Method for improving extraction efficiency of heparin sodium by using supercritical fluid extraction technology

Abstract

The invention provides a method for improving the extraction efficiency of heparin sodium by using a supercritical fluid extraction technology, relates to the technical field of polysaccharide preparation, and belongs to the IPC classification number C08B37. The invention provides a method for improving the extraction efficiency of heparin sodium by using a supercritical fluid extraction technology, which comprises the following steps: drying mucous membranes separated from fresh small intestines of pigs, carrying out superfine grinding to obtain mucous membrane powder, adding a sodium chloride solution for soaking, carrying out enzymolysis by using a supercritical compound enzyme, mixing enzymatic hydrolysate with an adsorbent, standing, carrying out alcohol precipitation on supernate, and collecting precipitate, thereby obtaining the heparin sodium. Heparin sodium is obtained. The porcine small intestinal mucosa is subjected to superfine grinding, dissolution of intracellular soluble substances is facilitated, and the reaction time is shortened; a supercritical and complex enzyme enzymolysis mode is adopted, so that sufficient enzymolysis reaction is promoted, and heparin sodium is promoted to be effectively dissociated from protein; protein can be further separated by adding the adsorbent, and the extraction efficiency of the heparin sodium is improved. All the steps have a synergistic effect, and the titer and the yield of the heparin sodium are improved.

Description

Technical Field

[0001] The invention relates to the technical field of polysaccharide preparation, belongs to IPC classification number C08B37, and specifically relates to a method for improving the extraction efficiency of heparin sodium by utilizing supercritical fluid extraction technology. Background Art

[0002] In the food processing industry, the preparation of pig casings involves discarding components such as mucosa, which become waste byproducts. However, from the perspective of biopharmaceuticals, intestinal mucosa is actually a very valuable raw material. It is a key source for extracting sodium heparin. As an anticoagulant drug with extensive and important clinical application value, sodium heparin occupies an indispensable position in the field of modern medicine. It can effectively prevent blood coagulation and plays a key role in the prevention and treatment of thrombotic diseases. It is of great significance to protect the life and health of patients and the smooth progress of medical operations such as surgery.

[0003] At present, common methods for extracting sodium heparin mainly include salting-ion exchange method, enzymatic hydrolysis-ion exchange method, etc. These methods are usually based on the solubility and ionic properties of sodium heparin in a specific solution environment to achieve its separation and extraction from raw materials such as animal tissues. For example, the salting-ion exchange method first uses a high-concentration sodium chloride solution to destroy the tissue cell structure, release sodium heparin into the solution, and then adsorbs and elutes sodium heparin through ion exchange resin to achieve the purpose of separation and purification. Traditional extraction methods usually use a large amount of chemical reagents, such as strong acids, strong bases, and organic solvents. If these chemical reagents are not handled properly after the extraction is completed, they will cause great pollution to the environment. For example, the discharge of a large amount of acidic or alkaline wastewater will cause the pH value of the water body to be unbalanced, affecting the living environment of aquatic organisms; the volatilization of organic solvents will pollute the atmospheric environment, and the recovery and treatment cost of organic solvents is high. If they are not handled properly, there will be safety hazards. In addition, traditional extraction methods often require multiple precipitation, centrifugation, elution and other operations, and the entire extraction process takes a long time. In these processes, the transfer of sodium heparin between steps and changes in processing conditions may lead to the loss of some sodium heparin, making the actual amount of sodium heparin extracted from the raw material relatively low, and the extraction efficiency difficult to be effectively improved. For example, during multiple precipitation processes, due to slight differences in precipitation conditions, some sodium heparin may not be completely precipitated and remain in the solution and be discarded, resulting in losses.

[0004] In order to overcome the many disadvantages of traditional heparin sodium extraction methods and give full play to the advantages of supercritical fluid extraction technology in improving extraction efficiency, improving product quality and environmental friendliness, it is of great practical significance and broad application prospects to develop a method for improving the extraction efficiency of heparin sodium using supercritical fluid extraction technology. Summary of the invention

[0005] The object of the present invention is to provide a method for improving the extraction efficiency of heparin sodium by using supercritical fluid extraction technology, so as to solve the problems of low extraction rate and low purity in the prior art.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a method for improving the extraction efficiency of sodium heparin by using supercritical fluid extraction technology, comprising: drying and ultrafinely grinding mucosa separated from fresh pig small intestine to obtain mucosa powder, soaking in sodium chloride solution, performing supercritical combined enzyme hydrolysis, mixing the hydrolyzate with an adsorbent, standing, precipitating the supernatant with alcohol, collecting the precipitate, and obtaining sodium heparin; the supercritical fluid for supercritical combined enzyme hydrolysis is CO2, the pressure is 25-35MPa, the temperature is 35-40℃, the pH value is 7-8, the CO2 flow rate is 10-15kg / h, and the time is 45-60min.

[0008] Preferably, the drying temperature is 30-40°C.

[0009] Preferably, the particle size of the ultrafine pulverization is 1000-1200 mesh.

[0010] Preferably, the concentration of the sodium chloride solution is 3-5 g / L.

[0011] Preferably, the weight ratio of the mucosal powder to the sodium chloride solution is 1:8-12, and the soaking time is 1-3 hours.

[0012] The complex enzyme is composed of serrapeptase, trypsin and pancreatic lipase in a weight ratio of 1:(2-5):(3-8); the added amount of the complex enzyme is 2‰-6‰ of the weight of the mucosal powder.

[0013] Preferably, the adsorbent is one or both of polyferric sulfate and polyaluminium chloride; the adsorbent accounts for 2%-5% of the weight of the enzymatic hydrolysate.

[0014] Preferably, the mixing and standing comprises: mixing the enzymatic hydrolyzate with the adsorbent, stirring at 300-400 r / min for 2-5 min, and standing for 2-4 h.

[0015] Preferably, the alcohol precipitation comprises: mixing the supernatant with an ethanol solution, letting it stand for 4-6 hours, and collecting the precipitate.

[0016] More preferably, the volume percentage of the ethanol solution is 80%-90%.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention provides a method for improving the extraction efficiency of sodium heparin by using supercritical fluid extraction technology, comprising: drying and ultrafine grinding the mucosa separated from fresh pig small intestine to obtain mucosa powder, soaking in sodium chloride solution, enzymolysis by supercritical and composite enzyme, mixing the enzymolysis solution with an adsorbent, standing, precipitating the supernatant with alcohol, collecting the precipitate, and obtaining sodium heparin. The present invention ultrafinely grinds the pig small intestine mucosa to break the mucosal cells, which is conducive to the dissolution of intracellular solubles and shortens the reaction time; after soaking in sodium chloride solution, supercritical + composite enzyme enzymolysis is adopted. On the one hand, supercritical pressure promotes the full reaction of the composite enzyme with the substrate, so that the protein and fat are decomposed into small molecules, and supercritical extraction can remove fat and part of the protein. On the other hand, under the condition of reducing the concentration of sodium chloride solution, it can also promote the effective dissociation of sodium heparin from protein; adding an adsorbent to the extraction solution can further separate the protein and improve the purity and extraction efficiency of sodium heparin. The present invention is carried out at low temperature to avoid the degradation of sodium heparin, and each step has a synergistic effect, which improves the titer and yield of sodium heparin.

[0019] The invention rationally utilizes the waste product mucosa in the preparation process of pig casings to prepare heparin sodium, thereby realizing efficient utilization of resources. DETAILED DESCRIPTION

[0020] The invention provides a method for improving the extraction efficiency of sodium heparin by using supercritical fluid extraction technology, comprising: drying and ultrafinely grinding mucosa separated from fresh pig small intestine to obtain mucosa powder, soaking in sodium chloride solution, performing supercritical combined enzyme enzymolysis, mixing the enzymolysis solution with an adsorbent, standing, precipitating the supernatant with alcohol, collecting the precipitate, and obtaining sodium heparin; the supercritical fluid for supercritical combined enzyme enzymolysis is CO2, the pressure is 25-35MPa, the temperature is 35-40℃, the pH value is 7-8, the CO2 flow rate is 10-15kg / h, and the time is 45-60min.

[0021] The drying temperature of the present invention is preferably 30-40° C., more preferably 35° C., until the moisture content of the mucous membrane reaches 3%-5%.

[0022] The particle size of the ultrafine grinding of the present invention is preferably 1000-1200 mesh, more preferably 1100 mesh. Ultrafine grinding can effectively destroy the cell structure of the pig small intestinal mucosa and various organelle membranes in the cell, so that the heparin sodium originally wrapped in the cell can be fully exposed and released, so that more heparin sodium can participate in the subsequent extraction process, and the pig small intestinal mucosa after ultrafine grinding has a greatly increased surface area, and the contact area with the extraction solvent is also correspondingly increased, so that the heparin sodium molecules are more easily dissolved in the sodium chloride solution. Under the same extraction time and conditions, more heparin sodium can be extracted, thereby improving the efficiency of the entire extraction process.

[0023] The concentration of the sodium chloride solution of the present invention is preferably 3-5 g / L, more preferably 4 g / L; the weight ratio of the mucosal powder to the sodium chloride solution is preferably 1:8-12, more preferably 1:10, and the immersion time is preferably 1-3 h, more preferably 2 h.

[0024] Heparin sodium is a negatively charged polysaccharide sulfate that has good solubility in sodium chloride solution of appropriate concentration. Salt ions can interact with the charges on the heparin sodium molecules, weakening the mutual attraction between the heparin sodium molecules, making it easier to disperse in the solution, and weakening the binding of heparin sodium to proteins.

[0025] The composite enzyme of the present invention is preferably composed of serrapeptase, trypsin and pancreatic lipase in a weight ratio of 1:(2-5):(3-8), more preferably 1:3:5; the added amount of the composite enzyme is preferably 2‰-6‰ of the weight of the mucosal powder, more preferably 4‰. The enzyme activity of the serrapeptase is preferably 100,000-200,000 U / g, more preferably 150,000 U / g, the enzyme activity of the trypsin is preferably 80,000-140,000 U / g, more preferably 100,000 U / g, and the enzyme activity of the pancreatic lipase is preferably 50,000-100,000 U / g, more preferably 80,000 U / g. The serrapeptase of the invention can decompose various proteins in the extracellular matrix and glycoproteins on the cell membrane, while trypsin can specifically hydrolyze peptide bonds and mainly act on proteins in the cell, such as cytoskeleton proteins, and the synergistic effect of the two can destroy the cell structure from the inside out and comprehensively, further weaken the combination of heparin sodium and protein, and separate the heparin sodium; the pancreatic lipase decomposes fat to weaken the connection between cells and destroy the lipid bilayer structure of the cell membrane, cooperates with serrapeptase and trypsin to decompose protein, accelerates the destruction of the cell structure, and makes the release of heparin sodium more efficient.

[0026] The present invention adopts a supercritical + complex enzyme enzymolysis method. On the one hand, supercritical pressure promotes the full reaction of the complex enzyme with the substrate, so that the protein and fat are decomposed into small molecules, and the fat and part of the protein are removed by supercritical extraction. On the other hand, under the condition of reducing the concentration of the sodium chloride solution, it can also promote the effective dissociation of sodium heparin from the protein.

[0027] The adsorbent of the present invention is preferably one or both of polyferric sulfate and polyaluminium chloride; the adsorbent is preferably 2%-5% by weight of the enzymatic solution, more preferably 3%. The mixing and standing preferably include: mixing the enzymatic solution with the adsorbent, stirring at 300-400 r / min for 2-5 min, and standing for 2-4 h.

[0028] The polyferric sulfate and polyaluminium chloride of the present invention can neutralize the charge of proteins in a wider pH range, reduce the electrostatic repulsion between protein molecules, and promote the proteins to approach and aggregate. The ferric hydroxide colloid produced by the hydrolysis of polyferric sulfate and the aluminium hydroxide colloid produced by the hydrolysis of polyaluminium chloride are intertwined in the solution to form flocs that are more stable and have a tighter structure, so that the proteins in the enzymatic hydrolysate are adsorbed and precipitated, thereby improving the extraction effect of heparin sodium.

[0029] The alcohol precipitation of the present invention preferably comprises: mixing the supernatant with an ethanol solution, standing for 4-6 hours, and collecting the precipitate, wherein the volume percentage of the ethanol solution is preferably 80%-90%, more preferably 85%. The present invention precipitates heparin sodium by the alcohol precipitation method, thereby further improving the purity of heparin sodium.

[0030] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0031] 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.

[0032] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0033] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0034] The polyacrylamide described in the present invention is anionic polyacrylamide, which was purchased from Gongyi Xinqi Polymer Co., Ltd. Polyaluminium chloride was purchased from Henan Shuanglong Water Treatment Materials Co., Ltd.

[0035] Example 1

[0036] (1) drying the mucosa separated from fresh porcine small intestine at 35° C. to a moisture content of 3%, ultrafinely grinding the mucosa powder, and passing the powder through a 1100-mesh sieve to obtain mucosa powder; mixing 100 parts by weight of the mucosa powder with 1000 parts by weight of a 4 g / L sodium chloride solution and soaking the mixture for 2 hours to obtain a soaking material;

[0037] (2) mixing the soaking material (mucous membrane powder and sodium chloride solution) with 0.4 parts by weight of the complex enzyme and placing it in an extraction kettle; injecting supercritical CO2 into the extraction kettle, extracting for 50 minutes under the conditions of pressure 30 MPa, temperature 37° C., pH 7.5, and CO2 flow rate 13 kg / h to obtain an extract;

[0038] The complex enzyme is composed of serrapeptase, trypsin and pancreatic lipase in a weight ratio of 1:3:5, the enzyme activity of serrapeptase is 150,000 U / g, the enzyme activity of trypsin is 100,000 U / g, and the enzyme activity of pancreatic lipase is 80,000 U / g;

[0039] (3) Mix the enzymatic hydrolyzate with the adsorbent, stir at 350 r / min for 3 min, let stand for 3 h, and remove the precipitate;

[0040] The adsorbent is composed of polyferric sulfate and polyaluminium chloride in a weight ratio of 1:1, and the amount of the adsorbent is 3% of the weight of the enzymatic hydrolysate;

[0041] (4) The supernatant was mixed with an 85% by volume ethanol solution at a volume ratio of 1:1, and the mixture was allowed to stand for 5 hours. The precipitate was collected and dried at 35° C. to a water content of 5%, thereby obtaining heparin sodium.

[0042] Example 2

[0043] (1) The mucosa separated from fresh pig small intestine is dried at 30° C. to a water content of 5%, ultrafinely ground, and passed through a 1000-mesh sieve to obtain mucosa powder. 100 parts by weight of the mucosa powder is mixed with 800 parts by weight of a 3 g / L sodium chloride solution and soaked for 3 hours;

[0044] (2) mixing the soaking material (mucous membrane powder and sodium chloride solution) with 0.2 parts by weight of the complex enzyme and placing it in an extraction kettle; injecting supercritical CO2 into the extraction kettle, extracting for 60 minutes under the conditions of pressure 25 MPa, temperature 35° C., pH 7, and CO2 flow rate 10 kg / h to obtain an extract;

[0045] The complex enzyme is composed of serrapeptase, trypsin and pancreatic lipase in a weight ratio of 1:2:3, the enzyme activity of serrapeptase is 100,000 U / g, the enzyme activity of trypsin is 80,000 U / g, and the enzyme activity of pancreatic lipase is 50,000 U / g;

[0046] (3) Mix the enzymatic hydrolyzate with the adsorbent, stir at 300 r / min for 5 min, and let stand for 4 h;

[0047] The adsorbent is composed of polyferric sulfate and polyaluminium chloride in a weight ratio of 1:2, and the amount of the adsorbent is 2% of the weight of the enzymatic hydrolysate;

[0048] (4) The supernatant was mixed with an 80% ethanol solution in a volume ratio of 1:1, and the mixture was allowed to stand for 4 hours. The precipitate was collected and dried at 30° C. to a water content of 5%, thereby obtaining heparin sodium.

[0049] Example 3

[0050] (1) The mucosa separated from fresh pig small intestine is dried at 40° C. to a water content of 2%, ultrafinely ground, and passed through a 1200-mesh sieve to obtain mucosa powder. 100 parts by weight of the mucosa powder is mixed with 1200 parts by weight of a 5 g / L sodium chloride solution and soaked for 1 hour;

[0051] (2) After the soaking material (mucous membrane powder and sodium chloride solution) is mixed with 0.6 parts by weight of the composite enzyme, it is placed in an extraction kettle; supercritical CO2 is injected into the extraction kettle, and extraction is performed for 45 minutes under the conditions of pressure 35 MPa, temperature 40°C, pH value 8, and CO2 flow rate 15 kg / h to obtain an enzymatic hydrolyzate;

[0052] The complex enzyme is composed of serrapeptase, trypsin and pancreatic lipase in a weight ratio of 1:5:8, the enzyme activity of serrapeptase is 200,000 U / g, the enzyme activity of trypsin is 140,000 U / g, and the enzyme activity of pancreatic lipase is 100,000 U / g;

[0053] (3) Mix the enzymatic hydrolyzate with the adsorbent, stir at 400 r / min for 2 min, let stand for 2 h, and remove the precipitate;

[0054] The adsorbent is composed of polyferric sulfate and polyaluminium chloride in a weight ratio of 2:1, and the amount of the adsorbent is 2% of the weight of the enzymatic hydrolysate;

[0055] (4) The supernatant was mixed with an 80% ethanol solution in a volume ratio of 1:1, and the mixture was allowed to stand for 4 hours. The precipitate was collected and dried at 40° C. to a water content of 3%, thereby obtaining heparin sodium.

[0056] Comparative Example 1

[0057] The specific implementation is the same as that of Example 1, except that step (1) is performed by conventional crushing through an 80-mesh sieve.

[0058] Comparative Example 2

[0059] The specific implementation method is the same as that of Example 1, except that the compound enzyme in step (2) is composed of pancreatic peptidase, papain and phosphatase in a weight ratio of 1:3:5, the enzyme activity of pancreatic peptidase is 150,000 U / g, the enzyme activity of papain is 100,000 U / g, the enzyme activity of phosphatase is 80,000 U / g, and the supercritical CO2 extraction parameters remain unchanged.

[0060] Comparative Example 3

[0061] The specific implementation method is the same as that of Example 1, except that the adsorbent in step (3) is polyferric sulfate, and the amount used is 3% of the weight of the enzymatic hydrolyzate.

[0062] Comparative Example 4

[0063] The specific implementation method is the same as that of Example 1, except that the adsorbent in step (3) is polyaluminium chloride, and the amount used is 3% of the weight of the enzymatic hydrolysate.

[0064] Test Example 1

[0065] The titer and yield of heparin sodium of Examples 1-3 of the present invention and Comparative Examples 1-4 were tested, and the specific results are shown in Table 1.

[0066] The titer of heparin sodium was determined by the azure A colorimetric method.

[0067] Heparin sodium yield (%) = heparin sodium mass ÷ mucosal powder mass × 100%.

[0068] Table 1 Titer and yield of heparin sodium in Examples 1-3 and Comparative Examples 1-4

[0069]

[0070]

[0071] It can be seen from the data in Table 1 that Examples 1-3 have higher titers and yields than the comparative examples. Comparison of the data of Example 1 and Comparative Example 1 shows that ultrafine grinding can significantly improve the titer and yield of heparin sodium. Comparison of the data of Example 1 and Comparative Example 2 shows that the use of a suitable composite enzyme to enzymolyze the pig small intestinal mucosa can significantly improve the titer and yield of heparin sodium. Comparison of the data of Example 1 and Comparative Example 2 shows that there is a synergistic effect between the adsorbents polyferric sulfate and polyaluminium chloride, which can adsorb and precipitate the protein in the enzymolysis solution, thereby improving the titer and yield of heparin sodium.

[0072] 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 improving the extraction efficiency of heparin sodium using supercritical fluid extraction technology, characterized in that: include: The mucosa separated from the fresh pig small intestine is dried and ultrafinely crushed to obtain mucosa powder, which is then soaked in sodium chloride solution and hydrolyzed by supercritical mixed enzyme. The hydrolyzate is mixed with an adsorbent and allowed to stand, the supernatant is precipitated with alcohol, and the precipitate is collected to obtain heparin sodium; The supercritical fluid for the supercritical combined enzyme hydrolysis is CO2, the pressure is 25-35MPa, the temperature is 35-40°C, the pH value is 7-8, the CO2 flow rate is 10-15kg / h, and the time is 45-60min.

2. The method according to claim 1, characterized in that The drying temperature is 30-40°C.

3. The method according to claim 1, characterized in that The particle size of ultrafine grinding is 1000-1200 mesh.

4. The method according to claim 1, characterized in that The concentration of the sodium chloride solution is 3-5 g / L.

5. The method according to claim 1, characterized in that The weight ratio of the mucosal powder to the sodium chloride solution is 1:8-12, and the soaking time is 1-3 hours.

6. The method according to claim 1, wherein the complex enzyme is composed of serrapeptase, trypsin and pancreatic lipase in a weight ratio of 1:(2-5):(3-8); and the added amount of the complex enzyme is 2‰-6‰ of the weight of the mucosal powder.

7. The method according to claim 1, characterized in that The adsorbent is one or both of polyferric sulfate and polyaluminium chloride; the adsorbent accounts for 2%-5% of the weight of the enzymatic hydrolysate.

8. The method according to claim 1, characterized in that The mixing and standing comprises: mixing the enzymatic hydrolysate with the adsorbent, stirring at 300-400 r / min for 2-5 min, and standing for 2-4 h.

9. The method according to claim 1, characterized in that: The alcohol precipitation comprises: mixing the supernatant with the ethanol solution, standing for 4-6 hours, and collecting the precipitate.

10. The method according to claim 9, characterized in that The volume percentage of the ethanol solution is 80%-90%.

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