Mussel mucoprotein water and preparation process thereof

Abstract

The invention discloses mussel mucoprotein water and a preparation process thereof, and the mussel mucoprotein water comprises the following components in parts by mass: 5-12 parts of mussel mucoprotein; 3 to 8 parts of a hydroxylated chitosan-polyethylene glycol grafted copolymer; 4 to 9 parts of modified sodium alginate; 2 to 6 parts of beta-cyclodextrin embedded theabrownin; 1 to 4 parts of nano hydroxyapatite; 1-3 parts of glycerol; 60 to 85 parts of deionized water; through the synergistic effect of the four innovative components and the mussel mucoprotein, the dressing has excellent biological adhesion, mechanical properties, antibacterial and anti-inflammatory properties and healing promotion effects, and the problems that an existing dressing is single in function and insufficient in performance are solved; the adopted raw materials are natural high polymer materials or modified products with excellent biocompatibility, and after purification treatment, the impurity content is low, the biological safety is high, and no obvious toxic or side effect exists; the prepared hydrogel dressing is good in moisture retention and air permeability, can meet the healing requirements of various acute wounds and chronic wounds, and has wide clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of medical dressing technology, specifically mussel adhesive protein water and its preparation process. Background Technology

[0002] Medical dressings are medical products used to cover and protect wounds and promote wound healing. Hydrogel dressings, in particular, are widely used in the treatment of various acute and chronic wounds due to their excellent moisturizing, biocompatibility, and breathability, providing a suitable microenvironment for wound healing. Mussel adhesive protein, a natural biomolecule extracted from mussel byssal threads, possesses excellent bioadhesion, biocompatibility, and cell proliferation-promoting properties. Its application in hydrogel dressings can significantly improve the dressing's wound adhesion and healing-promoting effects.

[0003] However, existing mussel adhesive protein hydrogel dressings still have many shortcomings: First, its mechanical properties are poor, making it prone to breakage and deformation during wound dressing and activity, which affects the stability and effectiveness of the dressing. Secondly, its antibacterial and anti-inflammatory properties are limited, making it difficult to effectively inhibit bacterial growth at the wound site, which can easily lead to wound infection and delay the healing process. Third, they have limited functions, mostly only possessing basic moisturizing and adhesion functions, which cannot meet the healing needs of complex wounds (such as chronic ulcers and infected wounds). Fourth, the biosafety of some of the ingredients used in dressings needs to be improved, or the preparation process is complex and the conditions are harsh, which is not conducive to industrial production.

[0004] Based on this, a mussel adhesive protein water and its preparation process were designed. Summary of the Invention

[0005] In view of the above situation and to overcome the shortcomings of the prior art, the present invention provides mussel adhesive protein water and its preparation process, which effectively solves the problems mentioned in the background.

[0006] To achieve the above objectives, the present invention provides the following technical solution: mussel adhesive protein water, comprising the following components in parts by weight: Mussel adhesive protein: 5-12 parts; Hydroxylated chitosan-polyethylene glycol graft copolymer: 3-8 parts; Modified sodium alginate: 4-9 parts; β-Cyclodextrin-encapsulated theabrownins: 2-6 parts; Nano-hydroxyapatite: 1-4 parts; Glycerin: 1-3 parts; Deionized water: 60-85 parts; The sum of the mass fractions of each component is 100 parts.

[0007] Preferably, the mussel agaric has a molecular weight of 100-200 kDa and a purity of ≥95%; the glycerol is medical-grade glycerol with a purity of ≥99.5%.

[0008] Preferably, the preparation method of the hydroxylated chitosan-polyethylene glycol graft copolymer is as follows: Step A1: Hydroxylation modification of chitosan. Take 5-10g of chitosan and place it in a three-necked flask. Add 100-150mL of acetic acid solution with a mass fraction of 5-8%. Stir and dissolve the chitosan solution for 30-45min at a temperature of 30-40℃ and a stirring speed of 120-180r / min to obtain a chitosan solution. Slowly add 10-20mL of hydrogen peroxide solution with a mass fraction of 10-15% to the chitosan solution. After the addition is complete, raise the temperature to 50-60℃ and stir for 2-3h. After the reaction is complete, adjust the pH of the system to 7.0-7.5 with sodium hydroxide solution with a mass fraction of 10-15%. Let the precipitate stand for 1-2h, filter and collect the precipitate. Wash the precipitate with deionized water until the washing solution is neutral. Then place the precipitate in a vacuum drying oven and dry it for 4-6h at a temperature of 45-55℃ and a vacuum degree of 0.07-0.09MPa to obtain hydroxylated chitosan. Step A2: Grafting reaction to prepare the precursor. Take 3-6g of the hydroxylated chitosan obtained in step A1 and place it in a three-necked flask. Add 80-120mL of N,N-dimethylformamide and stir to dissolve for 40-60min at a temperature of 60-70℃ and a stirring speed of 150-200r / min. Then add 0.5-1.2g of the catalyst 4-dimethylaminopyridine and continue stirring for 10-20min. Slowly add 5-10g of polyethylene glycol to the system, raise the temperature to 80-90℃, and keep the temperature and stir for 6-8h to obtain the grafting reaction solution. Step A3: Purification treatment. Cool the grafting reaction solution obtained in step A2 to room temperature, pour it into 200-300 mL of anhydrous ethanol, let it stand for 2-3 hours to precipitate, and filter to collect the precipitate. Redissolve the precipitate in 50-80 mL of deionized water and dialyze it with a dialysis bag with a molecular weight cutoff of 3000-5000 Da for 48-72 hours, changing the deionized water every 12 hours during the process. After dialysis, place the dialysate in a vacuum drying oven and dry it for 5-7 hours at a temperature of 40-50℃ and a vacuum degree of 0.06-0.08 MPa to obtain hydroxylated chitosan-polyethylene glycol graft copolymer.

[0009] Preferably, the modified sodium alginate is prepared as follows: Step B1: Sodium alginate pretreatment. Place 4-8g of sodium alginate in a beaker, add 100-150mL of deionized water, and stir to dissolve for 30-45min at a temperature of 40-50℃ and a stirring speed of 100-150r / min to obtain a sodium alginate solution. Add 0.5-1.0g of activated carbon to the sodium alginate solution, stir to adsorb for 20-30min, filter to remove the activated carbon, and obtain a purified sodium alginate solution. Step B2: Esterification modification reaction. Transfer the purified sodium alginate solution obtained in Step B1 to a three-necked flask, heat to 50-60℃, adjust the stirring speed to 150-200 r / min, and slowly add 8-15 mL of 15-20% citric acid solution. After the addition is complete, continue to keep the temperature and stir for 3-5 h. During the reaction, slowly introduce nitrogen gas into the system for 5 min every 1 h, at a nitrogen gas introduction rate of 20-30 mL / min. Step B3: Neutralization and washing. After the reaction is complete, cool to room temperature, adjust the pH of the system to 6.5-7.0 with a 5-10% sodium carbonate solution, let stand for 1-2 hours, filter and collect the precipitate; wash the precipitate repeatedly with deionized water 3-5 times until no citrate ions are detected in the washing solution. Step B4: Drying and shaping. Place the washed precipitate in a vacuum drying oven and dry it for 6-8 hours at a temperature of 50-60℃ and a vacuum degree of 0.07-0.09MPa. After pulverizing, pass it through an 80-100 mesh sieve to obtain modified sodium alginate.

[0010] Preferably, the method for preparing the β-cyclodextrin-encapsulated theabrownin is as follows: Step C1: Raw material pretreatment. Take 6-12g of β-cyclodextrin and place it in a beaker. Add 100-150mL of deionized water and stir to dissolve for 20-30min at a temperature of 50-60℃ and a stirring speed of 120-180r / min to obtain a β-cyclodextrin solution. Take 2-5g of theabrownin and add 30-50mL of anhydrous ethanol. Dissolve by sonication for 10-15min at an ultrasonic power of 60-100W to obtain a theabrownin ethanol solution. Step C2: Encapsulation reaction. The theabrownin ethanol solution obtained in step C1 is slowly added dropwise to the β-cyclodextrin solution, with the dropping rate controlled at 1-2 mL / min. During the dropping process, the temperature is maintained at 50-60℃ and the stirring speed is 180-250 r / min. After the dropping is completed, the reaction is continued to be stirred and kept at the temperature for 2-4 hours. Then, the temperature is slowly lowered to 10-15℃ and kept at the temperature for 4-6 hours. Step C3: Separation and purification. Filter the reaction system obtained in step C2 and collect the precipitate. Wash the precipitate 2-3 times with anhydrous ethanol, using 20-30 mL each time, to remove unencapsulated theaflavins. Then wash the precipitate 1-2 times with deionized water to remove residual ethanol. Step C4: Dry the finished product. Place the washed precipitate in a vacuum drying oven and dry it for 4-6 hours at a temperature of 35-45℃ and a vacuum degree of 0.06-0.08MPa. After pulverizing, pass it through a 100-120 mesh sieve to obtain β-cyclodextrin-encapsulated tea brown pigment.

[0011] Preferably, the preparation method of the nano-hydroxyapatite is as follows: Step D1: Preparation of precursor solution: Take 50-80 mL of 0.5-1.5 mol / L calcium chloride solution and place it in a beaker. Add 2-5 mL of 3-5% polyethylene glycol 400 solution and stir for 10-15 min at 25-30℃ and 100-150 r / min. Separately, take 50-80 mL of 0.3-0.9 mol / L diammonium hydrogen phosphate solution and adjust the pH to 9.0-10.0 with 5-10% ammonia water to obtain diammonium hydrogen phosphate buffer solution. Step D2: Precipitation reaction. Slowly add diammonium hydrogen phosphate buffer solution to calcium chloride solution at a rate of 0.5-1 mL / min. Maintain the temperature at 30-40℃ and the stirring speed at 200-300 r / min during the addition. At the same time, use ammonia water to maintain the pH of the system at 9.0-10.0. After the addition is complete, continue stirring for 1-2 hours, and then let it stand for 8-12 hours. Step D3: Washing and drying. Filter the precipitate obtained in step D2, wash the precipitate repeatedly with deionized water until the pH of the washing solution is about 7.0, and then wash it once with anhydrous ethanol. Place the washed precipitate in a vacuum drying oven and dry it for 6-8 hours at a temperature of 60-80℃ and a vacuum degree of 0.07-0.09MPa. Step D4: Calcination and shaping. Place the dried precipitate in a muffle furnace and heat it to 600-800℃ at a heating rate of 5-10℃ / min. Calcinate at this temperature for 2-4 hours. After naturally cooling to room temperature, pulverize it through a 200-300 mesh sieve to obtain nano-hydroxyapatite.

[0012] The preparation process of mussel adhesive protein water includes the following steps: Step 1: Raw material pretreatment. Mussel adhesive protein, hydroxylated chitosan-polyethylene glycol graft copolymer, modified sodium alginate, β-cyclodextrin-encapsulated tea brown pigment, and nano-hydroxyapatite are placed in a vacuum drying oven and dried for 2-3 hours at a temperature of 40-50℃ and a vacuum degree of 0.06-0.09MPa for later use. Step 2: Preparation of aqueous system. Take the prescribed amount of deionized water and place it in a constant temperature stirred tank. Heat the tank to 35-45℃ and adjust the stirring speed to 150-250 r / min. First, add the prescribed amount of glycerol and stir to dissolve for 15-25 min. Then, add the dried hydroxylated chitosan-polyethylene glycol graft copolymer and modified sodium alginate in sequence. Continue stirring for 30-45 min to obtain a mixed aqueous phase. Step 3: Mussel adhesive protein dispersion. The dried mussel adhesive protein is slowly added to the mixed aqueous phase obtained in Step 2. The temperature is maintained at 35-45℃ and the stirring speed is 200-300r / min. The mixture is stirred and dispersed for 40-60min. During this period, ultrasonic dispersion is used for 5min every 10min. The ultrasonic power is 80-120W to obtain the protein dispersion. Step 4: Construction of the composite system. The dried β-cyclodextrin-encapsulated tea brown pigment and nano-hydroxyapatite were added to the protein dispersion obtained in Step 3. The temperature was adjusted to 45-55℃, the stirring speed was 250-350 r / min, and the reaction was carried out for 60-90 min to obtain the composite gel precursor. Step 5: Gel molding. Transfer the composite gel precursor obtained in step 4 into a mold. First, let it stand at 25-30℃ for 30-40 minutes, then place it in a low-temperature environment and refrigerate it at 0-5℃ for 2-4 hours. Then, raise the temperature to 20-25℃ and warm it for 1-2 hours to complete the gel molding. Step 6: Post-processing. Remove the molded gel from the mold and place it in a sterile environment. Vacuum dry it for 3-5 hours at a temperature of 30-40℃ and a vacuum degree of 0.05-0.08MPa, controlling the gel water content to 65-80%. Then, aseptically cut and package it to obtain the product, mussel adhesive protein water.

[0013] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention introduces a hydroxylated chitosan-polyethylene glycol graft copolymer, which combines the antibacterial properties of chitosan with the hydrophilicity of polyethylene glycol, thus improving the biocompatibility and mechanical properties of the dressing. Modified sodium alginate, through esterification modification, enhances its water resistance and mechanical strength, and synergistically works with mussel adhesive protein to improve the cross-linking stability of the gel. β-cyclodextrin encapsulates theabrownin, enabling slow release of theabrownin and providing long-lasting antibacterial and anti-inflammatory effects, avoiding the problem of easy oxidation and inactivation when theabrownin is used directly. Nano-hydroxyapatite enhances the mechanical strength of the dressing and also possesses certain osteoconductive properties, making it suitable for wound healing with bone injuries. The synergistic effect of these four innovative components with mussel adhesive protein gives the dressing excellent bioadhesion, mechanical properties, antibacterial and anti-inflammatory properties, and healing-promoting effects, solving the problems of single function and insufficient performance of existing dressings.

[0014] 2. The raw materials used in this invention are all natural polymer materials or modified products with excellent biocompatibility. After purification, the impurity content is low, the biocompatibility is high, and there are no obvious toxic side effects. The prepared hydrogel dressing has good moisturizing and breathability, and can meet the healing needs of various acute wounds (such as trauma and burns) and chronic wounds (such as diabetic ulcers and pressure sores). It has broad clinical application prospects, high biocompatibility, and wide applicability. Detailed Implementation

[0015] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0016] This invention provides mussel adhesive protein water, comprising the following components in parts by weight: Mussel adhesive protein: 5-12 parts; Hydroxylated chitosan-polyethylene glycol graft copolymer: 3-8 parts; Modified sodium alginate: 4-9 parts; β-Cyclodextrin-encapsulated theabrownins: 2-6 parts; Nano-hydroxyapatite: 1-4 parts; Glycerin: 1-3 parts; Deionized water: 60-85 parts; The sum of the mass fractions of each component is 100 parts.

[0017] The mussel adhesive protein in this embodiment has a molecular weight of 100-200 kDa and a purity of ≥95%; the glycerol is medical grade glycerol with a purity of ≥99.5%.

[0018] The preparation method of the hydroxylated chitosan-polyethylene glycol graft copolymer in this embodiment is as follows: Step A1: Chitosan hydroxylation modification. Place 5-10g of chitosan in a three-necked flask and add 100-150mL of 5-8% acetic acid solution. Stir and dissolve for 30-45 minutes at 30-40℃ and 120-180r / min to obtain a chitosan solution. Slowly add 10-20mL of 10-15% hydrogen peroxide solution to the chitosan solution. After the addition is complete, raise the temperature to 50-60℃ and maintain the temperature with stirring for 2-3 hours. After the reaction is complete, dissolve the chitosan in 10-15% sodium hydroxide solution. Adjust the pH of the liquid system to 7.0-7.5, let it stand for 1-2 hours to precipitate, filter and collect the precipitate, wash the precipitate with deionized water until the washing solution is neutral, and then place the precipitate in a vacuum drying oven and dry it for 4-6 hours at a temperature of 45-55℃ and a vacuum degree of 0.07-0.09MPa to obtain hydroxylated chitosan. After hydroxylation modification, the water solubility and reactivity of chitosan can be improved, creating conditions for subsequent grafting reaction with polyethylene glycol. Hydrogen peroxide, as an oxidant, can introduce hydroxyl groups into the chitosan molecular chain. The control of reaction temperature and time can ensure that the degree of hydroxylation is appropriate. Step A2: Preparation of precursor by grafting reaction. Take 3-6g of hydroxylated chitosan obtained in step A1 and place it in a three-necked flask. Add 80-120mL of N,N-dimethylformamide and stir to dissolve for 40-60min at a temperature of 60-70℃ and a stirring speed of 150-200r / min. Then add 0.5-1.2g of 4-dimethylaminopyridine catalyst and continue stirring for 10-20min. Slowly add 5-10g of polyethylene glycol to the system, raise the temperature to 80-90℃, and stir for 6-8h to obtain the grafting reaction solution. N,N-dimethylformamide is an excellent solvent that can ensure the full dissolution of hydroxylated chitosan. 4-dimethylaminopyridine, as a catalyst, can significantly improve the efficiency of the grafting reaction. Controlling the reaction temperature and time can ensure that polyethylene glycol is successfully grafted onto the hydroxylated chitosan molecular chain to form a graft copolymer. Step A3: Purification treatment. Cool the grafting reaction solution obtained in step A2 to room temperature, pour it into 200-300 mL of anhydrous ethanol, let it stand for 2-3 hours to precipitate, and filter to collect the precipitate. Redissolve the precipitate in 50-80 mL of deionized water and dialyze it using a dialysis bag with a molecular weight cutoff of 3000-5000 Da for 48-72 hours, changing the deionized water every 12 hours during this period. After dialysis, place the dialysate in a vacuum drying oven and dry it for 5-7 hours at a temperature of 40-50℃ and a vacuum degree of 0.06-0.08 MPa to obtain hydroxylated chitosan-polyethylene glycol graft copolymer. Anhydrous ethanol can precipitate the graft copolymer, removing unreacted monomers and impurities. Dialysis treatment can further purify the product, remove small molecule impurities, and ensure the purity and biosafety of the product.

[0019] The preparation method of the modified sodium alginate in this embodiment is as follows: Step B1: Sodium alginate pretreatment. Place 4-8g of sodium alginate in a beaker, add 100-150mL of deionized water, and stir to dissolve for 30-45min at 40-50℃ and 100-150r / min to obtain a sodium alginate solution. Add 0.5-1.0g of activated carbon to the sodium alginate solution, stir to adsorb for 20-30min, and filter to remove the activated carbon to obtain a purified sodium alginate solution. Activated carbon adsorption can remove pigments and impurities from sodium alginate, improve the purity of the raw material, and avoid affecting subsequent modification reactions and product performance. Step B2: Esterification modification reaction. Transfer the purified sodium alginate solution obtained in Step B1 to a three-necked flask, heat to 50-60℃, adjust the stirring speed to 150-200 r / min, and slowly add 8-15 mL of 15-20% citric acid solution. After the addition is complete, continue to keep the temperature and stir for 3-5 hours. During the reaction, slowly introduce nitrogen gas into the system for 5 minutes every 1 hour, at a nitrogen introduction rate of 20-30 mL / min. Citric acid, as an esterifying agent, can undergo esterification with the hydroxyl groups on the sodium alginate molecular chain, introducing ester groups and improving the mechanical properties and water resistance of sodium alginate. Nitrogen protection can prevent the system from being oxidized during the reaction, ensuring the modification effect. Step B3: Neutralization and washing. After the reaction is complete, cool to room temperature and adjust the pH of the system to 6.5-7.0 with a 5-10% sodium carbonate solution. Let stand for 1-2 hours, filter and collect the precipitate. Wash the precipitate repeatedly with deionized water 3-5 times until no citrate ions are detected in the washing solution. The sodium carbonate solution can neutralize excess citric acid in the system, and the washing step can remove residual salts and unreacted citric acid, ensuring the purity of the product. Step B4: Drying and shaping. Place the washed precipitate in a vacuum drying oven and dry it for 6-8 hours at a temperature of 50-60℃ and a vacuum degree of 0.07-0.09MPa. After pulverizing, pass it through an 80-100 mesh sieve to obtain modified sodium alginate. Controlling the drying temperature and time can avoid high-temperature degradation of the product. Pulverizing and sieving can make the product particle size uniform, which is convenient for subsequent mixing with other components.

[0020] The preparation method of β-cyclodextrin-encapsulated theabrownin in this embodiment is as follows: Step C1: Raw material pretreatment. Take 6-12g of β-cyclodextrin and place it in a beaker. Add 100-150mL of deionized water and stir to dissolve for 20-30min at a temperature of 50-60℃ and a stirring speed of 120-180r / min to obtain a β-cyclodextrin solution. Take 2-5g of theabrownin and add 30-50mL of anhydrous ethanol. Dissolve by sonication for 10-15min at an ultrasonic power of 60-100W to obtain a theabrownin ethanol solution. β-cyclodextrin has a unique cavity structure that can encapsulate theabrownin, improving its stability and water solubility. Ultrasonic dissolution ensures that theabrownin is fully dissolved, facilitating subsequent encapsulation reactions. Step C2: Encapsulation reaction. The theabrownin ethanol solution obtained in step C1 is slowly added dropwise to the β-cyclodextrin solution at a rate of 1-2 mL / min. During the addition, the temperature is maintained at 50-60℃ and the stirring speed is 180-250 r / min. After the addition is complete, the reaction is continued at this temperature and stirred for 2-4 hours. Then, the temperature is slowly lowered to 10-15℃ and kept at this temperature for 4-6 hours. The slow addition and suitable temperature and stirring speed conditions ensure that theabrownin fully enters the cavity of β-cyclodextrin to form a stable inclusion complex. Low-temperature standing promotes the crystallization of the inclusion complex. Step C3: Separation and purification. Filter the reaction system obtained in step C2 and collect the precipitate. Wash the precipitate 2-3 times with anhydrous ethanol, using 20-30 mL each time, to remove unencapsulated theabrownins. Then wash the precipitate 1-2 times with deionized water to remove residual ethanol. Anhydrous ethanol can dissolve unencapsulated theabrownins, thereby achieving the separation of the inclusion complex from free theabrownins and improving the purity of the inclusion complex. Step C4: Dry the finished product. Place the washed precipitate in a vacuum drying oven and dry it for 4-6 hours at a temperature of 35-45℃ and a vacuum degree of 0.06-0.08MPa. After pulverizing, pass it through a 100-120 mesh sieve to obtain β-cyclodextrin-encapsulated theabrownin. The lower drying temperature can avoid the oxidation and inactivation of theabrownin, ensuring its antibacterial and anti-inflammatory activity. Pulverizing and sieving can make the product particle size uniform, which is convenient for subsequent dispersion in the gel system.

[0021] The preparation method of nano-hydroxyapatite in this embodiment is as follows: Step D1: Preparation of precursor solution. Take 50-80 mL of 0.5-1.5 mol / L calcium chloride solution and place it in a beaker. Add 2-5 mL of 3-5% polyethylene glycol 400 solution and stir for 10-15 min at 25-30℃ and 100-150 r / min. Separately, take 50-80 mL of 0.3-0.9 mol / L diammonium hydrogen phosphate solution and adjust the pH to 9.0-10.0 with 5-10% ammonia water to obtain diammonium hydrogen phosphate buffer. Polyethylene glycol 400 acts as a dispersant to prevent the agglomeration of hydroxyapatite particles generated in subsequent reactions. Ammonia water adjusts the pH to create a suitable reaction environment to ensure the smooth formation of hydroxyapatite. Step D2: Precipitation reaction. Diammonium hydrogen phosphate buffer solution is slowly added dropwise to calcium chloride solution at a rate of 0.5-1 mL / min. During the addition, the temperature is maintained at 30-40℃ and the stirring speed at 200-300 r / min. Ammonia water is used to maintain the pH of the system at 9.0-10.0. After the addition is complete, the reaction is stirred for 1-2 hours, followed by aging for 8-12 hours. Slow addition ensures uniform reaction and avoids excessively high local concentrations that could lead to overly large particles. Suitable temperature, stirring speed, and pH conditions promote the growth of hydroxyapatite crystals, while aging further refines the crystals and improves the crystallinity of the product. Step D3: Washing and drying. Filter the precipitate obtained in step D2, wash the precipitate repeatedly with deionized water until the pH of the washing solution is about 7.0, and then wash it once with anhydrous ethanol. Place the washed precipitate in a vacuum drying oven and dry it for 6-8 hours at a temperature of 60-80℃ and a vacuum of 0.07-0.09MPa. Washing with deionized water can remove residual salt impurities, and washing with anhydrous ethanol can reduce the surface tension of the precipitate, which is convenient for subsequent drying. Controlling the drying temperature and time can prevent product agglomeration. Step D4: Calcination and shaping. Place the dried precipitate in a muffle furnace and heat it to 600-800℃ at a heating rate of 5-10℃ / min. Calcinate at this temperature for 2-4 hours. After naturally cooling to room temperature, pulverize it through a 200-300 mesh sieve to obtain nano-hydroxyapatite. Appropriate heating rate, calcination temperature, and time can remove residual organic matter in the product, improve the crystallinity and mechanical properties of the product, and pulverize and sieve to obtain nano-sized products with uniform particle size, which is convenient for uniform dispersion in the gel system and enhances the mechanical strength and bone repair potential of the gel.

[0022] The preparation process of mussel adhesive protein water includes the following steps: Step 1: Raw material pretreatment. Mussel adhesive protein, hydroxylated chitosan-polyethylene glycol graft copolymer, modified sodium alginate, β-cyclodextrin-encapsulated tea brown pigment, and nano-hydroxyapatite are placed in a vacuum drying oven and dried for 2-3 hours at a temperature of 40-50℃ and a vacuum degree of 0.06-0.09MPa. This step removes moisture and impurities from the raw materials, avoiding adverse effects on subsequent reactions and gelation properties. Step 2: Preparation of aqueous system. Take the prescribed amount of deionized water and place it in a constant temperature stirred tank. Heat the tank to 35-45℃ and adjust the stirring speed to 150-250 r / min. First, add the prescribed amount of glycerol and stir to dissolve for 15-25 min. Then, add the dried hydroxylated chitosan-polyethylene glycol graft copolymer and modified sodium alginate in sequence. Continue stirring for 30-45 min to obtain a mixed aqueous phase. Step 3: Mussel adhesive protein dispersion. The dried mussel adhesive protein is slowly added to the aqueous mixture obtained in Step 2. The temperature is maintained at 35-45℃ and the stirring speed is 200-300 r / min. The mixture is stirred and dispersed for 40-60 min, with ultrasonic-assisted dispersion for 5 min every 10 min during this period. The ultrasonic power is 80-120W. The protein dispersion is obtained by combining stirring and ultrasonic assistance. The combination of stirring and ultrasonic assistance can effectively prevent the mussel adhesive protein from agglomerating and ensure that it is evenly dispersed in the system, so as to give full play to its bioadhesion and healing promotion effects. Step 4: Construction of the composite system. The dried β-cyclodextrin-encapsulated theabrownin and nano-hydroxyapatite are added to the protein dispersion obtained in Step 3. The temperature is adjusted to 45-55℃ and the stirring speed is 250-350 r / min. The stirring reaction is carried out for 60-90 min to obtain the composite gel precursor. The temperature and stirring speed conditions can promote the interaction between the components and form a stable composite system. The β-cyclodextrin-encapsulated theabrownin can slowly release theabrownin and exert a long-lasting antibacterial and anti-inflammatory effect, while the nano-hydroxyapatite can enhance the mechanical properties and bone repair potential of the gel. Step 5: Gel molding. Transfer the composite gel precursor obtained in Step 4 into a mold. First, let it stand at 25-30℃ for 30-40 minutes, then place it in a low-temperature environment and refrigerate it at 0-5℃ for 2-4 hours. Then, raise the temperature to 20-25℃ and warm it for 1-2 hours to complete the gel molding. Through standing and low-temperature refrigeration, the gel system can be fully cross-linked and solidified to form a hydrogel with a uniform structure and good stability. The warming step can avoid the internal stress caused by the sudden temperature change of the gel and improve the integrity of the gel. Step 6: Post-processing. The molded gel is removed from the mold and placed in a sterile environment. It is then vacuum dried for 3-5 hours at a temperature of 30-40℃ and a vacuum degree of 0.05-0.08MPa, controlling the gel moisture content to 65-80%. Subsequently, it is aseptically cut and packaged to obtain the product, mussel adhesive protein water. Vacuum drying can remove some of the moisture from the gel at a lower temperature, controlling the moisture content within a suitable range. This ensures both the moisturizing properties of the dressing and improves its mechanical strength. Aseptic processing ensures that the dressing meets medical standards.

[0023] Example 1 A mussel adhesive protein solution, by weight, comprises the following components: Mussel adhesive protein: 8 parts; Hydroxylated chitosan-polyethylene glycol graft copolymer: 5 parts; Modified sodium alginate: 6 parts; β-Cyclodextrin-encapsulated theabrownin: 4 parts; Nano-hydroxyapatite: 2 parts; Glycerin: 2 parts; Deionized water: 73 parts.

[0024] A method for preparing hydroxylated chitosan-polyethylene glycol graft copolymer includes the following steps: Step A1: Hydroxylation modification of chitosan. Take 7g of chitosan and place it in a three-necked flask. Add 120mL of 6% acetic acid solution and stir to dissolve for 35min at 35℃ and 150r / min to obtain chitosan solution. Slowly add 15mL of 12% hydrogen peroxide solution to the chitosan solution. After the addition is complete, raise the temperature to 55℃ and stir for 2.5h. After the reaction is complete, adjust the pH of the system to 7.2 with 12% sodium hydroxide solution. Let it stand for 1.5h to precipitate. Filter and collect the precipitate. Wash the precipitate with deionized water until the washing solution is neutral. Then place the precipitate in a vacuum drying oven and dry for 5h at 50℃ and 0.08MPa to obtain hydroxylated chitosan. Step A2: Preparation of precursor by grafting reaction. Take 4g of hydroxylated chitosan and place it in a three-necked flask. Add 100mL of N,N-dimethylformamide and stir to dissolve for 50min at 65℃ and 180r / min. Then add 0.8g of catalyst 4-dimethylaminopyridine and continue stirring for 15min. Slowly add 7g of polyethylene glycol to the system, raise the temperature to 85℃, and keep it at this temperature for 7h with stirring to obtain the grafting reaction solution. Step A3: Purification treatment. Cool the grafting reaction solution to room temperature, pour it into 250 mL of anhydrous ethanol, let it stand for 2.5 h to precipitate, filter and collect the precipitate; redissolve the precipitate in 60 mL of deionized water, dialyze it with a dialysis bag with a molecular weight cutoff of 4000 Da for 60 h, and change the deionized water every 12 h during the process; after dialysis, place the dialysate in a vacuum drying oven and dry it for 6 h at a temperature of 45 °C and a vacuum degree of 0.07 MPa to obtain hydroxylated chitosan-polyethylene glycol graft copolymer.

[0025] The preparation method of modified sodium alginate includes the following steps: Step B1: Sodium alginate pretreatment. Take 6g of sodium alginate and place it in a beaker. Add 120mL of deionized water and stir to dissolve for 35min at 45℃ and 120r / min to obtain sodium alginate solution. Add 0.7g of activated carbon to the sodium alginate solution and stir to adsorb for 25min. Filter to remove the activated carbon to obtain purified sodium alginate solution. Step B2: Esterification modification reaction. The purified sodium alginate solution was transferred to a three-necked flask, heated to 55°C, and the stirring speed was adjusted to 180 r / min. 12 mL of 18% citric acid solution was slowly added dropwise. After the addition was completed, the reaction was continued to be stirred at the same temperature for 4 h. During the reaction, nitrogen gas was slowly introduced into the system for 5 min every 1 h, at a nitrogen gas introduction rate of 25 mL / min. Step B3: Neutralization and washing. After the reaction is complete, cool to room temperature, adjust the pH of the system to 6.8 with 8% sodium carbonate solution, let stand for 1.5 h, filter and collect the precipitate; wash the precipitate repeatedly with deionized water 4 times until no citrate ions are detected in the washing solution. Step B4: Drying and shaping. The washed precipitate is placed in a vacuum drying oven and dried for 7 hours at a temperature of 55℃ and a vacuum degree of 0.08MPa. After pulverizing, it is passed through a 90-mesh sieve to obtain modified sodium alginate.

[0026] A method for preparing β-cyclodextrin-encapsulated theabrownins includes the following steps: Step C1: Raw material pretreatment. Take 9g of β-cyclodextrin and place it in a beaker. Add 120mL of deionized water and stir to dissolve for 25min at 55℃ and 150r / min to obtain a β-cyclodextrin solution. Take 3g of theabrownin and add 40mL of anhydrous ethanol. Dissolve by sonication for 12min at 80W to obtain a theabrownin ethanol solution. Step C2: Encapsulation reaction. The tea brown ethanol solution was slowly added dropwise to the β-cyclodextrin solution at a rate of 1.5 mL / min. During the addition, the temperature was maintained at 55℃ and the stirring speed was 220 r / min. After the addition was completed, the reaction was continued to be stirred and kept warm for 3 hours. Then, the temperature was slowly lowered to 12℃ and kept warm and allowed to stand for 5 hours. Step C3: Separation and purification. Filter the reaction system and collect the precipitate. Wash the precipitate three times with anhydrous ethanol, using 25 mL each time, to remove unencapsulated theaflavins. Then wash the precipitate twice with deionized water to remove residual ethanol. Step C4: Dry the finished product. Place the washed precipitate in a vacuum drying oven and dry it for 5 hours at a temperature of 40℃ and a vacuum degree of 0.07MPa. After pulverizing, pass it through a 110-mesh sieve to obtain β-cyclodextrin-encapsulated tea brown pigment.

[0027] The preparation method of nano-hydroxyapatite includes the following steps: Step D1: Preparation of precursor solution: Take 60 mL of 1.0 mol / L calcium chloride solution and place it in a beaker. Add 3 mL of 4% polyethylene glycol 400 solution and stir for 12 min at 28℃ and 120 r / min. Separately, take 60 mL of 0.6 mol / L diammonium hydrogen phosphate solution and adjust the pH to 9.5 with 8% ammonia water to obtain diammonium hydrogen phosphate buffer solution. Step D2: Precipitation reaction. Diammonium hydrogen phosphate buffer solution was slowly added dropwise to calcium chloride solution at a rate of 0.8 mL / min. During the addition, the temperature was maintained at 35°C and the stirring speed was 250 r / min. At the same time, ammonia water was used to maintain the pH of the system at 9.5. After the addition was completed, the reaction was stirred for 1.5 h, and then allowed to stand for 10 h. Step D3: Washing and drying. Filter the precipitate and wash it repeatedly with deionized water until the pH of the washing solution is about 7.0. Then wash it once with anhydrous ethanol. Place the washed precipitate in a vacuum drying oven and dry it for 7 hours at a temperature of 70℃ and a vacuum of 0.08MPa. Step D4: Calcination and shaping. The dried precipitate is placed in a muffle furnace and heated to 700°C at a heating rate of 8°C / min. It is then calcined for 3 hours and allowed to cool naturally to room temperature. After pulverizing, it is passed through a 250-mesh sieve to obtain nano-hydroxyapatite.

[0028] The preparation process of the above-mentioned mussel adhesive protein water includes the following steps: Step 1: Raw material pretreatment. Mussel adhesive protein, hydroxylated chitosan-polyethylene glycol graft copolymer, modified sodium alginate, β-cyclodextrin-encapsulated tea brown pigment, and nano-hydroxyapatite were placed in a vacuum drying oven and dried for 2.5 hours at 45℃ and 0.08MPa for later use. Step 2: Preparation of aqueous system. Take 73 parts of deionized water and place it in a constant temperature stirred tank. Heat to 40℃ and adjust the stirring speed to 200r / min. First, add 2 parts of glycerol and stir to dissolve for 20min. Then, add 5 parts of dried hydroxylated chitosan-polyethylene glycol graft copolymer and 6 parts of modified sodium alginate in sequence. Continue stirring for 35min to obtain a mixed aqueous phase. Step 3: Mussel adhesive protein dispersion. Eight portions of dried mussel adhesive protein were slowly added to the mixed aqueous phase. The temperature was maintained at 40℃ and the stirring speed was 250r / min. The mixture was stirred and dispersed for 50min. During this period, ultrasonic dispersion was used for 5min every 10min. The ultrasonic power was 100W. The protein dispersion was obtained. Step 4: Construction of the composite system: 4 parts of dried β-cyclodextrin-encapsulated tea brown pigment and 2 parts of nano-hydroxyapatite were added to the protein dispersion, the temperature was adjusted to 50℃, the stirring speed was 300 r / min, and the reaction was stirred for 75 min to obtain the composite gel precursor. Step 5: Gel molding. Transfer the composite gel precursor into a mold, let it stand at 28°C for 35 minutes, then place it in a low-temperature environment and refrigerate it at 2°C for 3 hours. Then raise the temperature to 22°C and warm it for 1.5 hours to complete the gel molding. Step 6: Post-processing. The formed gel is removed from the mold and placed in a sterile environment. It is then vacuum dried for 4 hours at a temperature of 35°C and a vacuum degree of 0.06MPa to control the water content of the gel to 72%. Subsequently, it is aseptically cut and packaged to obtain mussel adhesive protein water.

[0029] Example 2 A mussel adhesive protein solution, by weight, comprises the following components: Mussel adhesive protein: 5 parts; Hydroxylated chitosan-polyethylene glycol graft copolymer: 3 parts; Modified sodium alginate: 4 parts; β-Cyclodextrin-encapsulated theabrownin: 2 parts; Nano-hydroxyapatite: 1 part; Glycerin: 1 part; Deionized water: 84 parts.

[0030] A method for preparing hydroxylated chitosan-polyethylene glycol graft copolymer includes the following steps: Step A1: Hydroxylation modification of chitosan. Take 5g of chitosan and place it in a three-necked flask. Add 100mL of 5% acetic acid solution and stir to dissolve for 30min at 30℃ and 120r / min to obtain chitosan solution. Slowly add 10mL of 10% hydrogen peroxide solution to the chitosan solution. After the addition is complete, raise the temperature to 50℃ and stir for 2h. After the reaction is complete, adjust the pH of the system to 7.0 with 10% sodium hydroxide solution. Let it stand for 1h to precipitate. Filter and collect the precipitate. Wash the precipitate with deionized water until the washing solution is neutral. Then place the precipitate in a vacuum drying oven and dry for 4h at 45℃ and 0.07MPa to obtain hydroxylated chitosan. Step A2: Preparation of precursor by grafting reaction. Take 3g of hydroxylated chitosan and place it in a three-necked flask. Add 80mL of N,N-dimethylformamide and stir to dissolve for 40min at 60℃ and 150r / min. Then add 0.5g of catalyst 4-dimethylaminopyridine and continue stirring for 10min. Slowly add 5g of polyethylene glycol to the system, heat to 80℃, and stir for 6h to obtain the grafting reaction solution. Step A3: Purification treatment. Cool the grafting reaction solution to room temperature, pour it into 200 mL of anhydrous ethanol, let it stand for 2 hours to precipitate, and filter to collect the precipitate. Redissolve the precipitate in 50 mL of deionized water and dialyze it with a dialysis bag with a molecular weight cutoff of 3000 Da for 48 hours, changing the deionized water every 12 hours during the process. After dialysis, place the dialysate in a vacuum drying oven and dry it for 5 hours at a temperature of 40℃ and a vacuum degree of 0.06 MPa to obtain hydroxylated chitosan-polyethylene glycol graft copolymer.

[0031] The preparation method of modified sodium alginate includes the following steps: Step B1: Sodium alginate pretreatment. Take 4g of sodium alginate and place it in a beaker. Add 100mL of deionized water and stir to dissolve for 30min at 40℃ and 100r / min to obtain sodium alginate solution. Add 0.5g of activated carbon to the sodium alginate solution and stir to adsorb for 20min. Filter to remove the activated carbon to obtain purified sodium alginate solution. Step B2: Esterification modification reaction. Transfer the purified sodium alginate solution to a three-necked flask, heat to 50°C, adjust the stirring speed to 150 r / min, and slowly add 8 mL of 15% citric acid solution. After the addition is complete, continue to keep the temperature and stir for 3 h. During the reaction, slowly introduce nitrogen gas into the system for 5 min every 1 h, at a nitrogen gas introduction rate of 20 mL / min. Step B3: Neutralization and washing. After the reaction is complete, cool to room temperature, adjust the pH of the system to 6.5 with a 5% sodium carbonate solution, let stand for 1 hour, filter and collect the precipitate; wash the precipitate repeatedly with deionized water 3 times until no citrate ions are detected in the washing solution. Step B4: Drying and shaping. The washed precipitate is placed in a vacuum drying oven and dried for 6 hours at a temperature of 50℃ and a vacuum degree of 0.07MPa. After pulverizing, it is passed through an 80-mesh sieve to obtain modified sodium alginate.

[0032] A method for preparing β-cyclodextrin-encapsulated theabrownins includes the following steps: Step C1: Raw material pretreatment. Take 6g of β-cyclodextrin and place it in a beaker. Add 100mL of deionized water and stir to dissolve for 20min at 50℃ and 120r / min to obtain a β-cyclodextrin solution. Take 2g of theabrownin and add 30mL of anhydrous ethanol. Dissolve by sonication for 10min at 60W to obtain a theabrownin ethanol solution. Step C2: Encapsulation reaction. The tea brown ethanol solution was slowly added dropwise to the β-cyclodextrin solution at a rate of 1 mL / min. During the addition, the temperature was maintained at 50°C and the stirring speed was 180 r / min. After the addition was completed, the reaction was continued to be stirred and kept warm for 2 hours. Then, the temperature was slowly lowered to 10°C and kept warm and allowed to stand for 4 hours. Step C3: Separation and purification. Filter the reaction system and collect the precipitate. Wash the precipitate twice with anhydrous ethanol, using 20 mL each time, to remove unencapsulated theaflavins. Then wash the precipitate once with deionized water to remove residual ethanol. Step C4: Dry the finished product. Place the washed precipitate in a vacuum drying oven and dry it for 4 hours at a temperature of 35℃ and a vacuum degree of 0.06MPa. After pulverizing, pass it through a 100-mesh sieve to obtain β-cyclodextrin-encapsulated tea brown pigment.

[0033] The preparation method of nano-hydroxyapatite includes the following steps: Step D1: Preparation of precursor solution: Take 50 mL of 0.5 mol / L calcium chloride solution and place it in a beaker. Add 2 mL of 3% polyethylene glycol 400 solution and stir for 10 min at 25℃ and 100 r / min. Separately, take 50 mL of 0.3 mol / L diammonium hydrogen phosphate solution and adjust the pH to 9.0 with 5% ammonia water to obtain diammonium hydrogen phosphate buffer solution. Step D2: Precipitation reaction. Diammonium hydrogen phosphate buffer solution was slowly added dropwise to calcium chloride solution at a rate of 0.5 mL / min. During the addition, the temperature was maintained at 30°C and the stirring speed was 200 r / min. At the same time, ammonia water was used to maintain the pH of the system at 9.0. After the addition was completed, the reaction was stirred for 1 hour, and then allowed to stand for 8 hours. Step D3: Washing and drying. Filter the precipitate and wash it repeatedly with deionized water until the pH of the washing solution is about 7.0. Then wash it once with anhydrous ethanol. Place the washed precipitate in a vacuum drying oven and dry it for 6 hours at a temperature of 60℃ and a vacuum of 0.07MPa. Step D4: Calcination and shaping. The dried precipitate is placed in a muffle furnace and heated to 600°C at a heating rate of 5°C / min. It is then calcined for 2 hours and allowed to cool naturally to room temperature. After pulverizing, it is passed through a 200-mesh sieve to obtain nano-hydroxyapatite.

[0034] The preparation process of the above-mentioned mussel adhesive protein water includes the following steps: Step 1: Raw material pretreatment. Mussel adhesive protein, hydroxylated chitosan-polyethylene glycol graft copolymer, modified sodium alginate, β-cyclodextrin-encapsulated tea brown pigment, and nano-hydroxyapatite were placed in a vacuum drying oven and dried for 2 hours at 40℃ and 0.06MPa for later use. Step 2: Preparation of aqueous system. Take 84 parts of deionized water and place it in a constant temperature stirred tank. Heat to 35℃ and adjust the stirring speed to 150 r / min. First, add 1 part of glycerol and stir to dissolve for 15 min. Then, add 3 parts of dried hydroxylated chitosan-polyethylene glycol graft copolymer and 4 parts of modified sodium alginate in sequence. Continue stirring for 30 min to obtain a mixed aqueous phase. Step 3: Mussel adhesive protein dispersion. Slowly add 5 portions of dried mussel adhesive protein to the mixed aqueous phase, maintain the temperature at 35℃ and the stirring speed at 200r / min, and stir and disperse for 40min. During this period, use ultrasonic-assisted dispersion for 5min every 10min, with an ultrasonic power of 80W, to obtain the protein dispersion. Step 4: Construction of the composite system: Two parts of dried β-cyclodextrin-encapsulated tea brown pigment and one part of nano-hydroxyapatite were added to the protein dispersion. The temperature was adjusted to 45℃, the stirring speed was 250r / min, and the reaction was stirred for 60min to obtain the composite gel precursor. Step 5: Gel molding. Transfer the composite gel precursor into the mold, let it stand at 25°C for 30 minutes, then place it in a low temperature environment and refrigerate it at 0°C for 2 hours. Then raise the temperature to 20°C and warm it for 1 hour to complete the gel molding. Step 6: Post-processing. The formed gel is removed from the mold and placed in a sterile environment. It is then vacuum dried for 3 hours at a temperature of 30°C and a vacuum degree of 0.05MPa, controlling the water content of the gel to 65%. Subsequently, it is aseptically cut and packaged to obtain mussel adhesive protein water.

[0035] Example 3 A mussel adhesive protein solution, by weight, comprises the following components: Mussel adhesive protein: 12 parts; Hydroxylated chitosan-polyethylene glycol graft copolymer: 8 parts; Modified sodium alginate: 9 parts; β-Cyclodextrin-encapsulated theabrownin: 6 parts; Nano-hydroxyapatite: 4 parts; Glycerin: 3 parts; Deionized water: 60 parts.

[0036] A method for preparing hydroxylated chitosan-polyethylene glycol graft copolymer includes the following steps: Step A1: Hydroxylation modification of chitosan. Take 10g of chitosan and place it in a three-necked flask. Add 150mL of 8% acetic acid solution and stir to dissolve for 45min at 40℃ and 180r / min to obtain chitosan solution. Slowly add 20mL of 15% hydrogen peroxide solution to the chitosan solution. After the addition is complete, raise the temperature to 60℃ and stir for 3h. After the reaction is complete, adjust the pH of the system to 7.5 with 15% sodium hydroxide solution. Let it stand for 2h to precipitate. Filter and collect the precipitate. Wash the precipitate with deionized water until the washing solution is neutral. Then place the precipitate in a vacuum drying oven and dry for 6h at 55℃ and 0.09MPa to obtain hydroxylated chitosan. Step A2: Preparation of precursor by grafting reaction. Take 6g of hydroxylated chitosan and place it in a three-necked flask. Add 120mL of N,N-dimethylformamide and stir to dissolve for 60min at 70℃ and 200r / min. Then add 1.2g of catalyst 4-dimethylaminopyridine and continue stirring for 20min. Slowly add 10g of polyethylene glycol to the system, raise the temperature to 90℃, and stir for 8h to obtain the grafting reaction solution. Step A3: Purification treatment. Cool the grafting reaction solution to room temperature, pour it into 300 mL of anhydrous ethanol, let it stand for 3 h to precipitate, and filter to collect the precipitate. Redissolve the precipitate in 80 mL of deionized water and dialyze it with a dialysis bag with a molecular weight cutoff of 5000 Da for 72 h, changing the deionized water every 12 h during the process. After dialysis, place the dialysate in a vacuum drying oven and dry it for 7 h at a temperature of 50 °C and a vacuum degree of 0.08 MPa to obtain hydroxylated chitosan-polyethylene glycol graft copolymer.

[0037] The preparation method of modified sodium alginate includes the following steps: Step B1: Sodium alginate pretreatment. Take 8g of sodium alginate and place it in a beaker. Add 150mL of deionized water and stir to dissolve for 45min at 50℃ and 150r / min to obtain sodium alginate solution. Add 1.0g of activated carbon to the sodium alginate solution and stir to adsorb for 30min. Filter to remove the activated carbon to obtain purified sodium alginate solution. Step B2: Esterification modification reaction. The purified sodium alginate solution was transferred to a three-necked flask, heated to 60°C, and the stirring speed was adjusted to 200 r / min. 15 mL of 20% citric acid solution was slowly added dropwise. After the addition was completed, the reaction was continued to be stirred at the same temperature for 5 h. During the reaction, nitrogen gas was slowly introduced into the system for 5 min every 1 h, at a nitrogen gas introduction rate of 30 mL / min. Step B3: Neutralization and washing. After the reaction is complete, cool to room temperature, adjust the pH of the system to 7.0 with a 10% sodium carbonate solution, let stand for 2 hours, filter and collect the precipitate; wash the precipitate repeatedly with deionized water 5 times until no citrate ions are detected in the washing solution. Step B4: Drying and shaping. The washed precipitate is placed in a vacuum drying oven and dried for 8 hours at a temperature of 60℃ and a vacuum degree of 0.09MPa. After pulverizing, it is passed through a 100-mesh sieve to obtain modified sodium alginate.

[0038] A method for preparing β-cyclodextrin-encapsulated theabrownins includes the following steps: Step C1: Raw material pretreatment. Take 12g of β-cyclodextrin and place it in a beaker. Add 150mL of deionized water and stir to dissolve for 30min at 60℃ and 180r / min to obtain a β-cyclodextrin solution. Take 5g of theabrownin and add 50mL of anhydrous ethanol. Dissolve by sonication for 15min at 100W to obtain a theabrownin ethanol solution. Step C2: Encapsulation reaction. The tea brown ethanol solution was slowly added dropwise to the β-cyclodextrin solution at a rate of 2 mL / min. During the addition, the temperature was maintained at 60℃ and the stirring speed was 250 r / min. After the addition was completed, the reaction was continued to be stirred and kept warm for 4 hours. Then, the temperature was slowly lowered to 15℃ and kept warm and allowed to stand for 6 hours. Step C3: Separation and purification. Filter the reaction system and collect the precipitate. Wash the precipitate three times with anhydrous ethanol, using 30 mL each time, to remove unencapsulated theaflavins. Then wash the precipitate twice with deionized water to remove residual ethanol. Step C4: Dry the finished product. Place the washed precipitate in a vacuum drying oven and dry it for 6 hours at a temperature of 45℃ and a vacuum degree of 0.08MPa. After pulverizing, pass it through a 120-mesh sieve to obtain β-cyclodextrin-encapsulated tea brown pigment.

[0039] The preparation method of nano-hydroxyapatite includes the following steps: Step D1: Preparation of precursor solution: Take 80 mL of 1.5 mol / L calcium chloride solution and place it in a beaker. Add 5 mL of 5% polyethylene glycol 400 solution and stir for 15 min at 30℃ and 150 r / min. Separately, take 80 mL of 0.9 mol / L diammonium hydrogen phosphate solution and adjust the pH to 10.0 with 10% ammonia water to obtain diammonium hydrogen phosphate buffer solution. Step D2: Precipitation reaction. Diammonium hydrogen phosphate buffer solution was slowly added dropwise to calcium chloride solution at a rate of 1 mL / min. During the addition, the temperature was maintained at 40°C and the stirring speed was 300 r / min. At the same time, ammonia water was used to maintain the pH of the system at 10.0. After the addition was completed, the reaction was stirred for 2 hours and then allowed to stand for 12 hours. Step D3: Washing and drying. Filter the precipitate and wash it repeatedly with deionized water until the pH of the washing solution is about 7.0. Then wash it once with anhydrous ethanol. Place the washed precipitate in a vacuum drying oven and dry it for 8 hours at a temperature of 80℃ and a vacuum of 0.09MPa. Step D4: Calcination and shaping. The dried precipitate is placed in a muffle furnace and heated to 800°C at a heating rate of 10°C / min. It is then calcined for 4 hours and allowed to cool naturally to room temperature. After pulverizing, it is passed through a 300-mesh sieve to obtain nano-hydroxyapatite.

[0040] The preparation process of the above-mentioned mussel adhesive protein water includes the following steps: Step 1: Raw material pretreatment. Mussel adhesive protein, hydroxylated chitosan-polyethylene glycol graft copolymer, modified sodium alginate, β-cyclodextrin-encapsulated tea brown pigment, and nano-hydroxyapatite were placed in a vacuum drying oven and dried for 3 hours at 50℃ and 0.09MPa for later use. Step 2: Preparation of aqueous system. Take 60 parts of deionized water and place it in a constant temperature stirred tank. Heat to 45℃ and adjust the stirring speed to 250 r / min. First, add 3 parts of glycerol and stir to dissolve for 25 min. Then, add 8 parts of dried hydroxylated chitosan-polyethylene glycol graft copolymer and 9 parts of modified sodium alginate in sequence. Continue stirring for 45 min to obtain a mixed aqueous phase. Step 3: Mussel adhesive protein dispersion. 12 portions of dried mussel adhesive protein were slowly added to the mixed aqueous phase. The temperature was maintained at 45℃ and the stirring speed was 300r / min. The mixture was stirred and dispersed for 60min. During the process, ultrasonic dispersion was used for 5min every 10min. The ultrasonic power was 120W. The protein dispersion was obtained. Step 4: Construction of the composite system: 6 parts of dried β-cyclodextrin-encapsulated tea brown pigment and 4 parts of nano-hydroxyapatite were added to the protein dispersion, the temperature was adjusted to 55℃, the stirring speed was 350 r / min, and the reaction was stirred for 90 min to obtain the composite gel precursor. Step 5: Gel molding. Transfer the composite gel precursor into a mold, let it stand at 30°C for 40 minutes, then place it in a low-temperature environment and refrigerate it at 5°C for 4 hours. Then raise the temperature to 25°C and warm it for 2 hours to complete the gel molding. Step 6: Post-processing. The formed gel is removed from the mold and placed in a sterile environment. It is then vacuum dried for 5 hours at a temperature of 40℃ and a vacuum degree of 0.08MPa, controlling the water content of the gel to 80%. Subsequently, it is aseptically cut and packaged to obtain mussel adhesive protein water.

[0041] The performance of the mussel adhesive protein water prepared in Examples 1-3 was tested, and the results are shown in the table below: As shown in the table above, the mussel adhesive protein water prepared by this invention is significantly superior to existing products in terms of tensile strength, elongation at break, antibacterial properties, and wound healing promotion effect, which fully demonstrates the advanced nature and practicality of this invention.

[0042] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0043] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. Mussel adhesive protein water, characterized in that, The ingredients include the following parts by weight: Mussel adhesive protein: 5-12 parts; Hydroxylated chitosan-polyethylene glycol graft copolymer: 3-8 parts; Modified sodium alginate: 4-9 parts; β-Cyclodextrin-encapsulated theabrownins: 2-6 parts; Nano-hydroxyapatite: 1-4 parts; Glycerin: 1-3 parts; Deionized water: 60-85 parts; The sum of the mass fractions of each component is 100 parts.

2. The mussel adhesive protein water according to claim 1, characterized in that, The mussel adhesive protein has a molecular weight of 100-200 kDa and a purity of ≥95%; the glycerol is medical-grade glycerol with a purity of ≥99.5%.

3. The mussel adhesive protein water according to claim 1, characterized in that, The preparation method of the hydroxylated chitosan-polyethylene glycol graft copolymer is as follows: Step A1: Hydroxylation modification of chitosan. Take 5-10g of chitosan and place it in a three-necked flask. Add 100-150mL of acetic acid solution with a mass fraction of 5-8%. Stir and dissolve the chitosan solution for 30-45min at a temperature of 30-40℃ and a stirring speed of 120-180r / min to obtain a chitosan solution. Slowly add 10-20mL of hydrogen peroxide solution with a mass fraction of 10-15% to the chitosan solution. After the addition is complete, raise the temperature to 50-60℃ and stir for 2-3h. After the reaction is complete, adjust the pH of the system to 7.0-7.5 with sodium hydroxide solution with a mass fraction of 10-15%. Let the precipitate stand for 1-2h, filter and collect the precipitate. Wash the precipitate with deionized water until the washing solution is neutral. Then place the precipitate in a vacuum drying oven and dry it for 4-6h at a temperature of 45-55℃ and a vacuum degree of 0.07-0.09MPa to obtain hydroxylated chitosan. Step A2: Grafting reaction to prepare the precursor. Take 3-6g of the hydroxylated chitosan obtained in step A1 and place it in a three-necked flask. Add 80-120mL of N,N-dimethylformamide and stir to dissolve for 40-60min at a temperature of 60-70℃ and a stirring speed of 150-200r / min. Then add 0.5-1.2g of the catalyst 4-dimethylaminopyridine and continue stirring for 10-20min. Slowly add 5-10g of polyethylene glycol to the system, raise the temperature to 80-90℃, and keep the temperature and stir for 6-8h to obtain the grafting reaction solution. Step A3: Purification treatment. Cool the grafting reaction solution obtained in step A2 to room temperature, pour it into 200-300 mL of anhydrous ethanol, let it stand for 2-3 hours to precipitate, and filter to collect the precipitate. Redissolve the precipitate in 50-80 mL of deionized water and dialyze it with a dialysis bag with a molecular weight cutoff of 3000-5000 Da for 48-72 hours, changing the deionized water every 12 hours during the process. After dialysis, place the dialysate in a vacuum drying oven and dry it for 5-7 hours at a temperature of 40-50℃ and a vacuum degree of 0.06-0.08 MPa to obtain hydroxylated chitosan-polyethylene glycol graft copolymer.

4. The mussel adhesive protein water according to claim 1, characterized in that, The modified sodium alginate is prepared as follows: Step B1: Sodium alginate pretreatment. Place 4-8g of sodium alginate in a beaker, add 100-150mL of deionized water, and stir to dissolve for 30-45min at a temperature of 40-50℃ and a stirring speed of 100-150r / min to obtain a sodium alginate solution. Add 0.5-1.0g of activated carbon to the sodium alginate solution, stir to adsorb for 20-30min, filter to remove the activated carbon, and obtain a purified sodium alginate solution. Step B2: Esterification modification reaction. Transfer the purified sodium alginate solution obtained in Step B1 to a three-necked flask, heat to 50-60℃, adjust the stirring speed to 150-200 r / min, and slowly add 8-15 mL of 15-20% citric acid solution. After the addition is complete, continue to keep the temperature and stir for 3-5 h. During the reaction, slowly introduce nitrogen gas into the system for 5 min every 1 h, at a nitrogen gas introduction rate of 20-30 mL / min. Step B3: Neutralization and washing. After the reaction is complete, cool to room temperature, adjust the pH of the system to 6.5-7.0 with a 5-10% sodium carbonate solution, let stand for 1-2 hours, filter and collect the precipitate; wash the precipitate repeatedly with deionized water 3-5 times until no citrate ions are detected in the washing solution. Step B4: Drying and shaping. Place the washed precipitate in a vacuum drying oven and dry it for 6-8 hours at a temperature of 50-60℃ and a vacuum degree of 0.07-0.09MPa. After pulverizing, pass it through an 80-100 mesh sieve to obtain modified sodium alginate.

5. The mussel adhesive protein water according to claim 1, characterized in that, The preparation method of the β-cyclodextrin-encapsulated theabrownin is as follows: Step C1: Raw material pretreatment. Take 6-12g of β-cyclodextrin and place it in a beaker. Add 100-150mL of deionized water and stir to dissolve for 20-30min at a temperature of 50-60℃ and a stirring speed of 120-180r / min to obtain a β-cyclodextrin solution. Take 2-5g of theabrownin and add 30-50mL of anhydrous ethanol. Dissolve by sonication for 10-15min at an ultrasonic power of 60-100W to obtain a theabrownin ethanol solution. Step C2: Encapsulation reaction. The theabrownin ethanol solution obtained in step C1 is slowly added dropwise to the β-cyclodextrin solution, with the dropping rate controlled at 1-2 mL / min. During the dropping process, the temperature is maintained at 50-60℃ and the stirring speed is 180-250 r / min. After the dropping is completed, the reaction is continued to be stirred and kept at the temperature for 2-4 hours. Then, the temperature is slowly lowered to 10-15℃ and kept at the temperature for 4-6 hours. Step C3: Separation and purification. Filter the reaction system obtained in step C2 and collect the precipitate. Wash the precipitate 2-3 times with anhydrous ethanol, each time using 20-30 mL, to remove unencapsulated theabrownins. The precipitate was then washed 1-2 times with deionized water to remove residual ethanol. Step C4: Dry the finished product. Place the washed precipitate in a vacuum drying oven and dry it for 4-6 hours at a temperature of 35-45℃ and a vacuum degree of 0.06-0.08MPa. After pulverizing, pass it through a 100-120 mesh sieve to obtain β-cyclodextrin-encapsulated tea brown pigment.

6. The mussel adhesive protein water according to claim 1, characterized in that, The preparation method of the nano-hydroxyapatite is as follows: Step D1: Preparation of precursor solution: Take 50-80 mL of 0.5-1.5 mol / L calcium chloride solution and place it in a beaker. Add 2-5 mL of 3-5% polyethylene glycol 400 solution and stir for 10-15 min at 25-30℃ and 100-150 r / min. Separately, take 50-80 mL of 0.3-0.9 mol / L diammonium hydrogen phosphate solution and adjust the pH to 9.0-10.0 with 5-10% ammonia water to obtain diammonium hydrogen phosphate buffer solution. Step D2: Precipitation reaction. Slowly add diammonium hydrogen phosphate buffer solution to calcium chloride solution at a rate of 0.5-1 mL / min. Maintain the temperature at 30-40℃ and the stirring speed at 200-300 r / min during the addition. At the same time, use ammonia water to maintain the pH of the system at 9.0-10.

0. After the addition is complete, continue stirring for 1-2 hours, and then let it stand for 8-12 hours. Step D3: Washing and drying. Filter the precipitate obtained in step D2, wash the precipitate repeatedly with deionized water until the pH of the washing solution is about 7.0, and then wash it once with anhydrous ethanol. Place the washed precipitate in a vacuum drying oven and dry it for 6-8 hours at a temperature of 60-80℃ and a vacuum degree of 0.07-0.09MPa. Step D4: Calcination and shaping. Place the dried precipitate in a muffle furnace and heat it to 600-800℃ at a heating rate of 5-10℃ / min. Hold it at this temperature for 2-4 hours. After naturally cooling to room temperature, pulverize it through a 200-300 mesh sieve to obtain nano-hydroxyapatite.

7. A process for preparing mussel adhesive protein water, characterized in that, Includes the following steps: Step 1: Raw material pretreatment. Mussel adhesive protein, hydroxylated chitosan-polyethylene glycol graft copolymer, modified sodium alginate, β-cyclodextrin-encapsulated tea brown pigment, and nano-hydroxyapatite are placed in a vacuum drying oven and dried for 2-3 hours at a temperature of 40-50℃ and a vacuum degree of 0.06-0.09MPa for later use. Step 2: Preparation of aqueous system. Take the prescribed amount of deionized water and place it in a constant temperature stirred tank. Heat the tank to 35-45℃ and adjust the stirring speed to 150-250 r / min. First, add the prescribed amount of glycerol and stir to dissolve for 15-25 min. Then, add the dried hydroxylated chitosan-polyethylene glycol graft copolymer and modified sodium alginate in sequence. Continue stirring for 30-45 min to obtain a mixed aqueous phase. Step 3: Mussel adhesive protein dispersion. The dried mussel adhesive protein is slowly added to the mixed aqueous phase obtained in Step 2. The temperature is maintained at 35-45℃ and the stirring speed is 200-300r / min. The mixture is stirred and dispersed for 40-60min. During this period, ultrasonic dispersion is used for 5min every 10min. The ultrasonic power is 80-120W to obtain the protein dispersion. Step 4: Construction of the composite system. The dried β-cyclodextrin-encapsulated tea brown pigment and nano-hydroxyapatite were added to the protein dispersion obtained in Step 3. The temperature was adjusted to 45-55℃, the stirring speed was 250-350 r / min, and the reaction was carried out for 60-90 min to obtain the composite gel precursor. Step 5: Gel molding. Transfer the composite gel precursor obtained in step 4 into a mold. First, let it stand at 25-30℃ for 30-40 minutes, then place it in a low-temperature environment and refrigerate it at 0-5℃ for 2-4 hours. Then, raise the temperature to 20-25℃ and warm it for 1-2 hours to complete the gel molding. Step 6: Post-processing. Remove the molded gel from the mold and place it in a sterile environment. Vacuum dry it for 3-5 hours at a temperature of 30-40℃ and a vacuum degree of 0.05-0.08MPa, controlling the gel water content to 65-80%. Then, aseptically cut and package it to obtain the product, mussel adhesive protein water.

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