Controllable preparation method of high-activity earthworm protein peptide

Abstract

The invention discloses a controllable preparation method of high-activity earthworm protein peptide, and belongs to the technical field of earthworm processing. The preparation method comprises the following steps: (1) adding amylase and keratinase into earthworm pulp for hydrolysis; (2) filtering by using a filter; (3) hydrolyzing the filtrate by using a compound enzyme; (4) adding alkaline protease into the filter residues for hydrolysis, and then adding high-temperature protease for hydrolysis; (5) filtering the hydrolyzed slurry, and then carrying out filtering treatment by using a ceramic micro-filter to obtain low-molecular-weight earthworm protein peptide; and (6) mixing the obtained earthworm water-soluble protein hydrolysate and the obtained earthworm insoluble protein polypeptide, concentrating and drying to obtain the high-activity earthworm protein peptide. According to the method, the water-soluble protein and the water-insoluble protein in the lumbricus are subjected to step-by-step differential hydrolysis, so that protein resources in the lumbricus are utilized to the maximum extent, the polypeptide / protein yield can be 85% or above, waste is avoided, and the activity of the obtained protein peptide is guaranteed.

Description

Technical Field

[0001] The invention belongs to the technical field of earthworm processing, and particularly relates to a controllable preparation method of high-activity earthworm protein peptide. Background Art

[0002] Pteris protein peptide is a bioactive substance extracted from earthworms. In recent years, it has received extensive attention in the fields of medicine and health care, especially in the prevention and treatment of cardiovascular diseases. At present, the extraction of Pteris protein peptide in the prior art is mostly aimed at the hydrolysis of water-soluble proteins in Pteris, such as patent CN107760747A. However, studies have shown that Pteris also contains a large amount of water-insoluble proteins, which can also be obtained after hydrolysis of these water-insoluble proteins. Pteris protein peptides are also rich in lysine, arginine and other amino acids that are beneficial to the human body.

[0003] Therefore, the existing research on earth dragon protein, on the one hand, only focuses on the treatment of water-soluble protein, while ignoring the water-insoluble protein in earth dragon, resulting in a waste of earth dragon resources, and on the other hand, if only focusing on the yield of small molecule protein peptides, water-soluble protein and water-insoluble protein are usually mixed together for hydrolysis, and the problem of excessive hydrolysis of water-soluble protein that may be caused will be ignored in this process, resulting in a decrease in the activity of the obtained earth dragon protein peptide. Therefore, it is necessary to propose a treatment method that is directed to earth dragon water-soluble protein and pays attention to water-insoluble protein at the same time, so that the yield of earth dragon protein peptide is increased without reducing activity. Summary of the invention

[0004] Technical issues

[0005] Existing research on earthworm protein, on the one hand, only focuses on the processing of water-soluble proteins, while ignoring the water-insoluble proteins in earthworms, resulting in a waste of earthworm resources. On the other hand, if water-soluble proteins and water-insoluble proteins are mixed together for hydrolysis, the problem of excessive hydrolysis of water-soluble proteins will be ignored, resulting in reduced activity of earthworm protein peptides.

[0006] Technical content

[0007] In order to solve the above problems, the present invention designs a step-by-step differential hydrolysis method for water-soluble protein and water-insoluble protein of earth dragon, which can improve the yield and bioavailability of earth dragon protein peptides while maximizing the activity of water-soluble protein. For water-soluble protein, by wet grinding and crushing, cutinase and amylase hydrolysis, the dissolution of water-soluble protein is promoted, and then a small amount of endonuclease is used to moderately hydrolyze the water-soluble protein of earth dragon, release more effective active sites, and improve thrombolytic activity. For insoluble earth dragon protein, alkaline protease and high temperature protease are used for step-by-step hydrolysis, and protein hydrolysis is promoted under the dual action of alkali solution and high temperature, so as to improve the yield of protein peptides.

[0008] The present invention provides a method for preparing a high-activity earthworm protein peptide, the preparation method comprising the following steps:

[0009] (1) Compound enzyme solubilization: adding amylase and cutinase to the earthworm pulp for hydrolysis;

[0010] (2) multi-stage filtration: first, using a bag filter to filter the concentrated earthworm slurry after hydrolysis in step (1), collecting the filter residue and the effluent, then using a plate and frame filter to filter the effluent, taking the filtrate, and finally using an ultrafiltration membrane to ultrafilter the filtrate to obtain an ultrafiltration permeate;

[0011] (3) moderate hydrolysis by endonuclease: using a composite enzyme to hydrolyze the ultrafiltration permeate obtained in step (2) to obtain a water-soluble protein hydrolyzate of earthworm;

[0012] (4) Insoluble protein separation: Take the filter residue obtained in step (2), add water and stir, adjust the pH to 9.5-11.0, add alkaline protease for hydrolysis, then adjust the pH to 6.0-8.0, add high temperature protease for hydrolysis;

[0013] (5) Protein peptide purification: The slurry after high temperature protease hydrolysis in step (4) is filtered using a bag filter, the filtrate is taken, and then the filtrate is filtered using a ceramic microfilter to remove all insoluble matter to obtain low molecular weight earthworm protein peptide;

[0014] (6) Concentration and drying: The water-soluble protein hydrolysate of earthworm obtained in step (3) and the insoluble protein polypeptide of earthworm obtained in step (5) are mixed and then concentrated, and finally dried to obtain highly active earthworm protein peptides.

[0015] Furthermore, the preparation method of the earthworm slurry in step (1) is: killing the live earthworm, cleaning it, and then performing wet ultrafine grinding, with the grinding level being 300-400 meshes;

[0016] Alternatively, the dried earthworms are soaked and cleaned and then subjected to wet ultrafine grinding with a grinding grade of 300 to 400 meshes.

[0017] Furthermore, the soaking and cleaning of the dried earthworm is as follows: the dried earthworm is added with water at a solid-liquid ratio of 1:2-4, and then 1-2 wt% of flour by weight of the dried earthworm is added, and the dried earthworm is soaked at 40-50° C. for 8-10 hours, and then the dried earthworm is taken out, and the soaked dried earthworm is repeatedly washed with water at 40-50° C. until the washing water is clear and has no turbidity.

[0018] Furthermore, in step (1), the amount of amylase added is 3000-5000 U / g of dried earthworm.

[0019] Furthermore, in step (1), the amount of cutinase added is 3000-5000 U / g of dried earthworm.

[0020] Furthermore, in step (1), the hydrolysis temperature is 40-60° C., and the hydrolysis time is 3-5 h.

[0021] Furthermore, the ultrafiltration membrane in step (2) is a medium molecular weight ultrafiltration membrane, and the molecular weight of the retained substance is 8000 to 10000 Da.

[0022] Furthermore, in step (3), the complex enzyme includes one or more of papain, Amano PC10F protease, and bromelain.

[0023] Furthermore, in step (3), the complex enzyme is papain and bromelain.

[0024] Furthermore, the mass ratio of papain to bromelain is 1:0.8-1.2.

[0025] Furthermore, in step (3), the amount of the complex enzyme added is 1000 to 3000 U / g of dried earthworm.

[0026] Furthermore, in step (3), the hydrolysis temperature is 40-60° C., and the hydrolysis time is 2-3 h.

[0027] Furthermore, the amount of water added in step (4) is 80 to 150% of the mass of the filter residue.

[0028] Furthermore, in step (4), the amount of alkaline protease added is 4000-6000 U / g filter residue.

[0029] Furthermore, in step (4), the hydrolysis temperature of the alkaline protease is 40-60° C., and the hydrolysis time is 3-4 hours.

[0030] Furthermore, in step (4), the amount of high temperature protease added is 4000-6000 U / g filter residue.

[0031] Furthermore, in step (4), the hydrolysis temperature of the high-temperature protease is 60-80° C., and the hydrolysis time is 3-4 hours.

[0032] Furthermore, in step (5), the retention size of the ceramic microfilter is 50 to 80 nm.

[0033] Furthermore, the concentration treatment in step (6) is carried out using nanofiltration concentration equipment or vacuum evaporation concentration equipment.

[0034] Furthermore, the drying in step (6) is preferably low-temperature spray drying; the air inlet temperature during the drying process is 110-130°C, and the air outlet temperature is 60-70°C.

[0035] The present invention provides a high-activity earthworm protein peptide obtained according to the preparation method.

[0036] The invention provides an application of the highly active earthworm protein peptide in the field of health products, functional beverages or special medical formula foods.

[0037] Beneficial Effects

[0038] The product has high yield and good activity: the present invention maximizes the use of protein resources in earthworms by performing step-by-step differential hydrolysis on water-soluble proteins and water-insoluble proteins in earthworms, and can achieve a polypeptide / protein yield of more than 85%, thereby avoiding waste and ensuring the activity of the obtained protein peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The elution curves of the earthworm peptide gel chromatography electrophoresis in Example 1 and Comparative Examples 1-2 are shown.

[0040] Figure 2 The anticoagulant effect of the earthworm peptide in Example 1 and Comparative Examples 1-2. DETAILED DESCRIPTION

[0041] Source of raw materials

[0042] Amylase was purchased from Suzhou Huarui Biotechnology Co., Ltd.; cutinase was purchased from Suzhou Huarui Biotechnology Co., Ltd.; unless otherwise specified, all raw materials used were commercially available common products.

[0043] Testing process

[0044] 1. Pheretima protein / peptide yield: The total protein content m0 ​​of the dried worm sample and the total protein content m1 of the worm protein peptide product finally obtained in step (11) were determined by the Kjeldahl method, and the protein yield was calculated using the formula m0 / m1*100.

[0045] The operation of Kjeldahl nitrogen determination method is as follows: accurately weigh 1.0g solid sample, transfer it into a dry 100mL nitrogen determination bottle, add 0.2g copper sulfate, 6g potassium sulfate and 20ml sulfuric acid, shake it slightly and place a small funnel at the mouth of the bottle, place the bottle at a 45-degree angle on an asbestos net with small holes, heat it with a low fire, wait until the contents are completely carbonized and the foam stops completely, increase the firepower, and keep the liquid in the bottle slightly boiling until the liquid is blue-green, clear and transparent, and continue heating for 0.5 hours. Remove and cool, carefully add 20mL of water, cool it, transfer it into a 100mL volumetric flask, and wash the nitrogen determination bottle with a small amount of water, add the washing liquid into the volumetric flask, add water to the scale, mix well and set aside. At the same time, do a reagent blank test. Add a few drops of methyl red indicator and a few milliliters of sulfuric acid to about 2 / 3 of the water in the steam generator to keep the water acidic, and add a few glass beads to prevent violent boiling. Add 10mL of 2% boric acid solution and 1 drop of mixed indicator to the receiving bottle, and insert the lower end of the condenser under the liquid surface. Pipette 10.0mL of sample digestion solution from the small glass cup into the reaction chamber, and wash the small beaker with 10mL of water to make it flow into the reaction chamber, and plug the rod-shaped glass stopper of the small glass cup. Pour 10mL of 40% sodium hydroxide solution into the small glass cup, lift the glass stopper to make it slowly flow into the reaction chamber, and do not plug the glass cover immediately. It should be rinsed with distilled water first and then covered, and water should be added to the small glass cup to prevent air leakage. Clamp the spiral clamp and start distillation. Steam is passed into the reaction chamber to allow ammonia to pass through the condenser and enter the receiving bottle. Distill for 5 minutes. Move the receiving bottle so that the lower end of the condenser leaves the liquid dish, distill for another 1 minute, and then rinse the outside of the lower end of the condenser with a small amount of water. Remove the receiving bottle and titrate with 0.05mol / L sulfuric acid or 0.05mol / L hydrochloric acid standard solution until gray or blue-purple is the end point. At the same time, take 10.0mL of reagent blank digestion solution and titrate according to the above operation.

[0046] Finally, the absolute mass of protein in the sample was calculated according to the following formula:

[0047] m=(V-V0)×N×0.014×F

[0048] V is the volume of sulfuric acid or hydrochloric acid standard solution consumed by the sample; V0 is the volume of sulfuric acid or hydrochloric acid standard solution consumed by the reagent blank; N is the equivalent concentration of sulfuric acid or hydrochloric acid standard solution; F is the coefficient of nitrogen conversion to protein (calculated as 6.25).

[0049] 2. Molecular weight distribution: First, the sample was filtered using a 0.22 μm water filter membrane, and then its molecular weight distribution was analyzed using HPLC equipped with a TSK G2000SW (300 mm × 7.5 mm) gel chromatography column. The specific working conditions of the instrument are: acetonitrile, water and trifluoroacetic acid were mixed in a volume ratio of 60%, 39.95% and 0.05% as the mobile phase, the pump flow rate was set to 1 mL / min, the detection wavelength was selected to be 214 nm, and the volume of each injection was 10 μL.

[0050] To construct the relative molecular weight calibration curve, the following standards were used: cytochrome C (molecular weight 12384 Da), bovine insulin (molecular weight 5733.49 Da), bacitracin (molecular weight 1422.69 Da), reduced glutathione (molecular weight 307.32 Da), and glycine (molecular weight 75.07 Da).

[0051] 3. Thrombolytic activity: Rats were anesthetized by intraperitoneal injection of a mixed solution of chloral hydrate and urethane (the solvent was water, the proportion of chloral hydrate was 10wt%, and the proportion of urethane was 25wt%) at a dosage of 5mL / kg (rat body weight). After fixation, 5mL of blood was collected from the abdominal aorta of each rat and injected into a common vacuum blood collection tube.

[0052] Take the sterilized test tubes and add 16 mL of sterilized saline to each tube, then slowly inject 2 mL of blood along the tube wall, finally, add 2 mL of earthworm protein peptide solution (the solvent is water, and the concentration of earthworm protein peptide is 0.5 g / mL) to each tube, and treat it in a water bath at 37°C. Observe the complete coagulation of each tube of blood, and take pictures for comparison after 3000 seconds.

[0053] Example 1

[0054] (1) Treatment of earthworms: Using dried earthworms as raw material, water was added at a solid-liquid ratio of 1:3, and then 1 wt% of wheat flour was added to the dried earthworms. The dried earthworms were soaked at 40°C for 10 hours to fully swell the dried earthworms and dissolve the fishy substances. The dried earthworms were then taken out and repeatedly washed with 40°C water until the washing water was clear and free of turbidity.

[0055] (2) Ultrafine grinding: Use a wet ultrafine grinding instrument to grind the dried earthworm washed in step (1) to a grinding level of 350 meshes to obtain an earthworm slurry.

[0056] (3) Complex enzyme solubilization: adding amylase and keratinase to the earthworm slurry in step (2) for hydrolysis to obtain earthworm water-soluble protein hydrolysate, wherein the addition amount of both enzymes is 4000U / g (dry earthworm), the hydrolysis temperature is 50°C, and the hydrolysis time is 4h; the combination of protein and keratin and earthworm cell wall is destroyed by the bioenzymatic hydrolysis, and the dissolution of water-soluble protein is promoted. The water-soluble protein released after the reaction includes small molecules with a molecular weight of about 10,000Da, such as lumbrokinase and plasmin, and highly active water-soluble proteins.

[0057] (4) Multi-stage filtration: First, use a bag filter to filter the concentrated earthworm slurry after hydrolysis in step (3), collect the filter residue and the effluent, and then use a plate and frame filter to filter the effluent. Then, take the filtrate and use a 10,000Da ultrafiltration membrane to ultrafilter the filtrate to obtain an ultrafiltration permeate. Multi-stage filtration can remove small molecular weight polysaccharide compounds and minerals produced after hydrolysis by amylase and cutinase, so as to improve the purity of water-soluble protein and reduce heavy metal residues.

[0058] (5) Enzyme moderate hydrolysis: The ultrafiltration permeate obtained in step (4) is moderately hydrolyzed by protease using a composite enzyme of papain and bromelain to obtain a water-soluble protein hydrolyzate of earthworm with higher thrombolytic activity; the total amount of the composite enzyme added is 2000 U / g (dried earthworm), the mass ratio of the two enzymes is 1:1, the hydrolysis temperature is 55°C, and the hydrolysis time is 2 h.

[0059] (6) Separation of insoluble proteins: The filter residue separated by bag filtration in step (4) is subjected to gradient hydrolysis using alkaline protease and high-temperature protease to improve the protein yield. The protein yield and low molecular weight yield in this step are important indicators for determining the effectiveness of the process. The specific operation of the gradient hydrolysis is to first add 60°C warm water of equal weight to the filter residue, stir evenly, adjust the pH to 10, add 5000U / g (filter residue) alkaline protease, and hydrolyze at 50°C for 3.5h, then adjust the pH to 7, add 5000U / g (filter residue) high-temperature protease, and perform a second hydrolysis at 70°C for 3.5h. The method is characterized in that alkaline protease is first used to destroy the spatial structure of the protein under alkaline conditions to induce the unfolding and partial hydrolysis of the protein structure, and then a protease with high temperature resistance is used to perform deep hydrolysis at high temperature to improve the yield of earthworm protein peptides.

[0060] (7) Protein peptide purification: The slurry after the second hydrolysis by high-temperature protease in step (6) is filtered using a bag filter, and the filtrate is taken and then filtered using a ceramic microfilter with a retention size of 50 nm to remove all insoluble matter and obtain low molecular weight earthworm protein peptide.

[0061] (8) Nanofiltration concentration: The water-soluble protein hydrolysate of earthworm obtained in step (5) and the low molecular weight earthworm protein peptide obtained in step (7) are mixed, and then concentrated using a nanofiltration concentration device to a solid content of 25%.

[0062] (9) Vacuum evaporation concentration: The nanofiltration concentrate obtained in step (8) is further concentrated using a vacuum evaporation concentration device to a solid content of more than 45%.

[0063] (10) Pulse strong light low temperature sterilization: The concentrated solution obtained in step (9) is sterilized by a strong light pulse sterilization method, and the temperature of the strong light pulse is controlled within 50° C. and the time is 10 s.

[0064] (11) Low-temperature spray drying: The concentrated liquid sterilized in step (10) is subjected to low-temperature spray drying to obtain earthworm protein peptide; the air inlet temperature during the drying process is 120° C. and the air outlet temperature is 60° C., which is conducive to better retaining the activity of the earthworm protein peptide.

[0065] Comparative Example 1

[0066] The steps in Example 1 were followed, except that step (3) was omitted, i.e., the amylase and cutinase treatment was not performed, to finally obtain the earthworm protein peptide.

[0067] Comparative Example 2

[0068] The steps in Example 1 are referred to, wherein only steps (4) and (5) are omitted, and step (6) is directly performed, that is, the earthworm concentrate hydrolyzed in step (3) is directly subjected to gradient hydrolysis to finally obtain earthworm protein peptides.

[0069] The earthworm protein peptides prepared in Example 1 and Comparative Examples 1-2 were tested for polypeptide / protein yield, molecular weight distribution and thrombolytic activity. The test data are shown in Tables 1 and Figures 1-2 .

[0070] Table 1 Comparison of polypeptide / protein yields in Examples and Comparative Examples

[0071] sample Example 1 Comparative Example 1 Comparative Example 2 Peptide / protein yield 85.6％ 45.6％ 85.3％

[0072] As can be seen from the table above, the polypeptide / protein yield in Comparative Example 1 is much lower than that in Example 1, which fully proves that the benefit of step (3) is to promote the dissolution of protein in dried earthworms, thereby increasing the polypeptide / protein yield. Comparative Example 2 is different from Example 1 in that the water-soluble protein and the water-insoluble protein are not hydrolyzed step by step, which has little effect on the polypeptide / protein yield.

[0073] from Figure 1 It can be seen that Example 1 has an independent peak in the earlier elution time period, indicating that there is a certain content of relatively macromolecular substances in its composition, indicating that the water-soluble protein molecular structure is retained to the greatest extent in the earth dragon protein peptide product obtained according to the method of the present invention, while the insoluble earth dragon protein is hydrolyzed into a polypeptide with a smaller molecular weight. The molecular weight distribution of polypeptides in Comparative Examples 1 and 2 presents similar characteristics, showing small molecule polypeptide distribution characteristics. Comparative Example 2 differs from Example 1 in that water-soluble proteins and water-insoluble proteins are not hydrolyzed step by step, and small molecule polypeptides account for more in the overall molecular weight distribution, indicating that it is not conducive to selectively protecting the activity of water-soluble proteins.

[0074] from Figure 2 It can be seen that both Example 1 and Comparative Example 1 have significantly better anticoagulant effects, and still present a liquid state when the coagulation time reaches 3000s, without coagulation and sedimentation, indicating that the earthworm protein peptide prepared according to the method of the present invention has better anticoagulant and thrombolytic effects, and the characteristic of Comparative Example 1 is that it does not promote the dissolution of more proteins and polypeptides with anticoagulant activity through step (3), and the earthworm peptides extracted according to the method of Comparative Example 1 are mostly water-soluble protein peptides, so they also have high anticoagulant activity, but Table 1 shows that its earthworm peptide yield is extremely low, so Example 1 shows better advantages in terms of earthworm peptide yield and anticoagulant activity. In comparison, Comparative Example 2 has a poor anticoagulant effect, and the blood shows obvious aggregation and sedimentation, indicating that the water-soluble protein and water-insoluble protein of the protein are not distributed and hydrolyzed through steps (4) and (5), which will cause the water-soluble active protein function to drop sharply, thereby causing the anticoagulant activity of the obtained earthworm peptide to be lost.

[0075] In summary, it can be seen that the method of the present invention, on the one hand, greatly improves the yield of earthworm protein peptides, and on the other hand, effectively protects the anticoagulant activity of earthworm protein peptides during processing and hydrolysis.

[0076] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing a highly active earthworm protein peptide, characterized in that: The preparation method comprises the following steps: (1) Compound enzyme solubilization: adding amylase and cutinase to the earthworm slurry for hydrolysis; the amount of amylase added is 3000-5000U / g of dry earthworm; the amount of cutinase added is 3000-5000U / g of dry earthworm; (2) multi-stage filtration: first, using a bag filter to filter the concentrated earthworm slurry after hydrolysis in step (1), collecting the filter residue and the effluent, then using a plate and frame filter to filter the effluent, taking the filtrate, and finally using an ultrafiltration membrane to ultrafilter the filtrate to obtain an ultrafiltration permeate; (3) moderate hydrolysis with endonuclease: using a composite enzyme to hydrolyze the ultrafiltration permeate obtained in step (2) to obtain a water-soluble protein hydrolyzate of earthworm; the composite enzyme comprises one or more of papain, Amano PC10F protease, and bromelain; the amount of the composite enzyme added is 1000 to 3000 U / g of dry earthworm; (4) Insoluble protein separation: Take the filter residue obtained in step (2), add water and stir, adjust the pH to 9.5-11.0, add alkaline protease for hydrolysis, then adjust the pH to 6.0-8.0, add high temperature protease for hydrolysis; (5) Protein peptide purification: The slurry after high temperature protease hydrolysis in step (4) is filtered using a bag filter, the filtrate is taken, and then the filtrate is filtered using a ceramic microfilter to remove all insoluble matter to obtain a low molecular weight earthworm protein peptide; the amount of alkaline protease added is 4000-6000U / g filter residue; the amount of high temperature protease added is 4000-6000U / g filter residue; (6) Concentration and drying: The water-soluble protein hydrolysate of earthworm obtained in step (3) and the insoluble protein polypeptide of earthworm obtained in step (5) are mixed and then concentrated, and finally dried to obtain highly active earthworm protein peptides.

2. The preparation method according to claim 1, characterized in that The preparation method of the earthworm slurry in step (1) is: killing the living earthworm, cleaning it and then performing wet ultrafine grinding, the grinding level is 300-400 mesh; or, soaking and cleaning the dried earthworm and then performing wet ultrafine grinding, the grinding level is 300-400 mesh.

3. The preparation method according to claim 1, characterized in that: In step (1), the hydrolysis temperature is 40-60° C. and the hydrolysis time is 3-5 h.

4. The preparation method according to claim 1, characterized in that: In step (3), the composite enzyme is papain and bromelain; the mass ratio of papain to bromelain is 1:0.8-1.

2.

5. The preparation method according to claim 1, characterized in that: In step (3), the hydrolysis temperature is 40-60° C. and the hydrolysis time is 2-3 h.

6. The preparation method according to claim 1, characterized in that: In step (4), the hydrolysis temperature of the alkaline protease is 40-60°C, and the hydrolysis time is 3-4 hours; in step (4), the hydrolysis temperature of the high-temperature protease is 60-80°C, and the hydrolysis time is 3-4 hours.

7. The preparation method according to claim 1, characterized in that: The concentration treatment in step (6) is carried out using a nanofiltration concentration device or a vacuum evaporation concentration device.

8. The preparation method according to claim 1, characterized in that: The drying in step (6) is preferably low-temperature spray drying; the air inlet temperature during the drying process is 110-130°C, and the air outlet temperature is 60-70°C.

9. A highly active earthworm protein peptide, characterized in that: The highly active earthworm protein peptide is prepared according to the preparation method according to any one of claims 1 to 8.

10. Use of the highly active earthworm protein peptide described in claim 9 in the field of health products, functional beverages or special medical formula foods.

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