Salt-free production technology of heparin sodium crude product extracted by enzymolysis method of bio-enzyme fermentation

Abstract

The application discloses a salt-free production technology of heparin sodium crude product extracted by enzyme hydrolysis method of biological enzyme fermentation, and belongs to the technical field of production of heparin sodium crude product extracted from pig small intestinal mucosa. The application adopts controllable biological fermentation technology of bioactive enzyme combined with enzyme hydrolysis method to extract heparin sodium biological macromolecule with anticoagulant biological activity. The yield of anticoagulant activity in the obtained enzyme hydrolysis liquid is 96,000 USPU / root, and the yield of dried solid heparin sodium crude product is 82,000 US standard units / root. The technical scheme of the application greatly improves the yield of heparin sodium crude product, simultaneously reduces 73% of wastewater and 99% of chloride ion content, greatly reduces the difficulty of wastewater treatment, makes it possible to change waste into treasure by comprehensive recycling of wastewater, and is favorable for industrial application.

Description

TECHNICAL FIELD

[0001] The present application relates to a salt-free production technology of crude heparin sodium extracted by enzymatic hydrolysis of biological enzyme fermentation, and belongs to the technical field of production of crude heparin sodium extracted from pig small intestinal mucosa. BACKGROUND

[0002] Heparin sodium is a biological drug widely used in the medical field for anticoagulant and antithrombotic purposes. Heparin anticoagulants are widely used for (a) treatment of disseminated intravascular coagulation in early stage of various diseases; (b) prevention of venous and pulmonary embolism; (c) treatment of venous and pulmonary embolism, ischemic stroke, unstable angina (symptom relief, prevention of myocardial infarction), acute myocardial infarction (prevention of early reinfarction and infarction area extension, reduction of mortality); (d) as maintenance therapy for thrombolytic therapy; (e) used for blood coagulation prevention and blood bank preservation of fresh blood as an extracorporeal anticoagulant.

[0003] The molecular structure of heparin sodium is extremely complex, and no one has successfully synthesized heparin sodium. It can only be extracted from animal tissues. At present, the official drug administration recognizes the source of heparin sodium, which is extracted from pig small intestinal mucosa. Heparin sodium is an acidic mucopolysaccharide, which is a mucopolysaccharide sulfate alternatingly composed of glucosamine, L-iduronic acid, N-acetylglucosamine and D-glucuronic acid, with an average molecular weight of 15KD, mainly produced by mast cells and basophilic granulocytes.

[0004] The raw material for producing crude heparin sodium is pig small intestine. The intestinal mucosa is separated from the intestinal lining by a scraping machine. At present, the existing production technology of crude heparin sodium in the industry mainly includes high-salt salt dissolution method, medium-salinity and dilute mucosa liquid enzymatic hydrolysis method, salt dissolution and enzymatic hydrolysis complex method, and medium-salinity and dilute mucosa liquid complex enzyme double enzyme hydrolysis method. Especially in the high-salt salt dissolution method and the salt dissolution and enzymatic hydrolysis complex method, intestinal lining salt water and intestinal lining salt are added during the extraction process, resulting in a high salt concentration of 3.5% (medium salinity and dilute mucosa liquid), which makes wastewater treatment difficult and costly.

[0005] The wastewater after extracting crude heparin sodium contains a large amount of small intestinal mucosa protein and small molecule peptide nutrients, and the content is as high as 9-12%, which can be used as a high-quality nutrient additive for animal feed or a high-quality organic matter for organic compound fertilizer. However, the nutrient additive and organic matter have strict requirements on the content of chloride ions (≤0.1%, i.e. 1000 ppm or less), and accordingly, the concentration of chloride ions in the wastewater after extracting crude heparin sodium needs to be controlled below 0.3% (3000 ppm) to realize the transformation of waste into treasure. In order to reduce the content of sodium chloride in the wastewater after extracting heparin sodium, the existing technology has a method for extracting crude heparin sodium without salt, but the existing salt-free extraction method either relies on specific equipment to achieve specific effects or cannot achieve the expected effects. Patent CN 115448995A relates to a salt-free extraction method of crude heparin sodium. The heparin sodium content obtained from 2000 animal small intestines finally reaches 1075g, but the yield is low, and ultrasonic is needed in the extraction, which has high requirements for the equipment. Patent CN 103724456A provides a normal temperature salt-free extraction process of heparin sodium, which uses a composite enzyme preparation composed of protease, lipase and nuclease for enzymolysis under normal temperature and salt-free conditions, and then uses ion exchange resin to adsorb heparin sodium in the centrifugal liquid, and finally obtains crude heparin sodium through elution, alcohol precipitation and drying. However, the extraction effect of the salt-free process is not disclosed, and it is still in the laboratory test stage and far from large-scale production.

[0006] In addition, by adjusting the pig small intestinal mucosa liquid into thick slurry during the extraction process, the water consumption can be reduced, and the amount of wastewater can be significantly reduced, and the treatment difficulty can be reduced. The existing technology does not have strict requirements on the volume of pig small intestinal mucosa liquid, and the volume of pig small intestinal mucosa liquid reaches 10 liters per pig small intestine, which results in unsatisfactory heparin sodium crude product yield and wastewater discharge. The salt-free production technology requires the volume of pig small intestinal mucosa liquid to be 2-4L per pig small intestine in the form of extremely thick slurry, which is of practical significance and can improve the yield of heparin sodium crude product, facilitate wastewater recovery, and reduce wastewater discharge.

[0007] However, thick slurry and salt-free conditions are both unfavorable for mucosa tissue rupture and lysis. On the one hand, the pig small intestinal mucosa liquid is too thick, which is not conducive to the full contact between the tissue and the enzyme, resulting in incomplete lysis of the small intestinal tissue, less "heparin sodium-protein complex" substrate, and affecting the yield of heparin sodium crude product; on the other hand, sodium chloride salt plays multiple functions such as preservation and providing ionic strength stability, and under salt-free conditions, the stability of production cannot be guaranteed. Under the double unfavorable conditions of thick slurry and salt-free, the efficiency of free heparin sodium molecules is low. At present, the average heparin sodium crude product yield of the existing thick slurry salt-free technology scheme abroad is 45,000 American standard units per pig small intestine (2222 per billion), which cannot meet the needs of industrial production.

[0008] In summary, the existing salt-free enzymatic technology scheme has three fatal defects. Firstly, the volume of each pig small intestinal mucosa reaches 10 liters, resulting in a large amount of wastewater discharge; secondly, there is no ion strength stabilizer, and there is no suitable alternative preservative to preserve the intestinal mucosa, resulting in uncontrollable production results; thirdly, there is no suitable and effective intestinal mucosa tissue rupture technology, which causes incomplete rupture of the intestinal mucosa tissue, resulting in a small amount of "heparin-sodium-protein complex" substrate that can be enzymatically hydrolyzed, which significantly affects the yield of heparin sodium crude product. The existing concentrated slurry salt-free extraction of heparin sodium crude product technology cannot meet the industrial demand. SUMMARY

[0009] The present application aims at the defects of large wastewater discharge, uncontrollable production results and low extraction rate in the technology of extracting heparin sodium crude product from pig small intestinal mucosa, and provides a biological fermentation technology using bioactive enzymes and bioactive bacteria such as probiotics. In the condition of salt-free and extremely thick concentrated slurry intestinal mucosa liquid, the selective enzymatic hydrolysis of pancreatin is used to produce free mast cells in the intestinal mucosa tissue, and the free mast cells are lysed as much as possible to produce the free heparin-sodium precursor "heparin-protein complex" in the maximum amount, so as to improve the yield of heparin sodium crude product.

[0010] The technical scheme of the present application comprises four series of technical processes of biology, enzymology and purification and separation science, and is carried out in six steps.

[0011] The first step: for the extremely thick concentrated slurry intestinal mucosa tissue liquid, no sodium chloride salt from any source can be added, but a reasonable amount of sodium sulfate is added as a necessary ion strength electrolyte stabilizer;

[0012] The second step: for the extremely thick concentrated slurry intestinal mucosa tissue liquid, sodium pyrosulfite is added as a preservative;

[0013] The third step: the biological fermentation technology of bioactive bacteria such as probiotics is used to achieve the biological activity fermentation technology process of effectively rupturing the extremely thick concentrated slurry intestinal mucosa tissue, rupturing the intestinal mucosa tissue and freeing the mast cells;

[0014] The fourth step: the biological activity fermentation technology process of lysing the mast cells, using bioactive enzymes, controls the bioactive fermentation technology of the free mast cells, and the free mast cells are lysed as much as possible to produce the free heparin-sodium precursor "heparin-protein complex" in the maximum amount;

[0015] The fifth step: using pancreatin enzymolysis, the free heparin-sodium precursor "heparin-protein complex" is subjected to pancreatin site-specific cleavage enzymolysis technology, so that the chemical bond of "heparin-protein complex" is subjected to "precise protein site cutting" site-specific high-efficiency selective enzymolysis cutting, and the mixed solution of heparin sodium biological macromolecules with anticoagulant biological activity and protein molecules is released;

[0016] Step 6: Purification and separation technology process, the released heparin sodium and protein mixed solution, using macroporous anion resin (Dupont Lomond - USA FPA98Cl macroporous strong anion heparin sodium special resin), preferentially selective adsorption / desorption process, to achieve the separation of heparin sodium. Further use of alcohol precipitation and classification technology, ultimately get heparin sodium crude product (biological activity of macromolecules purification and separation technology).

[0017] In one embodiment, the macroporous anion resin comprises Dupont Lomond - USA FPA98Cl macroporous strong anion heparin sodium special resin.

[0018] The present application provides a method for extracting heparin sodium crude product from pig small intestinal mucosa, comprising the following steps:

[0019] (1) Preparation of pig small intestinal mucosa solution: Step (1) The pig small intestine is scraped with softened water to prepare mucosa solution, and the mucosa solution is adjusted with softened water so that the volume of each pig small intestinal mucosa solution reaches 2.7L, or the volume of each pig small intestinal mucosa solution reaches 2.0-4.0L, which reduces the wastewater volume by 60-80% compared with the existing method. Sodium sulfate electrolyte and sodium metabisulfite preservative are added to the obtained thick pig small intestinal mucosa solution in sequence, and the pH is adjusted to 6.0-8.0 with 30% sodium hydroxide lye.

[0020] (2) Biological fermentation: adding biological active enzyme and auxiliary agent to the pig small intestinal mucosa solution obtained in step (1), and heating to 25-50℃ for 90-300 minutes to obtain fermented small intestinal mucosa solution; the addition amount of the biological active enzyme is 0.05-0.50% of the mass of the pig small intestinal mucosa solution, and the addition amount of the auxiliary agent is 0.15-1.50% of the mass of the pig small intestinal mucosa solution;

[0021] (3) Enzymatic hydrolysis: adjusting the pH of the fermented small intestinal mucosa solution obtained in step (2) to 7-9, adding 0.05-0.30% of the mass of the pig small intestinal mucosa solution of trypsin, and heating to 50-60℃ for 120-300 minutes to obtain an enzymatic hydrolysate;

[0022] (4) Resin adsorption: heating the enzymatic hydrolysate obtained in step (3) to 70-85℃ for 10-60 minutes, then filtering and cooling to 56-60℃, and then performing resin adsorption and elution to obtain a heparin sodium crude product solution; the mass of the resin is 1.0-3.0% of the mass of the pig small intestinal mucosa solution;

[0023] (5) Resin desorption: collecting the resin after resin adsorption in step (4). The collected resin is washed with 5% dilute brine and then eluted with saturated brine. The mass of the saturated brine is 100-200% of the mass of the resin, i.e. a heparin sodium crude product solution is obtained;

[0024] (6) Alcohol precipitation: the crude heparin sodium solution obtained in step (5) is precipitated with 60-90% alcohol, the final concentration of alcohol precipitation is 25-55%, and the crude heparin sodium is precipitated and allowed to stand for 12-24 hours;

[0025] (7) Collection of crude heparin sodium: the crude heparin sodium obtained in step (6) is filtered, dehydrated and dried to obtain the crude heparin sodium product.

[0026] (8) Storage: the crude heparin sodium product obtained in step (7) is registered and stored.

[0027] In one embodiment, the viscous concentrated slurry pig small intestinal mucosa solution in step (1) is added with sodium sulfate electrolyte, and the addition amount is 0.10-0.60% of the mass of the pig small intestinal mucosa solution.

[0028] In one embodiment, the viscous concentrated slurry pig small intestinal mucosa solution in step (1) is added with sodium metabisulfite preservative, and the addition amount is 0.15-1.5% of the mass of the pig small intestinal mucosa solution.

[0029] In one embodiment, the viscous concentrated slurry pig small intestinal mucosa solution in step (1) is adjusted to pH 6.0-8.0 with 30% sodium hydroxide lye.

[0030] In one embodiment, the bioactive enzyme in step (2) includes probiotics and biological enzymes. The probiotics are lactobacillus bifidus, and the colony unit of lactobacillus bifidus is ≥10 billion CFU / g. The biological enzymes are alkaline protease, and the enzyme activity content is 30000 U / g. The addition amount of the bioactive enzyme is 0.05-0.50% of the mass of the pig small intestinal mucosa solution.

[0031] In one embodiment, the auxiliary agent in step (2) includes 10-50% sodium bicarbonate, 10-50% glucose, 10-50% citric acid and 10-30% sodium benzoate. The addition amount of the auxiliary agent is 0.10-1.00% of the mass of the pig small intestinal mucosa solution.

[0032] In one embodiment, the pancreatin in step (3) includes ≥3000 U / g trypsin, ≥40000 U / g pancreatic lipase and ≥60000 U / g pancreatic amylase. The amount of the pancreatin used is 0.05-0.30% of the mass of the pig small intestinal mucosa solution.

[0033] In one embodiment, the mass of the resin in step (4) is 1.0-3.0% of the mass of the pig small intestinal mucosa solution.

[0034] In one embodiment, the eluent used in step (5) is a sodium chloride solution with a mass fraction of 18-25%.

[0035] In one embodiment, the alcohol precipitation in step (6) refers to using alcohol with a concentration of 60-90% to a final concentration of 25-55%.

[0036] In one embodiment, the drying in step (7) is vacuum drying.

[0037] In one embodiment, the storage condition of the crude heparin sodium product in step (8) is room temperature and light protection.

[0038] The present application also provides heparin sodium prepared by the method.

[0039] The present application also provides the use of the method in the preparation of heparin sodium and heparin sodium-containing products.

[0040] Advantages:

[0041] The present application is a salt-free production technology for extracting crude heparin sodium from pig small intestinal mucosa fluid. Compared with the existing heparin sodium crude product production technology, the present application has three advantages:

[0042] (1) Salt-free: The present application does not add sodium chloride salt in the extraction of crude heparin sodium. Compared with the prior art, the amount of sodium chloride used is reduced by more than 99%, so that the content of sodium chloride ions in the wastewater is less than 0.03%, i.e. 300 ppm or less, meeting the needs of ecological environmental protection and waste utilization.

[0043] (2) Concentrated slurry: The present application uses extremely thick pig small intestine concentrated slurry mucosa fluid with a volume of 2.7 liters per root. Compared with the current situation of 10 liters of mucosa fluid after adding water to each pig small intestine in the prior art, the water content in the pig small intestine concentrated slurry mucosa fluid is reduced by more than 70%. The content of small intestinal mucosa protein and small molecule peptide nutrients in the wastewater after extraction of crude heparin sodium is as high as 9-12%, which ensures that the small intestinal mucosa protein and small molecule peptide nutrients in the final product meet the standards, especially the ash content does not exceed the standard, making it possible to turn waste into treasure. At present, the "intestinal membrane protein powder" animal protein nutritional feed additive product has been jointly developed with domestic feed additive companies.

[0044] (3) Bioactive enzyme biofermentation: The application of bioactive enzyme controllable biofermentation technology to the organization rupture and lysis of salt-free and extremely viscous pig small intestine concentrated mucosa solution is disclosed for the first time. Under the double adverse conditions of concentrated mucosa and salt-free, the application of the new technical scheme proves that, under the synergistic effect of bioactive enzyme controllable biofermentation technology and biological enzyme hydrolysis, the dry solid heparin sodium crude product yield is 82,000 USP units per root (1,220 per billion), which is 28.1% higher than the heparin sodium crude product yield of 64,000 USP units per root (1,563 per billion) without the implementation of bioactive enzyme hydrolysis technology, and 32.3% higher than the heparin sodium crude product yield of 62,000 USP units per root (1,613 per billion) using alkaline protease 2709 hydrolysis technology, and the heparin sodium crude product yield is significantly improved. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 The biological flow chart of the existing technical scheme, heparin sodium crude product enzymatic hydrolysis production process;

[0046] Figure 2 The biological flow chart of the new technology of salt-free biofermentation enzymatic hydrolysis production of heparin sodium crude product;

[0047] Figure 3 The heparin sodium crude product yield corresponding to different amounts of pancreatin. DETAILED DESCRIPTION

[0048] The experimental materials used in the application are as follows:

[0049] Bioactive enzyme: a mixture of self-made active probiotic bioactive enzyme and biological enzyme, probiotic bioactive enzyme raw material, food-grade lactobacillus bifidus purchased from Shaanxi Yunqi Biotechnology Co., Ltd., containing 10 billion live bacteria per gram, product number YQ202001101. Biological enzyme raw material purchased from Henan Xinyangshao Biological Enzyme Preparation Co., Ltd., food-grade alkaline protease, product number DBJ20200901, 200,000 units per gram. Before use, 1 part of lactobacillus bifidus, 1.5 parts of alkaline protease and 7.5 parts of soft water are stirred uniformly, and a self-made mixture of bioactive enzyme and biological enzyme is obtained immediately. Among them, lactobacillus bifidus contains 1 billion colony forming units per gram, and alkaline protease contains 30,000 units per gram. The addition amount of bioactive enzyme is 0.20% of the mass of the mucosa solution of the pig small intestine.

[0050] The auxiliary agent is a mixture containing 30% sodium bicarbonate, 30% glucose, 20% citric acid and 20% sodium benzoate by mass, and the addition amount is 0.20% of the mass of the mucosa solution of the pig small intestine.

[0051] Pancreatin: a complex enzyme extracted from pig pancreas, which contains more than 4000 units of trypsin activity per gram, more than 30000 units of lipase activity per gram, and more than 60000 units of amylase activity per gram. The pancreatin is purchased from Chongqing Xiangsheng Biopharmaceutical Co., Ltd. with product batch number C01200930. The amount of pancreatin is 0.15% of the mass of the mucosa solution of the pig small intestine.

[0052] The pancreatin enzymolysis conditions are as follows: pH value 8-8.5, 52℃±2 incubation for 150 minutes, and the pH value is maintained at about 8 during the incubation.

[0053] Adsorption resin: DuPont Lomond (USA) FPA98Cl macroporous strong anion heparin sodium special resin. The mass of the resin is 1.30% of the mass of the mucosa solution of the pig small intestine.

[0054] The experimental method used in the present application is as follows:

[0055] Definition of the anticoagulant activity potency of heparin sodium:

[0056] Heparin is a natural active substance, and its activity cannot be accurately measured by chemical or physical methods, but can only be measured by biological assay. Reinert et al. introduced that the anticoagulant activity unit is defined as the amount of heparin that can just inhibit the coagulation of 1 mL of recalcified bovine plasma within 4 hours at 37℃.

[0057] Definition of the anticoagulant activity potency of heparin sodium: the number of anticoagulant activity units per milligram of heparin sodium.

[0058] Detection method of the anticoagulant activity potency of heparin sodium:

[0059] The detection method of the anticoagulant activity potency of heparin sodium in the current national standard is the sheep plasma method. In the examples of the present application, the sheep plasma method is used to determine the potency of the enzymolysis solution and the heparin sodium crude product, and the heparin sodium standard used is the United States Pharmacopoeia standard (USP).

[0060] Definition of the yield of heparin sodium:

[0061] The yield of heparin sodium crude product described in the examples of the present application is defined as the number of anticoagulant activity units of heparin sodium produced per pig small intestine, which is also expressed as: 10,000 units per pig small intestine. The unit is the anticoagulant activity unit, and the higher the numerical value, the higher the yield.

[0062] The yield of heparin sodium crude product described in the examples of the present application can also be defined as the number of pig small intestines (roots) required to produce 1 billion anticoagulant activity units, which is also expressed as: roots per 1 billion units. The unit is the anticoagulant activity unit, and the higher the numerical value, the lower the yield.

[0063] The technical solutions in the embodiments of the present application will be described below with reference to the accompanying drawings. The technical method of the present application "Salt-free production technology of crude heparin sodium extracted by enzymatic method of biological enzyme fermentation" is not limited to the following described embodiments. Any modification, equivalent replacement, improvement, etc. within the scope of the biological scientific principles and basic logical ideas of the present application shall be included in the protection scope of the present application.

[0064] Example 1: Salt-free production technology of crude heparin sodium extracted by enzymatic method of biological enzyme fermentation

[0065] This embodiment relates to the technical method of "Salt-free production technology of crude heparin sodium extracted by enzymatic method of biological enzyme fermentation", which includes the following steps: under the condition of salt-free (sodium chloride salt), using the biological fermentation technology of biological active enzyme combined with the enzymatic technology of pancreatin to break the intestinal mucosa tissue and lyse the mast cell, so as to produce the heparin sodium precursor "heparin-protein complex". The free heparin sodium precursor "heparin-protein complex" substrate is subjected to the site-specific high-efficiency selective enzymolysis of "precise protein point cutting", so as to efficiently release the heparin sodium biological macromolecule with the anticoagulant biological activity. Then, the temperature is increased for inactivation; the residue is removed by filtration; the resin adsorption and desorption process is performed, so as to obtain the crude heparin sodium eluent. The alcohol precipitation / dewatering is performed to collect the heparin sodium wet solid. Finally, the vacuum drying is performed to obtain the crude heparin sodium solid. There are totally twelve key steps.

[0066] Step one, biological active enzyme biological fermentation under salt-free condition: 3500 fresh pig small intestines are scraped fresh, and the water volume is strictly controlled to be not more than 2.5 liters per pig small intestine mucosa liquid. The pig mucosa liquid is transferred into a 10000 liter enzymolysis tank, and soft water is added to 9500L (according to the dilution ratio of each pig small intestine mucosa liquid to 2.7L mucosa solution, the soft water is ion exchange resin softening water, which is pure water without calcium and magnesium ions).

[0067] Step two, 20 kilograms of sodium sulfate and 20 kilograms of sodium metabisulfite are added to the extremely thick pig small intestine mucosa liquid in step (1) under sufficient stirring.

[0068] Step three, the mucosa liquid in step (2) is finely adjusted to a pH value of 7.0 with 30% mass fraction sodium hydroxide lye under sufficient stirring, and is fully stirred.

[0069] Step four, 20 kilograms of auxiliary agent (a mixture containing sodium bicarbonate, glucose, citric acid, and sodium benzoate) are added to the mucosa liquid in step (3) under sufficient stirring. After fully stirring, 20 kilograms of biological active enzyme are added. The temperature is slowly increased to 39℃±2, and the temperature is maintained for 120±10 minutes under continuous stirring.

[0070] The controllable biological fermentation process used in the method can effectively digest and decompose the small intestinal mucosa tissue, thoroughly break and separate the mast cells, and effectively promote the high-efficiency lysis of the mast cells. On the other hand, excessive microbial fermentation will cause fermentation spoilage and decay of the small intestinal mucosa fluid, and further cause degradation of the heparin sodium biological macromolecules. Therefore, the controllability of the biological active enzyme fermentation process is very important. By using the formulation of auxiliary agents, components that can improve the microbial activity and components that can control the fermentation spoilage and decay are contained. Through the reasonable formulation of various components, the high-efficiency microbial activity and the effect of controlling the mucosa fluid spoilage and decay are achieved.

[0071] Step five, pancreatic enzyme hydrolysis is carried out: a small amount of dilute alkali is used to adjust the pH value to 8.5, the temperature is raised to 52°C±2, 14 kg of pancreatin (0.15% of the mass of the small intestinal mucosa fluid of the pig) is added, and stirring, heating and maintaining are continued. During the maintaining process, the pH value is adjusted to about 8.0, and the maintaining is performed for 150±10 minutes. After the maintaining is completed, the enzyme hydrolysis sample is immediately detected, and the titer is 35.3 USPU / ML (equivalent to a yield of 96,000 USPU / root). Then, the temperature is raised to 75°C±5, the temperature raising is stopped, and the temperature is maintained for 30 minutes to obtain the enzyme hydrolysis liquid.

[0072] Step six, the enzyme hydrolysis liquid obtained in step (5) is filtered through a 60-mesh filter screen to remove the dregs, and the filtered liquid is cooled to 58°C±3 and transferred into an adsorption tank. 120 kg of prepared Rohm & Haas (USA) macroporous strong anion resin is added, and stirring and adsorption are performed for about 8 hours.

[0073] Step seven, the adsorbed resin obtained in step (6) is filtered through a 100-mesh filter screen, and the collected resin is rinsed with a large amount of softened water.

[0074] Step eight, the rinsed resin obtained in step (7) is washed with 5% dilute brine at room temperature for 2 times. Then, the resin is eluted with saturated brine solution for 3 times, the mass of the saturated brine solution accounts for 100-200% of the mass of the resin, the elution time is 2 hours, the temperature is 55°C, and the heparin sodium elution liquid is collected.

[0075] Step nine, 85° food-grade alcohol is added to the elution liquid obtained in step (8), the ethanol content is adjusted to 45% after the addition, and the mixture is statically placed for 12 hours. The mixture is filtered through a 200-mesh filter screen to obtain wet heparin sodium solid.

[0076] Step ten, the wet heparin sodium solid obtained in step (9) is dehydrated with alcohol with a mass fraction of more than 90% for 2-3 hours. Then, the mixture is filtered again through a 200-mesh filter screen to obtain the wet heparin sodium solid after high-concentration alcohol dehydration.

[0077] Step eleven, the dehydrated wet sodium heparin solid obtained from step (10) was dried in vacuum, temperature ~ 60°C, time 12 hours, to obtain the dried crude sodium heparin light yellow solid 3120 grams.

[0078] Step twelve, the dried crude sodium heparin light yellow solid obtained from step (11) was tested for potency, the potency was 92.0 USPU / MG (yield 82,000 USPU / root). The crude sodium heparin product was registered and stored in food grade PE plastic bag sealed at room temperature in a dry and dark storage cabinet.

[0079] The test results showed that the sodium chloride ion content in the wastewater produced by the above process was less than 0.03%.

[0080] Comparative Example 1: Extraction of crude sodium heparin with different amounts of pancreatin

[0081] The specific implementation is referred to Example 1, the difference is that the amount of pancreatin in step five is changed to 7 kg and 20 kg, and the yield of crude sodium heparin is detected. The results show that when the amount of pancreatin is 7 kg, the potency of the enzyme solution in step five is 28.7 USPU / ML (equivalent to a yield of 78,000 USPU / root), and the dried crude sodium heparin light yellow solid obtained in steps eleven and twelve is 2776 grams, with a potency of 87.0 USPU / MG. The yield of crude sodium heparin is 69,000 USP units / root, and the amount of crude sodium heparin produced is reduced by 18.8%. When the amount of pancreatin is 20 kg, the potency of the enzyme solution in step five is 30.6 USPU / ML (equivalent to a yield of 83,000 USPU / root), and the dried crude sodium heparin light yellow solid obtained in steps eleven and twelve is 2807 grams, with a potency of 91.0 USPU / MG. The yield of crude sodium heparin is 73,000 USP units / root, and the amount of crude sodium heparin produced is reduced by 12.3%, indicating that the amount of pancreatin used has a direct impact on the amount of free sodium heparin in the enzyme solution, as shown in Table 1. Figure 3

[0082] Comparative Example 2: Production of crude sodium heparin by omitting the biological fermentation of bioactive enzymes

[0083] The specific implementation is referred to Example 1, the difference is that the biological fermentation technology of bioactive enzymes in step four is omitted, and only pancreatin enzymolysis technology is used. After the enzyme solution is incubated, the anticoagulant potency of the enzyme solution is measured to be 27.7 USPU / ML (equivalent to a yield of 75,000 USPU / root). Finally, the wet sodium heparin solid is dried in vacuum, the temperature is adjusted to ~ 65°C, and the time is 12 hours, to obtain the dried crude sodium heparin light yellow solid 2856 grams, with a potency of 78.0 USPU / MG, and the yield of crude sodium heparin is 64,000 USP units / root. The yield of solid crude sodium heparin in Example 1 is increased by 28.1% compared to the yield in Comparative Example 2. ​

[0084] Comparative Example 3: Enzymatic extraction of crude heparin sodium using alkaline protease 2709

[0085] The specific embodiment is in accordance with Example 1, except that in Step Five, trypsin is replaced with alkaline protease 2709 for enzymatic hydrolysis. The alkaline protease 2709 used has an activity of 200,000 units per gram, and is used in an amount of 17.5 kilograms. After the incubation is completed, the activity of the enzymatic hydrolysate is measured to be 21.4 USPU / ML (equivalent to a yield of 58,000 USPU per root). The final dried crude heparin sodium is obtained as a light yellow solid 2614 grams, with an activity of 83.0 USPU / MG, and a crude heparin sodium yield of 62,000 USPU per root. The yield of solid heparin sodium crude in Example 1 is increased by 32.3% compared to the yield in Comparative Example 3.

[0086] Comparative Example 4: Enzymatic extraction of crude heparin sodium using alkaline protease 2709 alone

[0087] The specific embodiment is in accordance with Example 1, except that the bio-fermentation technology of the bio-active enzyme in Step Four is omitted, and alkaline protease 2709 is used in Step Five for enzymatic hydrolysis. The alkaline protease 2709 used has an activity of 200,000 units per gram, and is used in an amount of 17.5 kilograms. The activity of the enzymatic hydrolysate is measured to be 18.8 USPU / ML (equivalent to a yield of 51,000 USPU per root). The final dried crude heparin sodium is obtained as a light yellow solid 1909 grams, with an activity of 88.0 USPU / MG, and a crude heparin sodium yield of 48,000 USPU per root. The yield of solid heparin sodium crude in Example 1 is increased by 70.8% compared to the yield in Comparative Example 4.

[0088] Comparative Example 5: Production of crude heparin sodium by adjusting the proportion of bio-active enzyme

[0089] The specific embodiment is in accordance with Example 1, except that in the bio-enzyme fermentation of Step Four, the content of Lactobacillus lactis in the bio-active enzyme is reduced by 50%. That is, 0.5 parts of Lactobacillus lactis, 1.5 parts of alkaline protease, and 8.0 parts of soft water are stirred uniformly. In the mixture of active probiotics and bio-enzyme thus prepared, Lactobacillus lactis contains 500 million colony-forming units per gram, and the alkaline protease has an activity content of 30,000 units per gram. The amount of bio-active enzyme added is still 20 kilograms. The results show that the activity of heparin sodium in the enzymatic hydrolysate is 28.6 USPU / ML (equivalent to a yield of 78,000 USPU per root), which is decreased by 23.1% compared to the 96,000 USPU per root in Example 1. Further, the final dried crude heparin sodium is obtained as a light yellow solid 2815 grams, with an activity of 86.0 USPU / MG, and a crude heparin sodium yield of 69,000 USPU per root. The yield of heparin sodium crude in Example 1 is increased by 18.8% compared to Comparative Example 5.

[0090] The results show that reducing the bio-fermentation process of the bio-active enzyme will result in a significant decrease in the yield of crude heparin sodium.

[0091] Comparative Example 6: Adjusting the ratio of bio-active enzyme to produce crude heparin sodium

[0092] The specific implementation refers to Example 1, with the difference that in the bio-enzyme fermentation of step four, the content of Bifidobacterium lactis in the bio-active enzyme is increased to 2 times, i.e. “2 parts of Bifidobacterium lactis: 1.5 parts of alkaline protease: 6.5 parts of softened water” are stirred uniformly. That is, in the mixture of the active probiotic bacteria and bio-enzyme prepared, Bifidobacterium lactis contains 2 billion colony units per gram, and the alkaline protease activity content is 30,000 units per gram. The addition amount of bio-active enzyme is still 20 kg. The results show that the activity titer of heparin sodium in the enzymatic hydrolysate is 26.8 USPU / ML (equivalent to a yield of 73,000 USPU / root), which is 31.5% lower than 96,000 USPU / root in Example 1. Further, the dried crude heparin sodium is obtained as a light yellow solid 2733 grams, with a titer of 81.0 USPU / MG, and the crude heparin sodium yield is 63,000 USPU / root. The crude heparin sodium yield in Example 1 is increased by 30.2% compared to the yield in Comparative Example 6.

[0093] The results show that too high bio-fermentation process of bio-active enzyme will result in a decrease in the yield of crude heparin sodium.

[0094] Comparative Example 7: Adjusting the fermentation time of bio-active enzyme to produce crude heparin sodium

[0095] The specific implementation refers to Example 1, with the difference that in the bio-enzyme fermentation of step four, the bio-fermentation time of bio-active enzyme is shortened by 60 minutes, and the incubation time is changed to 60 minutes. The results show that the activity titer of heparin sodium in the enzymatic hydrolysate is 31.4 USPU / ML (equivalent to a yield of 85,000 USPU / root), which is 12.9% lower than 96,000 USPU / root in Example 1. Further, the dried crude heparin sodium is obtained as a light yellow solid 2785 grams, with a titer of 93.0 USPU / MG, and the crude heparin sodium yield is 74,000 USPU / root. The crude heparin sodium yield in Example 1 is increased by 10.8% compared to Comparative Example 7.

[0096] The results show that shortening the bio-fermentation process time of bio-active enzyme results in incomplete bio-fermentation process, which leads to a decrease in the yield of crude heparin sodium.

[0097] Comparative Example 8: Adjusting the fermentation time of bio-active enzyme to produce crude heparin sodium

[0098] The procedure of Example 1 was followed except that in the bio-enzyme fermentation of Step Four, the bio-enzyme fermentation time was extended by 60 minutes and the incubation time was changed to 180 minutes. The results showed that the heparin sodium activity titer in the enzyme solution was 26.9 USPU / ML (equivalent to a yield of 73,000 USPU / roots), which was 31.5% lower than the 96,000 USPU / roots of Example 1. Further, the dried heparin sodium crude product was obtained as a light yellow solid 2613 grams with a titer of 81.7 USPU / MG, and the heparin sodium crude product yield was 61,000 USPU / roots. The heparin sodium crude product yield of Example 1 was increased by 34.4% compared to Comparative Example 8.

[0099] The results showed that extending the bio-enzyme fermentation process time caused an excess of the bio-enzyme fermentation process, and the heparin sodium crude product yield was significantly reduced.

[0100] Comparative Example 9: Producing heparin sodium crude product by adjusting the types and proportions of auxiliary agents

[0101] The procedure of Example 1 was followed except that in the bio-enzyme fermentation of Step Four, the proportion of auxiliary agents was adjusted to a mixture of 10% glucose, 40% sodium bicarbonate, 30% citric acid, and 20% sodium benzoate. The results showed that the heparin sodium activity titer in the enzyme solution was 29.1 USPU / ML (equivalent to a yield of 79,000 USPU / roots), which was 21.5% lower than the 96,000 USPU / roots of Example 1. Further, the dried heparin sodium crude product was obtained as a light yellow solid 2526 grams with a titer of 95.6 USPU / MG, and the heparin sodium crude product yield was 69,000 USPU / roots. The heparin sodium crude product yield of Example 1 was increased by 18.8% compared to Comparative Example 9.

[0102] The results showed that when the activating component glucose content of the bio-enzyme was reduced by two-thirds, the bio-enzyme fermentation process was insufficient, and the heparin sodium crude product yield was significantly reduced.

[0103] Comparative Example 10: Producing heparin sodium crude product by adjusting the types and proportions of auxiliary agents

[0104] The procedure of Example 1 was followed except that in the bio-enzyme fermentation of Step Four, the proportion of auxiliary agents was adjusted to a mixture of 50% glucose, 30% sodium bicarbonate, 10% citric acid, and 10% sodium benzoate. The results showed that the heparin sodium activity titer in the enzyme solution was 26.7 USPU / ML (equivalent to a yield of 72,000 USPU / roots), which was 33.3% lower than the 96,000 USPU / roots of Example 1. Further, the dried heparin sodium crude product was obtained as a light yellow solid 2665 grams with a titer of 78.8 USPU / MG, and the heparin sodium crude product yield was 60,000 USPU / roots. The heparin sodium crude product yield of Example 1 was increased by 36.7% compared to Comparative Example 10.

[0105] The results show that when the auxiliary ingredient glucose content is increased by two-thirds, the biological fermentation process is excessive, and the crude heparin yield is greatly reduced.

[0106] Comparative Example 11: Production of crude heparin without sodium sulfate electrolyte stabilizer

[0107] The specific implementation is similar to Example 1, except that the sodium sulfate electrolyte stabilizer in step two is omitted. After the enzyme hydrolysis incubation is complete, the anticoagulant potency of the enzyme hydrolysis solution is measured to be 28.7 USPU / ML (equivalent to a yield of 78,000 USPU / roots). Finally, the wet heparin sodium solid is vacuum dried, with the temperature adjusted to ~65°C for 12 hours, to obtain 2718 grams of dried crude heparin sodium light yellow solid with a potency of 86.5 USPU / MG, and a crude heparin sodium yield of 67,000 USPUs / roots. The crude heparin sodium solid yield in Example 1 is 22.4% higher than that in Comparative Example 11.

[0108] Comparative Example 12: Production of crude heparin by adjusting the amount of sodium sulfate electrolyte stabilizer

[0109] The specific implementation is similar to Example 1, except that the amount of sodium sulfate electrolyte stabilizer is adjusted, with the addition amount increased from 20 kg to 30 kg. After the enzyme hydrolysis incubation is complete, the anticoagulant potency of the enzyme hydrolysis solution is measured to be 31.5 USPU / ML (equivalent to a yield of 86,000 USPU / roots). Finally, the wet heparin sodium solid is vacuum dried, with the temperature adjusted to ~65°C for 12 hours, to obtain 2846 grams of dried crude heparin sodium light yellow solid with a potency of 91.0 USPU / MG, and a crude heparin sodium yield of 74,000 USPUs / roots. The crude heparin sodium solid yield in Example 1 is 10.8% higher than that in Comparative Example 12.

[0110] Although the present application has been disclosed with reference to the preferred embodiments above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. A salt-free production method for extracting crude heparin sodium by biological enzymatic hydrolysis method with biological enzyme fermentation promotion, characterized in that, The method comprises the following steps: (1) preparing pig small intestine mucosa solution: scraping pig small intestine to obtain pig small intestine mucosa solution; adding electrolyte and preservative to the pig small intestine mucosa solution, and adjusting pH value; the pig small intestine mucosa solution is diluted to 2.0-4.0 L of thick slurry solution; (2) biological fermentation: adding biological active enzyme and auxiliary agent to the pig small intestine mucosa solution obtained in step (1), and heating to 25-50 ℃ for 120 minutes to obtain fermented small intestine mucosa solution; the addition amount of the biological active enzyme is 0.05-0.50% of the mass of the pig small intestine mucosa solution, and the addition amount of the auxiliary agent is 0.10-1.00% of the mass of the pig small intestine mucosa solution; (3) enzymolysis: adjusting the pH value of the fermented small intestine mucosa solution obtained in step (2) to 7.0-9.0, adding 0.15% of the mass of the pig small intestine mucosa solution of trypsin, heating to 45-60 ℃, and keeping for 120-300 minutes to obtain an enzymolysis solution; (4) resin adsorption: heating the enzymolysis solution obtained in step (3) to 70-85 ℃ for 10-60 minutes, then filtering and cooling to 56-60 ℃, adding resin adsorption for at least 8 hours, and collecting the resin; the mass of the resin is 1.0-3.0% of the mass of the pig small intestine mucosa solution; (5) resin desorption: washing the resin obtained in step (4) with 5% dilute brine, and then eluting the resin with concentrated brine to obtain a crude heparin sodium solution; (6) alcohol precipitation: adding ethanol with a final concentration of 25-55% to the crude heparin sodium solution obtained in step (5), and standing for 12-24 hours to precipitate crude heparin sodium; (7) collecting crude heparin sodium: filtering, dehydrating and drying the crude heparin sodium obtained in step (6) to obtain crude heparin sodium product; The trypsin in step (3) comprises ≥3000 U / g of trypsin, ≥40000 U / g of pancreatic lipase and ≥60000 U / g of pancreatic amylase; The biological active enzyme in step (2) comprises probiotics and biological enzyme; the biological enzyme comprises ≥30000 U / g of protease; the probiotics are lactobacillus bifidus with a colony unit of 1 billion CFU / g; and the addition amount of the biological active enzyme is 0.15-0.25% of the mass of the pig small intestine mucosa solution; The auxiliary agent in step (2) comprises 30% sodium bicarbonate, 30% glucose, 20% citric acid and 20% sodium benzoate by mass fraction; and the addition amount of the auxiliary agent is 0.15-0.25% of the mass of the pig small intestine mucosa solution; The electrolyte in step (1) comprises sodium sulfate; and the addition amount of the electrolyte is 0.10-0.21% of the mass of the pig small intestine mucosa solution.

2. The method of claim 1, wherein, The slurry in step (1) is diluted to 2.5-3.0 L of thick slurry.

3. The method of claim 1, wherein, The preservative in step (1) comprises sodium metabisulfite; and the addition amount of the preservative is 0.15-1.50% of the mass of the pig small intestine mucosa solution.

4. The method of claim 1, wherein, The pH value of the pig small intestine mucosa solution in step (1) is adjusted to 6.0-8.

0.

5. Heparin sodium prepared by the method in any one of claims 1-4.

6. Use of the process according to any one of claims 1 to 4 for the preparation of a product containing sodium heparin.

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