Natural hirudin extraction method based on cell fermentation

Abstract

The invention discloses a natural hirudin extraction method based on cell fermentation. The natural hirudin extraction method comprises the following steps: S1, providing leeches, and carrying out starvation treatment on the leeches; s2, taking out the leeches, cleaning the leeches with a metaldehyde solution, and then putting the leeches into purified water for cleaning; s3, inducing and stimulating the leeches, and collecting secretory fluid; s4, extracting hirudin in the secretion on the basis of cell fermentation to obtain fermentation liquor; s5, carrying out separation and purification treatment on the fermentation liquor based on ammonium sulfate fractional precipitation salting-out; and S6, further separating and purifying the fermentation liquor based on ion exchange chromatography. According to the method, the components of the leeches are utilized, the extraction rate of the hirudin is further improved through cell fermentation, a two-stage separation and purification system is established, the purification process is optimized, and the recovery rate and the purification effect of the hirudin are further improved.

Description

Technical Field

[0001] The invention relates to the technical field of hirudin extraction, in particular to a method for extracting natural hirudin based on cell fermentation. Background Art

[0002] Natural hirudin is a protein contained in the secretion of the leech salivary glands. It exists in the salivary glands of blood-sucking leeches and the saliva they secrete. It is the strongest thrombin inhibitor discovered in the world so far and is widely used in anticoagulant and blood-dissolving drugs.

[0003] The invention patent with reference number CN201611241920.8 is a method for preparing hirudin by using microbial fermentation catalysis. A detailed analysis of the patent shows that it uses TB culture medium containing peptone, yeast extract and glycerol to perform anaerobic fermentation on ultrasonically treated leech solution to prepare hirudin. However, in actual operation, the aforementioned patent still uses external force to squeeze the leech, which makes it impossible to maintain the activity of the leech body and to extract and utilize natural hirudin for a long time. At the same time, the whole leech is used as the extraction stock solution. Since there are a large number of "pseudo-hirudin" components similar to hirudin but without biological activity in the leech body, this increases the difficulty of hirudin separation, affects the purification effect, and makes the activity recovery rate of natural hirudin poor. Summary of the invention

[0004] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a natural hirudin extraction method based on cell fermentation to optimize the purification process, improve the purification efficiency, and improve the purity of the extracted natural hirudin.

[0005] A method for extracting natural leech based on cell fermentation comprises the following steps: S1 provided leeches and starved them; S2 takes out the leech, washes it with metaldehyde solution, and then washes it in pure water; S3 induces stimulation to leeches and collects secretions; S4 extracts hirudin from the secretion fluid based on cell fermentation to obtain fermentation fluid; S5 separates and purifies the fermentation broth based on ammonium sulfate graded precipitation and salting out; S6 further separates and purifies the fermentation broth based on ion exchange chromatography.

[0006] As a further improvement of the above scheme, the method of starving leeches is specifically performed as follows: put the leeches into a container containing an appropriate amount of pure water, and place the container in a dry, ventilated and hygienic place with a temperature of 20-30°C for 5-7 days, and avoid contact with sunlight and rain. Considering that leeches have a strong ability to withstand hunger and can go without food for many days or even months after sucking blood, and leeches will only secrete a large amount of secretion liquid during the feeding process, in order to ensure the extraction amount of natural hirudin, the present invention pre-starves the leeches to obtain a secretion liquid containing a large amount of hirudin.

[0007] As a further improvement of the above scheme, the concentration of the metaldehyde solution is 0.1% to 0.5%. Too high a concentration will damage the leeches, while too low a concentration will not achieve a disinfection effect. Therefore, the present invention uses a 0.1% to 0.5% metaldehyde solution to disinfect leeches, thereby achieving the purpose of effectively inactivating various pathogenic microorganisms.

[0008] As a further improvement of the above scheme, the method for inducing stimulation of leeches is specifically performed as follows: S31 Add glutamate, arginine, citric acid and sodium chloride to fresh blood and mix well; S32 selects fresh animal intestines, injects blood into the intestines and ties them tightly; S33: placing leeches near the intestines filled with blood, under the conditions of 20-25°C and pH 7-8, to induce the leeches to suck the blood in the intestines; After S34 induced feeding for 30 to 40 minutes, the environmental temperature was changed to make the leeches spit out saliva, and the secretion fluid was collected.

[0009] As a further improvement of the above scheme, the fresh blood is any one of pig blood, cow blood, sheep blood, chicken blood and donkey blood. The amount of glutamic acid added accounts for 0.5w%~2w% of the fresh blood, and the ratio of glutamic acid, arginine, citric acid and sodium chloride is 1:1~10:1~4:0.4~2. The present invention attracts leeches to eat by adding glutamic acid, arginine and sodium chloride to fresh blood, and citric acid prevents blood from coagulating.

[0010] As a further improvement of the above scheme, the environmental temperature is changed to be instantaneously raised to 40-70° C. or slowly raised to 40-70° C. The present invention achieves the purpose of stimulating the leech to secrete a large amount of saliva by raising the temperature so that the front and rear temperature difference reaches more than 20° C.

[0011] As a further improvement of the above scheme, the cell fermentation is carried out under anaerobic conditions, and the method for extracting hirudin from the secretion fluid by cell fermentation is specifically performed as follows: S41 transfers the secretion fluid to a fermentation tank containing culture medium; S42 is fermented anaerobically for 80-100 hours at a fermentation temperature of 25-35°C to obtain the fermentation liquid which is then filtered.

[0012] As a further improvement of the above scheme, the culture medium has the following components: 100 ml buffer, 12 g peptone, 15 g yeast extract and 6 ml glycerol. The buffer is obtained by mixing 170 mol / L potassium dihydrogen phosphate and 0.72 mol / L potassium dihydrogen phosphate. In the present invention, the buffer plays a role in maintaining the pH value of the culture medium stable.

[0013] As a further improvement of the above scheme, the ammonium sulfate graded precipitation and salting-out uses 35% to 55% saturated (NH4)2SO4. The present invention removes protein from the fermentation broth based on ammonium sulfate graded precipitation and salting-out.

[0014] As a further improvement of the above scheme, the method for separating and purifying the fermentation broth by ammonium sulfate graded precipitation and salting out is specifically performed as follows: S51 Add saturated (NH4)2SO4 to the fermentation broth, and then stir on a magnetic stirrer for 4-6 hours; S52 was centrifuged for 25-30 min, and supernatant A was retained; S53: Continue to add saturated (NH4)2SO4 to the supernatant A, and then place it on a magnetic stirrer and stir for 4-6 hours; Centrifuge S54 for 30-40 min and retain supernatant B.

[0015] As a further improvement of the above scheme, the volume ratio of the fermentation broth and saturated (NH4)2SO4 is 0.5:1; the volume ratio of the supernatant A and saturated (NH4)2SO4 is 0.5:1.

[0016] As a further improvement of the above scheme, the resin used in the ion exchange chromatography method is diethylamino agarose gel CL-6B, the buffer used is 0.025 mol / mL phosphate buffer, the ion elution concentration is 0.2~1.0 mol / LNaC1, the sample volume is 100 mL, and the elution rate is 30 mL / h.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention extracts hirudin from the secretion fluid based on cell fermentation. In the above-mentioned mode, the production of hirudin not only utilizes the components of the leech itself, but also further improves the extraction rate of hirudin through the process of microbial fermentation.

[0018] The invention removes protein from the fermentation broth based on ammonium sulfate graded precipitation and salting out, the protein recovery rate can reach 34.37%, the hirudin recovery rate can reach 69.33%, the hirudin purification multiple is 2.02, the hirudin recovery rate is high, the purification effect is good, and the inconvenience of traditional extraction using a water-acetone mixture is overcome.

[0019] The present invention further separates and purifies the fermentation broth based on ion exchange chromatography, and forms a secondary separation and purification system with ammonium sulfate graded precipitation and salting out, which further improves the recovery rate and purification effect of hirudin. After ion exchange chromatography treatment, the protein recovery rate is 5.07%, the hirudin recovery rate is 77.76%, the hirudin purification factor is 15.34, and the purity of hirudin can reach more than 90% when detected by high performance liquid chromatography. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments. Additional aspects and advantages of the present invention will be given in part in the following description, and will become apparent from the following description, or will be understood through the practice of the present invention. It should be understood that the following description is only used to explain the present invention and is not intended to limit the present invention.

[0021] As used herein, the terms "comprises," "including," "contains," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0022] When an amount, concentration, or other value or parameter is expressed as a range, a preferred range, or a range limited by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted as including a range of "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5" etc. When a numerical range is described in this article, unless otherwise specified, the range is intended to include its end values ​​and all integers and fractions within the range.

[0023] The specific embodiments of the present invention are described in detail below.

[0024] Example 1 This embodiment provides a method for extracting natural hirudin based on cell fermentation, characterized in that it comprises the following steps: S1 provides leeches and starves them. The method of starving leeches is specifically performed as follows: put the leeches into a container containing an appropriate amount of pure water, and place the container in a dry, ventilated and hygienic place at a temperature of 25°C for 6 days, and avoid contact with sunlight and rain. Considering that leeches have a strong ability to withstand hunger and can go without food for many days or even months after sucking blood, and leeches will only secrete a large amount of secretion liquid during the feeding process, in order to ensure the extraction amount of natural hirudin, this embodiment pre-starves the leeches to obtain a secretion liquid containing a large amount of hirudin.

[0025] S2: taking out the leech, washing it with a 0.3% metaldehyde solution, and then washing it in pure water. In this embodiment, a 0.3% metaldehyde solution is used to disinfect the leech, thereby achieving the purpose of effectively inactivating various pathogenic microorganisms.

[0026] S3 induces stimulation to leeches and collects secretions.

[0027] The method for inducing stimulation of leeches is specifically performed as follows: S31: adding glutamic acid, arginine, citric acid and sodium chloride to pig blood and mixing them evenly, wherein the amount of glutamic acid added is 1w% of fresh blood, and the ratio of glutamic acid, arginine, citric acid and sodium chloride is 1:5:2:0.7; in this embodiment, glutamic acid, arginine and sodium chloride are added to pig blood to attract leeches to eat, and citric acid prevents blood coagulation.

[0028] S32 Select fresh animal intestines, inject pig blood into the intestines and tie them tightly.

[0029] S33 placed leeches near intestines filled with pig blood, and induced the leeches to suck the blood in the intestines at 24°C and pH 7.2.

[0030] S34: After inducing feeding for 35 minutes, the ambient temperature is changed to make the leech spit out saliva, and the secretion liquid is collected. The specific method of changing the ambient temperature is to slowly raise the temperature to 50°C. In this embodiment, the temperature difference between the front and the back is raised to more than 20°C, so as to stimulate the leech to secrete a large amount of saliva, thereby overcoming the traditional method of using the whole leech as the extraction stock solution. Since there are a large number of "pseudo-hirudin" components similar to hirudin but without biological activity in the leech body, this increases the difficulty of separating hirudin, affects the purification effect, and makes the activity recovery rate of natural hirudin poor.

[0031] S4 extracts hirudin from the secretion fluid based on cell fermentation to obtain fermentation fluid.

[0032] The cell fermentation is carried out under anaerobic conditions, and the method for extracting hirudin from the secretion fluid by cell fermentation is specifically performed as follows: S41 transfers the secretion liquid to a fermentation tank containing a culture medium. The culture medium has the following components: 100 ml buffer, 12 g peptone, 15 g yeast extract and 6 ml glycerol. The buffer is obtained by mixing 170 mol / L potassium dihydrogen phosphate and 0.72 mol / L potassium dihydrogen phosphate, and the buffer plays a role in maintaining the pH value of the culture medium stable.

[0033] S42 is anaerobically fermented for 90 hours at a fermentation temperature of 30° C., and a fermented liquid is obtained and filtered. In this embodiment, hirudin in the secretion liquid is extracted based on cell fermentation. In the above mode, the production of hirudin not only utilizes the components of the leech itself, but also further improves the extraction rate of hirudin through the process of microbial fermentation.

[0034] S5 is based on ammonium sulfate graded precipitation and salting out to separate and purify the fermentation broth. The present invention removes protein from the fermentation broth based on ammonium sulfate graded precipitation and salting out, and the protein recovery rate can reach 34.37%, the hirudin recovery rate can reach 69.33%, and the hirudin purification multiple is 2.02. The hirudin recovery rate is high and the purification effect is good, and the inconvenience of traditional extraction using a water-acetone mixture is overcome. The method for separating and purifying the fermentation broth by ammonium sulfate graded precipitation and salting out is specifically operated as follows: S51 added 40% saturated (NH4)2SO4 to the fermentation broth, the volume ratio of the fermentation broth to 40% saturated (NH4)2SO4 was 0.5:1, and then placed on a magnetic stirrer and stirred for 5 hours.

[0035] Centrifuge S52 for 25-30 minutes and retain supernatant A.

[0036] S53 continued to add 40% saturated (NH4)2SO4 to the supernatant A, the volume ratio of supernatant A and 40% saturated (NH4)2SO4 was 0.5:1, and then placed on a magnetic stirrer and stirred for 5 hours.

[0037] S54 was centrifuged for 35 min and supernatant B was retained.

[0038] S6 is further separated and purified based on ion exchange chromatography. The resin used in the ion exchange chromatography is diethylamino sepharose CL-6B, the buffer used is 0.025mol / mL phosphate buffer, the ion elution concentration is 0.6mol / L NaCl, the sample load is 100mL, and the elution rate is 30mL / h. In this embodiment, the fermentation broth is further separated and purified based on ion exchange chromatography, and a secondary separation and purification system is formed by ammonium sulfate graded precipitation and salting out, which further improves the recovery rate and purification effect of hirudin. After ion exchange chromatography, the protein recovery rate is 5.07%, the hirudin recovery rate is 77.76%, the hirudin purification multiple is 15.34, and the purity of hirudin can reach more than 90% by high performance liquid chromatography.

[0039] Example 2 This embodiment provides a method for extracting natural hirudin based on cell fermentation, characterized in that it comprises the following steps: S1 provides leeches and starves them. The method of starving leeches is specifically performed as follows: put the leeches into a container containing an appropriate amount of pure water, and place the container in a dry, ventilated and hygienic place at a temperature of 20°C for 5 days, and avoid contact with sunlight and rain. Considering that leeches have a strong ability to withstand hunger, they can go without food for many days or even months after sucking blood, and leeches will only secrete a large amount of secretion liquid during the feeding process. In order to ensure the extraction amount of natural hirudin, this embodiment pre-starves the leeches to obtain a secretion liquid containing a large amount of hirudin.

[0040] S2: taking out the leech, washing it with a 0.1% metaldehyde solution, and then washing it in pure water. In this embodiment, a 0.1% metaldehyde solution is used to disinfect the leech, thereby achieving the purpose of effectively inactivating various pathogenic microorganisms.

[0041] S3 induces stimulation to leeches and collects secretions.

[0042] The method for inducing stimulation of leeches is specifically performed as follows: S31: adding glutamate, arginine, citric acid and sodium chloride to the cow's blood and mixing them evenly, wherein the amount of glutamate added is 0.5w% of the cow's blood, and the ratio of glutamate, arginine, citric acid and sodium chloride is 1:1:1:0.4; in this embodiment, glutamate, arginine and sodium chloride are added to the cow's blood to attract leeches to eat, and citric acid prevents blood coagulation.

[0043] S32 uses fresh animal intestines, injects cow blood into the intestines and ties them tightly.

[0044] S33 places leeches near intestines filled with cow blood, and induces the leeches to suck the blood in the intestines at 20°C and pH 7.

[0045] S34: After inducing feeding for 30 minutes, the ambient temperature is changed to make the leech spit out saliva, and the secretion liquid is collected. The specific change of the ambient temperature is to instantly raise the temperature to 40°C. In this embodiment, the temperature difference between the front and the back is raised to more than 20°C, so as to stimulate the leech to secrete a large amount of saliva, thereby overcoming the traditional method of using the whole leech as the extraction stock solution. Since there are a large number of "pseudo-hirudin" components similar to hirudin but without biological activity in the leech body, this increases the difficulty of hirudin separation, affects the purification effect, and makes the activity recovery rate of natural hirudin poor.

[0046] S4 extracts hirudin from the secretion fluid based on cell fermentation to obtain fermentation fluid.

[0047] The cell fermentation is carried out under anaerobic conditions, and the method for extracting hirudin from the secretion fluid by cell fermentation is specifically performed as follows: S41 transfers the secretion liquid to a fermentation tank containing a culture medium. The culture medium has the following components: 100 ml buffer, 12 g peptone, 15 g yeast extract and 6 ml glycerol. The buffer is obtained by mixing 170 mol / L potassium dihydrogen phosphate and 0.72 mol / L potassium dihydrogen phosphate, and the buffer plays a role in maintaining the pH value of the culture medium stable.

[0048] S42 is anaerobically fermented for 80 hours at a fermentation temperature of 25° C., and a fermentation liquid is obtained and filtered. In this embodiment, hirudin in the secretion liquid is extracted based on cell fermentation. In the above mode, the production of hirudin not only utilizes the components of the leech itself, but also further improves the extraction rate of hirudin through the process of microbial fermentation.

[0049] S5 separates and purifies the fermentation broth based on ammonium sulfate graded precipitation and salting out.

[0050] The method for separating and purifying the fermentation broth by ammonium sulfate graded precipitation and salting out is specifically performed as follows: S51 Add 35% saturated (NH4)2SO4 to the fermentation broth, the volume ratio of the fermentation broth to 35% saturated (NH4)2SO4 is 0.5:1, and then place it on a magnetic stirrer and stir for 4~6h.

[0051] S52 was centrifuged for 25 min and supernatant A was retained.

[0052] S53 continued to add 35% saturated (NH4)2SO4 to the supernatant A, the volume ratio of supernatant A and 35% saturated (NH4)2SO4 was 0.5:1, and then placed on a magnetic stirrer and stirred for 4 hours.

[0053] S54 was centrifuged for 30 min and supernatant B was retained.

[0054] S6 further separates and purifies the fermentation broth based on ion exchange chromatography. The resin used in the ion exchange chromatography is diethylamino sepharose CL-6B, the buffer used is 0.025 mol / mL phosphate buffer, the ion elution concentration is 0.2 mol / L NaCl, the sample volume is 100 mL, and the elution rate is 30 mL / h.

[0055] Example 3 This embodiment provides a method for extracting natural hirudin based on cell fermentation, characterized in that it comprises the following steps: S1 provides leeches and starves them. The method of starving leeches is specifically performed as follows: put the leeches into a container containing an appropriate amount of pure water, and place the container in a dry, ventilated and hygienic place at a temperature of 30°C for 7 days, and avoid contact with sunlight and rain. Considering that leeches have a strong ability to withstand hunger and can go without food for many days or even months after sucking blood, and leeches will only secrete a large amount of secretion liquid during the feeding process, in order to ensure the extraction amount of natural hirudin, this embodiment pre-starves the leeches to obtain a secretion liquid containing a large amount of hirudin.

[0056] S2: taking out the leech, washing it with a 0.5% concentration of metaldehyde solution, and then washing it in pure water. In this embodiment, a 0.5% concentration of metaldehyde solution is used to disinfect the leech, thereby achieving the purpose of effectively inactivating various pathogenic microorganisms.

[0057] S3 induces stimulation to leeches and collects secretions.

[0058] The method for inducing stimulation of leeches is specifically performed as follows: S31: adding glutamate, arginine, citric acid and sodium chloride to chicken blood and mixing them evenly, wherein the amount of glutamate added is 2w% of fresh blood, and the ratio of glutamate, arginine, citric acid and sodium chloride is 1:10:4:2; in this embodiment, glutamate, arginine and sodium chloride are added to chicken blood to attract leeches to eat, and citric acid prevents blood coagulation.

[0059] S32 Select fresh animal intestines, inject chicken blood into the intestines and tie them tightly.

[0060] S33 places leeches near the intestines filled with chicken blood, and induces the leeches to suck the blood in the intestines at 25°C and pH 8.

[0061] S34: After inducing feeding for 40 minutes, the ambient temperature is changed to make the leech spit out saliva, and the secretion liquid is collected. The specific method of changing the ambient temperature is to slowly raise the temperature to 70°C. In this embodiment, the temperature difference between the front and the back is raised to more than 20°C, so as to stimulate the leech to secrete a large amount of saliva, thereby overcoming the traditional method of using the whole leech as the extraction stock solution. Since there are a large number of "pseudo-hirudin" components in the leech body that are similar to hirudin but have no biological activity, this increases the difficulty of separating hirudin, affects the purification effect, and makes the activity recovery rate of natural hirudin poor.

[0062] S4 extracts hirudin from the secretion fluid based on cell fermentation to obtain fermentation fluid.

[0063] The cell fermentation is carried out under anaerobic conditions, and the method for extracting hirudin from the secretion fluid by cell fermentation is specifically performed as follows: S41 transfers the secretion liquid to a fermentation tank containing a culture medium. The culture medium has the following components: 100 ml buffer, 12 g peptone, 15 g yeast extract and 6 ml glycerol. The buffer is obtained by mixing 170 mol / L potassium dihydrogen phosphate and 0.72 mol / L potassium dihydrogen phosphate, and the buffer plays a role in maintaining the pH value of the culture medium stable.

[0064] S42 is anaerobically fermented for 100 hours at a fermentation temperature of 35° C., and a fermentation liquid is obtained and filtered. In this embodiment, hirudin in the secretion liquid is extracted based on cell fermentation. In the above mode, the production of hirudin not only utilizes the components of the leech itself, but also further improves the extraction rate of hirudin through the process of microbial fermentation.

[0065] S5 is to separate and purify the fermentation broth based on ammonium sulfate graded precipitation and salting out. The method of separating and purifying the fermentation broth by ammonium sulfate graded precipitation and salting out is specifically performed as follows: S51 Add 55% saturated (NH4)2SO4 to the fermentation broth, the volume ratio of the fermentation broth to 55% saturated (NH4)2SO4 is 0.5:1, and then place it on a magnetic stirrer and stir for 6 hours.

[0066] Centrifuge S52 for 25-30 minutes and retain supernatant A.

[0067] S53 continued to add 55% saturated (NH4)2SO4 to the supernatant A, the volume ratio of supernatant A and 55% saturated (NH4)2SO4 was 0.5:1, and then placed on a magnetic stirrer and stirred for 6 hours.

[0068] S54 was centrifuged for 40 min and supernatant B was retained.

[0069] S6 further separates and purifies the fermentation broth based on ion exchange chromatography. The resin used in the ion exchange chromatography is diethylamino sepharose CL-6B, the buffer used is 0.025 mol / mL phosphate buffer, the ion elution concentration is 1.0 mol / L NaCl, the sample volume is 100 mL, and the elution rate is 30 mL / h.

[0070] The above embodiments are merely preferred implementations of the present invention. Any simple modification, amendment and substitution of the above embodiments based on the technical essence of the present invention shall fall within the scope of the technical solution of the present invention.

Claims

1. A method for extracting natural hirudin based on cell fermentation, characterized in that: The following steps are involved: S1 provided leeches and starved them; S2 takes out the leech, washes it with metaldehyde solution, and then washes it in pure water; S3 induces stimulation to leeches and collects secretions; S4 extracts hirudin from the secretion fluid based on cell fermentation to obtain fermentation fluid; S5 separates and purifies the fermentation broth based on ammonium sulfate graded precipitation and salting out; S6 further separates and purifies the fermentation broth based on ion exchange chromatography.

2. The method for extracting natural hirudin based on cell fermentation according to claim 1, characterized in that: The method for starving leeches is specifically performed as follows: the leeches are placed in a container containing an appropriate amount of pure water, and the container is placed in a dry, ventilated and sanitary place with a temperature of 20-30° C. for 5-7 days, and is kept away from sunlight and rain.

3. The method for extracting natural hirudin based on cell fermentation according to claim 1, characterized in that: The concentration of the metaldehyde solution is 0.1% to 0.5%.

4. The method for extracting natural hirudin based on cell fermentation according to claim 1, characterized in that: The method for inducing stimulation of leeches is specifically performed as follows: S31 Add glutamate, arginine, citric acid and sodium chloride to fresh blood and mix well; S32 selects fresh animal intestines, injects blood into the intestines and ties them tightly; S33: placing leeches near the intestines filled with blood, under the conditions of 20-25°C and pH 7-8, to induce the leeches to suck the blood in the intestines; After S34 induced feeding for 30 to 40 minutes, the environmental temperature was changed to make the leeches spit out saliva, and the secretion fluid was collected.

5. The method for extracting natural hirudin based on cell fermentation according to claim 4, characterized in that: The fresh blood is any one of pig blood, cow blood, sheep blood, chicken blood and donkey blood; The amount of glutamic acid added is 0.5w%~2w% of the fresh blood, and the ratio of glutamic acid, arginine, citric acid and sodium chloride is 1:1~10:1~4:0.4~2; The changing of the ambient temperature specifically includes instantly raising the temperature to 40-70°C; or slowly raising the temperature to 40-70°C.

6. The method for extracting natural hirudin based on cell fermentation according to claim 1, characterized in that: The cell fermentation is carried out under anaerobic conditions, and the method for extracting hirudin from the secretion fluid by cell fermentation is specifically performed as follows: S41 transfers the secretion fluid to a fermentation tank containing culture medium; S42 is fermented anaerobically for 80-100 hours at a fermentation temperature of 25-35°C to obtain the fermentation liquid which is then filtered.

7. The method for extracting natural hirudin based on cell fermentation according to claim 6, characterized in that: The culture medium has the following components: 100 ml buffer, 12 g peptone, 15 g yeast extract and 6 ml glycerol; The buffer solution is obtained by mixing 170 mol / L potassium dihydrogen phosphate and 0.72 mol / L dipotassium hydrogen phosphate.

8. The method for extracting natural hirudin based on cell fermentation according to claim 1, characterized in that: The ammonium sulfate graded precipitation and salting-out method uses 35% to 55% saturated (NH4)2SO4. The ammonium sulfate graded precipitation and salting-out method is used to separate and purify the fermentation broth. The specific operation is as follows: S51 Add saturated (NH4)2SO4 to the fermentation broth, and then stir on a magnetic stirrer for 4-6 hours; S52 was centrifuged for 25-30 min, and supernatant A was retained; S53: Continue to add saturated (NH4)2SO4 to the supernatant A, and then place it on a magnetic stirrer and stir for 4-6 hours; Centrifuge S54 for 30-40 min and retain supernatant B.

9. The method for extracting natural hirudin based on cell fermentation according to claim 8, characterized in that: The volume ratio of fermentation broth and saturated (NH4)2SO4 was 0.5:1; The volume ratio of supernatant A and saturated (NH4)2SO4 was 0.5:

1.

10. The method for extracting natural hirudin based on cell fermentation according to claim 1, characterized in that: The resin used in the ion exchange chromatography method is diethylamino agarose gel CL-6B, the buffer used is 0.025 mol / mL phosphate buffer, the ion elution concentration is 0.2-1.0 mol / L NaCl, the sample volume is 100 mL, and the elution rate is 30 mL / h.