Method for extracting and purifying mussel mucoprotein

Abstract

The invention discloses a mussel mucoprotein extraction and purification method, which comprises: 1) taking mussel foot glands, adding an extraction liquid 1, soaking, homogenizing, precipitating, taking the precipitate, adding an extraction liquid 2, re-suspending, homogenizing, carrying out centrifugal filtration, and taking the filtrate to obtain an extract; and 2) purifying the extract obtained in the step 1). The mussel mucoprotein obtained by extracting through a specific extracting solution and then adopting a specific purification method is high in protein and dopa content, can be compatible with pharmaceutical auxiliary materials such as anionic polymer components carbomer, sodium hyaluronate and sodium carboxymethyl cellulose, is used for preparing medicines or skin care products, and is wide in application prospect.

Description

Technical Field

[0001] The invention belongs to the field of drug preparation, and particularly relates to a method for extracting and purifying mussel mucin. Background Art

[0002] Mussel mucin is a viscous protein secreted by the foot gland of marine mussels, with a molecular weight distribution of 10 to 100 kDa and rich in dopa groups, which give it strong adhesion properties and biological activity. This protein can effectively adhere to various solid surfaces, including inorganic and organic surfaces, which makes it widely used in surface chemistry, biomedicine and marine engineering. In the field of biomedicine, mussel mucin is widely used for its anti-inflammatory, antioxidant, cell adhesion and crawling and wound healing effects.

[0003] At present, the extraction and purification of natural mussel mucin is obtained by using acetic acid combined with denaturants (urea, guanidine hydrochloride, etc.) for tissue homogenization, separation and purification. However, this traditional method generally has a low extraction yield, and the obtained mussel mucin is incompatible with the anionic polymer components in external coating skin care products and medical dressing products due to the charge and hydrophilic / hydrophobic properties of the amino acid residues in the molecular structure. It has been reported that in order to avoid physical or chemical interactions during the compatibility process and affect the efficacy of the product, alkaline substances are used to eliminate the precipitation after the compatibility by adjustment, but whether the structural characteristics of the complex of mussel mucin and anionic materials after adjustment are completely restored to the original state and play its function, there is no experimental data to support it. Therefore, it is necessary to provide a method for preparing mussel mucin with a high yield of mussel mucin and no incompatibility with anionic polymer components. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a method for extracting and purifying mussel mucin, comprising the following steps:

[0005] 1) Take the foot gland of mussel, soak it in extracting solution-1, homogenize it, precipitate it, take the precipitate, resuspend it in extracting solution-2, homogenize it, centrifuge and filter it, take the filtrate to obtain the extract;

[0006] 2) Purifying the extract obtained in step 1) to obtain;

[0007] The extract-1 is an aqueous solution containing 50-150 mM neutral buffer salt, 20-30 mM EDTA, 0.5-1.5 M sodium chloride, 0.5-1.5 mM phenylmethylsulfonyl fluoride, 5-15 mM N-ethylmaleimide, and 25-75 mM ascorbic acid;

[0008] The extracting solution-2 is an aqueous solution containing 1-10% acid, 4-10M urea, 25-75mM ascorbic acid, 20-50mM EDTA, 5-15mM EGTA, 0.5-1.5mM phenylmethylsulfonyl fluoride, 5-15mM N-ethylmaleimide, 10-30mM glutathione and 0.005-0.015% Triton X-100.

[0009] Furthermore, the extracting solution-1 is an aqueous solution containing 50 mM neutral buffer salt, 25 mM EDTA, 1 M sodium chloride, 1 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide, and 50 mM ascorbic acid.

[0010] Furthermore, the neutral buffer salt includes sodium phosphate, acetate, tris(hydroxymethylaminomethane) hydrochloride, sodium borate, sodium carbonate, preferably tris(hydroxymethylaminomethane) hydrochloride.

[0011] Furthermore, the extracting solution-2 is an aqueous solution containing 5% acid, 8M urea, 50mM ascorbic acid, 25mM EDTA, 10mM EGTA, 1mM phenylmethylsulfonyl fluoride, 10mM N-ethylmaleimide, 20mM glutathione and 0.01% Triton X-100.

[0012] Furthermore, the acid comprises citric acid or acetic acid, preferably acetic acid.

[0013] Furthermore, in step 1), the mass volume ratio of the mussel byssus to the extract-1 is 1 g: 5-20 mL; the mussel foot gland includes mussel byssus and / or byssus gland;

[0014] The soaking time is 30 to 120 minutes;

[0015] The homogenizing speed is 5000-20000 rpm, and the time is 5-30 minutes;

[0016] The mass volume ratio of the precipitate to the extract-2 is 1 g: 5 to 7.5 mL;

[0017] The centrifugal speed is above 10000g;

[0018] The filtration is carried out by using 5-100 micron filter paper and / or 0.5-5 micron filter element.

[0019] Furthermore, the purification method in step 2) is dialysis;

[0020] The dialysis method is to place the extract in a dialysis bag, dialyze with a dialysis solution, filter the solution in the bag after dialysis, and collect the filtrate;

[0021] The dialysate is composed of 1# to 5# dialysates; each dialysate is dialyzed at least twice, and each dialysis time is greater than 5 hours;

[0022] The 1# dialysate is an aqueous solution containing 3-7% acetic acid, 6M urea and auxiliary components; the 2# dialysate is an aqueous solution containing 3-7% acetic acid, 4M urea and auxiliary components; the 3# dialysate is an aqueous solution containing 3-7% acetic acid, 2M urea and auxiliary components; the 4# dialysate is an aqueous solution containing 3-7% acetic acid and auxiliary components; the 5# dialysate is an aqueous solution containing 0.5-1.5% citric acid and auxiliary components;

[0023] The auxiliary components in the dialysate are 5-15 mM EDTA, 6-10 mM EGTA, 0.5-1.5 mM phenylmethylsulfonyl fluoride, 10-30 mM N-ethylmaleimide, and 0.005-0.015% Triton X-100.

[0024] Furthermore, the volume ratio of the extract to the dialysate is 1:200-1000;

[0025] The 1# dialysate is an aqueous solution containing 5% acetic acid, 6M urea and auxiliary components; the 2# dialysate is an aqueous solution containing 5% acetic acid, 4M urea and auxiliary components; the 3# dialysate is an aqueous solution containing 5% acetic acid, 2M urea and auxiliary components; the 4# dialysate is an aqueous solution containing 5% acetic acid and auxiliary components; the 5# dialysate is an aqueous solution containing 1% citric acid and auxiliary components;

[0026] The auxiliary components in the dialysate are 10 mM EDTA, 8 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 20 mM N-ethylmaleimide, and 0.01% Triton X-100;

[0027] The filtration is carried out using a 0.5-5 micron polyethersulfone filter element.

[0028] Furthermore, the purification method in step 2) is a flushing method;

[0029] The flushing method is to place the extract in a tangential flow system, flush it with a flushing liquid, and take the solution in the system after flushing;

[0030] The flushing liquid is composed of flushing liquid-1 and flushing liquid-2; flushing with flushing liquid-1 for 1 to 6 hours, and then flushing with flushing liquid-2 for 0.5 to 3 hours;

[0031] The flushing solution-1 is an aqueous solution containing 3-8% acetic acid, 2-5M urea and auxiliary components, and the flushing solution-2 is an aqueous solution containing 0.5-1.5% citric acid, 10-30mM glutathione and auxiliary components;

[0032] The auxiliary components in the washing solution are 5-15 mM EDTA, 6-10 mM EGTA, 0.5-1.5 mM phenylmethylsulfonyl fluoride, 15-25 mM N-ethylmaleimide, and 0.03-0.08% Triton X-100.

[0033] Furthermore, the filter membrane in the tangential flow system is a polyethersulfone membrane, a polyvinylidene fluoride membrane or a polyamide membrane;

[0034] The volume ratio of the extract to the flushing liquid is 1:2-20;

[0035] The flushing solution-1 is an aqueous solution containing 5% acetic acid, 3M urea and auxiliary components, and the flushing solution-2 is an aqueous solution containing 1% citric acid, 20mM glutathione and auxiliary components;

[0036] The auxiliary components in the washing solution are 10 mM EDTA, 8 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 20 mM N-ethylmaleimide, and 0.05% Triton X-100.

[0037] Finally, the invention provides a mussel mucin extract, which is prepared according to the above extraction and purification method.

[0038] The method for extracting and purifying mussel mucin of the present invention is to extract with a specific extracting solution and then obtain the mussel mucin by a specific purification method, wherein the mussel mucin has high protein and DOPA contents and can be compatible with drug excipients such as anionic polymer components carbomer, sodium hyaluronate, sodium carboxymethyl cellulose, etc., and can be used for the preparation of medicines or skin care products, and has broad application prospects.

[0039] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0040] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1Mussel mucin compatibility test (A-1: mussel mucin of the present invention and carbomer (0.3%) compatibility results, A-2: mussel mucin of the present invention and sodium hyaluronate 20w (0.3%) compatibility results, A-3: mussel mucin of the present invention and sodium carboxymethyl cellulose (0.2%) compatibility results; B-1: the extract obtained by the method of CN_101348520_B and carbomer (0.3%) compatibility results, B-2: the extract obtained by the method of CN_101348520_B and The compatibility results of sodium hyaluronate 20w (0.3%), B-3: The compatibility results of the extract obtained by the method in CN_101348520_B and sodium carboxymethyl cellulose (0.2%); C-1: The compatibility results of "recombinant mussel mucin" purchased on the market and carbomer (0.3%), C-2: The compatibility results of "recombinant mussel mucin" purchased on the market and sodium hyaluronate 20w (0.3%), C-3: The compatibility results of "recombinant mussel mucin" purchased on the market and sodium carboxymethyl cellulose (0.2%)) DETAILED DESCRIPTION

[0042] The raw materials, reagents and equipment used in the specific embodiments of the present invention are all purchased from commercial sources.

[0043] Example 1 Extraction and purification of mussel mucin

[0044] (I) Preparation of solution

[0045] Extraction solution-1: Take tris(hydroxymethyl)aminomethane hydrochloride, EDTA, sodium chloride, phenylmethylsulfonyl fluoride, N-ethylmaleimide and ascorbic acid, and dissolve them in water to prepare an aqueous solution containing 50 mM tris(hydroxymethyl)aminomethane hydrochloride, 25 mM EDTA, 1 M sodium chloride, 1 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide and 50 mM ascorbic acid;

[0046] Extraction solution-2: Take acetic acid, urea, ascorbic acid, EDTA, EGTA, phenylmethylsulfonyl fluoride, N-ethylmaleimide, glutathione and Triton X-100, dissolve in water to prepare an aqueous solution containing 5% (v / v) acetic acid, 8M urea, 50mM ascorbic acid, 25mM EDTA, 10mM EGTA, 1mM phenylmethylsulfonyl fluoride, 10mM N-ethylmaleimide, 20mM glutathione and 0.01% (v / v) Triton X-100;

[0047] 1# Dialysate: Take acetic acid, urea, EDTA, EGTA, phenylmethylsulfonyl fluoride, N-ethylmaleimide and Triton X-100, add water to dissolve to prepare an aqueous solution containing 5% (v / v) acetic acid, 6M urea, 10mM EDTA, 8mM EGTA, 1mM phenylmethylsulfonyl fluoride, 20mM N-ethylmaleimide and 0.01% (v / v) Triton X-100;

[0048] 2# Dialysate: Take acetic acid, urea, EDTA, EGTA, phenylmethylsulfonyl fluoride, N-ethylmaleimide and Triton X-100, add water to dissolve to prepare an aqueous solution containing 5% (v / v) acetic acid, 4M urea, 10mM EDTA, 8mM EGTA, 1mM phenylmethylsulfonyl fluoride, 20mM N-ethylmaleimide and 0.01% (v / v) Triton X-100;

[0049] 3# Dialysate: Take acetic acid, urea, EDTA, EGTA, phenylmethylsulfonyl fluoride, N-ethylmaleimide and Triton X-100, add water to dissolve to prepare an aqueous solution containing 5% (v / v) acetic acid, 2M urea, 10mM EDTA, 8mM EGTA, 1mM phenylmethylsulfonyl fluoride, 20mM N-ethylmaleimide and 0.01% (v / v) Triton X-100;

[0050] 4# Dialysate: Take acetic acid, urea, EDTA, EGTA, phenylmethylsulfonyl fluoride, N-ethylmaleimide and Triton X-100, add water to dissolve to prepare an aqueous solution containing 5% (v / v) acetic acid, 10mM EDTA, 8mM EGTA, 1mM phenylmethylsulfonyl fluoride, 20mM N-ethylmaleimide and 0.01% (v / v) Triton X-100;

[0051] 5# Dialysate: Take citric acid, EDTA, EGTA, phenylmethylsulfonyl fluoride, N-ethylmaleimide and Triton X-100, add water to dissolve to prepare an aqueous solution containing 1% (w / v) citric acid, 10mM EDTA, 8mM EGTA, 1mM phenylmethylsulfonyl fluoride, 20mM N-ethylmaleimide and 0.01% (v / v) Triton X-100;

[0052] (II) Preparation of mussel mucin

[0053] (1) Clean mussel byssus glands were soaked in "extract solution-1" at a solid-liquid ratio of 1 g:20 ml for 60 min, and then homogenized at 10000 rpm for 20 min. After natural precipitation, the precipitate was resuspended in "extract solution-2" at a solid-liquid ratio of 1 g:7.5 ml, homogenized at 10000 rpm for 20 min, and then centrifuged at a speed of 15000 g. The supernatant was filtered with a filter paper with a pore size of 50 μm, and the filtrate was filtered with a filter element (made of polyethersulfone, pore size of 1 μm) to obtain the extract;

[0054] (2) The extract obtained in step (1) is placed in a dialysis bag with a molecular weight cutoff of 2500 Daltons, and dialyzed with 1# to 5# dialysates in sequence, with the volume ratio of the extract to the dialysate being 1:500. Each dialysate is dialyzed twice, and each dialysis time is 6 hours; the liquid in the dialysis bag after dialysis is filtered with a filter element (made of polyethersulfone, pore size 1 μm), and the filtrate is taken to obtain mussel mucin.

[0055] Example 2 Extraction and purification of mussel mucin

[0056] (I) Preparation of solution

[0057] Extraction solution-1: Take tris(hydroxymethylaminomethane) hydrochloride, EDTA, sodium chloride, phenylmethylsulfonyl fluoride, N-ethylmaleimide and ascorbic acid, and dissolve them in water to prepare an aqueous solution containing 50 mM tris(hydroxymethylaminomethane) hydrochloride, 25 mM EDTA, 1 M sodium chloride, 1 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide and 50 mM ascorbic acid;

[0058] Extraction solution-2: Take acetic acid, urea, ascorbic acid, EDTA, EGTA, phenylmethylsulfonyl fluoride, N-ethylmaleimide, glutathione and Triton X-100, dissolve in water to prepare an aqueous solution containing 5% (v / v) acetic acid, 8M urea, 50mM ascorbic acid, 25mM EDTA, 10mM EGTA, 1mM phenylmethylsulfonyl fluoride, 10mM N-ethylmaleimide, 20mM glutathione and 0.01% (v / v) Triton X-100;

[0059] Flushing solution-1: Take acetic acid, urea, EDTA, EGTA, phenylmethylsulfonyl fluoride, N-ethylmaleimide and Triton X-100, add water to dissolve to prepare an aqueous solution containing 5% (v / v) acetic acid, 3M urea, 10mM EDTA, 8mM EGTA, 1mM phenylmethylsulfonyl fluoride, 20mM N-ethylmaleimide and 0.05% (v / v) Triton X-100;

[0060] Washing solution-2: Take citric acid, glutathione, EDTA, EGTA, phenylmethylsulfonyl fluoride, N-ethylmaleimide and Triton X-100, add water to dissolve to prepare an aqueous solution containing 1% (w / v) citric acid, 20mM glutathione, 10mM EDTA, 8mM EGTA, 1mM phenylmethylsulfonyl fluoride, 20mM N-ethylmaleimide and 0.05% (v / v) Triton X-100;

[0061] (II) Preparation of mussel mucin

[0062] (1) clean mussel byssus glands were soaked in "extract solution-1" at a solid-liquid ratio of 1 g:20 ml for 60 min, and then homogenized at 10000 rpm for 20 min. After natural precipitation, the precipitate was resuspended in "extract solution-2" at a solid-liquid ratio of 1 g:7.5 ml, homogenized at 10000 rpm for 20 min, and then centrifuged at a speed of 15000 g. The supernatant was filtered with a filter paper with a pore size of 50 μm, and the filtrate was filtered with a filter element with a pore size of 1 μm to obtain an extract;

[0063] (2) The extract obtained in step (1) is placed in a tangential flow system with a polyethersulfone membrane as the filter membrane, and is washed and desalted with a washing liquid-1 for 4 hours, and then washed and concentrated with a washing liquid-2 for 2 hours, and the concentrated liquid is obtained to obtain mussel mucin.

[0064] The beneficial effects of the present invention are specifically described below through test examples.

[0065] Experimental Example 1 Study on the extraction and purification method of mussel mucin

[0066] 1. Methods

[0067] Based on the preliminary tests, two basic processes were determined:

[0068] Basic process①:

[0069] (1) Clean mussel byssus glands were soaked in "extract solution-1" at a solid-liquid ratio of 1 g:20 ml for 60 min, and then homogenized at 10000 rpm for 20 min. After natural precipitation, the precipitate was resuspended in "extract solution-2" at a solid-liquid ratio of 1 g:7.5 ml, homogenized at 10000 rpm for 20 min, and then centrifuged at a speed of 15000 g. The supernatant was filtered with a filter paper with a pore size of 50 μm, and the filtrate was filtered with a filter element (made of polyethersulfone, pore size of 1 μm) to obtain the extract;

[0070] (2) The extract obtained in step (1) is placed in a dialysis bag with a molecular weight cutoff of 2500 Daltons, and dialyzed with 1# to 5# dialysates in sequence, with the volume ratio of the extract to the dialysate being 1:500. Each dialysate is dialyzed twice, and each dialysis time is 6 hours; the liquid in the dialysis bag after dialysis is filtered through a filter element (made of polyethersulfone, pore size 1 μm), and the filtrate is taken to obtain mussel mucin.

[0071] Basic process ②:

[0072] (1) Same as step (1) in basic process ①

[0073] (2) The extract obtained in step (1) is placed in a tangential flow system with a polyethersulfone membrane as the filter membrane (the tangential flow system is purchased from Shandong Bona Biotechnology Group Co., Ltd.), and is rinsed and desalted with rinse solution-1 for 4 hours, and then concentrated with rinse solution-2 for 2 hours, and the concentrated liquid is obtained to obtain mussel mucin.

[0074] The basic formulas of the solutions used are:

[0075] Extraction solution-1: neutral buffer salt (50 mM tris(hydroxymethyl)aminomethane hydrochloride), auxiliary stabilizer (25 mM EDTA, 1 M sodium chloride, 1 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide, 50 mM ascorbic acid), water;

[0076] Extraction solution-2: acidic salt (5% acetic acid), auxiliary components (8M urea), auxiliary stabilizers (25mM EDTA, 8mM EGTA, 1mM phenylmethylsulfonyl fluoride, 10mM N-ethylmaleimide, 20mM glutathione, 0.01% Triton X-100), water;

[0077] 1# dialysate: 5% acetic acid, auxiliary components (10 mM EDTA, 8 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 20 mM N-ethylmaleimide, 0.01% Triton X-100), 6 M urea, water;

[0078] 2# dialysate: 5% acetic acid, auxiliary components (10mM EDTA, 8mM EGTA, 1mM phenylmethylsulfonyl fluoride, 20mM N-ethylmaleimide, 0.01% Triton X-100), 4M urea, water;

[0079] 3# dialysate: 5% acetic acid, auxiliary components (10 mM EDTA, 8 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 20 mM N-ethylmaleimide, 0.01% Triton X-100), 2 M urea, water;

[0080] 4# dialysate: 5% acetic acid, auxiliary components (10 mM EDTA, 8 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 20 mM N-ethylmaleimide, 0.01% Triton X-100), water;

[0081] 5# dialysate: 1% citric acid, auxiliary components (10 mM EDTA, 8 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 20 mM N-ethylmaleimide, 0.01% Triton X-100), water;

[0082] Flushing solution-1: 5% acetic acid, auxiliary components (10 mM EDTA, 8 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 20 mM N-ethylmaleimide, 0.05% Triton X-100), 3 M urea, water;

[0083] Washing solution-2: 1% citric acid, 20 mM glutathione, auxiliary components (10 mM EDTA, 8 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 20 mM N-ethylmaleimide, 0.05% Triton X-100), and water.

[0084] 2. Inspection indicators

[0085] 2.1 Extraction rate of mussel mucin

[0086] 2.1.1 Protein content in mussel mucin

[0087] The test was carried out using the second method of forlin phenol method in the protein content determination method 0731 of the Pharmacopoeia of the People's Republic of China (Volume 4 of the 2015 edition).

[0088] 2.1.2 DOPA content in mussel mucin

[0089] The DOPA content test in Appendix B of the Pharmaceutical Industry Standard of the People's Republic of China YY_T 1293.6-2020 Contact Wound Dressings Part 6: Mussel Mucin Dressings was used for determination.

[0090] 2.2 Mussel mucin incompatibility test

[0091] Sodium hyaluronate, carbomer and sodium carboxymethyl cellulose are selected as the "anion-containing polymer materials compatible with mussel mucin. The specific compatibility test steps are as follows:

[0092] a Weigh 200,000 molecular weight sodium hyaluronate dry powder, add purified water to dissolve, and prepare 2% sodium hyaluronate mother solution;

[0093] b. Weigh Carbomer 980 dry powder, add purified water to dissolve, and prepare 1% Carbomer stock solution;

[0094] c. Weigh sodium carboxymethyl cellulose powder, add purified water to dissolve, and prepare 2% sodium carboxymethyl cellulose mother solution;

[0095] d. Mix the prepared sodium hyaluronate, carbomer, sodium carboxymethyl cellulose mother solution and triethanolamine with the mussel mucin solution to be tested according to the final concentration of each polymer material in Table 2, observe the changes after mixing, and record the experimental results;

[0096] eAnalyze the experimental results to determine whether there are any incompatibility contraindications.

[0097] 3. Preparation process investigation

[0098] The mussel mucin obtained by different preparation processes was tested for protein content and DOPA content, and a compatibility test was performed, see Tables 1-2.

[0099] Table 1 Investigation of different preparation processes

[0100]

[0101] Table 2 Compatibility observation results

[0102]

[0103] From the above results, it can be seen that the protein content of the mussel mucin solution extracted by basic process ① is equivalent to that of basic process ②, but the proportion of DOPA is higher, so basic process ① is the best.

[0104] 4. Extraction process parameter screening

[0105] The extraction process is completed in two steps. First, the extracting solution-1 is used to dissolve and release the impurity protein in the mussel byssus gland as much as possible. When the solid-liquid ratio exceeds a certain proportion, it has no effect on the removal of impurity protein. Preliminary experiments have determined that when the ratio of mussel byssus gland to extracting solution-1 is 1g:20ml, the efficiency of removing impurity protein reaches the maximum. On this basis, increasing the ratio of extracting solution-1 has no effect on the removal of impurity protein.

[0106] The precipitate after removing some impurities contains rich target protein and residual impurities. These proteins are in the acidic extract-2. If the ratio of precipitate to extract-2 is not properly controlled, it will affect the extraction rate of the target protein. Therefore, the liquid-to-precipitate ratio of precipitate to extract-2 was further screened.

[0107] 4.1 Screening of material-liquid ratio

[0108] 4.1.1 Ratio of the precipitate of the byssin glands extracted with extract-1 to that of extract-2

[0109] On the basis of basic process ①, the liquid-to-precipitate ratio of precipitate and extract-2 was adjusted to 1g:5ml, 1g:7.5mL, 1g:10ml, 1g:20ml, respectively. The protein and DOPA contents of the finally extracted mussel mucin and the observation results of incompatibility are shown in Table 3.

[0110] Table 3 Effect of the ratio of precipitate to extract-2 on mussel mucin

[0111]

[0112] As can be seen from Table 3, according to basic process ① extraction, when the solid-liquid ratio of precipitate to extract-2 is less than 1g:7.5ml, the protein and DOPA contents are significantly reduced. Therefore, it is necessary to control the solid-liquid ratio of precipitate to extract-2 in the range of 1g:5~7.5mL, with 1g:7.5mL being the best.

[0113] 4.2. Extraction solvent formulation screening

[0114] 4.2.1 Extraction Solution-1 Optimal Design

[0115] Based on step (1) of the basic process, the formula components and ratios of the extract-1 were adjusted. According to the preliminary experimental results, a total of 8 formulas were formulated, as shown in Table 4.

[0116]

[0117] Table 5 Results of investigation on extract content of different extracts-1 in the extraction process

[0118]

[0119] From the above results, it can be seen that among the 8 formulations of extract-1, formulation 4 has the highest protein content. Since the second stage of extraction and subsequent purification process need to continue to remove the impurity protein in the raw material, more protein should be retained as much as possible during the initial extraction process. Therefore, formulation 4, i.e., an aqueous solution prepared from neutral buffer salt (50mM tris(hydroxymethyl)aminomethane hydrochloride), auxiliary stabilizer (25mM EDTA, 1M sodium chloride, 1mM phenylmethylsulfonyl fluoride, 10mM N-ethylmaleimide, 50mM ascorbic acid) and water, is the best extraction solvent for preliminary removal of impurity protein and extraction of target protein.

[0120] 4.2.2 Optimal design of extract-2

[0121] Based on basic process ① and basic process ②, the optimal extract-1 formula determined in "4.2.1 Optimal design of extract-1" was used, and only the formula components and ratios of extract-2 were adjusted. According to the preliminary experimental results, a total of 6 formulas were proposed, as shown in Table 6.

[0122] Table 6 Extraction Solution-2 Formula (g / L)

[0123]

[0124] Using the extract-2 in Table 6 and the optimal extract-1 formula determined in "4.2.1 Optimal Design of Extract-1", extraction and purification were performed according to the preparation process of basic process ① and basic process ②, and the mussel mucin harvested in step (2) of the basic process was tested for protein content and dopa content, as well as incompatibility test. The results are shown in Tables 7 to 10.

[0125] Table 7 Investigation results of different extracts based on basic process ①-2

[0126]

[0127] Table 8 Observation results based on basic process ①

[0128]

[0129] Table 9 Investigation results of different extracts-2 based on basic process ②

[0130] Extract-2 No. Protein content ug / mL DOPA content ug / mL Dopa ratio (%) 1 5508 87.5 1.59 2 5453 245.3 4.50 3 2543 110.2 4.33 4 5537 253.2 4.57 5 4568 187.5 4.10 6 4238 176.2 4.16

[0131] Table 10 Observation results based on basic process ②

[0132]

[0133] From the above results, it can be seen that among the six formulations of extract-2, formulation 4 has a higher protein content and a higher DOPA content, has no incompatibility, and is suitable for extracting the target protein. Therefore, formulation 4, i.e., an aqueous solution prepared from acid (5% acetic acid), auxiliary stabilizer (8M urea, 50mM ascorbic acid), auxiliary components (25mM EDTA, 10mM EGTA, 1mM phenylmethylsulfonyl fluoride, 10mM N-ethylmaleimide, 20mM glutathione, 0.01% Triton X-100) and water, is the best extraction solvent to avoid the loss of target protein and DOPA in the byssus gland extract and the incompatibility with polymer materials.

[0134] 4.3. Purification solvent formulation screening

[0135] 4.3.1 Optimal design of 1#-5# dialysis fluid

[0136] On the basis of basic process ①, the optimal extract-1 formula and the optimal extract-2 formula were used, and only the 1#-5# dialysate (gradient) components and ratios were adjusted. According to the preliminary experimental results, a total of 4 formulas were formulated, as shown in Table 11.

[0137] Table 11 Dialysate formulation

[0138]

[0139] The dialyzate in Table 11 was used to extract and purify mussel mucin according to the preparation process of basic process ①, and the obtained mussel mucin was tested for incompatibility. The results are shown in Table 12.

[0140] Table 12 Observation results based on basic process ①

[0141]

[0142] From the above results, it can be seen that the mussel mucin obtained by purification using 1#-5# dialysate in Formula 1 has no incompatibility with common pharmaceutical excipients such as carbomer, sodium hyaluronate, and sodium carboxymethyl cellulose, indicating that the mussel byssus extract needs to be purified by gradient dialysis using 1#-5# dialysate.

[0143] 4.3.2 Optimal design of flushing fluid

[0144] Based on basic process ②, the optimal extract-1 formula and the optimal extract-2 formula were used to adjust the formula components and ratios of the flushing solution. According to the preliminary experimental results, a total of 5 formulas were formulated, as shown in Table 13.

[0145] Table 13 Formula of Flushing Liquid-1 and Flushing Liquid-2

[0146]

[0147] Using the flushing solution in Table 13, the mussel mucin was extracted and purified according to the preparation process of basic process ②, and the incompatibility test of the mussel mucin was performed. The results are shown in Table 14.

[0148] Table 14 Observation results based on basic process ②

[0149]

[0150] From the above results, it can be seen that when formula 1 is used as the flushing liquid in the tangential flow system to purify the byssus gland extract, the mussel mucin finally obtained will not have any incompatibility with the anionic polymer material, and can be used as the optimal flushing liquid for purifying the byssus gland extract in the tangential flow system.

[0151] From the above screening tests, it can be seen that:

[0152] 1) The process of extracting mussel mucin from mussel byssus significantly affects the protein and DOPA content in mussel mucin. When extracting mussel byssus glands that have been preliminarily deproteinized with extracting solution-2, the ratio of the extracted precipitate to the extracting solution-2 is controlled in the range of 1g:5-7.5mL, which can maximize the protein and DOPA content in the extract after the second stage of extraction, among which 1g:7.5mL is the best.

[0153] The experiment also found that the formula of the extract not only affects the extraction efficiency of protein and DOPA, but also affects the compatibility with anionic polymer materials. The formula of the most suitable extract determined by screening is:

[0154] Extraction solution-1: neutral buffer salt (50 mM Tris hydrochloride) and auxiliary stabilizers (25 mM EDTA, 1 M sodium chloride, 1 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide, 50 mM ascorbic acid), and water.

[0155] Extraction solution-2 is: acid (5% acetic acid), auxiliary stabilizer (8M urea, 50mM ascorbic acid), auxiliary components (25mM EDTA, 10mM EGTA, 1mM phenylmethylsulfonyl fluoride, 10mM N-ethylmaleimide, 20mM glutathione, 0.01% Triton X-100).

[0156] 2) The purification and concentration of mussel byssus extract can be carried out using a dialysis bag or a tangential flow system. The two purification and concentration processes have similar effects, but considering the proportion of DOPA, the dialysis effect of using a dialysis bag is better. The dialysate and flushing fluid used in the purification and concentration process have been found to affect the compatibility of the final mussel mucin product with the anionic polymer material.

[0157] When purifying and concentrating the mussel byssus extract by dialysis, 1#-5# dialysates are required to ensure that the mussel mucin final product does not have incompatibility with the anionic polymer material. The best formulas of 1#-5# dialysates are 5% 6M urea acetate, 5% 4M urea acetate, 5% 2M urea acetate, 5% acetic acid, and 1% citric acid, respectively. Auxiliary components (10mM EDTA, 8mM EGTA, 1mM phenylmethylsulfonyl fluoride, 20mM N-ethylmaleimide, and 0.01% Triton X-100) are added to each dialysate.

[0158] When purifying and concentrating the extract of mussel byssus through a tangential flow system, it is necessary to use flushing solution-1 and flushing solution-2 to ensure that the final product of mussel mucin will not have incompatibility with anionic polymer materials. The formula of flushing solution-1 is 5% acetic acid and 3M urea, and the formula of flushing solution-2 is 1% citric acid and 20mM glutathione. Auxiliary components (10mM EDTA, 8mM EGTA, 1mM phenylmethylsulfonyl fluoride, 20mM N-ethylmaleimide, 0.05% Triton X-100) are added to each flushing solution.

[0159] 5. Verification experiment

[0160] Since basic process ① has a higher proportion of DOPA in the mussel mucin solution obtained during extraction and purification than basic process ②, the optimal process obtained after optimizing basic process ①, namely Example 1, was further verified.

[0161] The experiment was repeated 5 times, and the prepared mussel mucin was a light yellow liquid solution. The test results are as follows:

[0162] Table 15 Verification results

[0163]

[0164] From the above results, it can be seen that the average protein content of mussel mucin obtained by the optimal process obtained after optimization of basic process ① is 5413.8ug / mL, the average DOPA content is 283.56ug / mL, and there is no incompatibility with anionic polymer materials.

[0165] 6. Comparative test

[0166] The mussel mucin extracted and purified according to the verified optimal process (Example 1), the extract extracted and purified by the method disclosed in CN_101348520_B, and the recombinant mussel mucin purchased on the market were tested for protein, DOPA and compatibility. The specific results are shown in Tables 16 to 17 and Figure 1 :

[0167] Table 16 Comparison of protein and dopa content in mussel mucin

[0168]

[0169] Table 17 Mussel mucin compatibility test results

[0170]

[0171] From the above, it can be seen that the mussel mucin obtained by the extraction and purification process of the present invention has advantages over the currently disclosed mussel mucin products in terms of both protein content and DOPA content, and solves the problem that the existing mussel mucin has incompatibility with anionic polymer materials and that mussel mucin cannot be directly used as a raw material for compounding with anionic polymer materials.

[0172] In summary, the mussel mucin obtained by the present invention through extraction with a specific extract and then purification by a specific method has high protein and DOPA content and can be compatible with anionic polymer components such as carbomer, sodium hyaluronate, sodium carboxymethyl cellulose and other pharmaceutical excipients for the preparation of medicines or skin care products, and has broad application prospects.

Claims

1. A method for extracting and purifying mussel mucin, characterized in that: The steps include: 1) Take the foot gland of mussel, soak it in extracting solution-1, homogenize it, precipitate it, take the precipitate, resuspend it in extracting solution-2, homogenize it, centrifuge and filter it, take the filtrate to obtain the extract; 2) Purifying the extract obtained in step 1) to obtain; The extract-1 is an aqueous solution containing 50-150 mM neutral buffer salt, 20-30 mM EDTA, 0.5-1.5 M sodium chloride, 0.5-1.5 mM phenylmethylsulfonyl fluoride, 5-15 mM N-ethylmaleimide, and 25-75 mM ascorbic acid; The extracting solution-2 is an aqueous solution containing 1-10% acid, 4-10M urea, 25-75mM ascorbic acid, 20-50mM EDTA, 5-15mM EGTA, 0.5-1.5mM phenylmethylsulfonyl fluoride, 5-15mM N-ethylmaleimide, 10-30mM glutathione and 0.005-0.015% Triton X-100.

2. The extraction and purification method according to claim 1, characterized in that: The extracting solution-1 is an aqueous solution containing 50 mM neutral buffer salt, 25 mM EDTA, 1 M sodium chloride, 1 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide, and 50 mM ascorbic acid.

3. The extraction and purification method according to claim 2, characterized in that: The neutral buffer salt includes sodium phosphate, acetate, tris(hydroxymethylaminomethane) hydrochloride, sodium borate, sodium carbonate, preferably tris(hydroxymethylaminomethane) hydrochloride.

4. The extraction and purification method according to claim 1, characterized in that: The extracting solution-2 is an aqueous solution containing 5% acid, 8M urea, 50mM ascorbic acid, 25mM EDTA, 10mM EGTA, 1mM phenylmethylsulfonyl fluoride, 10mM N-ethylmaleimide, 20mM glutathione and 0.01% Triton X-100.

5. The extraction and purification method according to claim 4, characterized in that: The acid includes citric acid and acetic acid, preferably acetic acid.

6. The extraction and purification method according to claim 1, characterized in that: Step 1) the mass volume ratio of the mussel byssus to the extract-1 is 1 g: 5-20 mL; the mussel foot gland includes mussel byssus and / or byssus gland; The soaking time is 30 to 120 minutes; The homogenizing speed is 5000-20000 rpm, and the time is 5-30 minutes; The mass volume ratio of the precipitate to the extract-2 is 1 g: 5 to 7.5 mL; The centrifugal speed is above 10000g; The filtration is carried out by using 5-100 micron filter paper and / or 0.5-5 micron filter element.

7. The extraction and purification method according to claim 1, characterized in that: Step 2) The purification method is dialysis; The dialysis method is to place the extract in a dialysis bag, dialyze with a dialysis solution, filter the solution in the bag after dialysis, and collect the filtrate; The dialysate is composed of 1# to 5# dialysates; each dialysate is dialyzed at least twice, and each dialysis time is greater than 5 hours; The 1# dialysate is an aqueous solution containing 3-7% acetic acid, 6M urea and auxiliary components; the 2# dialysate is an aqueous solution containing 3-7% acetic acid, 4M urea and auxiliary components; the 3# dialysate is an aqueous solution containing 3-7% acetic acid, 2M urea and auxiliary components; the 4# dialysate is an aqueous solution containing 3-7% acetic acid and auxiliary components; the 5# dialysate is an aqueous solution containing 0.5-1.5% citric acid and auxiliary components; The auxiliary components in the dialysate are 5-15 mM EDTA, 6-10 mM EGTA, 0.5-1.5 mM phenylmethylsulfonyl fluoride, 10-30 mM N-ethylmaleimide, and 0.005-0.015% Triton X-100.

8. The extraction and purification method according to claim 7, characterized in that: The volume ratio of the extract to the dialysate is 1:200-1000; The 1# dialysate is an aqueous solution containing 5% acetic acid, 6M urea and auxiliary components; the 2# dialysate is an aqueous solution containing 5% acetic acid, 4M urea and auxiliary components; the 3# dialysate is an aqueous solution containing 5% acetic acid, 2M urea and auxiliary components; the 4# dialysate is an aqueous solution containing 5% acetic acid and auxiliary components; the 5# dialysate is an aqueous solution containing 1% citric acid and auxiliary components; The auxiliary components in the dialysate are 10 mM EDTA, 8 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 20 mM N-ethylmaleimide, and 0.01% Triton X-100; The filtration is carried out using a 0.5-5 micron polyethersulfone filter element.

9. The extraction and purification method according to claim 1, characterized in that: Step 2) The purification method is a flushing method; The flushing method is to place the extract in a tangential flow system, flush it with a flushing liquid, and take the solution in the system after flushing; The flushing liquid is composed of flushing liquid-1 and flushing liquid-2; flushing with flushing liquid-1 for 1 to 6 hours, and then flushing with flushing liquid-2 for 0.5 to 3 hours; The flushing solution-1 is an aqueous solution containing 3-8% acetic acid, 2-5M urea and auxiliary components, and the flushing solution-2 is an aqueous solution containing 0.5-1.5% citric acid, 10-30mM glutathione and auxiliary components; The auxiliary components in the flushing solution are 5-15 mM EDTA, 6-10 mM EGTA, 0.5-1.5 mM phenylmethylsulfonyl fluoride, 15-25 mM N-ethylmaleimide, and 0.03-0.08% Triton X-100; The filter membrane in the tangential flow system is a polyethersulfone membrane, a polyvinylidene fluoride membrane or a polyamide membrane; The volume ratio of the extract to the flushing solution is 1:2 to 20; Preferred: The flushing solution-1 is an aqueous solution containing 5% acetic acid, 3M urea and auxiliary components, and the flushing solution-2 is an aqueous solution containing 1% citric acid, 20mM glutathione and auxiliary components; The auxiliary components in the washing solution are 10 mM EDTA, 8 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 20 mM N-ethylmaleimide, and 0.05% Triton X-100.

10. A mussel mucin extract, characterized in that: The product is prepared according to the extraction and purification method described in any one of claims 1 to 9.

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