A preparation method of jellyfish collagen active protein, a composition capable of regulating skin microecology, and a film patch capable of improving skin inflammation

Abstract

The present invention discloses a method for preparing jellyfish collagen active protein, a composition capable of regulating skin microecology, and a film patch capable of improving skin inflammation. The method for preparing jellyfish collagen active protein comprises the following steps: desalting, pulping, degreasing, alkalization, enzymatic hydrolysis, salting out, purification, and drying. The method for preparing jellyfish collagen active protein of the present invention has a wide range of material sources, a simple method, and is suitable for large-scale production. At the same time, the jellyfish collagen active protein prepared by the method for preparing jellyfish collagen active protein of the present invention has lower allergic reactivity and higher safety than that of bovine-derived materials. The jellyfish collagen active protein prepared by the method for preparing jellyfish collagen active protein of the present invention can maintain a triple helix structure.

Description

Technical Field

[0001] The invention relates to the technical field of medical biomaterials, and in particular to a method for preparing jellyfish collagen active protein, a composition capable of regulating skin microecology, and a film patch capable of improving skin inflammation. Background Art

[0002] In recent years, with the rapid development of biotechnology, more biological materials have been used in cosmetics. Representative raw materials are various proteins and peptides, and collagen is one of the most widely used ones. Collagen has good moisturizing effects. It has the effects of filling the skin and anti-aging, and has always been concerned by consumers with skin care needs and has great commercial value. Collagen can be extracted from various animal tissues, such as pig skin, cow bones, etc., and its sources are wide.

[0003] In recent years, with the good development of biotechnology, more biological raw materials have been applied to cosmetics, among which the representative raw materials are various proteins and peptides, and collagen is one of the most widely used ones. Collagen has good effects of moisturizing, filling the skin, and anti-aging. It has always been concerned by consumers with skin care needs and has great commercial value. Collagen can be extracted from various animal tissues, such as pig skin, cow bones, etc., which have a wide range of sources and are easy to obtain. In addition, recombinant collagen can be obtained by genetic recombination and microbial fermentation technology, which has the advantages of high purity and strong editability. However, the extraction of collagen from traditional animals such as pigs and cattle has always been very controversial, with religious restrictions or BSE / TSE risks. The technical solution of obtaining collagen by bioengineering has a relatively high technical threshold, and the time and cost invested will also be correspondingly high.

[0004] Collagen repair materials derived from marine organisms are a kind of biomaterial with broad development prospects. They will not cause risks of animal-borne diseases and pathogen transmission, and will not involve religious beliefs. The ocean is a special environment with high osmotic pressure and high ultraviolet radiation. Its unique ecological environment gives marine organisms more efficient bioactive substances, including antimicrobial peptides, antioxidant proteins and other new functional proteins with good development prospects. Marine plankton lives in the surface layer of seawater at a depth of 4-6 meters and lives in a strong light radiation environment for a long time. Oxidative damage is one of the important mechanisms for ultraviolet radiation to cause damage to the body. Studies have shown that in order to avoid or reduce the damage caused by ultraviolet radiation, plankton has produced a wide variety of antioxidant active substances with specific structures and strong activity in its body after long-term adaptive selection. The developed antioxidant system plays an important role in protecting cells and organisms from ultraviolet radiation damage. It can resist the radiation of strong ultraviolet rays to organisms and remove free radicals caused by radiation.

[0005] As a marine organism, jellyfish contains a large amount of collagen in its body, and its sources are wide. Compared with ordinary collagen, jellyfish collagen active protein is very easy to absorb, because its molecular size is 50-100 times smaller than that of collagen extracted from ordinary animals or plants, and its absorption rate is 40 times higher than that of ordinary collagen, so jellyfish collagen active protein has great application prospects in skin care products.

[0006] Jellyfish belongs to coelenterates and is rich in resources. Due to its high water content, only a small part of jellyfish is pickled as food, which has low added value and causes waste of resources. Jellyfish contains rich protein. Using enzymatic hydrolysis technology to convert protein in jellyfish into polypeptides is the key to processing and high-value utilization of jellyfish resources. Moreover, the current research on jellyfish peptides mainly focuses on jellyfish collagen active protein peptides, that is, after extracting jellyfish collagen active protein by acid, alkali, hot water or enzyme dissolution, collagen peptides are prepared by enzymatic hydrolysis of collagen using protease. In the process of extracting jellyfish collagen active protein, a large amount of acid-soluble or alkali-soluble protein is discarded, resulting in low utilization of jellyfish protein. Using enzymatic hydrolysis technology to convert protein in jellyfish into polypeptides and broaden the application field of jellyfish resources is the key to processing and high-value utilization of jellyfish resources. The collagen obtained by the existing preparation method of jellyfish collagen active protein is prone to instability, difficulty in preservation, and low activity. Summary of the invention

[0007] In order to overcome the deficiencies of the prior art, the first object of the present invention is to provide a method for preparing jellyfish collagen active protein, which has a wide range of material sources, is simple and convenient, and is easy to achieve large-scale production.

[0008] The second object of the present invention is to provide a composition capable of regulating skin microecology, comprising the jellyfish collagen active protein prepared by the above-mentioned preparation method of the jellyfish collagen active protein.

[0009] The third object of the present invention is to provide a patch that can improve skin inflammation and contains the jellyfish collagen active protein prepared by the above-mentioned preparation method of the jellyfish collagen active protein.

[0010] One of the purposes of the present invention can be achieved by adopting the following technical solution:

[0011] A method for preparing jellyfish collagen active protein comprises the following steps:

[0012] The desalted jellyfish is pulped to obtain freeze-dried powder. The pulping process is to filter the desalted jellyfish, then pre-freeze, mix with liquid nitrogen and cool after pre-freezing, and then crush to obtain freeze-dried powder; then the freeze-dried powder is successively defatted, alkalized, enzymatically hydrolyzed, salted out, and purified twice to obtain a secondary purified liquid, and then the secondary purified liquid is mixed with a protective liquid and dried at low temperature to obtain jellyfish collagen active protein.

[0013] Furthermore, the protective solution is one or a combination of two or more of L-ascorbic acid, glucose, mannitol, trehalose, polyethylene glycol, and bovine serum albumin; the volume ratio of the secondary purification solution to the protective solution is 1:(0.01-0.20).

[0014] Furthermore, the defatting treatment is as follows: adding an organic solvent to the freeze-dried powder, soaking, centrifuging to obtain a lower protein solution after extraction, washing the lower protein solution with purified water for 3 to 5 times, filtering, removing water to obtain jellyfish protein; the organic solvent is one or a combination of two or more of acetone, butanone or chloroform.

[0015] Furthermore, the alkalinization treatment is as follows: adding a mixed salt solution of disodium hydrogen phosphate and sodium dihydrogen phosphate to the defatted jellyfish protein to a final concentration of 0.02 to 0.2 mol / L, stirring for 1 to 3 days, centrifuging to obtain a lower precipitate, washing the lower precipitate with purified water for 5 to 8 times until the pH of the washing solution is neutral, filtering, and removing water to obtain a jellyfish protein solid.

[0016] Furthermore, the enzymatic hydrolysis treatment is: enzymatically hydrolyzing the jellyfish protein solid obtained by alkalinization treatment with 0.05 mol / L citric acid solution or 0.2 mol / L glacial acetic acid solution containing 1.5% pepsin, stirring, and centrifuging to extract the supernatant.

[0017] Further, the secondary purification treatment is as follows: (1) First purification: the complex solution obtained after the salting-out treatment is placed in a 50-100KD dialysis bag and dialyzed for 2-3 days using a 0.02 mol / L sulfate buffer solution as an external liquid. The external liquid is replaced every 8 hours during the dialysis process. After the dialysis is completed, the material in the bag is collected as a primary purified solution;

[0018] (2) Second purification: The first purified liquid is dialyzed with purified water as the external liquid for 2 to 3 days. The external liquid is replaced every 3 to 4 hours during the dialysis process. At the end of the dialysis, the material in the collection bag is the second purified liquid, which is packaged and stored.

[0019] The second purpose of the present invention can be achieved by adopting the following technical solutions:

[0020] A composition capable of regulating skin microecology, comprising the following components by weight:

[0021] The jellyfish collagen active protein prepared by any of the above-mentioned methods comprises 0.1 to 20 parts of jellyfish collagen active protein, 0.1 to 20 parts of Propionibacterium acnes bacteriophage, and 0.1 to 20 parts of a first thickener.

[0022] Furthermore, the first thickener is one or a combination of two or more of starch, gum arabic, pectin, agar, gelatin, alginate, carrageenan or dextrin.

[0023] The third object of the present invention can be achieved by adopting the following technical solutions:

[0024] A film patch capable of improving skin inflammation comprises the following components in parts by weight:

[0025] The jellyfish collagen active protein prepared by any of the above-mentioned methods comprises 0.05-0.35 parts of jellyfish collagen active protein, 0.1-10 parts of Propionibacterium acnes bacteriophage, 0.01-0.5 parts of moisturizer, 0.1-20 parts of second thickener and 0.05-5 parts of dispersant.

[0026] Furthermore, the moisturizing agent is sodium hyaluronate; the second thickener is one or a combination of two or more of starch, gum arabic, pectin, agar, gelatin, alginate, carrageenan, dextrin, carbomer, ethyl cellulose, hydroxypropyl methylcellulose or polyvinyl alcohol; the dispersant is one or a combination of two or more of hexylene glycol, propylene glycol, caprylyl glycol or glycerol.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The preparation method of the jellyfish collagen active protein of the present invention has a wide range of material sources, a simple method, and is suitable for large-scale production. At the same time, the jellyfish collagen active protein prepared by the preparation method of the jellyfish collagen active protein of the present invention has lower allergic reactivity and higher safety than that of bovine-derived materials. The freeze-drying protection method of the present invention can make the film prepared from the jellyfish collagen active protein smoother, more evenly thick, and more delicate without defects such as cracks. The jellyfish collagen active protein prepared by the preparation method of the jellyfish collagen active protein of the present invention can maintain a triple helix structure.

[0029] 2. The jellyfish collagen active protein prepared by the preparation method of the jellyfish collagen active protein of the present invention will not cause animal-borne diseases and pathogen transmission risks; it does not involve religious beliefs, has good water solubility, can be used in products for improving and controlling skin inflammation, and will not affect its liquid absorption performance. It can also be used in products for improving skin rejuvenation and correcting wrinkles and filling.

[0030] 3. The composition capable of regulating skin microecology of the present invention does not contain any chemical preservatives. The jellyfish collagen active protein can form a film to play a sealing role, thereby enhancing the effect of bacteriophages on Propionibacterium. At the same time, the jellyfish collagen active protein can also adsorb and clean the residual substances after the bacteria are removed, thereby strengthening the cleaning of hair follicles, regulating the secretion of sebaceous glands, improving the level of local healthy microenvironment, and maintaining the balance of skin microecology.

[0031] 4. The film patch for improving skin inflammation of the present invention does not contain any chemical preservatives, thus avoiding the instability of jellyfish active collagen due to the precipitation of preservatives, and reducing the incidence of allergic reactions. The bacteriophage can directly and specifically remove Propionibacterium acnes, and can have a good inhibitory and killing effect on Propionibacterium acnes and Staphylococcus epidermidis, and has a good inhibitory effect on the inflammatory reaction and the secretion of inflammatory factors caused by Propionibacterium acnes. At the same time, the active protein of jellyfish collagen can form a film to play a sealing role, enhance the effect of bacteriophage on Propionibacterium, and improve and control skin inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a before and after comparison of the film patch capable of improving skin inflammation according to Example 8 of the present invention. DETAILED DESCRIPTION

[0033] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only partial embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0034] Unless otherwise specified, the experimental methods used in the following experimental examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0035] Jellyfish contain abundant proteins. Using enzymatic hydrolysis technology to convert proteins in jellyfish into polypeptides is the key to processing and high-value utilization of jellyfish resources. Moreover, the current research on jellyfish peptides mainly focuses on jellyfish collagen active protein peptides, that is, after extracting jellyfish collagen active protein by acid, alkali, hot water or enzyme dissolution, collagen peptides are prepared by enzymatic hydrolysis of collagen protein using protease. In the process of extracting jellyfish collagen active protein, a large amount of acid-soluble or alkali-soluble proteins are discarded, resulting in low utilization rate of jellyfish protein. Jellyfish collagen active protein can be used in products that improve and control skin inflammation. Therefore, the present invention provides a method for preparing jellyfish collagen active protein.

[0036] A method for preparing jellyfish collagen active protein comprises the following steps:

[0037] The desalted jellyfish is pulped to obtain freeze-dried powder. The pulping process is to filter the desalted jellyfish, then pre-freeze, mix with liquid nitrogen and cool after pre-freezing, and then crush to obtain freeze-dried powder; then the freeze-dried powder is successively defatted, alkalized, enzymatically hydrolyzed, salted out, and purified twice to obtain a secondary purified liquid, and then the secondary purified liquid is mixed with a protective liquid and dried at low temperature to obtain jellyfish collagen active protein.

[0038] As one embodiment, the protective solution is one or a combination of two or more of L-ascorbic acid, glucose, mannitol, trehalose, polyethylene glycol, and bovine serum albumin; the volume ratio of the secondary purification solution to the protective solution is 1: (0.01-0.20). Preferably, the volume ratio of the secondary purification solution to the protective solution is 1: (0.05-0.10)

[0039] As one embodiment, the desalination treatment is to rinse the jellyfish's oral arm and umbrella with water, and then soak it in purified water to obtain the desalted jellyfish. Preferably, in the desalination treatment, the mass ratio of jellyfish to purified water is 1: (5-20); the soaking time is 2-7 days, wherein the purified water is changed every 2-6 hours, and the soaking temperature is 4-15°C. Further preferably, in the desalination treatment, the mass ratio of jellyfish to purified water is 1: (8-10); the soaking time is 3-5 days, wherein the purified water is changed every 3-4 hours, and the soaking temperature is 8-10°C.

[0040] As one embodiment, the pre-freezing temperature in the beating process is 80 to 0°C, the mass volume ratio of jellyfish to liquid nitrogen is 1 kg: (2 to 10) L, and the mixing time is 5 to 15 minutes. Preferably, the pre-freezing temperature in the beating process is -15 to -20°C.

[0041] As one embodiment, the defatting treatment is: adding an organic solvent to the freeze-dried powder, soaking, centrifuging, obtaining the lower protein solution after extraction, washing the lower protein solution with purified water for 3 to 5 times, filtering, removing water to obtain jellyfish protein; the organic solvent is one or a combination of two or more of acetone, butanone or chloroform. Preferably, in the defatting treatment, the soaking temperature is 15 to 25°C, the soaking time is 8 to 24 hours, and the process conditions of the centrifugal treatment are: centrifugal treatment at 2000 to 5000 rpm for 10 to 30 minutes, and filtering is performed using a 60 to 80 mesh nylon filter bag; the volume ratio of the freeze-dried powder to the organic solvent is 1: (5 to 10); the volume ratio of the lower protein solution to purified water is 1: (5 to 10). Further preferably, in the defatting treatment, the soaking temperature is 20°C and the soaking time is 15 to 20 hours.

[0042] As one embodiment, the alkalization treatment is: adding a mixed salt solution of disodium hydrogen phosphate and sodium dihydrogen phosphate to the jellyfish protein obtained by the defatting treatment to a final concentration of 0.02-0.2 mol / L, stirring for 1-3 days, centrifuging to obtain a lower precipitate, washing the lower precipitate with purified water for 5-8 times until the pH of the washing liquid is neutral, filtering, and removing water to obtain a jellyfish protein solid. Preferably, in the alkalization treatment, a mixed salt solution of disodium hydrogen phosphate and sodium dihydrogen phosphate is added to the jellyfish protein obtained by the defatting treatment to a final concentration of 0.1 mol / L; the stirring process conditions are stirring at a temperature of 25-60 rpm for 2 days, the stirring temperature is 4-25°C, the centrifugal treatment process conditions are stirring at 2000-5000 rpm for 30 minutes, and the filtration is carried out using a double-layer 60-80 mesh nylon filter bag; the volume ratio of jellyfish protein to the mixed salt solution of disodium hydrogen phosphate and sodium dihydrogen phosphate is 1: (3-6). More preferably, the volume ratio of the jellyfish protein to the mixed salt solution of disodium hydrogen phosphate and sodium dihydrogen phosphate is 1:(4-5); the stirring process conditions are stirring at 35-50 rpm for 2 days, and the stirring temperature is 8-10°C.

[0043] As one embodiment, the enzymatic hydrolysis treatment is: using 0.05mol / L citric acid solution or 0.2mol / L acetic acid solution containing 1.5% pepsin to enzymatically hydrolyze the jellyfish protein solid obtained by alkalinization treatment, stirring, centrifuging and extracting the supernatant. Preferably, in the enzymatic hydrolysis treatment, the volume ratio of protein solids to citric acid or acetic acid is 1: (2-3), the stirring process conditions are stirring at 30-60rpm for 12-32h, the stirring temperature is 20-33°C, and the centrifugal treatment process conditions are 4-28°C, 13000g centrifugation for 20-30min. Further preferably, in the enzymatic hydrolysis treatment, the stirring process conditions are stirring at 40-50rpm for 18-24h, the stirring temperature is 28-30°C, and the centrifugal treatment process conditions are 20-25°C, 13000g centrifugation for 22-38min.

[0044] As one embodiment, the salting-out treatment is: adding sodium chloride to the supernatant and stirring, centrifuging, collecting the precipitate, and re-dissolving with purified water to obtain a reconstituted solution. Preferably, in the salting-out treatment, sodium chloride is added to the supernatant to a final concentration of 2.0 mol / L, the stirring process conditions are stirring at 30-60 rpm for 8-12 hours, and the process conditions for the centrifugal treatment are centrifugation at 13000g for 20-30 minutes at 4-28°C. Further preferably, in the salting-out treatment, the process conditions for the centrifugal treatment are centrifugation at 13000g for 22-28 minutes at 20-25°C.

[0045] As one embodiment, the secondary purification treatment is as follows: (1) First purification: the reconstituted solution obtained after the salting-out treatment is placed in a 50-100KD dialysis bag and dialyzed for 2-3 days using a 0.02 mol / L sulfate buffer solution as an external liquid. The external liquid is replaced every 8 hours during the dialysis process. After the dialysis is completed, the material in the bag is collected as a primary purified liquid;

[0046] (2) Second purification: The purified liquid is dialyzed for 2 to 3 days with purified water as the external liquid. The external liquid is replaced every 3 to 4 hours during the dialysis process. After the dialysis is completed, the material in the collection bag is the second purified liquid, which is packaged and stored. Preferably, in the purification process, (1) First purification: The reconstituted solution is placed in a dialysis bag of 80 to 100 KD and dialyzed. The dialysis temperature is 15 to 25 ° C, and the volume ratio of the reconstituted solution in the bag to the sulfate buffer solution is 1: (5 to 15); (2) Second purification: The dialysis temperature is 15 to 25 ° C, the volume ratio of the purified liquid in the bag to the purified water is 1: (5 to 10), and the temperature of the package storage is -20 ° C to 25 ° C. More preferably, in the purification process, the volume ratio of the reconstituted solution in the first purification dialysis bag to the sulfate buffer solution is 1: (8 to 10).

[0047] As one embodiment, the secondary purified solution is mixed with the protective solution and then placed in a vacuum freeze dryer for low-temperature drying.

[0048] A composition capable of regulating skin microecology, comprising the following components by weight:

[0049] The jellyfish collagen active protein prepared by any of the above-mentioned methods comprises 0.1-20 parts of jellyfish collagen active protein, 0.1-20 parts of Propionibacterium acnes bacteriophage, and 0.1-20 parts of the first thickener.

[0050] As one embodiment, the first thickener is one or a combination of two or more of starch, gum arabic, pectin, agar, gelatin, alginate, carrageenan or dextrin.

[0051] A film patch capable of improving skin inflammation comprises the following components in parts by weight:

[0052] The jellyfish collagen active protein prepared by any of the above-mentioned methods comprises 0.05-0.35 parts of jellyfish collagen active protein, 0.1-10 parts of Propionibacterium acnes bacteriophage, 0.01-0.5 parts of moisturizer, 0.1-20 parts of second thickener and 0.05-5 parts of dispersant.

[0053] As one embodiment, the moisturizing agent is sodium hyaluronate; the second thickener is one or a combination of two or more of starch, gum arabic, pectin, agar, gelatin, alginate, carrageenan, dextrin, carbomer, ethyl cellulose, hydroxypropyl methylcellulose or polyvinyl alcohol; the dispersant is one or a combination of two or more of hexylene glycol, propylene glycol, caprylyl glycol or glycerol.

[0054] The present invention is further described below in conjunction with specific implementation methods;

[0055] Example 1

[0056] The preparation method of jellyfish collagen active protein comprises the following steps:

[0057] Desalination: rinse the jellyfish's mouth arm and umbrella with purified water, then soak it in purified water. The mass ratio of purified water to jellyfish is 1:5. Soak it at 4°C for 7 days, changing the purified water every 2 hours to obtain the desalinated jellyfish.

[0058] Beating: Filter the desalted jellyfish, pre-freeze at -80°C, mix the pre-frozen jellyfish with liquid nitrogen at a mass volume ratio of 1kg:2L, mix for 5 minutes, take out the jellyfish and crush them to obtain freeze-dried powder;

[0059] Degreasing: add 5 volumes of acetone to the freeze-dried powder, soak at 15°C for 24 hours, centrifuge at 2000 rpm for 30 minutes to obtain the extracted lower protein solution, rinse with 5 volumes of purified water for 5 times, filter with a 60-mesh nylon filter bag to remove moisture and obtain the drained jellyfish protein;

[0060] Alkalization: add 3 times the volume of a mixed salt solution of disodium hydrogen phosphate and sodium dihydrogen phosphate to the drained jellyfish protein to a final concentration of 0.02 mol / L, stir at 4°C and 25 rpm for 3 days, centrifuge at 5000 rpm for 10 min to obtain the lower precipitate, rinse with purified water 5 times until the pH of the protein mixture is neutral, filter with a 60-mesh nylon filter bag to remove water to obtain the jellyfish protein solid;

[0061] Enzymatic hydrolysis: add 0.05 mol / L citric acid solution containing 1.5% pepsin to the drained jellyfish protein solids, with the volume ratio of protein solids to citric acid being 1:2, stir at 20°C, 60 rpm for 12 h, then centrifuge at 4°C, 13000g for 20 min, and extract the supernatant;

[0062] Salting out: add sodium chloride to the supernatant to a final concentration of 2.0 mol / L, stir at 30 rpm for 12 h, centrifuge at 28 °C, 13000 g for 30 min, collect the precipitate, and re-dissolve it with purified water to obtain a reconstituted solution;

[0063] Purification: (1) First purification: put the reconstituted solution into a 50KD dialysis bag and dialyze at 15°C using 0.02 mol / L sulfate buffer solution as the external liquid for 2 days. The volume ratio of the reconstituted solution in the bag to the sulfate buffer solution is 1:5. The external liquid is replaced every 8 hours during the dialysis process. After the dialysis is completed, the material in the bag is collected as the first purified solution.

[0064] (2) Second purification: The first purified solution was dialyzed with purified water as the external liquid at 25°C for 3 days. The volume ratio of the first purified solution to purified water in the bag was 1:10. The external liquid was replaced every 3 hours during the dialysis process. After the dialysis was completed, the material in the bag was collected as the second purified solution, which was packaged and stored at -20°C.

[0065] Drying: placing the secondary purified solution and the protective solution in a vacuum freeze dryer for low-temperature drying, the volume ratio of the secondary purified solution to the protective solution being 1:0.20, to obtain jellyfish collagen active protein.

[0066] Example 2

[0067] The preparation method of jellyfish collagen active protein comprises the following steps:

[0068] Desalination: rinse the jellyfish's mouth arm and umbrella with purified water, then soak it with purified water. The mass ratio of purified water to jellyfish is 1:10. Soak it at 15°C for 2 days. Change the purified water every 6 hours to obtain the desalinated jellyfish.

[0069] Beating: Filter the desalted jellyfish, pre-freeze at 0°C, mix the pre-frozen jellyfish with liquid nitrogen at a mass volume ratio of 1kg:10L, mix for 15 minutes, take out the jellyfish and crush them to obtain freeze-dried powder;

[0070] Degreasing: add 10 times the volume of acetone to the freeze-dried powder, soak at 25°C for 8 hours, centrifuge at 5000rpm for 10 minutes to obtain the extracted lower protein solution, rinse with 10 times the volume of purified water for 3 times, filter with an 80-mesh nylon filter bag to remove moisture and obtain the drained jellyfish protein;

[0071] Alkalization: add 6 times the volume of a mixed salt solution of disodium hydrogen phosphate and sodium dihydrogen phosphate to the drained jellyfish protein to a final concentration of 0.2 mol / L, stir at 25°C and 60 rpm for 1 day, centrifuge at 2000 rpm for 30 min to obtain the lower precipitate, rinse with purified water 8 times until the pH of the protein mixture is neutral, filter with an 80-mesh nylon filter bag to remove water to obtain the jellyfish protein solid;

[0072] Enzymatic hydrolysis: add 0.2 mol / L glacial acetic acid solution containing 1.5% pepsin to the drained jellyfish protein solids, the volume ratio of protein solids to glacial acetic acid is 1:3, stir at 33°C, 30 rpm for 32 h, then centrifuge at 28°C, 13000g for 30 min, and extract the supernatant;

[0073] Salting out: add sodium chloride to the supernatant to a final concentration of 2.0 mol / L, stir at 60 rpm for 8 h, centrifuge at 4 °C, 13000 g for 20 min, collect the precipitate, and re-dissolve it with purified water to obtain a reconstituted solution;

[0074] Purification: (1) First purification: The reconstituted solution was placed in a 100KD dialysis bag and dialyzed at 25°C using a 0.02 mol / L sulfate buffer solution as the external liquid for 3 days. The volume ratio of the reconstituted solution in the bag to the sulfate buffer solution was 1:15. The external liquid was replaced every 8 hours during the dialysis process. After the dialysis was completed, the material in the bag was collected as the first purified solution.

[0075] (2) Second purification: The first purified solution was dialyzed with purified water as the external liquid at 15°C for 2 days. The volume ratio of the first purified solution to purified water in the bag was 1:5. The external liquid was replaced every 4 hours during the dialysis process. At the end of the dialysis, the material in the bag was collected as the second purified solution, which was packaged and stored at 25°C.

[0076] Drying: placing the secondary purified solution and the protective solution in a vacuum freeze dryer for low-temperature drying, the volume ratio of the secondary purified solution to the protective solution being 1:0.01, to obtain jellyfish collagen active protein.

[0077] Example 3

[0078] The method for preparing jellyfish collagen active protein comprises the following steps:

[0079] Desalination: rinse the jellyfish's mouth arm and umbrella with purified water, then soak it in purified water. The mass ratio of purified water to jellyfish is 1:8. Soak it at 10℃ for 4 days, changing the purified water every 4 hours to obtain the desalted jellyfish.

[0080] Beating: Filter the desalted jellyfish, pre-freeze at -20°C, mix the pre-frozen jellyfish with liquid nitrogen at a mass volume ratio of 1kg:6L, mix for 10 minutes, take out the jellyfish and crush them to obtain freeze-dried powder;

[0081] Degreasing: add 7 volumes of acetone to the freeze-dried powder, soak at 20°C for 16 hours, centrifuge at 3500rpm for 20 minutes to obtain the extracted lower protein solution, rinse with 7 volumes of purified water for 4 times, filter with a 70-mesh nylon filter bag to remove moisture and obtain the drained jellyfish protein;

[0082] Alkalization: add 4 times the volume of a mixed salt solution of disodium hydrogen phosphate and sodium dihydrogen phosphate to the drained jellyfish protein to a final concentration of 0.1 mol / L, stir at 9°C and 40 rpm for 2 days, centrifuge at 3500 rpm for 20 min to obtain the lower layer of precipitate, rinse with purified water 7 times until the pH of the protein mixture is neutral, filter with a 70-mesh nylon filter bag to remove water to obtain the jellyfish protein solid;

[0083] Enzymatic hydrolysis: add 0.05 mol / L citric acid solution containing 1.5% pepsin to the drained jellyfish protein solids, with the volume ratio of protein solids to citric acid being 1:3, stir at 29°C, 45 rpm for 21 h, then centrifuge at 22°C, 13000g for 25 min, and extract the supernatant;

[0084] Salting out: add sodium chloride to the supernatant to a final concentration of 2.0 mol / L, stir at 45 rpm for 10 h, centrifuge at 22 °C, 13000 g for 25 min, collect the precipitate, and re-dissolve it with purified water to obtain a reconstituted solution;

[0085] Purification: (1) First purification: The reconstitution solution was placed in an 80KD dialysis bag and dialyzed at 20°C using 0.02 mol / L sulfate buffer solution as the external liquid for 2 days. The volume ratio of the reconstitution solution in the bag to the sulfate buffer solution was 1:10. The external liquid was replaced every 8 hours during the dialysis process. After the dialysis was completed, the material in the bag was collected as the first purified solution.

[0086] (2) Second purification: The first purified solution was dialyzed with purified water as the external liquid at 20°C for 3 days. The volume ratio of the first purified solution to purified water in the bag was 1:8. The external liquid was replaced every 3 hours during the dialysis process. After the dialysis was completed, the material in the bag was collected as the second purified solution, which was packaged and stored at 0°C.

[0087] Drying: placing the secondary purified solution and the protective solution in a vacuum freeze dryer for low-temperature drying, the volume ratio of the secondary purified solution to the protective solution being 1:0.10, to obtain jellyfish collagen active protein.

[0088] Examples 1-3 are jellyfish collagen active proteins prepared by the preparation method of jellyfish collagen active protein of the present invention. Examples 1-3 can all maintain a triple helix structure and will not cause animal-borne diseases and pathogen transmission risks; at the same time, the jellyfish collagen active proteins of Examples 1-3 have good water solubility and can be used in products for improving and controlling skin inflammation, and can also be used in products for improving skin rejuvenation and correcting wrinkles and filling.

[0089] Table 1 Raw material composition of a composition capable of regulating skin microecology

[0090]

[0091]

[0092] As can be seen from Table 1, Table 1 is the raw material composition of the composition capable of regulating skin microecology in the present invention. The compositions capable of regulating skin microecology in Examples 4-6 do not contain any chemical preservatives. The jellyfish collagen active protein can form a film to play a sealing role, thereby enhancing the effect of bacteriophage on Propionibacterium. At the same time, the jellyfish collagen active protein can also adsorb and clean the residual substances after the bacteria are removed, thereby strengthening the cleaning of hair follicles, regulating the secretion of sebaceous glands, improving the level of local healthy microenvironment, and maintaining the balance of skin microecology.

[0093] Table 2 Raw material composition of a film patch that can improve skin inflammation

[0094]

[0095] Table 3 Raw material composition of different comparative examples

[0096]

[0097] Effectiveness Evaluation

[0098] The patch for improving skin inflammation of Example 8 was used to treat subjects with skin inflammation. The frequency of use was 2-3 times a week, 15-20 minutes each time, for 3 consecutive weeks. No other care products were used during this period. The test results are as follows: Figure 1 shown.

[0099] Antibacterial performance evaluation

[0100] The antibacterial performance of Example 8 and Comparative Examples 1-3 was evaluated, and the changes in the number of Escherichia coli, Staphylococcus aureus and Propionibacterium acnes in Example 8 and Comparative Examples 1-3 within 0-36 hours were observed.

[0101] Table 4 Changes in the number of Escherichia coli in Comparative Examples 1-3 and Example 8

[0102]

[0103] Table 5 Changes in the number of Staphylococcus aureus in Comparative Examples 1-3 and Example 8

[0104]

[0105] Table 6 Changes in the number of Propionibacterium acnes in Comparative Examples 1-3 and Example 8

[0106]

[0107]

[0108] from Figure 1 It can be seen that Figure 1 The figure is a before-and-after comparison of the skin inflammation test subject treated with the skin inflammation patch of Example 8. After the test subject was treated with the skin inflammation patch of Example 8, the number of rashes was significantly reduced, the inflammation was significantly alleviated, and there was no erythema. In addition, it can be seen from Table 2 that Table 2 is the raw material composition of the skin inflammation patch of the present invention. The skin inflammation patches of Examples 7-10 do not have any chemical preservatives added, which avoids the instability of jellyfish active collagen due to the precipitation of preservatives and reduces the incidence of allergic reactions. In addition, Table 4-6 shows the changes in the number of Escherichia coli, Staphylococcus aureus and Propionibacterium acnes in Example 8 and Comparative Examples 1-3 within 0-36h. It can be seen from Table 4-6 that the number of Escherichia coli, Staphylococcus aureus and Propionibacterium acnes in Example 8 is much smaller than that in Comparative Examples 1-3. The addition of bacteriophage in Example 8 can directly and specifically remove Propionibacterium acnes, and can have a good inhibitory and killing effect on Propionibacterium acnes and Staphylococcus epidermidis, and has a good inhibitory effect on the inflammatory reaction caused by Propionibacterium acnes and the secretion of inflammatory factors. At the same time, the active protein of jellyfish collagen can form a film to play a sealing role, enhance the effect of bacteriophage on Propionibacterium, and improve and control skin inflammation.

[0109] For those skilled in the art, various other corresponding changes and deformations can be made according to the technical solutions and concepts described above, and all of these changes and deformations should fall within the protection scope of the claims of the present invention.

Claims

1. A raw material composition for preparing a film patch for treating skin inflammation, characterized in that: The composition is composed of the following components in parts by weight: 0.1 parts of jellyfish collagen active protein, 2 parts of Propionibacterium acnes bacteriophage, 0.2 parts of sodium hyaluronate, 0.5 parts of starch, 1 part of gum arabic, 1 part of ethyl cellulose, 2.5 parts of polyvinyl alcohol, 1 part of propylene glycol, and 1 part of glycerol; The jellyfish collagen active protein is prepared by the following preparation method: The desalted jellyfish is pulped to obtain freeze-dried powder. The pulping process is to filter the desalted jellyfish, then pre-freeze, mix with liquid nitrogen and cool after pre-freezing, and then crush to obtain freeze-dried powder; then the freeze-dried powder is sequentially defatted, alkalized, enzymatically hydrolyzed, and salted out, and purified twice to obtain a secondary purified solution, and then the secondary purified solution is mixed with a protective solution and low-temperature dried to obtain jellyfish collagen active protein; The temperature of the pre-freezing treatment in the pulping process is -80 to 0°C, the mass volume ratio of jellyfish to liquid nitrogen is 1 kg: (2 to 10) L, and the mixing time is 5 to 15 minutes.

2. The raw material composition according to claim 1, characterized in that The protective solution is one or a combination of two or more of L-ascorbic acid, glucose, mannitol, trehalose, polyethylene glycol and bovine serum albumin; the volume ratio of the secondary purification solution to the protective solution is 1: (0.01-0.20).

3. The raw material composition according to claim 1, characterized in that The defatting treatment comprises: adding an organic solvent to the freeze-dried powder, soaking, centrifuging to obtain a lower protein solution after extraction, washing the lower protein solution with purified water for 3 to 5 times, filtering, removing water to obtain jellyfish protein; the organic solvent is one or a combination of two or more of acetone, butanone or chloroform.

4. The raw material composition according to claim 1, characterized in that The alkalization treatment is as follows: adding a mixed salt solution of disodium hydrogen phosphate and sodium dihydrogen phosphate to the defatted jellyfish protein to a final concentration of 0.02 to 0.2 mol / L, stirring for 1 to 3 days, centrifuging to obtain a lower precipitate, washing the lower precipitate with purified water for 5 to 8 times until the pH of the washing solution is neutral, filtering, and removing water to obtain a jellyfish protein solid.

5. The raw material composition according to claim 1, characterized in that The enzymatic hydrolysis treatment is as follows: the jellyfish protein solid obtained by alkalinization treatment is enzymatically hydrolyzed using a 0.05 mol / L citric acid solution or a 0.2 mol / L glacial acetic acid solution containing 1.5% pepsin, followed by stirring and centrifugation to extract the supernatant.

6. The raw material composition according to claim 1, characterized in that The two purification processes are as follows: (1) First purification: the reconstituted solution obtained after the salting-out treatment is placed in a 50-100KD dialysis bag and dialyzed for 2-3 days using a 0.02 mol / L sulfate buffer solution as the external liquid. The external liquid is replaced every 8 hours during the dialysis process. After the dialysis is completed, the material in the bag is collected as the first purified solution; (2) Second purification: The first purified liquid is dialyzed with purified water as the external liquid for 2 to 3 days. The external liquid is replaced every 3 to 4 hours during the dialysis process. At the end of the dialysis, the material in the collection bag is the second purified liquid, which is packaged and stored.

Images