Earthworm protein as well as preparation method and application thereof

Abstract

The invention provides earthworm protein as well as a preparation method and application thereof, and the method comprises the following steps: S1, mixing earthworm powder and ethanol, treating, and collecting a solid phase; s2, mixing the solid phase with a citric acid solution, extracting, and collecting a liquid phase; s3, mixing the liquid phase obtained in the step S2 with dopamine modified activated carbon, and collecting the liquid phase; and S4, performing ultrafiltration on the liquid phase obtained in the step S4, and performing freeze drying. The earthworm powder treated by ethanol is mixed with citric acid for extraction, so that the full dissolution of active substances in the earthworm powder is improved; substances such as micromolecular inorganic substances and pigments are adsorbed through dopamine modified activated carbon, so that the purity and the activity of the earthworm protein are further improved; finally, impurities are further removed through ultrafiltration, and the purity and activity of the earthworm protein are further improved.

Description

Technical Field

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

[0002] Geosaurus, also known as earthworm, belongs to the class Oligochaeta of the phylum Annelida and is an animal-based Chinese medicinal material. Related studies have shown that geosaurus has the effects of promoting blood circulation and removing blood stasis, dissolving blood clots and lowering blood pressure, anti-tumor, relieving asthma and cough, and anti-inflammatory and analgesic.

[0003] Earthworm protein is extracted from earthworms and contains collagenase, plasmin, lumbrokinase, nucleic acid, trace elements and other ingredients. Its molecular weight is between 5000 and 10000 and it is a short-chain small molecule substance.

[0004] Earth dragon protein contains collagenase, plasmin, nucleic acid, trace elements and other components, which have a wide range of effects on the coagulation system and fibrinolytic system in the body. It can significantly reduce the platelet adhesion rate, prolong the thrombosis and dissolve the thrombus in the body, increase cerebral blood flow, reduce cerebrovascular resistance, and improve the deformability of red blood cells, thereby improving blood rheology and microcirculation disorders. Its medicinal value is highly valued. However, due to the instability of earth dragon protein, the traditional method of decoction in water affects its efficacy. Summary of the invention

[0005] The present application is made in view of the above-mentioned 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, 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 post-processing, collecting the solid phase;

[0008] S2, extracting after mixing the solid phase and 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, ultrafiltration of the liquid phase obtained in step S4 and freeze-drying.

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

[0012] In the present application, ethanol-treated earthworm powder and citric acid are mixed for extraction to improve the full dissolution of active substances in the earthworm powder; small molecule inorganic substances and pigments are then adsorbed by dopamine-modified activated carbon to further improve the purity and activity of earthworm protein; finally, impurities are further removed by ultrafiltration to further improve the purity and activity of earthworm protein.

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

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

[0015] According to some embodiments of the present application, the processing 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 the present application, activated carbon is modified by carboxyl groups. By modifying the activated carbon by 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 them; 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 and liquid are separated, 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 also 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 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 the 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 ultrafiltration membrane cutoff molecular weight of the first ultrafiltration is 10 kDa to 20 kDa.

[0050] According to some embodiments of the present application, the ultrafiltration membrane cutoff molecular weight 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 blood 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 the present application clearer, the present application is described and illustrated below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. Based on the embodiments provided in the present application, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present application.

[0055] Obviously, the following descriptions are only some examples or embodiments of the present application. For ordinary technicians in this field, the present application can also be applied to other similar scenarios without creative work. In addition, it can also be understood that although the efforts made in this development process may be complicated and lengthy, for ordinary technicians in the field related to the content disclosed in this application, some changes in design, manufacturing or production based on the technical content disclosed in this application are just conventional technical means, and should not be understood as insufficient content disclosed in this application.

[0056] If there is no special explanation, the "include" and "comprising" mentioned in this application are open-ended or closed-ended. For example, the "include" and "comprising" may mean that other components not listed may also be included or only the listed components may be included or only the listed components may be included.

[0057] If not specifically stated, in this application, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, any of the following conditions satisfies the condition "A or B": 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, mixing earthworm powder and ethanol (the mass volume ratio of earthworm powder to ethanol is 1 g: 10 mL) and treating (temperature is 4°C, time is 20 h), and collecting the solid phase;

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

[0065] S3, mixing 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 collecting the liquid phase;

[0066] S4. The liquid phase obtained in step S4 is ultrafiltered and 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 (the 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 1g:10mL), react at 25°C for 1h, 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 to 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 is 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, mixing earthworm powder and ethanol (the mass volume ratio of earthworm powder to ethanol is 1 g: 10 mL) and treating (temperature is 4°C, time is 20 h), and collecting the solid phase;

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

[0085] S3, mixing 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 collecting the liquid phase;

[0086] S4. The liquid phase obtained in step S4 is ultrafiltered and 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 (the 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 1g:10mL), react at 25°C for 1h, 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 to 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 is 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, mixing earthworm powder and ethanol (the mass volume ratio of earthworm powder to ethanol is 1 g: 10 mL) and treating (temperature is 4°C, time is 20 h), and collecting the solid phase;

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

[0105] S3, mixing 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 collecting the liquid phase;

[0106] S4. The liquid phase obtained in step S4 is ultrafiltered and 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 (the 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 1g:10mL), react at 25°C for 1h, 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 to 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 is 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, mixing earthworm powder and ethanol (the mass volume ratio of earthworm powder to ethanol is 1 g: 10 mL) and treating (temperature is 4°C, time is 20 h), and collecting the solid phase;

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

[0125] S3, mixing 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 collecting the liquid phase;

[0126] S4. The liquid phase obtained in step S4 is ultrafiltered and 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 (the 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 1g:10mL), react at 25°C for 1h, 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 to 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 is 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, mixing earthworm powder and ethanol (the mass volume ratio of earthworm powder to ethanol is 1 g: 10 mL) and treating (temperature is 4°C, time is 20 h), and collecting the solid phase;

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

[0145] S3, mixing 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 collecting the liquid phase;

[0146] S4. The liquid phase obtained in step S4 is ultrafiltered and 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 (the 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 1g:10mL), react at 25°C for 1h, 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 to NHS is 2:1;

[0158] The mass ratio of carboxyl-modified activated carbon and dopamine-PEG-NH2 is 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 is 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 obtain 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 obtain 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 to NHS is 2:1;

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

[0185] The preparation method of carboxyl-modified activated carbon is carried out in accordance with 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 embodiments and comparative examples was tested by the following method:

[0192] 1. Test methods

[0193] 1.1 Determination of thrombin time (TT)

[0194] Take thrombin and dissolve it with physiological saline (5 μL / mL), then take 0.2 mL of the plasma to be tested, keep it at 37°C for 3 min, add 0.2 mL of the dissolved thrombin, add an equal amount of the earthworm protein obtained in the embodiment and the comparative example (0.1 mL of 100 μg / mL earthworm protein physiological saline solution) to the above plasma, shake well, and start timing. Each group of experiments was repeated 5 times.

[0195] 1.2 Determination of the yield of earthworm protein

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

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

[0198] Among them, r in the formula 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 only examples, and the embodiments having the same structure as the technical idea and exerting the same effect within the scope of the technical solution of the present application are all included in the technical scope of the present application. In addition, without departing from the scope of the main purpose of the present application, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining 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, mixing earthworm powder and ethanol and post-processing, collecting the solid phase; S2, extracting after mixing the solid phase and the citric acid solution, and collecting the liquid phase; S3, mixing the liquid phase obtained in step S2 with the dopamine-modified activated carbon, and collecting the liquid phase; S4, ultrafiltration of the liquid phase obtained in step S4 and freeze-drying.

2. The preparation method according to claim 1, characterized in that: The mass volume ratio of the earthworm powder and ethanol is 1g:5mL-10mL; And / or, the treatment temperature in step S1 is 0°C to 4°C; And / or, the processing time in step S1 is 16 hours to 24 hours.

3. The preparation method according to claim 1, characterized in that: The molar concentration of the citric acid solution is 0.4 mol / L to 0.6 mol / L; and / or, the mass volume ratio of the solid phase to the citric acid solution is 1 g: 10 mL to 20 mL; And / or, the extraction temperature in step S2 is 10°C to 20°C; And / or, the extraction time in step S2 is 4h to 8h.

4. The preparation method according to claim 1, characterized in that: The mass volume ratio of the dopamine-modified activated carbon and the liquid phase in step S2 is 0.02 g to 0.04 g: 1 L; And / or, the method for preparing the dopamine-modified activated carbon comprises the following steps: reacting carboxyl-modified activated carbon with dopamine-PEG-NH2; And / or, the carboxyl-modified activated carbon is prepared by oxidizing activated carbon with an oxidant; and / or, the oxidant comprises a nitric acid solution; And / or, the molar concentration of the nitric acid solution is 5 mol / L to 10 mol / L; And / or, the mass volume ratio of the activated carbon to the nitric acid solution is 1 g: 10 mL to 20 mL; And / or, the oxidation temperature is 60°C to 80°C; And / or, the oxidation time is 6h to 10h; And / or, the mass ratio of the carboxyl-modified activated carbon to dopamine-PEG-NH2 is 10:1-3.

5. The preparation method according to claim 1, characterized in that: The ultrafiltration includes a first ultrafiltration and a second ultrafiltration; And / or, the first ultrafiltration collects the filtrate; And / or, the second ultrafiltration collects a concentrate.

6. The preparation method according to claim 1, characterized in that: The ultrafiltration membrane cut-off molecular weight of the first ultrafiltration is 10 kDa to 20 kDa.

7. The preparation method according to claim 1, characterized in that: The ultrafiltration membrane cut-off molecular weight of the first ultrafiltration is 4 kDa to 5 kDa.

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

9. Pheretima protein obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the earthworm protein as claimed in claim 9 in the preparation of any one of a thrombus prevention drug, a blood pressure lowering drug, a blood lipid lowering drug, a lipid metabolism promoting drug, an antibacterial drug and an immunomodulatory drug.