Earthworm protein extraction method and application thereof in heart and cerebral vessels

Abstract

The invention relates to the field of animal extraction, and provides an earthworm protein extraction method and application thereof in heart and cerebral vessels, the earthworm protein extraction method comprises the following steps: S1, placing living earthworms in a container, adding an ethanol solution, soaking, taking out the earthworms, and cleaning to obtain emetic living earthworms for later use; s2, putting the living earthworm for promoting vomiting into warm water at 30-40 DEG C, adding a protective agent and papain, stirring and soaking, then adding bromelain, stirring and soaking to obtain a mixed solution for later use; s3, grinding the mixed solution, adding modified activated carbon, filtering, taking filtrate 1, and centrifuging to obtain filtrate 2 for later use; and S4, carrying out primary membrane separation on the filtrate 2 to obtain a filtrate 3, carrying out secondary membrane separation, and drying to obtain the earthworm protein. The earthworm protein extraction method provided by the invention has the characteristics of simple operation, low cost, high purity, high activity and high content, and has good commercial application value.

Description

Technical Field

[0001] The invention relates to the field of animal extraction, and in particular to a method for extracting earthworm protein and application thereof in cardiovascular and cerebrovascular diseases. Background Art

[0002] Pheromone, also known as earthworm, is an important animal medicinal material with the supernatural ability to regenerate blood vessels, nerves and muscles. The pheromone protein extracted from pheromone contains collagenase, plasmin, lumbrokinase, nucleic acid and trace elements and other ingredients. It is the "nemesis" of various blood clots and has the effects of dissolving blood clots, treating cardiovascular and cerebrovascular diseases, lowering blood lipids and blood viscosity, promoting nerve repair and regeneration, and anti-cancer.

[0003] Traditional methods of extracting earthworm protein mostly involve drying, steaming, and boiling live earthworms, and then crushing them with a grinder. However, it takes a long time for fresh earthworms to be naturally dried and dried, and during this period, the tissues and cells of the earthworms will gradually be destroyed, resulting in the degradation of protein active ingredients. In addition, earthworm protein is easily denatured under adverse external physical and chemical environments, resulting in loss of biological activity. The high-speed rotating grinder will also generate high temperatures during the crushing process, which will also cause protein denaturation and loss of biological activity. In recent years, researchers have also begun to use liquid nitrogen freezing or enzymatic hydrolysis to extract earthworm protein from earthworms, but these extraction methods still have the problems of high cost or complex operation.

[0004] Patent CN 114990182A discloses a method for extracting active earthworm protein by rapid freezing with liquid nitrogen, including sterilization, liquid nitrogen freezing, crushing and grinding, spray drying and other steps. The method for extracting active earthworm protein in this application can freeze the biological activity of internal tissue cells in a short time, effectively prevent the denaturation of earthworm biological enzymes, avoid the degradation and denaturation of protein components, and maintain the high activity of earthworm protein. However, this application has many steps, cumbersome operations and multiple uses of liquid nitrogen, which greatly increases the production cost.

[0005] Therefore, there is an urgent need on the market for a method for extracting earthworm protein that is simple to operate, low-cost, and produces earthworm protein with high purity, high activity, and high content, and the earthworm protein obtained by this extraction method can be widely used in cardiovascular and cerebrovascular diseases. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention has developed a method for extracting earthworm protein. Fresh and living earthworms are selected, and the bio-enzyme double hydrolysis technology is adopted under the condition of 30-40°C. A protective agent and modified activated carbon are designed for the extraction of earthworm protein. The problems of complex operation, high cost, low purity, activity and content of earthworm protein in the existing extraction methods of earthworm protein are solved.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] On the one hand, the present invention provides a method for extracting earthworm protein, comprising the following steps:

[0009] S1, placing the living earthworm in a container, adding ethanol solution, soaking, taking out the earthworm, washing, obtaining the emetic living earthworm for standby use;

[0010] S2, placing the vomiting live earthworm of step S1 in 30-40° C. warm water, adding a protective agent and papain, stirring, soaking, then adding bromelain, stirring, soaking, and obtaining a mixed solution for standby use;

[0011] S3, grinding the mixed solution of step S2, adding modified activated carbon, filtering, taking filtrate 1, centrifuging, and obtaining filtrate 2 for standby use;

[0012] S4, subjecting the filtrate 2 of step S3 to a first membrane separation to obtain a filtrate 3, and then subjecting the filtrate to a second membrane separation and drying to obtain earthworm protein.

[0013] In some embodiments of the present invention, the mass ratio of the emetic live earthworm, the protective agent, papain and bromelain in step S2 is 1:(3-5):(0.3-0.5):(0.2-0.4).

[0014] Preferably, the mass ratio of the emetic live earthworm, the protective agent, papain and bromelain in step S2 is 1:4:0.4:0.3.

[0015] The applicant firstly hydrolyzes the earthworm with papain, and then hydrolyzes the earthworm with bromelain. The synergistic effect of the secondary enzymolysis can make the extracted earthworm protein content higher.

[0016] In some embodiments of the present invention, the method for preparing the protective agent in step S2 comprises the following steps:

[0017] (1) adding chitosan to a mixed solution of an acetic acid aqueous solution and a glycerol solution, stirring, adding Span 80, stirring, and obtaining a mixed solution 1 for later use;

[0018] (2) adding tea polyphenols and L-cysteine ​​into deionized water and stirring to obtain a mixed solution 2 for later use;

[0019] (3) adding the mixed solution 2 of step (2) dropwise into the mixed solution 1 of step (1) while homogenizing, and continuing to homogenize to obtain a mixed solution 3 for later use;

[0020] (4) The mixed solution 3 of step (3) is spray-dried and sealed and stored at 2-6° C. to obtain a protective agent.

[0021] In some embodiments of the present invention, the mass ratio of chitosan, tea polyphenols and L-cysteine ​​is 1:(0.4-0.6):(0.2-0.4).

[0022] Preferably, the mass ratio of chitosan, tea polyphenols and L-cysteine ​​is 1:0.5:0.3.

[0023] During the extraction process of earthworm protein, adverse factors such as light, oxygen or bacteria may cause the earthworm protein to undergo oxidation and other deterioration phenomena, resulting in the destruction of the structural integrity and activity of the earthworm protein.

[0024] The applicant uses tea polyphenols and L-cysteine ​​as core materials and chitosan as wall materials to prepare a protective agent with a microcapsule structure. On the one hand, tea polyphenols is a natural plant extract with antioxidant and antibacterial properties, which can protect earth dragon protein from oxidative stress and bacterial damage, and maintain its activity and stability. Furthermore, the applicant uses L-cysteine ​​and tea polyphenols in combination. L-cysteine ​​has antioxidant and reducing effects, which can effectively protect earth dragon protein from oxidation and deterioration, thereby maintaining the structural integrity and activity of earth dragon protein. In addition, the acidity of L-cysteine ​​itself can also play a role in regulating pH, which can make the system The total number of bacteria in the mixture is maintained at a low level, and the combination with tea polyphenols has a synergistic antioxidant and antibacterial effect. On the other hand, the applicant selected chitosan as the wall material to encapsulate tea polyphenols and L-cysteine. Chitosan itself has good antibacterial properties and promotes the repair and regeneration of tissues after autolysis of earthworms. Furthermore, chitosan enables tea polyphenols and L-cysteine ​​to maintain their original chemical properties and biological activities in a microscopic state, thereby improving the protective effect of tea polyphenols and L-cysteine ​​on earthworm protein. In addition, there is a hydrogen bond between tea polyphenols and L-cysteine ​​and the wall material chitosan, which can enable the protective agent to play a more stable role.

[0025] In some embodiments of the present invention, the mass ratio of the emetic living earthworm to the modified activated carbon is 1:(0.04-0.06).

[0026] Preferably, the mass ratio of the emetic living earthworm to the modified activated carbon is 1:0.05.

[0027] In some embodiments of the present invention, the method for preparing modified activated carbon in step S3 comprises the following steps:

[0028] 1) washing the poultry feathers with deionized water, removing impurities, drying, shearing and crushing to obtain pre-treated poultry feathers for later use;

[0029] 2) The poultry feathers pretreated in step 1) are mixed with a 40-50 wt% phosphoric acid aqueous solution, stirred for 25-35 min at 70-80 kPa and 115-118° C., heated to 550-650° C. for carbonization for 25-35 min, washed, and dried to obtain modified activated carbon.

[0030] In some embodiments of the present invention, in step 2), the mass ratio of poultry feathers to aqueous phosphoric acid solution is 1:(2-3).

[0031] Preferably, in step 2), the mass ratio of poultry feathers to the aqueous phosphoric acid solution is 1:2.5.

[0032] The introduction of protective agents during the extraction of earthworm protein plays a good role in protecting the structure of the earthworm itself from being destroyed and inhibiting the growth of bacteria. However, the tea polyphenols in the protective agent will introduce pigment components such as anthocyanins into the system, which will affect the purity of earthworm protein to a certain extent.

[0033] The applicant uses poultry feathers as raw materials, which are sufficient and low-priced, and can achieve the purpose of waste resource utilization. Further, by controlling the mass ratio of poultry feathers and aqueous phosphoric acid solution and the carbonization temperature, modified activated carbon is prepared. The modified activated carbon has the characteristics of high carbon content, low ash content and developed voids, and can effectively adsorb pigment impurities, thereby improving the purity and content of earthworm protein.

[0034] In some embodiments of the present invention, the molecular weight cutoff of the primary membrane separation in step S4 is 9000-10000 Daltons; the molecular weight cutoff of the secondary membrane separation is 1000-2000 Daltons.

[0035] In some embodiments of the present invention, the content of earthworm protein obtained by the extraction method described in the above technical solution is not less than 83.5%.

[0036] Another aspect of the present invention provides application of the earthworm protein extraction method in cardiovascular and cerebrovascular diseases.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] (1) The present invention provides a method for extracting earthworm protein, which selects fresh and living earthworms, adopts bio-enzyme double hydrolysis technology at 30-40°C, and designs protective agents and modified activated carbon for use in the extraction of earthworm protein, so that the earthworm protein extraction method is simple to operate and low-cost, and the obtained earthworm protein is high in purity, high in activity, and high in content, and can be widely used in cardiovascular and cerebrovascular diseases.

[0039] (2) The present invention adopts tea polyphenols and L-cysteine ​​as core materials and chitosan as wall material to prepare a protective agent with a microcapsule structure, so that the protective agent has good antioxidant and antibacterial properties and good stability, thereby making the extracted earthworm protein have the characteristics of high purity, high activity and high content.

[0040] (3) The present invention uses poultry feathers as raw materials and prepares modified activated carbon by controlling the mass ratio of poultry feathers and phosphoric acid aqueous solution and the carbonization temperature. The modified activated carbon can effectively adsorb pigment impurities and improve the purity and content of earthworm protein.

[0041] (4) The content of earthworm protein obtained by the extraction method of the present invention is not less than 83.5%. DETAILED DESCRIPTION

[0042] The present invention will be described below in conjunction with specific embodiments. It should be noted that the following embodiments are examples of the present invention and are only used to illustrate the present invention, but not to limit the present invention. Other combinations and various modifications within the concept of the present invention may be performed without departing from the spirit or scope of the present invention.

[0043] In the following examples, except for the protective agent and modified activated carbon, the other compound monomers and related reagents used can be purchased from the market, among which chitosan was purchased from Guangdong Mingcheng Biotechnology Co., Ltd.; poultry feathers were taken from discarded chicken feathers in the poultry market.

[0044] Preparation Example 1

[0045] The synthetic method of protective agent A comprises the following steps:

[0046] (1) Add 9 g of chitosan to a mixed solution of 250 ml of 1 wt % acetic acid aqueous solution and 250 ml of 0.5 wt % glycerol solution, stir for 30 min, add 1 ml of Span 80, stir for 5 min, and obtain a mixed solution 1 for later use;

[0047] (2) Add 4.5 g of tea polyphenols and 3 g of L-cysteine ​​to 50 ml of deionized water and stir for 1 h to obtain a mixed solution 2 for later use;

[0048] (3) adding the mixed solution 2 of step (2) dropwise into the mixed solution 1 of step (1) while homogenizing, and continuing the homogenization for 10 minutes to obtain a mixed solution 3 for later use;

[0049] (4) The mixed solution 3 of step (3) is spray-dried (the conditions of spray drying are: nozzle diameter: 0.7 mm, air inlet temperature: 45° C., material flow rate: 180 mL / h), and sealed and stored at 4° C. to obtain protective agent A.

[0050] Preparation Example 2

[0051] Protective agent B, the specific implementation method is the same as that of protective agent A, except that: in step (2), the mass of tea polyphenols is replaced with 3g.

[0052] Preparation Example 3

[0053] Protective agent C has the same specific implementation as protective agent A, except that in step (2), the mass of L-cysteine ​​is replaced with 1.6 g.

[0054] Preparation Example 4

[0055] The synthesis method of modified activated carbon A comprises the following steps:

[0056] 1) 5 g of poultry hair was washed with 200 ml of deionized water to remove impurities, dried at 105° C. for 4 h, sheared and crushed to obtain pretreated poultry hair for later use;

[0057] 2) 4 g of the poultry hair pretreated in step 1) and 10 g of a 45 wt% phosphoric acid aqueous solution were mixed, stirred at 75 kPa and 117° C. for 30 min, heated to 600° C. for carbonization for 30 min, washed with deionized water to pH = 7, and dried at 105° C. for 4 h to obtain modified activated carbon A.

[0058] Preparation Example 5

[0059] The specific implementation manner of modified activated carbon B is the same as that of modified activated carbon A, except that in step 2), the mass of the phosphoric acid aqueous solution is replaced with 7.5 g.

[0060] Example 1

[0061] A method for extracting earthworm protein comprises the following steps:

[0062] S1, 50g of live earthworms are placed in a container, 100g of 5wt% ethanol solution is added, and the earthworms are soaked for 30min, and the earthworms are taken out and cleaned with water to obtain the emetic live earthworms for standby use;

[0063] S2, placing 30g of the vomiting live earthworm of step S1 in 35°C warm water, adding 120g of protective agent A and 12g of papain, stirring for 30min, soaking for 24h, then adding 9g of bromelain, stirring for 30min, soaking for 24h, and obtaining a mixed solution for standby use;

[0064] S3, put the mixed solution of step S2 into a colloid mill for grinding, add 1.5g of modified activated carbon A, filter with a 60-mesh screen, take filtrate 1, centrifuge at 5000r for 30min, and obtain filtrate 2 for standby use;

[0065] S4, the filtrate 2 of step S3 is subjected to a membrane separation using an ultrafiltration membrane with a molecular weight cutoff of 9500 Daltons to obtain a filtrate 3, and then a secondary membrane separation is performed using an ultrafiltration membrane with a molecular weight cutoff of 1500 Daltons, the filtrate is removed, and dried at room temperature to constant weight to obtain earthworm protein.

[0066] Example 2

[0067] A method for extracting earthworm protein comprises the following steps:

[0068] S1, 50g of live earthworms are placed in a container, 100g of 5wt% ethanol solution is added, and the earthworms are soaked for 30min, and the earthworms are taken out and cleaned with water to obtain the emetic live earthworms for standby use;

[0069] S2, placing 30g of the vomiting live earthworm of step S1 in 30°C warm water, adding 90g of protective agent A and 9g of papain, stirring for 30min, soaking for 24h, then adding 6g of bromelain, stirring for 30min, soaking for 24h, and obtaining a mixed solution for standby use;

[0070] S3, put the mixed solution of step S2 into a colloid mill for grinding, add 1.2g of modified activated carbon A, filter with a 60-mesh screen, take filtrate 1, centrifuge at 5000r for 30min, and obtain filtrate 2 for standby use;

[0071] S4, the filtrate 2 of step S3 is subjected to a membrane separation using an ultrafiltration membrane with a molecular weight cutoff of 9000 Daltons to obtain a filtrate 3, and then a secondary membrane separation is performed using an ultrafiltration membrane with a molecular weight cutoff of 1000 Daltons, the filtrate is removed, and dried at room temperature to constant weight to obtain earthworm protein.

[0072] Example 3

[0073] A method for extracting earthworm protein comprises the following steps:

[0074] S1, 50g of live earthworms are placed in a container, 100g of 5wt% ethanol solution is added, and the earthworms are soaked for 30min, and the earthworms are taken out and cleaned with water to obtain the emetic live earthworms for standby use;

[0075] S2, placing 30g of the vomiting live earthworm of step S1 in 40°C warm water, adding 150g of protective agent A and 15g of papain, stirring for 30min, soaking for 24h, then adding 12g of bromelain, stirring for 30min, soaking for 24h, and obtaining a mixed solution for standby use;

[0076] S3, put the mixed solution of step S2 into a colloid mill for grinding, add 1.88g of modified activated carbon A, filter with a 60-mesh screen, take filtrate 1, centrifuge at 5000r for 30min, and obtain filtrate 2 for standby use;

[0077] S4, the filtrate 2 of step S3 is subjected to a membrane separation using an ultrafiltration membrane with a molecular weight cutoff of 10,000 Daltons to obtain a filtrate 3, and then a secondary membrane separation is performed using an ultrafiltration membrane with a molecular weight cutoff of 2,000 Daltons, the filtrate is removed, and dried at room temperature to constant weight to obtain earthworm protein.

[0078] Example 4

[0079] This embodiment provides a method for extracting earthworm protein. The specific implementation method is the same as that of Example 1, except that the mass of papain in step S1 is 8.4 g.

[0080] Example 5

[0081] This embodiment provides a method for extracting earthworm protein. The specific implementation method is the same as that of Example 1, except that the mass of bromelain in step S1 is 5.4 g.

[0082] Example 6

[0083] This embodiment provides a method for extracting earthworm protein. The specific implementation method is the same as that of Example 1, except that the protective agent A is replaced by an equal amount of protective agent B.

[0084] Example 7

[0085] This embodiment provides a method for extracting earthworm protein. The specific implementation method is the same as that of Example 1, except that the protective agent A is replaced by an equal amount of protective agent C.

[0086] Example 8

[0087] This embodiment provides a method for extracting earthworm protein. The specific implementation method is the same as that of Example 1, except that modified activated carbon A is replaced by modified activated carbon B in equal amounts.

[0088] Example 9

[0089] This embodiment provides a method for extracting earthworm protein. The specific implementation method is the same as that of Example 1, except that 5 wt%

[0090] An equal amount of L-cysteine ​​aqueous solution replaces the protective agent A.

[0091] Comparative Example 1

[0092] This comparative example provides a method for extracting earthworm protein. The specific implementation method is the same as that of Example 1, except that the modified activated carbon A is replaced by an equal amount of activated carbon.

[0093] Performance Testing

[0094] The content of earthworm protein obtained by the extraction method described in the above Examples 1-9 and Comparative Example 1 was tested, and the test results are shown in Table 1.

[0095] The powder products (earth dragon protein products) obtained by the extraction methods described in Examples 1-9 and Comparative Example 1 can be used to determine the content of earth dragon protein by measuring the content of acid-soluble protein and the content of free amino acids.

[0096] Principle: Protein is easily precipitated under acidic conditions, and substances with lower molecular weight (molecular weight below 10,000 Daltons), namely acid-soluble proteins, can be dissolved in trichloroacetic acid (TCA) solution. After the sample is acidified, the acid-soluble protein content in the filtrate minus the free amino acid content is the content of earthworm protein.

[0097] The specific steps for testing the content of earthworm protein are as follows:

[0098] (1) Determination of acid-soluble protein content: 1 g of extracted earthworm protein was added with 5 ml of 15 wt% TCA solution, stirred for 20 min, allowed to stand for 10 min, centrifuged at 4000 r / min for 10 min, and the supernatant was taken. The protein conversion factor was 6.25 according to GB / T 5009.5-2003. The test result was converted to dry basis according to the drying loss of earthworm protein, and the acid-soluble protein content was P1;

[0099] (2) Determination of free amino acid content: 1 g of extracted earthworm protein was added with 5 ml of 15 wt% TCA solution, stirred for 20 min, allowed to stand for 10 min, centrifuged at 4000 rpm for 10 min, and the supernatant was taken and determined according to GB / T 5009.124-2003. The free amino acid content was P2;

[0100] (3) Calculation of earthworm protein content (P): P = P1-P2.

[0101] Table 1

[0102] Group Pheretima protein content (%) Example 1 84.9 Example 2 84.2 Example 3 83.9 Example 4 81.6 Example 5 81.2 Example 6 82.5 Example 7 82.2 Example 8 82.0 Example 9 81.7 Comparative Example 1 81.4

[0103] As can be seen from the data in Table 1, the earth dragon protein extracted in Examples 1-3 of the present invention has the characteristics of high content as a whole. Among them, Examples 4-5 respectively change the addition ratio of papain and bromelain in step S1, so that the earth dragon cannot be efficiently hydrolyzed, resulting in a significant decrease in the content of earth dragon protein; Examples 6-7 change the addition ratio of the key ingredients tea polyphenols and L-cysteine ​​during the synthesis of the protective agent, so that the earth dragon protein is not well protected, and then show the phenomenon of decreased earth dragon protein content; Example 8 changes the modification ratio of the phosphoric acid aqueous solution during the synthesis of modified activated carbon, so that the adsorption capacity of the modified activated carbon to the pigment decreases, resulting in a decrease in the content of earth dragon protein to a certain extent; Example 9 and Comparative Example 1 are 5wt% L-cysteine ​​aqueous solution and activated carbon respectively replace the protective agent A and the modified activated carbon A in equal amounts, and the test finds that the content of earth dragon protein shows poor results.

[0104] The above implementation modes are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the content of the present invention and implement it, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for extracting earthworm protein, characterized in that: The following steps are involved: S1, placing the living earthworm in a container, adding ethanol solution, soaking, taking out the earthworm, washing, obtaining the emetic living earthworm for standby use; S2, placing the vomiting live earthworm of step S1 in 30-40° C. warm water, adding a protective agent and papain, stirring, soaking, then adding bromelain, stirring, soaking, and obtaining a mixed solution for standby use; S3, grinding the mixed solution of step S2, adding modified activated carbon, filtering, taking filtrate 1, centrifuging, and obtaining filtrate 2 for standby use; S4, subjecting the filtrate 2 of step S3 to a first membrane separation to obtain a filtrate 3, and then subjecting the filtrate to a second membrane separation and drying to obtain earthworm protein.

2. The method for extracting earthworm protein according to claim 1, characterized in that: The mass ratio of the emetic living earthworm, the protective agent, papain and bromelain in step S2 is 1:(3-5):(0.3-0.5):(0.2-0.4).

3. The method for extracting earthworm protein according to claim 1, characterized in that: The method for preparing the protective agent in step S2 comprises the following steps: (1) adding chitosan to a mixed solution of an acetic acid aqueous solution and a glycerol solution, stirring, adding Span 80, stirring, and obtaining a mixed solution 1 for later use; (2) adding tea polyphenols and L-cysteine ​​into deionized water and stirring to obtain a mixed solution 2 for later use; (3) adding the mixed solution 2 of step (2) dropwise into the mixed solution 1 of step (1) while homogenizing, and continuing to homogenize to obtain a mixed solution 3 for later use; (4) The mixed solution 3 of step (3) is spray-dried and sealed and stored at 2-6° C. to obtain a protective agent.

4. The method for extracting earthworm protein according to claim 3, characterized in that: The mass ratio of chitosan, tea polyphenols and L-cysteine ​​is 1:(0.4-0.6):(0.2-0.4).

5. The method for extracting earthworm protein according to claim 1, characterized in that: The mass ratio of the living earthworm for inducing emesis to the modified activated carbon is 1:(0.04-0.06).

6. The method for extracting earthworm protein according to claim 1, characterized in that: The method for preparing the modified activated carbon in step S3 comprises the following steps: 1) washing the poultry feathers with deionized water, removing impurities, drying, shearing and crushing to obtain pre-treated poultry feathers for later use; 2) The poultry feathers pretreated in step 1) are mixed with a 40-50 wt% phosphoric acid aqueous solution, stirred for 25-35 min at 70-80 kPa and 115-118° C., heated to 550-650° C. for carbonization for 25-35 min, washed, and dried to obtain modified activated carbon.

7. The method for extracting earthworm protein according to claim 6, characterized in that: In the step 2), the mass ratio of the poultry feathers to the phosphoric acid aqueous solution is 1:(2-3).

8. The method for extracting earthworm protein according to claim 1, characterized in that: The molecular weight cutoff of the primary membrane separation in step S4 is 9000-10000 Daltons; the molecular weight cutoff of the secondary membrane separation is 1000-2000 Daltons.

9. The method for extracting earthworm protein according to any one of claims 1 to 8, characterized in that: The content of the prepared earthworm protein is not less than 83.5%.

10. Use of earthworm protein obtained by the earthworm protein extraction method according to any one of claims 1 to 9 in cardiovascular and cerebrovascular applications.