Earthworm protein peptide with in-vitro thrombin activity and preparation method thereof

Abstract

This invention discloses an earthworm protein peptide with in vitro thrombin-inhibiting activity and its preparation method, belonging to the field of functional food technology. The method first pre-treats earthworm raw materials to obtain earthworm protein; then, it uses pepsin and alkaline protease for stepwise enzymatic hydrolysis under varying temperature and pH, and ultrafiltration to collect short peptide solutions with a molecular weight below 3 kDa; the short peptide solution is specifically captured using an affinity chromatography material coupled with bovine thrombin, followed by elution, nanofiltration desalting concentration, and spray drying to obtain the final product. This invention fully releases the peptides through a dual enzymatic hydrolysis process and utilizes thrombin affinity chromatography to directionally enrich peptides with specific inhibitory activity, effectively solving the problems of low content and poor targeting of active ingredients in earthworm protein peptides in existing technologies, and significantly improving the purity and in vitro anticoagulant activity of the product.

Description

Technical Field

[0001] This invention relates to the field of functional food technology, and more specifically, to an earthworm protein peptide with in vitro thrombin-inhibiting activity and its preparation method. Background Technology

[0002] Earthworm protein and its hydrolysates have long been studied for their role in cardiovascular health regulation, particularly in anticoagulation and improving microcirculation. Traditional research has shown that serine peptidase and various small peptides with active sequences contained in earthworms can intervene in thrombin activity. Therefore, the enzymatic hydrolysis of earthworm protein to prepare low molecular weight peptides for use in functional foods, nutritional supplements, and special dietary applications has gradually become a research hotspot in recent years.

[0003] Existing earthworm protein peptide preparation technologies mainly rely on traditional enzymatic hydrolysis or chemical extraction, which lacks specificity, resulting in low content of active peptides, wide molecular weight distribution, unstable anticoagulant effect, and insufficient functionality in the products. In view of this, we propose an earthworm protein peptide with in vitro thrombin inhibition activity and its preparation method. Summary of the Invention

[0004] The purpose of this invention is to provide an earthworm protein peptide with in vitro thrombin-inhibiting activity and its preparation method, in order to solve the problems mentioned in the background art that the existing earthworm protein peptide preparation technology mainly relies on traditional enzymatic hydrolysis or chemical extraction, which is not targeted, resulting in low content of active peptides, wide molecular weight distribution, unstable anticoagulant effect and insufficient functionality in the product.

[0005] This invention provides a method for preparing earthworm protein peptides with in vitro thrombin-inhibiting activity, comprising the following steps: S1.1 Pre-treat the earthworm raw material to obtain earthworm protein; S1.2. Under nitrogen protection, at pH 2.0-2.5 and 40-50℃, add pepsin to hydrolyze the earthworm protein for 1-2 hours; slowly add 1-4 mol / L NaOH solution to adjust the pH to 8.0-9.0, add alkaline protease at 50-55℃ and continue hydrolysis for 1-4 hours, heat at 95℃ for 10 minutes to inactivate the enzyme, and cool to ≤30℃ to obtain the hydrolysate. S1.3. The hydrolysate is filtered and separated by passing it through 10kDa and 3kDa ultrafiltration membranes in sequence, and short peptide solutions with molecular weight below 3kDa are collected. S1.4. Pass the short peptide solution through an affinity chromatography material coupled with thrombin and elute to collect the peptide fragments; S1.5. Spray dry the peptide segments and add 10-30% maltodextrin by dry weight of the peptide segments to obtain earthworm protein peptides with in vitro thrombin inhibitory activity.

[0006] Preferably, in step S1.1, the preprocessing includes the following steps: The earthworm raw material was washed with deionized water at 4℃ and cut into 0.5-1.0cm segments. It was then added to a sodium chloride solution pre-cooled to 4℃ with a mass concentration of 0.9% and shaken at 100-200rpm for 10-20min. The earthworm was then filtered to obtain the purified earthworm. After removing impurities, the earthworms were extracted with anhydrous ethanol at a material-to-liquid ratio of 1:5-10 at 40-50℃ with heating and stirring for 1-2 times, each time for 20-40 minutes. The extracts were combined and the ethanol was recovered under reduced pressure. The mixture was then filtered to obtain defatted earthworm tissue. The defatted earthworm tissue was treated with hot water at 90℃ for 20 minutes and then cooled to room temperature to obtain earthworm protein.

[0007] Preferably, in step S1.2, the mass ratio of pepsin to earthworm protein is 0.5-2.0%, and the mass ratio of alkaline protease to earthworm protein is 1-3%.

[0008] Preferably, in step S1.4, the step of passing the short peptide solution through an affinity chromatography material and eluting to collect the peptide fragments is as follows: Bovine thrombin was dissolved in coupling buffer to obtain an enzyme solution with a concentration of 1-10 mg / mL; The N-hydroxysuccinimide-activated agarose gel was equilibrated three times with coupling buffer to obtain a pre-equilibrated agarose gel. The enzyme solution was added to a pre-equilibrated agarose gel and the reaction was carried out with shaking at 4°C for 4-12 h. After the reaction was completed, the gel was washed 2-3 times alternately with coupling buffer and a solution of 0.1 mol / L Tris-HCl, 0.5 mol / L sodium chloride, pH 8.3. The reaction was then blocked with 0.1 mol / L ethanolamine solution at pH 8.3 for 2 h. The gel was washed again until neutral and then equilibrated with TBS buffer to obtain the coupled affinity medium. The coupled affinity medium was packed into a column with a height-to-diameter ratio of 5-10:1 and equilibrated with 5-10 column volumes of TBS buffer. The short peptide solution in S1.3 was filtered through a 0.22 μm filter and passed through the affinity column at a rate of 0.1-0.2 column volumes per minute. The eluent was collected and washed with 5-10 column volumes of TBS buffer until the baseline was stable. Elute with a gradient of 3-5 column volumes of 0.1 mol / L citric acid solution at pH 3.0-3.5, collecting the elution peak with absorbance >0.2 AU at 280 nm. Adjust the pH of the eluent to 7.0-7.4 using a pH adjuster. Circulate the pH-adjusted eluent through a nanofiltration membrane with a molecular weight cutoff of 100-500 Da for desalting and concentration. Control the operating pressure at 0.5-2.5 MPa until the permeate conductivity drops below 200 μS / cm, then collect the retentate to obtain the peptides.

[0009] Preferably, the thrombin is a bovine thrombin that is biologically extracted or recombinantly expressed, with a specific activity ≥1000 U / mg.

[0010] Preferably, the coupling buffer solution is a solution of 0.1 mol / L sodium bicarbonate, 0.5 mol / L sodium chloride, and pH 8.3.

[0011] Preferably, the enzyme solution is added to the pre-equilibrated agarose gel at a ratio of 2-10 mg thrombin / mL sedimentation gel.

[0012] Preferably, the TBS buffer solution is a solution of 20 mmol / L Tris-HCl, 150 mmol / L sodium chloride, and pH 7.4.

[0013] Preferably, in step S1.5, the inlet air temperature for spray drying is 130-140℃, and the outlet air temperature is 80-85℃.

[0014] On the other hand, the present invention provides a terrestrial protein peptide with in vitro thrombin-inhibiting activity, which is prepared by any of the above-described methods for preparing a terrestrial protein peptide with in vitro thrombin-inhibiting activity.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention discloses an earthworm protein peptide with in vitro thrombin-inhibiting activity and its preparation method. The method employs stepwise enzymatic hydrolysis with pepsin and alkaline protease, which fully cleaves earthworm protein under different optimal pH and temperature conditions, significantly improving the yield of small-molecule active peptides and better preserving their biological activity. Secondly, by utilizing thrombin as an affinity ligand, based on the highly specific affinity recognition mechanism between enzymes and inhibitors, the thrombin on the stationary phase directly captures inhibitory peptides in the mobile phase that can bind to its active site. Through specific adsorption, the capture and enrichment of target peptides with thrombin-inhibiting potential in the hydrolysate are achieved, significantly improving the purity and specific activity of the functional components. Detailed Implementation

[0016] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0017] The dried body of *Eisenia fetida*, the raw material for earthworms, was purchased from Bozhou Gaoshouchen Agricultural Development Co., Ltd.

[0018] Pepsin (CAS No.: 9001-75-6, Product No.: S10027-5g), maltodextrin (CAS No.: 9050-36-6, Purity BR, Product No.: S11157-500g), citric acid (CAS No.: 77-92-9, Purity ≥98%, Product No.: B23391-20mg), and alkaline protease (CAS No.: 9014-01-1, Product No.: S10051-50mg) were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0019] Preparation steps of N-hydroxysuccinimide-activated agarose: Take a certain amount of agarose gel 4B and wash it with a large amount of pre-cooled deionized water; then continue washing with an equal volume of 0.5 mol / L sodium chloride solution, and finally rinse with 0.1 mol / L cold hydrochloric acid solution and dry to obtain the pretreated gel; quickly transfer the pretreated gel to a reaction vessel containing 0.1 mol / L, pH 6.0 MES buffer to form a slurry; under ice bath and continuous slow stirring conditions, at a rate of per milliliter The following were added sequentially to the sedimentation gel: N-hydroxysuccinimide to a final concentration of approximately 15-30 mM; 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to a final concentration of approximately 50-100 mM; the reaction system temperature was maintained at 0-4℃, and the reaction was continued for 30-60 minutes; after the reaction was completed, the gel was rapidly washed several times with a large amount of pre-cooled 0.1 mol / L, pH 7.0 phosphate buffer; the resulting NHS-activated agarose gel was stored in isopropanol at 4℃ for later use.

[0020] The agarose gel 4B (sedimentation volume water content approximately 96%) was purchased from Shanghai Huzheng Biotechnology Co., Ltd.

[0021] Bovine thrombin (CAS No.: 9002-04-4, enzyme activity ≥2000 U / mg) was purchased from Sigma-Aldrich.

[0022] Ethanolamine (CAS No.: 141-43-5, purity 99%) was purchased from Nanjing Zhongchun Biotechnology Co., Ltd.

[0023] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, CAS No.: 25952-53-8, purity ≥98%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0024] The coupling buffer solution is a solution of 0.1 mol / L sodium bicarbonate, 0.5 mol / L sodium chloride, and pH 8.3.

[0025] The TBS buffer solution is a solution of 20 mmol / L Tris-HCl, 150 mmol / L sodium chloride, and pH 7.4.

[0026] Example 1: A method for preparing earthworm protein peptide with in vitro thrombin-inhibiting activity, comprising the following steps: S1.1 Wash the earthworm raw material with 4℃ deionized water and cut it into 1.0cm segments. Add it to a sodium chloride solution pre-cooled to 4℃ with a mass concentration of 0.9% and shake it at 150rpm for 15min. Filter to obtain the earthworm after removing impurities. The cleaned earthworms were extracted twice with anhydrous ethanol at a material-to-liquid ratio of 1:7 under heating and stirring conditions at 45°C for 30 minutes each time. The extracts were combined and the ethanol was recovered under reduced pressure. The mixture was then filtered to obtain defatted earthworm tissue. Defatted earthworm tissue was treated with hot water at 90℃ for 20 minutes and then cooled to room temperature to obtain earthworm protein. S1.2. Under nitrogen protection, at pH 2.5 and 45°C, add pepsin (1.5% by mass of earthworm protein) and hydrolyze for 2 hours; adjust the pH to 8.5, add alkaline protease (2% by mass of earthworm protein) at 55°C and continue hydrolysis for 2 hours; heat at 95°C for 10 minutes to inactivate the enzyme; cool to ≤30°C to obtain hydrolysate. S1.3. The hydrolysate is filtered and separated by passing it through 10kDa and 3kDa ultrafiltration membranes in sequence, and short peptide solutions with molecular weight below 3kDa are collected. S1.4 Dissolve bovine thrombin in coupling buffer to obtain an enzyme solution with a concentration of 5 mg / mL; The N-hydroxysuccinimide-activated agarose gel was equilibrated three times with coupling buffer to obtain a pre-equilibrated agarose gel. The enzyme solution was added to the pre-equilibrated agarose gel at a ratio of 4 mg thrombin / mL sedimentation gel, and the reaction was carried out with shaking at 4 °C for 10 h. After the reaction was completed, the gel was washed three times alternately with coupling buffer and a solution of 0.1 mol / L Tris-HCl, 0.5 mol / L sodium chloride, pH 8.3. The reaction was then blocked with 0.1 mol / L ethanolamine solution, pH 8.3, for 2 h. The gel was washed again until neutral and then equilibrated with TBS buffer to obtain the coupled affinity medium. The coupled affinity medium was packed into a column with a height-to-diameter ratio of 8:1 and equilibrated with 8 column volumes of TBS buffer. The short peptide solution in S1.3 was filtered through a 0.22 μm filter membrane and passed through the affinity column at a rate of 0.2 column volumes per minute. The eluent was collected and washed with 7 column volumes of TBS buffer until the baseline was stable. Elution was performed using a gradient of 4 column volumes of 0.1 mol / L citric acid solution at pH 3.5, collecting elution peaks with absorbance > 0.2 AU at 280 nm. The eluent was collected, and 1 mol / L NaOH solution was slowly added to adjust the pH to 7.0-7.4. The pH-adjusted eluent was pumped into a nanofiltration unit, using a spiral-wound nanofiltration membrane module with a molecular weight cutoff (MWCO) of 200 Da. Constant-volume percolation desalination was performed at an operating pressure of 1.5 MPa and a feed temperature of 25 °C. Deionized water was continuously added until the conductivity of the permeate was below 150 μS / cm. Water addition was then stopped, and the solution was concentrated to 1 / 5 of its original volume. The retentate was collected to obtain peptides. S1.5. Spray-dry the peptide fragments and add maltodextrin accounting for 20% of the dry weight of the peptide fragments. The inlet air temperature is 135℃ and the outlet air temperature is 85℃ to obtain earthworm protein peptides with in vitro thrombin inhibitory activity.

[0027] Example 2: The difference between this example and Example 1 is that the mass ratio of pepsin to earthworm protein is 0.5%.

[0028] Example 3: The difference between this example and Example 1 is that the mass ratio of pepsin to earthworm protein is 2.0%.

[0029] Example 4: The difference between this example and Example 1 is that the mass ratio of alkaline protease to earthworm protein is 1%.

[0030] Example 5: The difference between this example and Example 1 is that the mass ratio of alkaline protease to earthworm protein is 3%.

[0031] Example 6: The difference between this example and Example 1 is that the enzyme solution was added to the pre-equilibrated agarose gel at a ratio of 2 mg thrombin / mL sedimentation gel.

[0032] Example 7: The difference between this example and Example 1 is that the enzyme solution was added to the pre-equilibrated agarose gel at a ratio of 6 mg thrombin / mL sedimentation gel.

[0033] Example 8: A method for preparing earthworm protein peptide with in vitro thrombin-inhibiting activity, comprising the following steps: S1.1 Wash the earthworm raw material with 4℃ deionized water and cut it into 0.5cm segments. Add it to a sodium chloride solution pre-cooled to 4℃ with a mass concentration of 0.9% and shake it at 100rpm for 10min. Filter to obtain the earthworm after removing impurities. The cleaned earthworms were extracted once with anhydrous ethanol at a material-to-liquid ratio of 1:5 under heating and stirring conditions at 40°C for 20 minutes each time. The extracts were combined and the ethanol was recovered under reduced pressure. The mixture was then filtered to obtain defatted earthworm tissue. Defatted earthworm tissue was treated with hot water at 90℃ for 20 minutes and then cooled to room temperature to obtain earthworm protein. S1.2. Under nitrogen protection, at pH 2.0 and 40°C, add pepsin (1.5% by mass of earthworm protein) and hydrolyze for 1 hour; adjust the pH to 8.0, add alkaline protease (2% by mass of earthworm protein) at 50°C and continue hydrolysis for 1 hour; heat at 95°C for 10 minutes to inactivate the enzyme; cool to ≤30°C to obtain hydrolysate; S1.3. The hydrolysate is filtered and separated by passing it through 10kDa and 3kDa ultrafiltration membranes in sequence, and short peptide solutions with molecular weight below 3kDa are collected. S1.4 Dissolve bovine thrombin in coupling buffer to obtain an enzyme solution with a concentration of 5 mg / mL; The N-hydroxysuccinimide-activated agarose gel was equilibrated three times with coupling buffer to obtain a pre-equilibrated agarose gel. The enzyme solution was added to the pre-equilibrated agarose gel at a ratio of 5 mg thrombin / mL sedimentation gel, and the reaction was carried out with shaking at 4 °C for 4 h. After the reaction was completed, the gel was washed twice alternately with coupling buffer and a solution of 0.1 mol / L Tris-HCl, 0.5 mol / L sodium chloride, pH 8.3. The reaction was then blocked with 0.1 mol / L ethanolamine solution, pH 8.3, for 2 h. The gel was washed again until neutral and then equilibrated with TBS buffer to obtain the coupled affinity medium. The coupled affinity medium was packed into a column with a height-to-diameter ratio of 5:1 and equilibrated with 5 column volumes of TBS buffer. The short peptide solution in S1.3 was filtered through a 0.22 μm filter membrane and passed through the affinity column at a rate of 0.1 column volumes per minute. The eluent was collected and washed with 5 column volumes of TBS buffer until the baseline was stable. Elution was performed using a gradient of 3 column volumes of 0.1 mol / L citric acid solution at pH 3.0, collecting elution peaks with absorbance > 0.2 AU at 280 nm. The eluent was collected and the pH was adjusted to 7.0 with 1 mol / L Tris-HCl solution. The pH-adjusted eluent was pumped into a nanofiltration unit, using a nanofiltration membrane module with a molecular weight cutoff (MWCO) of 200 Da. Constant-volume percolation desalination was performed at an operating pressure of 1.5 MPa and a feed temperature of 25 °C. Deionized water was added during the process until the permeate conductivity was below 150 μS / cm. Water addition was then stopped, and the solution was concentrated to 1 / 5 of its original volume. The retentate was collected to obtain peptides. S1.5. Spray-dry the peptide fragments and add maltodextrin accounting for 10% of the dry weight of the peptide fragments. The inlet air temperature is 130℃ and the outlet air temperature is 80℃ to obtain earthworm protein peptides with in vitro thrombin inhibitory activity.

[0034] Example 9: A method for preparing earthworm protein peptide with in vitro thrombin-inhibiting activity, comprising the following steps: S1.1 Wash the earthworm raw material with 4℃ deionized water and cut it into 1.0cm segments. Add it to a sodium chloride solution pre-cooled to 4℃ with a mass concentration of 0.9% and shake at 200rpm for 20min. Filter to obtain the earthworm after impurity removal. The earthworms after impurity removal were extracted twice with anhydrous ethanol at a material-to-liquid ratio of 1:10 under heating and stirring conditions at 50°C for 40 minutes each time. The extracts were combined and the ethanol was recovered under reduced pressure. The mixture was then filtered to obtain defatted earthworm tissue. Defatted earthworm tissue was treated with hot water at 90℃ for 20 minutes and then cooled to room temperature to obtain earthworm protein. S1.2. Under nitrogen protection, at pH 2.5 and 50°C, pepsin (1.5% by mass of earthworm protein) was added and hydrolyzed for 2 hours. The pH was adjusted to 9.0, and alkaline protease (2% by mass of earthworm protein) was added at 55°C and hydrolysis continued for 4 hours. The enzyme was inactivated by heating at 95°C for 10 minutes, and the solution was cooled to ≤30°C to obtain the hydrolysate. S1.3. The hydrolysate is filtered and separated by passing it through 10kDa and 3kDa ultrafiltration membranes in sequence, and short peptide solutions with molecular weight below 3kDa are collected. S1.4 Dissolve bovine thrombin in coupling buffer to obtain an enzyme solution with a concentration of 5 mg / mL; The N-hydroxysuccinimide-activated agarose gel was equilibrated three times with coupling buffer to obtain a pre-equilibrated agarose gel. The enzyme solution was added to the pre-equilibrated agarose gel at a ratio of 4 mg thrombin / mL sedimentation gel, and the reaction was carried out with shaking at 4 °C for 12 h. After the reaction was completed, the gel was washed three times alternately with coupling buffer and a solution of 0.1 mol / L Tris-HCl, 0.5 mol / L sodium chloride, and pH 8.3. The reaction was then blocked with 0.1 mol / L ethanolamine solution at pH 8.3 for 2 h. The gel was washed again until neutral and then equilibrated with TBS buffer to obtain the coupled affinity medium. The coupled affinity medium was packed into a column with a height-to-diameter ratio of 10:1 and equilibrated with 10 column volumes of TBS buffer. The short peptide solution in S1.3 was filtered through a 0.22 μm filter membrane and passed through the affinity column at a rate of 0.2 column volumes per minute. The eluent was collected and washed with 10 column volumes of TBS buffer until the baseline was stable. Elution was performed using a gradient of 5 column volumes of 0.1 mol / L citric acid solution at pH 3.5, collecting elution peaks with absorbance > 0.2 AU at 280 nm. The eluent was immediately collected, and 1 mol / L NaOH solution was slowly added to adjust the pH to 7.0-7.4 to prevent ligand detachment or product denaturation under acidic conditions. The pH-adjusted eluent was pumped into a nanofiltration unit, using a nanofiltration membrane module with a molecular weight cutoff (MWCO) of 200 Da. Constant-volume percolation desalination was performed at an operating pressure of 1.5 MPa and a feed temperature of 25 °C. During this process, deionized water was added until the permeate conductivity was below 150 μS / cm. Subsequently, water addition was stopped, and the solution was concentrated to 1 / 5 of its original volume. The retentate was collected to obtain peptides. S1.5. Spray-dry the peptide fragments and add maltodextrin accounting for 30% of the dry weight of the peptide fragments. The inlet air temperature is 140℃ and the outlet air temperature is 85℃ to obtain earthworm protein peptides with in vitro thrombin inhibitory activity.

[0035] In vitro anticoagulant activity assay procedure: Dilute the earthworm protein peptide sample to be tested to a series of concentrations with Tris-HCl buffer (containing 0.1% BSA, pH 8.4); add 25 μL of diluted sample and 25 μL of thrombin solution (0.5 NIHU / mL) sequentially to a 96-well plate; mix well and incubate at 37℃ for 5 minutes; quickly add 150 μL of chloromozym TH substrate solution (0.3 mM); immediately place in a microplate reader and continuously monitor the absorbance value at a wavelength of 405 nm. Changes in enzyme activity over 5 minutes; Calculation: Wells without sample were used as positive controls (100% enzyme activity), and wells without sample and thrombin were used as blanks; Calculate the enzyme activity inhibition rate of the sample wells relative to the positive control; Inhibition rate (%) = [1 - (sample OD growth rate / positive control OD growth rate)] × 100%; Usually, the half-maximal inhibitory concentration (MCC) is used. The activity level is indicated by ( ). The lower the value, the stronger the antithrombin activity.

[0036] The procedure for determining peptide content is as follows: It utilizes the principle of the reaction between peptide bonds and biuret, which has higher specificity than the determination of free amino acids; usually, casein is used as a standard, and the absorbance at around 750 nm is measured to calculate the total peptide content in the sample; the peptide content reflects the total peptide content.

[0037] Solubility determination procedure: Weigh a certain amount of sample, dissolve it in deionized water, centrifuge, determine the solid content in the supernatant, and calculate the solubility.

[0038] Table 1. Performance data of earthworm protein peptides with in vitro thrombin-inhibiting activity. In Examples 2 and 4, the enzyme dosage was insufficient, leading to incomplete enzymatic hydrolysis and a significant decrease in anticoagulant activity. In Example 3, the enzyme dosage was increased compared to Example 1, but the activity decreased slightly, possibly due to excessive hydrolysis causing further degradation of the active peptides. Example 5 exhibited better activity, indicating that appropriately increasing the alkaline protease dosage is beneficial for the release of active peptides.

[0039] The coupling density of the affinity ligand determines the purification effect. When the enzyme coupling density is too low (Example 6), the chromatographic medium provides insufficient active binding sites, resulting in the loss of some target peptides and a relatively increased proportion of non-specific adsorption. The activity of the final product is lower than that of Example 1. Example 7 (enzyme coupling density 6 mg / mL) showed the best overall performance with the lowest IC50 value (14.5 μg / mL) under the same excipient addition ratio, indicating that the optimized affinity purification step can efficiently enrich highly active peptides.

[0040] Regarding the effect of excipient addition (comparison of Examples 1, 8, and 9): Example 8 (addition of 10% maltodextrin) had the lowest excipient addition, resulting in the highest peptide content (88.5%) in the final product, exhibiting strong anticoagulant activity (IC50 of 15.4 μg / mL) at the same mass concentration. Example 9 (addition of 30% maltodextrin) showed a slight increase in solubility, although the peptide content decreased to 75.4% due to dilution and the IC50 value increased to 18.2 μg / mL (relatively weakened activity). This indicates that while the addition of maltodextrin physically diluted the active ingredient, it did not destroy the inherent biological activity of earthworm protein peptides and improved powder properties. Considering cost, solubility, and activity, the process conditions of Example 7 and the excipient formulation of Example 1 were more suitable. Comparative Example 1: This example differs from Example 7 in that pepsin was not added; alkaline protease was used directly.

[0041] Comparative Example 2: The difference between this example and Example 7 is that the short peptide solution was not specifically captured by affinity chromatography material. Instead, the short peptide solution obtained in S1.3 was directly desalted and concentrated according to the nanofiltration process in the latter half of S1.4 in Example 7, and then spray dried.

[0042] Stability test (activity retention): The sample was stored in a constant temperature and humidity chamber at 40℃ and 75% relative humidity for 7 days, and the anticoagulant activity was measured every 24 hours. ).

[0043] Table 2 Performance data of earthworm protein peptides with in vitro thrombin-inhibiting activity. Compared with Example 7, Comparative Example 1 (single-enzyme hydrolysis) showed a significant decrease in anticoagulant activity (IC50 of 28.6 μg / mL). At the same time, due to incomplete enzymatic hydrolysis and a large amount of impurities, the specific activity after the same purification steps was still low.

[0044] Under acidic conditions, pepsin can specifically cleave earthworm protein macromolecules into intermediate peptides that are more suitable for further action by alkaline proteases. Without this pretreatment step, alkaline proteases alone cannot fully release the active fragments encapsulated inside the protein, resulting in a reduction in the yield and variety of target active peptides, and ultimately a significant reduction in the product's functionality.

[0045] Compared to Example 7, Comparative Example 2 (without affinity purification) showed extremely low anticoagulant activity (IC50 of 52.3 μg / mL). Because the affinity chromatography step was omitted, the product was essentially a crude peptide mixture with a molecular weight <3 kDa, containing a large number of non-anticoagulant peptides. Therefore, although the total peptide content (68.7%) showed the presence of peptides, the proportion of effectively active peptides was extremely low, resulting in a significant decrease in the bioactivity per unit mass of product. Furthermore, the solubility of Comparative Example 2 (93.5%) was significantly lower than that of Example 7, indicating that the affinity chromatography step effectively removed some hydrophobic impurities.

[0046] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely preferred examples and are not intended to limit the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing earthworm protein peptides with in vitro thrombin-inhibiting activity, characterized in that, Includes the following steps: S1.1 Pre-treat the earthworm raw material to obtain earthworm protein; S1.

2. Under nitrogen protection, at pH 2.0-2.5 and 40-50℃, add pepsin to hydrolyze the earthworm protein for 1-2 hours; slowly add 1-4 mol / L NaOH solution to adjust the pH to 8.0-9.0, add alkaline protease at 50-55℃ and continue hydrolysis for 1-4 hours, heat at 95℃ for 10 minutes to inactivate the enzyme, and cool to ≤30℃ to obtain the hydrolysate. S1.

3. The hydrolysate is filtered and separated by passing it through 10kDa and 3kDa ultrafiltration membranes in sequence, and short peptide solutions with molecular weight below 3kDa are collected. S1.

4. Pass the short peptide solution through an affinity chromatography material coupled with thrombin and elute to collect the peptide fragments; S1.

5. Spray dry the peptide fragments and add 10-30% maltodextrin by dry weight of the peptide fragments to obtain earthworm protein peptides with in vitro thrombin inhibitory activity.

2. The method for preparing earthworm protein peptide with in vitro thrombin-inhibiting activity according to claim 1, characterized in that, In step S1.1, the preprocessing includes the following steps: The earthworm raw material was washed with deionized water at 4℃ and cut into 0.5-1.0cm segments. It was then added to a sodium chloride solution pre-cooled to 4℃ with a mass concentration of 0.9% and shaken at 100-200rpm for 10-20min. The earthworm was then filtered to obtain the purified earthworm. After removing impurities, the earthworms were extracted with anhydrous ethanol at a material-to-liquid ratio of 1:5-10 at 40-50℃ with heating and stirring for 1-2 times, each time for 20-40 minutes. The extracts were combined and the ethanol was recovered under reduced pressure. The mixture was then filtered to obtain defatted earthworm tissue. The defatted earthworm tissue was treated with hot water at 90℃ for 20 minutes and then cooled to room temperature to obtain earthworm protein.

3. The method for preparing earthworm protein peptide with in vitro thrombin-inhibiting activity according to claim 1, characterized in that, In step S1.2, the mass ratio of pepsin to earthworm protein is 0.5-2.0%, and the mass ratio of alkaline protease to earthworm protein is 1-3%.

4. The method for preparing earthworm protein peptide with in vitro thrombin-inhibiting activity according to claim 1, characterized in that, In step S1.4, the process of passing the short peptide solution through an affinity chromatography material and eluting to collect the peptide fragments is as follows: Bovine thrombin was dissolved in coupling buffer to obtain an enzyme solution with a concentration of 1-10 mg / mL; The N-hydroxysuccinimide-activated agarose gel was equilibrated three times with coupling buffer to obtain a pre-equilibrated agarose gel. The enzyme solution was added to a pre-equilibrated agarose gel and the reaction was carried out with shaking at 4°C for 4-12 h. After the reaction was completed, the gel was washed 2-3 times alternately with coupling buffer and a solution of 0.1 mol / L Tris-HCl, 0.5 mol / L sodium chloride, pH 8.

3. The reaction was then blocked with 0.1 mol / L ethanolamine solution at pH 8.3 for 2 h. The gel was washed again until neutral and then equilibrated with TBS buffer to obtain the coupled affinity medium. The coupled affinity medium was packed into a column with a height-to-diameter ratio of 5-10:1 and equilibrated with 5-10 column volumes of TBS buffer. The short peptide solution in S1.3 was filtered through a 0.22 μm filter and passed through the affinity column at a rate of 0.1-0.2 column volumes per minute. The eluent was collected and washed with 5-10 column volumes of TBS buffer until the baseline was stable. Elute with a gradient of 3-5 column volumes of 0.1 mol / L citric acid solution at pH 3.0-3.5, collecting the elution peak with absorbance >0.2 AU at 280 nm. Adjust the pH of the eluent to 7.0-7.4 using a pH adjuster. Circulate the pH-adjusted eluent through a nanofiltration membrane with a molecular weight cutoff of 100-500 Da for desalting and concentration. Control the operating pressure at 0.5-2.5 MPa until the permeate conductivity drops below 200 μS / cm, then collect the retentate to obtain the peptides.

5. The method for preparing earthworm protein peptide with in vitro thrombin-inhibiting activity according to claim 4, characterized in that, The thrombin is bovine thrombin extracted or recombinantly expressed, with a specific activity ≥1000 U / mg.

6. The method for preparing earthworm protein peptide with in vitro thrombin-inhibiting activity according to claim 4, characterized in that, The coupling buffer solution is a solution of 0.1 mol / L sodium bicarbonate, 0.5 mol / L sodium chloride, and pH 8.

3.

7. The method for preparing earthworm protein peptide with in vitro thrombin-inhibiting activity according to claim 4, characterized in that, The enzyme solution was added to the pre-equilibrated agarose gel at a ratio of 2-10 mg thrombin / mL sedimentation gel.

8. The method for preparing earthworm protein peptide with in vitro thrombin-inhibiting activity according to claim 4, characterized in that, The TBS buffer solution is a solution of 20 mmol / L Tris-HCl, 150 mmol / L sodium chloride, and pH 7.

4.

9. The method for preparing earthworm protein peptide with in vitro thrombin-inhibiting activity according to claim 1, characterized in that, In S1.5, the inlet air temperature for spray drying is 130-140℃, and the outlet air temperature is 80-85℃.

10. A type of earthworm protein peptide with in vitro thrombin-inhibiting activity, characterized in that, It is prepared by the method for preparing earthworm protein peptide with in vitro thrombin inhibitory activity as described in any one of claims 1-9.

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