Method for extracting and purifying mussel mucoprotein

Abstract

The invention belongs to the field of biological material extraction, and particularly relates to a mussel mucoprotein extraction and purification method which comprises the following steps: S1, immersing mussel byssus gland tissues into a buffer solution containing ascorbic acid and ethylenediamine tetraacetic acid for cleaning, and removing connective tissues; s2, carrying out pulse type negative pressure-positive pressure alternate treatment on the pretreated byssus gland tissue, and simultaneously carrying out shearing treatment in a positive pressure treatment stage, so as to obtain the cell disruption liquid. And S3, separating the cell disruption liquid through a hollow fiber membrane to obtain a concentrated solution. And S4, loading the concentrated solution into a chromatographic column filled with agarose microspheres of which the surfaces are loaded with polydopamine-iron ion composite ligands, and carrying out adsorption, washing and elution operations to obtain the high-purity mussel mucoprotein. Through cooperation of multiple steps, the problems of low cell disruption efficiency, damaged mussel mucoprotein activity, long desalination time, serious active group oxidation, low chromatographic purification adsorption rate and low product purity in the traditional process are effectively solved.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterial extraction, and in particular to a method for extracting and purifying mussel mucin. Background Art

[0002] Mussel Adhesive Protein (MAP) has unique strong adhesion properties mediated by 3,4-dihydroxyphenylalanine (DOPA). It has the functions of promoting cell adhesion and crawling, promoting wound healing, inhibiting itching, broad-spectrum adhesion, and forming a water-resistant protective film. It can be widely used in surface chemistry, biomedicine, marine engineering, daily chemical products and other fields.

[0003] The existing industrial extraction and purification of mussel mucin relies primarily on the traditional process of "mechanical disruption-salting precipitation-ion exchange chromatography." This process first places mussel foot gland tissue in a homogenizer for high-speed shearing, disrupting cells and releasing intracellular proteins. Saturated ammonium sulfate solution is then added to the disrupted liquid, and the precipitated protein is collected by centrifugation. The precipitated protein is then redissolved in a buffer solution and dialyzed to remove the ammonium sulfate. Finally, the target protein is eluted through ion exchange chromatography to obtain the mussel mucin.

[0004] Although the above process is widely used, the following problems still exist in its various operation links:

[0005] During the mechanical disruption stage, homogenization can only disrupt some cells, and the operation needs to be repeated 3-4 times to increase the protein release rate. However, repeated application of high-speed shear force will lead to loss of protein activity. In the salting out and dialysis stages, dialysis desalination treatment is time-consuming and will accelerate the oxidation of the active group DOPA, resulting in an increase in the oxidation rate of the active groups of mussel mucin. In the chromatographic purification stage, the anion exchange medium has a low adsorption rate for mussel mucin, and multiple cycles of loading are required to improve the recovery rate. In addition, high salt elution is generally used in this stage, which induces protein aggregation and results in low product purity.

[0006] Therefore, it is necessary to provide a mussel mucin extraction and purification scheme that can improve cell disruption efficiency, reduce mussel mucin activity loss, shorten desalination time, reduce the oxidation of the active group DOPA, and at the same time improve the chromatographic purification adsorption rate and product purity. Summary of the Invention

[0007] (1) Technical issues to be resolved

[0008] In order to solve the problems of low cell disruption efficiency, impaired mussel mucin activity, long desalting time, easy oxidation of the active group DOPA, low chromatographic purification adsorption rate and product purity in the existing technology, the present invention provides a method for extracting and purifying mussel mucin.

[0009] (2) Technical solution

[0010] In order to achieve the above objectives, the main technical solutions adopted by the present invention include:

[0011] The present invention provides a method for extracting and purifying mussel mucin, comprising the following steps:

[0012] S1: Immerse the mussel's byssal gland tissue in a buffer solution containing ascorbic acid and ethylenediaminetetraacetic acid for cleaning, remove the connective tissue, and obtain the pretreated byssal gland tissue;

[0013] S2: The pretreated foot silk gland tissue is subjected to a pulsed negative pressure-positive pressure alternating treatment, and a shear treatment is performed simultaneously during the positive pressure treatment stage to obtain a cell disruption solution;

[0014] S3: Separate the cell lysate through a hollow fiber membrane to obtain a concentrated solution;

[0015] S4: The concentrated solution is loaded onto a chromatography column filled with a biomimetic dopamine ligand affinity chromatography medium, and adsorption, washing, and elution operations are performed to obtain high-purity mussel mucin;

[0016] The biomimetic dopamine ligand affinity chromatography medium is agarose microspheres with polydopamine-iron ion composite ligands loaded on the surface.

[0017] In the above-mentioned method for extracting and purifying mussel mucin, preferably, in step S1, the buffer is a phosphate buffer with a pH of 6.5-7.0, wherein the concentration of ascorbic acid is 3-5 mmol / L, and the concentration of ethylenediaminetetraacetic acid is 0.5-1.5 mmol / L;

[0018] In step S1, the byssal gland tissue of the mussel is ultrasonically cleaned for 10-20 minutes.

[0019] In the above-mentioned method for extracting and purifying mussel mucin, preferably, in step S2, in the pulsed negative pressure-positive pressure alternating treatment, the negative pressure is (-80kPa)-(-90kPa), the positive pressure is 50kPa-60kPa, the alternating frequency is 8-12 times / min, and the number of cycles of the pulsed negative pressure-positive pressure alternating treatment is 25-35 times;

[0020] The rotation speed of the shearing process is 1000-3000 rpm.

[0021] In the above-mentioned method for extracting and purifying mussel mucin, preferably, in step S3, the molecular weight cutoff of the hollow fiber membrane is 30-40 kDa, the transmembrane pressure is 0.1-0.2 MPa, and the flow rate of the cell disruption solution is 8-12 L / min.

[0022] In the above-mentioned method for extracting and purifying mussel mucin, preferably, in step S3, during the separation process, the feeding direction of the cell disruption solution is switched every 3-5 minutes, and Tris-HCl buffer containing 1-2 mmol / L ascorbic acid is supplemented.

[0023] In the above-mentioned method for extracting and purifying mussel mucin, preferably, in step S4, in the biomimetic dopamine ligand affinity chromatography medium, the loading amount of the polydopamine-iron ion complex ligand on the surface of the agarose microspheres is 1-3 mg / g.

[0024] In the above-mentioned method for extracting and purifying mussel mucin, preferably, the preparation method of the biomimetic dopamine ligand affinity chromatography medium is as follows:

[0025] A1: Wash the agarose microspheres and then add deionized water to obtain an agarose microsphere suspension;

[0026] A2: Add dopamine hydrochloride to Tris-HCl buffer, stir to obtain a dopamine polymerization reaction solution, add the dopamine polymerization reaction solution to the agarose microsphere suspension, and stir to obtain agarose microspheres coated with polydopamine;

[0027] A3: Adding agarose microspheres coated with a polydopamine coating to a trivalent iron salt solution, stirring and reacting to obtain agarose microspheres loaded with a polydopamine-iron ion complex ligand on the surface.

[0028] In the above-mentioned method for extracting and purifying mussel mucin, preferably, in step A2, the concentration of dopamine hydrochloride in the dopamine polymerization reaction solution is 5-15 mg / mL; the dopamine polymerization reaction solution is added to the agarose microsphere suspension, and stirred for 10-16 hours to obtain agarose microspheres with a surface coated with polydopamine;

[0029] In step A3, the ferric salt solution is a ferric chloride solution, a ferric sulfate solution or a ferric nitrate solution, the concentration of iron ions in the ferric salt solution is 0.05-0.2 mol / L, and the stirring reaction time is 3-6 hours.

[0030] In the above-mentioned method for extracting and purifying mussel mucin, preferably, in step S4, the eluent used in the elution operation is a citric acid-sodium citrate buffer containing 40-60 mmol / L ascorbic acid and 0.1-0.2 mol / L sodium citrate, the pH of the buffer is 4.5-5.5, and the flow rate of the eluent is 1.0-2.0 mL / min.

[0031] (3) Beneficial effects

[0032] First, the buffer solution containing ascorbic acid and ethylenediaminetetraacetic acid used in the present invention is used to clean the byssal gland tissue of the mussel. Ascorbic acid can prevent the oxidation of mussel mucin, and ethylenediaminetetraacetic acid can chelate metal ions. The two work together to protect the activity of mussel mucin.

[0033] Second, the present invention adopts pulsed negative pressure-positive pressure alternating treatment combined with shear treatment in the positive pressure stage, which can significantly improve the efficiency of cell disruption, avoid the problem of damage to mussel mucin activity caused by repeated operations, and at the same time avoid the inactivation of mussel mucin caused by continuous high-speed shearing.

[0034] Third, the present invention uses hollow fiber membranes to separate the cell lysate, resulting in a gentler separation process that further reduces the risk of damaging the activity of mussel mucin. Furthermore, compared to traditional dialysis, hollow fiber membrane separation significantly shortens desalination time and reduces oxidation of the active DOPA group.

[0035] Fourth, the present invention uses agarose microspheres with surface-loaded polydopamine-iron ion complex ligands as chromatography media. Its biomimetic design can specifically bind to mussel mucin. The iron ions form specific coordination bonds with the catechol groups of the mussel mucin, achieving targeted adsorption, significantly improving the adsorption rate, and reducing impurities, thereby obtaining mussel mucin with higher purity. DETAILED DESCRIPTION

[0036] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below in conjunction with specific embodiments.

[0037] The present invention provides a method for extracting and purifying mussel mucin, comprising the following steps:

[0038] S1: Immerse the mussel byssal gland tissue in a buffer containing ascorbic acid and ethylenediaminetetraacetic acid for cleaning, remove the connective tissue, and obtain the pretreated byssal gland tissue. Ascorbic acid prevents the oxidation of mussel mucin, while ethylenediaminetetraacetic acid chelates metal ions, preventing metal ion-catalyzed oxidation of mussel mucin. The two work together to protect the activity of mussel mucin.

[0039] S2: The pretreated byssal gland tissue is subjected to alternating pulsed negative and positive pressure, and shearing is performed simultaneously during the positive pressure phase to produce a cell disruption solution. Using alternating pulsed negative and positive pressure combined with shearing during the positive pressure phase significantly improves cell disruption efficiency, avoiding the potential for damage to mussel mucin activity caused by repeated manipulations and the inactivation of mussel mucin caused by continuous high-speed shearing.

[0040] S3: The cell lysate is separated and processed using a hollow fiber membrane to obtain a concentrated solution. This separation process is gentle and can further reduce the risk of damage to the mussel mucin activity. Furthermore, compared to traditional dialysis, hollow fiber membrane separation significantly shortens desalination time and reduces oxidation of the active DOPA group.

[0041] S4: The concentrated liquid is loaded onto a chromatography column filled with a biomimetic dopamine ligand affinity chromatography medium, and adsorption, washing, and elution operations are performed to obtain high-purity mussel mucin. The biomimetic dopamine ligand affinity chromatography medium is agarose microspheres with a surface-loaded polydopamine-iron ion complex ligand. The biomimetic design of agarose microspheres loaded with a surface-loaded polydopamine-iron ion complex ligand is used as a chromatography medium. The biomimetic design of the agarose microspheres can specifically bind to mussel mucin. The iron ions form specific coordination bonds with the catechol groups of the mussel mucin, achieving targeted adsorption, significantly improving the adsorption rate, and reducing impurities, thereby obtaining mussel mucin of higher purity.

[0042] Preferably, in step S1, the buffer is a phosphate buffer with a pH of 6.5-7.0, wherein the concentration of ascorbic acid is 3-5 mmol / L and the concentration of ethylenediaminetetraacetic acid is 0.5-1.5 mmol / L. Furthermore, in step S1, the mussel's byssal gland tissue is ultrasonically cleaned for 10-20 minutes. Ultrasonic cleaning utilizes the cavitation effect of ultrasound to more thoroughly remove connective tissue and surface impurities.

[0043] Preferably, in the above-mentioned step S2, in the pulsed negative pressure-positive pressure alternating process, negative pressure is (-80kPa)-(-90kPa), positive pressure is 50kPa-60kPa, and alternating frequency is 8-12 times / min, and the number of cycles of the pulsed negative pressure-positive pressure alternating process is 25-35 times, and the rotating speed of shearing process is 1000-3000rpm. The above-mentioned pressure range can form an effective pressure difference on the cell surface, so that the cell membrane is subjected to periodic stretching and extrusion, destroying the structure of the cell, thereby achieving cell fragmentation. The alternating frequency of 8-12 times / min can both ensure that the rhythm of pressure change is moderate, so that the cell fragmentation effect is best, and will not cause the cell fragmentation to be too violent and impaired due to the excessive frequency, and through 25-35 cycles, it can be ensured that the thoroughness of cell fragmentation. In addition, shearing process is carried out in the positive pressure treatment stage, and the rotating speed is 1000-3000rpm. Appropriate rotating speed can further assist cell fragmentation without destroying the protein activity, so that the fragmentation effect is more ideal.

[0044] Preferably, in step S3, the hollow fiber membrane has a molecular weight cutoff of 30-40 kDa, a transmembrane pressure of 0.1-0.2 MPa, and the cell lysis solution has a flow rate of 8-12 L / min. During the separation process, the feed direction of the cell lysis solution is switched every 3-5 minutes, and Tris-HCl buffer containing 1-2 mmol / L ascorbic acid is supplemented.

[0045] The hollow fiber membrane has a molecular weight cutoff of 30-40 kDa, allowing small molecules such as salt ions and small impurities to pass through the membrane while retaining the larger mussel mucin molecules, thereby separating and concentrating the cell lysate. Switching the feed direction of the cell lysate every 3-5 minutes prevents clogging of the membrane surface with cell debris and impurities, ensuring the continuity and stability of the separation process. Supplementing the solution with Tris-HCl buffer containing ascorbic acid provides antioxidant properties, protecting the mussel mucin from oxidation.

[0046] Preferably, in the above step S4, in the biomimetic dopamine ligand affinity chromatography medium, the loading amount of the polydopamine-iron ion complex ligand on the surface of the agarose microspheres is 1-3 mg / g.

[0047] The particle size of agarose microspheres is 50-150 μm.

[0048] The appropriate loading allows the polydopamine-iron complex ligand to form a uniform and stable distribution on the surface of the agarose microspheres. When a sample containing mussel mucin passes through the chromatography column, the iron ions form specific coordination bonds with the catechol groups of the mussel mucin, achieving targeted adsorption. A loading range of 1-3 mg / g can maintain adsorption efficiency while reducing nonspecific adsorption, improving separation selectivity and purity.

[0049] In the present invention, the effects of polydopamine are as follows:

[0050] First, polydopamine has good biocompatibility, which ensures that the chromatography medium will not negatively affect the structure and activity of mussel mucin when in contact with it.

[0051] Second, polydopamine has strong adhesion and can spontaneously adsorb to the surface of agarose microspheres. This adhesion allows polydopamine to stably adhere to the matrix of the chromatography medium, providing a stable foundation for the subsequent loading of iron ions. If only iron ions are loaded without polydopamine, the iron ions will not be firmly bound to the agarose microspheres, and problems such as iron ion shedding may occur, affecting the performance and stability of the chromatography medium.

[0052] Third, the catechol structure on polydopamine can form a stable coordination bond with iron ions. This coordination effect results in a specific distribution and orientation of iron ions on the polydopamine surface. Mussel mucin contains catechol groups. The polydopamine-iron ion composite ligand can achieve efficient targeted adsorption of mussel mucin through the specific interaction between iron ions and the catechol groups on mussel mucin. This synergistic effect greatly enhances the adsorption capacity of the chromatography medium for the target protein, enabling more effective enrichment and purification of mussel mucin compared to loading only iron ions.

[0053] Fourth, polydopamine can also provide protection for iron ions, reducing their oxidation and precipitation in the environment. Specifically, polydopamine can stabilize the existence of iron ions by complexing with them, reducing the possibility of their oxidation and precipitation, thereby improving the stability and service life of the chromatography medium.

[0054] Further preferably, the preparation method of the biomimetic dopamine ligand affinity chromatography medium is as follows:

[0055] A1: Wash the agarose microspheres to remove impurities on the surface of the microspheres, and then add deionized water to obtain an agarose microsphere suspension.

[0056] A2: Add dopamine hydrochloride to Tris-HCl buffer and stir to obtain a dopamine polymerization reaction solution. Add the dopamine polymerization reaction solution to the agarose microsphere suspension and stir for 10-16 hours to uniformly coat the agarose microspheres with polydopamine, thereby obtaining agarose microspheres coated with polydopamine. The concentration of dopamine hydrochloride in the dopamine polymerization reaction solution is 5-15 mg / mL.

[0057] A3: Add polydopamine-coated agarose microspheres to a ferric salt solution and stir for 3-6 hours. The ferric ions react with the polydopamine to form a polydopamine-iron ion complex ligand, resulting in agarose microspheres loaded with the polydopamine-iron ion complex ligand. The ferric salt solution can be ferric chloride, ferric sulfate, or ferric nitrate. The iron concentration in the ferric salt solution is 0.05-0.2 mol / L.

[0058] Preferably, in the above step S4, the eluent used in the elution operation is a citric acid-sodium citrate buffer containing 40-60 mmol / L ascorbic acid and 0.1-0.2 mol / L sodium citrate, the pH of the buffer is 4.5-5.5, and the flow rate of the eluent is 1.0-2.0 mL / min.

[0059] Ascorbic acid in the eluent acts as a strong reducing agent, reducing ferric iron to ferrous iron, breaking its coordination bond with mussel mucin. Furthermore, ascorbic acid simultaneously scavenges free radicals in the system, preventing DOPA oxidation. Sodium citrate provides citrate ions, which form a stable tetracoordinate complex with ferrous iron, competitively replacing the catechol group of DOPA and completely blocking the rebinding of ferrous iron to DOPA in mussel mucin.

[0060] In order to further clarify the solution of the present invention and its technical advancement, the following description is made in conjunction with specific embodiments and technical effects.

[0061] Example 1

[0062] This embodiment provides a method for extracting and purifying mussel mucin, comprising the following steps:

[0063] S1: Immerse the mussel byssal gland tissue in a buffer solution and ultrasonically clean it for 15 minutes to remove connective tissue, thereby obtaining pretreated byssal gland tissue. In this step, the buffer solution is a phosphate buffer solution with a pH of 6.8, wherein the concentration of ascorbic acid is 4 mmol / L and the concentration of ethylenediaminetetraacetic acid is 1.0 mmol / L.

[0064] S2: The pretreated foot silk gland tissue was subjected to alternating pulsed negative and positive pressure treatment, and shearing was performed simultaneously during the positive pressure treatment to obtain a cell disruption solution. In this step, the negative pressure was -85 kPa, the positive pressure was 55 kPa, the alternating frequency was 10 times / min, the number of pulsed negative and positive pressure alternating cycles was 30, and the shearing speed was 2000 rpm.

[0065] S3: Separate the cell lysate using a hollow fiber membrane to obtain a concentrate. The hollow fiber membrane has a molecular weight cutoff of 35 kDa, a transmembrane pressure of 0.15 MPa, and a flow rate of 10 L / min. During the separation process, the feed direction of the cell lysate is switched every 4 minutes and supplemented with Tris-HCl buffer containing 1.5 mmol / L ascorbic acid.

[0066] S4: The concentrated solution is loaded onto a chromatography column filled with agarose microspheres loaded with a polydopamine-iron ion complex ligand on the surface, and adsorption, washing and elution operations are performed to obtain high-purity mussel mucin. The loading amount of polydopamine-iron ion complex ligand on the surface of the agarose microspheres is 2 mg / g. The particle size of the agarose microspheres is 100 μm. In this step, the eluent is a citric acid-sodium citrate buffer containing 50 mmol / L ascorbic acid and 0.15 mol / L sodium citrate, the pH of the buffer is 5, and the flow rate of the eluent is 1.5 mL / min.

[0067] In step S4, the preparation method of agarose microspheres loaded with polydopamine-iron ion complex ligands on the surface is as follows:

[0068] A1: Wash the agarose microspheres, then add deionized water to obtain an agarose microsphere suspension.

[0069] A2: Add dopamine hydrochloride to Tris-HCl buffer and stir to obtain a dopamine polymerization reaction solution. Add the dopamine polymerization reaction solution to the agarose microsphere suspension and stir for 14 hours to obtain agarose microspheres coated with polydopamine. The concentration of dopamine hydrochloride in the dopamine polymerization reaction solution is 8 mg / mL.

[0070] A3: Add polydopamine-coated agarose microspheres to a ferric salt solution and stir for 5 hours to obtain agarose microspheres loaded with polydopamine-iron ion complex ligands. The ferric salt solution is a 0.12 mol / L ferric chloride solution.

[0071] Example 2

[0072] This embodiment provides a method for extracting and purifying mussel mucin, comprising the following steps:

[0073] S1: Immerse the mussel byssal gland tissue in a buffer solution and ultrasonically clean it for 10 minutes to remove connective tissue, thereby obtaining pretreated byssal gland tissue. In this step, the buffer solution is a phosphate buffer solution with a pH of 6.5, wherein the concentration of ascorbic acid is 3 mmol / L and the concentration of ethylenediaminetetraacetic acid is 0.5 mmol / L.

[0074] S2: The pretreated foot silk gland tissue was subjected to alternating pulsed negative and positive pressure treatment, and shearing was performed simultaneously during the positive pressure treatment to obtain a cell disruption solution. In this step, the negative pressure was -80 kPa, the positive pressure was 50 kPa, the alternating frequency was 8 times / min, the number of pulsed negative and positive pressure alternating cycles was 25, and the shearing speed was 1000 rpm.

[0075] S3: Separate the cell lysate using a hollow fiber membrane to obtain a concentrate. The hollow fiber membrane has a molecular weight cutoff of 30 kDa, a transmembrane pressure of 0.1 MPa, and a flow rate of 8 L / min. During the separation process, the feed direction of the cell lysate is switched every 3 minutes and supplemented with Tris-HCl buffer containing 1 mmol / L ascorbic acid.

[0076] S4: The concentrated solution is loaded onto a chromatography column filled with agarose microspheres loaded with a polydopamine-iron ion complex ligand on the surface, and adsorption, washing and elution operations are performed to obtain high-purity mussel mucin. The loading amount of polydopamine-iron ion complex ligand on the surface of the agarose microspheres is 1 mg / g. The particle size of the agarose microspheres is 150 μm. In this step, the eluent is a citric acid-sodium citrate buffer containing 40 mmol / L ascorbic acid and 0.1 mol / L sodium citrate, the pH of the buffer is 5.5, and the flow rate of the eluent is 1.0 mL / min.

[0077] In step S4, the preparation method of agarose microspheres loaded with polydopamine-iron ion complex ligands on the surface is as follows:

[0078] A1: Wash the agarose microspheres, then add deionized water to obtain an agarose microsphere suspension.

[0079] A2: Add dopamine hydrochloride to Tris-HCl buffer and stir to obtain a dopamine polymerization reaction solution. Add the dopamine polymerization reaction solution to the agarose microsphere suspension and stir for 10 hours to obtain agarose microspheres coated with polydopamine. The concentration of dopamine hydrochloride in the dopamine polymerization reaction solution is 5 mg / mL.

[0080] A3: Add the polydopamine-coated agarose microspheres to a ferric salt solution and stir for 3 hours to obtain agarose microspheres loaded with a polydopamine-iron complex ligand. The ferric salt solution is a 0.05 mol / L ferric sulfate solution.

[0081] Example 3

[0082] This embodiment provides a method for extracting and purifying mussel mucin, comprising the following steps:

[0083] S1: Immerse the mussel byssal gland tissue in a buffer solution and ultrasonically clean it for 20 minutes to remove connective tissue, thereby obtaining pretreated byssal gland tissue. In this step, the buffer solution is a phosphate buffer solution with a pH of 7.0, wherein the concentration of ascorbic acid is 5 mmol / L and the concentration of ethylenediaminetetraacetic acid is 1.5 mmol / L.

[0084] S2: The pretreated foot silk gland tissue was subjected to alternating pulsed negative and positive pressure treatment, and shearing was performed simultaneously during the positive pressure treatment to obtain a cell disruption solution. In this step, the negative pressure was -90 kPa, the positive pressure was 60 kPa, the alternating frequency was 12 times / min, the number of pulsed negative and positive pressure alternating cycles was 35, and the shearing speed was 3000 rpm.

[0085] S3: The cell lysate was separated using a hollow fiber membrane to obtain a concentrate. The hollow fiber membrane had a molecular weight cutoff of 40 kDa, a transmembrane pressure of 0.2 MPa, and a flow rate of 12 L / min. During the separation process, the feed direction of the cell lysate was switched every 5 minutes and supplemented with Tris-HCl buffer containing 2 mmol / L ascorbic acid.

[0086] S4: The concentrated solution is loaded onto a chromatography column filled with agarose microspheres loaded with a polydopamine-iron ion complex ligand on the surface, and adsorption, washing and elution operations are performed to obtain high-purity mussel mucin. The loading amount of polydopamine-iron ion complex ligand on the surface of the agarose microspheres is 3 mg / g. The particle size of the agarose microspheres is 50 μm. In this step, the eluent is a citric acid-sodium citrate buffer containing 60 mmol / L ascorbic acid and 0.2 mol / L sodium citrate, the pH of the buffer is 4.5, and the flow rate of the eluent is 2.0 mL / min.

[0087] In step S4, the preparation method of agarose microspheres loaded with polydopamine-iron ion complex ligands on the surface is as follows:

[0088] A1: Wash the agarose microspheres, then add deionized water to obtain an agarose microsphere suspension.

[0089] A2: Add dopamine hydrochloride to Tris-HCl buffer and stir to obtain a dopamine polymerization reaction solution. Add the dopamine polymerization reaction solution to the agarose microsphere suspension and stir for 16 hours to obtain agarose microspheres coated with polydopamine. The concentration of dopamine hydrochloride in the dopamine polymerization reaction solution is 15 mg / mL.

[0090] A3: Add polydopamine-coated agarose microspheres to a 0.2 mol / L ferric nitrate solution and stir for 6 hours to obtain polydopamine-iron complex ligand-loaded agarose microspheres.

[0091] The purity, DOPA oxidation rate and molecular weight of the mussel mucin prepared in Examples 1-3 were tested to obtain Table 1.

[0092] Table 1 Statistics of purity, DOPA oxidation rate and molecular weight of mussel mucin in Examples 1-3

[0093]

[0094]

[0095] As shown in Table 1, the present invention solves the problems of low purity, severe oxidation, and large activity loss of mussel mucin in traditional processes through the technology of antioxidant pretreatment-high-efficiency and low-loss crushing-dynamic membrane separation-bionic affinity chromatography. The purity of the prepared mussel mucin is ≥95%, the activity retention rate of DOPA is >95%, and the molecular weight is close to that of the natural state, providing reliable technical support for the large-scale production and application of mussel mucin.

[0096] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for extracting and purifying mussel mucin, characterized in that: The steps include: S1: Immerse the mussel's byssal gland tissue in a buffer solution containing ascorbic acid and ethylenediaminetetraacetic acid for cleaning, remove the connective tissue, and obtain the pretreated byssal gland tissue; S2: The pretreated foot silk gland tissue is subjected to a pulsed negative pressure-positive pressure alternating treatment, and a shear treatment is performed simultaneously during the positive pressure treatment stage to obtain a cell disruption solution; S3: Separate the cell lysate through a hollow fiber membrane to obtain a concentrated solution; S4: The concentrated solution is loaded onto a chromatography column filled with a biomimetic dopamine ligand affinity chromatography medium, and adsorption, washing, and elution operations are performed to obtain high-purity mussel mucin; The biomimetic dopamine ligand affinity chromatography medium is agarose microspheres with polydopamine-iron ion composite ligands loaded on the surface.

2. The method for extracting and purifying mussel mucin according to claim 1, wherein In step S1, the buffer solution is a phosphate buffer solution with a pH of 6.5-7.0, wherein the concentration of ascorbic acid is 3-5 mmol / L and the concentration of ethylenediaminetetraacetic acid is 0.5-1.5 mmol / L; In step S1, the byssal gland tissue of the mussel is ultrasonically cleaned for 10-20 minutes.

3. The method for extracting and purifying mussel mucin according to claim 1, wherein In step S2, in the pulsed negative pressure-positive pressure alternating treatment, the negative pressure is (-80 kPa)-(-90 kPa), the positive pressure is 50 kPa-60 kPa, the alternating frequency is 8-12 times / min, and the number of cycles of the pulsed negative pressure-positive pressure alternating treatment is 25-35 times; The rotation speed of the shearing process is 1000-3000 rpm.

4. The method for extracting and purifying mussel mucin according to claim 1, wherein In step S3, the molecular weight cut-off of the hollow fiber membrane is 30-40 kDa, the transmembrane pressure is 0.1-0.2 MPa, and the flow rate of the cell disruption solution is 8-12 L / min.

5. The method for extracting and purifying mussel mucin according to claim 4, wherein In step S3, during the separation process, the feeding direction of the cell disruption solution is switched every 3-5 minutes, and a Tris-HCl buffer containing 1-2 mmol / L ascorbic acid is added.

6. The method for extracting and purifying mussel mucin according to claim 1, wherein In step S4, in the biomimetic dopamine ligand affinity chromatography medium, the loading amount of the polydopamine-iron ion complex ligand on the surface of the agarose microspheres is 1-3 mg / g.

7. The method for extracting and purifying mussel mucin according to claim 6, wherein The preparation method of the biomimetic dopamine ligand affinity chromatography medium is as follows: A1: Wash the agarose microspheres and then add deionized water to obtain an agarose microsphere suspension; A2: Add dopamine hydrochloride to Tris-HCl buffer, stir to obtain a dopamine polymerization reaction solution, add the dopamine polymerization reaction solution to the agarose microsphere suspension, and stir to obtain agarose microspheres coated with polydopamine; A3: Adding agarose microspheres coated with a polydopamine coating to a trivalent iron salt solution, stirring and reacting to obtain agarose microspheres loaded with a polydopamine-iron ion complex ligand on the surface.

8. The method for extracting and purifying mussel mucin according to claim 7, wherein In step A2, the concentration of dopamine hydrochloride in the dopamine polymerization reaction solution is 5-15 mg / mL; the dopamine polymerization reaction solution is added to the agarose microsphere suspension, and stirred for 10-16 hours to obtain agarose microspheres coated with a polydopamine coating; In step A3, the ferric salt solution is a ferric chloride solution, a ferric sulfate solution or a ferric nitrate solution, the concentration of iron ions in the ferric salt solution is 0.05-0.2 mol / L, and the stirring reaction time is 3-6 hours.

9. The method for extracting and purifying mussel mucin according to claim 1, wherein In step S4, the eluent used in the elution operation is a citric acid-sodium citrate buffer containing 40-60 mmol / L ascorbic acid and 0.1-0.2 mol / L sodium citrate, the pH of the buffer is 4.5-5.5, and the flow rate of the eluent is 1.0-2.0 mL / min.

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