New technology of extracting crude heparin sodium by low-salt complex enzyme two-step incubation enzymolysis

Abstract

The application discloses a new technology for extracting crude heparin sodium by low-salt composite enzyme two-step incubation enzymolysis method, and belongs to the technical field of production process of extracting crude heparin sodium from pig small intestinal mucosa fluid. In the application, the composite enzyme enzymolysis technology is used under low-salt condition, so that the protein molecules connected with heparin sodium macromolecules are cut at multiple sites and saturated, thereby effectively releasing free heparin sodium macromolecules with anticoagulant biological activity; the potency of heparin sodium in the obtained enzymolysis liquid is 14.5 USPU / ML, which is equivalent to a yield of 116,000 USPU / root; and the yield of the obtained solid crude heparin sodium is 98,000 American standard units / root. Compared with the existing production technology, the total amount of wastewater discharge is reduced by 20%, and the total salt content in the wastewater is reduced by 78%. The application meets the needs of industrial production in two aspects of improving the yield of crude heparin sodium product and greatly reducing the content of wastewater pollutants.

Description

TECHNICAL FIELD

[0001] The present application relates to a new technology of low-salt complex enzyme two-step incubation enzymolysis method for extracting crude heparin sodium, and belongs to the technical field of production process for extracting crude heparin sodium from pig small intestinal mucosa fluid. BACKGROUND

[0002] Heparin sodium is an acidic mucopolysaccharide, and after further processing and purification, it is the fine heparin sodium specified in the pharmacopoeia. The crude heparin sodium is a yellow, light yellow, white or off-white powder without odor, has hygroscopicity, is easily soluble in water, is insoluble in organic solvents, has an optical rotation of +42 to +56, and has a characteristic absorption peak at a wavelength of 185 to 220 nm. Heparin sodium is widely used for preventing and treating various cardiovascular and cerebrovascular diseases, and is the most effective anticoagulant drug, and is mainly used for: (a) treating disseminated intravascular coagulation in the early stage of various diseases; (b) preventing venous and pulmonary embolism; (c) treating venous and pulmonary embolism, ischemic stroke, unstable angina pectoris (reducing symptoms and preventing myocardial infarction), acute myocardial infarction (preventing early reinfarction and infarction area extension, and reducing mortality); (d) as a maintenance therapy for thrombolytic therapy; (e) used as an extracorporeal anticoagulant for preventing blood coagulation during blood transfusion and for preserving fresh blood in blood banks.

[0003] The molecular structure of heparin sodium is extremely complex, and at present, it can only be obtained by extraction from animal tissues. At present, the official drug administration of Europe and the United States recognizes pig small intestinal mucosa fluid as the source of heparin sodium.

[0004] The main production technical schemes of the existing crude heparin sodium production industry include high-salt salt dissociation method, enzymolysis method, and salt dissociation and enzymolysis complex method. Patent CN 103755837A relates to a method for preparing heparin sodium from pig small intestine by using salt dissociation and enzymolysis complex method, the salt dissociation conditions used are 2% to 2.5% salinity, and the enzyme used is alkaline protease 2709. The method produces 1 billion units of heparin sodium, and 1400 to 1600 pig small intestines are needed. Due to the specific enzymolysis cleavage site of the single enzyme, the protein molecules linked to the heparin sodium macromolecules cannot be completely and effectively cleaved off, so that the heparin sodium macromolecules cannot be completely released, which affects the yield of the crude heparin sodium. In addition, in the existing enzymolysis production process, the salt concentration is about 2.5%, which causes the chloride ion in the wastewater to be seriously over-standard, and the environmental protection problem of handling is faced. SUMMARY

[0005] The present application aims to provide a two-step incubation enzymolysis method using bioactive complex enzymes, to provide multiple enzymolysis cleavage sites, to effectively cleave the protein molecules linked to the heparin sodium macromolecules, to maximally release the heparin sodium macromolecules, and to improve the yield (yield) and quality of the crude heparin sodium.

[0006] The application provides a high-efficiency production method for extracting crude heparin sodium from pig small intestinal mucosa under low-salt conditions, which comprises the following seven steps.

[0007] (1) scraping intestines and preparing pig small intestinal mucosa liquid volume: fresh pig small intestines are scraped with softened water to obtain mucosa liquid, which is diluted with softened water to achieve a volume of 8.0 L per pig small intestinal mucosa, which is reduced by more than 20% compared with the volume of 10.0-12.0 L per pig small intestinal mucosa in the prior art;

[0008] (2) low-salt treatment of pig small intestinal mucosa liquid: food-grade casing salt sodium chloride or casing salt concentrated brine is added to the pig small intestinal mucosa solution obtained in step (1) to achieve a final concentration of ≤0.7%, which is reduced by 77.0% compared with the volume of 10.0 L per pig small intestinal mucosa and the mucosa liquid salt concentration of 2.5% in the prior art;

[0009] (3) two-step heat preservation enzymolysis: the pH of the low-salt pig small intestinal mucosa liquid obtained in step (2) is adjusted to 8.5, and a composite enzyme is added, and the enzymolysis is carried out at 35-45 ℃ for 60-150 minutes; then the pH is adjusted to 7-9, and the enzymolysis is carried out at 50-60 ℃ for 60-150 minutes to obtain a complete enzymolysis liquid; the addition amount of the composite enzyme is 0.05-0.10% of the mass of the pig small intestinal mucosa solution;

[0010] (4) inactivation of the enzymolysis liquid: the enzymolysis liquid in step (3) is incubated at 60-85 ℃ for 10-60 minutes;

[0011] (5) adsorption: the inactivated enzymolysis liquid in step (4) is filtered and the temperature is adjusted to 48-65 ℃ and the pH is adjusted to 7-9, and resin adsorption is carried out to obtain a crude heparin sodium solution; the mass of the resin accounts for 0.3-0.5% of the mass of the pig small intestinal mucosa solution;

[0012] (6) resin washing and resin desorption: the resin for adsorbing heparin sodium obtained in step (5) is washed with 4.0-6.0% dilute brine for 2 times, and then, heparin sodium is desorbed from the resin by using 16-25% concentrated brine to obtain a desorption liquid containing crude heparin sodium;

[0013] (7) collection of crude heparin sodium: the desorption liquid containing crude heparin sodium obtained in step (6) is subjected to alcohol precipitation and drying to obtain dried crude heparin sodium solid.

[0014] In one embodiment, the complex enzyme in step (3) comprises 10-30% trypsin, 20-60% papain, 10-50% alkaline 2709 protease and 5-30% collagenase; the activity of the trypsin is ≥4000 U / g, the activity of the papain is ≥300000 U / g, the activity of the alkaline 2709 protease is ≥200000 U / g, and the activity of the collagenase is ≥100000 U / g.

[0015] In one embodiment, the complex enzyme comprises 15-25% trypsin, 35-45% papain, 25-35% alkaline 2709 protease and 5-15% collagenase.

[0016] In one embodiment, the amount of the complex enzyme added in step (3) is 0.050-0.090% of the mass of the pig small intestinal mucosa solution.

[0017] In one embodiment, the final concentration of sodium chloride in step (2) is 0.5-0.7%.

[0018] In one embodiment, the dilution in step (1) is to dilute each pig small intestinal mucosa solution to 7.5-8.5 L.

[0019] In one embodiment, the mass of the resin in step (5) is 0.4-0.5% of the mass of the pig small intestinal mucosa solution.

[0020] In one embodiment, the alcohol precipitation in step (6) is performed using ethanol with a final concentration of 25-55%, and the solution is allowed to stand for 12-24 hours.

[0021] In one embodiment, the heparin sodium precipitation in step (6) is performed by dehydrating the heparin sodium precipitation using alcohol with a mass percentage concentration of 90% or above, and the dehydrating time is 2-3 hours.

[0022] In one embodiment, the dehydrated heparin sodium precipitation in step (7) is filtered using a 200-mesh filter screen, and then dried using a vacuum drying system at a vacuum degree of 65 Pa and a constant temperature of 65°C for 12 hours to obtain the dried heparin sodium crude product.

[0023] The present application also provides a heparin sodium crude product prepared by the method.

[0024] The present application also provides the use of the method in the preparation of a product containing heparin sodium.

[0025] Beneficial effects:

[0026] 1、The present application uses the volume ratio of pig small intestinal mucosa liquid, compared with the volume ratio (10-12 L / root) of prior art, reduces 20%, reaches 20% reduction of wastewater discharge amount. The present application uses ≤0.7% salt solution, reaches low salt condition. In the generated wastewater, the mass percentage concentration of chloride ion is less than 0.7%, compared with the high salt concentration 2.5% of prior art, the chloride ion concentration reduction rate reaches 70%. Superimposed 20% intestinal mucosa liquid volume reduction, the total chloride ion content reduction rate reaches 77%. Greatly reduces the environmental protection pressure of wastewater discharge, reduces the cost.

[0027] 2、The present application uses the new technology of composite enzyme enzymolysis under low salt condition, saturated cutting of multiple sites of protein molecules, reaches maximum removal of protein molecules linked on heparin sodium macromolecule, thereby releasing free heparin sodium macromolecule with anticoagulant biological activity. The potency of heparin sodium in the obtained enzymolysis liquid is 14.5 USPU / ML, equivalent to yield 116,000 USPU / root; the yield of the obtained heparin sodium crude product is 980,000 American standard units / root (i.e. 1020 roots / billion units, American standard USPU). Compared with the crude heparin sodium yield of prior art of 1320-1360 roots / billion, the improvement degree is 29.4-33.3%, greatly improves the yield.

[0028] The present application technology greatly meets the needs of industrial production in two aspects of wastewater discharge index and heparin sodium crude product yield. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The heparin sodium anticoagulant activity potency corresponding to different composite enzyme dosages;

[0030] Figure 2 The heparin sodium crude product yield corresponding to different composite enzyme dosages. DETAILED DESCRIPTION

[0031] The experimental materials used in the present application are as follows:

[0032] The composite enzyme is a mixture of multiple biological proteases, wherein 20% trypsin, 40% papain, 30% alkaline 2709 protease and 10% collagenase are contained in mass fraction.

[0033] The pancreatin is a complex enzyme extracted from pig pancreas, containing 4000 active units / g of trypsin, purchased from Chongqing Xiangsheng Biopharmaceutical Co., Ltd., with the item number C01210310. The papain activity is 300,000 units / g, purchased from Xuzhou Baishun Biotechnology Co., Ltd., with the item number 1101. The alkaline 2709 protease activity is 200,000 units / g, purchased from Henan Xinyangzhao Biozyme Co., Ltd., with the item number DBJ20210318. The collagenase activity is 100,000 units / g, purchased from Hangzhou Qiancheng Biotechnology Co., Ltd., with the item number 3-07.

[0034] The amount of the complex enzyme is 0.075% of the mucosa liquid mass of pig small intestine.

[0035] The suitable conditions for the complex enzyme hydrolysis include adjusting the pH value to 8.0-8.5, incubating at a low temperature of 39℃±2 for 90 minutes, and incubating at a temperature of 55℃±2 for 120 minutes.

[0036] The adsorption resin is DuPont Lomond (USA) resin, FPA98Cl macroporous strong anion heparin sodium special resin. The mass of the resin is 0.40% of the mucosa liquid mass of pig small intestine.

[0037] The experimental method used in the present application is as follows:

[0038] Definition of the anticoagulant activity unit of heparin sodium: The definition of the heparin activity unit by Howell is that the amount of heparin required to just prevent 1 mL of fresh pigeon blood from coagulating within 24 hours at 0℃ is 1 active unit. Reinert et al. introduced that the anticoagulant activity unit is defined as the amount of heparin required to just prevent 1 mL of recalcified bovine plasma from coagulating within 4 hours at 37℃.

[0039] Definition of the anticoagulant activity potency of heparin sodium: The anticoagulant activity unit per milligram of heparin sodium.

[0040] Detection method of the anticoagulant activity potency of heparin sodium:

[0041] The detection method of the anticoagulant activity potency of heparin sodium in the current national standard is the sheep plasma method. The examples of the present application use the sheep plasma method to determine the potency of the enzyme hydrolysate and the heparin sodium crude product, and the heparin sodium standard used is the United States Pharmacopoeia standard (USP).

[0042] Definition of the heparin sodium yield:

[0043] The crude heparin sodium yield described in the examples of the present application is defined as the number of anti-coagulant activity units of heparin sodium produced per piglet small intestine, also expressed as: 10,000 units / piglet (unit is USP, USPU), the higher the value, the higher the yield. It can also be defined as the number of piglet small intestines needed to produce 100 million anti-coagulant activity units, also expressed as: piglet / 100 million units (unit is USP, USPU), the higher the value, the lower the yield.

[0044] 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 "new technology for extracting crude heparin sodium from piglet small intestine mucosa by biological fermentation and enzymatic hydrolysis" is not limited to the following described examples. Any modification, equivalent replacement, improvement, etc. made 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.

[0045] Example 1: New technology for extracting crude heparin sodium by low-salt complex enzyme two-step incubation enzymatic hydrolysis method

[0046] The technical method of the present application relates to a complex enzyme composed of multiple biological active proteases according to the principle that different biological active proteases have different "cutting" sites for protein macromolecules, which provides multiple enzymatic cleavage sites to effectively cut the protein molecules connected to the heparin sodium macromolecules, and releases heparin sodium macromolecules with anti-coagulant biological activity. The technical solution uses complex enzyme enzymatic hydrolysis technology under low-salt conditions to implement efficient saturation enzymatic hydrolysis on free "heparin-protein complex" substrate, thereby efficiently releasing heparin sodium biological macromolecules with anti-coagulant biological activity. Then, the temperature is increased for inactivation; residual substances are filtered out; resin adsorption and desorption process is performed to obtain crude heparin sodium eluate. Alcohol precipitation / dewatering is used to collect heparin sodium wet solid. Finally, vacuum drying is used to obtain heparin sodium crude product solid. There are a total of eight key steps.

[0047] Step one, adjust low salt:

[0048] 1000 fresh piglet small intestines are softened with ion exchange resin method, freshly scraped, and piglet mucosa liquid is transferred into an 8000 liter enzymatic hydrolysis tank, softening water is added to 8000L (diluted to 8L mucosa solution per piglet small intestine mucosa liquid volume ratio). Under sufficient stirring, food grade casing salt or concentrated brine is added to make the final concentration of sodium chloride 0.7%.

[0049] The volume ratio of the average pig small intestine mucosa fluid is more than 10L per pig small intestine according to the prior art production technology method, and the amount of salt used accounts for 2.5% of the mass percentage concentration of the intestinal mucosa fluid, reaching 250g of salt in the intestinal mucosa fluid per pig small intestine. According to the technical scheme of the present application, the volume of the mucosa solution of the pig small intestine mucosa fluid is 8L per pig small intestine, the amount of salt used accounts for 0.7% of the mass percentage concentration of the intestinal mucosa fluid, and the amount of salt in the intestinal mucosa fluid per pig small intestine is 56g. Compared with the amount of salt in the intestinal mucosa fluid, the technical scheme of the present application is reduced by 78%. Therefore, the total salt content in the wastewater is reduced by 78% compared with the total salt amount in the prior art. The total amount of wastewater is reduced by 20%.

[0050] Step two, complex enzyme enzymolysis:

[0051] Complex enzyme: a mixture of various biological proteases, containing 20% trypsin, 40% papain, 30% alkaline 2709 protease and 10% collagenase.

[0052] Complex enzyme enzymolysis (I): the pig small intestine mucosa fluid obtained in step one is finely adjusted to a pH value of 8.5 with a small amount of 30% mass fraction sodium hydroxide lye. Then, slowly warm up to 39℃±2, continue to stir, add 6kg of complex enzyme at a mass percentage concentration of 0.075% of the pig small intestine mucosa fluid for enzymolysis. Keep warm for 90 minutes. After the warm-up is completed, adjust the pH value to about 8 with a small amount of 30% mass fraction sodium hydroxide lye to obtain a primary enzymolysis liquid.

[0053] Complex enzyme enzymolysis (II): under the condition of maintaining pH value 8.0, warm the above-mentioned primary enzymolysis liquid to a temperature of 55℃±2, and keep warm for 120 minutes. Adjust and maintain the pH value to about 8 during the warm-up process to obtain a completely enzymolyzed enzymolysis liquid. Immediately take a sample of the completely enzymolyzed enzymolysis liquid to measure the anticoagulant activity of the contained heparin sodium, and the titer is 14.5 USPU / ML (equivalent to a yield of 116,000 USPU per root).

[0054] Step three, inactivation by increasing temperature:

[0055] Warm the above-mentioned completely enzymolyzed enzymolysis liquid to 75℃ for inactivation. After stopping the temperature rise, keep warm for 30 minutes.

[0056] Step four, filtration, temperature adjustment, and acid-base adjustment:

[0057] Filter the inactivated enzymolysis liquid in step three with a 60-mesh filter screen to remove residues. On the transmission pipeline of the filtrate, an online condenser is connected to adjust the temperature of the filtered enzymolysis liquid to 58℃. The temperature-adjusted enzymolysis liquid is sent into an adsorption tank, and 10% sodium hydroxide dilute lye is used to adjust the pH value to 8.

[0058] Step five, resin adsorption:

[0059] Add prepared 32 kg of LCM (USA) macroporous strong anion resin, the mass of the resin is 0.4% of the mass of the pig small intestinal mucosa liquid. The adsorption time is 8 hours with stirring.

[0060] Step six, resin desorption:

[0061] Collect the resin of step five, first wash with 5% mass fraction of dilute brine for 3 times. Then wash the resin with saturated brine solution of 22% mass fraction, fully stirred at 55°C, desorption for 2 hours. Repeat once, a total of two desorption. Collect the two desorption liquid and combine, leave for precipitation.

[0062] Step seven, alcohol precipitation:

[0063] Alcohol precipitation (I): to the combined two desorption liquid, add food grade alcohol of 85% mass fraction of ethanol, so that the mass fraction of ethanol in the desorption liquid is 45%, at room temperature for 12h. 200 mesh filter screen to obtain wet sodium heparin solid.

[0064] Alcohol precipitation (II): to the wet sodium heparin solid obtained by filtration, add alcohol of more than 92% mass fraction of ethanol, dehydrate at room temperature for 3 hours. 200 mesh filter screen to obtain good dehydrated wet sodium heparin solid.

[0065] Step eight, vacuum drying:

[0066] To the good dehydrated wet sodium heparin solid, vacuum drying for 12 hours, temperature 65°C, vacuum degree 30-70pa. Get dry crude heparin sodium light yellow solid 1056 grams, the potency of 93 USPU / MG, heparin sodium crude yield 98000 American standard units per root.

[0067] Comparative example 1: different amount of enzyme extraction of heparin sodium crude

[0068] The specific embodiments refer to Example 1, with the difference that the amount of complex enzyme is changed to 2 kg, 4 kg, 8 kg and 10 kg, corresponding to 0.025%, 0.050%, 0.100% and 0.125% of the mass of pig small intestine mucosa, respectively, and the potency of the enzymatic hydrolysate and the yield of the crude heparin sodium are detected. The results show that when the amount of complex enzyme is 2 kg, the potency of the enzymatic hydrolysate obtained in step two is 9.5 USPU / ML (equivalent to a yield of 76,000 USPU / root), and the dried crude heparin sodium obtained in step eight is a light yellow solid 829 g, with a potency of 76 USPU / MG, and the yield of the crude heparin sodium is 63,000 USP units / root. When the amount of complex enzyme is 4 kg, the potency of the enzymatic hydrolysate obtained in step two is 12.6 USPU / ML (equivalent to a yield of 101,000 USPU / root), and the dried crude heparin sodium obtained in step eight is a light yellow solid 901 g, with a potency of 91 USPU / MG, and the yield of the crude heparin sodium is 82,000 USP units / root. When the amount of complex enzyme is 8 kg, the potency of the enzymatic hydrolysate obtained in step two is 13.3 USPU / ML (equivalent to a yield of 106,000 USPU / root), and the dried crude heparin sodium obtained in step eight is a light yellow solid 1061 g, with a potency of 82 USPU / MG, and the yield of the crude heparin sodium is 87,000 USP units / root. When the amount of complex enzyme is 10 kg, the potency of the enzymatic hydrolysate obtained in step two is 10.9 USPU / ML (equivalent to a yield of 87,000 USPU / root), and the dried crude heparin sodium obtained in step eight is a light yellow solid 986 g, with a potency of 73 USPU / MG, and the yield of the crude heparin sodium is 72,000 USP units / root.

[0069] The above results show that the amount of complex enzyme used has a direct impact on the content of heparin sodium with anticoagulant activity released in the enzymatic hydrolysate, such as Figure 1 As shown in the table (the decrease rate is calculated based on the yield of the comparative example), when the amount of complex enzyme is 6 kg, the yield of the crude heparin sodium is the highest; when the amount of complex enzyme is 2 kg, the amount of crude heparin sodium produced is reduced by 55.6%; when the amount of complex enzyme is 4 kg, the amount of crude heparin sodium produced is reduced by 19.5%; when the amount of complex enzyme is 8 kg, the amount of crude heparin sodium produced is reduced by 12.6%; and when the amount of complex enzyme is 10 kg, the amount of crude heparin sodium produced is reduced by 36.1%. This shows that too much or too little complex enzyme is not conducive to the yield of the crude heparin sodium.

[0070] Comparative Example 2: Extraction of crude heparin sodium with different types and ratios of complex enzymes

[0071] The specific embodiment is the same as that of Example 1, except that the proportion of the complex enzyme is changed, the mass fraction of trypsin is increased from 20% to 30%, the mass fraction of papain is decreased from 40% to 30%, and the mass fractions of alkaline 2709 protease (30%) and collagenase (10%) remain unchanged. The added amount of the complex enzyme is still 6 kg. The potency of the enzyme solution and the yield of the crude heparin sodium are detected, and the results show that the potency of heparin sodium in the completely enzymolyzed enzyme solution obtained in Step Two is 12.7 USPU / ML (equivalent to a yield of 102,000 USPU / root), and 1024 grams of light yellow solid of dried crude heparin sodium is obtained in Step Eight, with a potency of 85 USPU / MG, and the yield of the crude heparin sodium is 87,000 American Standard Units / root. Compared with Example 1, the yield of the crude heparin sodium decreases by 12.6% (the decrease rate is calculated based on the yield of the comparative example).

[0072] Comparative Example 3: Extraction of crude heparin sodium by different types of complex enzymes

[0073] The specific embodiment is the same as that of Example 1, except that the proportion of the complex enzyme is changed, the mass fraction of trypsin is increased from 20% to 30%, the mass fraction of papain is decreased from 40% to 30%, and the mass fractions of alkaline 2709 protease (30%) and collagenase (10%) remain unchanged. The added amount of the complex enzyme is still 6 kg. The potency of the enzyme solution and the yield of the crude heparin sodium are detected, and the results show that the potency of heparin sodium in the completely enzymolyzed enzyme solution obtained in Step Two is 12.7 USPU / ML (equivalent to a yield of 102,000 USPU / root), and 1024 grams of light yellow solid of dried crude heparin sodium is obtained in Step Eight, with a potency of 85 USPU / MG, and the yield of the crude heparin sodium is 87,000 American Standard Units / root. Compared with Example 1, the yield of the crude heparin sodium decreases by 12.6% (the decrease rate is calculated based on the yield of the comparative example).

[0074] Comparative Example 4: Extraction of crude heparin sodium by different types of complex enzymes

[0075] The specific embodiment is in reference to Example 1, the difference is that the ratio of the complex enzyme is changed, the mass fraction of trypsin (20%) and papain (40%) is kept unchanged, the mass fraction of alkaline 2709 protease is reduced from 30% to 10%, and the mass fraction of collagenase is increased from 10% to 30%. The added amount of the complex enzyme is still 6 kg. The potency of the enzyme solution and the yield of the crude heparin sodium are detected, and the results show that the potency of heparin sodium in the completely enzymolyzed enzyme solution obtained in step two is 10.8 USPU / ML (equivalent to a yield of 86,000 USPU / root), and the dry crude heparin sodium obtained in step eight is 926 grams of light yellow solid with a potency of 81 USPU / MG, and the yield of the crude heparin sodium is 75,000 American standard units / root. Compared with Example 1, the yield of the crude heparin sodium is decreased by 30.7% (the decrease rate is calculated based on the yield of the comparative example).

[0076] Comparative Example 5: Extraction of crude heparin sodium by different types of complex enzymes

[0077] The specific embodiment is in reference to Example 1, the difference is that the ratio of the complex enzyme is changed, the mass fraction of trypsin (20%) and papain (40%) is kept unchanged, the mass fraction of alkaline 2709 protease is reduced from 30% to 10%, and the mass fraction of collagenase is increased from 10% to 30%. The added amount of the complex enzyme is still 6 kg. The potency of the enzyme solution and the yield of the crude heparin sodium are detected, and the results show that the potency of heparin sodium in the completely enzymolyzed enzyme solution obtained in step two is 10.8 USPU / ML (equivalent to a yield of 86,000 USPU / root), and the dry crude heparin sodium obtained in step eight is 926 grams of light yellow solid with a potency of 81 USPU / MG, and the yield of the crude heparin sodium is 75,000 American standard units / root. Compared with Example 1, the yield of the crude heparin sodium is decreased by 30.7% (the decrease rate is calculated based on the yield of the comparative example).

[0078] Comparative Example 6: Extraction of crude heparin sodium by using alkaline protease 2709 for enzymolysis

[0079] The specific embodiment is in reference to Example 1, the difference is that the ratio of the complex enzyme is changed, the mass fraction of trypsin (20%) and papain (40%) is kept unchanged, the mass fraction of alkaline 2709 protease is reduced from 30% to 10%, and the mass fraction of collagenase is increased from 10% to 30%. The added amount of the complex enzyme is still 6 kg. The potency of the enzyme solution and the yield of the crude heparin sodium are detected, and the results show that the potency of heparin sodium in the completely enzymolyzed enzyme solution obtained in step two is 10.8 USPU / ML (equivalent to a yield of 86,000 USPU / root), and the dry crude heparin sodium obtained in step eight is 926 grams of light yellow solid with a potency of 81 USPU / MG, and the yield of the crude heparin sodium is 75,000 American standard units / root. Compared with Example 1, the yield of the crude heparin sodium is decreased by 30.7% (the decrease rate is calculated based on the yield of the comparative example).

[0080] Although the present application has been disclosed in its preferred embodiments with reference to the accompanying drawings, it is not intended to limit the present application thereto, and various modifications and alterations can be made thereto by those skilled in the art without departing from the spirit and scope of the present application, and the scope of protection of the present application should be defined by the appended claims.

Claims

1. A method for extracting crude heparin sodium from porcine small intestinal mucosa, characterized in that, Includes the following steps: (1) Preparation of porcine small intestinal mucosal solution: scrape the porcine small intestine to obtain mucosal solution, and dilute the volume of mucosal solution of each porcine small intestine with water to 6.0-10.0L; (2) Adjusting the salt concentration: Add concentrated salt water to the porcine small intestinal mucosal solution obtained in step (1) to reach a final concentration of sodium chloride ≤0.7% to obtain a porcine small intestinal mucosal solution with low salt content. (3) Two-step enzymatic hydrolysis with compound enzyme: Adjust the pH of the porcine small intestinal mucosal fluid obtained in step (2) to 8.5, add compound enzyme, and enzymatically hydrolyze at 25-50℃ for 50-150 minutes; then adjust the pH to 7-9, and enzymatically hydrolyze at 50-60℃ for 60-180 minutes to obtain complete enzymatic hydrolysate; (4) Adsorption: After inactivating the complete enzymatic hydrolysate described in step (3), filter it, adjust the temperature to 48-65℃ and pH to 7-9, add resin for adsorption for at least 8 hours, and collect the resin. The mass of the resin accounts for 0.3-0.5% of the mass of the porcine small intestinal mucosa solution. (5) Resin washing and resin desorption: The resin obtained in step (4) is washed with dilute saline solution with a mass fraction of 4.0 to 6.0%, and then eluted with concentrated saline solution with a mass fraction of 16 to 25% to obtain a desorption solution containing crude heparin sodium. (6) Collect crude heparin sodium: The desorption solution obtained in step (5) is subjected to alcohol precipitation and drying to obtain crude heparin sodium; The complex enzyme in step (3) comprises 20% trypsin, 40% papain, 30% alkaline 2709 protease and 10% collagenase; the activity of the trypsin is ≥4000U / g, the activity of the papain is ≥300000U / g, the activity of the alkaline 2709 protease is ≥200000U / g, and the activity of the collagenase is ≥100000U / g. The amount of the compound enzyme added in step (3) is 0.075% of the mass of the porcine small intestinal mucosa solution.

2. The method as described in claim 1, characterized in that, The final concentration of sodium chloride in step (2) is 0.5~0.7%.

3. The method as described in claim 2, characterized in that, The dilution in step (1) is to dilute the volume of mucosal fluid in each pig small intestine to 7.5-8.5L.

4. The method as described in claim 3, characterized in that, The mass of the resin in step (5) accounts for 0.4 to 0.5% of the mass of the porcine small intestinal mucosa solution.

5. The method as described in claim 4, characterized in that, The alcohol precipitation in step (6) involves using ethanol with a final concentration of 25-55% and allowing it to stand for 12-24 hours.

6. The use of the method according to any one of claims 1 to 5 in the preparation of products containing heparin sodium.

Images