A kind of earthworm protein and its preparation method and application

Abstract

The present application provides a kind of earth dragon protein and its preparation method and application, and the method comprises the following steps: S1, mixing earth dragon powder and ethanol and post-processing, collecting solid phase; S2, mixing the solid phase and citric acid solution and extracting, collecting liquid phase; S3, mixing the liquid phase obtained in step S2 and dopamine-modified activated carbon, collecting liquid phase; S4, ultrafiltration and freeze-drying of the liquid phase obtained in step S4. The present application extracts earth dragon powder treated with ethanol and citric acid, thereby improving the full dissolution of active substances in the earth dragon powder; then, dopamine-modified activated carbon is used to adsorb small molecule inorganic substances and pigments, thereby further improving the purity and activity of earth dragon protein; finally, impurities are further removed by ultrafiltration, thereby further improving the purity and activity of earth dragon protein.

Description

Technical Field

[0001] The present application relates to the technical field of protein extraction, and in particular to earthworm protein and its preparation method and application. Background Art

[0002] Geosaurus, also known as earthworm, belongs to the class Oligochaeta in the phylum Annelida and is an animal-based traditional Chinese medicine. Studies have shown that earthworms have the potential to promote blood circulation and remove blood stasis, dissolve blood clots and lower blood pressure, fight tumors, relieve asthma and cough, and alleviate inflammation and pain.

[0003] Earthworm protein is extracted from earthworms and contains collagenase, plasmin, lumbrokinase, nucleic acid, trace elements and other components. Its molecular weight is between 5,000 and 10,000, and it is a short-chain small molecule substance.

[0004] Earthworm protein contains a variety of components, including collagenase, plasmin, nucleic acids, and trace elements. It has a wide range of effects on the body's coagulation and fibrinolytic systems, significantly reducing platelet adhesion, prolonging thrombosis, and dissolving thrombi. It also increases cerebral blood flow, reduces cerebral vascular resistance, and enhances red blood cell deformability, thereby improving hemorheology and microcirculatory disorders. Its medicinal value is highly valued. However, due to its instability, earthworm protein is traditionally boiled in water, which limits its effectiveness. Summary of the Invention

[0005] The present application is made in view of the above problems, and its purpose is to provide a method for preparing earthworm protein. The earthworm protein prepared by this method has good anticoagulant effect.

[0006] Specifically as follows, the first aspect of the present application provides a method for preparing earthworm protein, comprising the following steps:

[0007] S1, mixing earthworm powder and ethanol and then processing, collecting the solid phase;

[0008] S2, extracting the solid phase by mixing the solid phase with the citric acid solution and collecting the liquid phase;

[0009] S3, mixing the liquid phase obtained in step S2 with the dopamine-modified activated carbon, and collecting the liquid phase;

[0010] S4. The liquid phase obtained in step S4 is ultrafiltered and then freeze-dried.

[0011] According to one of the technical solutions of this application, at least the following beneficial effects are achieved:

[0012] This application mixes ethanol-treated earthworm powder and citric acid for extraction, thereby improving the full dissolution of active substances in the earthworm powder; then, dopamine-modified activated carbon is used to adsorb small molecule inorganic substances, pigments and other substances, thereby further improving the purity and activity of earthworm protein; finally, impurities are further removed through ultrafiltration, thereby further improving the purity and activity of earthworm protein.

[0013] According to some embodiments of the present application, the mass-to-volume ratio of the earthworm powder and ethanol is 1 g: 5 mL to 10 mL.

[0014] According to some embodiments of the present application, the temperature of the treatment in step S1 is 0°C to 4°C.

[0015] According to some embodiments of the present application, the treatment time in step S1 is 16 hours to 24 hours.

[0016] According to some embodiments of the present application, the molar concentration of the citric acid solution is 0.4 mol / L to 0.6 mol / L.

[0017] According to some embodiments of the present application, the mass volume ratio of the solid phase and the citric acid solution is 1 g: 10 mL to 20 mL.

[0018] According to some embodiments of the present application, the extraction temperature in step S2 is 10°C to 20°C.

[0019] According to some embodiments of the present application, the extraction time in step S2 is 4 hours to 8 hours.

[0020] According to some embodiments of the present application, the mass volume ratio of the dopamine-modified activated carbon and the liquid phase in step S2 is 0.02g-0.04g:1L.

[0021] According to some embodiments of the present application, the method for preparing dopamine-modified activated carbon comprises the following steps:

[0022] The carboxyl-modified activated carbon was reacted with dopamine-PEG-NH2.

[0023] In this application, activated carbon is modified with carboxyl groups. By modifying the activated carbon with carboxyl groups, acid-center carboxyl groups are added to the surface of the activated carbon. The carboxyl groups are then reacted with the amino groups in dopamine-PEG-NH2 to connect dopamine-PEG-NH2 to the surface of the carboxyl-modified activated carbon, thereby improving the adsorption effect and further improving the purity.

[0024] According to some embodiments of the present application, the carboxyl-modified activated carbon is prepared by oxidizing activated carbon with an oxidant.

[0025] According to some embodiments of the present application, the oxidant includes a nitric acid solution.

[0026] According to some embodiments of the present application, the molar concentration of the nitric acid solution is 5 mol / L to 10 mol / L.

[0027] According to some embodiments of the present application, the mass volume ratio of the activated carbon and the nitric acid solution is 1 g: 10 mL to 20 mL.

[0028] According to some embodiments of the present application, the oxidation temperature is 60°C to 80°C.

[0029] According to some embodiments of the present application, the oxidation time is 6 hours to 10 hours.

[0030] According to some embodiments of the present application, the mass ratio of the carboxyl-modified activated carbon to dopamine-PEG-NH2 (amino-polyethylene glycol-dopamine) is 10:1-3.

[0031] According to some embodiments of the present application, the Mw of the dopamine-PEG-NH2 (CAS No.: 1383691-49-3) is 1000-3000.

[0032] According to some embodiments of the present application, the method for preparing dopamine-modified activated carbon comprises the following steps:

[0033] S01, mixing carboxyl-modified activated carbon and water to form a first mixed solution;

[0034] S02, adding EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) to the first mixed solution to prepare a second mixed solution;

[0035] The dopamine-PEG-NH2 and water are mixed to prepare a third mixed solution;

[0036] S03, mixing the second mixed solution and the third mixed solution and reacting; after the reaction is completed, collecting the solid phase;

[0037] The solid phase is added to an acetic acid / sodium acetate buffer solution for reaction, the solid-liquid separation is performed, and the solid phase is collected and freeze-dried.

[0038] According to some embodiments of the present application, the mass ratio of dopamine-PEG-NH2 to EDC is 8 to 12:1.

[0039] According to some embodiments of the present application, the mass ratio of EDC to NHS is 1 to 3:1.

[0040] According to some embodiments of the present application, the acetic acid / sodium acetate buffer solution further contains ferrous ions.

[0041] According to some embodiments of the present application, the pH of the acetic acid / sodium acetate buffer solution is 4-6.

[0042] According to some embodiments of the present application, the concentration of ferrous ions is 5*10 -5 mol / L~15*10 -5 mol / L.

[0043] According to some embodiments of the present application, the mass-to-volume ratio of the solid phase and the buffer solution in step S03 is 1 g: 5 mL to 15 mL.

[0044] According to some embodiments of the present application, the reaction temperature in step S03 is 20°C to 30°C.

[0045] According to some embodiments of the present application, the reaction time of step S03 is 0.5 h to 1.5 h.

[0046] According to some embodiments of the present application, the ultrafiltration includes a first ultrafiltration and a second ultrafiltration.

[0047] According to some embodiments of the present application, the first ultrafiltration collects filtrate.

[0048] According to some embodiments of the present application, the second ultrafiltration collects the concentrated liquid.

[0049] According to some embodiments of the present application, the molecular weight cutoff of the ultrafiltration membrane of the first ultrafiltration is 10 kDa to 20 kDa.

[0050] According to some embodiments of the present application, the molecular weight cutoff of the ultrafiltration membrane of the first ultrafiltration is 4 kDa to 5 kDa.

[0051] According to some embodiments of the present application, the freeze-drying temperature is -60°C to -40°C.

[0052] The second aspect of the present application provides earthworm protein prepared by the preparation method described in the first aspect of the present application.

[0053] The third aspect of the present application provides the use of the earthworm protein as described in the second aspect of the present application in the preparation of any one of a thrombosis prevention drug, a blood pressure lowering drug, a lipid-lowering drug, a lipid metabolism promoting drug, an antibacterial drug and an immunomodulatory drug. DETAILED DESCRIPTION

[0054] In order to make the purpose, technical solutions and advantages of this application more clearly understood, the present application is described and illustrated below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are merely used to explain this application and are not intended to limit this application. Based on the embodiments provided in this application, all other embodiments obtained by those of ordinary skill in the art without making any creative work are within the scope of protection of this application.

[0055] Obviously, the following descriptions are merely some examples or embodiments of the present application. Those skilled in the art can apply the present application to other similar scenarios without inventive effort. Furthermore, it is also understood that, although the effort involved in such a development process may be complex and lengthy, for those skilled in the art related to the content disclosed in the present application, changes in design, manufacturing, or production based on the technical content disclosed in the present application are merely conventional technical means and should not be construed as an insufficiency of the content disclosed in the present application.

[0056] Unless otherwise specified, the terms "include" and "comprising" used in this application may be open-ended or closed-ended. For example, "include" and "comprising" may mean that other components not listed may also be included or that only the listed components are included.

[0057] Unless otherwise specified, the term "or" is used in this application to be inclusive. For example, the phrase "A or B" means "A, B, or both A and B." More specifically, the condition "A or B" is satisfied if any of the following conditions are met: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0058] The Mw of dopamine-PEG-NH2 selected in the embodiment of the present application is 2000.

[0059] The mesh number of the activated carbon selected in the embodiment of the present application is 325 mesh.

[0060] The earthworm powder selected in the embodiment of the present application is prepared by freezing the earthworm with liquid nitrogen and then crushing it.

[0061] Example 1

[0062] This embodiment is a method for preparing earthworm protein, which consists of the following steps:

[0063] S1. Mix earthworm powder and ethanol (the mass volume ratio of earthworm powder to ethanol is 1 g:10 mL) and perform post-treatment (temperature 4°C, time 20 h), and collect the solid phase;

[0064] S2. The solid phase obtained in step S1 was mixed with a citric acid solution (molar concentration of 0.5 mol / L, mass volume ratio of solid phase to citric acid solution of 1 g:15 mL) and then extracted (extraction temperature of 20° C. for 6 h), and the liquid phase was collected;

[0065] S3. Mix the liquid phase obtained in step S2 with dopamine-modified activated carbon (the mass volume ratio of dopamine-modified activated carbon to liquid phase is 0.028 g:1 L), and collect the liquid phase;

[0066] S4. The liquid phase obtained in step S4 is ultrafiltered and then freeze-dried (freeze-drying temperature is -50°C).

[0067] The ultrafiltration consists of a first ultrafiltration and a second ultrafiltration;

[0068] The filtrate was collected by the first ultrafiltration (molecular weight cut-off of 10 kDa);

[0069] The concentrate was collected by a second ultrafiltration (molecular weight cut-off of 5 kDa).

[0070] The preparation method of dopamine-modified activated carbon consists of the following steps:

[0071] S01, mixing carboxyl-modified activated carbon and water (the mass volume ratio of carboxyl-modified activated carbon to water is 1 g:10 mL) to form a first mixed solution;

[0072] S02, adding EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) to the first mixed solution to prepare a second mixed solution;

[0073] Dopamine-PEG-NH2 and water (mass volume ratio of dopamine-PEG-NH2 to water is 1 g:10 mL) are mixed to prepare a third mixed solution;

[0074] S03, mixing the second mixed solution and the third mixed solution and reacting them at 25° C. for 6 hours; after the reaction is completed, collecting the solid phase;

[0075] The solid phase was added to an acetic acid / sodium acetate buffer solution (pH 5, to which ferrous ions were added at a concentration of 10 -4 mol / L; the mass volume ratio of the solid phase and the buffer solution is 1 g:10 mL), react at 25 ° C for 1 h, separate the solid and liquid, collect the solid phase, and freeze-dry.

[0076] The mass ratio of dopamine-PEG-NH2 and EDC was 10:1;

[0077] The mass ratio of EDC and NHS is 2:1;

[0078] The mass ratio of carboxyl-modified activated carbon and dopamine-PEG-NH2 is 10:2.5.

[0079] The preparation method of carboxyl modified activated carbon consists of the following steps:

[0080] Activated carbon and nitric acid solution (7 mol / L, mass volume ratio of activated carbon to nitric acid solution is 1 g:15 mL) were mixed and refluxed (temperature was 70°C) for 8 h, washed and dried at 110°C to obtain carboxyl-modified activated carbon.

[0081] Example 2

[0082] This embodiment is a method for preparing earthworm protein, which consists of the following steps:

[0083] S1. Mix earthworm powder and ethanol (the mass volume ratio of earthworm powder to ethanol is 1 g:10 mL) and perform post-treatment (temperature 4°C, time 20 h), and collect the solid phase;

[0084] S2. The solid phase obtained in step S1 was mixed with a citric acid solution (molar concentration of 0.4 mol / L, mass volume ratio of solid phase to citric acid solution of 1 g:20 mL) and then extracted (extraction temperature of 20° C. for 6 h), and the liquid phase was collected;

[0085] S3. Mix the liquid phase obtained in step S2 with dopamine-modified activated carbon (the mass volume ratio of dopamine-modified activated carbon to liquid phase is 0.02 g:1 L), and collect the liquid phase;

[0086] S4. The liquid phase obtained in step S4 is ultrafiltered and then freeze-dried (freeze-drying temperature is -50°C).

[0087] The ultrafiltration consists of a first ultrafiltration and a second ultrafiltration;

[0088] The filtrate was collected by the first ultrafiltration (molecular weight cut-off of 10 kDa);

[0089] The concentrate was collected by a second ultrafiltration (molecular weight cut-off of 5 kDa).

[0090] The preparation method of dopamine-modified activated carbon consists of the following steps:

[0091] S01, mixing carboxyl-modified activated carbon and water (the mass volume ratio of carboxyl-modified activated carbon to water is 1 g:10 mL) to form a first mixed solution;

[0092] S02, adding EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) to the first mixed solution to prepare a second mixed solution;

[0093] Dopamine-PEG-NH2 and water (mass volume ratio of dopamine-PEG-NH2 to water is 1 g:10 mL) are mixed to prepare a third mixed solution;

[0094] S03, mixing the second mixed solution and the third mixed solution and reacting them at 25° C. for 6 hours; after the reaction is completed, collecting the solid phase;

[0095] The solid phase was added to an acetic acid / sodium acetate buffer solution (pH 5, to which ferrous ions were added at a concentration of 10 -4 mol / L; the mass volume ratio of the solid phase and the buffer solution is 1 g:10 mL), react at 25 ° C for 1 h, separate the solid and liquid, collect the solid phase, and freeze-dry.

[0096] The mass ratio of dopamine-PEG-NH2 and EDC was 10:1;

[0097] The mass ratio of EDC and NHS is 2:1;

[0098] The mass ratio of carboxyl-modified activated carbon and dopamine-PEG-NH2 is 10:3.

[0099] The preparation method of carboxyl modified activated carbon consists of the following steps:

[0100] Activated carbon and nitric acid solution (7 mol / L, mass volume ratio of activated carbon to nitric acid solution is 1 g:15 mL) were mixed and refluxed (temperature was 70°C) for 8 h, washed and dried at 110°C to obtain carboxyl-modified activated carbon.

[0101] Example 3

[0102] This embodiment is a method for preparing earthworm protein, which consists of the following steps:

[0103] S1. Mix earthworm powder and ethanol (the mass volume ratio of earthworm powder to ethanol is 1 g:10 mL) and perform post-treatment (temperature 4°C, time 20 h), and collect the solid phase;

[0104] S2. The solid phase obtained in step S1 was mixed with a citric acid solution (molar concentration of 0.6 mol / L, mass volume ratio of solid phase to citric acid solution of 1 g:10 mL) and then extracted (extraction temperature of 20° C. for 6 h), and the liquid phase was collected;

[0105] S3. Mix the liquid phase obtained in step S2 with dopamine-modified activated carbon (the mass volume ratio of dopamine-modified activated carbon to liquid phase is 0.04 g:1 L), and collect the liquid phase;

[0106] S4. The liquid phase obtained in step S4 is ultrafiltered and then freeze-dried (freeze-drying temperature is -50°C).

[0107] The ultrafiltration consists of a first ultrafiltration and a second ultrafiltration;

[0108] The filtrate was collected by the first ultrafiltration (molecular weight cut-off of 10 kDa);

[0109] The concentrate was collected by a second ultrafiltration (molecular weight cut-off of 5 kDa).

[0110] The preparation method of dopamine-modified activated carbon consists of the following steps:

[0111] S01, mixing carboxyl-modified activated carbon and water (the mass volume ratio of carboxyl-modified activated carbon to water is 1 g:10 mL) to form a first mixed solution;

[0112] S02, adding EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) to the first mixed solution to prepare a second mixed solution;

[0113] Dopamine-PEG-NH2 and water (mass volume ratio of dopamine-PEG-NH2 to water is 1 g:10 mL) are mixed to prepare a third mixed solution;

[0114] S03, mixing the second mixed solution and the third mixed solution and reacting them at 25° C. for 6 hours; after the reaction is completed, collecting the solid phase;

[0115] The solid phase was added to an acetic acid / sodium acetate buffer solution (pH 5, to which ferrous ions were added at a concentration of 10 -4 mol / L; the mass volume ratio of the solid phase and the buffer solution is 1 g:10 mL), react at 25 ° C for 1 h, separate the solid and liquid, collect the solid phase, and freeze-dry.

[0116] The mass ratio of dopamine-PEG-NH2 and EDC was 10:1;

[0117] The mass ratio of EDC and NHS is 2:1;

[0118] The mass ratio of carboxyl-modified activated carbon and dopamine-PEG-NH2 is 10:3.

[0119] The preparation method of carboxyl modified activated carbon consists of the following steps:

[0120] Activated carbon and nitric acid solution (7 mol / L, mass volume ratio of activated carbon to nitric acid solution is 1 g:15 mL) were mixed and refluxed (temperature was 70°C) for 8 h, washed and dried at 110°C to obtain carboxyl-modified activated carbon.

[0121] Example 4

[0122] This embodiment is a method for preparing earthworm protein, which consists of the following steps:

[0123] S1. Mix earthworm powder and ethanol (the mass volume ratio of earthworm powder to ethanol is 1 g:10 mL) and perform post-treatment (temperature 4°C, time 20 h), and collect the solid phase;

[0124] S2. The solid phase obtained in step S1 was mixed with a citric acid solution (molar concentration of 0.4 mol / L, mass volume ratio of solid phase to citric acid solution of 1 g:20 mL) and then extracted (extraction temperature of 20° C. for 6 h), and the liquid phase was collected;

[0125] S3. Mix the liquid phase obtained in step S2 with dopamine-modified activated carbon (the mass volume ratio of dopamine-modified activated carbon to liquid phase is 0.04 g:1 L), and collect the liquid phase;

[0126] S4. The liquid phase obtained in step S4 is ultrafiltered and then freeze-dried (freeze-drying temperature is -50°C).

[0127] The ultrafiltration consists of a first ultrafiltration and a second ultrafiltration;

[0128] The filtrate was collected by the first ultrafiltration (molecular weight cut-off of 10 kDa);

[0129] The concentrate was collected by a second ultrafiltration (molecular weight cut-off of 5 kDa).

[0130] The preparation method of dopamine-modified activated carbon consists of the following steps:

[0131] S01, mixing carboxyl-modified activated carbon and water (the mass volume ratio of carboxyl-modified activated carbon to water is 1 g:10 mL) to form a first mixed solution;

[0132] S02, adding EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) to the first mixed solution to prepare a second mixed solution;

[0133] Dopamine-PEG-NH2 and water (mass volume ratio of dopamine-PEG-NH2 to water is 1 g:10 mL) are mixed to prepare a third mixed solution;

[0134] S03, mixing the second mixed solution and the third mixed solution and reacting them at 25° C. for 6 hours; after the reaction is completed, collecting the solid phase;

[0135] The solid phase was added to an acetic acid / sodium acetate buffer solution (pH 5, to which ferrous ions were added at a concentration of 10 -4mol / L; the mass volume ratio of the solid phase and the buffer solution is 1 g:10 mL), react at 25 ° C for 1 h, separate the solid and liquid, collect the solid phase, and freeze-dry.

[0136] The mass ratio of dopamine-PEG-NH2 and EDC was 10:1;

[0137] The mass ratio of EDC and NHS is 2:1;

[0138] The mass ratio of carboxyl-modified activated carbon and dopamine-PEG-NH2 is 10:3.

[0139] The preparation method of carboxyl modified activated carbon consists of the following steps:

[0140] Activated carbon and nitric acid solution (7 mol / L, mass volume ratio of activated carbon to nitric acid solution is 1 g:15 mL) were mixed and refluxed (temperature was 70°C) for 8 h, washed and dried at 110°C to obtain carboxyl-modified activated carbon.

[0141] Example 5

[0142] This embodiment is a method for preparing earthworm protein, which consists of the following steps:

[0143] S1. Mix earthworm powder and ethanol (the mass volume ratio of earthworm powder to ethanol is 1 g:10 mL) and perform post-treatment (temperature 4°C, time 20 h), and collect the solid phase;

[0144] S2. The solid phase obtained in step S1 was mixed with a citric acid solution (molar concentration of 0.4 mol / L, mass volume ratio of solid phase to citric acid solution of 1 g:10 mL) and then extracted (extraction temperature of 20° C. for 6 h), and the liquid phase was collected;

[0145] S3. Mix the liquid phase obtained in step S2 with dopamine-modified activated carbon (the mass volume ratio of dopamine-modified activated carbon to liquid phase is 0.02 g:1 L), and collect the liquid phase;

[0146] S4. The liquid phase obtained in step S4 is ultrafiltered and then freeze-dried (freeze-drying temperature is -50°C).

[0147] The ultrafiltration consists of a first ultrafiltration and a second ultrafiltration;

[0148] The filtrate was collected by the first ultrafiltration (molecular weight cut-off of 10 kDa);

[0149] The concentrate was collected by a second ultrafiltration (molecular weight cut-off of 5 kDa).

[0150] The preparation method of dopamine-modified activated carbon consists of the following steps:

[0151] S01, mixing carboxyl-modified activated carbon and water (the mass volume ratio of carboxyl-modified activated carbon to water is 1 g:10 mL) to form a first mixed solution;

[0152] S02, adding EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) to the first mixed solution to prepare a second mixed solution;

[0153] Dopamine-PEG-NH2 and water (mass volume ratio of dopamine-PEG-NH2 to water is 1 g:10 mL) are mixed to prepare a third mixed solution;

[0154] S03, mixing the second mixed solution and the third mixed solution and reacting them at 25° C. for 6 hours; after the reaction is completed, collecting the solid phase;

[0155] The solid phase was added to an acetic acid / sodium acetate buffer solution (pH 5, to which ferrous ions were added at a concentration of 10 -4 mol / L; the mass volume ratio of the solid phase and the buffer solution is 1 g:10 mL), react at 25 ° C for 1 h, separate the solid and liquid, collect the solid phase, and freeze-dry.

[0156] The mass ratio of dopamine-PEG-NH2 and EDC was 10:1;

[0157] The mass ratio of EDC and NHS is 2:1;

[0158] The mass ratio of carboxyl-modified activated carbon and dopamine-PEG-NH2 was 10:1.

[0159] The preparation method of carboxyl modified activated carbon consists of the following steps:

[0160] Activated carbon and nitric acid solution (7 mol / L, mass volume ratio of activated carbon to nitric acid solution is 1 g:15 mL) were mixed and refluxed (temperature was 70°C) for 8 h, washed and dried at 110°C to obtain carboxyl-modified activated carbon.

[0161] Comparative Example 1

[0162] This comparative example is a method for preparing earthworm protein, which differs from Example 5 in that:

[0163] The preparation method of dopamine-modified activated carbon consists of the following steps:

[0164] S01, mixing activated carbon and water to form a first mixed liquid;

[0165] S02, mixing dopamine and water to prepare a second mixed solution;

[0166] S03, mixing the first mixed solution and the second mixed solution and reacting them at 25° C. for 6 hours; after the reaction is completed, collecting the solid phase to obtain dopamine-modified activated carbon;

[0167] The mass ratio of activated carbon to dopamine is 10:1.

[0168] Comparative Example 2

[0169] This comparative example is a method for preparing earthworm protein, which differs from Example 5 in that:

[0170] The preparation method of dopamine-modified activated carbon consists of the following steps:

[0171] S01, mixing activated carbon and water to form a first mixed liquid;

[0172] S02, mixing dopamine-PEG-NH2 and water to prepare a second mixed solution;

[0173] S03, mixing the first mixed solution and the second mixed solution and reacting them at 25° C. for 6 hours; after the reaction is completed, collecting the solid phase to obtain dopamine-modified activated carbon;

[0174] The mass ratio of activated carbon to dopamine-PEG-NH2 was 10:1.

[0175] Comparative Example 3

[0176] This comparative example is a method for preparing earthworm protein, which differs from Example 5 in that:

[0177] The preparation method of dopamine-modified activated carbon consists of the following steps:

[0178] S01, mixing carboxyl-modified activated carbon and water to form a first mixed solution;

[0179] S02, adding EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) to the first mixed solution to prepare a second mixed solution;

[0180] The dopamine-PEG-NH2 and water are mixed to prepare a third mixed solution;

[0181] S03, mixing the second mixed solution and the third mixed solution and reacting them at 25° C. for 6 hours; after the reaction is completed, collecting the solid phase to obtain dopamine-modified activated carbon;

[0182] The mass ratio of dopamine-PEG-NH2 and EDC was 10:1;

[0183] The mass ratio of EDC and NHS is 2:1;

[0184] The mass ratio of carboxyl-modified activated carbon and dopamine-PEG-NH2 was 10:1.

[0185] The preparation method of carboxyl-modified activated carbon is carried out with reference to Example 5.

[0186] Comparative Example 4

[0187] This comparative example is a method for preparing earthworm protein, which differs from Example 5 in that dopamine-modified activated carbon is replaced by activated carbon.

[0188] Comparative Example 5

[0189] This comparative example is a method for preparing earthworm protein, which differs from Example 5 in that: dopamine-modified activated carbon is replaced with carboxyl-modified activated carbon;

[0190] The preparation method of carboxyl-modified activated carbon is carried out with reference to Example 5.

[0191] The extraction effect of the earth dragon protein obtained by the embodiment and the comparative example was tested by the following method:

[0192] 1. Test method

[0193] 1.1 Determination of thrombin time (TT)

[0194] Thrombin was dissolved in saline (5 μL / mL), and then 0.2 mL of the plasma to be tested was taken and maintained at 37°C for 3 min. 0.2 mL of the dissolved thrombin was added. Equal amounts of the earthworm protein obtained in the Examples and Comparative Examples (0.1 mL of a 100 μg / mL earthworm protein solution in saline) were added to the plasma, shaken, and the timer was started. Each experiment was repeated 5 times.

[0195] 1.2 Determination of earthworm protein yield

[0196] The water content of living earthworms was determined according to the method in the Chinese Pharmacopoeia. The earthworm protein yield was calculated as follows:

[0197] Protein yield = total amount of earthworm protein obtained / [mass of living earthworm × (1-r)];

[0198] Wherein, r is the water content of fresh earthworms.

[0199] 2. Test results

[0200] The thrombin time and yield are shown in Table 1. From the results, it can be seen that the earthworm protein prepared in the embodiment of the present invention can significantly prolong the thrombin time compared with the comparative example.

[0201] Table 1

[0202] - Thrombin time (s) Yield (%) Example 1 92 24.6 Example 2 89 24.8 Example 3 85 25.1 Example 4 84 25.3 Example 5 81 25.7 Comparative Example 1 49 26.9 Comparative Example 2 52 27.8 Comparative Example 3 66 27.1 Comparative Example 4 38 28.9 Comparative Example 5 43 28.3

[0203] It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are merely examples, and any embodiments having substantially the same structure and effect as the technical concept within the scope of the present application are all included in the technical scope of the present application. In addition, without departing from the scope of the present application, any other embodiments that can be conceived by those skilled in the art and that combine some of the constituent elements in the embodiments are also included in the scope of the present application.

Claims

1. A method for preparing earthworm protein, characterized in that: The following steps are involved: S1. Mix earthworm powder and ethanol at a mass-to-volume ratio of 1 g:10 mL; incubate at 4°C for 20 h, and collect the solid phase; S2. Mix the solid phase obtained in step S1 with 0.4 mol / L to 0.6 mol / L citric acid solution at a mass volume ratio of 1 g: 10 mL to 15 mL; extract at 20° C. for 6 h, and collect the liquid phase; S3, mixing the dopamine-modified activated carbon and the liquid phase obtained in step S2 at a mass volume ratio of 0.02 g to 0.04 g:1 L, and collecting the liquid phase; S4, ultrafiltration of the liquid phase obtained in step S3 followed by freeze drying; The ultrafiltration consists of a first ultrafiltration and a second ultrafiltration; First ultrafiltration: molecular weight cut-off is 10 kDa, and the filtrate is collected; Second ultrafiltration: molecular weight cut-off 5kDa, collect the concentrate; The preparation method of dopamine-modified activated carbon consists of the following steps: S01, mixing carboxyl-modified activated carbon and water in a mass volume ratio of 1 g:10 mL to form a first mixed solution; S02, adding EDC and NHS to the first mixed solution to prepare a second mixed solution; Dopamine-PEG-NH2 and water were mixed at a mass volume ratio of 1 g:10 mL to prepare a third mixed solution; S03, mixing the second mixed solution and the third mixed solution, reacting at 25° C. for 6 h; after the reaction is completed, collecting the solid phase; The mass ratio of dopamine-PEG-NH2 and EDC was 10:1; The mass ratio of EDC and NHS is 2:1; The mass ratio of the carboxyl-modified activated carbon to dopamine-PEG-NH2 is 10:2.5-3; The solid phase obtained in step S03 was added to an acetic acid / sodium acetate buffer solution at a mass volume ratio of 1 g:10 mL, reacted at 25°C for 1 hour, solid-liquid separation was performed, the solid phase was collected, and freeze-dried; The pH of the acetic acid / sodium acetate buffer solution is 5; Ferrous ions are also added to the buffer solution, and the concentration of ferrous ions is 10 -4 mol / L; The preparation method of carboxyl modified activated carbon consists of the following steps: Activated carbon and 7 mol / L nitric acid solution were mixed in a mass volume ratio of 1 g:15 mL, refluxed at 70°C for 8 h, washed, and dried at 110°C to obtain carboxyl-modified activated carbon.

2. The preparation method according to claim 1, characterized in that The freeze-drying temperature in step S4 is -60°C to -40°C.