C, and the time for inactivating the enzyme is 10 min-20 min.

9. The anti-aging and anti-oxidation composite peptide of claim 1 is applied to cosmetics.