Earthworm protein peptide as well as preparation method and application thereof

Abstract

The invention provides earthworm protein peptide as well as a preparation method and application thereof, and belongs to the technical field of medicines. The preparation method comprises the following steps: drying and crushing earthworms to obtain earthworm powder, adding the earthworm powder into water, adding immobilized compound enzyme for enzymolysis, carrying out magnet separation, adjusting the pH value of enzymatic hydrolysate for precipitation, collecting solids, adding the solids into water, adding a purifying agent for adsorption separation, adding the solids into a deep eutectic solvent, heating for desorption, carrying out magnet separation, carrying out ultrafiltration on residual liquid, and collecting protein peptide to obtain the earthworm protein peptide. The immobilized compound enzyme is prepared by immobilizing modified snail protease and alkaline protease on chitosan modified MOF / magnetic microspheres; the purifying agent is a passivated angiotensin converting enzyme immobilized magnetic agar ball. The high-activity earthworm protein peptide has the advantages of simple preparation method, high purity, good inhibition effect on angiotensin converting enzyme, safety, high efficiency, no side effect, good antihypertensive effect on hypertensive patients, good patient compliance, and wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceuticals, and particularly relates to a earthworm protein peptide, a preparation method thereof, and an application thereof. Background Art

[0002] Earthworm is a traditional Chinese medicine in China, which has the effects of clearing heat and calming endogenous wind, relieving asthma and diuresis, activating blood circulation and dredging collaterals. The 2020 edition of the Chinese Pharmacopoeia includes the dried bodies of Pheretima aspergillum, P. vulgaris, P. guillelmi or P. pectinifera of the family Megascolecidae as the source of earthworm. The chemical components of earthworm mainly include amino acids, proteins, polypeptides, nucleosides, organic acids, lipids, etc. In particular, earthworm protein accounts for 55%-68% of the dried earthworm body. Modern research has found that earthworm protein has various pharmacological effects such as anticoagulation, antibacterial, and neuroprotection, and is commonly used to treat cardiovascular and cerebrovascular diseases such as stroke.

[0003] Bioactive peptides have beneficial effects on the body and play a role in human health, and are attracting more and more attention. Since many pharmacological effects of proteins in the body are achieved by the polypeptides released by proteins. These bioactive peptides have effects such as antihypertensive, antibacterial, antithrombotic, immunomodulatory, opioid, antioxidant, etc. With the rapid development of enzyme engineering technology, enzymatic modification of proteins to improve their functional properties, obtain high-activity substances, and prepare bioactive peptides has also become a research hotspot in recent years.

[0004] In the prior art, when separating protein components in earthworm, biochemical separation methods such as salting out with homogenate, ion exchange chromatography, and gel filtration chromatography are usually used. Although it has a certain feasibility, the steps are complicated, the experimental conditions are demanding, it is not suitable for large-scale production, and it is very different from the traditional Chinese medicine syndrome differentiation and medication. Protein polypeptide components are the main active components of earthworm. Due to the unstable nature of protein polypeptide components and the high requirements for the environment, acids, alkalis, ionic strength, temperature, or light will all affect their activity. Therefore, the traditional method of decocting with water affects the exertion of its medicinal efficacy.

[0005] In addition, preparing bioactive peptides by protein hydrolysis enzyme hydrolysis or microbial fermentation has the advantages of high safety, mild conditions, and easy process control. The enzymatic hydrolysis method is to carry out enzymatic treatment of proteins at a specific pH value and temperature. Protein hydrolases commonly used to prepare bioactive peptides include alkaline protease, trypsin, pepsin, papain, etc. The selection of the protease used and the enzyme hydrolysis time has a certain influence on the type of bioactive peptides released. For example, compared with the proteins hydrolyzed by subtilisin, the rice proteins hydrolyzed by lysin show stronger anti-inflammatory and anti-tyrosinase activities than those hydrolyzed by subtilisin. Summary of the Invention

[0006] The object of the present invention is to provide a earthworm protein peptide and a preparation method thereof. The preparation method is simple, with high purity, has a good inhibitory effect on angiotensin converting enzyme, is safe and efficient, has no side effects, has a good blood pressure lowering effect on hypertensive patients, has good patient compliance, and has broad application prospects.

[0007] The technical solution of the present invention is realized as follows:

[0008] The present invention provides a preparation method of earthworm protein peptide. Earthworms are dried and crushed to obtain earthworm powder, which is added to water, and immobilized composite enzyme is added for enzymatic hydrolysis, followed by magnetic separation. The pH value of the enzymatic hydrolysate is adjusted for precipitation, and the solid is collected. The solid is added to water, and a purification agent is added for adsorption separation. The solid is added to a deep eutectic solvent for heating desorption, followed by magnetic separation. The remaining liquid is ultrafiltered, and the protein peptide is collected to obtain earthworm protein peptide; the immobilized composite enzyme is modified snail protease and alkaline protease immobilized on chitosan-modified MOF / magnetic microspheres; the purification agent is magnetic agarose beads immobilized with inactivated angiotensin converting enzyme.

[0009] As a further improvement of the present invention, the mass ratio of the earthworm powder, the immobilized composite enzyme, and the purification agent is 10-15:0.5-1:1-2; the deep eutectic solvent is a mixture of betaine and maltose, and the molar ratio of the two is 3-5:1; the temperature of the heating desorption is 25-35 °C, and the time is 20-40 min; the ultrafiltration collects protein substances with a molecular weight of 10-100 Kda; the adjusted pH value is 4-5; the temperature of the enzymatic hydrolysis is 40-50 °C, the pH value is 7.5-8.5, and the time is 3-5 h.

[0010] As a further improvement of the present invention, the preparation method of the immobilized composite enzyme is as follows:

[0011] S1. Nano-magnetic tetroxide is added to a mixed solution of acetic acid and N,N-dimethylformamide, zirconium tetrachloride and 2-aminoterephthalic acid are added, and ultrasonic dispersion is carried out until uniform, followed by hydrothermal reaction, centrifugation, washing, and drying to obtain MOF / magnetic microspheres;

[0012] S2. Chitosan is dissolved in an acid solution, MOF / magnetic microspheres are added, and heating and stirring reaction is carried out, followed by centrifugation, washing, and drying to obtain chitosan-modified MOF / magnetic microspheres;

[0013] S3. Streptococcus thermophilus, Acidithiobacillus ferrooxidans, and Bacillus alkalophilus are mixed and added to water, lysozyme is added for enzymatic hydrolysis, filtration is carried out, and the pH value of the filtrate is adjusted for precipitation to obtain acid / alkali / heat-resistant protein peptide;

[0014] S4. Dissolve snail protease and alkaline protease in water, add NHS and EDC, stir for activation, add acid / alkali / heat-resistant protein peptide and hydrophobic amino acid, stir for reaction, add acetone for precipitation, centrifuge, wash, and dry to obtain modified snail protease and alkaline protease;

[0015] S5. Add the modified snail protease and alkaline protease into water, add NHS and EDC, stir for activation, add chitosan-modified MOF / magnetic microspheres, stir for reaction, centrifuge, wash, and dry to obtain immobilized composite enzyme.

[0016] As a further improvement of the present invention, in step S1, the mass ratio of the nano-magnetic iron tetroxide, zirconium tetrachloride, and 2-aminoterephthalic acid is 4-6: 1.5-1.7: 1.2-1.4, the volume ratio of acetic acid and N,N-dimethylformamide in the mixed solution of acetic acid and N,N-dimethylformamide is 1:18-20, the temperature of the hydrothermal reaction is 120-140 °C, and the time is 20-24 h; in step S2, the acid solution is 1-3 wt% acetic acid or lactic acid solution, the mass ratio of chitosan and MOF / magnetic microspheres is 3-5:10, the temperature of the heating and stirring reaction is 40-50 °C, and the time is 20-40 min.

[0017] As a further improvement of the present invention, in step S3, the mass ratio of Streptococcus thermophilus, Acidithiobacillus ferrooxidans, Bacillus alkalophilus, and lysozyme is 3-5: 1-3: 5-7: 0.5-1, the temperature of the enzymatic hydrolysis is 25-30 °C, the pH value is 6.5-8, the time is 2-4 h, and the pH value is adjusted to 4-5; in step S5, the mass ratio of the modified snail protease and alkaline protease, NHS, EDC, and chitosan-modified MOF / magnetic microspheres is 3-5: 0.5-1: 0.5-1: 8-10, the stirring activation time is 20-40 min, and the stirring reaction time is 7-10 h.

[0018] As a further improvement of the present invention, in step S4, the mass ratio of snail protease, alkaline protease, NHS, EDC, acid / alkali / heat-resistant protein peptide, and hydrophobic amino acid is 5-7: 8-10: 3-5: 3-5: 2-4: 1-3, the hydrophobic amino acid is selected from at least one of tryptophan, phenylalanine, valine, leucine, isoleucine, alanine, proline, and methionine, the stirring activation time is 20-40 min, and the stirring reaction time is 5-8 h.

[0019] As a further improvement of the present invention, the preparation method of the purifying agent is as follows:

[0020] T1. Add angiotensin converting enzyme to water, add a complexing agent, stir and react, add acetone for precipitation, centrifuge, wash, and dry to obtain passivated angiotensin converting enzyme;

[0021] T2. Add agarose magnetic microspheres to Tris-HCl solution, add dopamine hydrochloride, heat and stir to react, centrifuge, wash, and dry to obtain modified agarose magnetic microspheres;

[0022] T3. Add passivated angiotensin converting enzyme to water, add NHS and EDC, stir for activation, add modified agarose magnetic microspheres, stir and react, centrifuge, wash, and dry to obtain a purifying agent.

[0023] As a further improvement of the present invention, in step T1, the mass ratio of the angiotensin converting enzyme to the complexing agent is 10:3-5, the complexing agent is selected from at least one of EDTA, disodium EDTA, citric acid, oxalic acid, and malic acid, and the stirring reaction time is 0.5-1 h; in step T2, the pH value of the Tris-HCl solution is 8.5-9.5, the mass ratio of the agarose magnetic microspheres to dopamine hydrochloride is 10:3-6, the temperature of the heating and stirring reaction is 45-65 °C, and the time is 5-7 h; in step T3, the mass ratio of the passivated angiotensin converting enzyme, NHS, EDC, and modified agarose magnetic microspheres is 3-5:0.5-1:0.5-1:8-10.

[0024] The present invention further protects a lumbrokinase peptide prepared by the above preparation method.

[0025] The present invention further protects the application of the above lumbrokinase peptide in the preparation of antihypertensive drugs.

[0026] The present invention has the following beneficial effects:

[0027] The present invention prepares an immobilized composite enzyme, which uses nano-magnetic iron tetroxide as the core and has the function of magnetic separation, can be reused, reduces the production cost, and is coated with a MOF material (UiO-66-NH2) with a high specific surface area. Then, chitosan is immobilized through hydrogen bonding. The MOF material increases the specific surface area of the carrier, improves the thermal stability and operational stability of the enzyme, and can provide more binding sites for the enzyme and the substrate, thus improving the enzymatic hydrolysis efficiency. Thermophilic bacteria (Streptococcus thermophilus), acidophilic bacteria (Acidithiobacillus ferrooxidans), and alkaliphilic bacteria (Bacillus alkalophilus) are enzymatically hydrolyzed by lysozyme to obtain acid / alkali / heat-resistant protein peptides. These proteins can significantly improve the acid, alkali, and heat resistance of snail protease and alkaline protease, and enhance their conformational stability, so that the immobilized composite enzyme can play a better enzymatic hydrolysis role under mild neutral conditions and at a relatively high temperature, and the enzymatic hydrolysis efficiency is improved. In addition, snail protease and alkaline protease are also conjugated with hydrophobic amino acids. In the presence of hydrophobic amino acids, the affinity of the enzyme for the phospholipid bilayer of the animal cell membrane can be improved, thereby increasing the binding force between the substrate and the enzyme and promoting the progress of the enzymatic reaction.

[0028] Angiotensin-converting enzyme (ACE) is an enzyme that plays a key role in the pathogenesis of hypertension. Inhibiting the activity of ACE is an important way to treat hypertension. Hypotensive peptides derived from animal and plant proteins have attracted increasing attention due to their obvious hypotensive effect and mild adverse reactions.

[0029] The present invention prepares a purifying agent in which angiotensin-converting enzyme is passivated by a complexing agent. The complexing agent can fix the Zn2+ site at the active center of angiotensin-converting enzyme, block the catalytic reaction between angiotensin-converting enzyme and earthworm protein peptides, while retaining its binding site with earthworm protein peptides, does not interfere with the initial adsorption of earthworm protein peptides, and can well adsorb and purify the peptide component in earthworm protein peptides that can catalytically inhibit the activity of angiotensin-converting enzyme. The agarose magnetic microsphere carrier increases the contact area between the substrate and the enzyme, and has the function of magnetic separation, can be reused, and reduces the production cost.

[0030] The highly active earthworm protein peptides separated by the purifying agent of the present invention are added to a deep eutectic solvent. Under the action of forming hydrogen bonds and heating, it can promote the desorption of the highly active earthworm protein peptides on the purifying agent into the solvent. Its molecular weight is between 10 - 100 Kda, which is much higher than the molecules of the deep eutectic solvent, so that highly active earthworm protein peptides can be obtained by molecular weight cut-off ultrafiltration.

[0031] The preparation method of the highly active earthworm protein peptides of the present invention is simple, with high purity, has a good inhibitory effect on angiotensin-converting enzyme, is safe and efficient, has no side effects, has a good hypotensive effect on hypertensive patients, has good patient compliance, and has broad application prospects. Detailed implementation mode

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0033] Nano-magnetic iron oxide (Fe₃O₄), with an average particle size of 500 nm; chitosan, with a molecular weight of 50 - 60 kDa; snail protease, snail mucus extract, with a content > 98%; alkaline protease, 200,000 U / g; angiotensin-converting enzyme, 20,000 U / g; lysozyme, 20,000 U / mg; Streptococcus thermophilus, 20 billion cfu / g; Acidithiobacillus ferrooxidans, 10 billion cfu / g; Bacillus alkalophilus, 20 billion cfu / g; NHS, N-hydroxysuccinimide; EDC, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; agarose magnetic microspheres, 6% cross-linked magnetic agarose, magnetic bead concentration 25% (v / v) in 20% ethanol, with an average particle size of 25 μm.

[0034] Preparation Example 1 Preparation of immobilized composite enzyme

[0035] The method is as follows:

[0036] S1. Add 4 g of nano-magnetic iron oxide (Fe₃O₄) to 100 mL of a mixed solution of acetic acid and N,N-dimethylformamide, add 1.5 g of zirconium tetrachloride and 1.2 g of 2-aminoterephthalic acid, ultrasonically disperse for 15 min at 1000 W, perform hydrothermal reaction at 120 °C for 20 h, centrifuge, wash, and dry to obtain MOF / magnetic microspheres;

[0037] In the mixed solution of acetic acid and N,N-dimethylformamide, the volume ratio of acetic acid to N,N-dimethylformamide is 1:18;

[0038] S2. Dissolve 3 g of chitosan in 200 mL of 1 wt% acetic acid solution, add 10 g of MOF / magnetic microspheres, heat to 40 °C, stir and react for 20 min, centrifuge, wash, and dry to obtain chitosan-modified MOF / magnetic microspheres;

[0039] S3. Mix 3 g of Streptococcus thermophilus, 1 g of Acidithiobacillus ferrooxidans, and 5 g of Bacillus alkalophilus and add them to 200 mL of water, add 0.5 g of lysozyme, keep the temperature at 25 °C, pH value at 6.5, enzymatically hydrolyze for 2 h, filter, adjust the pH value of the filtrate to 4, and precipitate for 1 h to obtain acid / alkali / heat-resistant protein peptides;

[0040] S4. Dissolve 5 g of snail protease and 8 g of alkaline protease in 500 mL of water, add 3 g of NHS and 3 g of EDC, stir and activate for 20 min, add 2 g of acid / alkali / heat-resistant protein peptide and 1 g of isoleucine, stir and react for 5 h, add acetone for precipitation, centrifuge, wash, and dry to obtain modified snail protease and alkaline protease;

[0041] S5. Add 3 g of modified snail protease and alkaline protease to 200 mL of water, add 0.5 g of NHS and 0.5 g of EDC, stir and activate for 20 min, add 8 g of chitosan-modified MOF / magnetic microspheres, stir and react for 7 h, centrifuge, wash, and dry to obtain immobilized composite enzyme.

[0042] Preparation of the immobilized composite enzyme in Preparation Example 2

[0043] The method is as follows:

[0044] S1. Add 6 g of nano-magnetic iron tetroxide to 100 mL of a mixed solution of acetic acid and N,N-dimethylformamide, add 1.7 g of zirconium tetrachloride and 1.4 g of 2-aminoterephthalic acid, ultrasonically disperse at 1000 W for 15 min, carry out hydrothermal reaction at 140 °C for 24 h, centrifuge, wash, and dry to obtain MOF / magnetic microspheres;

[0045] The volume ratio of acetic acid to N,N-dimethylformamide in the mixed solution of acetic acid and N,N-dimethylformamide is 1:20;

[0046] S2. Dissolve 5 g of chitosan in 200 mL of 3 wt% lactic acid solution, add 10 g of MOF / magnetic microspheres, heat to 50 °C, stir and react for 40 min, centrifuge, wash, and dry to obtain chitosan-modified MOF / magnetic microspheres;

[0047] S3. Mix 5 g of Streptococcus thermophilus, 3 g of Acidithiobacillus ferrooxidans, and 7 g of Bacillus alkalophilus, add them to 200 mL of water, add 1 g of lysozyme, keep the temperature at 30 °C, pH value at 8, carry out enzymatic hydrolysis for 4 h, filter, adjust the pH value of the filtrate to 5, and precipitate for 1 h to obtain acid / alkali / heat-resistant protein peptide;

[0048] S4. Dissolve 7 g of snail protease and 10 g of alkaline protease in 500 mL of water, add 5 g of NHS and 5 g of EDC, stir and activate for 40 min, add 4 g of acid / alkali / heat-resistant protein peptide and 3 g of tryptophan, stir and react for 8 h, add acetone for precipitation, centrifuge, wash, and dry to obtain modified snail protease and alkaline protease;

[0049] S5. Add 5 g of modified snail protease and alkaline protease to 200 mL of water, add 1 g of NHS and 1 g of EDC, stir and activate for 40 min, add 10 g of chitosan-modified MOF / magnetic microspheres, stir and react for 10 h, centrifuge, wash, and dry to obtain immobilized composite enzyme.

[0050] Preparation Example 3 Preparation of Immobilized Composite Enzyme

[0051] The method is as follows:

[0052] S1. Add 5 g of nano-magnetic iron tetroxide to a mixed solution of 100 mL of acetic acid and N,N-dimethylformamide, add 1.6 g of zirconium tetrachloride and 1.3 g of 2-aminoterephthalic acid, ultrasonically disperse at 1000 W for 15 min, carry out hydrothermal reaction at 130 °C for 22 h, centrifuge, wash, and dry to obtain MOF / magnetic microspheres;

[0053] The volume ratio of acetic acid to N,N-dimethylformamide in the mixed solution of acetic acid and N,N-dimethylformamide is 1:19;

[0054] S2. Dissolve 4 g of chitosan in 200 mL of 2 wt% acetic acid solution, add 10 g of MOF / magnetic microspheres, heat to 45 °C, stir and react for 30 min, centrifuge, wash, and dry to obtain chitosan-modified MOF / magnetic microspheres;

[0055] S3. Mix 4 g of Streptococcus thermophilus, 2 g of Acidithiobacillus ferrooxidans, and 6 g of Bacillus alkalophilus, add them to 200 mL of water, add 7 g of lysozyme, keep the temperature at 27 °C, pH value at 7, enzymatically hydrolyze for 3 h, filter, adjust the pH value of the filtrate to 4.5, and precipitate for 1 h to obtain acid / alkali / heat-resistant protein peptides;

[0056] S4. Dissolve 6 g of snail protease and 9 g of alkaline protease in 500 mL of water, add 4 g of NHS and 4 g of EDC, stir and activate for 30 min, add 3 g of acid / alkali / heat-resistant protein peptides and 2 g of phenylalanine, stir and react for 7 h, add acetone for precipitation, centrifuge, wash, and dry to obtain modified snail protease and alkaline protease;

[0057] S5. Add 4 g of modified snail protease and alkaline protease to 200 mL of water, add 0.7 g of NHS and 0.7 g of EDC, stir and activate for 30 min, add 9 g of chitosan-modified MOF / magnetic microspheres, stir and react for 8 h, centrifuge, wash, and dry to obtain immobilized composite enzyme.

[0058] Comparative Preparation Example 1

[0059] Compared with Preparation Example 3, the difference is that step S1 is not carried out.

[0060] Specifically as follows:

[0061] S1. Dissolve 4 g of chitosan in 200 mL of 2 wt% acetic acid solution, add 10 g of nano-magnetic iron tetroxide, heat to 45 °C, stir and react for 30 min, centrifuge, wash, and dry to obtain chitosan-modified magnetic microspheres;

[0062] S2. Mix 4 g of Streptococcus thermophilus, 2 g of Acidithiobacillus ferrooxidans, and 6 g of Bacillus alkalophilus, add them to 200 mL of water, add 7 g of lysozyme, keep the temperature at 27 °C, pH value at 7, enzymatically hydrolyze for 3 h, filter, adjust the pH value of the filtrate to 4.5, and precipitate for 1 h to obtain acid / alkali / heat-resistant protein peptides;

[0063] S3. Dissolve 6 g of snail protease and 9 g of alkaline protease in 500 mL of water, add 4 g of NHS and 4 g of EDC, stir and activate for 30 min, add 3 g of acid / alkali / heat-resistant protein peptides and 2 g of phenylalanine, stir and react for 7 h, add acetone to precipitate, centrifuge, wash, and dry to obtain modified snail protease and alkaline protease;

[0064] S4. Add 4 g of modified snail protease and alkaline protease to 200 mL of water, add 0.7 g of NHS and 0.7 g of EDC, stir and activate for 30 min, add 9 g of chitosan-modified magnetic microspheres, stir and react for 8 h, centrifuge, wash, and dry to obtain immobilized composite enzyme.

[0065] Comparative Preparation Example 2

[0066] Compared with Preparation Example 3, the difference is that step S2 is not carried out.

[0067] Specifically as follows:

[0068] S1. Add 5 g of nano-magnetic iron tetroxide to a mixed solution of 100 mL of acetic acid and N,N-dimethylformamide, add 1.6 g of zirconium tetrachloride and 1.3 g of 2-aminoterephthalic acid, ultrasonically disperse at 1000 W for 15 min, carry out hydrothermal reaction at 130 °C for 22 h, centrifuge, wash, and dry to obtain MOF / magnetic microspheres;

[0069] The volume ratio of acetic acid to N,N-dimethylformamide in the mixed solution of acetic acid and N,N-dimethylformamide is 1:19;

[0070] S2. Mix 4 g of Streptococcus thermophilus, 2 g of Acidithiobacillus ferrooxidans, and 6 g of Bacillus alkalophilus, add them to 200 mL of water, add 7 g of lysozyme, keep the temperature at 27 °C, pH value at 7, enzymatically hydrolyze for 3 h, filter, adjust the pH value of the filtrate to 4.5, and precipitate for 1 h to obtain acid / alkali / heat-resistant protein peptides;

[0071] S3. Dissolve 6 g of snail protease and 9 g of alkaline protease in 500 mL of water, add 4 g of NHS and 4 g of EDC, stir and activate for 30 min, add 3 g of acid / alkali / heat-resistant protein peptide and 2 g of phenylalanine, stir and react for 7 h, add acetone for precipitation, centrifuge, wash, and dry to obtain modified snail protease and alkaline protease;

[0072] S4. Add 4 g of modified snail protease and alkaline protease to 200 mL of water, add 0.7 g of NHS and 0.7 g of EDC, stir and activate for 30 min, add 9 g of MOF / magnetic microspheres, stir and react for 8 h, centrifuge, wash, and dry to obtain immobilized composite enzyme.

[0073] Comparative Preparation Example 3

[0074] Compared with Preparation Example 3, the difference lies in that Streptococcus thermophilus was not added in step S3.

[0075] Specifically as follows:

[0076] S3. Mix 2 g of Thiobacillus ferrooxidans and 6 g of Bacillus alkalophilus and add them to 200 mL of water, add 7 g of lysozyme, the temperature is 27 °C, the pH value is 7, enzymolysis is carried out for 3 h, filter, adjust the pH value of the filtrate to 4.5, and precipitate for 1 h to obtain acid / alkali-resistant protein peptide.

[0077] Comparative Preparation Example 4

[0078] Compared with Preparation Example 3, the difference lies in that Thiobacillus ferrooxidans was not added in step S3.

[0079] Specifically as follows:

[0080] S3. Mix 4 g of Streptococcus thermophilus and 6 g of Bacillus alkalophilus and add them to 200 mL of water, add 7 g of lysozyme, the temperature is 27 °C, the pH value is 7, enzymolysis is carried out for 3 h, filter, adjust the pH value of the filtrate to 4.5, and precipitate for 1 h to obtain alkali / heat-resistant protein peptide.

[0081] Comparative Preparation Example 5

[0082] Compared with Preparation Example 3, the difference lies in that Bacillus alkalophilus was not added in step S3.

[0083] Specifically as follows:

[0084] S3. Mix 4 g of Streptococcus thermophilus and 2 g of Thiobacillus ferrooxidans and add them to 200 mL of water, add 7 g of lysozyme, the temperature is 27 °C, the pH value is 7, enzymolysis is carried out for 3 h, filter, adjust the pH value of the filtrate to 4.5, and precipitate for 1 h to obtain acid / heat-resistant protein peptide.

[0085] Comparative Preparation Example 6

[0086] Compared with Preparation Example 3, the difference lies in that no acid / alkali / heat-resistant protein peptide was added in step S4.

[0087] Specifically as follows:

[0088] S4. Dissolve 6 g of snail protease and 9 g of alkaline protease in 500 mL of water, add 4 g of NHS and 4 g of EDC, stir and activate for 30 min, add 5 g of phenylalanine, stir and react for 7 h, add acetone for precipitation, centrifuge, wash, and dry to obtain modified snail protease and alkaline protease.

[0089] Comparative Preparation Example 7

[0090] Compared with Preparation Example 3, the difference lies in that no phenylalanine was added in step S4.

[0091] Specifically as follows:

[0092] S4. Dissolve 6 g of snail protease and 9 g of alkaline protease in 500 mL of water, add 4 g of NHS and 4 g of EDC, stir and activate for 30 min, add 5 g of acid / alkali / heat-resistant protein peptide, stir and react for 7 h, add acetone for precipitation, centrifuge, wash, and dry to obtain modified snail protease and alkaline protease.

[0093] Preparation of the purifying agent of Preparation Example 4

[0094] The method is as follows:

[0095] T1. Add 10 g of angiotensin-converting enzyme to 200 mL of water, add 3 g of EDTA, stir and react for 0.5 h, add acetone for precipitation, centrifuge, wash, and dry to obtain passivated angiotensin-converting enzyme;

[0096] T2. Add 10 g of agarose magnetic microspheres to 200 mL of Tris-HCl solution with a pH value of 8.5, add 3 g of dopamine hydrochloride, heat to 45 °C, stir and react for 5 h, centrifuge, wash, and dry to obtain modified agarose magnetic microspheres;

[0097] T3. Add 3 g of passivated angiotensin-converting enzyme to 200 mL of water, add 0.5 g of NHS and 0.5 g of EDC, stir and activate for 30 min, add 8 g of modified agarose magnetic microspheres, stir and react for 6 h, centrifuge, wash, and dry to obtain the purifying agent.

[0098] Preparation of the purifying agent of Preparation Example 5

[0099] The method is as follows:

[0100] T1. Add 10 g of angiotensin-converting enzyme to 200 mL of water, add 5 g of citric acid, stir and react for 1 h, add acetone for precipitation, centrifuge, wash, and dry to obtain passivated angiotensin-converting enzyme;

[0101] T2. Add 10 g of agarose magnetic microspheres to 200 mL of Tris-HCl solution with a pH value of 9.5, add 6 g of dopamine hydrochloride, heat to 65 °C, stir and react for 7 h, centrifuge, wash, and dry to obtain modified agarose magnetic microspheres;

[0102] T3. Add 5 g of passivated angiotensin-converting enzyme to 200 mL of water, add 1 g of NHS and 1 g of EDC, stir and activate for 30 min, add 10 g of modified agarose magnetic microspheres, stir and react for 6 h, centrifuge, wash, and dry to obtain the purifying agent.

[0103] Preparation Example 6 Preparation of the purifying agent

[0104] The method is as follows:

[0105] T1. Add 10 g of angiotensin-converting enzyme to 200 mL of water, add 4 g of disodium EDTA, stir and react for 1 h, add acetone for precipitation, centrifuge, wash, and dry to obtain passivated angiotensin-converting enzyme;

[0106] T2. Add 10 g of agarose magnetic microspheres to 200 mL of Tris-HCl solution with a pH value of 9, add 4.5 g of dopamine hydrochloride, heat to 55 °C, stir and react for 6 h, centrifuge, wash, and dry to obtain modified agarose magnetic microspheres;

[0107] T3. Add 4 g of passivated angiotensin-converting enzyme to 200 mL of water, add 0.7 g of NHS and 0.7 g of EDC, stir and activate for 30 min, add 9 g of modified agarose magnetic microspheres, stir and react for 6 h, centrifuge, wash, and dry to obtain the purifying agent.

[0108] Comparative Preparation Example 8

[0109] Compared with Preparation Example 6, the difference is that step T1 is not carried out.

[0110] Specifically as follows:

[0111] T1. Add 10 g of agarose magnetic microspheres to 200 mL of Tris-HCl solution with a pH value of 9, add 4.5 g of dopamine hydrochloride, heat to 55 °C, stir and react for 6 h, centrifuge, wash, and dry to obtain modified agarose magnetic microspheres;

[0112] T2. Add 4 g of angiotensin converting enzyme to 200 mL of water, add 0.7 g of NHS and 0.7 g of EDC, stir and activate for 30 min, add 9 g of modified agarose magnetic microspheres, stir and react for 6 h, centrifuge, wash, and dry to obtain the purifying agent.

[0113] Comparative Preparation Example 9

[0114] Compared with Preparation Example 6, the difference lies in that step T2 is not carried out.

[0115] Specifically as follows:

[0116] T1. Add 10 g of angiotensin converting enzyme to 200 mL of water, add 4 g of disodium EDTA, stir and react for 1 h, add acetone for precipitation, centrifuge, wash, and dry to obtain passivated angiotensin converting enzyme;

[0117] T2. Add 4 g of passivated angiotensin converting enzyme to 200 mL of water, add 0.7 g of NHS and 0.7 g of EDC, stir and activate for 30 min, add 9 g of agarose magnetic microspheres, stir and react for 6 h, centrifuge, wash, and dry to obtain the purifying agent.

[0118] Comparative Preparation Example 10

[0119] Compared with Preparation Example 6, the difference lies in that steps T2 and T3 are not carried out.

[0120] Specifically as follows:

[0121] T1. Add 10 g of angiotensin converting enzyme to 200 mL of water, add 4 g of disodium EDTA, stir and react for 1 h, add acetone for precipitation, centrifuge, wash, and dry to obtain passivated angiotensin converting enzyme, which is the purifying agent.

[0122] Example 1

[0123] This example provides a method for preparing earthworm protein peptide, including the following steps:

[0124] Dry and crush earthworms to obtain earthworm powder. Add 10 g of earthworm powder to 200 mL of water, add 0.5 g of the immobilized composite enzyme prepared in Preparation Example 1, at a temperature of 40 °C and a pH value of 7.5, enzymatically hydrolyze for 3 h, separate with a magnet, adjust the pH value of the enzymatic hydrolysate to 4.5, precipitate, collect the solid, add it to 100 mL of water, add 1 g of the purifying agent prepared in Preparation Example 4 for adsorption and separation, add the solid to 50 mL of deep eutectic solvent, heat to 25 °C, desorb for 20 min, separate with a magnet, ultrafilter the remaining solution, collect the protein peptide with a molecular weight of 10 - 100 Kda, recycle the deep eutectic solvent for reuse, and freeze-dry to obtain earthworm protein peptide;

[0125] The deep eutectic solvent is a mixture of betaine and maltose, and the molar ratio of the two is 3:1.

[0126] Example 2

[0127] This example provides a method for preparing earthworm protein peptide, which includes the following steps:

[0128] The earthworms are dried and crushed to obtain earthworm powder. 15 g of the earthworm powder is added to 200 mL of water, and 1 g of the immobilized composite enzyme prepared in Preparation Example 2 is added. The temperature is 50 °C, the pH value is 8.5, and enzymatic hydrolysis is carried out for 5 h. Magnet separation is carried out. The pH value of the enzymatic hydrolysate is adjusted to 4.5 for precipitation. The solid is collected and added to 100 mL of water. 2 g of the purifying agent prepared in Preparation Example 5 is added for adsorption separation. The solid is added to 50 mL of the deep eutectic solvent, heated to 35 °C, and desorbed for 40 min. Magnet separation is carried out. The remaining liquid is ultrafiltered, and the protein peptide with a molecular weight of 10 - 100 Kda is collected. The deep eutectic solvent is recovered and reused. After freeze-drying, earthworm protein peptide is obtained;

[0129] The deep eutectic solvent is a mixture of betaine and maltose, and the molar ratio of the two is 5:1.

[0130] Example 3

[0131] This example provides a method for preparing earthworm protein peptide, which includes the following steps:

[0132] The earthworms are dried and crushed to obtain earthworm powder. 12 g of the earthworm powder is added to 200 mL of water, and 0.7 g of the immobilized composite enzyme prepared in Preparation Example 1 is added. The temperature is 45 °C, the pH value is 8, and enzymatic hydrolysis is carried out for 4 h. Magnet separation is carried out. The pH value of the enzymatic hydrolysate is adjusted to 4.5 for precipitation. The solid is collected and added to 100 mL of water. 1.5 g of the purifying agent prepared in Preparation Example 6 is added for adsorption separation. The solid is added to 50 mL of the deep eutectic solvent, heated to 30 °C, and desorbed for 30 min. Magnet separation is carried out. The remaining liquid is ultrafiltered, and the protein peptide with a molecular weight of 10 - 100 Kda is collected. The deep eutectic solvent is recovered and reused. After freeze-drying, earthworm protein peptide is obtained;

[0133] The deep eutectic solvent is a mixture of betaine and maltose, and the molar ratio of the two is 4:1.

[0134] Comparative Example 1

[0135] Compared with Example 3, the difference is that the immobilized composite enzyme is prepared from Comparative Preparation Example 1.

[0136] Comparative Example 2

[0137] Compared with Example 3, the difference is that the immobilized composite enzyme is prepared from Comparative Preparation Example 2.

[0138] Comparative Example 3

[0139] Compared with Example 3, the difference lies in that the immobilized composite enzyme is prepared from Comparative Preparation Example 3.

[0140] Comparative Example 4

[0141] Compared with Example 3, the difference lies in that the immobilized composite enzyme is prepared from Comparative Preparation Example 4.

[0142] Comparative Example 5

[0143] Compared with Example 3, the difference lies in that the immobilized composite enzyme is prepared from Comparative Preparation Example 5.

[0144] Comparative Example 6

[0145] Compared with Example 3, the difference lies in that the immobilized composite enzyme is prepared from Comparative Preparation Example 6.

[0146] Comparative Example 7

[0147] Compared with Example 3, the difference lies in that the immobilized composite enzyme is prepared from Comparative Preparation Example 7.

[0148] Comparative Example 8

[0149] Compared with Example 3, the difference lies in that the purifying agent is prepared from Comparative Preparation Example 8.

[0150] Comparative Example 9

[0151] Compared with Example 3, the difference lies in that the purifying agent is prepared from Comparative Preparation Example 9.

[0152] Comparative Example 10

[0153] Compared with Example 3, the difference lies in that the purifying agent is prepared from Comparative Preparation Example 10.

[0154] Test Example 1

[0155] The earthworm protein peptides prepared in Examples 1-3 and Comparative Examples 8-10 were dissolved in 0.1 mol / L sodium borate buffer (containing 0.3 mol / L NaCl, pH = 8.3) at a concentration of 1 mg / mL to obtain samples. Take 25 μL of the sample and mix it with 50 μL of 4.7 mmol / L hippuryl-histidyl-leucine (Sigma, USA) solution (the solvent is 0.1 mol / L sodium borate buffer containing 0.3 mol / L NaCl, pH = 8.3). After incubating at 37 °C for 5 min, add 50 μL of 25 U / L ACE (25 U / L, Yuanye Bio-Technology Co., Ltd., Shanghai) solution and incubate at 37 °C for 30 min. Finally, add 750 μL of 0.3 mol / L NaOH to terminate the reaction. Mix the mixture with 50 μL of 2% (mass fraction) o-phthalaldehyde and 100 μL of 3 mol / L HCl. Measure the fluorescence at the excitation and emission wavelengths of 340 nm and 455 nm, respectively.

[0156] ACE inhibition rate (%) = 1 - (F b - F c ) / F a , where: F a , the fluorescence value of sample a (sample + buffer); F b , the fluorescence value of sample b (sample); F c , the fluorescence value of sample c (buffer).

[0157] The results are shown in Table 1.

[0158] Table 1

[0159] Group ACE Inhibition Rate (%) Example 1 83.0 Example 2 82.7 Example 3 83.5 Comparative Example 8 52.9 Comparative Example 9 75.4 Comparative Example 10 64.3

[0160] As can be seen from the above table, the earthworm protein peptides prepared in Examples 1-3 of the present invention have good ACE inhibitory activity.

[0161] Test Example 2

[0162] Calculate the extraction rate of the earthworm protein peptides prepared in Examples 1-3 and Comparative Examples 1-10. The calculation formula is as follows: Extraction rate (%) = earthworm protein peptide / earthworm powder × 100%.

[0163] The results are shown in Table 2.

[0164] Table 2

[0165] Group Extraction Rate (%) Example 1 51.9 Example 2 50.2 Example 3 52.5 Comparative Example 1 40.2 Comparative Example 2 42.7 Comparative Example 3 46.1

[0166] Comparative Example 4 45.8 Comparative Example 5 45.1 Comparative Example 6 41.3 Comparative Example 7 43.5 Comparative Example 8 32.5 Comparative Example 9 45.6 Comparative Example 10 41.5

[0167] As can be seen from the above table, the extraction rate of the earthworm protein peptides prepared in Examples 1-3 of the present invention is relatively high.

[0168] Test Example 3

[0169] After adaptive feeding, male SPF-grade C57BL / 6J mice, with a body weight of 18 - 20 g and 7 weeks old, were randomly selected, and 10 of them were divided into a normal control group. The normal control group was intragastrically administered with drinking water for 8 weeks, and the remaining mice were used to construct a hypertension model by using 0.5 mg / mL L-NAME solution instead of drinking water. The successful standard for modeling was that the systolic blood pressure (SBP) of the mice increased by 20 mmHg. Four weeks later, the successfully modeled mice were randomly divided into 5 groups: hypertension model group, lisinopril drug group (6.1 mg / kg), Example 1 - 3 groups, and Comparative Example 8 - 10 groups (30 mg / kg), with 10 mice in each group. All mice continued to drink L-NAME-containing drinking water, and the lisinopril drug group, Example 1 - 3 groups, and Comparative Example 8 - 10 groups were intragastrically administered with lisinopril aqueous solution and the corresponding prepared earthworm protein peptide aqueous solution respectively. The experiment was carried out for 4 weeks.

[0170] Measurement of mouse blood pressure: The blood pressure of the mice was measured using a small animal sphygmomanometer. Blood pressure was measured once at the same time before the experiment and after 4 weeks. Before the measurement, all mice were allowed to adapt to the sphygmomanometer for 5 min, and the temperature was set at 37°C. The results are shown in Table 3.

[0171] Table 3

[0172]

[0173]

[0174] As can be seen from the above table, the earthworm protein peptides prepared in Examples 1 - 3 of the present invention have good blood pressure lowering effects.

[0175] Measurement of mouse plasma biochemical indexes:

[0176] After the mice were anesthetized, blood was taken from the orbital cavity, and plasma was obtained by centrifugation. Angiotensin-converting enzyme (ACE), angiotensin-2 (AngⅡ), endothelin-1 (ET-1), and interleukin-6 (IL-6) in the plasma were measured using an enzyme-linked immunosorbent assay kit. The results are shown in Table 4.

[0177] Table 4

[0178]

[0179] Note: * Compared with the normal control group, P < 0.05; # compared with the hypertension model group, P < 0.05.

[0180] As can be seen from the above table, the earthworm protein peptides prepared in Examples 1 - 3 of the present invention can significantly reduce the contents of angiotensin-converting enzyme (ACE), angiotensin-2 (AngⅡ), endothelin-1 (ET-1), and interleukin-6 (IL-6) in the blood.

[0181] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of earthworm protein peptide, characterized in that, The earthworms are dried and crushed to obtain earthworm powder, which is added to water, and immobilized composite enzyme is added for enzymatic hydrolysis. Then, magnetic separation is carried out. The pH value of the enzymatic hydrolysate is adjusted for precipitation, and the solid is collected. The solid is added to water, and a purifying agent is added for adsorption separation. The solid is added to a deep eutectic solvent for heating desorption, and magnetic separation is carried out. The remaining liquid is ultrafiltered, and the protein peptide is collected to obtain earthworm protein peptide; the immobilized composite enzyme is modified snail protease and alkaline protease immobilized on chitosan-modified MOF / magnetic microspheres; the purifying agent is magnetic agarose beads immobilized with inactivated angiotensin converting enzyme.

2. The preparation method according to claim 1, characterized in that, The mass ratio of the earthworm powder, the immobilized composite enzyme, and the purifying agent is 10-15:0.5-1:1-2; the deep eutectic solvent is a mixture of betaine and maltose, and the molar ratio of the two is 3-5:1; the temperature of the heating desorption is 25-35 °C, and the time is 20-40 min; the molecular weight of the protein substances collected by ultrafiltration is 10-100 Kda; the pH value is adjusted to 4-5; the temperature of the enzymatic hydrolysis is 40-50 °C, the pH value is 7.5-8.5, and the time is 3-5 h.

3. The preparation method according to claim 1, characterized in that The preparation method of the immobilized composite enzyme is as follows: S1. Nano-magnetic iron tetroxide is added to a mixed solution of acetic acid and N,N-dimethylformamide, zirconium tetrachloride and 2-aminoterephthalic acid are added, and ultrasonic dispersion is carried out until uniform. Then, hydrothermal reaction is carried out, followed by centrifugation, washing, and drying to obtain MOF / magnetic microspheres. S2. Chitosan is dissolved in an acid solution, MOF / magnetic microspheres are added, and heating and stirring reaction is carried out. Then, centrifugation, washing, and drying are carried out to obtain chitosan-modified MOF / magnetic microspheres. S3. Streptococcus thermophilus, Acidithiobacillus ferrooxidans, and Bacillus alkalophilus are mixed and added to water, and lysozyme is added for enzymatic hydrolysis. Then, filtration is carried out, and the pH value of the filtrate is adjusted for precipitation to obtain acid / alkali / heat-resistant protein peptides. S4. Snail protease and alkaline protease are dissolved in water, NHS and EDC are added, and stirring activation is carried out. Then, acid / alkali / heat-resistant protein peptides and hydrophobic amino acids are added, and stirring reaction is carried out. Acetone is added for precipitation, followed by centrifugation, washing, and drying to obtain modified snail protease and alkaline protease. S5. Modified snail protease and alkaline protease are added to water, NHS and EDC are added, and stirring activation is carried out. Then, chitosan-modified MOF / magnetic microspheres are added, and stirring reaction is carried out. Centrifugation, washing, and drying are carried out to obtain the immobilized composite enzyme.

4. The preparation method according to claim 3, characterized in that, In step S1, the mass ratio of the nano-magnetic iron tetroxide, zirconium tetrachloride, and 2-aminoterephthalic acid is 4-6:1.5-1.7:1.2-1.4, the volume ratio of acetic acid and N,N-dimethylformamide in the mixed solution of acetic acid and N,N-dimethylformamide is 1:18-20, the temperature of the hydrothermal reaction is 120-140 °C, and the time is 20-24 h; in step S2, the acid solution is 1-3 wt% acetic acid or lactic acid solution, the mass ratio of chitosan and MOF / magnetic microspheres is 3-5:10, and the temperature of the heating and stirring reaction is 40-50 °C, and the time is 20-40 min.

5. The preparation method according to claim 3, characterized in that, In step S3, the mass ratio of Streptococcus thermophilus, Acidithiobacillus ferrooxidans, Bacillus alkalophilus, and lysozyme is 3-5:1-3:5-7:0.5-1. The temperature of enzymatic hydrolysis is 25-30 °C, the pH value is 6.5-8, and the time is 2-4 h. The pH value is adjusted to 4-5. In step S5, the mass ratio of modified snail protease, alkaline protease, NHS, EDC, and chitosan-modified MOF / magnetic microspheres is 3-5:0.5-1:0.5-1:8-10. The time for stirring activation is 20-40 min, and the time for stirring reaction is 7-10 h.

6. The preparation method according to claim 3, wherein, In step S4, the mass ratio of snail protease, alkaline protease, NHS, EDC, acid / alkali / heat-resistant protein peptide, and hydrophobic amino acid is 5-7:8-10:3-5:3-5:2-4:1-3. The hydrophobic amino acid is selected from at least one of tryptophan, phenylalanine, valine, leucine, isoleucine, alanine, proline, and methionine. The time for stirring activation is 20-40 min, and the time for stirring reaction is 5-8 h.

7. The preparation method according to claim 1, characterized in that, The preparation method of the purifying agent is as follows: T1. Add angiotensin-converting enzyme into water, add a complexing agent, stir and react, add acetone for precipitation, centrifuge, wash, and dry to obtain passivated angiotensin-converting enzyme. T2. Add agarose magnetic microspheres into Tris-HCl solution, add dopamine hydrochloride, heat and stir to react, centrifuge, wash, and dry to obtain modified agarose magnetic microspheres. T3. Add passivated angiotensin-converting enzyme into water, add NHS and EDC, stir and activate, add modified agarose magnetic microspheres, stir and react, centrifuge, wash, and dry to obtain the purifying agent.

8. The preparation method according to claim 7, wherein In step T1, the mass ratio of angiotensin-converting enzyme to the complexing agent is 10:3-5. The complexing agent is selected from at least one of EDTA, disodium EDTA, citric acid, oxalic acid, and malic acid. The time for stirring reaction is 0.5-1 h. In step T2, the pH value of the Tris-HCl solution is 8.5-9.

5. The mass of agarose magnetic microspheres to dopamine hydrochloride is 10:3-6. The temperature for heat stirring reaction is 45-65 °C, and the time is 5-7 h. In step T3, the mass ratio of passivated angiotensin-converting enzyme, NHS, EDC, and modified agarose magnetic microspheres is 3-5:0.5-1:0.5-1:8-10.

9. An earthworm protein peptide prepared by the preparation method according to any one of claims 1-8.

10. Use of the earthworm protein peptide according to claim 9 in the preparation of a drug for lowering blood pressure.

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