Leech polypeptide extraction method, obtained product and application

Abstract

The invention discloses a leech polypeptide extraction method, an obtained product and application, leeches are used as a raw material, a pretreatment process of soaking in rice water in combination with slow-fire processing of lard is innovatively adopted, uniform powder is obtained through a liquid nitrogen quick-freezing and low-temperature ball milling technology, protein is dissolved by using a betaine-urea eutectic system, and the leech polypeptide is obtained. According to the leech polypeptide freeze-dried powder and the preparation method thereof, the breakthrough of improving the polypeptide extraction rate and improving the antithrombin activity is realized by combining a green purification method under the assistance of glycerol synergy and a segmented temperature control enzymolysis technology, meanwhile, the fishy smell of the extracted leech polypeptide freeze-dried powder is greatly reduced, and an efficient and environment-friendly industrial solution is provided for development of anticoagulant drugs and health-care products.

Description

Technical Field

[0001] The present invention relates to a method for extracting leech polypeptides, the obtained products and applications, and in particular to a method for efficiently extracting leech polypeptides, the obtained products and applications, belonging to the technical field of biomedicine. Background Art

[0002] Leeches, as a traditional Chinese medicinal material, have a long history of medicinal use. The polypeptide components contained in them have been proven to have multiple biological activities, including anticoagulant, antithrombotic, anti-inflammatory and angiogenesis-promoting effects. Therefore, they have extremely high application value in the field of biomedicine. Traditional leech extraction processes mainly include water decoction and organic solvent extraction. The water decoction method is relatively simple to operate, but it has the problem of low extraction efficiency. Many effective ingredients are difficult to fully dissolve, resulting in waste of resources. Although the organic solvent extraction method can improve the extraction rate of some components, the problem of organic solvent residue is serious, which may affect the safety and quality of the extract. In addition, the subsequent impurity removal and solvent recovery steps are cumbersome and costly. Summary of the Invention

[0003] The present invention aims to provide a method for extracting leech extracts, more specifically, a method for extracting leech polypeptides. This method addresses the technical bottlenecks of traditional leech polypeptide extraction processes, such as low efficiency, high cost, and severe pollution. By innovatively constructing an extraction route of "pretreatment optimization - low-temperature pulverization - deep eutectic solvent synergistic enzymatic extraction - green purification," the extraction rate of leech polypeptides is improved.

[0004] The specific technical solutions adopted in the present invention are as follows: A method for extracting a leech extract, wherein the extract is mainly leech polypeptides, the method comprising the following steps: (1) Soak the dried leeches in rice washing water, then take them out and dry them; (2) Frying the leeches processed in step (1) in lard until browned; (3) freezing the leeches treated in step (2) with liquid nitrogen, then cryogenically grinding and sieving to obtain leech powder; (4) Mixing leech powder with a binary eutectic solvent aqueous solution, adjusting the pH and then adding trypsin, controlling the temperature for enzymatic hydrolysis, and then adding a ternary eutectic solvent aqueous solution to the mixture, mixing well, and then continuing to control the temperature for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, adjusting the pH and inactivating the enzyme; (5) adding a flocculant to the enzymatic hydrolyzate of step (4), adjusting the pH to the isoelectric point, and precipitating a precipitate; (6) Collect the precipitate and freeze-dry it to obtain leech polypeptide.

[0005] Furthermore, in step (1), the rice washing water is rice swill obtained by soaking washed rice in water. Wash the rice, add water and stir gently, then let it stand and soak for a period of time, and filter to obtain the rice washing water. The rice can be various varieties of rice, preferably long-grain fragrant rice. The rice swill obtained from long-grain fragrant rice can remove the fishy smell of leeches to the greatest extent and reduce the taste of leech polypeptide products. The mass ratio of rice to water is 1:5-10, for example, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10. Preferably, the rice is first gently stirred in water for 15-20 turns, and then left to soak for 15-20 minutes. After soaking, the leeches are removed and dried to a moisture content of ≤8%. The drying is carried out at a temperature of 65°C or less.

[0006] Furthermore, in step (1), the leech is a dried leech, which may be a species such as, but not limited to, Hirudo styraciflua, Hirudo japonica, Hirudo philippinosus, Hirudo styraciflua, etc. The dried leech is soaked in rice washing water for 7 to 10 hours.

[0007] Furthermore, in step (2), lard can be freshly prepared from lard, or pre-refined lard paste can be used directly. When frying leeches, the oil temperature is maintained at 75-80°C, for example, 75°C, 76°C, 77°C, 78°C, 79°C, or 80°C. Fry until browned, crispy, with no white core on the cross section, and not too light or too heavy. Lard can be reused 2-3 times. This method can not only dissolve fat-soluble odorous substances in leeches, such as aldehydes and volatile fatty acids, but also alleviate the irritation of the drug and reduce side effects.

[0008] Furthermore, in step (3), the decocted leeches are placed in liquid nitrogen for freezing for 5-10 minutes.

[0009] Furthermore, in step (3), the frozen leeches are ground at low temperatures and in the presence of liquid nitrogen to ensure a low temperature during grinding. The specific operation is as follows: the grinding apparatus and operating tools are pre-cooled in liquid nitrogen, and then the frozen leeches are placed in the grinding apparatus for grinding. The grinding apparatus can be a vibrating ball mill, and the grinding is performed in a liquid nitrogen environment. The grinding is preferably performed in multiple times to ensure a low temperature environment. The single grinding time is 2 to 3 minutes, and the vibration frequency of the grinding in the vibrating ball mill is 20 to 30 Hz. A total of 3 to 5 cycles are performed until the sample is completely crushed. The total grinding time is controlled within the range of 6 to 15 minutes. Liquid nitrogen is added to the grinding apparatus after each grinding to maintain a low temperature environment. This process effectively avoids protein denaturation caused by mechanical heat generation in the traditional crushing process through the synergistic effect of liquid nitrogen quick freezing and low-temperature grinding, while achieving precise control of particle size, significantly improving the dissolution rate of active ingredients, and providing an efficient quality control solution for biological sample pretreatment.

[0010] Furthermore, in step (3), the powder obtained after grinding is sequentially passed through an 80-mesh sieve and a 100-mesh sieve to obtain a powder with a size of 80-100 mesh. The purpose of sieving is to remove larger particles and improve the extraction rate of leech polypeptides. During the leech crushing and screening process, sieves with different apertures are usually used to achieve this goal. After multiple experimental verifications, the particle size range of 80-100 mesh has the highest extraction rate and is more suitable for process production.

[0011] Furthermore, in step (4), the binary deep eutectic solvent is a mixture of betaine and urea or a mixture of betaine and butanediol in a molar ratio of 5-8:1, for example, 5:1, 6:1, 7:1, 8:1, the deep eutectic solvent solution is a mixed solution formed by the deep eutectic solvent and water, and the concentration of the binary deep eutectic solvent solution is 40-60 wt%, for example, 40%, 45%, 50%, 55%, 60%.

[0012] Furthermore, in step (4), the hydrogen bond acceptor of the ternary deep eutectic solvent is betaine, and the hydrogen bond donor is urea and glycerol, or butanediol and glycerol. The molar ratio of betaine, urea and glycerol is 5-8:1-3:2, and the molar ratio of betaine, butanediol and glycerol is also 5-8:1-3:2. The ternary deep eutectic solvent solution is a mixture of the ternary deep eutectic solvent and water, and the concentration of the ternary deep eutectic solvent solution is 50-65wt%, for example, 50%, 55%, 60%, 65%. The synergistic effect of betaine with urea (or butanediol) and glycerol can form a more stable hydrogen bond network, significantly broaden the solvent polarity range, enhance the solubility of non-polar polypeptides, and greatly increase the extraction rate of leech polypeptides.

[0013] Furthermore, in step (4), the ratio of leech powder to the binary deep eutectic solvent aqueous solution is 1 g:15-25 ml, for example, 1:15, 1:20, or 1:25. After the ternary deep eutectic solvent aqueous solution is added, the ratio of leech powder to the total amount of solution in the system is 1 g:30-35 ml, for example, 1:30, 1:31, 1:32, 1:33, 1:34, or 1:35.

[0014] Furthermore, in step (4), trypsin is added after adjusting the pH to 6-8. When the activity of trypsin is 6000-8000 u / mg, the amount of trypsin used is 3-6% of the mass of the leech powder.

[0015] Furthermore, in step (4), after adding trypsin, the temperature is first raised to 30-40°C at a rate of 1-5°C / min, and enzymatic hydrolysis is carried out for 1-2 hours. Then, an aqueous solution of a ternary low eutectic solvent is added, and the temperature is then raised to 45-55°C at a rate of 1-5°C / min, and enzymatic hydrolysis is carried out for 3-5 hours. This enzymatic hydrolysis method allows trypsin to first hydrolyze the easily degradable part of the substrate (such as loosely structured proteins), reducing the complexity of the substrate in the subsequent high-temperature stage and improving the overall hydrolysis efficiency.

[0016] Furthermore, in step (4), after the enzymatic hydrolysis is completed, acid is added to adjust the pH to 1.5-2.5 to inactivate the enzyme. This method of enzyme inactivation can avoid the phenomenon of reduced activity of leech polypeptides caused by high-temperature enzyme inactivation.

[0017] Furthermore, in step (5), the flocculant is ethanol or chitosan. When the flocculant is chitosan, the amount of chitosan used is 0.1-0.3% of the mass of the leech powder. When the flocculant is ethanol, the amount of ethanol used is such that the ethanol concentration in the enzymatic hydrolyzate is 70-75 wt%.

[0018] Furthermore, in step (5), after adding the flocculant, the pH is adjusted to the isoelectric point using an acid or base such as HCL or sodium hydroxide. The isoelectric point is pH 3.3-4.8, and the polypeptide is precipitated at the isoelectric point.

[0019] This method uses leeches as raw material, removes the fishy smell with rice water, and pre-treats them with slow-cooking lard. Liquid nitrogen quick-freezing and low-temperature ball milling are used to ensure the activity of the active ingredients. A betaine-urea eutectic system is used to dissolve the protein, supplemented by glycerol enhancement and segmented temperature-controlled enzymatic hydrolysis to improve protein dissolution efficiency and polypeptide yield. Flocculants and isoelectric precipitation are used to achieve environmentally friendly separation and reduce wastewater discharge. The resulting leech polypeptide has high anticoagulant activity, providing a green and efficient solution for the development of anticoagulant drugs.

[0020] The present invention also provides a leech polypeptide prepared according to the above method and the use of the leech polypeptide in preparing an anticoagulant product.

[0021] Compared with the prior art, the present invention has the following significant advantages: 1. This invention innovatively constructs a three-dimensional quality control system: rice water pretreatment, low-temperature drying, and lard processing. First, the medicinal materials are immersed in rice water, where the rich starch colloidal network absorbs the fishy-smelling terpenes and fat-soluble toxins in leeches, simultaneously activating rice water enzymes to enhance the biological activity of leech polypeptides. After precise temperature-controlled drying to lock in the active ingredients, the medicinal materials are then gently fried in lard. This affinity for animal fats and fats selectively removes non-polar impurities from the medicinal materials, further converting odorous molecules during the caramelization reaction. This process represents an intelligent upgrade to traditional processing techniques. The treated medicinal materials are significantly effective in removing fishy and odorous substances while also preserving their antithrombin activity to the greatest extent possible, demonstrating excellent toxicity-reducing and synergistic effects.

[0022] 2. This invention innovatively incorporates ultra-low-temperature embrittlement technology, using liquid nitrogen flash freezing (<-196°C) to transform leech samples into a glassy, ​​brittle structure, effectively preventing mechanical damage to peptides caused by ice crystal growth. Within the cryogenic ball milling system, liquid nitrogen circulation maintains a grinding chamber temperature of ≤-100°C, keeping heat input below 50 J / g throughout the process. This process not only fully preserves the three-dimensional conformation of heat-sensitive proteins such as leech peptides, but also significantly improves subsequent extraction efficiency by increasing the specific surface area through milling, providing key technical support for the high-value utilization of leech resources.

[0023] 3. The present invention utilizes binary and ternary deep eutectic solvents as extraction solvents. These solvents offer multiple advantages for protein and peptide extraction, including environmental friendliness, high extraction efficiency, significant cost-effectiveness, ease of operation and optimization, and protection of peptide activity. They represent a promising green extraction technology. Furthermore, the combination of binary and ternary deep eutectic solvents maximizes the extraction rate of leech peptides.

[0024] 4. The present invention uses trypsin to hydrolyze leech proteins, achieving higher yields and higher solubility than traditional methods, and yielding more bioactive peptides, making it highly suitable for industrial production. Furthermore, the enzymatic hydrolysis and deep eutectic solvent extraction work in tandem. Due to the significant enhancement of enzyme activity by the deep eutectic solvent system, more leech proteins are hydrolyzed into peptides and dissolved in the solvent, achieving a synergistic effect.

[0025] 5. The present invention adopts a one-pot process, which greatly simplifies the process flow, avoids the separation and purification steps of intermediate products, reduces losses, and significantly improves the output and yield of the final product.

[0026] 6. The leech polypeptide of the present invention has a high extraction rate, greatly reduced fishy odor, and high antithrombin activity, which can reach more than 800 U / g, providing an efficient and environmentally friendly industrial solution for the development of anticoagulant drugs and health products. DETAILED DESCRIPTION

[0027] The following is a clear and complete description of the technical solutions of the present invention in conjunction with specific embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0028] Unless otherwise specified, the following concentrations are all mass percentages.

[0029] In the following examples and comparative examples, the leech used was Hirudo spp.

[0030] Example 1 S1. Place 0.1 kg of long-grain fragrant rice in 0.5 L of water, gently stir to remove impurities, discard the turbid water, add 0.8 L of water again, gently stir 15 times, let stand for 15 minutes, filter to obtain rice water, completely immerse the dried leeches in the rice water for 9 hours, remove, and initially dry at 50°C for 2 hours, then dry at 65°C for 4 hours, and then slowly dry at 40°C until the moisture content is ≤8%. S2. Melt the lard over low heat, add the dried leeches, and stir-fry repeatedly. Keep the frying temperature at 76°C and fry until golden brown, crispy, and with no white core on the cross section. It should not be too light or too heavy.

[0031] S3. Quickly freeze the decocted leech sample in liquid nitrogen for 6 minutes. Place the empty grinding jar and grinding balls in liquid nitrogen to fully pre-cool them, and start the pre-cooling process of the low-temperature vibrating ball mill. Subsequently, use pre-cooled tweezers to quickly transfer the frozen leech pieces to the pre-cooled grinding jar. Next, pour a small amount of liquid nitrogen into the grinding jar, ensuring that the liquid nitrogen can cover the surface of the sample. Immediately afterwards, quickly and firmly tighten the sealing lid of the grinding jar. Adjust the grinding vibration frequency to 25Hz, the single grinding time to 2 minutes, and grind in sections: replenish liquid nitrogen after each grinding, and grind for a total of 4 cycles, with a total grinding time of 8 minutes. Pour the powder into a pre-cooled stainless steel vibrating sieve, pass it through an 80-mesh and 100-mesh sieves, and accurately collect the leech powder with a particle size of 80-100 mesh.

[0032] S4. Prepare a deep eutectic solvent solution. Weigh 140.58 g of betaine, 12.01 g of urea, and 152.59 g of deionized water, mix them together, heat and stir them in a 60°C water bath for 30 minutes, until the solution is transparent and free of particles. Cool the heated solution to room temperature to obtain a binary deep eutectic solvent solution with a concentration of 50 wt%. Weigh 70.29 g of betaine, 12.01 g of urea, 18.4 g of glycerol, and 100.72 g of deionized water, mix them together, heat and stir them in a 60°C water bath for 30 minutes, until the solution is transparent and free of particles. Cool the heated solution to room temperature to obtain a ternary deep eutectic solvent solution with a concentration of 50 wt%.

[0033] S5. Weigh 5g of leech powder at a solid-liquid ratio of 1:20 (g / mL) and mix thoroughly with 100ml of the binary deep eutectic solvent solution, ensuring complete immersion of the leech powder in the solution. While stirring, add 0.1mol / L sodium hydroxide solution to adjust the pH of the mixture to 6.8. Add trypsin (enzyme activity 7000u / mg) at a concentration of 5% by weight of the leech powder. Heat the mixture to 35°C at a rate of 2°C / min. Enzymatically digest for 2 hours. Add 75ml of the ternary deep eutectic solvent solution to achieve a solid-liquid ratio of 1:35. Heat the mixture to 50°C at a rate of 2°C / min. Enzymatically digest for 4 hours. After completion of the enzymatic digestion, add HCl to adjust the pH to 2.0 and maintain for 10 minutes to inactivate the enzyme.

[0034] S6. After inactivating the enzyme, chitosan was added to the enzymatic hydrolysate as a flocculant at a concentration of 0.1% by weight of the leech powder. The pH was then adjusted to an isoelectric point of 4.0 using 1 mol / L HCl, at which the polypeptide was precipitated.

[0035] S7. After the enzymatic hydrolyzate was cooled to room temperature, centrifuged at 6000 rpm for 15 minutes. S8. Place the collected precipitate into a freeze-drying bottle, place the freeze-drying bottle in a freeze dryer, and freeze-dry for 48 hours until the precipitate is completely dried into a freeze-dried product, thereby obtaining the leech polypeptide.

[0036] Example 2 Leech polypeptides were extracted according to the method of Example 1, except that: in step S4, 93.72 g of betaine, 6.01 g of urea, and 99.73 g of deionized water were weighed and mixed together, placed in a 60°C water bath, heated and stirred for 30 minutes, until the solution became transparent and free of particles, and the heated solution was cooled to room temperature to obtain a binary deep eutectic solvent solution with a concentration of 50 wt%; 93.76 g of betaine, 18.02 g of urea, 18.42 g of glycerol, and 130.2 g of deionized water were weighed and mixed together, placed in a 60°C water bath, heated and stirred for 30 minutes, until the solution became transparent and free of particles, and the heated solution was cooled to room temperature to obtain a ternary deep eutectic solvent solution with a concentration of 50%.

[0037] Example 3 Leech polypeptides were extracted according to the method of Example 1, except that: in step S4, 70.29 g of betaine, 9.01 g of 1,4-butanediol, and 79.3 g of deionized water were mixed together, heated in a 60°C water bath, and stirred for 30 minutes until the solution became transparent and free of particles. The heated solution was cooled to room temperature to obtain a binary deep eutectic solvent solution with a concentration of 50 wt%; 70.29 g of betaine, 18.02 g of 1,4-butanediol, 18.41 g of glycerol, and 106.72 g of deionized water were mixed together, heated in a 60°C water bath, and stirred for 30 minutes until the solution became transparent and free of particles. The heated solution was cooled to room temperature to obtain a ternary deep eutectic solvent solution with a concentration of 50 wt%.

[0038] Example 4 Leech polypeptides were extracted according to the method of Example 1, except that in step S5, 5 g of leech powder was weighed at a solid-liquid ratio of 1:20 (g / mL) and mixed evenly with 100 ml of a binary deep eutectic solvent solution to ensure that the leech powder was completely immersed in the solution. 0.01 mol / L sodium hydroxide solution was added while stirring, and the pH of the mixture was adjusted to 6.8. Trypsin (enzyme activity 7000 u / mg) was added at a concentration of 5% by weight of the leech powder. The enzymatic hydrolysis temperature was first increased by 5°C / min to 30°C, and enzymatic hydrolysis was carried out for 2 hours. The ternary deep eutectic solvent was then added to adjust the solid-liquid ratio to 1:35. The temperature was then increased by 5°C / min to 45°C, and enzymatic hydrolysis was carried out for 4 hours. HCl was added to adjust the pH to 2.0, maintained for 10 minutes, and the enzyme was inactivated.

[0039] Example 5 Leech polypeptides were extracted according to the method of Example 1, except that in step S5, 5 g of leech powder was weighed at a solid-liquid ratio of 1:15 (g / mL) and mixed evenly with 75 ml of the binary deep eutectic solvent solution, ensuring that the leech powder was completely immersed in the solution. 0.1 mol / L sodium hydroxide solution was added while stirring, and the pH of the mixture was adjusted to 6.8. Trypsin (enzyme activity 7000 u / mg) was added at 3% of the leech powder mass. The temperature was then raised to 35°C at a rate of 2°C / min for 2 hours of enzymatic hydrolysis. 75 ml of the ternary deep eutectic solvent solution was then added to adjust the solid-liquid ratio to 1:30. The temperature was then raised to 50°C at a rate of 2°C / min for 4 hours of enzymatic hydrolysis. After the enzymatic hydrolysis was completed, HCl was added to adjust the pH to 2.0 and maintained for 10 minutes to inactivate the enzyme.

[0040] Example 6 Leech polypeptides were extracted according to the method of Example 1, except that in step S6, the amount of chitosan added was 0.3% of the mass of the leech powder.

[0041] Example 7 Leech polypeptides were extracted according to the method of Example 1, except that: in step S1, the dried leeches were completely immersed in rice water for 9 hours, and then dried at 60° C. for 8 hours to ensure that the water content was ≤8%.

[0042] Comparative Example 1 Leech polypeptides were extracted according to the method of Example 1, except that: in step S4, 117.15 g of betaine, 60.06 g of urea, and 177.21 g of deionized water were weighed and mixed together, placed in a 60°C water bath, heated and stirred for 30 minutes, until the solution became transparent and free of particles, and the heated solution was cooled to room temperature to obtain a binary deep eutectic solvent solution with a concentration of 50 wt%; 117.15 g of betaine, 60.06 g of urea, 92.09 g of glycerol, and 269.3 g of deionized water were weighed and mixed together, placed in a 60°C water bath, heated and stirred for 30 minutes, until the solution became transparent and free of particles, and the heated solution was cooled to room temperature to obtain a ternary deep eutectic solvent solution with a concentration of 50 wt%.

[0043] Comparative Example 2 Leech polypeptides were extracted according to the method of Example 1, except that: in step S4, 83.78 g of choline chloride, 13.40 g of malic acid, and 97.18 g of deionized water were weighed and mixed together, placed in a 60°C water bath, heated and stirred for 30 minutes, until the solution became transparent and free of particles, and the heated solution was cooled to room temperature to obtain a binary deep eutectic solvent solution with a concentration of 50%; 83.78 g of choline chloride, 26.42 g of malic acid, 18.42 g of glycerol, and 128.62 g of deionized water were weighed and mixed together, placed in a 60°C water bath, heated and stirred for 30 minutes, until the solution became transparent and free of particles, and the heated solution was cooled to room temperature to obtain a ternary deep eutectic solvent solution with a concentration of 50 wt%.

[0044] Comparative Example 3 Leech polypeptides were extracted according to the method of Example 1, except that: in step S4, 70.29 g of betaine, 6.21 g of ethylene glycol, and 76.5 g of deionized water were weighed and mixed together, and the mixture was heated and stirred in a 60°C water bath for 30 minutes until the solution became transparent and free of particles. The heated solution was cooled to room temperature to obtain a binary deep eutectic solvent solution with a concentration of 50 wt %. 70.29 g of betaine, 12.41 g of ethylene glycol, 18.42 g of glycerol, and 101.12 g of deionized water were weighed and mixed together, and the mixture was heated and stirred in a 60°C water bath for 30 minutes until the solution became transparent and free of particles. The heated solution was cooled to room temperature to obtain a ternary deep eutectic solvent solution with a concentration of 50 wt %.

[0045] Comparative Example 4 Leech polypeptides were extracted according to the method of Example 1, except that: in step S4, 140 g of choline chloride, 10 g of urea, and 150 g of deionized water were weighed and mixed together, and the mixture was heated and stirred in a 60°C water bath for 30 minutes until the solution became transparent and free of particles. The heated solution was cooled to room temperature to obtain a binary deep eutectic solvent solution with a concentration of 50 wt %; 83.78 g of choline chloride, 12.01 g of urea, 18.41 g of glycerol, and 114.2 g of deionized water were weighed and mixed together, and the mixture was heated and stirred in a 60°C water bath for 30 minutes until the solution became transparent and free of particles. The heated solution was cooled to room temperature to obtain a ternary deep eutectic solvent solution with a concentration of 50 wt %.

[0046] Comparative Example 5 Leech polypeptides were extracted according to the method of Example 1, except that in step S5, 75 ml of the ternary deep eutectic solvent solution was replaced with 75 ml of the binary deep eutectic solvent solution.

[0047] Comparative Example 6 Leech polypeptides were extracted according to the method of Example 1, except that in step S5, 100 ml of the binary deep eutectic solvent solution was replaced with 100 ml of the ternary deep eutectic solvent solution.

[0048] Comparative Example 7 Leech polypeptides were extracted according to the method of Example 1, except that in step S5, 5 g of leech powder was weighed at a solid-liquid ratio of 1:20 (g / mL) and mixed evenly with 100 ml of the binary deep eutectic solvent solution, ensuring that the leech powder was completely immersed in the solution. While stirring, 0.1 mol / L sodium hydroxide solution was added to adjust the pH of the mixture to 6.8. Trypsin (enzyme activity 7000 u / mg) was added to the mixture at a concentration of 5% by weight of the leech powder. The mixture was then heated to 50°C at a rate of 5°C / min and enzymatic hydrolysis was continued for 2 h. 75 ml of the ternary deep eutectic solvent solution was then added to bring the solid-liquid ratio to 1:35, and enzymatic hydrolysis was continued at 50°C for 4 h. After the enzymatic hydrolysis was completed, HCl was added to adjust the pH to 2.0 and maintained for 10 min to inactivate the enzyme.

[0049] The leech polypeptides obtained in the above examples and comparative examples were evaluated in the following manner: 1. Sensory evaluation A blind sensory evaluation of the leech polypeptide odor was conducted by sealing 5g of freeze-dried leech polypeptide in an odorless glass bottle and labeling the treatment process. A panel of 10 reviewers who had undergone standardized training evaluated the odor of the freeze-dried powder after different treatments in a constant temperature and humidity environment (25°C ± 2°C, RH 50%).

[0050] Table 1 Evaluation criteria The sensory evaluation results of each sample were averaged to form the score of the sample. The results are shown in Table 2 below: Table 2 As shown in Table 2, the sensory evaluation results show that the freeze-dried leech polypeptide prepared by the method of the present invention exhibits significant advantages in odor control.

[0051] 2. Determination of polypeptide content in leech polypeptide ① Preparation of Coomassie Brilliant Blue G250 solution Weigh 50 mg of Coomassie Brilliant Blue G250, add 25 mL of anhydrous ethanol, and 50 mL of 85% (W / V) phosphoric acid. Dissolve the Coomassie Brilliant Blue powder in anhydrous ethanol, mix well with the phosphoric acid, and add water to make up to 500 mL.

[0052] ②Drawing the standard curve Protein content was determined using the Coomassie Brilliant Blue method, and a standard curve was drawn. Accurately pipette 0 μL, 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, and 100 μL of a 1 mg / mL BSA standard solution into a test tube, add ultrapure water to 1 mL, and mix thoroughly to obtain standard solutions with concentrations of 0.00 mg / mL, 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, and 0.10 mg / mL. Accurately pipette 1 mL of each standard sample into a test tube, then pipette 5 mL of Coomassie Brilliant Blue G250 solution into the test tube, mix thoroughly, let stand for 5 minutes, measure the absorbance at 595 nm, draw a standard curve of concentration and absorbance, and obtain the regression equation.

[0053] Y = 0.0071X + 0.0552, R 2 = 0.9997. This indicates that the protein has a good linear relationship with absorbance in the concentration range of 0-100 μg / mL.

[0054] ③ Preparation of sample solution to be tested 5 mg of the leech polypeptide powder prepared in each example and comparative example was added to a 50 mL volumetric flask, and water was added to the volume. After the powder was fully dissolved, a sample solution was obtained.

[0055] ④Determination of sample solution Take 1 mL of the sample solution in a test tube, add 5 mL of the prepared Coomassie Brilliant Blue G250 solution, shake evenly, let it stand for 5 minutes, and measure the absorbance at 595 nm.

[0056] ⑤Substitute the absorbance into the above regression equation to calculate the solution concentration, and then calculate the purity (D) of the leech polypeptide according to the following formula: Where: D is the purity of leech polypeptide,%; C—mass concentration of leech polypeptide in sample solution, μg / mL; V—total volume of sample solution, mL; M—mass of leech polypeptide freeze-dried powder.

[0057] The leech polypeptide extraction rate (Y) was calculated according to the following formula: D is the purity of leech polypeptide,%; G is the mass of leech polypeptide freeze-dried powder, g; S is the mass of leech powder, g.

[0058] ⑥ The experimental results are shown in Table 3 below: Table 3 According to the results of the Coomassie Brilliant Blue method, Example 1 showed significant advantages in polypeptide purity and extraction rate. Its leech polypeptide purity was as high as 95.0%, and the extraction rate was 32.0%, ranking first among all samples.

[0059] 3. Antithrombin activity assay ① Preparation of the sample solution to be tested: Take 1 g of the leech polypeptide powder prepared in each example and comparative example and put it into a 5 mL volumetric flask, add water to make up to volume, and after it is fully dissolved, the sample solution can be obtained.

[0060] ② Preparation of Tris-HCl buffer solution containing 0.5% (bovine) fibrinogen: Take 25 ml of 0.2 mol / L Tris solution and about 40 ml of 0.1 mol / L hydrochloric acid solution, add water to 100 ml, adjust the pH to 7.4, and then add 0.5 g (bovine) fibrinogen.

[0061] ③ Preparation of thrombin solution: Take an appropriate amount of thrombin reagent and add normal saline to make 40 units of thrombin per 1 ml (prepared immediately).

[0062] ④ Sampling: Take 100 μl of sample solution.

[0063] ⑤ Add buffer: Add 200 μl of Tris-HCl buffer containing 0.5% (bovine) fibrinogen.

[0064] ⑥ Warm soak: Place in a water bath (37℃±0.5℃) and soak for 5 minutes.

[0065] ⑦ Titration of thrombin: Add 40 units of thrombin solution per 1 ml to the above solution until coagulation occurs, and record the volume of thrombin solution consumed.

[0066] ⑧ Calculation of antithrombin activity: Calculate the thrombin activity units per 1g of sample according to the formula.

[0067] U is the unit of thrombin activity per 1g of sample, U / g; C1 is the concentration of thrombin solution, U / ml; C2 is the concentration of the sample solution, g / ml; V1 is the volume of consumed thrombin solution, μl; V2 is the amount of test solution added, μl.

[0068] The amount of thrombin that neutralizes one unit is one unit of antithrombin activity. The antithrombin activities in each sample obtained according to the above formula are shown in Table 4 below.

[0069] Table 4 The results of the antithrombin activity assay showed that the leech polypeptide of Example 1 had significant biological activity advantages, with an antithrombin activity of up to 823 U / g, demonstrating the excellent performance of the process of the present invention in retaining active ingredients.

Claims

1. A method for extracting leech polypeptides, characterized by The following steps are involved: (1) Soak the dried leeches in rice washing water, then take them out and dry them; (2) Frying the leeches processed in step (1) in lard until browned; (3) freezing the leeches treated in step (2) with liquid nitrogen, then cryogenically grinding and sieving to obtain leech powder; (4) Mixing leech powder with a binary eutectic solvent aqueous solution, adjusting the pH and then adding trypsin, controlling the temperature for enzymatic hydrolysis, and then adding a ternary eutectic solvent aqueous solution to the mixture, mixing well, and then continuing to control the temperature for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, adjusting the pH and inactivating the enzyme; (5) adding a flocculant to the enzymatic hydrolyzate of step (4), adjusting the pH to the isoelectric point, and precipitating a precipitate; (6) Collect the precipitate and freeze-dry it to obtain leech polypeptide.

2. The extraction method according to claim 1, wherein: In step (1), the rice-washing water is obtained by soaking rice in water 5-10 times its mass for 15-20 minutes; preferably, in step (1), the dried leeches are soaked in the rice-washing water for 7-10 hours.

3. The extraction method according to claim 1, wherein: In step (2), the oil temperature is maintained at 75-80°C during frying.

4. The extraction method according to claim 1, wherein: In step (3), the leech is frozen in liquid nitrogen for 5-10 minutes; preferably, in step (3), the leech is ground in a liquid nitrogen environment, the grinding vibration frequency is 20-30 Hz, the single grinding time is 2-3 minutes, and a total of 3-5 cycles of grinding are performed; preferably, in step (3), the particle size of the leech powder is 80-100 mesh.

5. The extraction method according to claim 1, wherein: In step (4), the binary deep eutectic solvent is a mixture of betaine and urea in a molar ratio of 5-8:1 or a mixture of betaine and butanediol in a molar ratio of 5-8:1, and the concentration of the binary deep eutectic solvent aqueous solution is 40-60wt%; the ternary deep eutectic solvent is a mixture of betaine, urea and glycerol in a molar ratio of 5-8:1-3:2 or a mixture of betaine, butanediol and glycerol in a molar ratio of 5-8:1-3:2, and the concentration of the ternary deep eutectic solvent aqueous solution is 50-65wt%.

6. The extraction method according to claim 1, wherein: In step (4), the ratio of leech powder to binary eutectic solvent aqueous solution is 1 g:15-25 ml, and the ratio of leech powder to the total amount of binary eutectic solvent aqueous solution and ternary eutectic solvent aqueous solution is 1 g:30-35 ml.

7. The extraction method according to claim 1, wherein: In step (4), trypsin is added after adjusting the pH to 6-8; preferably, the amount of trypsin used is 3-6% of the mass of the leech powder, and the enzyme activity of trypsin is 6000-8000u / mg.

8. The extraction method according to claim 1, wherein: In step (4), after adding trypsin, the temperature is first raised to 30-40°C at a rate of 1-5°C / min, and enzymatic hydrolysis is carried out for 1-2 hours. Then, an aqueous solution of a ternary low eutectic solvent is added, and the temperature is then raised to 45-55°C at a rate of 1-5°C / min, and enzymatic hydrolysis is carried out for 3-5 hours. Preferably, in step (4), the pH is adjusted to 1.5-2.5 to inactivate the enzyme.

9. The extraction method according to claim 1, wherein: In step (5), the flocculant is chitosan or ethanol, the amount of chitosan is 0.1-0.3% of the mass of the leech powder, and the amount of ethanol is such that the ethanol concentration in the enzymatic hydrolyzate is 70-75 wt %. Preferably, the pH of the isoelectric point is 3.3-4.

8.

10. A leech polypeptide obtained by the extraction method according to any one of claims 1 to 9, and use of the leech polypeptide in the preparation of an anticoagulant product.

Images