Method for preparing earthworm peptide by adopting eutectic solvent in combination with electric field effect

Abstract

The invention belongs to the technical field of protein, and provides a method for preparing earthworm peptide by adopting a deep-eutectic solvent in combination with an electric field effect, the method comprises the following steps: (1) mixing an earthworm protein raw material with the deep-eutectic solvent to form a uniform reaction system; (2) an external electric field with preset parameters is applied to the reaction system for a reaction, so that earthworm protein molecules are broken under the synergistic effect of the electric field and DES, and earthworm peptide is generated; and (3) after the reaction is finished, separating and purifying a reaction product to obtain the target earthworm peptide. The method is mild in condition, green, environment-friendly, short in reaction time and low in energy consumption, protease does not need to be added, and the molecular weight of the product can be controlled by adjusting parameters such as electric field intensity. The average molecular weight of the obtained earthworm peptide is lower than 1000 Da, the DES residual quantity is low, the peptide fragment composition is unique, the function of remarkably improving insulin resistance is achieved, and the earthworm peptide is superior to a product prepared through a traditional enzymolysis or acidolysis method and suitable for industrial large-scale production.

Description

Technical Field

[0001] This application belongs to the field of protein technology. Specifically, this application provides a method for preparing earthworm peptide using a eutectic solvent combined with an electric field effect. Background Technology

[0002] Earthworms, or earthworms, have a long history of medicinal use in my country. Modern pharmacological studies have shown that the main active component of earthworms is earthworm peptide, obtained from the hydrolysis of earthworm protein, which possesses significant fibrinolytic, anticoagulant, anti-inflammatory, antioxidant, and immunomodulatory activities. Recent research also suggests that earthworm peptide has potential value in improving insulin resistance and regulating glucose metabolism. Therefore, the efficient and green preparation of highly active earthworm peptide has significant application value.

[0003] Currently, the main methods for industrial-scale preparation of earthworm peptides are enzymatic hydrolysis and chemical hydrolysis. Enzymatic hydrolysis offers mild conditions and high specificity, but it suffers from drawbacks such as high enzyme costs, long reaction times, susceptibility to introducing exogenous protein allergens, and limited peptide diversity due to fixed enzymatic sites. Chemical hydrolysis (acid or alkaline methods), while less expensive, involves harsh reaction conditions (strong acids, strong alkalis, high temperatures), which can easily damage amino acid structures, leading to reduced product activity and generating large amounts of saline wastewater, thus being environmentally unfriendly. Earthworm peptides prepared using these traditional methods often exhibit unstable or insufficiently pronounced specific biological activities, such as improving insulin resistance.

[0004] Eutectic solvents are eutectic mixtures formed by hydrogen bond donors and acceptors through hydrogen bonding. They are known as "green solvents" due to their advantages such as simple preparation, low cost, low vapor pressure, biodegradability, and good solubility for biomacromolecules. In recent years, some studies have explored the use of DES to assist in the extraction of bioactive components, but its application in the directed hydrolysis of proteins to prepare bioactive peptides, especially its synergistic effect with physical field technology, remains largely unexplored.

[0005] Electric fields, as a physical field technology, can influence the conformation, polarity, and chemical bond stability of molecules in solution. If the energy of an electric field could be used to selectively break peptide bonds in proteins, it would be a clean hydrolysis technique that requires no chemical reagents or biological enzymes.

[0006] Therefore, developing a new method that combines the superior solubility of eutectic solvents with the green catalytic effect of electric fields to achieve efficient and controllable hydrolysis of earthworm protein and prepare highly active earthworm peptides with higher biological activity, especially significantly improving insulin resistance, has important innovative significance and practical application prospects. Summary of the Invention

[0007] The primary objective of this invention is to overcome the shortcomings of existing technologies and provide a novel, mild, efficient, and environmentally friendly method for preparing earthworm peptides. Another objective is to provide a low-eutectic solvent composition specifically for the above method. A further objective is to provide an earthworm peptide product obtained by the above method, which has low solvent residue, a unique peptide composition, and significant bioactivity in improving insulin resistance. A further objective is to provide a method for preparing low-uric acid skipjack tuna peptide powder. This method uses low-temperature plasma-activated water and a pulsed electric field to synergistically pretreat skipjack tuna raw materials, gently disrupting the protein structure and significantly improving subsequent enzymatic hydrolysis efficiency. Simultaneously, by adding a purine-reducing enzyme during the enzymatic hydrolysis stage, targeted degradation of nucleic acid substances is achieved, reducing the purine content of the product from the source. The entire process is green, mild, and controllable, achieving multiple objectives simultaneously: high-efficiency production, low uric acidification of the product, and high yield of small molecule peptides.

[0008] On one hand, this application provides a method for preparing earthworm peptide using a eutectic solvent combined with an electric field effect, the method comprising: (1) Mix earthworm protein raw material with a eutectic solvent to form a homogeneous reaction system; (2) Apply an external electric field with preset parameters to the reaction system to carry out the reaction, so that the earthworm protein molecules break down under the synergistic effect of the electric field and DES to generate earthworm peptide; (3) After the reaction is completed, the reaction product is separated and purified to obtain the target earthworm peptide.

[0009] Further, the eutectic solvent is composed of hydrogen bond acceptors and hydrogen bond donors. The hydrogen bond acceptor is selected from at least one of choline chloride, betaine, and L-carnitine; the hydrogen bond donor is selected from at least one of urea, glycerol, ethylene glycol, lactic acid, citric acid, glucose, and sorbitol, and the molar ratio of hydrogen bond donor to hydrogen bond acceptor is 1:1 to 5:1; preferably, the hydrogen bond acceptor is choline chloride, and the hydrogen bond donor is glycerol or lactic acid, and the molar ratio of hydrogen bond donor to hydrogen bond acceptor is 1:2 to 1:3; more preferably, the method for preparing the eutectic solvent includes: mixing choline chloride and lactic acid in a molar ratio of 1:2, adding water at 0.25 times the total mass of choline chloride and lactic acid, and stirring at 80°C until a colorless and transparent liquid is formed.

[0010] Furthermore, the mass-to-volume ratio of the earthworm protein raw material to the eutectic solvent, calculated in g:mL, is 1:10 to 1:50.

[0011] Further, the parameters of the applied electric field are: electric field strength 1-50 V / cm, reaction time 0.5-24 hours, and reaction temperature controlled at 20-80°C; preferably, the electric field strength is 50 V / cm, and the reaction temperature is controlled at 30-50°C. Furthermore, the earthworm protein raw material is the dried body powder, homogenate, or crude protein extract of Eisenia fetida, Pheretima filamentosa, or Pheretima spp.

[0012] Further, the separation and purification steps in step (3) include: adding water to dilute the reaction product, centrifuging, membrane filtration and drying (such as freeze drying, spray drying) to obtain earthworm peptide powder with an average molecular weight of less than 1000 Daltons.

[0013] Furthermore, the membrane filtration uses an ultrafiltration membrane with a molecular weight cutoff of less than 1000 Da.

[0014] On the other hand, this application provides earthworm peptide prepared using the above method.

[0015] On the other hand, this application provides the use of the above-mentioned earthworm peptide in the preparation of drugs for treating or preventing thrombosis.

[0016] On the other hand, this application provides the use of the above-mentioned earthworm peptide in the preparation of health products or drugs that help with antioxidation.

[0017] On the other hand, this application provides the use of the above-mentioned earthworm peptide in the preparation of a drug for treating insulin resistance.

[0018] Compared with existing technologies, the advantages of this invention are as follows: This technology uses a green and environmentally friendly eutectic solvent (DES) to replace the strong acids and bases in traditional chemical hydrolysis, effectively avoiding related pollution. Simultaneously, the entire reaction process does not require the addition of proteases, eliminating the introduction of exogenous proteins. Its reaction conditions are mild (conducted under low-intensity electric fields and medium-low temperatures), maximizing the protection of the bioactivity of earthworm peptides and avoiding the racemization and activity degradation that may occur under harsh conditions. In terms of efficiency and cost, DES itself is inexpensive and simple to prepare. Combined with an electric field that can directly act on protein molecules to accelerate their hydrolysis, it significantly shortens the reaction time while reducing overall energy consumption and cost. The obtained product is unique and of high quality: this method can break some peptide bonds that are difficult to break using enzymatic methods, thereby producing peptides with novel structures and unique activities, especially exhibiting a significant improvement in insulin resistance that is not found in traditional methods. Furthermore, simple post-processing can reduce DES residues to extremely low levels, ensuring high product safety. The process is highly controllable. By precisely adjusting key parameters such as electric field strength, reaction time, and DES composition, the molecular weight distribution and degree of hydrolysis of earthworm peptides can be effectively regulated to meet diverse application needs. Detailed Implementation

[0019] The following embodiments are provided to help to better understand the present invention, but are not limited thereto. These embodiments are for illustrative purposes only and do not limit the scope of protection of the invention.

[0020] Example 1: Validation of Basic Methods and Proof of Synergistic Effect This embodiment aims to verify that the synergistic effect of the combination of DES and electric field is the key to the success of this invention.

[0021] DES preparation: Mix choline chloride and lactic acid in a molar ratio of 1:2, then add water at a mass ratio of 0.25 times that of the total mass of choline chloride and lactic acid. Stir at 80°C until a colorless and transparent liquid is formed, then cool and set aside.

[0022] Experimental Groups: The present invention group: Weigh 1.0 g of dried Eisenia fetida powder (passed through an 80-mesh sieve) and mix it with 20 mL of the above-mentioned DES. Place the reaction system in a 40°C water bath, insert platinum electrodes (2 cm apart), apply a DC electric field of 10 V / cm, and react for 6 hours.

[0023] Comparative Example A (DES alone): Except for the absence of an applied electric field, the conditions were exactly the same as those of the present invention group, and the mixture was left to stand at 40°C for 6 hours.

[0024] Comparative Example B (isolated electric field): Weigh 1.0 g of earthworm protein powder and add 20 mL of deionized water. Treat under the same electric field conditions (10 V / cm, 40°C, 6 h).

[0025] Comparative Example C (Traditional Enzymatic Hydrolysis): Weigh 1.0 g of earthworm protein powder, add 20 mL of phosphate buffer (0.1 M, pH 7.5), add 2% (w / w) of trypsin, and inactivate the enzyme after reacting at 37°C for 4 hours.

[0026] Post-processing and detection: After the reaction of all groups was completed, 100 mL of ultrapure water was added for dilution, centrifuged at 8000 rpm for 15 minutes, and the supernatant was filtered through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da. The filtrate was then freeze-dried. The peptide yield (biuret method), average molecular weight (GPC method, before ultrafiltration), and DES residue (HPLC method) were determined.

[0027] Conclusion: (See Table 1) The peptide yields of Comparative Examples A and B were extremely low, proving that DES or electric field alone cannot effectively hydrolyze earthworm protein under mild conditions. However, our group achieved yields comparable to or even better than those of the traditional enzymatic hydrolysis method (Comparative Example C), and the molecular weight was smaller, fully demonstrating that there is a significant synergistic effect between DES and electric field, and both are indispensable.

[0028] Table 1 Comparison of the effects of different methods in preparing earthworm peptides .

[0029] Example 2: Screening of hydrolysis efficiency for different DES combinations This embodiment aims to verify the superiority of the DES formulation described in the claims.

[0030] Experimental design: Under fixed reaction conditions (1.0 g earthworm protein, 20 mL solvent, 10 V / cm DC electric field, 40°C, 6 h), the hydrolysis efficiency of different DES combinations was investigated. Three parallel experiments were set up for each group.

[0031] Group 1: Choline chloride / lactic acid (1:2) (Preferred ratio in this invention) Group 2: Choline chloride / glycerol (1:3) (Preferred ratio in this invention) Group 3: Choline chloride / urea (1:2) Group 4: Betaine / Lactic Acid (1:2) Group 5: Choline chloride / glucose (1:2) (Preparation temperature 100°C) Conclusion: (See Table 2) The DES composed of choline chloride and lactic acid or glycerol (Group 1 and Group 2) showed significantly higher hydrolysis efficiency (higher yield and smaller molecular weight) than other combinations, which provides solid data support for the DES composition protected in claim 9.

[0032] Table 2 Comparison of hydrolysis efficiencies of different DES .

[0033] Example 3: Effect of electric field strength on degree of hydrolysis and molecular weight of products This embodiment aims to demonstrate that the molecular weight of the product can be controlled by adjusting the electric field strength.

[0034] Experimental design: Based on the ChCl / La (1:2) DES system of Example 1, other conditions were fixed (1.0 g earthworm protein, 20 mL DES, 40°C, 6 h) to investigate the effect of different electric field strengths (5, 10, 20, 50 V / cm).

[0035] Conclusion: (See Table 3) With increasing electric field strength, the degree of protein hydrolysis deepens, resulting in increased peptide yield and decreased average molecular weight. This indicates that the process of this invention is highly controllable, and earthworm peptides of different molecular weight ranges can be directionally prepared by simply adjusting the electric field strength to meet the needs of different application scenarios.

[0036] Table 3 Effect of different electric field strengths on hydrolysis .

[0037] Example 4: Investigation of Reaction Time Progress This embodiment aims to demonstrate reaction kinetics and provide a basis for optimizing production cycles.

[0038] Experimental design: Under the conditions of Example 1 (ChCl / La DES, 10 V / cm, 40°C), samples were taken at different time points (1, 2, 4, 6, 8 hours) to determine the peptide yield.

[0039] Conclusion: (See Table 4) The reaction proceeds rapidly within the first 6 hours, with a significant increase in yield; after 6 hours, the yield increase tends to level off. This indicates that the method of the present invention is highly efficient, achieving the desired hydrolysis effect in a short time, which is beneficial for energy conservation and consumption reduction.

[0040] Table 4 Relationship between reaction time and hydrolysis efficiency .

[0041] Further verification revealed that the optimal response time was approximately 8 hours when the electric field strength was 5 V / cm; approximately 4 hours when the electric field strength was 20 V / cm; and approximately 2 hours when the electric field strength was 50 V / cm. These results indicate that the optimal response time is inversely proportional to the electric field strength.

[0042] Example 5: Verification of the universality of different earthworm raw materials and different processing methods Experimental Design: Variable factors: Factor 1 (Species): I. Eisenia fetida, II. Vermicomyces William, III. Vermicomyces chinensis Factor Two (Physical Form): A. Dry powder: The dried body of the corresponding species is pulverized through an 80-mesh sieve.

[0043] B. Homogenate: Take fresh individuals of the corresponding species and add 3 times their weight of pure water to homogenize them.

[0044] C. Crude protein extract: prepared by centrifugation, ammonium sulfate precipitation, dialysis, and freeze drying of homogenates of the corresponding species (protein content >80%).

[0045] Controlled variables: To ensure comparability of results, the protein input for each experimental combination was precisely controlled to 1.0 gram. Based on the protein content of each ingredient (pre-determined), the corresponding mass or volume of the ingredient was calculated and weighed or measured.

[0046] Reaction conditions: A uniform choline chloride / lactic acid (1:2) DES solution was used, with the DES volume controlled at 20 mL. The reaction conditions were: 10 V / cm DC electric field, 40°C, and 6 hours. Three parallel experiments were conducted for each combination.

[0047] Detection indicators: peptide yield (calculated based on the quality of the input protein) and average molecular weight.

[0048] Conclusion: (See Table 5) Regardless of the common earthworm species used, and regardless of whether the raw material is simply processed powder, coarse homogenate, or semi-refined crude extract, this method can stably and efficiently produce high-quality earthworm peptides.

[0049] Table 5. Effects of different earthworm raw materials and different processing methods on enzymatic hydrolysis .

[0050] Example 6: Evaluation of product bioactivity and uniqueness This embodiment aims to demonstrate the superiority of the product of the present invention in terms of bioactivity and peptide composition through comparative experiments.

[0051] Sample preparation: Sample I: Earthworm peptide (DES-E) prepared in Example 1. Sample II: Earthworm peptide (Enz) prepared by Comparative Example C (conventional enzymatic hydrolysis). Sample III: Commercially available acid-hydrolyzed earthworm peptide (Acid).

[0052] Detection and Analysis: 1) Peptide fingerprinting (HPLC) 2) In vitro fibrinolytic activity (U / mg) 3) Antioxidant activity (DPPH free radical scavenging rate) Conclusion: (See Table 6) The data show that the earthworm peptide (sample I) prepared by the method of the present invention not only has a unique peptide composition, but its key biological activities (fibrinolytic activity and antioxidant activity) are also significantly better than those of traditional enzymatic hydrolysis and acid hydrolysis methods, demonstrating the outstanding progress of the present invention in terms of product superiority.

[0053] Table 6. Bioactivity and peptide composition of the products .

[0054] Example 7: Process Scale-up Experiment This embodiment aims to verify the scalability of the process of the present invention and provide data support for industrial production.

[0055] Experimental steps: 1) DES preparation: Scale up the preparation of choline chloride / lactic acid (1:2) DES 10 L.

[0056] 2) Electric field reaction: 500 g of earthworm protein powder and 10 L of DES were added to a 20 L reaction vessel with a jacketed insulation and stainless steel electrodes. The reaction temperature was controlled at 40°C, the average electric field strength was applied at 10 V / cm, and the reaction was carried out for 6 hours with low-speed stirring.

[0057] 3) Post-processing: After the reaction is completed, 50 L of pure water is added for dilution, and the mixture is separated by a tubular centrifuge. The clear liquid is desalted and concentrated by a hollow fiber ultrafiltration system (molecular weight cutoff of 1000 Da), and finally spray dried.

[0058] Results: 415 g of white earthworm peptide powder was obtained, with a yield of 83%. The product was tested and found to have an average molecular weight of 870 Da and a DES residue of 0.20%. All indicators were highly consistent with the results of the small-scale test.

[0059] Conclusion: (See Table 7) The key indicators (yield, molecular weight, and residual amount) of the product obtained from the scale-up test are highly consistent with the results of the small-scale test, indicating that the process of the present invention has good repeatability, is easy to scale up, has high stability, and has the potential for industrial application.

[0060] Table 7. Parameters and Results of Process Scale-up Tests .

[0061] Example 8: The effect of earthworm peptide product on improving an insulin-resistant cell model Objective: To evaluate the activity of the earthworm peptide of the present invention in improving insulin resistance.

[0062] Samples: Earthworm peptide (DES-E) prepared in Example 1 was used as the experimental group. A conventional enzymatic hydrolysis of earthworm peptide (Enz) was also performed. The process involved: 1. Enzymatic hydrolysis: Weigh 1.0 g of earthworm protein powder and add 20 mL of phosphate buffer (0.1 M, pH 7.5) and mix well. 2. Enzyme addition: Add trypsin at 2% w / w of the earthworm protein mass. 3. Reaction: Stir and react at 37°C for 4 hours. 4. Enzyme inactivation: After the reaction, heat the system to 90°C and maintain this temperature for 10 minutes to inactivate the trypsin and terminate the reaction. 5. Post-treatment: Centrifuge the reaction solution, collect the supernatant, and filter it through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da. The final filtrate was freeze-dried to obtain the conventional enzymatic hydrolyzed earthworm peptide (Enz) sample.

[0063] ( ) and positive control drugs (such as metformin) were used as controls.

[0064] Model: A dexamethasone-induced insulin-resistant HepG2 human liver cancer cell model was used.

[0065] Detection: Different concentrations of earthworm peptide were co-incubated with model cells, and the following parameters were detected: glucose consumption (GOD-POD method), glycogen synthesis, and expression levels of key proteins in the insulin signaling pathway (such as p-AKT / AKT) (Western Blot).

[0066] Conclusion: (See Table 8) The data show that the earthworm peptide (DES-E) prepared in this invention can significantly increase glucose consumption and glycogen synthesis in insulin-resistant cells and upregulate p-AKT expression. Its effect is better than that of traditional enzymatically hydrolyzed earthworm peptide, proving that it has a significant function in improving insulin resistance.

[0067] Table 8. Effects of earthworm peptide products on insulin-resistant cell models. .

Claims

1. A method for preparing a Pheretima peptide by using a deep eutectic solvent combined with an electric field effect, characterized in that, The method comprises: (1) mixing earthworm protein raw materials with deep eutectic solvent to form a uniform reaction system; (2) applying a preset parameter electric field to the reaction system for reaction, so that the earthworm protein molecules are broken under the synergistic action of the electric field and the DES to generate earthworm peptides; (3) After the reaction is completed, the reaction product is separated and purified to obtain the target earthworm peptide.

2. The method of claim 1, wherein the deep eutectic solvent is composed of a hydrogen bond acceptor and a hydrogen bond donor, the hydrogen bond acceptor is selected from at least one of choline chloride, betaine, L-carnitine; the hydrogen bond donor is selected from at least one of urea, glycerol, ethylene glycol, lactic acid, citric acid, glucose, sorbitol, the molar ratio of hydrogen bond donor to hydrogen bond acceptor is 1:1 to 5:1; preferably, the hydrogen bond acceptor is choline chloride, the hydrogen bond donor is glycerol or lactic acid, the molar ratio of hydrogen bond donor to hydrogen bond acceptor is 1:2 to 1:3; more preferably, the method for preparing the deep eutectic solvent comprises: Choline chloride and lactic acid are mixed at a molar ratio of 1:2, then water is added at 0.25 times the total mass of choline chloride and lactic acid, and stirred at 80°C until a colorless transparent liquid is formed.

3. The method of claim 1 or 2, wherein the mass-volume ratio of the earthworm protein raw material to deep eutectic solvent is 1:10 to 1:

50.

4. The method of any one of claims 1-3, wherein the parameters of the applied electric field are: electric field strength 1-50 V / cm, reaction time 0.5-24 hours, and the reaction temperature is controlled at 20-80°C; preferably, the electric field strength is 50 V / cm, and the reaction temperature is controlled at 30-50°C.

5. The method of any one of claims 1-4, wherein the earthworm protein raw material is dried body powder, homogenate liquid or crude protein extract of Eisenia foetida, Pheretima vulgaris or Perionyx excavatus.

6. The method of claim 1, the separation purification step in step (3) comprising: Water is added to dilute the reaction product, and after centrifugation, membrane filtration and drying, earthworm peptide powder with an average molecular weight of less than 1000 daltons is obtained.

7. Earthworm peptides prepared using the method of any one of claims 1-6.

8. Use of earthworm peptides prepared using the method of any one of claims 1-6 in the preparation of a drug for treating or preventing thrombosis.

9. Use of earthworm peptides prepared using the method of any one of claims 1-6 in the preparation of health care products or drugs that help against oxidation.

10. Use of earthworm peptides prepared using the method of any one of claims 1-6 in the preparation of a drug for treating insulin resistance.