Application of solid alkaline protease preparation in improving yield of crude heparin sodium

Abstract

The invention belongs to application of an enzyme preparation, and particularly relates to application of a solid alkaline protease preparation in improving the yield of crude heparin sodium. Comprising the steps of preparation of fermentation liquor containing alkaline protease, flocculation and filtration of the fermentation liquor, ultrafiltration and concentration, preparation of a solid enzyme preparation, drying and screening to prepare solid or granular alkaline protease, enzymolysis of small intestines of pigs, heparin adsorption, heparin elution, acquisition of crude heparin sodium and the like. The method provided by the invention solves the problems of difficult dissolution of an enzyme preparation, unstable product quality, high subsequent environment-friendly treatment cost and the like in the prior art, and has the advantages of high yield of crude heparin sodium, simple subsequent environment-friendly treatment, low cost, stable process conditions, easy realization and control and the like.

Description

Technical Field

[0001] The present invention belongs to the application of enzyme preparations, and particularly relates to the application of a solid alkaline protease preparation in improving the yield of crude heparin sodium. Background Art

[0002] At present, the preparation of crude heparin sodium mainly includes the salting-out method and the enzymatic hydrolysis method. The process of preparing crude heparin sodium by enzymatic hydrolysis is as follows: The mucosa of fresh pig small intestine is stirred with water into a paste, sodium chloride is added to the total volume to a salt concentration of 3%, the pH is adjusted to 11 with sodium hydroxide, 2709 alkaline protease is quantitatively added according to the number of small intestines and the mass of mucosa, and the temperature is raised for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the temperature is quickly raised to inactivate the enzyme. After the enzymatic hydrolysate is filtered, it is adsorbed by an anion exchange resin, and the heparin adsorbed by the anion exchange resin is eluted with a sodium chloride solution. After the eluates are combined, they are precipitated with an ethanol solution, and the precipitate is dried to obtain crude heparin sodium.

[0003] In the enzymatic hydrolysis method, protease can activate the heparin activity by decomposing the protein bound to heparin. However, if the protein is not decomposed well, the heparin activity cannot be fully activated. Excessive decomposition will damage the structure of the heparin molecule. Both will affect the yield of heparin. Therefore, whether heparin sodium is completely dissociated from the protein and does not damage the effective components of heparin sodium during production is the most important difficulty in the enzymatic hydrolysis process.

[0004] The advantage of the enzymatic hydrolysis method is that the yield is higher than that of the salting-out method, the titer is high and the weight is low. The reason is that heparin exists in the form of glycoprotein by binding to protein in the small intestine mucosa. The bound protein has no anticoagulant activity of heparin. Only with the removal of protein, the anticoagulant activity increases. By using protease to decompose the protein, the protein content of crude heparin sodium can be reduced, the heparin titer can be increased, and the weight of the dried product is also reduced accordingly, which is more beneficial for the subsequent refining process. The sodium chloride content in water is low, which is more beneficial for the subsequent sewage treatment of sodium chloride. Therefore, the enzymatic hydrolysis method has better effect than the salting-out method.

[0005] At present, almost all domestic enterprises producing crude heparin sodium by enzymatic hydrolysis use alkaline protease in the form of spray-dried powder. The spray-dried powder has inevitable inherent defects. Firstly, it has problems such as incomplete dissolution, easy moisture absorption, and extremely difficult melting after moisture absorption and caking, which will lead to difficult control of enzyme dosage. In industrial production, it will cause the enzymatic hydrolysis effect to be very unstable when the enzyme participates in the substrate reaction, and the quality of similar products on the market also varies, resulting in differences in yield. Secondly, the COD content of the organic wastewater in the enzymatic hydrolysis process is about 50,000 mg / L. When directly discharged to the factory's sewage treatment station, the treatment cycle of this kind of organic sewage requires 7 to 10 days. This will greatly limit the production capacity of crude heparin sodium. If we want to not affect the previous production, we need to expand the treatment capacity of the sewage station, which will virtually increase the investment in construction and operation and maintenance costs. As far as the applicant's understanding is concerned, spray-dried powder is usually divided into industrial grade and food grade. The problems with industrial-grade powder alkaline protease are as follows: Firstly, there is no quantitative limit on the viable bacteria count. As the national standard for industrial-grade enzyme preparations, GB / T 23527.1-2023 does not quantitatively limit the viable bacteria. Its production process is direct spray drying of the fermentation broth, and the bacteria in the fermentation broth are not removed and survive in the product. This will lead to easy microbial contamination of the enzymatic hydrolysis substrate, reduce the quality of the enzymatic hydrolysis solution, and also increase the dosage of sodium hydroxide and the neutralization salt content of the enzymatic hydrolysis solution, having a negative impact on the production results. Secondly, there are no restrictive requirements for the use of excipients. The national standard GB / T 23527.1-2023 does not put forward restrictive requirements for the use of excipients, so excipients such as sodium sulfate, ammonium sulfate, and rice husk powder, which are cheap products on the market, are added. Sodium sulfate is the preferred excipient due to its low price, and its content is between 25% and 60%. This leads to an increase in the soluble salt components in the intestinal enzymatic hydrolysis solution, affecting resin adsorption. The problems with food-grade powder alkaline protease are as follows: Firstly, the national standard GB 1886.174-2024 - Enzyme Preparations for Food Industry makes restrictive requirements for carriers, viable bacteria count, and excipients. However, enzyme preparations with carriers and excipients such as starch and dextrin added have strong hygroscopicity and cannot solve the problem of difficult dissolution of enzyme preparations. Secondly, because the carriers and excipients are starch sugars, they are extremely prone to Maillard reactions with the nitrogen source in the intestinal enzymatic hydrolysis solution during the temperature rise for enzyme inactivation, deepening the color, and the pigment will affect the adsorption of heparin by the resin.

[0006] For the above reasons, currently available powder products on the market cannot well solve the problem of improving the quality of the enzymatic hydrolysis solution. The applicant has not retrieved domestic patent literature reports identical or similar to the present application. Summary of the Invention

[0007] The object of the present invention is to provide an application of a solid alkaline protease preparation in improving the yield of crude heparin sodium. By optimizing the fermentation process, an alkaline protease can be obtained for use in the enzymatic hydrolysis to obtain crude heparin sodium. At the same time, by optimizing the enzymatic hydrolysis process conditions, it is possible to effectively improve the yield of crude heparin sodium, reduce the solid content in the enzymatic hydrolysate, reduce the sewage treatment time and lower the treatment cost.

[0008] The main technical concept of the present invention is:

[0009] The application of a solid alkaline protease preparation in improving the yield of crude heparin sodium is to add the alkaline protease to a pig small intestine mucosa solution stirred into a paste, heat it for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, inactivate the enzyme preparation and filter the enzymatic hydrolysate. Adsorb the filtrate with an anion resin, elute the anion resin adsorbed with the filtrate with a sodium chloride solution, and extract the eluate with an ethanol solution to obtain crude heparin sodium; it includes the following technological steps:

[0010] A. Preparation of solid or granular alkaline protease

[0011] A1. Preparation of fermentation broth containing alkaline protease

[0012] Select Bacillus licheniformis with the product number BNCC360660 produced by Beijing NaChuangLian Biotechnology Co., Ltd. as the production strain; inoculate the production strain into a sterile fermentation medium containing a carbon source, a nitrogen source and necessary growth factors, and conduct aeration stirring fermentation to make a fermentation broth containing alkaline protease;

[0013] A2. Flocculation and filtration of the fermentation broth;

[0014] A3. Ultrafiltration and concentration;

[0015] A4. Preparation of solid enzyme preparation;

[0016] A5. Dry and screen to make solid or granular alkaline protease;

[0017] Steps A2 - A5 are carried out according to the method described in the patent number 202211187843.8;

[0018] B. Preparation of crude heparin sodium

[0019] B1. Enzymatic hydrolysis of pig small intestine

[0020] Adjust the sodium chloride concentration in the stirred and mashed porcine small intestine mucosa solution to 0-3% and pH = 7-9. According to the dosage of adding 3-10 g of solid or granular alkaline protease prepared in step A per small intestine, add the solid or granular alkaline protease to the mashed porcine small intestine solution, heat it to 45°C-55°C and maintain for 60-90 minutes for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, heat up to inactivate the alkaline protease;

[0021] B2. Adsorb heparin

[0022] Filter the enzymatic hydrolysate and cool the filtrate to 50°C-60°C. Adsorb the cooled filtrate with anion exchange resin for 8-10 hours and then collect the resin;

[0023] B3. Heparin elution

[0024] Elute the anion exchange resin adsorbed with heparin in step B2 with sodium chloride solution;

[0025] B4. Obtain crude heparin sodium

[0026] Precipitate the eluate with an ethanol solution with a volume percentage concentration of 40%-55%, and dry the precipitate to obtain crude heparin sodium.

[0027] The specific technical concept in the present invention also includes:

[0028] To facilitate the growth of the strain and the production of secondary metabolites, the preferred technical implementation means is that the fermentation medium in step A1 is composed of the following raw materials:

[0029] Corn flour 10%-12%, soybean meal 8%-10%, disodium hydrogen phosphate 0.3%-0.5%, sodium carbonate 0.1%-0.3%, soybean oil 0.1%-0.3%, and the balance is water.

[0030] Furthermore, the aeration and stirring fermentation conditions in step A1 are as follows: the medium loading is 63%-70%, the inoculation amount is 9.5%-10.5%, the temperature is 37°C ± 0.5°C, pH = 6.6-7.1, the ventilation volume is 800-1300 m 3 / h, the rotation speed is 110-130 revolutions per minute, and the fermentation period is 35-40 hours.

[0031] To facilitate industrial production, the preferred technical implementation means is that it also includes an amplification culture step, which is to make a seed solution by amplifying the culture of Bacillus licheniformis with the product number BNCC360660 produced by Beijing NaChuangLian Biotechnology Co., Ltd., and inoculate the seed solution into the fermentation medium and ferment it by aeration and stirring to make a fermentation broth containing alkaline protease.

[0032] Furthermore, the enlarged culture includes the preparation of shake flask seeds and fermentation seeds. The shake flask seeds are prepared by inoculating Bacillus licheniformis produced by Beijing NaChuangLian Biotechnology Co., Ltd. with product number BNCC360660 into a shake flask medium and culturing it by shaking. The shake flask medium is composed of the following raw materials: 0.9% - 1.3% beef extract, 0.9% - 1.3% peptone, 0.3% - 0.6% sodium chloride, and the balance is water. A shake flask with a capacity of 2000 ml is used to make the volume of the shake flask medium 550 ml. The conditions for shaking culture are pH = 7 - 7.5, temperature 37°C ± 0.5°C, and time 8 - 12 hours.

[0033] The preparation of fermentation seeds is to inoculate the shake flask seeds into a seed medium and culture them by aeration to make fermentation seeds. The seed medium is composed of the following raw materials: 3% - 4% corn flour, 5% - 6% soybean meal, 0.2% - 0.3% disodium hydrogen phosphate, 0.08% - 0.1% sodium carbonate, 0.4% - 0.6% soybean oil, and the balance is water. A seed tank with a capacity of 2 tons is used to make the volume of the seed medium 1.1 - 1.3 tons. The conditions for aeration culture are pH = 6.8 - 7.1, temperature 37°C ± 0.5°C, ventilation volume 800 - 1300 m 3 / h, stirring speed 110 - 130 revolutions per minute, and time 35 - 40 hours.

[0034] In step B1, the pH is adjusted using sodium hydroxide or a sodium hydroxide solution.

[0035] Washing with a low - concentration sodium chloride solution is to remove proteins and impurities. Elution is to elute heparin from the ion - exchange column using a high - concentration sodium chloride solution, which requires a strong ionic strength to displace the sodium heparin on the resin. To further improve the washing and elution effects and thus increase the yield of sodium heparin, the preferred technical implementation means is that in step B3, the resin adsorbed with heparin is rinsed with water to wash away the suspended matter and then filtered dry; elute 2 - 3 times and filter dry with a sodium chloride aqueous solution with a volume 1.5 - 2 times the resin volume and a concentration of 3.5% - 4.5%; elute 2 - 3 times with a sodium chloride aqueous solution with a volume 1.5 - 2 times the resin volume and a concentration of 18% - 24%, and preserve and combine the eluates of 2 - 3 times.

[0036] The solubility of sodium heparin in ethanol is low, and it is easier to precipitate, which helps to further remove proteins. Ethanol has little effect on the activity titer of sodium heparin and is easy to operate in industrial - scale production. Ethanol is volatile and can be removed by evaporation in subsequent processes, and it can be recycled with relatively low cost. In step B4, the eluate is precipitated with an ethanol solution with a volume percentage concentration of 40% - 55% for 12 hours.

[0037] The substantial features and remarkable technical progress of the present invention lie in:

[0038] 1. By optimizing the preparation process of alkaline protease, the prepared solid alkaline protease has a pH tolerance of 7-9, which can reduce the dosage of sodium hydroxide in the production process of heparin sodium. The addition of sodium hydroxide, sodium sulfate or ammonium sulfate, etc. will cause the increase of OH - and SO 4 2- content in the enzymatic hydrolysate. Since the adsorption resin selected is an anion resin and heparin itself is a weakly charged anion. OH - and SO 4 2- have a greater electronegativity than heparin anions, so they are more easily adsorbed by the anion resin preferentially. This will cause a large number of adsorption sites of the anion resin to be occupied by these interfering ions, resulting in the ineffective adsorption of heparin anions, and finally manifested as a decrease in the yield of crude heparin sodium and an increase in the impurity content. Therefore, reducing the interfering factors related to resin adsorption can effectively improve the yield of heparin.

[0039] 2. The alkaline protease fermentation broth is made into a solid enzyme preparation after sterilization and impurity removal, reducing the probability of microbial contamination in the production of heparin sodium. The solid enzyme preparation only contains enzyme preparation components and no excipients are added to the finished product. Its preparation is to carry out a specific ratio on the concentrated solution and then prepare it into small particles through low-temperature drying, and the product has high purity.

[0040] 3. The prepared enzyme preparation has good fluidity and low hygroscopicity. The enzyme preparation can be quickly and evenly dispersed in the reaction material, effectively guaranteeing the enzymatic hydrolysis efficiency, significantly and stably improving the yield of crude heparin sodium, reducing the dosage of the enzyme preparation, and at the same time being able to stably control the production process.

[0041] 4. Since the solid alkaline protease in the present invention can effectively control the number of viable bacteria, through the applicant's experiments, the enzyme preparation prepared by the method of the present invention can control the total number of colonies within 1000-10000 CFU / g, far lower than the national standard requirement of 50000 CFU / g. And it has a good enzymatic hydrolysis effect on organic matter, so the enzymatic hydrolysis residue can be extracted in stages, and the sewage after separating heparin is directly subjected to desalination treatment. Through the applicant's experiments, the COD in the water can be reduced by 85%-95%, and the treatment time is shortened to 1-3 days. Compared with the existing sewage COD index, it is greatly reduced, the clarity is significantly improved, and it can be easily removed of solids through plate and frame filtration, membrane filtration, etc. and is not easy to block, greatly reducing the subsequent treatment difficulty and the treatment time is greatly shortened. Through the applicant's experiments, it is confirmed that it can effectively reduce the treatment time and treatment cost of sewage.

[0042] 5. The enzyme preparation prepared by the present invention has little change in enzyme activity in an environment where the sodium chloride concentration decreases and is alkaline, and the enzyme activity performance still meets the use requirements even in a neutral environment where the sodium chloride concentration decreases to zero. Therefore, the dosage of sodium chloride and sodium hydroxide can be effectively reduced, further reducing the difficulty of subsequent sewage treatment and shortening the treatment time. Detailed implementation manners

[0043] The following further describes the embodiments of the present invention, but does not further limit the present invention. The protection scope of the present invention is subject to the content recorded in the claims. Any equivalent technical means substitution based on the description of the specification shall not deviate from the protection scope of the present invention.

[0044] Example 1

[0045] Preparation of solid or granular alkaline protease, including the following process steps:

[0046] A1. Preparation of fermentation broth containing alkaline protease

[0047] Bacillus licheniformis with the product number BNCC360660 produced by Beijing NaChuangLian Biotechnology Co., Ltd. is selected as the production strain; the production strain is inoculated into a sterile fermentation medium containing a carbon source, a nitrogen source, and necessary growth factors, and aerated and stirred for fermentation to prepare a fermentation broth containing alkaline protease;

[0048] A2. Flocculation and filtration of the fermentation broth;

[0049] A3. Ultrafiltration and concentration;

[0050] A4. Preparation of solid enzyme preparation;

[0051] A5. Drying and screening to make solid or granular alkaline protease;

[0052] Steps A2 to A5 are carried out according to the method described in the patent number 202211187843.8.

[0053] The fermentation medium in step A1 is composed of the following raw materials:

[0054] Corn flour 10% - 12%, soybean meal 8% - 10%, disodium hydrogen phosphate 0.3% - 0.5%, sodium carbonate 0.1% - 0.3%, soybean oil 0.1% - 0.3%, and the balance is water.

[0055] The aeration and stirring fermentation conditions in step A1 are as follows: the medium filling amount is 63% - 70%, the inoculation amount is 9.5% - 10.5%, the temperature is 37°C ± 0.5°C, pH = 6.6 - 7.1, and the ventilation volume is 800 - 1300m 3 / h, rotation speed is 110 - 130 revolutions per minute, and the fermentation cycle is 35 - 40 hours.

[0056] The step of enlarged cultivation is to make a seed liquid by enlarging the cultivation of Bacillus licheniformis with product number BNCC360660 produced by Beijing Nacreative Biotech Co., Ltd., and inoculate the seed liquid into the fermentation medium to make a fermentation liquid containing alkaline protease through aerated stirring fermentation.

[0057] Among them, the shake flask seed is to inoculate Bacillus licheniformis with product number BNCC360660 produced by Beijing Nacreative Biotech Co., Ltd. into the shake flask medium and make the shake flask seed through oscillating cultivation; the shake flask medium is composed of the following raw materials: beef extract 0.9% - 1.3%, peptone 0.9% - 1.3%, sodium chloride 0.3% - 0.6%, and the balance is water. A shake flask with a capacity of 2000 ml is used to make the volume of the shake flask medium 550 ml; the conditions for oscillating cultivation are pH = 7 - 7.5, temperature is 37°C ± 0.5°C, and time is 8 - 12 hours.

[0058] The preparation of the fermentation seed is to inoculate the shake flask seed into the seed medium and make the fermentation seed through aerated cultivation; the seed medium is composed of the following raw materials: corn flour 3% - 4%, soybean meal 5% - 6%, disodium hydrogen phosphate 0.2% - 0.3%, sodium carbonate 0.08% - 0.1%, soybean oil 0.4% - 0.6%, and the balance is water. A seed tank with a capacity of 2 tons is used to make the volume of the seed medium 1.1 - 1.3 tons; the conditions for aerated cultivation are pH = 6.8 - 7.1, temperature is 37°C ± 0.5°C, the ventilation volume is 800 - 1300 m 3 / h, stirring speed is 110 - 130 revolutions per minute, and the time is 35 - 40 hours.

[0059] Comparison of the survival rate of alkaline protease at 55°C in Example 2

[0060] The applicant conducted the following tests to detect the application performance of the alkaline protease preparation prepared in Example 1. The test conditions were: sodium chloride 0 - 3%, pH = 7 - 9, time 0 - 90 minutes, temperature 55°C, simulating the protease survival rate under the enzymatic hydrolysis conditions in heparin sodium production. The specific results are shown in Table 1.

[0061] Table 1 Survival rate of alkaline protease prepared in Example 1 at 55°C

[0062]

[0063]

[0064] As can be seen from Table 1, under the conditions of 3% sodium chloride content, pH = 7, and temperature of 55°C, the protease survival rate is the highest within 90 minutes. Currently, the generally selected conditions in most enzymatic hydrolysis processes are: sodium chloride concentration of 3%, pH = 11, and temperature of 55°C. When using enzymatic hydrolysis conditions of 2% sodium chloride concentration, pH = 7 - 9, and temperature of 55°C, the difference in protease survival rate from the current enzymatic hydrolysis process conditions is not obvious. From the perspective of the protease survival rate index, the usage of sodium chloride and sodium hydroxide can be reduced. Under the enzymatic hydrolysis conditions of 0% sodium chloride concentration, pH = 7 - 9, and temperature of 55°C, compared with the protease survival rate under the current enzymatic hydrolysis process conditions, it decreases by 30% - 45%. In the case of 0% sodium chloride concentration, although the performance of the enzyme preparation prepared in Example 1 decreases, it can still be used.

[0065] Comparative Analysis of the Yield of Crude Heparin Sodium Prepared in Example 3

[0066] To compare the differences in the effects of the enzyme preparation in Example 1 and the enzyme preparation in the existing enzymatic hydrolysis process in the preparation of crude heparin sodium, the applicant conducted the following tests, and the specific results are shown in Tables 2, 3, 4, 5, and 6.

[0067] 1. Test basis: Use the production process parameters to simulate small tanks to calculate the yields of the existing enzyme preparation and the enzyme preparation in Example 1 for preparing crude heparin sodium;

[0068] 2. Test location: Changshan Kaikude Biotechnology Co., Ltd.;

[0069] 3. Test time: June 2024 - September 2024;

[0070] 4. Test materials: Pig small intestines (provided by Hebei Hongdu Industrial Group Co., Ltd., Linyi Xinshengyuan Food Co., Ltd., Linyi Junda Livestock Products Co., Ltd., Linyi Shunfa Food Co., Ltd., Shaanxi Pucheng Dahongmen Meat Food Co., Ltd., Fuxin City Food Co., Ltd., Daqing Xinqingji Meat Industry Co., Ltd., Panjin Hexiang Wangsheng Meat Food Co., Ltd., Heilongjiang Tiancaogang Agricultural Group Co., Ltd., Hulunbuir Dahongmen Meat Food Co., Ltd., Hebei Anping Dahongmen Food Co., Ltd. respectively), 2 - ton reaction tank, sodium chloride, ethanol, sodium hydroxide, 5 kg of resin, water, oven; the food - grade granular alkaline protease prepared in Example 1 (represented by "new enzyme" hereinafter), and the commercially available alkaline protease preparation (manufactured by Henan Xinyangshao Biotechnology Co., Ltd., with an enzyme activity of 200,000 units / gram for all products, represented by "old enzyme" hereinafter);

[0071] 5. Test procedure: Add sodium chloride to a concentration of 3% by total volume. Adjust the pH to 9 or 11 with sodium hydroxide, pH = 11 for the old enzyme, and pH = 9 for the new enzyme. Add 5 g / root of alkaline protease with an enzyme activity of 200000 u / g directly into the slurry. For small intestines whose mucosal quality does not meet the requirements, production makes a quality conversion for them, and adds enzymes according to the converted number of roots. The small intestines for which the number is not converted are those whose small intestine mucosal quality meets the enzyme usage condition of 5 g. The enzymatic hydrolysis temperature is 55 °C. The enzymatic hydrolysis time for the old enzyme is 100 and 120 minutes respectively, and the enzymatic hydrolysis time for the new enzyme is 45, 60, 75, 90, and 120 minutes respectively. The inactivation condition of the enzyme preparation is a temperature of 75 °C and a time of 15 minutes. Adsorb with 5 kg of resin at 55 °C - 60 °C for 8 hours. Wash the resin adsorbed with heparin to remove the suspended matter with water and filter it dry. Wash the resin 3 times with an aqueous sodium chloride solution with a concentration of 3.8% and a volume 1.5 times the volume of the resin, and filter it dry. Then elute the resin 2 times with an aqueous sodium chloride solution with a concentration of 18% - 20% and a volume 1.5 times the volume of the resin, and combine the two eluates. Add ethanol to the eluate until the ethanol concentration reaches 40% - 55% for precipitation for 12 hours, and filter the precipitate. Dry at 75 °C and assay the weight and potency of the crude heparin sodium.

[0072] 6. Glossary: Overnight refers to the test conducted the next day; The yield refers to the number of small intestines consumed for producing every 100 million heparins, that is, the number of small intestine roots required for producing 100 million heparins. Yield = (calculated number of roots ÷ heparin weight ÷ heparin potency) × 1000 × 100; The lower the number, the less raw material is consumed.

[0073] Table 2 Statistics of the yield of crude heparin sodium prepared with the old enzyme

[0074]

[0075] All the test raw materials used in Table 2 were sampled from the production workshop before the start of production. Based on the actual production plan arrangement, the small intestines used in the test were from different suppliers. Production personnel would first evaluate the quality of the small intestine mucosa. Usually, the mucosal quality of each small intestine meets the enzyme usage standard of 5 g / root, and the enzymatic hydrolysis process is carried out accordingly. When encountering the situation of scarce small intestine mucosa, production will adjust the enzyme usage standard: convert the number of small intestines with insufficient quality into the number of roots with normal quality, count this as the number of enzyme-added roots, and multiply by the enzyme usage standard to adjust the enzyme amount. Thus, it avoids the problem of excessive enzyme usage due to insufficient mucosal quality. Therefore, the test process is closer to the real situation of enterprise production, ensuring the authenticity of the test and the results.

[0076] Based on the provided test data and analysis, the old enzyme showed obvious instability during application, resulting in a large fluctuation in the yield of crude heparin sodium. The following is a detailed analysis:

[0077] The test data under the same supplier, test method, mucosal quality, enzyme dosage standard, enzymolysis time, and similar number of small intestines are as follows:

[0078] 1. Shunfa: YF240628, 113 pieces, yield 1625; YF240712, 100 pieces, yield 2045. The number of small intestine raw materials from the same supplier differs by 13 pieces (113 - 100), but the yield differs by 420 pieces (2045 - 1625).

[0079] 2. Under the same test method, mucosal quality, number of small intestines, enzyme dosage standard, and enzymolysis time, tests were conducted on small intestine raw materials from different suppliers. The specific data are as follows:

[0080] Shunfa: YF240628, 113 pieces, yield 1625;

[0081] Pucheng: YF240624, 113 pieces, yield 1276;

[0082] Theoretically, even if the suppliers are different, under the same mucosal quality, quantity, and method, the yield should be basically the same. However, the results show that the yield difference between the two is as high as 349 pieces (1625 - 1276).

[0083] 3. When the scope is extended to three different suppliers, for small intestines with similar quantities and other conditions remaining unchanged, the yield difference further expands (1276 - 2119). The specific data are as follows:

[0084] Shunfa: YF240628, 113 pieces, yield 1625;

[0085] Pucheng: YF240624, 113 pieces, yield 1276;

[0086] Fuxin: YF240718, 120 pieces, yield 2119;

[0087] 4. When the scope is extended to five different suppliers, for small intestines with similar quantities and other conditions remaining unchanged, the yield gap is even more significant (1276 - 2390). The specific data are as follows:

[0088] Shunfa: YF240628, 113 pieces, yield 1625;

[0089] YF240712, 100 pieces, yield 2045;

[0090] Pucheng: YF240624, 113 pieces, yield 1276;

[0091] Fuxin: YF240718, 120 pieces, yield 2119;

[0092] Xinqingji: YF240716, 130 pieces, yield 2390;

[0093] 5. Analyzing from the differences in results, the application performance of traditional enzyme preparations (powder, industrial grade) shows significant instability. Even when conditions such as the number of small intestine roots, mucosal quality, enzyme usage standards, and test methods are similar or the same, it is still impossible to obtain stable and similar results, and there are large deviations. Judging from the test data and the yield of the enzymatic hydrolysis process in the industry, this significant difference truly exists. This indicates that the application performance of the old enzyme is extremely unstable, resulting in large fluctuations in the yield under the same conditions.

[0094] To sum up, the instability of the application performance of the old enzyme is the key factor leading to large fluctuations in the yield of crude heparin sodium, which in turn affects the stability of production. In the current enzymatic hydrolysis process, the performance of the enzyme plays a crucial role in the yield. The dosage of the enzyme preparation needs to be adjusted according to the small intestine mucosal quality, solubility, and the quality of the enzyme preparation, etc. These variables bring many uncertainties and instabilities to the production process control. Therefore, to effectively improve the stability of the enzymatic hydrolysis process, it is particularly crucial to use enzyme preparations with more stable and reliable performance.

[0095] Table 3 Statistical table of the yield of crude heparin sodium prepared with the alkaline protease particles prepared in Example 1

[0096]

[0097] 1. In Table 3, the application stability of the new enzyme: Under the same conditions of test method, mucosal quality, number of small intestines, enzymatic hydrolysis time, and supplier, etc., compared with the old enzyme, the yield gap of each group of the new enzyme is very small, showing its extremely high application stability.

[0098] 2. Yields of small intestines from the same supplier but different batches

[0099] Under different batches, but the same conditions of supplier, mucosal quality, enzymatic hydrolysis time, similar number of roots, etc., the yield still maintains extremely high stability. This indicates that the raw material quality of the supplier is relatively stable. On the premise that the mucosal quality meets the production requirements, it can be seen that the difference in mucosal quality has a slight impact. The specific data is as follows:

[0100] Shunfa (120 min): YF240627, 113 pieces, yield 1906;

[0101] YF240711, 100 pieces, yield 1846.

[0102] Fuxin (60 min): YF240719, 120 pieces, yield 1937;

[0103] YF240829, 123 pieces, yield 2068.

[0104] The difference in Shunfa is 60 roots (1906 - 1846), and the difference in Fuxin is 131 roots (2068 - 1937).

[0105] 3. Small intestine recovery rates of different suppliers

[0106] Under the conditions of the same time (60 minutes), mucosal quality, and similar number of roots, among the five recovery rates of different suppliers, the recovery rate performances are basically the same. The specific data are as follows:

[0107] Xinshengyuan, Tiancaogang: YF240911, 115 roots, recovery rate 2266;

[0108] YF240911, 115 roots, recovery rate 2182;

[0109] Fuxin: YF240719, 120 roots, recovery rate 1937;

[0110] YF240829, 123 roots, recovery rate 2068;

[0111] Shunfa, Hexiang, Anping: YF240824, 117 roots, recovery rate 2028;

[0112] Compared with the five recovery rates of the old enzyme under the same conditions, the recovery rate difference of the new enzyme is significantly reduced (1937 - 2266), which indicates that the application performance of the new enzyme is more stable. From the perspective of mucosal quality, there are natural individual differences in the mucosal quality of the small intestine, and this difference needs to be controlled within an acceptable range, and the differences in production results also need to conform to the situation. When using the new enzyme, under the condition of meeting the production conditions, the small intestine quality of different suppliers has less impact on the recovery rate difference, and this difference may be due to the difference in mucosal quality.

[0113] 4. Small intestine recovery rates at different enzymatic hydrolysis times

[0114] By adjusting the time, when the enzymatic hydrolysis reaction is relatively sufficient, the recovery rate will be stable and increase significantly. The specific data are as follows:

[0115] Fuxin: YF240719, 120 roots, 60 min, recovery rate 1937;

[0116] YF240829, 123 roots, 60 min, recovery rate 2068;

[0117] YF240910, 106 roots, 75 min, recovery rate 1325;

[0118] YF240909, 106 roots, 90 min, recovery rate 1399;

[0119] It can be seen from this that the new enzyme performs excellently in application, with excellent stability and reliability. Under the condition of the same mucosal quality and proper process control, using the new enzyme can not only ensure stable production but also significantly improve the yield. With this advantage, users can formulate the best production process by adjusting the enzymolysis time according to the mucosal quality of the small intestine. It can not only ensure precise control of the process and stable production operation, but also greatly reduce raw material consumption and achieve the goal of economic and efficient production.

[0120] Table 4 Statistical results of the influence of the new enzyme on the yield at different times when the same small intestine manufacturer uses a similar quantity

[0121]

[0122] As can be seen from Table 4, with the increase of the enzymolysis time, the yield shows an obvious upward trend, indicating that the enzymolysis time has a significant impact on the yield. The experimental data reveals the controllability of the enzymolysis degree: with the prolongation of the enzymolysis time, the enzymolysis degree gradually deepens, the reaction is more thorough, and thus the yield is improved. It provides an important reference basis for precisely controlling the enzymolysis process.

[0123] However, at the enzymolysis time of 75 minutes, the yield shows a certain degree of instability, which may be related to factors such as mucosal quality or details of the test operation. Therefore, it is necessary to appropriately extend the enzymolysis time to ensure the thoroughness of the reaction.

[0124] When observing the last two data, the applicant noticed that a higher yield was obtained with less small intestine used. This phenomenon also occurred in the tests of Shunfa. Under the condition that the mucosal quality of the small intestine remains stable, it is speculated that this result is due to the improvement of the quality of the enzymolysis solution by the enzyme preparation in Example 1. The decrease in substrate concentration promotes a more sufficient reaction between the enzyme and the substrate, thus enhancing the enzymolysis effect. In addition, the use of the enzyme preparation in Example 1 improves the quality of the enzymolysis solution, reduces the interfering factors (such as pigments, other ions, etc.) that affect resin adsorption in the enzymolysis solution, enables the resin to have more space to adsorb heparin, and thus improves the yield. If this speculation holds, it can not only reduce raw material consumption but also increase the yield, which not only helps to improve production efficiency but also reduces costs, bringing greater economic benefits to heparin production enterprises.

[0125] Table 5 Comparison of the yields of the new enzyme and the old enzyme

[0126]

[0127] On the premise that all experimental conditions remain the same, a comparative analysis of the usage effects of the new enzyme and the old enzyme is carried out to reveal the differences in their application performance.

[0128] By comparing the test data in Table 5, the application effect of the new enzyme is more significant than that of the old enzyme, and it has better stability.

[0129] Using small intestines from the same supplier, different batches, and similar numbers of roots for the test, the yield difference of the new enzyme is only 60 roots (1846 - 1906), while the yield difference of the old enzyme is as high as 420 roots (1625 - 2045). This not only confirms the stability of the new enzyme in application but also indicates that when the raw material quality meets the production requirements, the yield fluctuations are mainly caused by the performance of the enzyme, rather than the differences in raw material batches or suppliers.

[0130] The problem of the low yield of the new enzyme is mainly due to excessive enzymatic hydrolysis. Given the characteristics of the new enzyme and its performance in other data, it is necessary to shorten the enzymatic hydrolysis time to achieve a better yield.

[0131] In the enzymatic hydrolysis process, if the application performance of the enzyme does not meet or does not match the industry control key points, the advantages of enzymatic hydrolysis cannot be exerted. This not only involves the variety of the enzyme but also the matching of other aspects of the enzyme with the technological key points. After all, the decomposition of proteins by proteases is its specificity, and the quality of the decomposition effect depends on the application performance of the enzyme, and the two are not the same concept.

[0132] In summary, the application effect of the new enzyme is more significant and it has better stability. Under the experimental conditions of the same raw material quality, maintaining a stable yield is crucial for stable production.

[0133] Table 6: Tests of Shunfa's New Enzyme and Old Enzyme

[0134]

[0135] Remarks: The small intestine supplier is Linyi Shunfa Food Co., Ltd., abbreviated as Shunfa.

[0136] After analyzing the test data of Shunfa when using the new enzyme and the old enzyme in Table 6, the following relevant situations can be comprehensively elaborated:

[0137] 1. Protein removal performance: By comparing the test data of 113 roots and 100 roots of the new and old enzymes, the details are as follows:

[0138] 2. Change in heparin weight from 113 roots to 100 roots of the new enzyme: (114 - 98.5) ÷ 114 = 13.6%.

[0139] 3. Heparin produced per root of small intestine by the new enzyme: 5928 ÷ 113 = 52.46, 5417.5 ÷ 100 = 54.18.

[0140] 4. Heparin produced per small intestine by the old enzyme: 6952÷113 = 61.52, 4890÷100 = 48.90. On the premise that the reaction conditions remain unchanged, when the number of small intestines decreases by 11.5% (13÷113), the amount of product produced with the same quality should also decrease accordingly. The weight of heparin obtained with the new enzyme decreased by 13.6%, and the titer remained stable. The old enzyme did not show such stability. When comparing the heparin produced per small intestine, the results of the new enzyme were consistent, which also confirmed that the new enzyme was basically consistent in the protein removal effect. To sum up, the protein removal performance of the new enzyme is stable.

[0141] Example 3

[0142] Impact of heparin weight and titer after enzymatic hydrolysis reaction using new and old enzyme preparations on cost

[0143] In the group standard "Quality Management Guidelines for the Production of Crude Heparin" (T / CBPIA 0001—2022, issued by the China Biochemical Pharmaceutical Industry Association), it is pointed out regarding the titer of crude heparin sodium: "Calculated on the dried product, the anticoagulant titer of this product per 1 mg shall not be less than 50 IU. Or the acceptable minimum anticoagulant titer of this product shall be determined by the crude heparin producer according to the registration process of the MAH and the downstream heparin API producer." In the Chinese Pharmacopoeia, regarding the titer standard of heparin sodium API, it is proposed that calculated on the dried product, the titer per 1 mg shall not be less than 150 IU.

[0144] The titer level and yield stability of crude heparin sodium directly affect the production cost. A higher titer indicates that the protein content in the enzymatic hydrolysate is relatively low. By increasing the resin dosage, more heparin can be adsorbed. In this way, the enterprise can not only improve the overall production efficiency but also reduce the relevant production costs, bringing higher economic benefits and market competitiveness to the enterprise.

[0145] Table VII Test data of the dried product titer of the old enzyme

[0146]

[0147] It can be seen from the test data in Table VII that under the same process conditions, using the old enzyme not only leads to large fluctuations in the yield but also relatively high heparin weight, and most of the heparin titers fail to reach the minimum titer standard (50 IU) specified in the "Quality Management Guidelines for the Production of Crude Heparin". This indicates that using the old enzyme may result in unqualified product quality, which will increase the production costs in the purification process, such as raw material consumption, energy, and the time and labor costs of re-precipitation and purification, thus having an adverse impact on the overall production efficiency and economic benefits, whether for the enterprise's own use or selling the product to downstream API enterprises.

[0148] Table VIII Test data of the dried product titer of the new enzyme

[0149]

[0150]

[0151] As can be seen from the data in Table VIII, in the tests using the new enzyme, all titers were greater than the minimum standard (50 IU), and this result provided a strong guarantee that the crude product met the quality standard. At the same time, the heparin weight decreased compared with the old enzyme. If the yield difference was not significant, this had a significant advantage in reducing the extraction cost of the active pharmaceutical ingredient.

[0152] The following is a detailed analysis of the data:

[0153] 1. Comparative analysis of the crude product quality differences between the new and old enzymes

[0154] To objectively and accurately evaluate the crude product quality differences between the new and old enzymes, referring to Table VI, five test data of the old enzyme (120 min) and the new enzyme (60 min) with consistent test conditions and similar numbers of roots were selected for analysis. The heparin weights and total titers of the five tests were comprehensively calculated respectively, and then the average titer was obtained. The relevant calculations are as follows:

[0155] (1) Old enzyme

[0156] Total heparin weight 651 g (163 + 158 + 111 + 96 + 123);

[0157] Total titer 31801 IU (4890 + 6952 + 5439 + 5664 + 8856);

[0158] Average heparin titer 48.85 IU (31801 ÷ 651);

[0159] Number of small intestine roots: 576 roots (100 + 113 + 130 + 120 + 113).

[0160] (2) New enzyme

[0161] Total heparin weight 381.76 g (105 + 70.5 + 72.2 + 69.96 + 64.1);

[0162] Total titer 28257.2 IU (6195 + 5079 + 5271 + 5947 + 5769);

[0163] Average heparin titer 74.02 IU (28257.2 ÷ 381.76);

[0164] Number of small intestine roots: 590 roots (120 + 115 + 115 + 123 + 117).

[0165] (3) Comparison of weights

[0166] (651 - 381.76)÷651 = 41.36%,

[0167] (4) Comparative titer

[0168] (74.02 - 48.85)÷48.85 = 51.53%.

[0169] It can be clearly seen from the comparison data that the total titer of the new enzyme is lower than that of the old enzyme, and 14 more small intestine roots are consumed. However, the total heparin weight is reduced by 41.36%, and the average heparin titer is increased by 51.53%. It should be emphasized that this result was obtained even when the enzymatic hydrolysis reaction was not sufficient.

[0170] 2. Comparison of crude product quality differences after extending the enzymatic hydrolysis time

[0171] With similar numbers of small intestine roots, when the time is extended (75 and 90 min), the yield is significantly increased. Referring to Table VIII, the data of these four tests with 126 and 106 roots were analyzed in the same way, and the relevant calculations are as follows:

[0172] (1) New enzyme

[0173] Total heparin weight: 403.88 g (108.18 + 102.6 + 108.9 + 84.2),

[0174] Total titer: 31861.3 IU (8221.7 + 8003 + 8058.6 + 7578),

[0175] Average heparin titer: 78.89 IU (31861.3÷403.88),

[0176] Number of small intestine roots: 464 (126 + 126 + 106 + 106).

[0177] (2) Comparative weight

[0178] (651 - 403.88)÷651 = 37.96%,

[0179] (3) Comparative titer

[0180] (78.89 - 48.85)÷48.85 = 61.49%.

[0181] It can be seen by comparing the total titers that the total titer of the new enzyme is slightly higher than that of the old enzyme this time, but the small intestine consumption is reduced by 112 roots. As the protein decomposes, the heparin activity increases. When the enzymatic hydrolysis reaction is sufficient, the new enzyme can significantly increase the yield. Under these four groups of tests this time, the new enzyme is already equivalent to the results of five tests with the old enzyme. At the same time, with basically the same total titer, the total weight obtained is reduced by 37.96%, and the average heparin titer is increased by 61.49%.

[0182] In summary, after comprehensive comparison of various data such as stability, heparin weight, heparin potency, and yield, the new enzyme has very significant advantages in terms of the stability of product quality and the control of the extraction cost of the active pharmaceutical ingredient.

[0183] Example 4: Reduction of the COD of the enzymatic hydrolysis residue

[0184] The reduction of the COD of the enzymatic hydrolysis residue is due to fractional extraction. The following is the process of fractional extraction:

[0185] Currently, intestinal residue protein is produced as a by-product and sold under the salting-out process. Through in-depth investigation, the applicant found that if an enzymatic hydrolysis residue recovery process is introduced, the enterprise can extract a variety of high-value-added by-products from a large amount of enzymatic hydrolysis residue:

[0186] 1. Feed protein raw material: The solid residue in the enzymatic hydrolysis residue is recovered and dried to prepare a feed protein raw material. Compared with the intestinal residue protein produced by the traditional salting-out process, this product has higher nutritional value and functionality.

[0187] 2. Crude heparin sodium: Heparin in the enzymatic hydrolysis residue is recovered through a membrane system, which can significantly improve the heparin sodium yield. The test data shows that the potency of the enzymatic hydrolysis residue is usually between 0.5 and 1. Retaining the residue can not only reduce the volume and increase the potency, but also facilitate operation and treatment. For example, taking 400 tons of enzymatic hydrolysis residue as an example, at least 25 tons of concentrated heparin residue can be obtained, and its value cannot be ignored.

[0188] 3. Enteric-coated peptides: The remaining residue after adsorption can be added with a carrier and dried to prepare enteric-coated peptide powder (industrial grade and food grade), or used for extracting other related products.

[0189] 4. Compound amino acids: Compound amino acids are extracted from the small molecule liquid, added with a carrier and dried, and can be used for special feeds for animals (young) or food-grade compound amino acid raw materials with higher added value, and their output is quite large.

[0190] 5. The above residue recovery process is based on the fact that the solid enzyme preparation in the present invention can better decompose proteins into small molecule substances. Using the solid enzyme preparation in the present invention can significantly reduce the heparin weight and increase the heparin potency. This means that more small peptides, compound amino acids and other small molecule substances will be decomposed in the enzymatic hydrolysis solution. By effectively recovering the above substances, the enterprise can not only achieve the full utilization of resources, but also significantly improve economic benefits.

Claims

1. The application of a solid alkaline protease preparation in improving the yield of crude heparin sodium comprises adding alkaline protease to a porcine small intestinal mucosa solution stirred into a mushy state and heating the solution for enzymolysis, inactivating the enzyme preparation after the enzymolysis is completed, filtering the enzymolysis solution, adsorbing the filtrate with an anion resin, eluting the anion resin after the adsorption of the filtrate with a sodium chloride solution, and extracting the eluate with an ethanol solution to obtain the crude heparin sodium; characterized in that The process steps include: A. Preparation of solid or granular alkaline protease A1. Preparation of fermentation broth containing alkaline protease Bacillus licheniformis (Bacillus licheniformis) produced by Beina Chuanglian Biotechnology Co., Ltd. with a product number of BNCC360660 was selected as the production strain; the production strain was inoculated into a sterile fermentation medium containing a carbon source, a nitrogen source and necessary growth factors, and a fermentation liquid containing alkaline protease was prepared by aeration and stirring. A2, flocculation and filtration of fermentation liquid; A3, ultrafiltration concentration; A4. Preparation of solid enzyme preparation; A5, drying and sieving to form solid or granular alkaline protease; Steps A2 to A5 are performed according to the method described in Patent No. 202211187843.8; B. Preparation of crude heparin sodium B1. Enzymatic hydrolysis of pig small intestine The sodium chloride concentration in the porcine small intestine mucosa solution stirred into a mushy state is adjusted to 0-3% and the pH value is 7-9, and 3-10 g of the solid or granular alkaline protease prepared in step A is added to each small intestine, and the solid or granular alkaline protease is added to the mushy porcine small intestine solution and heated to 45° C.-55° C. and maintained for 60-90 minutes for enzymolysis, and after the enzymolysis is completed, the alkaline protease is inactivated by heating; B2. Adsorbed heparin Filter the enzymatic hydrolyzate and cool the filtrate to 50°C to 60°C, adsorb the cooled filtrate using an anion exchange resin for 8 to 10 hours, and then collect the resin; B3, heparin elution Using sodium chloride solution to elute the anion exchange resin adsorbed with heparin in step B2; B4. Obtaining crude heparin sodium The eluate is precipitated with an ethanol solution having a volume percentage concentration of 40% to 55%, and the precipitate is dried to obtain crude heparin sodium.

2. The use of the solid alkaline protease preparation according to claim 1 in improving the yield of crude heparin sodium, characterized in that The fermentation medium in step A1 is composed of the following raw materials: Corn flour 10%-12%, soybean meal 8%-10%, disodium hydrogen phosphate 0.3%-0.5%, sodium carbonate 0.1%-0.3%, soybean oil 0.1%-0.3%, and the balance is water.

3. Use of the solid alkaline protease preparation according to claim 1 or 2 in improving the yield of crude heparin sodium, characterized in that The aeration and stirring fermentation conditions in step A1 are as follows: the culture medium loading is 63% to 70%, the inoculation amount is 9.5% to 10.5%, the temperature is 37°C ± 0.5°C, the pH is 6.6 to 7.1, and the ventilation volume is 800 to 1300 m 3 / h, the rotation speed is 110-130 rpm, and the fermentation period is 35-40 hours.

4. Use of the solid alkaline protease preparation according to any one of claims 1 to 3 in improving the yield of crude heparin sodium, characterized in that The method also includes an expansion culture step, which is to expand and culture Bacillus licheniformis (Bacillus licheniformis) produced by Beina Chuanglian Biotechnology Co., Ltd. and with a product number of BNCC360660 to prepare a seed liquid, inoculate the seed liquid into a fermentation medium, and ferment with ventilation and stirring to prepare a fermentation liquid containing alkaline protease.

5. Use of the solid alkaline protease preparation according to claim 4 in improving the yield of crude heparin sodium, characterized in that The enlarged culture comprises the preparation of shaking flask seeds and fermentation seeds, wherein the shaking flask seeds are prepared by inoculating Bacillus licheniformis produced by Beina Chuanglian Biotechnology Co., Ltd. and having a product number of BNCC360660 into a shaking flask medium, and then vibrating the medium to prepare the shaking flask seeds; the shaking flask medium is composed of the following raw materials: 0.9% to 1.3% beef extract, 0.9% to 1.3% peptone, 0.3% to 0.6% sodium chloride, and the balance is water; a shaking flask with a capacity of 2000 ml is used to set the shaking flask medium to 550 ml; the vibrating culture is carried out under the conditions of pH=7 to 7.5, temperature of 37°C±0.5°C, and time of 8 to 12 hours.

6. Use of the solid alkaline protease preparation according to claim 5 in improving the yield of crude heparin sodium, characterized in that The preparation of fermented seeds is to inoculate shake flask seeds into seed culture medium and then culture them through ventilation to prepare fermented seeds; the seed culture medium is composed of the following raw materials: 3% to 4% corn flour, 5% to 6% soybean meal, 0.2% to 0.3% disodium hydrogen phosphate, 0.08% to 0.1% sodium carbonate, 0.4% to 0.6% soybean oil, and the balance is water; a seed tank with a capacity of 2 tons is used to set the seed culture medium to 1.1 to 1.3 tons; the conditions for aeration culture are pH = 6.8 to 7.1, temperature is 37°C ± 0.5°C, and ventilation volume is 800 to 1300m 3 / h, stirring speed 110-130 rpm, time 35-40 hours.

7. Use of the solid alkaline protease preparation according to claim 1 in improving the yield of crude heparin sodium, characterized in that In step B1, the pH is adjusted using sodium hydroxide or sodium hydroxide solution.

8. Use of the solid alkaline protease preparation according to claim 1 in improving the yield of crude heparin sodium, characterized in that The step B3 is to rinse the heparin-adsorbed resin with water to remove the suspended matter and then filter it; elute it 2 to 3 times with a sodium chloride aqueous solution with a concentration of 3.5% to 4.5% and filter it; elute it 2 to 3 times with a sodium chloride aqueous solution with a concentration of 18% to 24% and 1.5 to 2 times the resin volume, and save and combine the 2 to 3 eluates.

9. Use of the solid alkaline protease preparation according to claim 1 in improving the yield of crude heparin sodium, characterized in that In the step B4, the eluent is an ethanol solution with a concentration of 40% to 55% and the precipitation is carried out for 12 hours.

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