A method for efficient extraction and full resource utilization of lumbrokinase
Through potassium bicarbonate-ammonium acetate combined with ethanol salting method and buffer solution treatment, the problems of environmental pollution and high cost during the extraction process of lutekinase in the prior art are solved, efficient extraction and waste liquid resource utilization are achieved, and green A new path to environmental protection.
Patent Information
- Application Number
- CN202211653322.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-12-22
- Publication Date
- 2025-01-03
- Estimated Expiration
- 2042-12-22
AI Technical Summary
The existing lumonkinase extraction methods have problems of environmental pollution and high costs, and cannot effectively recycle and utilize waste liquid during the extraction process.
The potassium bicarbonate-ammonium acetate combined with ethanol salting method was used to extract lumonkinase, and efficient extraction was achieved through buffer solution, membrane filtration, salting and protein purification steps, and the waste liquid was converted into ecological fertilizer to achieve full resource recycling.
It realizes efficient extraction of lutekinase and resource utilization of waste liquid, reduces production costs, provides a new path to green and environmental protection, and avoids "zero" emissions.
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biological extraction, and in particular to a method for efficiently extracting and fully utilizing lumbrokinase. Background Art
[0002] Earthworms are one of the largest common groups of soil animals. They play multiple roles in the ecosystem, both as "consumers" and "creators". They contribute to the material cycle and energy transfer of soil processes through activities such as feeding, digestion, excretion (worm castings), secretion (mucus) and burrowing, and can have an important impact on multiple processes that determine soil fertility. Earthworms are high-protein feeds, and earthworm castings are organic fertilizer raw materials with good economic value. Earthworms are also Chinese medicinal materials with great medicinal value. The lumbrokinase in earthworms has an anti-thrombotic effect, can track thrombolysis, effectively dissolve microthrombi, improve microcirculation, strengthen collateral circulation of heart and brain vessels, openly repair damaged endothelial cells in blood vessels, increase vascular elasticity, improve vascular oxygen supply function, reduce blood viscosity, reduce platelet aggregation rate, and inhibit the re-formation of thrombi. Repair necrotic brain cells around thrombi after thrombus formation and save the penumbra.
[0003] At present, the existing lumbrokinase extraction methods include: NH 4 The crude enzyme solution is obtained by homogenization with Ac-HAc buffer, phosphate buffer or Tris-HCl buffer, ammonium sulfate salting out, ion exchange chromatography or dialysis. However, the buffer and salting out solution used in this technology cannot be recycled or reused, which brings risks to the environment and also increases the cost of lumbrokinase extraction. Summary of the invention
[0004] The purpose of the present invention is to provide a method for efficiently extracting and fully recycling lumbrokinase, which can achieve efficient extraction, separation and full resource recycling of lumbrokinase with low cost.
[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0006] The present invention provides a method for efficiently extracting and fully utilizing lumbrokinase, comprising the following steps:
[0007] After soaking the earthworms in the first buffer solution, homogenizing the earthworms, and filtering the homogenized material through a membrane to obtain a protein solution;
[0008] The protein solution is mixed with ethanol and subjected to salting out to obtain a supernatant and a salting out product;
[0009] The salting out product is mixed with a second buffer solution, and the obtained mixed solution is subjected to protein purification and freeze-drying to obtain lumbrokinase;
[0010] After the supernatant is subjected to reduced pressure distillation, the obtained residual liquid is mixed with the earthworm residue obtained by homogenization, and then fermented to obtain ecological fertilizer;
[0011] The first buffer solution and the second buffer solution are both potassium bicarbonate-ammonium acetate mixed solutions;
[0012] The pH values of the first buffer solution and the second buffer solution are independently 7.5-8.0.
[0013] Preferably, the pH values of the first buffer solution and the second buffer solution are independently 7.8.
[0014] Preferably, the soaking time is 30 to 60 minutes.
[0015] Preferably, the homogenization speed is 20000-25000 rpm, and the homogenization time is 3-5 min.
[0016] Preferably, the molecular weight cutoff of the membrane used for the membrane filtration is 50KD, and the molecular weight of the protein in the protein solution is ≤50KD.
[0017] Preferably, the volume ratio of the ethanol to the protein solution is (0.5-3):1.
[0018] Preferably, the mass ratio of the second buffer solution to the salting-out product is (10-100):1.
[0019] Preferably, the temperature of the reduced pressure distillation is 50-60°C.
[0020] Preferably, the fermentation temperature is 30-35° C. and the fermentation time is 10-15 days.
[0021] Preferably, the fermentation is carried out under anaerobic closed conditions.
[0022] The invention provides a method for efficient extraction and full resource utilization of lumbrokinase, comprising the following steps: soaking earthworms in a first buffer solution, homogenizing, and filtering the obtained homogenized material through a membrane to obtain a protein solution; mixing the protein solution with ethanol, performing salting out, and obtaining a supernatant and a salting-out product; mixing the salting-out product with a second buffer solution, purifying the obtained mixed solution through protein purification, and freeze-drying to obtain lumbrokinase; performing reduced pressure distillation on the supernatant, mixing the obtained residual solution with earthworm residues obtained through homogenization, and fermenting to obtain an ecological fertilizer; the first buffer solution and the second buffer solution are both potassium bicarbonate-ammonium acetate mixed solutions, and the pH values of the first buffer solution and the second buffer solution are independently 7.5-8.0. The present invention adopts a potassium bicarbonate-ammonium acetate combined with ethanol salting-out method to extract lumbrokinase protein. The pH condition of 7.5-8.0 provided by the potassium bicarbonate and ammonium acetate buffer solution can maintain the high activity of the lumbrokinase protein and improve the extraction rate. Ethanol can reduce the electrolytic constant of the solution, thereby increasing the attraction of different charges on the protein molecules, resulting in a decrease in solubility. The action of ethanol and water can destroy the hydration membrane of the protein. The protein is separated and precipitated in ethanol due to the difference in solubility, thereby realizing efficient extraction and separation of lumbrokinase. In addition, the present invention converts the waste liquid obtained after the lumbrokinase extraction into liquid fertilizer, comprehensively utilizes the residual substances, solves the problem of resource utilization of the waste liquid, and realizes the green environmental protection and full resource recycling of the earthworm extraction process.
[0023] The present invention uses buffer solution dissolution, membrane filtration, salting out and protein purification steps to achieve the extraction of high-quality lumbrokinase; the waste residue and waste solution generated during the extraction process can be processed into ecological fertilizers after deep treatment, thereby achieving "zero" emissions in the entire earthworm extraction process, providing a new green path for the production of lumbrokinase. Compared with the prior art, the method of the present invention can achieve the recovery and resource utilization of the extraction waste liquid, has the advantages of simple method, easy operation, low cost, etc., and has significant economic and environmental benefits. DETAILED DESCRIPTION
[0024] The present invention provides a method for efficiently extracting and fully utilizing lumbrokinase, comprising the following steps:
[0025] After soaking the earthworms in the first buffer solution, homogenizing the earthworms, and filtering the homogenized material through a membrane to obtain a protein solution;
[0026] The protein solution is mixed with ethanol and subjected to salting out to obtain a supernatant and a salting out product;
[0027] The salting out product is mixed with a second buffer solution, and the obtained mixed solution is subjected to protein purification and freeze-drying to obtain lumbrokinase;
[0028] After the supernatant is subjected to reduced pressure distillation, the obtained residual liquid is mixed with the earthworm residue obtained by homogenization, and then fermented to obtain ecological fertilizer;
[0029] The first buffer solution and the second buffer solution are both potassium bicarbonate-ammonium acetate mixed solutions;
[0030] The pH values of the first buffer solution and the second buffer solution are independently 7.5-8.0.
[0031] In the present invention, unless otherwise specified, the required materials or reagents are commercially available products well known to those skilled in the art.
[0032] The invention soaks earthworms in a first buffer solution, then homogenizes the earthworms, and performs membrane filtration on the homogenized material to obtain a protein solution.
[0033] The present invention has no particular limitation on the source of the earthworms, and the earthworms can be obtained in a manner well known in the art.
[0034] In the present invention, the first buffer solution is a potassium bicarbonate-ammonium acetate mixed solution, and the pH value of the first buffer solution is 7.5-8.0, preferably 7.8. The present invention has no particular limitation on the amount of the first buffer solution, and it only needs to be able to completely immerse the corresponding earthworms.
[0035] In the present invention, the soaking time is preferably 30 to 60 minutes, more preferably 40 to 50 minutes.
[0036] After the soaking is completed, the present invention homogenizes the earthworms with a homogenizer; the speed of the homogenization is preferably 20000-25000 rpm, more preferably 23000 rpm; the time is preferably 3-5 minutes, more preferably 4 minutes. The present invention has no special limitation on the homogenizer, and any corresponding equipment known in the art can be used.
[0037] After completing the homogenization, the present invention preferably centrifuges the obtained material, takes the supernatant and transfers it to a suction filtration bottle for suction filtration, and retains the obtained earthworm residue (insoluble earthworm residue produced by centrifugal separation); the solution (uniform slurry) obtained after suction filtration is subjected to membrane filtration. In the present invention, the molecular weight cutoff of the membrane used for the membrane filtration is preferably 50KD, and the molecular weight of the protein in the protein solution is preferably ≤50KD. The membrane used for the membrane filtration membrane is preferably a flat membrane, and the protein separation equipment used for the membrane filtration is from Qingdao Fulham Company; the present invention uses membrane filtration to retain proteins with a molecular weight above 50KD and flow out proteins below 50KD to separate and form a protein solution. The present invention has no special limitation on the flat membrane, as long as it can achieve the above-mentioned retention effect.
[0038] After obtaining the protein solution, the present invention mixes the protein solution with ethanol and performs salting out to obtain a supernatant and a salting out product.
[0039] In the present invention, the ethanol is preferably anhydrous ethanol; the volume ratio of the ethanol to the protein solution is preferably (0.5-3):1, more preferably (1.5-2):1. In the present invention, the protein in the protein solution is precipitated by salting out, and the obtained salting out includes lumbrokinase and other proteins, and the lumbrokinase is extracted by subsequent protein purification.
[0040] After obtaining the salting-out product, the present invention mixes the salting-out product with a second buffer solution, purifies the obtained mixed solution through protein purification, and then freeze-dries it to obtain lumbrokinase.
[0041] In the present invention, the second buffer solution is a potassium bicarbonate-ammonium acetate mixed solution, and the pH value of the second buffer solution is 7.5-8.0, preferably 7.8; the mass ratio of the second buffer solution to the salting out product is preferably (10-100):1, more preferably (10-50):1.
[0042] In the present invention, the protein purification is preferably carried out in a protein purification device. The present invention has no special limitation on the protein purification device, and any corresponding equipment known in the art can be used. In the embodiment of the present invention, the protein purification device used is a protein purification device manufactured by Shanghai Shanpu. The present invention separates proteins with a molecular weight greater than 20KD through a protein purification device. Lumbrokinase is a complex protein product with a molecular weight range of 20 to 45KD. The protein with a molecular weight greater than 20KD extracted from earthworms in the present invention is lumbrokinase. The present invention has no special limitation on the specific conditions for the protein purification, and it can be carried out according to the process known in the art. The present invention preferably uses a separation column to obtain a protein of the desired molecular weight, and the separation column is preferably Sephadex G-100 or Sephadex G-50.
[0043] After the protein purification is completed, the present invention freeze-dries the obtained lumbrokinase solution to obtain lumbrokinase; the present invention has no special limitation on the freeze-drying, and the process can be carried out according to a process well known in the art.
[0044] After obtaining the supernatant, the present invention performs vacuum distillation on the supernatant, mixes the obtained residual liquid with the earthworm residue obtained by homogenization, and then ferments to obtain ecological fertilizer. In the present invention, the supernatant includes water, ethanol and protein that cannot be salted out.
[0045] In the present invention, the temperature of the reduced pressure distillation is preferably 50 to 60° C., more preferably 55 to 57° C. The present invention recovers anhydrous ethanol while performing reduced pressure distillation.
[0046] After completing the reduced pressure distillation, the present invention mixes the obtained residual liquid (protein solution with a molecular weight greater than 50KD retained by membrane filtration) with the earthworm residue obtained by homogenization, and puts them into a sealed container; maintains the sealed container at normal pressure, releases the pressure through a pressure reducing valve, and ferments to obtain ecological fertilizer.
[0047] In the present invention, the sealed container is preferably a fermentation tank; the fermentation temperature is preferably 30-35°C, more preferably 33°C; the fermentation time is preferably 10-15 days, more preferably 12 days; and the fermentation is preferably carried out under anaerobic sealed conditions.
[0048] In the fermentation process, under anaerobic conditions, the earthworm residues and the protein solution in the residual liquid are fermented to form enzymes.
[0049] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0050] In the following examples, the molecular weight cut-off of the flat membrane used for membrane filtration is 50KD, and the protein separation equipment used is from Qingdao Fulham Company;
[0051] The protein purification device used was manufactured by Shanghai Shanpu, and the separation column was Sephadex G-50.
[0052] Example 1
[0053] The earthworms were soaked in potassium bicarbonate-ammonium acetate buffer solution with pH = 7.8 for 40 minutes, and then homogenized with a homogenizer at 25000 rpm for 4 minutes;
[0054] The homogenized solution is centrifuged, the supernatant is transferred to a suction filtration bottle for suction filtration, and the earthworm residue is retained;
[0055] The filtered solution is subjected to membrane filtration to obtain a protein solution with a protein molecular weight of less than 50 KD;
[0056] The protein solution is mixed with anhydrous ethanol in a volume ratio of 1:2 to perform salting out to obtain a salting out substance and a supernatant;
[0057] The obtained salting out product is dissolved in a potassium bicarbonate-ammonium acetate buffer solution with a pH value of 7.8 at a mass ratio of 1:10; the dissolved solution is separated into proteins with a molecular weight greater than 20 KD by a protein purification device, and the obtained lumbrokinase solution is freeze-dried to obtain lumbrokinase powder;
[0058] The supernatant obtained after salting out is subjected to reduced pressure distillation at 55°C to recover anhydrous ethanol, and the residual liquid obtained is mixed with the earthworm residue after filtration and sent to a fermentation tank for anaerobic closed fermentation; the fermentation tank is maintained at normal pressure, the pressure is released through a pressure reducing valve, and fermented at 35°C for 10 days to obtain ecological fertilizer.
[0059] Example 2
[0060] The earthworms were soaked in potassium bicarbonate-ammonium acetate buffer solution with pH = 7.8 for 50 minutes, and then homogenized with a homogenizer at 20000 rpm for 5 minutes;
[0061] The homogenized solution is centrifuged, the supernatant is transferred to a suction filtration bottle for suction filtration, and the earthworm residue is retained;
[0062] The filtered solution is subjected to membrane filtration to obtain a protein solution with a protein molecular weight of less than 50 KD;
[0063] The protein solution is mixed with anhydrous ethanol at a volume ratio of 1:1.5, and salting out is performed to obtain a salting out product and a supernatant;
[0064] The obtained salting out product was dissolved in a potassium bicarbonate-ammonium acetate buffer solution with a pH value of 7.7 at a mass ratio of 1:100; the dissolved solution was separated into proteins with a molecular weight greater than 20KD by a protein purification device, and the obtained lumbrokinase solution was freeze-dried to obtain lumbrokinase powder;
[0065] The supernatant obtained after salting out is subjected to reduced pressure distillation at 60°C to recover anhydrous ethanol, and the residual liquid obtained is mixed with the earthworm residue after filtration and sent to a fermentation tank for anaerobic closed fermentation; the fermentation tank is maintained at normal pressure, the pressure is released through a pressure reducing valve, and fermented at 30°C for 15 days to obtain ecological fertilizer.
[0066] Example 3
[0067] The earthworms were soaked in potassium bicarbonate-ammonium acetate buffer solution with pH = 7.8 for 30 minutes, and then homogenized with a homogenizer at 23000 rpm for 3 minutes;
[0068] The homogenized solution is centrifuged, the supernatant is transferred to a suction filtration bottle for suction filtration, and the earthworm residue is retained;
[0069] The filtered solution is subjected to membrane filtration to obtain a protein solution with a protein molecular weight of less than 50 KD;
[0070] The protein solution is mixed with anhydrous ethanol in a volume ratio of 1:3 to perform salting out to obtain a salting out substance and a supernatant;
[0071] The obtained salting out product was dissolved in a potassium bicarbonate-ammonium acetate buffer solution with a pH value of 7.9 at a mass ratio of 1:50; the dissolved solution was separated into proteins with a molecular weight greater than 20 KD by a protein purification device, and the obtained lumbrokinase solution was freeze-dried to obtain lumbrokinase powder;
[0072] The supernatant obtained after salting out is subjected to reduced pressure distillation at 57°C to recover anhydrous ethanol, and the residual liquid obtained is mixed with the earthworm residue after filtration and sent to a fermentation tank for anaerobic closed fermentation; the fermentation tank is maintained at normal pressure, the pressure is released through a pressure reducing valve, and fermented at 33°C for 12 days to obtain ecological fertilizer.
[0073] Lumbrokinase activity test
[0074] 1 mL of the lumbrokinase solution obtained in Examples 1 to 3 was added to a clean glass dish, and then the fibrinogen solution kept at 40°C was mixed with the melted agarose solution, and then quickly poured into the dish with lumbrokinase added and shaken to mix, and placed at room temperature for 1 hour. According to the conventional method, 10 μL of lumbrokinase standard solution and sample solution of different concentrations were set, and they were respectively spotted in the same dish, covered, and placed in a 37°C constant temperature box for 18 hours. After taking out, use a vernier caliper to measure the two mutually perpendicular diameters of the lysis circle, and use the number of lumbrokinase standard units as the horizontal coordinate and the product of the two perpendicular diameters as the vertical coordinate to draw a standard curve and calculate the regression equation. Substitute the product of the two perpendicular diameters of the sample lumbrokinase solution into the regression equation to calculate the number of sample potency units.
[0075] The results showed that the lumbrokinase activity obtained in Example 1 was 30,000 IU / mg, the lumbrokinase activity obtained in Example 2 was 24,000 IU / mg, and the lumbrokinase activity obtained in Example 3 was 27,000 IU / mg.
[0076] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A method for efficient extraction and full resource utilization of lumbrokinase, characterized in that: The following steps are involved: After soaking the earthworms in the first buffer solution, homogenizing the earthworms, and filtering the homogenized material through a membrane to obtain a protein solution; The protein solution is mixed with ethanol and subjected to salting out to obtain a supernatant and a salting out product; The salting out product is mixed with a second buffer solution, and the obtained mixed solution is subjected to protein purification and freeze-drying to obtain lumbrokinase; After the supernatant is subjected to reduced pressure distillation, the obtained residual liquid is mixed with the earthworm residue obtained by homogenization, and then fermented to obtain ecological fertilizer; The first buffer solution and the second buffer solution are both potassium bicarbonate-ammonium acetate mixed solutions; The pH values of the first buffer solution and the second buffer solution are independently 7.5-8.
0.
2. The method for efficient extraction and full resource utilization of lumbrokinase according to claim 1, characterized in that: The pH value of the first buffer solution and the second buffer solution is 7.
8.
3. The method for efficient extraction and full resource utilization of lumbrokinase according to claim 1, characterized in that: The soaking time is 30 to 60 minutes.
4. The method for efficient extraction and full resource utilization of lumbrokinase according to claim 1, characterized in that: The rotation speed of the homogenization is 20000-25000 rpm, and the time is 3-5 minutes.
5. The method for efficient extraction and full resource utilization of lumbrokinase according to claim 1, characterized in that: The molecular weight cutoff of the membrane used for the membrane filtration is 50KD, and the molecular weight of the protein in the protein solution is ≤50KD.
6. The method for efficient extraction and full resource utilization of lumbrokinase according to claim 1 or 5, characterized in that: The volume ratio of the ethanol to the protein solution is (0.5-3):
1.
7. The method for efficient extraction and full resource utilization of lumbrokinase according to claim 1, characterized in that: The mass ratio of the second buffer solution to the salting-out product is (10-100):
1.
8. The method for efficient extraction and full resource utilization of lumbrokinase according to claim 1, characterized in that: The temperature of the reduced pressure distillation is 50-60°C.
9. The method for efficient extraction and full resource utilization of lumbrokinase according to claim 1, characterized in that: The fermentation temperature is 30-35°C and the fermentation time is 10-15 days.
10. The method for efficient extraction and full resource utilization of lumbrokinase according to claim 9, characterized in that: The fermentation is carried out under anaerobic closed conditions.