Preparation method of high-activity earthworm protein peptide

Abstract

The invention relates to the technical field of earthworm protein peptides, and discloses a high-activity earthworm protein peptide preparation method which comprises the following steps: pretreating living earthworms to obtain homogenate, and removing impurities from the homogenate to retain a middle-layer soluble protein solution; treating the soluble protein solution to obtain a protein concentrated solution; carrying out ultrafiltration desalination grading on the protein concentrated solution to obtain a desalted peptide solution, and then carrying out double-enzyme directional enzymolysis on the desalted peptide solution to obtain an enzymatic hydrolysate; filtering the enzymatic hydrolysate to obtain a graded peptide solution, adsorbing by using activated carbon, and decolorizing by using a resin double column to obtain an effluent; the effluent liquid is subjected to vacuum low-temperature concentration, concentrated peptide liquid is obtained, the concentrated peptide liquid is freeze-dried, and high-activity earthworm protein peptide powder is obtained.The obtained earthworm peptide is light in color, light in fishy smell and good in fluidity, can be directly used for functional food, special dietary and young animal feed, and has the physiological functions of oxidation resistance, fatigue resistance, immunoregulation and the like.

Description

Technical Field

[0001] This invention relates to the field of earthworm protein peptide technology, and more specifically, to a method for preparing highly active earthworm protein peptides. Background Technology

[0002] Research on earthworm protein peptides began in the late 20th century, initially focusing on the extraction and activity verification of single enzymes such as fibrinolytic enzymes and collagenases from earthworms. Due to the high water content, rapid autolysis, and complex odor components of earthworm tissue, traditional drying-pulverizing-organic solvent extraction processes not only result in significant activity loss but also suffer from solvent residues, dark color, strong fishy odor, and poor palatability, failing to meet the requirements of the food, feed, and biopharmaceutical industries for high-activity, low-odor, and residue-free protein peptides. In recent years, with the development of enzyme engineering, membrane separation, and freeze-drying technologies, researchers have attempted to combine proteolytic enzyme hydrolysis with membrane fractionation to improve the utilization rate of earthworm protein and reduce its molecular weight, thereby obtaining more easily absorbed small peptides. However, existing technologies generally employ single-enzyme one-step hydrolysis, resulting in a single cleavage site, leading to a bitter taste and insufficient exposure of active sequences in the product. Simultaneously, the decolorization and deodorization processes often rely on large doses of activated carbon or multiple extractions with organic solvents, resulting in high peptide loss, cumbersome processes, and significant environmental impact. Furthermore, some processes directly use high-temperature spray drying after enzymatic hydrolysis. The instantaneous high temperature causes oxidation or aggregation of heat-sensitive active sequences, ultimately leading to decreased product activity and reduced solubility. Moreover, existing published methods mostly focus on optimizing specific parameters such as hydrolysis time, temperature, and enzyme dosage, or limiting activity indicators such as ACE inhibition rate and DPPH scavenging rate. They lack a systematic design for the entire process chain and fail to address the scale-up production fluctuations caused by the strong coupling between parameters and activity. Therefore, we propose a method for preparing highly active earthworm protein peptides. Summary of the Invention

[0003] To overcome the above-mentioned defects of the prior art, embodiments of the present invention provide a method for preparing highly active earthworm protein peptides.

[0004] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing highly active earthworm protein peptides, the method comprising: S1: Pre-treat live earthworms to obtain a homogenate, and remove impurities from the homogenate, retaining the middle layer of soluble protein solution; S2: The soluble protein solution is processed to obtain a protein concentrate; S3: The protein concentrate is subjected to ultrafiltration desalting and fractionation to obtain desalted peptide solution, and then the desalted peptide solution is subjected to dual-enzyme targeted enzymatic hydrolysis to obtain enzymatic hydrolysate; S4: Filter the enzymatic hydrolysate to obtain graded fetal fluid, and then use activated carbon adsorption and resin dual-column decolorization to obtain the effluent. S5: The effluent is concentrated under vacuum at low temperature to obtain concentrated peptide solution, and the concentrated peptide solution is freeze-dried to obtain highly active earthworm protein peptide powder.

[0005] Furthermore, the live earthworm undergoes pretreatment to obtain a homogenate, including, After weighing healthy, undamaged live earthworms, place them in a clean temporary holding tank. After the temporary holding is completed, take them out and rinse them quickly twice with deionized water. After draining, lay the earthworms flat on a nylon mesh screen, rinse them with sterile water in a counter-current manner, remove any damaged individuals, soak them in peracetic acid for surface sterilization, and then immediately rinse them with sterile water until there is no odor. Use gauze to absorb the surface moisture. Place the cleaned earthworms into a stainless steel mesh basket and quickly immerse them in an ice-water mixture until they are completely inactivated, their color lightens but they remain soft. After removing them, place them on a pre-cooling workbench, use sterile absorbent paper to dry the surface free water, and add pre-cooled buffer solution according to the material-to-liquid ratio.

[0006] Using a high-speed tissue homogenizer, a coarse homogenate is obtained by intermittent homogenization. The coarse homogenate is then poured into a circulating cooling high-pressure homogenizer. After each homogenization, the temperature is lowered to ≤10 ℃ in an ice bath. The final homogenate is collected and quickly transferred to sterile centrifuge tubes for storage or for subsequent extraction processes.

[0007] Further, the process of removing impurities from the homogenate and retaining the middle layer of soluble protein solution includes: The homogenate was centrifuged at low temperature to remove the fat layer and residue layer, retaining the middle layer of soluble protein solution.

[0008] Furthermore, the process of treating the soluble protein solution to obtain a protein concentrate includes: adjusting the pH value of the soluble protein solution, and then allowing it to stand for 15-30 minutes to allow some of the impurities to fully denature and precipitate. Slowly add the solid neutral salt, stirring thoroughly after each addition until completely dissolved, until the target saturation is reached, allowing the target protein to be fully extracted; Pre-cool and centrifuge the salted-out solution, remove the supernatant, and retain the protein precipitate.

[0009] Further, the step of performing dual-enzyme targeted enzymatic hydrolysis on the desalted peptide solution to obtain the enzymatic hydrolysate includes: The protein concentrate was subjected to constant-volume ultrafiltration through a first ultrafiltration membrane, and the low molecular weight permeate was collected. Continue to add deionized water at the same temperature and pH until the conductivity drops to the set range to obtain desalted peptide solution; After adjusting the protein concentration of the desalted peptide solution, alkaline protease was added for the first enzymatic hydrolysis, followed by the addition of compound flavor enzymes for the second enzymatic hydrolysis. The entire enzymatic hydrolysis process was accompanied by ultrasonic pulse treatment to obtain the enzymatic hydrolysate.

[0010] Further, the process of obtaining the effluent by adsorption with activated carbon followed by decolorization and decolorization via a resin dual-column system includes: The enzyme hydrolysate was rapidly heated and then kept at that temperature for 15 minutes to inactivate the protease activity. Then, the cooling cycle was switched to cool the enzyme hydrolysate. After cooling, the liquid is first passed through a nylon mesh to remove large particles, and then filtered through a microfiltration membrane to collect the clarified peptide solution. The clarified peptide solution is passed sequentially through a second ultrafiltration membrane and a third ultrafiltration membrane to collect peptide components within the target molecular weight range, thus obtaining a fractionated peptide solution.

[0011] Furthermore, the freeze-drying of the concentrated peptide solution to obtain highly active earthworm protein peptide powder comprises: The effluent was concentrated under reduced pressure until the solids concentration increased, and then filtered to obtain a concentrated peptide solution. Add a freeze-drying protectant to the concentrated peptide solution, and after it is completely dissolved, pre-freeze the peptide solution. The pre-frozen sample was quickly transferred to a freeze dryer and vacuumed for sublimation drying. The sublimated and dried sample was then analyzed and dried. After drying, the powder is automatically sealed under high-purity nitrogen protection. Before leaving the box, an aluminum-plastic cap is affixed to obtain highly active earthworm protein peptide powder. The powder is then quickly transferred into an aluminum foil bag, where desiccant and deoxidizer are added. After heat sealing, the bag is labeled.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: This application discloses a method for preparing highly active earthworm protein peptides. The method employs a gentle pretreatment process involving "temporary intestinal cleansing—ice water shock—circulating cooling homogenization" to preserve the complete structure of the earthworm protein. Three-phase centrifugation coupled with isoelectric point salting-out simultaneously removes lipids, impurities, and reduces viscosity, achieving green concentration. Constant-volume ultrafiltration completes desalting and fractionation in a single step, achieving low-temperature energy saving. Dual-enzyme segmented enzymatic hydrolysis combined with ultrasonic pulses precisely cleaves the hydrophobic / hydrophilic regions, reducing bitterness and enhancing activity. A microfiltration-secondary ultrafiltration combination clarifies and locks in the target molecular weight range, allowing the product to dissolve rapidly in cold water. Activated carbon-resin... The process involves a dual-column tandem decolorization and deodorization method, with online resin regeneration, resulting in a significant reduction in solid waste. Vacuum low-temperature concentration combined with freeze-drying using a protective agent ensures low-heat exposure throughout the process, producing a loose, porous powder that reconstitutes quickly and maintains its active conformation. The entire process utilizes food-grade materials, with closed-loop water and salt solution circulation, eliminating organic solvent emissions, ensuring self-control compatibility, and robust scale-up. The method is free from specific numerical constraints, exhibiting minimal batch-to-batch variation. The resulting earthworm peptides are light in color, have a mild odor, and excellent flowability, making them suitable for direct use in functional foods, special diets, and feed for young animals, imparting physiological functions such as antioxidant, anti-fatigue, and immune regulation. Attached Figure Description

[0013] Figure 1 The flowchart illustrates a method for preparing highly active earthworm protein peptides provided by this invention. Detailed Implementation

[0014] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0015] Example 1 Please see Figure 1 As shown in the figure, this embodiment discloses a method for preparing highly active earthworm protein peptides, the method comprising: S1: Pre-treat live earthworms to obtain a homogenate, and remove impurities from the homogenate, retaining the middle layer of soluble protein solution; S2: The soluble protein solution is processed to obtain a protein concentrate; S3: The protein concentrate is subjected to ultrafiltration desalting and fractionation to obtain desalted peptide solution, and then the desalted peptide solution is subjected to dual-enzyme targeted enzymatic hydrolysis to obtain enzymatic hydrolysate; S4: Filter the enzymatic hydrolysate to obtain graded fetal fluid, and then use activated carbon adsorption and resin dual-column decolorization to obtain the effluent. S5: The effluent is concentrated under vacuum at low temperature to obtain concentrated peptide solution, and the concentrated peptide solution is freeze-dried to obtain highly active earthworm protein peptide powder.

[0016] In this embodiment, the live earthworm undergoes pretreatment to obtain a homogenate, including, After weighing healthy, undamaged live earthworms, place them in a clean temporary holding tank. After the temporary holding is completed, take them out and rinse them quickly twice with deionized water. After draining, lay the earthworms flat on a nylon mesh screen, rinse them with sterile water in a counter-current manner, remove any damaged individuals, soak them in peracetic acid for surface sterilization, and then immediately rinse them with sterile water until there is no odor. Use gauze to absorb the surface moisture. Place the cleaned earthworms into a stainless steel mesh basket and quickly immerse them in an ice-water mixture until they are completely inactivated, their color lightens but they remain soft. After removing them, place them on a pre-cooling workbench, use sterile absorbent paper to dry the surface free water, and add pre-cooled buffer solution according to the material-to-liquid ratio.

[0017] Using a high-speed tissue homogenizer, a coarse homogenate is obtained by intermittent homogenization. The coarse homogenate is then poured into a circulating cooling high-pressure homogenizer. After each homogenization, the temperature is lowered to ≤10 ℃ in an ice bath. The final homogenate is collected and quickly transferred to sterile centrifuge tubes for storage or for subsequent extraction processes.

[0018] In this embodiment, the process of removing impurities from the homogenate and retaining the soluble protein solution in the middle layer includes: The homogenate was centrifuged at low temperature to remove the fat layer and residue layer, retaining the middle layer of soluble protein solution.

[0019] In this embodiment, the process of treating the soluble protein solution to obtain the protein concentrate includes: adjusting the pH value of the soluble protein solution, and then letting it stand for 15-30 minutes to allow some of the impurity proteins to fully denature and precipitate. Slowly add the solid neutral salt, stirring thoroughly after each addition until completely dissolved, until the target saturation is reached, allowing the target protein to be fully extracted; Pre-cool and centrifuge the salted-out solution, remove the supernatant, and retain the protein precipitate.

[0020] In this embodiment, the step of performing dual-enzyme targeted enzymatic hydrolysis on the desalted peptide solution to obtain the enzymatic hydrolysate includes: The protein concentrate was subjected to constant-volume ultrafiltration through a first ultrafiltration membrane, and the low molecular weight permeate was collected. Continue to add deionized water at the same temperature and pH until the conductivity drops to the set range to obtain desalted peptide solution; After adjusting the protein concentration of the desalted peptide solution, alkaline protease was added for the first enzymatic hydrolysis, followed by the addition of compound flavor enzymes for the second enzymatic hydrolysis. The entire enzymatic hydrolysis process was accompanied by ultrasonic pulse treatment to obtain the enzymatic hydrolysate.

[0021] In this embodiment, the process of obtaining the effluent by adsorption with activated carbon followed by decolorization and decolorization via a resin dual-column system includes: The enzyme hydrolysate was rapidly heated and then kept at that temperature for 15 minutes to inactivate the protease activity. Then, the cooling cycle was switched to cool the enzyme hydrolysate. After cooling, the liquid is first passed through a nylon mesh to remove large particles, and then filtered through a microfiltration membrane to collect the clarified peptide solution. The clarified peptide solution is passed sequentially through a second ultrafiltration membrane and a third ultrafiltration membrane to collect peptide components within the target molecular weight range, thus obtaining a fractionated peptide solution.

[0022] In this embodiment, the step of freeze-drying the concentrated peptide solution to obtain highly active earthworm protein peptide powder includes: The effluent was concentrated under reduced pressure until the solids concentration increased, and then filtered to obtain a concentrated peptide solution. Add a freeze-drying protectant to the concentrated peptide solution, and after it is completely dissolved, pre-freeze the peptide solution. The pre-frozen sample was quickly transferred to a freeze dryer and vacuumed for sublimation drying. The sublimated and dried sample was then analyzed and dried. After drying, the powder is automatically sealed under high-purity nitrogen protection. Before leaving the box, an aluminum-plastic cap is affixed to obtain highly active earthworm protein peptide powder. The powder is then quickly transferred into an aluminum foil bag, where desiccant and deoxidizer are added. After heat sealing, the bag is labeled.

[0023] It should be noted that healthy earthworms are obtained, cleaned, and temporarily kept. After the temporarily kept earthworms are cleaned and damaged individuals are removed, they are then sterilized and dehydrated. The earthworms are then killed with an ice-water mixture. A high-speed tissue homogenizer is used for intermittent homogenization to obtain a coarse slurry. The final homogenate is collected and centrifuged at low temperature, retaining the soluble protein solution in the middle layer. The protein solution is salted out, and the supernatant is removed, retaining the protein precipitate. The concentrated protein solution is ultrafiltered, and deionized water is added to obtain a deionized solution. The deionized solution is then enzymatically hydrolyzed with alkaline protease and compound flavor enzyme to inactivate the protease activity in the hydrolysate. After removing large particles, it is filtered through a microfiltration membrane, and the clear peptide solution is collected. Then, it is filtered through a second and third ultrafiltration membrane to obtain a fractionated peptide solution. The peptide solution is concentrated, a preservative is added, and it is then sublimated, dried, and desorbed.

[0024] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

[0025] In conclusion, the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing high-activity dilm protein peptide, characterized in that, The method comprises: S1: pretreating the live earthworm to obtain a homogenate, and removing impurities from the homogenate to retain a middle-layer soluble protein solution; S2: treating the soluble protein solution to obtain a protein concentrate; S3: subjecting the protein concentrate to ultrafiltration desalting and fractionation to obtain a desalted peptide solution, and then subjecting the desalted peptide solution to double-enzyme directional enzymolysis to obtain an enzymolysis solution; S4: filtering the enzymolysis solution to obtain a fractionated peptide solution, adsorbing the fractionated peptide solution by activated carbon, and then decolorizing and removing impurities from the fractionated peptide solution by a resin double-column to obtain an effluent; S5: vacuum low-temperature concentrating the effluent to obtain a concentrated peptide solution, and freeze-drying the concentrated peptide solution to obtain a high-activity earthworm peptide powder.

2. The method for preparing highly active earthworm protein peptides according to claim 1, characterized in that, The pretreatment of the live earthworm to obtain the homogenate comprises: After the healthy and undamaged live earthworm is weighed, it is placed in a clean temporary breeding tank, and after temporary breeding, it is fished out and rinsed with deionized water for two times; After the earthworm is drained, it is laid on a nylon mesh screen and rinsed with sterile water in countercurrent, and damaged individuals are picked out, and the earthworm is immersed in peracetic acid for surface sterilization, and then immediately rinsed with sterile water until no odor is generated, and the surface moisture is absorbed by gauze; The washed earthworm is loaded into a stainless steel mesh basket, quickly immersed in an ice water mixture, until it is completely inactivated, the body color is lightened, but remains soft, and after being fished out, it is placed on a pre-cooling operation table, and the surface free water is absorbed by sterile water-absorbing paper, and the pre-cooled buffer is added according to the solid-liquid ratio.

3. A high-speed tissue homogenizer is used to obtain a coarse slurry by intermittent homogenization, the coarse slurry is poured into a circulating cooling high-pressure homogenizer, the temperature of the homogenizer is reduced to ≤10 ℃ after each homogenization, the final homogenate is collected, quickly transferred into a sterile centrifuge tube, and stored for standby or subsequent extraction process.

4. The method for preparing highly active earthworm protein peptides according to claim 2, characterized in that, The removal of impurities from the homogenate to retain the middle-layer soluble protein solution comprises: The homogenate is centrifuged at low temperature to remove the fat layer and the residue layer, and the middle-layer soluble protein solution is retained.

5. The method for preparing highly active earthworm protein peptides according to claim 3, characterized in that, The treatment of the soluble protein solution to obtain the protein concentrate comprises: The pH value of the soluble protein solution is adjusted, and then the solution is left to stand for 15-30 minutes to allow partial impure proteins to be denatured and precipitated; Solid neutral salt is slowly added, and after each addition, the solution is fully stirred until completely dissolved, until the target protein is allowed to be eluted, and the target protein is allowed to be eluted; The salted-out solution is pre-cooled and centrifuged to remove the supernatant, and the protein precipitate is retained.

6. The method for preparing highly active earthworm protein peptides according to claim 4, characterized in that, The double-enzyme directional enzymolysis of the desalted peptide solution to obtain the enzymolysis solution comprises: The protein concentrate is subjected to constant-volume ultrafiltration through a first ultrafiltration membrane, and low-molecular-weight permeate is collected; Deionized water at the same temperature and pH is continuously added until the conductivity decreases to a set range, to obtain a desalted peptide solution; After the protein concentration of the desalted peptide solution is adjusted, alkaline protease is first added for first enzymolysis, and then compound flavor enzyme is added for second enzymolysis, and the whole enzymolysis process is accompanied by ultrasonic pulse treatment, to obtain an enzymolysis solution.

7. The method for preparing highly active earthworm protein peptides according to claim 1, characterized in that, The adsorption of the enzymolysis solution by activated carbon and then decolorization and removal of impurities by a resin double-column to obtain an effluent comprises: The enzymolysis solution is quickly warmed and then kept warm for 15 minutes to kill the protease activity, and then the solution is cooled by switching the cooling cycle; The cooled solution is first filtered through a nylon mesh to remove large particles, and then filtered through a microfiltration membrane to collect a clear peptide solution; The clear peptide solution is sequentially passed through a second ultrafiltration membrane and a third ultrafiltration membrane to collect peptide components in a target molecular weight range, to obtain a fractionated peptide solution.

8. The method for preparing highly active earthworm protein peptides according to claim 1, characterized in that, The concentrated peptide solution is freeze-dried to obtain high-activity liuhe notopterygii peptide powder, comprising: The effluent is concentrated under reduced pressure to increase the solid concentration, and then filtered to obtain a concentrated peptide solution; A freeze-drying protective agent is added to the concentrated peptide solution, and after complete dissolution, the peptide solution is pre-frozen; The pre-frozen sample is quickly transferred to a freeze dryer for vacuum pumping and sublimation drying; The sublimation-dried sample is desiccated, After drying is completed, automatic plugging is performed under high-purity nitrogen protection, an aluminum plastic cover is attached before the box is taken out, high-activity liuhe notopterygii peptide powder is obtained, and the powder is quickly transferred to an aluminum foil bag, a drying agent and a deoxidizing agent are added, and the bag is heat-sealed and labeled.

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