A method for preparing a mussel adhesive protein

Abstract

The application relates to the technical field of protein extraction, and particularly discloses a preparation method of mussel mucin, which comprises the following steps: step S1, defatting treatment; step S2, deodorization treatment; step S3, acid extraction; step S4, enzymolysis; and step S5, fine extraction. In the technical scheme, aluminum citrate and salicylic acid are added in the acid extraction process, and the polypeptide components in the secondary enzymolysis liquid are subjected to ultrafiltration treatment through a modified polyether sulfone filter membrane in the fine extraction process. Through the synergistic effect of the above processes, the purity and yield of the mussel mucin are improved.

Description

Technical Field

[0001] The present application relates to the technical field of protein extraction, and more specifically, to a method for preparing mussel mucin. Background Art

[0002] Mussel mucin is the most representative natural adhesive substance. It not only has excellent adhesion, flexibility and good biocompatibility, but also has low toxicity and does not trigger an immune response in the human body. Therefore, it has broad application prospects in the biomedicine field.

[0003] In the prior art, the preparation of mussel mucin is mainly based on natural extraction and genetic engineering methods. Among them, the natural extraction method for preparing mussel mucin can retain the structure and activity of natural protein, has the advantages of good biocompatibility and functional diversity, and has broad application prospects. However, in the prior art, the natural extraction method of mussel mucin mostly relies on salting out, centrifugation and chromatographic purification methods, which are cumbersome steps, resulting in low extraction rate and purity of extracted protein. Based on the above statement, the present application provides a preparation method of mussel mucin with high extraction rate and high purity. Summary of the Invention

[0004] In order to solve the problems mentioned in the above background technology, the present application provides a method for preparing mussel mucin.

[0005] A method for preparing mussel mucin comprises the following steps:

[0006] Step S1, defatting: clean the fresh mussels, remove the shells, retain the meat and mince it to obtain chopped mussel meat, then defatted, crushed, and sieved to obtain defatted mussel powder;

[0007] Step S2, deodorizing treatment: adding mussel defatted powder to ethanol aqueous solution, homogenizing, maintaining the temperature at 20-30° C., centrifuging, collecting the precipitate, and drying at 36-40° C. to constant weight to obtain deodorized mussel defatted powder, wherein the mass ratio of mussel defatted powder to ethanol aqueous solution is 1:28-36;

[0008] Step S3, acid extraction: dissolving deodorized mussel defatted powder, aluminum citrate, and salicylic acid in deionized water, adding an acid value regulating solution to adjust the pH to 1.8-2.2, heating to 42-46° C., stirring and extracting for 1-2 hours, centrifuging, collecting the supernatant, adjusting the pH of the supernatant to 4-5, centrifuging, collecting the precipitate, washing, and freeze-drying to obtain a crude mussel mucin extract, wherein the mass ratio of the deodorized mussel defatted powder, aluminum citrate, salicylic acid, and deionized water is 1-3:0.04-0.08:0.02-0.05:60-80;

[0009] Step S4, enzymatic hydrolysis: adding the crude mussel mucin extract to deionized water, adjusting the pH to 7.0-7.2 with a Tris-HCl buffer having a mass fraction of 0.5-1.5%, then adding neutral protease and papain, performing a primary enzymatic hydrolysis, centrifuging, and taking the supernatant to obtain a primary enzymatic hydrolyzate; then adjusting the pH of the primary enzymatic hydrolyzate to 6.5-6.8, then adding trypsin and a composite protease, and performing a secondary enzymatic hydrolysis to obtain a secondary enzymatic hydrolyzate, wherein the mass ratio of the crude mussel mucin extract, deionized water, neutral protease, and papain is 2-4:80-100:0.008-0.026:0.005-0.011, and the mass ratio of the primary enzymatic hydrolyzate, trypsin, and composite protease is 80-100:0.01-0.014:0.018-0.022;

[0010] Step S5, refined extraction: the secondary enzymatic hydrolysate is concentrated by ultrafiltration and freeze-dried to obtain mussel mucin.

[0011] Preferably, in step S1, the defatting process comprises: placing the chopped mussel meat in a 76-80% (w / v) ethanol aqueous solution at a temperature of 46-52° C. for 4.8-5.4 hours, with a material-liquid mass ratio of 1:25-35, and washing with deionized water until neutral.

[0012] Preferably, in step S2, the homogenization time is 4-6 min, and the mass fraction of the ethanol aqueous solution is 46-52%.

[0013] Preferably, in step S3, the acid value regulating liquid is a citric acid aqueous solution with a mass fraction of 18-22%.

[0014] Preferably, in step S4, the enzymatic hydrolysis temperature of the first enzymatic hydrolysis is 42-46° C., the enzymatic hydrolysis time is 2.2-2.6 h, and the enzymatic hydrolysis temperature of the second enzymatic hydrolysis is 44-50° C., the enzymatic hydrolysis time is 2.8-3.2 h.

[0015] Preferably, in step S5, ultrafiltration concentration refers to: ultrafiltration of the secondary enzymatic hydrolysate using a modified polyethersulfone filter membrane.

[0016] Preferably, the modified polyethersulfone filter membrane has a molecular weight cutoff of 10-12 kDa, a pressure of 0.1-0.3 MPa, a temperature of 12-24° C., a pH of 6.5-7.5, a concentration multiple of 6-8 times, and a cross-flow velocity of 1-3 m / s.

[0017] Preferably, the modified polyethersulfone filter membrane is prepared by the following steps:

[0018] Step A1, placing a polyethersulfone filter membrane in a mixed solution of ethanol aqueous solution and sodium hydroxide, soaking for reaction for 2.6-3.4 hours, removing the polyethersulfone membrane, washing and drying it to obtain a hydroxylated polyethersulfone filter membrane, wherein the mass ratio of the polyethersulfone filter membrane, ethanol aqueous solution and sodium hydroxide is 0.1-0.3:30-40:10;

[0019] Step A2, adding the hydroxylated polyethersulfone filter membrane and the esterified chitosan to an ethanol aqueous solution, heating to 45-55°C, stirring and reacting for 1.4-2.6 hours, and after the reaction is completed, washing and drying to obtain a modified polyethersulfone filter membrane, wherein the mass ratio of the hydroxylated polyethersulfone filter membrane, the esterified chitosan and the ethanol aqueous solution is 5-6:0.42-0.54:120-160. During the above reaction process, the hydroxylated polyethersulfone membrane and the esterified chitosan are combined through the hydrogen bonding between the amino group and the hydroxyl group, and the esterified chitosan is grafted to the surface of the polyethersulfone membrane to obtain a modified polyethersulfone membrane.

[0020] Preferably, the mass fraction of the ethanol aqueous solution in step A1 and step A2 is 18-22%.

[0021] Preferably, the esterified chitosan is prepared by the following steps:

[0022] Step B1, adding a silane coupling agent and succinic anhydride to anhydrous DMF, heating to 35-45°C, stirring for 0.5-0.8h to obtain a modified solution, ultrasonically dispersing the cerium-doped oxide in anhydrous DMF, adding the modified solution and deionized water, stirring and reacting for 4.2-5.4h, and after the reaction is completed, filtering with suction, washing and drying the filter cake to obtain a carboxylated cerium-doped oxide, wherein the mass ratio of the silane coupling agent, succinic anhydride and anhydrous DMF in the modified solution is 2.2- 2.6:1.3-1.5:26-30, the mass ratio of cerium-doped oxide, anhydrous DMF, modification liquid and deionized water is 3-5:84-90:2.6-3:5-7, firstly, KH-550 and succinic anhydride are subjected to an amidation reaction to obtain a silane coupling agent containing an amide bond and a terminal carboxyl group, and then the hydrolysis product of the silane coupling agent is subjected to a condensation reaction with the surface hydroxyl group of the cerium-doped oxide to obtain a carboxylated cerium-doped oxide having a surface rich in carboxyl groups and amide bonds;

[0023] Step B2, ultrasonically disperse hydroxymethyl chitosan in acetic acid aqueous solution, then add phytic acid and p-toluenesulfonic acid, heat to 64-72 ° C, stir and react for 0.6-1.0h, then heat to 74-80 ° C, dropwise add a mixture of carboxylated cerium doped oxide and anhydrous DMF, control the dripping within 10min, after the dripping is completed, continue stirring and reacting for 1.6-2.2h, rotary evaporation, washing, and drying to obtain esterified chitosan, wherein hydroxymethyl chitosan, acetic acid aqueous solution The mass ratio of the solution, phytic acid, p-toluenesulfonic acid and mixed solution a is 4-6:110-140:1.2-1.6:0.06-0.12:30-40. In the mixed solution a, the mass ratio of the carboxylated cerium doped oxide and anhydrous DMF is 0.8-1:35-45. In the above reaction process, with p-toluenesulfonic acid as a catalyst, hydroxymethyl chitosan can undergo esterification reaction with the phytic acid and the carboxylated cerium doped oxide respectively to obtain esterified chitosan.

[0024] Preferably, in step B1, the silane coupling agent is γ-aminopropyltriethoxysilane or γ-aminopropyltrimethoxysilane.

[0025] Preferably, in step B2, the mass fraction of the acetic acid aqueous solution is 1-3%.

[0026] Preferably, the cerium-doped oxide is prepared by the following steps:

[0027] Zirconium oxychloride octahydrate, yttrium nitrate hexahydrate and cerium nitrate are added to an ethanol aqueous solution, stirred evenly, and then added dropwise to an ammonia aqueous solution, stirred for 0.4-0.8 hours, allowed to stand for 1.2-1.6 hours, washed, dried, ground, sieved, and then calcined at 790-820°C for 1.6-2.2 hours to obtain a cerium-doped oxide, wherein the mass ratio of the ethanol aqueous solution, zirconium oxychloride octahydrate, yttrium nitrate hexahydrate, cerium nitrate and ammonia aqueous solution is 160-200:12:1-2:1.2-2.4:24-30, the mass fraction of the ethanol aqueous solution is 42-48%, and the mass fraction of the ammonia aqueous solution is 24-26%. During the above reaction process, the cerium-doped oxide is prepared by a coprecipitation method.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) In the technical scheme of the present application, aluminum citrate and salicylic acid are added in the acidic extraction process. The aluminum ions generated by the hydrolysis of aluminum citrate can not only neutralize the negative charges on the surface of protein molecules, reduce the electrostatic repulsion between protein molecules, and thus promote the aggregation and precipitation of proteins, but also can coordinate with the carboxyl and amino groups in the mussel adhesive protein molecules to form a cross-linked structure, thereby enhancing the interaction between protein molecules and improving the extraction efficiency. The salicylic acid can not only act as a ligand, but also can form a dense cross-linked network with the DOPA-Al 3+ The salicylic acid can not only act as a ligand, but also can form a dense cross-linked network with the DOPA-Al

[0030] (2) In the technical scheme of the present application, in order to improve the extraction rate and purity of mussel adhesive protein, the polypeptide components in the secondary enzymatic hydrolysate are subjected to ultrafiltration treatment by using a modified polyether sulfone filter membrane. The surface of the modified polyether sulfone filter membrane contains chitosan structure, phytic acid structure and cerium-doped oxide structure. The hydroxyl and amino groups in the chitosan structure can further form hydrogen bonds and hydrophobic interactions with the carboxyl and amino groups in the mussel adhesive protein molecules, thereby promoting the aggregation and precipitation of protein molecules. The phytic acid structure contains a large number of phosphate groups, and the oxygen atoms on the phosphate groups can form hydrogen bonds with the hydrogen atoms in the mussel adhesive protein molecules, thereby further stabilizing the complex between the phosphate groups and the mussel adhesive protein. The zirconium oxide in the cerium-doped oxide structure has high adsorption capacity, and the cerium ions on the zirconium oxide can form stable complexes with the carboxyl groups in the mussel adhesive protein molecules, thereby improving the selective adsorption of the mussel adhesive protein. Through the synergistic effect of the above structures, the extraction rate and purity of the mussel adhesive protein are further improved. DETAILED DESCRIPTION

[0031] In order to make the embodiments of the present application easier to understand, the present application will be described in detail below in conjunction with specific examples, which are only illustrative and do not limit the scope of application of the present application.

[0032] In the examples, the specific techniques or conditions not specified are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The reagents or instruments not specified by the manufacturer are conventional products that can be purchased through regular channels.

[0033] Fresh mussels were produced in Shengsi Mussel Farming Base.

[0034] Preparation Examples 1-3 and Comparative Preparation Examples 1-3 provide a cerium-doped oxide.

[0035] Preparation Example 1

[0036] This preparation example provides a cerium-doped oxide, which is prepared by the following steps:

[0037] Zirconium oxychloride octahydrate, yttrium nitrate hexahydrate and cerium nitrate were added to a 42% mass fraction ethanol aqueous solution, the rotation speed was controlled at 540 rpm, and the mixture was stirred for 20 minutes until uniform. The mixture was then added dropwise to a 24% mass fraction ammonia aqueous solution, stirred for 0.4 hours, and allowed to stand for 1.2 hours. The mixture was washed three times with a 12% mass fraction ammonia aqueous solution and anhydrous ethanol, dried at 60°C, ground, passed through a 200 mesh sieve, and then calcined at 790°C for 1.6 hours to obtain a cerium-doped oxide, wherein the mass ratio of the ethanol aqueous solution, zirconium oxychloride octahydrate, yttrium nitrate hexahydrate, cerium nitrate and ammonia aqueous solution was 160:12:1:1.2:24.

[0038] Preparation Example 2

[0039] This preparation example provides a cerium-doped oxide, which is prepared by the following steps:

[0040] Zirconium oxychloride octahydrate, yttrium nitrate hexahydrate and cerium nitrate were added to a 45% mass fraction ethanol aqueous solution, the rotation speed was controlled at 560 rpm, and the mixture was stirred for 22 minutes until uniform. The mixture was then added dropwise to a 25% mass fraction ammonia aqueous solution, stirred for 0.6 hours, and allowed to stand for 1.4 hours. The mixture was washed with a 12% mass fraction ammonia aqueous solution and anhydrous ethanol four times each, dried at 64°C, ground, passed through a 220-mesh sieve, and then calcined at 805°C for 1.9 hours to obtain a cerium-doped oxide, wherein the mass ratio of the ethanol aqueous solution, zirconium oxychloride octahydrate, yttrium nitrate hexahydrate, cerium nitrate and ammonia aqueous solution was 180:12:1.5:1.8:27.

[0041] Preparation Example 3

[0042] This preparation example provides a cerium-doped oxide, which is prepared by the following steps:

[0043] Zirconium oxychloride octahydrate, yttrium nitrate hexahydrate and cerium nitrate were added to a 48% ethanol aqueous solution, the rotation speed was controlled at 580 rpm, and the mixture was stirred for 24 minutes until uniform. The mixture was then added dropwise to a 26% ammonia aqueous solution, stirred for 0.8 hours, and allowed to stand for 1.6 hours. The mixture was washed 5 times with a 16% ammonia aqueous solution and anhydrous ethanol, dried at 68°C, ground, passed through a 240-mesh sieve, and calcined at 820°C for 2.2 hours to obtain a cerium-doped oxide, wherein the mass ratio of the ethanol aqueous solution, zirconium oxychloride octahydrate, yttrium nitrate hexahydrate, cerium nitrate and ammonia aqueous solution was 200:12:2:2.4:30.

[0044] Comparative Preparation Example 1

[0045] This comparative preparation example provides a cerium-doped oxide, which is prepared by the following steps:

[0046] Calcium nitrate, yttrium nitrate hexahydrate and cerium nitrate were added to a 42% mass fraction ethanol aqueous solution, the rotation speed was controlled at 540 rpm, and the mixture was stirred for 20 minutes until uniform. The mixture was then added dropwise to a 24% mass fraction ammonia aqueous solution, stirred for 0.4 hours, and allowed to stand for 1.2 hours. The mixture was washed three times with a 12% mass fraction ammonia aqueous solution and anhydrous ethanol, dried at 60°C, ground, passed through a 200 mesh sieve, and then calcined at 790°C for 1.6 hours to obtain a cerium-doped oxide, wherein the mass ratio of the ethanol aqueous solution, calcium nitrate, yttrium nitrate hexahydrate, cerium nitrate and ammonia aqueous solution was 160:12:1:1.2:24.

[0047] Comparative Preparation Example 2

[0048] This comparative preparation example provides a cerium-doped oxide, which is prepared by the following steps:

[0049] Zirconium oxychloride octahydrate, magnesium nitrate and cerium nitrate were added to a 42% mass fraction ethanol aqueous solution, the rotation speed was controlled at 540 rpm, and the mixture was stirred for 20 minutes until uniform. The mixture was then added dropwise to a 24% mass fraction ammonia aqueous solution, stirred for 0.4 hours, and allowed to stand for 1.2 hours. The mixture was washed three times with a 12% mass fraction ammonia aqueous solution and anhydrous ethanol, dried at 60°C, ground, passed through a 200 mesh sieve, and then calcined at 790°C for 1.6 hours to obtain a cerium-doped oxide, wherein the mass ratio of the ethanol aqueous solution, zirconium oxychloride octahydrate, magnesium nitrate, cerium nitrate and ammonia aqueous solution was 160:12:1:1.2:24.

[0050] Preparation Examples 4-6 and Comparative Preparation Examples 3-5 provide an esterified chitosan.

[0051] Preparation Example 4

[0052] This preparation example provides an esterified chitosan, which is prepared by the following steps:

[0053] Step B1, adding γ-aminopropyltriethoxysilane and succinic anhydride to anhydrous DMF, heating to 35°C, controlling the speed to 420rpm, stirring for 0.5h to obtain a modified solution, ultrasonically dispersing the cerium-doped oxide prepared in Preparation Example 1 in anhydrous DMF, controlling the ultrasonic frequency to 30kHz, the ultrasonic power to 500w, and ultrasonication for 16min, adding the modified solution and deionized water, maintaining the speed unchanged, and continuing to stir and react for 4.2h. After the reaction is completed, filtering, the filter cake is washed with anhydrous ethanol and deionized water three times each, and dried at 50°C to constant weight to obtain a carboxylated cerium-doped oxide, wherein the mass ratio of γ-aminopropyltriethoxysilane, succinic anhydride and anhydrous DMF in the modified solution is 2.2:1.3:26, and the mass ratio of cerium-doped oxide, anhydrous DMF, modifying solution and deionized water is 3:84:2.6:5;

[0054] Step B2, ultrasonically disperse the hydroxymethyl chitosan in an aqueous solution of acetic acid with a mass fraction of 1%, control the ultrasonic frequency to 40 kHz, the ultrasonic power to 600 w, and ultrasonicate for 12 min, then add phytic acid and p-toluenesulfonic acid, heat to 64 ° C, stir and react at a speed of 540 rpm for 0.6 h, then heat to 74 ° C, add a mixture of carboxylated cerium doped oxide and anhydrous DMF dropwise, control the dripping within 10 min, and after the dripping, maintain the speed unchanged, continue stirring and reacting for 1.6 h, control the rotary evaporation temperature to 66 ° C, rotary evaporation to remove the solution, sequentially wash with anhydrous ethanol and deionized water 3 times each, and dry at 62 ° C to constant weight to obtain esterified chitosan, wherein the mass ratio of hydroxymethyl chitosan, acetic acid aqueous solution, phytic acid, p-toluenesulfonic acid and mixed solution a is 4:110:1.2:0.06:30, and the mass ratio of carboxylated cerium doped oxide and anhydrous DMF in the mixed solution a is 0.8:35.

[0055] Preparation Example 5

[0056] This preparation example provides an esterified chitosan, which is prepared by the following steps:

[0057] Step B1, adding γ-aminopropyltrimethoxysilane and succinic anhydride to anhydrous DMF, heating to 40°C, controlling the speed to 460rpm, stirring for 0.65h to obtain a modified solution, ultrasonically dispersing the cerium-doped oxide prepared in Preparation Example 2 in anhydrous DMF, controlling the ultrasonic frequency to 35kHz, the ultrasonic power to 550w, and ultrasonicating for 20min, adding the modified solution and deionized water, maintaining the speed unchanged, and continuing to stir and react for 4.8h. After the reaction is completed, filtering, the filter cake is washed with anhydrous ethanol and deionized water 4 times each, and dried at 55°C to constant weight to obtain a carboxylated cerium-doped oxide, wherein the mass ratio of γ-aminopropyltrimethoxysilane, succinic anhydride and anhydrous DMF in the modified solution is 2.4:1.4:28, and the mass ratio of cerium-doped oxide, anhydrous DMF, modifying solution and deionized water is 4:87:2.8:6;

[0058] Step B2, ultrasonically disperse the hydroxymethyl chitosan in an aqueous solution of acetic acid with a mass fraction of 2%, control the ultrasonic frequency to 35kHz, the ultrasonic power to 550w, and ultrasonicate for 15min, then add phytic acid and p-toluenesulfonic acid, raise the temperature to 68°C, stir and react for 0.8h, then raise the temperature to 77°C, add a mixture of carboxylated cerium-doped oxide and anhydrous DMF dropwise, control the dripping within 10min, and after the dripping, maintain the speed unchanged, continue stirring and reacting for 1.9h, control the rotary evaporation temperature to 70°C, rotary evaporation to remove the solution, sequentially wash with anhydrous ethanol and deionized water 4 times each, and dry at 66°C to constant weight to obtain esterified chitosan, wherein the mass ratio of hydroxymethyl chitosan, acetic acid aqueous solution, phytic acid, p-toluenesulfonic acid and mixed solution a is 5:125:1.4:0.09:35, and the mass ratio of carboxylated cerium-doped oxide and anhydrous DMF in the mixed solution a is 0.9:40.

[0059] Preparation Example 6

[0060] This preparation example provides an esterified chitosan, which is prepared by the following steps:

[0061] γ-Aminopropyltriethoxysilane and succinic anhydride were added to anhydrous DMF, the temperature was raised to 45°C, the rotation speed was controlled at 500 rpm, and the mixture was stirred for 0.8 h to obtain a modified solution. The cerium-doped oxide prepared in Preparation Example 3 was ultrasonically dispersed in anhydrous DMF, the ultrasonic frequency was controlled at 40 kHz, the ultrasonic power was 600 W, and the ultrasonication was carried out for 24 min. The modified solution and deionized water were added, the rotation speed was maintained unchanged, and the stirring reaction was continued for 5.4 h. After the reaction was completed, the filter cake was filtered, and the filter cake was washed with anhydrous ethanol and deionized water 5 times each, and dried at 60°C to constant weight to obtain a carboxylated cerium-doped oxide. In the modified solution, the mass ratio of γ-aminopropyltriethoxysilane, succinic anhydride and anhydrous DMF was 2.6:1.5:30, and the mass ratio of cerium-doped oxide, anhydrous DMF, modified solution and deionized water was 5:90:3:7;

[0062] Step B2, ultrasonically disperse the hydroxymethyl chitosan in a 3% mass fraction of acetic acid aqueous solution, control the ultrasonic frequency to 30kHz, the ultrasonic power to 500w, and ultrasonicate for 18min, then add phytic acid and p-toluenesulfonic acid, raise the temperature to 72°C, stir and react for 1.0h, then raise the temperature to 80°C, add a mixture of carboxylated cerium doped oxide and anhydrous DMF dropwise, control the dripping within 10min, and after the dripping, maintain the speed unchanged, continue stirring and reacting for 2.2h, control the rotary evaporation temperature to 74°C, rotary evaporation to remove the solution, sequentially wash with anhydrous ethanol and deionized water 5 times each, and dry at 70°C to constant weight to obtain esterified chitosan, wherein the mass ratio of hydroxymethyl chitosan, acetic acid aqueous solution, phytic acid, p-toluenesulfonic acid and mixed solution a is 6:140:1.6:0.12:40, and the mass ratio of carboxylated cerium doped oxide and anhydrous DMF in the mixed solution a is 1:45.

[0063] Comparative Preparation Example 3

[0064] This comparative preparation example provides an esterified chitosan, which is prepared by the following steps:

[0065] Step B1, γ-aminopropyltriethoxysilane and succinic anhydride were added to anhydrous DMF, the temperature was raised to 35°C, the speed was controlled to 420 rpm, and the mixture was stirred for 0.5 h to obtain a modified solution. The cerium-doped oxide prepared in Comparative Preparation Example 1 was ultrasonically dispersed in anhydrous DMF, the ultrasonic frequency was controlled to 30 kHz, the ultrasonic power was 500 w, and the ultrasound was performed for 16 min. The modified solution and deionized water were added, the speed was maintained unchanged, and the stirring reaction was continued for 4.2 h. After the reaction was completed, the filter cake was filtered, and the filter cake was washed with anhydrous ethanol and deionized water 3 times each, and dried at 50°C to constant weight to obtain a carboxylated cerium-doped oxide, wherein the mass ratio of γ-aminopropyltriethoxysilane, succinic anhydride and anhydrous DMF in the modified solution was 2.2:1.3:26, and the mass ratio of cerium-doped oxide, anhydrous DMF, modifying solution and deionized water was 3:84:2.6:5;

[0066] Step B2, ultrasonically disperse the hydroxymethyl chitosan in an aqueous solution of acetic acid with a mass fraction of 1%, control the ultrasonic frequency to 40 kHz, the ultrasonic power to 600 w, and ultrasonicate for 12 min, then add phytic acid and p-toluenesulfonic acid, heat to 64 ° C, stir and react at a speed of 540 rpm for 0.6 h, then heat to 74 ° C, add a mixture of carboxylated cerium doped oxide and anhydrous DMF dropwise, control the dripping within 10 min, and after the dripping, maintain the speed unchanged, continue stirring and reacting for 1.6 h, control the rotary evaporation temperature to 66 ° C, rotary evaporation to remove the solution, sequentially wash with anhydrous ethanol and deionized water 3 times each, and dry at 62 ° C to constant weight to obtain esterified chitosan, wherein the mass ratio of hydroxymethyl chitosan, acetic acid aqueous solution, phytic acid, p-toluenesulfonic acid and mixed solution a is 4:110:1.2:0.06:30, and the mass ratio of carboxylated cerium doped oxide and anhydrous DMF in the mixed solution a is 0.8:35.

[0067] Comparative Preparation Example 4

[0068] This comparative preparation example provides an esterified chitosan, which is prepared by the following steps:

[0069] Step B1, γ-aminopropyltriethoxysilane and succinic anhydride were added to anhydrous DMF, the temperature was raised to 35 ° C, the speed was controlled to 420 rpm, and the mixture was stirred for 0.5 h to obtain a modified solution. The cerium-doped oxide prepared in Comparative Preparation Example 2 was ultrasonically dispersed in anhydrous DMF, the ultrasonic frequency was controlled to 30 kHz, the ultrasonic power was 500 w, and the ultrasound was performed for 16 min. The modified solution and deionized water were added, the speed was maintained unchanged, and the stirring reaction was continued for 4.2 h. After the reaction was completed, the filter cake was filtered, and the filter cake was washed with anhydrous ethanol and deionized water 3 times each, and dried at 50 ° C to constant weight to obtain a carboxylated cerium-doped oxide, wherein the mass ratio of γ-aminopropyltriethoxysilane, succinic anhydride and anhydrous DMF in the modified solution was 2.2:1.3:26, and the mass ratio of cerium-doped oxide, anhydrous DMF, modifying solution and deionized water was 3:84:2.6:5;

[0070] Step B2, ultrasonically disperse the hydroxymethyl chitosan in an aqueous solution of acetic acid with a mass fraction of 1%, control the ultrasonic frequency to 40 kHz, the ultrasonic power to 600 w, and ultrasonicate for 12 min, then add phytic acid and p-toluenesulfonic acid, heat to 64 ° C, stir and react at a speed of 540 rpm for 0.6 h, then heat to 74 ° C, add a mixture of carboxylated cerium doped oxide and anhydrous DMF dropwise, control the dripping within 10 min, and after the dripping, maintain the speed unchanged, continue stirring and reacting for 1.6 h, control the rotary evaporation temperature to 66 ° C, rotary evaporation to remove the solution, sequentially wash with anhydrous ethanol and deionized water 3 times each, and dry at 62 ° C to constant weight to obtain esterified chitosan, wherein the mass ratio of hydroxymethyl chitosan, acetic acid aqueous solution, phytic acid, p-toluenesulfonic acid and mixed solution a is 4:110:1.2:0.06:30, and the mass ratio of carboxylated cerium doped oxide and anhydrous DMF in the mixed solution a is 0.8:35.

[0071] Comparative Preparation Example 5

[0072] This comparative preparation example provides an esterified chitosan, which is prepared by the following steps:

[0073] Step B1, adding γ-aminopropyltriethoxysilane and succinic anhydride to anhydrous DMF, heating to 35°C, controlling the speed to 420rpm, stirring for 0.5h to obtain a modified solution, ultrasonically dispersing the cerium-doped oxide prepared in Preparation Example 1 in anhydrous DMF, controlling the ultrasonic frequency to 30kHz, the ultrasonic power to 500w, and ultrasonication for 16min, adding the modified solution and deionized water, maintaining the speed unchanged, and continuing to stir and react for 4.2h. After the reaction is completed, filtering, the filter cake is washed with anhydrous ethanol and deionized water three times each, and dried at 50°C to constant weight to obtain a carboxylated cerium-doped oxide, wherein the mass ratio of γ-aminopropyltriethoxysilane, succinic anhydride and anhydrous DMF in the modified solution is 2.2:1.3:26, and the mass ratio of cerium-doped oxide, anhydrous DMF, modifying solution and deionized water is 3:84:2.6:5;

[0074] Step B2, the hydroxymethyl chitosan is ultrasonically dispersed in 1% acetic acid aqueous solution, the ultrasonic frequency is controlled to be 40 kHz, the ultrasonic power is 600 w, ultrasonic is performed for 12 min, then lactic acid and p-toluenesulfonic acid are added, the temperature is increased to 64 DEG C, stirring is performed at 540 rpm for 0.6 h, then the temperature is increased to 74 DEG C, a mixed solution a of carboxylated cerium doped oxide and anhydrous DMF is added dropwise in 10 min, after dropping, stirring is continuously performed at the same speed for 1.6 h, the rotary evaporation temperature is controlled to be 66 DEG C, rotary evaporation is performed until the solution is removed, then the obtained product is washed with anhydrous ethanol and deionized water respectively for 3 times, and drying is performed at 62 DEG C until the weight is constant, thereby obtaining esterified chitosan, wherein the mass ratio of hydroxymethyl chitosan, acetic acid aqueous solution, lactic acid, p-toluenesulfonic acid and the mixed solution a is 4:110:1.2:0.06:30, and the mass ratio of carboxylated cerium doped oxide and anhydrous DMF in the mixed solution a is 0.8:35.

[0075] Preparation Examples 7-9 and Comparative Preparation Examples 6-8 provide a modified polyether sulfone filter membrane.

[0076] Preparation Example 7

[0077] The present preparation example provides a modified polyether sulfone filter membrane, which is prepared by the following steps:

[0078] Step A1, the polyether sulfone filter membrane is placed in a mixed solution of 18% ethanol aqueous solution and sodium hydroxide, and immersed for 2.6 h, then the polyether sulfone membrane is taken out, washed with deionized water for 3 times, and dried at 56 DEG C, thereby obtaining a hydroxylated polyether sulfone filter membrane, wherein the mass ratio of the polyether sulfone filter membrane, the ethanol aqueous solution and the sodium hydroxide is 0.1:30:10.

[0079] Step A2, the hydroxylated polyether sulfone filter membrane and the esterified chitosan prepared in Preparation Example 4 are added into 18% ethanol aqueous solution, the temperature is increased to 45 DEG C, stirring is performed at a speed of 320 rpm for 1.4 h, after the reaction is completed, the obtained product is washed with deionized water for 3 times, and dried at 52 DEG C until the weight is constant, thereby obtaining a modified polyether sulfone filter membrane, wherein the mass ratio of the hydroxylated polyether sulfone filter membrane, the esterified chitosan and the ethanol aqueous solution is 5:0.42:120.

[0080] Preparation Example 8

[0081] The present preparation example provides a modified polyether sulfone filter membrane, which is prepared by the following steps:

[0082] Step A1, the polyether sulfone filter membrane is placed in a mixed solution of 20% ethanol aqueous solution and sodium hydroxide, and immersed for 3.0 h, then the polyether sulfone membrane is taken out, washed with deionized water for 4 times, and dried at 58 DEG C, thereby obtaining a hydroxylated polyether sulfone filter membrane, wherein the mass ratio of the polyether sulfone filter membrane, the ethanol aqueous solution and the sodium hydroxide is 0.2:35:10.

[0083] Step A2, add the hydroxylated polyethersulfone filter membrane and the esterified chitosan prepared in Preparation Example 5 to a 20% mass fraction of ethanol aqueous solution, heat to 50°C, control the speed to 360 rpm, and stir to react for 2.0 hours. After the reaction is completed, wash with deionized water 4 times and dry at 56°C to constant weight to obtain a modified polyethersulfone filter membrane, wherein the mass ratio of the hydroxylated polyethersulfone filter membrane, the esterified chitosan and the ethanol aqueous solution is 5.5:0.48:140.

[0084] Preparation Example 9

[0085] This preparation example provides a modified polyethersulfone filter membrane, which is prepared by the following steps:

[0086] Step A1, placing a polyethersulfone filter membrane in a mixed solution of 22% by mass ethanol aqueous solution and sodium hydroxide, soaking for reaction for 3.4 hours, removing the polyethersulfone membrane, washing it with deionized water five times, and drying it at 60°C to obtain a hydroxylated polyethersulfone filter membrane, wherein the mass ratio of the polyethersulfone filter membrane, the ethanol aqueous solution, and the sodium hydroxide is 0.3:40:10;

[0087] Step A2, add the hydroxylated polyethersulfone filter membrane and the esterified chitosan prepared in Preparation Example 6 to a 22% mass fraction of ethanol aqueous solution, heat to 55 ° C, control the speed to 400 rpm, stir and react for 2.6 hours. After the reaction is completed, wash with deionized water 5 times and dry at 60 ° C to constant weight to obtain a modified polyethersulfone filter membrane, wherein the mass ratio of the hydroxylated polyethersulfone filter membrane, esterified chitosan and ethanol aqueous solution is 6:0.54:160.

[0088] Comparative Preparation Example 6

[0089] This preparation example provides a modified polyethersulfone filter membrane, which is prepared by the following steps:

[0090] Step A1, placing a polyethersulfone filter membrane in a mixed solution of 18% by mass ethanol aqueous solution and sodium hydroxide, soaking for reaction for 2.6 hours, removing the polyethersulfone membrane, washing it three times with deionized water, and drying it at 56°C to obtain a hydroxylated polyethersulfone filter membrane, wherein the mass ratio of the polyethersulfone filter membrane, ethanol aqueous solution, and sodium hydroxide is 0.1:30:10;

[0091] Step A2, add the hydroxylated polyethersulfone filter membrane and the esterified chitosan prepared in Comparative Preparation Example 3 to an ethanol aqueous solution with a mass fraction of 18%, heat to 45 ° C, control the speed to 320 rpm, stir and react for 1.4 hours, after the reaction is completed, wash with deionized water 3 times, and dry at 52 ° C to constant weight to obtain a modified polyethersulfone filter membrane, wherein the mass ratio of the hydroxylated polyethersulfone filter membrane, esterified chitosan and ethanol aqueous solution is 5:0.42:120.

[0092] Comparative Preparation Example 7

[0093] This preparation example provides a modified polyethersulfone filter membrane, which is prepared by the following steps:

[0094] Step A1, placing a polyethersulfone filter membrane in a mixed solution of 18% by mass ethanol aqueous solution and sodium hydroxide, soaking for reaction for 2.6 hours, removing the polyethersulfone membrane, washing it three times with deionized water, and drying it at 56°C to obtain a hydroxylated polyethersulfone filter membrane, wherein the mass ratio of the polyethersulfone filter membrane, ethanol aqueous solution, and sodium hydroxide is 0.1:30:10;

[0095] Step A2, add the hydroxylated polyethersulfone filter membrane and the esterified chitosan prepared in Comparative Preparation Example 4 to an ethanol aqueous solution with a mass fraction of 18%, heat to 45 ° C, control the speed to 320 rpm, stir and react for 1.4 hours, after the reaction is completed, wash with deionized water 3 times, and dry at 52 ° C to constant weight to obtain a modified polyethersulfone filter membrane, wherein the mass ratio of the hydroxylated polyethersulfone filter membrane, esterified chitosan and ethanol aqueous solution is 5:0.42:120.

[0096] Comparative Preparation Example 8

[0097] This preparation example provides a modified polyethersulfone filter membrane, which is prepared by the following steps:

[0098] Step A1, placing a polyethersulfone filter membrane in a mixed solution of 18% by mass ethanol aqueous solution and sodium hydroxide, soaking for reaction for 2.6 hours, removing the polyethersulfone membrane, washing it three times with deionized water, and drying it at 56°C to obtain a hydroxylated polyethersulfone filter membrane, wherein the mass ratio of the polyethersulfone filter membrane, ethanol aqueous solution, and sodium hydroxide is 0.1:30:10;

[0099] Step A2, add the hydroxylated polyethersulfone filter membrane and the esterified chitosan prepared in Comparative Preparation Example 5 to an ethanol aqueous solution with a mass fraction of 18%, heat to 45 ° C, control the speed to 320 rpm, stir and react for 1.4 hours, after the reaction is completed, wash with deionized water 3 times, and dry at 52 ° C to constant weight to obtain a modified polyethersulfone filter membrane, wherein the mass ratio of the hydroxylated polyethersulfone filter membrane, esterified chitosan and ethanol aqueous solution is 5:0.42:120.

[0100] Examples 1-3 and Comparative Examples 1-5 provide a method for preparing mussel mucin. Example 1

[0101] This embodiment provides a method for preparing mussel mucin, comprising the following steps:

[0102] Step S1, defatting: fresh mussels are cleaned and shelled, the meat is retained and minced to obtain chopped mussel meat, which is then defatted and crushed to pass through a 260-mesh sieve to obtain defatted mussel powder, wherein the defatting process is specifically as follows: the chopped mussel meat is placed in a 76% (w / v) ethanol aqueous solution at a temperature of 46° C. for 4.8 hours, with a material-liquid mass ratio of 1:25, and then washed with deionized water until neutral;

[0103] Step S2, deodorization treatment: adding the defatted mussel powder to a 46% ethanol aqueous solution by mass, homogenizing at 8000 rpm for 4 minutes, maintaining the temperature at 20°C, centrifuging at 4000 rpm for 5 minutes, collecting the precipitate, and drying at 36°C to constant weight to obtain deodorized mussel defatted powder, wherein the mass ratio of the defatted mussel powder to the ethanol aqueous solution is 1:28;

[0104] Step S3, acid extraction: dissolving the deodorized mussel defatted powder, aluminum citrate and salicylic acid in deionized water, adding an 18% mass fraction citric acid aqueous solution to adjust the pH to 1.8, heating to 42°C, controlling the speed to 550 rpm, stirring and extracting for 1 hour, centrifuging, collecting the supernatant, adding a 3% mass fraction sodium hydroxide aqueous solution, adjusting the pH of the supernatant to 4, centrifuging at a speed of 4000 rpm for 6 minutes, collecting the precipitate, and then washing it with anhydrous ethanol and deionized water three times each, controlling the freeze-drying temperature to -32°C, the vacuum degree to 12 Pa, and the drying time to 8 hours to obtain a crude mussel mucin extract, wherein the mass ratio of the deodorized mussel defatted powder, aluminum citrate, salicylic acid and deionized water is 1:0.04:0.02:60;

[0105] Step S4, enzymatic hydrolysis: adding the crude mussel mucin extract to deionized water, adjusting the pH to 7.0 with a Tris-HCl buffer with a mass fraction of 0.5%, then adding neutral protease and papain, performing a primary enzymatic hydrolysis, controlling the enzymatic hydrolysis temperature to 42° C., and the enzymatic hydrolysis time to 2.2 h, centrifuging, and taking the supernatant to obtain a primary enzymatic hydrolyzate; adjusting the pH of the primary enzymatic hydrolyzate to 6.5 with a 0.02% acetic acid aqueous solution, then adding trypsin and composite protease, performing a secondary enzymatic hydrolysis, controlling the enzymatic hydrolysis temperature to 44° C., and the enzymatic hydrolysis time to 2.8 h, to obtain a secondary enzymatic hydrolyzate, wherein the mass ratio of the crude mussel mucin extract, deionized water, neutral protease and papain is 2:80:0.008:0.005, and the mass ratio of the primary enzymatic hydrolyzate, trypsin and composite protease is 80:0.01:0.018;

[0106] Step S5, fine extraction: The secondary enzymatic hydrolysate was concentrated by ultrafiltration and freeze-dried, the freeze-drying temperature was controlled at -34°C, the vacuum degree was 11 Pa, and the drying time was 10 hours to obtain mussel mucin, wherein the ultrafiltration concentration refers to: sequentially using three groups of modified polyethersulfone filter membranes prepared in Preparation Example 7 to ultrafilter the secondary enzymatic hydrolysate three times, wherein the modified polyethersulfone filter membrane has a molecular weight cutoff of 10 kDa, a pressure of 0.1 MPa, a temperature of 12°C, a pH of 6.5, a concentration multiple of 6 times, and a cross-flow velocity of 1 m / s. Example 2

[0107] This embodiment provides a method for preparing mussel mucin, comprising the following steps:

[0108] Step S1, defatting: clean the fresh mussels, remove the shells, retain the meat and mince it to obtain chopped mussel meat, defatted it, and crushed it to pass through a 280-mesh sieve to obtain defatted mussel powder, wherein the defatting process is specifically as follows: put the chopped mussel meat into 78% (w / v) ethanol aqueous solution at a temperature of 49° C. for 5.1 hours, with a material-liquid mass ratio of 1:30, and rinse with deionized water until neutral;

[0109] Step S2, deodorization treatment: adding mussel defatted powder to ethanol aqueous solution, homogenizing at 9000 rpm for 5 minutes, maintaining the temperature at 25°C, centrifuging at 5000 rpm for 7 minutes, collecting the precipitate, and drying at 38°C to constant weight to obtain deodorized mussel defatted powder, wherein the mass ratio of mussel defatted powder to ethanol aqueous solution is 1:32;

[0110] Step S3, acid extraction: dissolving the deodorized mussel defatted powder, aluminum citrate and salicylic acid in deionized water, adding a 20% mass fraction of citric acid aqueous solution to adjust the pH value to 2.0, heating to 44°C, controlling the speed to 580 rpm, stirring and extracting for 1.5 hours, centrifuging, collecting the supernatant, adding a 4% mass fraction of sodium hydroxide aqueous solution to adjust the pH of the supernatant to 4.5, centrifuging at a speed of 4200 rpm for 7 minutes, collecting the precipitate, and then washing it with anhydrous ethanol and deionized water 4 times each, controlling the freeze-drying temperature to -36°C, the vacuum degree to 14 Pa, and the drying time to 10 hours to obtain a crude mussel mucin extract, wherein the mass ratio of the deodorized mussel defatted powder, aluminum citrate, salicylic acid and deionized water is 2:0.06:0.035:70;

[0111] Step S4, enzymatic hydrolysis: adding the crude mussel mucin extract to deionized water, adjusting the pH to 7.1 with 1% Tris-HCl buffer by mass, adding neutral protease and papain, performing a primary enzymatic hydrolysis, controlling the enzymatic hydrolysis temperature to 44° C., and the enzymatic hydrolysis time to 2.4 h, centrifuging, and taking the supernatant to obtain a primary enzymatic hydrolyzate; adjusting the pH of the primary enzymatic hydrolyzate to 6.7 with a 0.04% acetic acid aqueous solution, and then adding trypsin and compound protease to perform a secondary enzymatic hydrolysis, controlling the enzymatic hydrolysis temperature to 47° C., and the enzymatic hydrolysis time to 3 h to obtain a secondary enzymatic hydrolyzate, wherein the mass ratio of the crude mussel mucin extract, deionized water, neutral protease and papain is 3:90:0.017:0.008, and the mass ratio of the primary enzymatic hydrolyzate, trypsin and compound protease is 90:0.012:0.02;

[0112] Step S5, fine extraction: The secondary enzymatic hydrolysate was concentrated by ultrafiltration and freeze-dried, the freeze-drying temperature was controlled at -36°C, the vacuum degree was 14 Pa, and the drying time was 11 hours to obtain mussel mucin, wherein the ultrafiltration concentration refers to: sequentially using the modified polyethersulfone filter membrane prepared in Preparation Example 8 to ultrafilter the secondary enzymatic hydrolysate three times, wherein the modified polyethersulfone filter membrane has a molecular weight cutoff of 11 kDa, the pressure is 0.2 MPa, the temperature is 18°C, the pH is 7.0, the concentration ratio is 7 times, and the cross flow velocity is 2 m / s. Example 3

[0113] This embodiment provides a method for preparing mussel mucin, comprising the following steps:

[0114] Step S1, defatting: fresh mussels are cleaned and shelled, the meat is retained and minced to obtain chopped mussel meat, which is then defatted and crushed to pass through a 300-mesh sieve to obtain defatted mussel powder, wherein the defatting process is specifically as follows: the chopped mussel meat is placed in an 80% (w / v) ethanol aqueous solution at a temperature of 52° C. for 5.4 hours, with a material-liquid mass ratio of 1:35, and then washed with deionized water until neutral;

[0115] Step S2, deodorization treatment: adding mussel defatted powder to ethanol aqueous solution, homogenizing at a speed of 10000 rpm for 6 minutes while maintaining the temperature at 25°C, centrifuging at a speed of 6000 rpm for 9 minutes, collecting the precipitate, and drying at 40°C to constant weight to obtain deodorized mussel defatted powder, wherein the mass ratio of mussel defatted powder to ethanol aqueous solution is 1:36;

[0116] Step S3, acid extraction: dissolving the deodorized mussel defatted powder, aluminum citrate and salicylic acid in deionized water, adding a 22% mass fraction citric acid aqueous solution to adjust the pH to 2.2, heating to 46°C, controlling the speed to 610 rpm, stirring and extracting for 2 hours, centrifuging, collecting the supernatant, adding a 5% mass fraction sodium hydroxide aqueous solution, adjusting the pH of the supernatant to 5, centrifuging at a speed of 4400 rpm for 8 minutes, collecting the precipitate, and then washing it with anhydrous ethanol and deionized water 5 times each in sequence, controlling the freeze-drying temperature to -40°C, the vacuum degree to 16 Pa, and the drying time to 12 hours to obtain a crude mussel mucin extract, wherein the mass ratio of the deodorized mussel defatted powder, aluminum citrate, salicylic acid and deionized water is 3:0.08:0.05:80;

[0117] Step S4, enzymatic hydrolysis: adding the crude mussel mucin extract to deionized water, adjusting the pH to 7.2 with a Tris-HCl buffer with a mass fraction of 1.5%, then adding neutral protease and papain, performing a primary enzymatic hydrolysis, controlling the enzymatic hydrolysis temperature to 46°C, and the enzymatic hydrolysis time to 2.6 hours, centrifuging, and taking the supernatant to obtain a primary enzymatic hydrolyzate; adjusting the pH of the primary enzymatic hydrolyzate to 6.8 with a 0.06% acetic acid aqueous solution, then adding trypsin and compound protease, performing a secondary enzymatic hydrolysis, controlling the enzymatic hydrolysis temperature to 50°C, and the enzymatic hydrolysis time to 3.2 hours to obtain a secondary enzymatic hydrolyzate, wherein the mass ratio of the crude mussel mucin extract, deionized water, neutral protease and papain is 4:100:0.026:0.011, and the mass ratio of the primary enzymatic hydrolyzate, trypsin and compound protease is 100:0.014:0.022;

[0118] Step S5, fine extraction: The secondary enzymatic hydrolysate was concentrated by ultrafiltration and freeze-dried, the freeze-drying temperature was controlled at -38°C, the vacuum degree was 17 Pa, and the drying time was 12 hours to obtain mussel mucin, wherein the ultrafiltration concentration refers to: sequentially using the modified polyethersulfone filter membrane prepared in Preparation Example 9 to ultrafilter the secondary enzymatic hydrolysate three times, wherein the modified polyethersulfone filter membrane has a molecular weight cutoff of 12 kDa, the pressure is 0.3 MPa, the temperature is 24°C, the pH is 7.5, the concentration multiple is 8 times, and the cross flow velocity is 3 m / s.

[0119] Comparative Example 1

[0120] Comparative Example 1 is the same as Example 1, except that the modified polyethersulfone filter membrane in Example 1 is replaced by the modified polyethersulfone filter membrane prepared in Comparative Preparation Example 6.

[0121] Comparative Example 2

[0122] Comparative Example 2 is the same as Example 1, except that the modified polyethersulfone filter membrane in Example 1 is replaced by the modified polyethersulfone filter membrane prepared in Comparative Preparation Example 7.

[0123] Comparative Example 3

[0124] Comparative Example 3 is the same as Example 1, except that the modified polyethersulfone filter membrane in Example 1 is replaced by the modified polyethersulfone filter membrane prepared in Comparative Preparation Example 8.

[0125] Comparative Example 4

[0126] Comparative Example 4 is the same as Example 1, except that the aluminum citrate in step S3 of Example 1 is replaced by citric acid.

[0127] Comparative Example 5

[0128] Comparative Example 5 is the same as Example 1, except that salicylic acid in step S3 of Example 1 is replaced by benzoic acid.

[0129] Performance testing

[0130] The comprehensive properties of the mussel mucins prepared in Examples 1-3 and Comparative Examples 1-5 of the present application are as follows:

[0131] The protein nitrogen content in the supernatant and the enzymatic hydrolyzate was determined by Kjeldahl method, and the content of the purified mussel mucin in Examples 1-2 and Comparative Examples 1-5 was calculated, and the yield was calculated according to the following formula:

[0132]

[0133] Purity: Determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE);

[0134] The above specific test results are shown in Table 1 below.

[0135] Table 1 Performance parameters of mussel mucin prepared in Examples 1-3 and Comparative Examples 1-5

[0136]

[0137] As can be seen from Table 1, compared with Comparative Examples 1-5, the mussel mucin prepared by the preparation method of mussel mucin provided by Examples 1-3 has higher yield and purity.

[0138] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A method for preparing mussel mucin, characterized in that: The following steps are involved: Step S1, defatting: clean the fresh mussels, remove the shells, retain the meat and mince it to obtain chopped mussel meat, then defatted, crushed, and sieved to obtain defatted mussel powder; Step S2, deodorizing treatment: adding mussel defatted powder to ethanol aqueous solution, homogenizing, centrifuging, collecting precipitate, and drying to constant weight to obtain deodorized mussel defatted powder; Step S3, acid extraction: dissolving deodorized mussel defatted powder, aluminum citrate, and salicylic acid in deionized water, adding an acid value regulating solution to adjust the pH to 1.8-2.2, stirring and extracting, centrifuging, collecting the supernatant, adjusting the pH of the supernatant to 4-5, centrifuging, collecting the precipitate, washing, and freeze-drying to obtain a crude mussel mucin extract; Step S4, enzymatic hydrolysis: adding the crude mussel mucin extract to deionized water, adjusting the pH to 7.0-7.2, then adding neutral protease and papain, performing a primary enzymatic hydrolysis, centrifuging, and collecting the supernatant to obtain a primary enzymatic hydrolyzate; then adjusting the pH of the primary enzymatic hydrolyzate to 6.5-6.8, then adding trypsin and compound protease, and performing a secondary enzymatic hydrolysis to obtain a secondary enzymatic hydrolyzate; Step S5, refined extraction: the secondary enzymatic hydrolysate is concentrated by ultrafiltration and freeze-dried to obtain mussel mucin; In step S5, ultrafiltration concentration refers to: ultrafiltration of the secondary enzymatic hydrolysate using a modified polyethersulfone filter membrane; The modified polyethersulfone filter membrane is prepared by the following steps: Step A1, placing a polyethersulfone filter membrane in a mixed solution of ethanol aqueous solution and sodium hydroxide, soaking for reaction for 2.6-3.4 hours, removing the polyethersulfone membrane, washing and drying it to obtain a hydroxylated polyethersulfone filter membrane; Step A2, adding the hydroxylated polyethersulfone filter membrane and the esterified chitosan to an ethanol aqueous solution, heating to 45-55° C., stirring and reacting for 1.4-2.6 hours, and after the reaction is completed, washing and drying to obtain a modified polyethersulfone filter membrane; The esterified chitosan is prepared by the following steps: Step B1, adding a silane coupling agent and succinic anhydride to anhydrous DMF, heating to 35-45°C, stirring for 0.5-0.8h to obtain a modified solution, ultrasonically dispersing the cerium-doped oxide in anhydrous DMF, adding the modified solution and deionized water, stirring and reacting for 4.2-5.4h, and after the reaction is completed, filtering with suction, washing and drying the filter cake to obtain a carboxylated cerium-doped oxide; Step B2, ultrasonically dispersing hydroxymethyl chitosan in an acetic acid aqueous solution, then adding phytic acid and p-toluenesulfonic acid, heating to 64-72°C, stirring and reacting for 0.6-1.0h, then heating to 74-80°C, and dropwise adding a mixture a of the carboxylated cerium-doped oxide and anhydrous DMF, controlling the dripping to be completed within 10min, and continuing to stir and react for 1.6-2.2h after completion of the dripping, rotary evaporation, washing, and drying to obtain esterified chitosan; The cerium-doped oxide is prepared by the following steps: Add zirconium oxychloride octahydrate, yttrium nitrate hexahydrate and cerium nitrate to the ethanol aqueous solution, stir evenly, then add dropwise to the ammonia aqueous solution, stir for 0.4-0.8 hours, let stand for 1.2-1.6 hours, wash, dry, grind, sieve, and then calcine at 790-820°C for 1.6-2.2 hours to obtain cerium-doped oxide.

2. The method for preparing mussel mucin according to claim 1, wherein In step S1, the defatting process includes: placing the chopped mussel meat in a 76-80% (w / v) ethanol aqueous solution at a temperature of 46-52° C. for 4.8-5.4 hours, with a material-liquid mass ratio of 1:25-35, and washing with deionized water until neutral.

3. The method for preparing mussel mucin according to claim 1, wherein In step S2, the mass fraction of the ethanol aqueous solution is 46-52%. In step S3, the acid value regulating liquid is a citric acid aqueous solution with a mass fraction of 18-22%. In step S4, the enzymatic hydrolysis temperature of the first enzymatic hydrolysis is 42-46° C., the enzymatic hydrolysis time is 2.2-2.6 h, and the enzymatic hydrolysis temperature of the second enzymatic hydrolysis is 44-50° C., and the enzymatic hydrolysis time is 2.8-3.2 h.

4. The method for preparing mussel mucin according to claim 1, wherein In step A1, the mass ratio of the polyethersulfone filter membrane, the ethanol aqueous solution and the sodium hydroxide is 0.1-0.3:30-40:

10. In step A2, the mass ratio of the hydroxylated polyethersulfone filter membrane, the esterified chitosan and the ethanol aqueous solution is 5-6:0.42-0.54:120-160.

5. The method for preparing mussel mucin according to claim 1, wherein In the step B1, in the modification liquid, the mass ratio of the silane coupling agent, succinic anhydride and anhydrous DMF is 2.2-2.6:1.3-1.5:26-30, the mass ratio of the cerium-doped oxide, anhydrous DMF, the modification liquid and deionized water is 3-5:84-90:2.6-3:5-7, and in the step B2, the mass ratio of hydroxymethyl chitosan, acetic acid aqueous solution, phytic acid, p-toluenesulfonic acid and mixed solution a is 4-6:110-140:1.2-1.6:0.06-0.12:30-40, and in the mixed solution a, the mass ratio of the carboxylated cerium-doped oxide and anhydrous DMF is 0.8-1:35-45.

6. The method for preparing mussel mucin according to claim 1, wherein The mass ratio of the ethanol aqueous solution, zirconium oxychloride octahydrate, yttrium nitrate hexahydrate, cerium nitrate and ammonia aqueous solution is 160-200:12:1-2:1.2-2.4:24-30, and the mass fraction of the ammonia aqueous solution is 24-26%.

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