A method for improving the extraction efficiency of heparin sodium by using supercritical fluid extraction technology

Abstract

The application provides a method for improving the extraction efficiency of heparin sodium by using supercritical fluid extraction technology, relates to the technical field of polysaccharide preparation, and belongs to the IPC classification number C08B37. The application provides a method for improving the extraction efficiency of heparin sodium by using supercritical fluid extraction technology, which comprises the following steps: drying and ultrafine grinding mucosa separated from fresh pig small intestine to obtain mucosa powder, soaking the mucosa powder in a sodium chloride solution, carrying out enzyme hydrolysis on the mucosa powder by using supercritical fluid and composite enzymes, mixing the enzyme hydrolysis liquid with an adsorbent and standing, alcohol precipitation of supernatant, collecting the precipitate, and obtaining heparin sodium. The pig small intestine mucosa is ultrafine ground, which is beneficial to the dissolution of intracellular solutes and shortens the reaction time; the enzyme hydrolysis mode of supercritical fluid and composite enzymes is adopted, which promotes the full reaction of enzyme hydrolysis and effectively dissociates heparin sodium from proteins; the addition of the adsorbent can further separate proteins and improve the extraction efficiency of heparin sodium. The method has a synergistic effect between the steps, improves the titer and yield of heparin sodium.

Description

Technical Field

[0001] This invention relates to the field of polysaccharide preparation technology, belonging to IPC classification number C08B37, and specifically to a method for improving the extraction efficiency of heparin sodium using supercritical fluid extraction technology. Background Technology

[0002] In the food processing industry, the preparation of pig casings involves discarding components such as the mucosa, rendering them waste byproducts. However, from a biopharmaceutical perspective, the intestinal mucosa is actually a highly valuable raw material, a key source for extracting heparin sodium. Heparin sodium, as an anticoagulant with wide and significant clinical applications, occupies an indispensable position in modern medicine. It effectively prevents blood clotting, playing a crucial role in the prevention and treatment of thrombotic diseases, and is of great importance in ensuring patients' health and the smooth progress of surgical and other medical procedures.

[0003] Currently, common methods for extracting heparin sodium mainly include salting-ion exchange and enzymatic hydrolysis-ion exchange. These methods typically rely on the solubility and ionic properties of heparin sodium in specific solution environments to separate and extract it from raw materials such as animal tissues. For example, the salting-ion exchange method first uses a high-concentration sodium chloride solution to disrupt the structure of tissue cells, releasing heparin sodium into the solution. Then, the heparin sodium is adsorbed and eluted using an ion exchange resin, achieving separation and purification. Traditional extraction methods usually use large amounts of chemical reagents, such as strong acids, strong bases, and organic solvents. Improper handling of these chemical reagents after extraction can cause significant environmental pollution. For example, the discharge of large amounts of acidic or alkaline wastewater can lead to pH imbalance in water bodies, affecting the living environment of aquatic organisms; the volatilization of organic solvents can pollute the atmosphere, and the recovery and treatment of organic solvents are costly and pose safety hazards if not handled properly. Furthermore, traditional extraction methods often require multiple precipitation, centrifugation, and elution operations, making the entire extraction process time-consuming. During these processes, the transfer of heparin sodium between each step and changes in processing conditions can lead to the loss of some heparin sodium, resulting in a relatively low actual amount of heparin sodium extracted from the raw material and making it difficult to effectively improve the extraction efficiency. For example, during multiple precipitation processes, slight differences in precipitation conditions may cause some heparin sodium to fail to precipitate completely and remain in the solution, resulting in loss.

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

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

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for improving the extraction efficiency of heparin sodium using supercritical fluid extraction technology, comprising: drying and ultra-finely pulverizing the mucosa separated from fresh pig small intestine to obtain mucosa powder, soaking it in sodium chloride solution, enzymatically hydrolyzing it with a supercritical combined enzyme, mixing the hydrolysate with an adsorbent, allowing it to stand, precipitating the supernatant with alcohol, collecting the precipitate, and obtaining heparin sodium; wherein the supercritical fluid used for enzymatic hydrolysis is CO2, the pressure is 25-35 MPa, the temperature is 35-40℃, the pH value is 7-8, the CO2 flow rate is 10-15 kg / h, and the time is 45-60 min.

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

[0009] Preferably, the particle size of the ultrafine pulverizer 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 serrata peptides, trypsin and pancreatic lipase in a weight ratio of 1:(2-5):(3-8); the amount of the complex enzyme added is 2‰-6‰ of the weight of the mucosal powder.

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

[0014] Preferably, the mixing and settling process includes: mixing the enzymatic hydrolysate with the adsorbent, stirring at 300-400 r / min for 2-5 min, and then settling for 2-4 h.

[0015] Preferably, the alcohol precipitation includes: mixing the supernatant with an ethanol solution, allowing it to 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] This invention provides a method for improving the extraction efficiency of heparin sodium using supercritical fluid extraction technology, comprising: drying and ultra-finely pulverizing the mucosa separated from fresh pig small intestine to obtain mucosa powder, soaking in sodium chloride solution, enzymatic hydrolysis using a supercritical combined enzyme, mixing the hydrolysate with an adsorbent, allowing it to stand, precipitating the supernatant with alcohol, collecting the precipitate, and obtaining heparin sodium. The ultra-fine pulverization of the pig small intestine mucosa in this invention breaks down the mucosal cells, facilitating the dissolution of intracellular solutes and shortening the reaction time; the supercritical + combined enzyme enzymatic hydrolysis method after soaking in sodium chloride solution, on the one hand, the supercritical pressure promotes the full reaction between the combined enzyme and the substrate, decomposing proteins and fats into smaller molecules, and supercritical extraction can remove fats and some proteins; on the other hand, by reducing the concentration of sodium chloride solution, it can also promote the effective dissociation of heparin sodium from proteins; adding an adsorbent to the extraction solution can further separate proteins, improving the purity and extraction efficiency of heparin sodium. This invention is carried out at low temperature to avoid the degradation of heparin sodium, and the steps have a synergistic effect, improving the potency and yield of heparin sodium.

[0019] This invention makes efficient use of the waste product mucosa from the preparation of pig intestine casings to prepare heparin sodium, thus achieving efficient resource utilization. Detailed Implementation

[0020] This invention provides a method for improving the extraction efficiency of heparin sodium using supercritical fluid extraction technology, comprising: drying and ultra-finely pulverizing the mucosa separated from fresh pig small intestine to obtain mucosa powder, soaking it in sodium chloride solution, enzymatically hydrolyzing it with a supercritical combined enzyme, mixing the enzymatic hydrolysate with an adsorbent, allowing it to stand, precipitating the supernatant with alcohol, collecting the precipitate, and obtaining heparin sodium; wherein the supercritical fluid used for the supercritical combined enzyme enzymatic hydrolysis is CO2, the pressure is 25-35 MPa, the temperature is 35-40℃, the pH value is 7-8, the CO2 flow rate is 10-15 kg / h, and the time is 45-60 min.

[0021] The drying temperature described in this invention is preferably 30-40°C, more preferably 35°C, until the moisture content of the mucosa is 3%-5%.

[0022] The preferred particle size of the ultrafine pulverizer in this invention is 1000-1200 mesh, more preferably 1100 mesh. Ultrafine pulverization effectively disrupts the cellular structure and organelle membranes of the porcine small intestinal mucosa, allowing heparin sodium, originally encapsulated within the cells, to be fully exposed and released. This enables more heparin sodium to participate in the subsequent extraction process. Furthermore, the surface area of ​​the ultrafine-pulverized porcine small intestinal mucosa is significantly increased, correspondingly increasing the contact area with the extraction solvent, making heparin sodium molecules more easily soluble in 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 in this 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 soaking time is preferably 1-3 h, more preferably 2 h.

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

[0025] The composite enzyme of this invention is preferably composed of serratiptase, trypsin, and pancreatic lipase in a weight ratio of 1:(2-5):(3-8), more preferably 1:3:5; the amount of the composite enzyme added is preferably 2‰-6‰ of the weight of the mucosal powder, more preferably 4‰. The enzyme activity of the serratiptase 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. This invention utilizes serratiptase, which can decompose various proteins in the extracellular matrix and glycoproteins on the cell membrane, while trypsin can specifically hydrolyze peptide bonds and mainly acts on intracellular proteins, such as cytoskeletal proteins. The synergistic effect of the two can comprehensively destroy cell structure from the inside out, further weakening the binding of heparin sodium to proteins and causing heparin sodium to dissociate. Pancreatic lipase decomposes fats, which can weaken intercellular connections and destroy the lipid bilayer structure of the cell membrane. Combined with the protein decomposition by serratiptase and trypsin, it accelerates the destruction of cell structure and makes the release of heparin sodium more efficient.

[0026] This invention employs a supercritical + complex enzyme hydrolysis method. On the one hand, supercritical pressure promotes the full reaction between the complex enzyme and the substrate, causing proteins and fats to decompose into smaller molecules. Fats and some proteins are removed through supercritical extraction. On the other hand, by reducing the concentration of sodium chloride solution, it can also promote the effective dissociation of heparin sodium from proteins.

[0027] The adsorbent of the present invention is preferably one or two of polyferric sulfate and polyaluminum chloride; the adsorbent is preferably 2%-5% of the weight of the enzymatic hydrolysate, more preferably 3%. The mixing and settling process preferably includes: mixing the enzymatic hydrolysate with the adsorbent, stirring at 300-400 r / min for 2-5 min, and then settling for 2-4 h.

[0028] This invention utilizes polyferric sulfate and polyaluminum chloride to neutralize protein charges over a wider pH range, reducing electrostatic repulsion between protein molecules and promoting protein aggregation. The ferric hydroxide colloid produced by the hydrolysis of polyferric sulfate and the aluminum hydroxide colloid produced by the hydrolysis of polyaluminum chloride intertwine in solution to form more stable and compact flocs, leading to protein adsorption and precipitation in the enzymatic hydrolysate, thereby improving the extraction efficiency of heparin sodium.

[0029] The preferred method of alcohol precipitation in this invention includes: mixing the supernatant with an ethanol solution, allowing it to stand for 4-6 hours, and collecting the precipitate. The volume percentage of the ethanol solution is preferably 80%-90%, more preferably 85%. This invention uses alcohol precipitation to precipitate heparin sodium, further improving the purity of heparin sodium.

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

[0031] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0032] Unless otherwise specified, the following embodiments are all conventional methods.

[0033] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0034] The polyacrylamide used in this invention is anionic polyacrylamide, purchased from Gongyi Xinqi Polymer Co., Ltd. Polyaluminum chloride was purchased from Henan Shuanglong Water Treatment Materials Co., Ltd.

[0035] Example 1

[0036] (1) The mucosa separated from fresh pig small intestine is dried at 35°C until the moisture content is 3%, then ultra-finely pulverized and passed through an 1100-mesh sieve to obtain mucosa powder. 100 parts by weight of mucosa powder is mixed with 1000 parts by weight of sodium chloride solution with a concentration of 4 g / L and soaked for 2 hours to obtain soaked material.

[0037] (2) After mixing the soaking material (mucosal powder and sodium chloride solution) with 0.4 parts by weight of the compound enzyme, it is placed in the extraction vessel; supercritical CO2 is injected into the extraction vessel and extracted for 50 min under the conditions of pressure 30 MPa, temperature 37℃, pH value 7.5 and CO2 flow rate 13 kg / h to obtain the extract.

[0038] The complex enzyme consists of serrata peptides, trypsin, and pancreatic lipase in a weight ratio of 1:3:5. The enzyme activity of serrata peptides 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 hydrolysate 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 polyaluminum chloride in a weight ratio of 1:1, and the amount of adsorbent used is 3% of the weight of the enzymatic hydrolysate.

[0041] (4) Mix the supernatant with an 85% ethanol solution at a volume ratio of 1:1, let stand for 5 hours, collect the precipitate, and dry it at 35°C until the water content is 5% to obtain heparin sodium.

[0042] Example 2

[0043] (1) The mucosa separated from fresh pig small intestine is dried at 30°C until the moisture content is 5%, then ultra-finely pulverized 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 3g / L sodium chloride solution and soaked for 3 hours.

[0044] (2) After mixing the soaking material (mucosal powder and sodium chloride solution) with 0.2 parts by weight of the compound enzyme, it is placed in the extraction vessel; supercritical CO2 is injected into the extraction vessel and extracted for 60 min under the conditions of pressure 25 MPa, temperature 35℃, pH value 7 and CO2 flow rate 10 kg / h to obtain the extract.

[0045] The complex enzyme consists of serrata peptides, trypsin, and pancreatic lipase in a weight ratio of 1:2:3. The enzyme activity of serrata peptides 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 hydrolysate 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 polyaluminum chloride in a weight ratio of 1:2, and the amount of adsorbent used is 2% of the weight of the enzymatic hydrolysate.

[0048] (4) Mix the supernatant with an 80% ethanol solution at a volume ratio of 1:1, let stand for 4 hours, collect the precipitate, and dry it at 30°C until the water content is 5% to obtain heparin sodium.

[0049] Example 3

[0050] (1) The mucosa separated from fresh pig small intestine is dried at 40°C until the moisture content is 2%, then ultra-finely pulverized 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 mixing the soaking material (mucosal powder and sodium chloride solution) with 0.6 parts by weight of the compound enzyme, it is placed in the extraction vessel; supercritical CO2 is injected into the extraction vessel and extracted for 45 min under the conditions of pressure 35 MPa, temperature 40℃, pH value 8 and CO2 flow rate 15 kg / h to obtain the enzymatic hydrolysate.

[0052] The complex enzyme consists of serrata peptides, trypsin, and pancreatic lipase in a weight ratio of 1:5:8. The enzyme activity of serrata peptides 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 hydrolysate 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 polyaluminum chloride in a weight ratio of 2:1, and the amount of adsorbent used is 2% of the weight of the enzymatic hydrolysate.

[0055] (4) Mix the supernatant with an 80% ethanol solution at a volume ratio of 1:1, let stand for 4 hours, collect the precipitate, and dry it at 40°C until the water content is 3% to obtain heparin sodium.

[0056] Comparative Example 1

[0057] The specific implementation method is the same as that in Example 1, except that step (1) involves conventional pulverization and passing through an 80-mesh sieve.

[0058] Comparative Example 2

[0059] The specific implementation method is the same as in Example 1, except that the complex 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 in Example 1, except that the adsorbent in step (3) is polyferric sulfate, and the amount used is 3% of the weight of the enzymatic hydrolysate.

[0062] Comparative Example 4

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

[0064] Experimental Example 1

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

[0066] The heparin sodium potency was determined using the azurite A colorimetric method.

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

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

[0069]

[0070]

[0071] As shown in Table 1, Examples 1-3 exhibit higher potency and yield compared to the Comparative Example. A comparison of the data from Example 1 and Comparative Example 1 reveals that ultrafine grinding significantly improves the potency and yield of heparin sodium. A comparison of the data from Example 1 and Comparative Example 2 shows that using a suitable composite enzyme to enzymatically hydrolyze the porcine small intestinal mucosa can significantly improve the potency and yield of heparin sodium. A comparison of the data from Example 1 and Comparative Example 2 indicates that the adsorbents polyferric sulfate and polyaluminum chloride have a synergistic effect, adsorbing and precipitating proteins in the enzymatic hydrolysate, thereby improving the potency and yield of heparin sodium.

[0072] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within 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 fresh pig small intestine is dried and ultra-finely pulverized to obtain mucosa powder. It is soaked in sodium chloride solution, enzymatically hydrolyzed by supercritical combined enzyme, and the hydrolysate is mixed with adsorbent and allowed to stand. The supernatant is precipitated with alcohol, and the precipitate is collected to obtain sodium heparin. The supercritical fluid used for enzymatic hydrolysis by the supercritical combined enzyme is CO2, with a pressure of 25-35 MPa, a temperature of 35-40℃, a pH of 7-8, a CO2 flow rate of 10-15 kg / h, and a time of 45-60 min. The complex enzyme is composed of serrata peptides, trypsin, and pancreatic lipase in a weight ratio of 1:(2-5):(3-8); the amount of the complex enzyme added is 2‰-6‰ of the weight of the mucosal powder. The adsorbent is a combination of polyferric sulfate and polyaluminum chloride; the adsorbent accounts for 2%-5% of the weight of the enzymatic hydrolysate.

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

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, characterized in that, The mixing and settling process includes: mixing the enzymatic hydrolysate with the adsorbent, stirring at 300-400 r / min for 2-5 min, and then settling for 2-4 h.

7. The method according to claim 1, characterized in that, The alcohol precipitation process involves mixing the supernatant with an ethanol solution, allowing it to stand for 4-6 hours, and then collecting the precipitate.

8. The method according to claim 7, characterized in that, The volume percentage of the ethanol solution is 80%-90%.

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