A controllable preparation method of high-activity earthworm protein peptide

Abstract

The application 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 cutinase to earthworm paste for hydrolysis; (2) filtering by using a filter; (3) hydrolyzing the filtrate by using a composite enzyme; (4) adding alkaline protease to the filter residue for hydrolysis, and then adding high-temperature protease for hydrolysis; (5) filtering the hydrolyzed paste, and then filtering and treating the paste by using a ceramic microfilter 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, and then concentrating and drying to obtain high-activity earthworm protein peptide. According to the application, the water-soluble protein and the water-insoluble protein in the earthworm are step-by-step differentially hydrolyzed, the protein resources in the earthworm are maximally utilized, the polypeptide / protein yield can be more than 85%, waste is avoided, and the activity of the obtained protein peptide is ensured.

Description

Technical Field

[0001] This invention belongs to the field of earthworm processing technology, specifically relating to a controllable preparation method of highly active earthworm protein peptides. Background Technology

[0002] Earthworm protein peptides are bioactive substances extracted from earthworms and have received widespread attention in the medical and health fields in recent years, particularly in the prevention and treatment of cardiovascular diseases. Currently, existing technologies for extracting earthworm protein peptides mostly focus on the hydrolysis of water-soluble proteins in earthworms, such as patent CN107760747A. However, research has shown that earthworms also contain a large amount of water-insoluble proteins, which can also yield earthworm protein peptides after hydrolysis. These peptides are rich in lysine, arginine, and other amino acids beneficial to the human body.

[0003] Therefore, existing research on earthworm proteins focuses solely on the treatment of water-soluble proteins, neglecting the water-insoluble proteins in earthworms, thus wasting earthworm resources. Furthermore, if only the yield of small-molecule protein peptides is considered, water-soluble and water-insoluble proteins are often hydrolyzed together. This process overlooks the potential for excessive hydrolysis of water-soluble proteins, leading to reduced activity of the resulting earthworm protein peptides. Therefore, a method is needed that addresses both water-soluble and water-insoluble proteins in earthworms, increasing the yield of earthworm protein peptides without reducing their activity. Summary of the Invention

[0004] Technical issues

[0005] Existing research on earthworm proteins focuses only on the treatment of water-soluble proteins while neglecting the water-insoluble proteins in earthworms, resulting in a waste of earthworm resources. On the other hand, if water-soluble and water-insoluble proteins are mixed together for hydrolysis, the potential problem of excessive hydrolysis of water-soluble proteins will be overlooked, leading to a decrease in the activity of earthworm protein peptides.

[0006] Technical content

[0007] To address the aforementioned issues, this invention employs a stepwise differentiated hydrolysis method for both water-soluble and water-insoluble earthworm proteins. This method maximizes the activity of water-soluble proteins while simultaneously increasing the yield and bioavailability of earthworm protein peptides. For water-soluble proteins, wet grinding and hydrolysis with keratinase and amylase promote dissolution. A small amount of endonuclease is then used for moderate hydrolysis, releasing more effective active sites and enhancing thrombolytic activity. For insoluble earthworm proteins, stepwise hydrolysis using alkaline protease and thermolytic protease promotes protein hydrolysis under the combined effects of alkaline solution and high temperature, thereby increasing the yield of protein peptides.

[0008] This invention provides a method for preparing highly active earthworm protein peptides, the preparation method comprising the following steps:

[0009] (1) Complex enzyme solubilization: Add amylase and keratinase to earthworm slurry for hydrolysis;

[0010] (2) Multi-stage filtration: First, use a bag filter to filter the earthworm slurry after hydrolysis in step (1), collect the filter residue and effluent, then use a plate and frame filter to filter the effluent, take the filtrate, and finally use an ultrafiltration membrane to ultrafilter the filtrate to obtain the ultrafiltration permeate.

[0011] (3) Moderate hydrolysis with endonuclease: The ultrafiltration permeate obtained in step (2) was hydrolyzed using a complex enzyme to obtain earthworm water-soluble protein hydrolysate;

[0012] (4) Separation of insoluble proteins: 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, and then adjust the pH to 6.0-8.0, add high temperature protease for hydrolysis;

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

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

[0015] Furthermore, the preparation method of earthworm slurry in step (1) is as follows: after killing and washing the live earthworms, wet ultrafine grinding is performed, and the grinding level is 300-400 mesh.

[0016] Alternatively, the dried earthworms can be soaked and washed, then subjected to wet ultrafine grinding to a grinding level of 300-400 mesh.

[0017] Furthermore, the soaking and cleaning of dried earthworms is as follows: add dried earthworms to water at a solid-liquid ratio of 1:2 to 4, then add flour at 1 to 2 wt% of the weight of the dried earthworms, soak at 40 to 50°C for 8 to 10 hours, then remove the dried earthworms and repeatedly wash them with water at 40 to 50°C until the washing water is clear and free of 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 keratinase 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, in step (2), the ultrafiltration membrane is a medium molecular weight ultrafiltration membrane, and the molecular weight of the retained substances is 8000-10000 Da.

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

[0023] Furthermore, the complex enzymes in step (3) are 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 compound enzyme added is 1000-3000 U / g of dried earthworm.

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

[0027] Furthermore, in step (4), the amount of water added is 80-150% 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 h.

[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℃, and the hydrolysis time is 3-4h.

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

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

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

[0035] This invention provides a highly active earthworm protein peptide obtained according to the above preparation method.

[0036] The application of the highly active earthworm protein peptide provided by this invention in the fields of health products, functional beverages, or special medical purpose formula foods.

[0037] Beneficial effects

[0038] High product yield and good activity: This invention maximizes the utilization of protein resources in earthworms by performing stepwise differentiated hydrolysis of water-soluble and water-insoluble proteins, achieving a peptide / protein yield of over 85%, thus avoiding waste and ensuring the activity of the obtained protein peptides. Attached Figure Description

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

[0040] Figure 2 The anticoagulant effect of earthworm peptide in Example 1 and Comparative Examples 1-2 is shown. Detailed Implementation

[0041] Source of raw materials

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

[0043] Testing process

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

[0045] The procedure for the Kjeldahl method is as follows: Accurately weigh 1.0 g of solid sample and transfer it to a dry 100 mL nitrogen determination flask. Add 0.2 g of copper sulfate, 6 g of potassium sulfate, and 20 mL of sulfuric acid. Shake slightly and place a small funnel at the mouth of the flask. Support the flask at a 45-degree angle against a perforated asbestos mesh and heat over a low flame. Once the contents are completely carbonized and the foaming has completely stopped, increase the heat and maintain a gentle boil until the liquid in the flask turns a clear, blue-green color. Continue heating for another 0.5 hours. Remove from heat and allow to cool. Carefully add 20 mL of water. After cooling, transfer the solution to a 100 mL volumetric flask and rinse the nitrogen determination flask with a small amount of water. Add the rinsing solution to the volumetric flask and then add water to the mark. Mix well and set aside. Simultaneously, perform a reagent blank test. Fill a steam generator with water to about 2 / 3 full, add a few drops of methyl red indicator and a few mL of sulfuric acid to maintain the acidity of the water, and add a few glass beads to prevent bumping. Add 10 mL of 2% boric acid solution and 1 drop of mixed indicator to the receiving flask, and insert the lower end of the condenser tube below the liquid surface. Pipette 10.0 mL of the sample digest into the reaction chamber through a small glass beaker, and rinse the beaker with 10 mL of water to allow the liquid to flow into the reaction chamber. Tightly stopper the small glass beaker with the rod-shaped glass stopper. Pour 10 mL of 40% sodium hydroxide solution into the small glass beaker, lift the glass stopper to allow it to slowly flow into the reaction chamber. Do not immediately tighten the glass stopper; rinse with distilled water first, then cover. Add water to the small glass beaker to prevent leakage. Clamp the screw clamp and begin distillation. The vapor will pass into the reaction chamber, allowing ammonia to pass through the condenser tube and into the receiving flask. Distill for 5 minutes. Move the receiving flask so that the lower end of the condenser tube is removed from the liquid dish, and distill for another 1 minute. Then rinse the outside of the lower end of the condenser tube with a small amount of water. Remove the receiving flask and titrate with 0.05 mol / L sulfuric acid or 0.05 mol / L hydrochloric acid standard solution until a gray or blue-purple endpoint is reached. Simultaneously, titrate 10.0 mL of reagent blank digest according to the above procedure.

[0046] Finally, the absolute mass of protein in the sample is calculated using 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 for converting nitrogen to protein (calculated as 6.25).

[0049] 2. Molecular Weight Distribution: The sample was first filtered through a 0.22 μm aqueous filter membrane, and then its molecular weight distribution was analyzed by HPLC using a TSK G2000SW (300 mm × 7.5 mm) gel chromatography column. The specific operating conditions were as follows: acetonitrile, water, and trifluoroacetic acid were mixed at a volume ratio of 60%, 39.95%, and 0.05% respectively as the mobile phase; the pump flow rate was set to 1 mL / min; the detection wavelength was selected as 214 nm; and the injection volume was 10 μL per sample.

[0050] To construct relative molecular weight calibration curves, 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 (solvent: water, chloral hydrate 10 wt%, urethane 25 wt%) at a dose of 5 mL / kg (rat body weight). After fixation, 5 mL of blood was collected from the abdominal aorta of each rat and injected into a regular vacuum blood collection tube.

[0052] Take a test tube that has been sterilized by high temperature and add 16 mL of sterilized physiological saline to each tube. Then, slowly inject 2 mL of blood along the tube wall. Finally, add 2 mL of earthworm protein peptide solution (solvent is water, and the concentration of earthworm protein peptide is 0.5 g / mL) to each tube. Incubate the tube in a water bath at 37°C and observe the complete coagulation of the blood in each tube. Take pictures after 3000 seconds for comparison.

[0053] Example 1

[0054] (1) Earthworm intervention treatment: using dried earthworms as raw material, add water at a solid-liquid ratio of 1:3, then add wheat flour of 1wt% of the mass of dried earthworms, soak at 40℃ for 10 hours to fully swell the dried earthworms and dissolve the fishy substances, then take out the dried earthworms and repeatedly wash the soaked dried earthworms with 40℃ water until the washing water is clear and free of turbidity.

[0055] (2) Ultrafine grinding: The earthworm dried after being washed in step (1) was ground using a wet ultrafine grinding instrument to a grinding level of 350 mesh to obtain earthworm slurry.

[0056] (3) Compound enzyme solubilization: Add amylase and keratinase to the earthworm slurry in step (2) for hydrolysis to obtain earthworm water-soluble protein hydrolysate. The amount of both enzymes added is 4000U / g (earthworm dry), the hydrolysis temperature is 50℃, and the hydrolysis time is 4h. The combination of protein and keratin and earthworm cell wall is destroyed by biological enzyme hydrolysis, which promotes the dissolution of water-soluble protein. The water-soluble protein released after the reaction includes small molecules and highly active water-soluble proteins with a molecular weight of about 10000Da, such as lumbrokinase and plasmin.

[0057] (4) Multi-stage filtration: First, use a bag filter to filter the earthworm slurry after hydrolysis in step (3), collect the filter residue and effluent, then use a plate and frame filter to filter the effluent, then take the filtrate, and use a 10000Da ultrafiltration membrane to ultrafilter the filtrate to obtain the ultrafiltration permeate; multi-stage filtration can remove small molecular weight polysaccharide compounds and minerals produced after hydrolysis by amylase and keratinase, so as to improve the purity of water-soluble protein and reduce heavy metal residue.

[0058] (5) Moderate hydrolysis with endopeptides: The ultrafiltration permeate obtained in step (4) was moderately hydrolyzed with papain and bromelain complex enzymes to obtain earthworm water-soluble protein hydrolysate with higher thrombolytic activity; the total amount of complex enzyme added was 2000 U / g (earthworm dry), the mass ratio of the two enzymes was 1:1, the hydrolysis temperature was 55℃, and the hydrolysis time was 2h.

[0059] (6) Insoluble protein separation: 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 that determine the effectiveness of the process. The specific operation of gradient hydrolysis is as follows: First, add 60°C warm water of the same weight as 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 feature of this method is that alkaline protease is first used to destroy the spatial structure of the protein under alkaline conditions, induce the protein structure to unfold and partially hydrolyze, 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. The filtrate is then filtered again using a ceramic microfilter with a cutoff size of 50 nm to remove all insoluble matter and obtain low molecular weight earthworm protein peptides.

[0061] (8) Nanofiltration concentration: The earthworm water-soluble protein hydrolysate 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 until the solid content is 25%.

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

[0063] (10) Pulsed high-intensity light low-temperature sterilization: The concentrated liquid obtained in step (9) is sterilized by the high-intensity light pulse sterilization method. The temperature of the high-intensity light pulse is controlled within 50℃ and the time is 10s.

[0064] (11) Low-temperature spray drying: The concentrated liquid after sterilization in step (10) is spray dried at low temperature to obtain earthworm protein peptides; the air inlet temperature during the drying process is 120℃ and the air outlet temperature is 60℃, which is conducive to better preserving the activity of earthworm protein peptides.

[0065] Comparative Example 1

[0066] The steps in Example 1 are followed, except that step (3) is omitted, i.e., the amylase and keratinase are not used to obtain earthworm protein peptides.

[0067] Comparative Example 2

[0068] The steps in Example 1 are followed, except that steps (4) and (5) are omitted and step (6) is performed directly, that is, the earthworm concentrate after hydrolysis in step (3) is 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 analyzed for peptide / protein yield, molecular weight distribution, and thrombolytic activity. The data are shown in Table 1 below. Figures 1-2 .

[0070] Table 1 Comparison of peptide / protein yields in the 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 peptide / protein yield in Comparative Example 1 is much lower than that in Example 1, which fully demonstrates that the benefit of step (3) lies in promoting the dissolution of proteins in earthworm dregs, thereby increasing the peptide / protein yield. Comparative Example 2 differs from Example 1 in that it does not perform stepwise hydrolysis of water-soluble and water-insoluble proteins, and has little impact on the peptide / protein yield.

[0073] from Figure 1 As can be seen, Example 1 exhibits an independent peak in the earlier elution period, indicating the presence of a certain amount of relatively large molecules in its composition. This suggests that the earthworm protein peptide product obtained according to the method described in this invention retains the water-soluble protein molecular structure to the greatest extent, while the insoluble earthworm protein is hydrolyzed into peptides with smaller molecular weights. Comparative Examples 1 and 2 show similar peptide molecular weight distribution characteristics, exhibiting a small molecule peptide distribution. Comparative Example 2 differs from Example 1 in that it did not perform stepwise hydrolysis of the water-soluble and water-insoluble proteins; the overall molecular weight distribution shows a higher proportion of small molecule peptides, indicating that it is not conducive to selectively protecting the activity of water-soluble proteins.

[0074] from Figure 2 As can be seen from the table, both Example 1 and Comparative Example 1 have significantly better anticoagulant effects. Even when the clotting time reaches 3000s, they remain in a liquid state without aggregation or sedimentation, indicating that the earthworm protein peptides prepared according to the method of this invention have better anticoagulant and thrombolytic effects. Comparative Example 1 is characterized by not undergoing step (3) to promote the dissolution of more proteins and peptides with anticoagulant activity. The earthworm peptides extracted according to the method of Comparative Example 1 are mostly water-soluble protein peptides, thus also possessing high anticoagulant activity. However, Table 1 shows that its earthworm peptide yield is extremely low. Therefore, Example 1 shows a better advantage in both earthworm peptide yield and anticoagulant activity. In comparison, Comparative Example 2 has a poorer anticoagulant effect, with obvious aggregation and sedimentation of the blood. This indicates that the absence of steps (4) and (5) for the distributed hydrolysis of water-soluble and water-insoluble proteins leads to a sharp decline in the function of water-soluble active proteins, resulting in the loss of anticoagulant activity of the obtained earthworm peptides.

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

[0076] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for preparing highly active earthworm protein peptides, characterized in that, The preparation method includes the following steps: (1) Compound enzyme solubilization: Add amylase and keratinase to earthworm slurry for hydrolysis; the amount of amylase added is 3000~5000 U / g dried earthworm; the amount of keratinase added is 3000~5000 U / g dried earthworm. (2) Multi-stage filtration: First, use a bag filter to filter the earthworm slurry after hydrolysis in step (1), collect the filter residue and effluent, then use a plate and frame filter to filter the effluent, take the filtrate, and finally use an ultrafiltration membrane to ultrafilter the filtrate to obtain the ultrafiltration permeate. (3) Moderate hydrolysis with endopeptidase: The ultrafiltration permeate obtained in step (2) is hydrolyzed with a compound enzyme to obtain earthworm water-soluble protein hydrolysate; the compound enzyme is papain and bromelain; the mass ratio of papain to bromelain is 1:0.8~1.2; the amount of compound enzyme added is 1000~3000 U / g dried earthworm; (4) Separation of insoluble proteins: 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 refining: The slurry after high-temperature protease hydrolysis in step (4) is filtered using a bag filter, and the filtrate is collected. Then, the filtrate is filtered using a ceramic microfilter to remove all insoluble matter and obtain low molecular weight earthworm protein peptides. The amount of alkaline protease added is 4000~6000 U / g filter residue; the amount of high-temperature protease added is 4000~6000 U / g filter residue. (6) Concentration and drying: The earthworm water-soluble protein hydrolysate obtained in step (3) and the earthworm insoluble protein peptide obtained in step (5) are mixed and then concentrated, and finally dried to obtain highly active earthworm protein peptide.

2. The preparation method according to claim 1, characterized in that, The preparation method of earthworm slurry in step (1) is as follows: after soaking and washing the dried earthworm, wet ultrafine grinding is carried out, and 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℃ and the hydrolysis time is 3~5 h.

4. The preparation method according to claim 1, characterized in that, In step (3), the hydrolysis temperature is 40~60℃ and the hydrolysis time is 2~3 h.

5. The preparation method according to claim 1, characterized in that, In step (4), the hydrolysis temperature of alkaline protease is 40~60℃ and the hydrolysis time is 3~4 h; in step (4), the hydrolysis temperature of high temperature protease is 60~80℃ and the hydrolysis time is 3~4 h.

6. The preparation method according to claim 1, characterized in that, In step (6), the concentration process is carried out using nanofiltration or vacuum evaporation equipment.

7. The preparation method according to claim 1, characterized in that, In step (6), the drying process is low-temperature spray drying; the inlet air temperature is 110~130℃ and the outlet air temperature is 60~70℃.

8. A highly active earthworm protein peptide, characterized in that, The highly active earthworm protein peptide is prepared according to the preparation method described in any one of claims 1 to 7.

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