Bio-fermentation and enzymatic production technology of crude heparin sodium from pig small intestinal mucosa

Abstract

The present invention discloses a bio-fermentation and enzymatic hydrolysis production technology for extracting crude sodium heparin from the mucosa of the pig small intestine, belonging to the technical field of production for extracting crude sodium heparin. The present invention uses a bio-active enzyme controlled bio-fermentation technology combined with an enzymatic hydrolysis method to extract heparin sodium biomacromolecules with anticoagulant biological activity. The anticoagulant activity yield in the resulting enzymatic hydrolyzate is 114,000 USPU / root, and the yield of the dried solid crude sodium heparin obtained is 88,843 USPU / root, or 1,126 roots / 100 million units. Compared with the process of enzymatic hydrolysis alone without the combined bio-active enzyme bio-fermentation technology, the yield is increased by 22.2%. This demonstrates that the bio-active enzyme controlled bio-fermentation technology combined with the enzymatic hydrolysis extraction technology has significant advantages in industrial production. At the same time, the salt usage in production is 1.2%, which is a 66% decrease compared to the common salt usage in existing industry methods, reducing the difficulty of wastewater treatment and facilitating industrial application.

Description

Technical Field

[0001] The invention relates to a biological fermentation and enzymatic hydrolysis production technology for extracting crude heparin sodium from pig small intestinal mucosa, belonging to the technical field of production of crude heparin sodium extraction. Background Art

[0002] Heparin, commonly used medically as its sodium salt, is also commonly called heparin sodium. Heparin is an acidic mucopolysaccharide, a sulfated mucopolysaccharide composed of alternating components of glucosamine, L-iduronide, N-acetylglucosamine, and D-glucuronic acid. It has an average molecular weight of 15 kDa and is primarily produced by mast cells and basophils. It is abundant in tissues such as the lungs, heart, liver, and muscle, but its concentration in plasma is very low under physiological conditions. Heparin belongs to the class of mucopolysaccharide sulfate anticoagulants. Its anticoagulant effect is very strong both in vitro and in vivo, leading to its widespread clinical use as an anticoagulant. Heparin anticoagulants have a wide range of medical uses: (a) treatment of early disseminated intravascular coagulation (DIC) associated with various diseases; (b) prevention of arterial and venous thrombosis and pulmonary embolism; (c) treatment of arterial and venous thrombosis and pulmonary embolism, ischemic stroke, unstable angina (to alleviate symptoms and prevent myocardial infarction), and acute myocardial infarction (to prevent early reinfarction and extension of the infarcted area, and reduce mortality); (d) as an anticoagulant during artificial heart-lung, peritoneal dialysis, or hemodialysis; (e) as maintenance therapy for thrombolytic therapy; (f) as an in vitro anticoagulant for preventing blood coagulation during transfusion and for preserving fresh blood in blood banks; (g) recent studies have shown that heparin also has multiple medical uses, including lipid-lowering and anti-tumor effects.

[0003] Because of the extremely complex molecular structure of sodium heparin, there are currently no successful examples of artificially synthesizing it, and it can only be extracted from animal tissue. Currently, the only officially approved source of sodium heparin in Europe and the United States is from porcine small intestinal mucosal fluid. Crude sodium heparin is produced from porcine small intestine. The intestine is processed using a scraper to separate the casing from the mucosa. The casing is then salted with food-grade casing salt to sterilize and preserve freshness.

[0004] Currently, the industry's existing crude heparin sodium production technologies primarily include high-salt salting, enzymatic hydrolysis, a combined salting-enzymatic hydrolysis method, and a complex enzyme-enzymatic hydrolysis method. In particular, the high-salt salting and combined salting-enzymatic hydrolysis methods require the addition of enteric brine and salt during the extraction process, resulting in salt concentrations as high as 3.5% by mass. High salt concentrations exacerbate the aging of intestinal mucosal fluid and the mast cells contained therein, making mucosal tissue breakdown more difficult and hindering mast cell lysis, resulting in reduced extraction efficiency and a direct reduction in crude heparin sodium yield. Furthermore, wastewater salt concentrations generally exceed 3.5%, making wastewater treatment difficult and costly. Patent CN110437348A provides a method for extracting crude heparin sodium from porcine small intestinal mucosa using a combined salting-enzymatic hydrolysis method, using alkaline protease 2709. The salt concentration used was 5%, reducing the number of pig intestines required to produce 100 million units of heparin from 1,873 to 1,166. While this significantly improved production efficiency, the high salt concentration of 5% during production replicated the inherent flaws of the salt hydrolysis process, causing the chloride ion content in wastewater to exceed the standard and irreversible environmental pollution. This method is no longer practical in actual production.

[0005] Patent CN112159486B provides a method for extracting crude heparin sodium using low-salt composite enzymatic hydrolysis, using a salt concentration of 0.8-1.0%. Compared to conventional enzymatic hydrolysis production methods, this method reduces salt usage by 70%, providing an alternative approach to partially addressing the problem of excessive chloride ion content in wastewater discharge. However, the extraction yield of crude heparin sodium in existing methods is generally low. This is due to the biological and molecular biology principles of crude heparin sodium extraction, which focus solely on enzymatic hydrolysis of the heparin sodium precursor, the "heparin sodium-protein complex," to cleave the protein molecules and release the heparin sodium molecules with anticoagulant biological activity. However, no research reports or patents exist in the current state of the art on how to maximize the production of the "heparin sodium-protein complex" precursor of heparin sodium. The existing art lacks a method for efficiently extracting crude heparin sodium under low-salt conditions. Summary of the Invention

[0006] The present invention adopts biologically active enzymes and actively uses the bio-fermentation technology of bioactive bacteria such as probiotics to rupture the pig small intestinal mucosal tissue, thereby producing a maximum amount of free mast cells. Moreover, under the synergistic effect of the active biological enzymes, the mast cells are lysed as much as possible to produce a free sodium heparin precursor - "heparin-protein complex", which is used for further enzymatic cleavage of protein molecules, freeing sodium heparin molecules and increasing as much raw material as possible. Thus, the purpose of increasing the yield of crude sodium heparin is achieved to the greatest extent possible. At the same time, the different effects of the ionic strength of different sodium chloride concentrations on the small intestinal mucosal fluid are fully considered, and the appropriate sodium chloride ionic strength is selected, which not only stabilizes the enzymatic hydrolyzate protein macromolecular solution system, ensures the purification and separation of stable sodium heparin bioactive macromolecules, but also avoids the decrease in the yield of crude sodium heparin caused by too low salt concentration.

[0007] The technical solution of the present invention includes a series of complex technical processes across four interdisciplinary disciplines: biological histology, molecular biology, enzymology, and purification and separation science. The first step is the bioactive fermentation technical process for intestinal mucosal tissue rupture; the second step is the bioactive fermentation technical process for mast cell lysis; the third step is the enzymatic hydrolysis technical process; and the fourth step is the purification and separation technical process. Among them, the first and second steps of the present invention, the bioactive fermentation technical process, are applied to the new technical method of extracting sodium heparin from pig small intestinal mucosal fluid, which has never been reported in research or patent applications. The method of the present invention is the first to be conceived, invented and studied, and has achieved remarkable results in actual application.

[0008] The process of bioactive fermentation technology is described as follows:

[0009] The first step is to use pig small intestinal mucosal fluid and controllable bio-fermentation technology with bioactive enzymes to decompose and rupture the pig small intestinal mucosal tissue as much as possible. In this process, the final crude heparin sodium yield is increased by 22.2%, which is used as an indirect quantitative evaluation method to increase the production of free mast cells.

[0010] In the second step, free mast cells are lysed using bioactive enzyme controlled biofermentation technology to produce free heparin sodium precursor - "heparin-protein complex"; this process increases the final crude heparin sodium yield by 18%, which serves as an indirect quantitative evaluation method for the production of free heparin sodium precursor - "heparin-protein complex".

[0011] The third step is to use a separate pancreatic enzyme enzymatic method to perform a targeted enzymatic cleavage of the free heparin sodium precursor, the "heparin-protein complex," to cause the linking chemical bonds of the "heparin-protein complex" to undergo targeted, efficient, and selective enzymatic cleavage at "precise protein cleavage sites," releasing the heparin sodium biomacromolecule with anticoagulant biological activity.

[0012] In the fourth step, the released heparin sodium and protein mixture is selectively adsorbed and desorbed using a large-pore anion resin to separate the heparin sodium. Alcohol precipitation and fractionation techniques are then used to obtain the crude heparin sodium product (purification and separation technology for bioactive macromolecules).

[0013] The present invention provides a method for extracting crude heparin sodium from pig small intestinal mucosa, comprising the following steps:

[0014] (1) Preparation of pig small intestinal mucosal solution: dilute the pig small intestinal mucosal solution to a volume of 6-12 L per pig small intestinal mucosal solution, adjust the sodium chloride salt concentration to 0.8-1.6%, and adjust the pH to 6-8;

[0015] (2) Biological fermentation: adding a bioactive enzyme and an adjuvant to the pig small intestinal mucosal solution obtained in step (1), heating the solution to 25-50° C., and keeping the temperature for 60-300 minutes to obtain a fermented small intestinal mucosal solution; the amount of the bioactive enzyme added is 0.05-0.30% of the mass of the pig small intestinal mucosal solution, and the amount of the adjuvant added is 0.05-0.30% of the mass of the pig small intestinal mucosal solution;

[0016] (3) Enzymatic hydrolysis: the pH of the fermented small intestinal mucosal fluid obtained in step (2) is adjusted to 6-8, pancreatic enzyme is added at 0.01-0.15% of the mass of the pig small intestinal mucosal fluid, the temperature is raised to 50-55°C, and the temperature is kept for 120-300 minutes to obtain an enzymatic hydrolyzate;

[0017] (4) Adsorption: The enzymatic hydrolysate obtained in step (3) is heated to 70-95°C, kept warm for 10-60 minutes, then filtered and cooled to 56-60°C, subjected to resin adsorption and elution, and a crude heparin sodium solution is obtained; the mass of the resin accounts for 0.10-0.50% of the mass of the pig small intestinal mucosal fluid;

[0018] (5) Collecting crude heparin sodium: The crude heparin sodium solution obtained in step (4) is subjected to alcohol precipitation and drying to obtain crude heparin sodium.

[0019] In one embodiment, the salt concentration in step (1) is 1.2-1.6%.

[0020] In one embodiment, the pancreatic enzymes in step (3) include ≥3000 U / g of trypsin, ≥30000 U / g of pancreatic lipase, and ≥60000 U / g of pancreatic amylase, and the amount of the pancreatic enzymes added is 0.01-0.04% of the mass of the mucosal fluid of the pig small intestine.

[0021] In one embodiment, the bioactive enzyme in step (2) comprises probiotics and biological enzymes; the probiotics are Bifidobacterium lactis, and the colony count of Bifidobacterium lactis is ≥1×10 9CFU / g; the biological enzyme is alkaline protease, and the enzyme activity content is 30000U / g; the added amount of the biologically active enzyme is 0.05~0.15% of the mass of the pig small intestine mucosal solution.

[0022] In one embodiment, the auxiliary agent in step (2) includes, by mass fraction, 20-50% sodium bicarbonate, 5-30% glucose, 15-50% citric acid, and 30-60% sodium metabisulfite; the amount of the auxiliary agent added is 0.05-0.25% of the mass of the porcine small intestinal mucosal solution.

[0023] In one embodiment, after adding the bioactive enzyme and the fermentation agent in step (2), the mixture is kept warm for 45 to 150 minutes.

[0024] In one embodiment, the pH in step (1) is 7-8.

[0025] In one embodiment, the eluent used in step (4) is a sodium chloride solution with a mass fraction of 18 to 25%.

[0026] In one embodiment, the alcohol precipitation in step (5) refers to adding 70-90% alcohol to the eluent to reach a final ethanol concentration of 25-55%, and allowing the solution to settle for at least 12 hours.

[0027] The present invention also provides heparin sodium prepared by the method.

[0028] The present invention also provides application of the method in preparing a product containing heparin sodium.

[0029] Beneficial effects:

[0030] 1. The present invention utilizes controlled biofermentation of bioactive enzymes combined with pancreatic enzymatic hydrolysis technology. In an embodiment, under suitable low-salt concentrations, the resulting enzymatic hydrolyzate exhibited an anticoagulant activity yield of 114,000 USPU / root. The crude heparin sodium produced 1,338 grams of a pale yellow solid with an anticoagulant activity titer of 83 USPU / mg, yielding a crude heparin sodium yield of 88,843 USPU / root, demonstrating its significant industrial application value. Compared to extraction techniques without controlled bioactive enzyme fermentation for heparin sodium, the enzymatic hydrolyzate obtained in the present invention exhibited a 23.9% increase in anticoagulant activity yield and a 22.2% increase in crude product yield. Compared to extraction techniques using alkaline protease 2709 for heparin sodium, the crude product yield in the present invention was increased by 21.7%.

[0031] 2. The suitable low salt concentration used in the present invention is 1.2% sodium chloride mass concentration. Compared with the high salt concentration of 3.5%, the chloride ion concentration decreases by 66%, which greatly reduces the difficulty of environmental protection treatment of wastewater discharge and reduces costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The following is a biological flow chart of the enzymatic hydrolysis production process of crude heparin sodium in the existing technical solution;

[0033] Figure 2 This is a biological flow chart of the new biological fermentation and enzymatic hydrolysis production technology for extracting crude heparin sodium from pig small intestinal mucosa;

[0034] Figure 3 is the anticoagulant activity of heparin sodium in enzymatic hydrolysate at different salt concentrations;

[0035] Figure 4 is the crude yield of heparin sodium corresponding to different salt concentrations. DETAILED DESCRIPTION

[0036] The experimental materials used in the present invention are as follows:

[0037] Bioactive enzyme: A mixture of homemade active probiotics and bio-enzymes, containing 1 billion colony-forming units (CFUs) of active probiotics and 30,000 units of bio-enzymes per gram. The bioactive enzyme is added in an amount of 0.05-0.30% by weight of the porcine small intestinal mucosal solution. Preferably, the bioactive enzyme is added in an amount of 0.10% by weight of the porcine small intestinal mucosal solution.

[0038] The active probiotic raw material is purchased from Shaanxi Yunqi Biotechnology Co., Ltd. and is food-grade Bifidobacterium lactis, containing 10 billion live bacteria / gram, item number YQ20210901. The bio-enzyme raw material is purchased from Henan Xinyangshao Bio-enzyme Preparation Co., Ltd. and is food-grade alkaline protease, item number 00211008, with a concentration of 200,000 units / gram. Before use, mix 1 part Bifidobacterium lactis, 1.5 parts alkaline protease, and 7.5 parts softened water (purified water obtained by ion exchange) and mix well. This mixture of active probiotics and bio-enzymes is prepared immediately before use.

[0039] The auxiliary agent is a mixture containing, by mass, 30% sodium bicarbonate, 10% glucose, 20% citric acid, and 40% sodium metabisulfite, and the amount added is 0.20% of the mass of the porcine small intestine mucosal solution.

[0040] Pancreatin: This complex enzyme is extracted from porcine pancreas, containing trypsin activity greater than 4,000 units / g, pancreatic lipase activity greater than 30,000 units / g, and pancreatic amylase activity greater than 60,000 units / g. Pancreatin was purchased from Chongqing Xiangsheng Biopharmaceutical Company, batch number C01210820. The dosage is 0.04% of the mass of the porcine small intestinal mucosal solution.

[0041] The conditions for trypsin hydrolysis are: pH 8-8.5, 52℃±2, insulation for 150 minutes, and maintaining the pH value at around 8 during the insulation process.

[0042] Adsorption resin: DuPont Rohm and Haas (USA) FPA98Cl large-pore strong anion heparin sodium resin. The mass of the resin accounts for 0.40% of the mass of the porcine small intestinal mucosal fluid.

[0043] The following describes the technical solutions in detail in the embodiments of the present invention, in conjunction with the accompanying drawings. The technical method of the present invention, "New Technology for the Bio-fermentation and Enzymatic Hydrolysis Production of Crude Heparin Sodium from Porcine Small Intestinal Mucosa," is not limited to the following embodiments. Any modifications, equivalent substitutions, and improvements within the biological principles and basic logical thinking of the present invention are intended to be included within the scope of protection of the present invention.

[0044] Example 1: Bio-fermentation and enzymatic production method for extracting crude heparin sodium from pig small intestinal mucosa

[0045] This embodiment relates to a fermentation and enzymatic hydrolysis technology for extracting crude sodium heparin from porcine small intestinal mucosa. The method comprises controlled fermentation using bioactive enzymes under appropriate sodium chloride concentrations to rupture intestinal mucosal tissue and lyse mast cells, producing a heparin sodium precursor, a "heparin-protein complex." Furthermore, the free heparin sodium precursor, a "heparin-protein complex," substrate, is subjected to targeted, efficient, and selective enzymatic hydrolysis using pancreatic enzymes, maximizing the number of protein chains attached to the heparin molecules, thereby releasing the heparin sodium biomacromolecules with anticoagulant bioactivity. The method then undergoes inactivation by heating, followed by filtration to remove residue. Finally, the heparin sodium in the fermentation / enzymatic hydrolysis mixture is subjected to adsorption and desorption using a wide-pore strong anion exchange resin to obtain a crude sodium heparin eluate. The collected heparin sodium wet solid is then precipitated and dehydrated using alcohol. Finally, vacuum drying is performed to obtain a crude sodium heparin solid. This totals eight steps.

[0046] Step 1: Controlled Biofermentation with Bioactive Enzymes: 1,250 fresh pig small intestines were scraped with softened water. 7,000 L of pig mucosal fluid was transferred to a 10,000 L enzymatic hydrolysis tank. 500 L of 24% casing saline and tap water were added to adjust the salinity of the intestinal mucosal fluid. Using a high-precision digital electronic salinity meter, the salinity reached ~1.2 (equivalent to 1.2% by mass). Softened water was then added to 10,000 L to obtain a pig small intestinal mucosal solution (dilute the volume of each pig small intestinal solution to 8.0 L). With thorough stirring, the pH was carefully adjusted to 7.5 using a 1 M dilute sodium hydroxide solution. After thorough mixing, 10,000 g of bioactive enzyme and 20,000 g of auxiliary agents were added. The temperature was slowly raised to 39°C ± 2°C. With continued stirring, the temperature was maintained for 120 minutes to obtain the fermented small intestinal mucosal fluid.

[0047] The controlled biofermentation process employed in this method effectively digests and decomposes the intestinal mucosal tissue, thoroughly rupturing and releasing mast cells, and promoting efficient mast cell lysis. However, excessive microbial fermentation can cause fermentation, rancidity, and decay of the intestinal mucosal fluid, further degrading the heparin sodium biomolecule. Therefore, the controllability of the bioactive enzyme fermentation process is crucial. This controlled biofermentation process is achieved through two steps. First, the sodium chloride concentration is controlled at approximately 1.2%, effectively enhancing microbial activity and improving the fermentation efficiency. Excessive salt concentrations can strongly inhibit the bioactivity of probiotics, compromising the fermentation efficiency. Second, the adjuvant, a mixture of sodium bicarbonate, glucose, citric acid, and sodium metabisulfite, contains both components that enhance microbial activity and preservatives that control fermentation, rancidity, and decay. Through the optimal blend of these components, efficient microbial activity is achieved while controlling rancidity and decay in the intestinal mucosal fluid.

[0048] Step 2, the above-mentioned bioactive enzyme is subjected to controllable biofermentation to obtain a biofermented small intestinal mucosal fluid, which contains a key heparin sodium precursor - "heparin-protein complex" substrate, and further implements pancreatic enzymatic hydrolysis of the free heparin sodium precursor - "heparin-protein complex" substrate: adjust the pH value to 8-8.5 with a small amount of 1M dilute sodium hydroxide lye, raise the temperature to 52°C ± 2, add 4000 grams of pancreatic enzyme, continue stirring, and keep warm for 150 minutes. During the insulation process, the pH value is adjusted to about 8. After the insulation is completed, an enzymatic solution containing heparin sodium with anticoagulant biological activity is obtained. The anticoagulant activity titer of heparin sodium in the enzymatic solution is 14.2 USPU / ml (equivalent to a yield of 114,000 USPU / root).

[0049] Step 3: heating the above-obtained biologically active heparin sodium solution to 80°C ± 5°C and keeping the temperature for 30 minutes.

[0050] Step 4: Filter the enzymatic hydrolysate to remove residue using a 60-mesh filter. Cool the filtrate to 58°C ± 2°C and transfer it to an adsorption tank.

[0051] Step 5: Add 40 kg of Rohm and Haas (USA) wide-pore strong anion resin and allow to adsorb for 8-10 hours with stirring. Filter and collect the resin, and the filtrate is sent to a wastewater treatment system. The resin dosage is 0.40% of the mass of the porcine small intestinal mucosal solution.

[0052] Step 6: Wash the resin collected in step 5 twice with 5% by mass dilute brine, and then desorb it three times with saturated brine solution. The desorption time is 2 hours and the temperature is 55° C. to collect the sodium heparin desorbed liquid.

[0053] Step 7: Add food-grade alcohol at approximately 85% ethanol to the heparin sodium desorption solution obtained in Step 6 until the ethanol content reaches 45%. Allow to stand for 12 hours. Filter to obtain wet heparin sodium solid. Dehydrate with ethanol at or above 93% ethanol for 2-3 hours. Then, filter through a 200-mesh filter to obtain wet heparin sodium solid.

[0054] In step 8, the wet heparin sodium solid obtained in step 7 was vacuum dried at 65° C. for 12 hours to obtain 1,338 g of dry crude heparin sodium as a light yellow solid with a potency of 83 USPU / mg and a crude heparin sodium yield of 88,843 USPU / root.

[0055] The concentration of sodium chloride in the wastewater generated by the above extraction method is 1.2%.

[0056] Example 2: Adjusting the Ratio of Bioactive Enzymes to Produce Crude Heparin Sodium

[0057] Specific implementation method: Refer to Example 1, except that the content of the biological enzyme in the biologically active enzyme is adjusted. Mix "1 part of Bifidobacterium lactis: 3 parts of alkaline protease: 6 parts of softened water" and stir evenly. That is, the mixture of active probiotics and biological enzymes prepared contains 1×10 9 Colony unit, alkaline protease activity content per gram 60000 units. The amount of bioactive enzyme added is still 0.10% of the mass of the mucosal solution of the pig small intestine. The results show that the activity titer of heparin sodium in the enzymatic hydrolyzate is 13.6USPU / ml (equivalent to a yield of 109,000 USPU / root), which is 4.6% lower than the 114,000 USPU / root of Example 1. Further, 1287 grams of dry crude heparin sodium light yellow solid are obtained with a titer of 82.0USPU / mg and a crude heparin sodium yield of 84427 US standard units / root. The crude heparin sodium yield of Comparative Example 1 is 88843 US standard units / root, a decrease of 5.2%.

[0058] Example 3: Adjusting the fermentation degree of biological enzymes to produce crude heparin sodium

[0059] Specific embodiments refer to Example 1, except that the fermentation time of the biologically active enzyme is adjusted to 45 minutes. The results show that the activity titer of heparin sodium in the enzymatic hydrolyzate is 12.6 USPU / ml (equivalent to a yield of 101,000 USPU / root), which is 11.4% lower than the 114,000 USPU / root of Example 1. Further, 1139 grams of dried crude heparin sodium light yellow solid was obtained with a titer of 89.0 USPU / mg and a crude heparin sodium yield of 81,097 American Standard units / root. The crude heparin sodium yield of Comparative Example 1 was 88,843 American Standard units / root, a decrease of 9.6%.

[0060] Comparative Example 1: Extraction of crude heparin sodium at different salt concentrations

[0061] Specific implementation method Referring to Example 1, the difference is that the mass concentration of sodium chloride is changed to 0.4%, 0.8%, 1.6%, 2.0%, and 3.5%, and the anticoagulant activity titer of heparin sodium in the enzymatic hydrolysis solution obtained in step 2 and the quality, titer, and yield of the heparin sodium product obtained in step 8 are tested. The results are shown in Table 1. Figure 3 and Figure 4 shown.

[0062] When the mass concentration of sodium chloride was 0.4%, 0.8%, 1.6%, 2.0%, and 3.5%, the anticoagulant activity titers of heparin sodium in the obtained enzymatic hydrolysis solution were 6.5, 10.6, 12.6, 11.2, and 9.2 USPU / ml, respectively (equivalent to yields of 5.2, 8.5, 10.1, 9.0, and 74,000 USPU / root), and the dried crude heparin sodium light yellow solids obtained were 761 g, 1093 g, 1198 g, 1093 g, and 927 g, respectively, with titers of 66.5, 75.5, 84.1, 85.4, and 86.3 USPU / mg, respectively. The yields of crude heparin sodium were 40,485, 66,017, 80,601, 74,674, and 640 million American Standard units / root.

[0063] Table 1 Anticoagulant activity and yield of heparin sodium in enzymatic hydrolysate and crude product at different salt concentrations

[0064]

[0065] From Table 1 and Figure 3 、 Figure 4 As can be seen, the anticoagulant activity of sodium heparin in the enzymatic hydrolysate and the yield of crude sodium heparin gradually increased as the salt concentration increased from 0.4% to 0.8%. The highest anticoagulant activity and crude sodium heparin yield were achieved at a salt concentration of 1.2%. This indicates that the efficiency of biofermentation gradually increases from low to high salt concentrations, reaching its peak at a salt concentration of 1.2%. As the salt concentration further increased, from 1.6% to 3.5%, the anticoagulant activity and crude sodium heparin yield decreased. This suggests that increasing salt concentration inhibits the biofermentation efficiency of probiotics.

[0066] from Figure 3The relationship between sodium chloride concentration and the enzymatic hydrolysis of crude heparin sodium provides a good understanding of the impact of sodium chloride concentration on the efficiency of bio-fermentation and the overall process of enzymatic hydrolysis and extraction of crude heparin sodium. On the one hand, high salt concentrations inhibit the probiotics involved in bio-fermentation, reducing the efficiency of bio-fermentation. Simultaneously, the "aging" of intestinal mucosal tissue and mast cells at high salt concentrations also affects the rupture of intestinal mucosal tissue and the lysis of mast cells. These two-way interactions reduce the efficiency of bio-fermentation. On the other hand, appropriately low salt concentrations enhance the activity of microbial probiotics, more efficiently rupturing intestinal mucosal tissue and mast cells, further maximizing their lysis and thereby maximizing the release of the free heparin sodium precursor—the "heparin-protein complex" substrate. Furthermore, when the salt concentration drops to a certain level, the salt's ionic strength also decreases, reducing the stabilizing effect of the ionic strength on the macromolecular system of the enzymatic hydrolyzate, thereby reducing the yield of heparin sodium.

[0067] As can be seen from Table 1 and Figure 4, the highest crude heparin sodium yield was obtained at a salt concentration of 1.2%. As the salt concentration increased from 0.4% to 0.8%, the crude heparin sodium yield gradually increased. At 1.2%, the highest yield was obtained. This shows that from low salt concentration to high salt concentration, the efficiency of biological fermentation gradually increases, and further, the crude heparin sodium yield increases synchronously. When the salt concentration reaches 1.2%, it reaches the highest. When the salt concentration further increases from 1.6% to 3.5%, the crude heparin sodium yield continues to decrease. This shows that from a salt concentration of 1.6% to 3.5%, the efficiency of biological fermentation continues to decrease, and the crude heparin sodium yield decreases synchronously.

[0068] Comparative Example 2: Production of crude heparin sodium by enzymatic hydrolysis with alkaline protease

[0069] Specific embodiments refer to Example 1, except that the pancreatic enzyme in step 2 is replaced with Bacillus subtilis alkaline protease 2709, with an enzyme activity of 200,000 units / gram, and the amount of Bacillus subtilis alkaline protease 2709 is 0.04% of the mass of the mucosal fluid of the pig small intestine. The results show that the activity titer of heparin sodium in the enzymatic hydrolyzate obtained in step 2 is 10.6 USPU / ml (equivalent to a yield of 85,000 USPU / root), which is 34.1% lower than the titer in Example 1. Further, 1098 grams of dried crude heparin sodium light yellow solid are obtained, with a titer of 83.1 USPU / mg and a crude heparin sodium yield of 72,995 American Standard units / root. Compared with the crude heparin sodium yield in Example 1, the yield decreased by 21.7% after replacing pancreatic enzyme with Bacillus subtilis alkaline protease 2709.

[0070] Comparative Example 3: Production of crude heparin sodium by enzymatic hydrolysis with pancreatic enzyme and alkaline protease

[0071] Specific embodiments refer to Example 1, except that the pancreatic enzyme in step 2 is replaced with a mixed enzyme of 4000 grams of pancreatic enzyme and 4000 grams of subtilisin alkaline protease 2709. The enzyme activity of the subtilisin alkaline protease 2709 is 200,000 units / gram, and the dosage of the pancreatic enzyme and subtilisin alkaline protease 2709 is 0.04% of the mass of the mucosal fluid of the pig small intestine. The results show that the activity titer of heparin sodium in the enzymatic hydrolyzate obtained in step 2 is 9.9 USPU / ml (equivalent to a yield of 79,000 USPU / root), which is 43.4% lower than the titer in Example 1. Further, 1036 grams of dried crude heparin sodium light yellow solid are obtained, with a titer of 83.8 USPU / mg and a crude heparin sodium yield of 69453 American Standard units / root. Compared with the crude heparin sodium yield of Example 1, after replacing pancreatic enzyme with a mixed enzyme of pancreatic enzyme and subtilisin alkaline protease 2709, the yield decreases by 27.9%.

[0072] Comparative Example 4: Production of crude heparin sodium by fermentation without bioactive enzymes

[0073] Specific implementation method Referring to Example 1, the difference is that the bioactive enzyme controllable biological fermentation process in step one is omitted, and steps 2 to 8 are directly implemented. The results show that the activity titer of heparin sodium in the enzymatic hydrolyzate is 11.5 USPU / ml (equivalent to a yield of 92,000 USPU / root), which is 23.9% lower than the 114,000 USPU / root in Example 1. Further, 1108 grams of dried crude heparin sodium light yellow solid are obtained with a titer of 82.0 USPU / mg and a crude heparin sodium yield of 72,685 American Standard units / root. The crude heparin sodium yield of Comparative Example 1 is 88,843 American Standard units / root, a decrease of 22.2%.

[0074] Comparative Example 5: Adjusting the ratio of biologically active enzymes to produce crude heparin sodium

[0075] The specific embodiment is with reference to Example 1, the difference is, adjust the content of Bifidobacterium lactis in the biologically active enzyme. By "0.5 part of Bifidobacterium lactis: 1.5 parts of alkaline protease: 6.5 parts of softened water", stir evenly. In the mixture of active probiotics and biological enzymes obtained, Bifidobacterium lactis contains 500 million colony units per gram, and the alkaline protease activity content is 30,000 units per gram. The addition amount of the biologically active enzyme is still 0.10% of the mass of the mucosal solution of the pig small intestine. The result shows that the activity titer of heparin sodium in the enzymolysis solution is 12.2USPU / ml (equivalent to a yield of 98,000 USPU / root), which is compared with the 114,000 USPU / root of Example 1, a decrease of 14.0%. Further, 1207 grams of dry crude heparin sodium light yellow solids are obtained, with a titer of 81.0USPU / mg and a crude heparin sodium yield of 78214 American Standard Units / root. The crude heparin sodium yield of Comparative Example 1 was 88,843 American Standard units per piece, a decrease of 13.6%.

[0076] The results showed that reducing the bio-fermentation process in the bioactive enzyme would lead to a decrease in the yield of crude heparin sodium.

[0077] Comparative Example 6: Adjusting the ratio of biologically active enzymes to produce crude heparin sodium

[0078] With reference to Example 1, the difference is that the content of Bifidobacterium lactis in the biologically active enzyme is adjusted. Before use, by " 2 parts of Bifidobacterium lactis: 1.5 parts of alkaline proteases: 6.5 parts of softened water ", stir evenly. In the mixture of the active probiotics and the biological enzyme obtained, 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 the biologically active enzyme is still 0.10% of the mass of the mucosal solution of the pig small intestine. The result shows that the activity titer of heparin sodium in the enzymolysis solution is 10.8USPU / ml (equivalent to 86,000 USPU / root of yield), which is compared with 114,000 USPU / root of Example 1, and decreases by 24.6%. Further, 982 grams of dry crude heparin sodium light yellow solids are obtained, with a titer of 86.0USPU / mg and a crude heparin sodium yield of 67,562 American Standard Units / root. The crude heparin sodium yield of Comparative Example 1 was 88,843 American Standard units per piece, a decrease of 31.5%.

[0079] The results showed that the yield of crude heparin sodium decreased instead of increased in the fermentation process with too high bioactive enzyme.

[0080] Comparative Example 7: Adjusting the ratio of biologically active enzymes to produce crude heparin sodium

[0081] The specific embodiment refers to Example 1, the difference is that the bio-enzyme content in the bioactive enzyme is adjusted. By "1 part of Bifidobacterium lactis: 0.5 part of alkaline protease: 8.5 parts of softened water", stir evenly. In the mixture of active probiotics and bio-enzymes obtained, Bifidobacterium lactis contains 1 billion colony units per gram, and the alkaline protease activity content is 10,000 units per gram. The addition amount of the bioactive enzyme is still 0.10% of the mass of the mucosal solution of the pig small intestine. The results show that the activity titer of heparin sodium in the enzymolysis solution is 11.8USPU / ml (equivalent to a yield of 94,000 USPU / root), which is 21.3% lower than the 114,000 USPU / root of Example 1. Further, 1152 grams of dry crude heparin sodium light yellow solids are obtained, with a titer of 86.0USPU / mg and a crude heparin sodium yield of 79,258 American Standard Units / root. The crude heparin sodium yield of Comparative Example 1 was 88,843 American Standard units per piece, a decrease of 12.1%.

[0082] The results showed that the yield of crude heparin sodium decreased significantly in the biofermentation process with three times lower enzyme activity.

[0083] Comparative Example 8: Adjusting the fermentation degree of biological enzymes to produce crude heparin sodium

[0084] Specific embodiments refer to Example 1, except that the fermentation time of the biologically active enzyme is adjusted to 180 minutes. The results show that the activity titer of heparin sodium in the enzymatic hydrolyzate is 11.0 USPU / ml (equivalent to a yield of 88,000 USPU / root), which is 29.5% lower than the 114,000 USPU / root of Example 1. Further, 1093 grams of dried crude heparin sodium light yellow solid was obtained with a titer of 75.0 USPU / mg and a crude heparin sodium yield of 65,580 US Standard units / root. The crude heparin sodium yield of Comparative Example 1 was 88,843 US Standard units / root, a decrease of 35.5%.

[0085] The results showed that extending the fermentation time of the bioactive enzymes increased the risk of over-fermentation and spoilage, and the yield of crude heparin sodium decreased. When the fermentation time was doubled to 180 minutes, the yield of crude heparin sodium decreased by 35.5%.

[0086] Comparative Example 9: Adjusting the Type and Ratio of Auxiliary Agents to Produce Crude Heparin Sodium

[0087] The specific implementation method refers to Example 1. The adjuvant in Example 1 is a homemade mixture containing 30% sodium bicarbonate, 10% glucose, 20% citric acid, and 40% sodium metabisulfite. The difference in Example 9 is that the type and ratio of the adjuvants are adjusted. Under the conditions where both 20% citric acid and 30% sodium bicarbonate remain unchanged, the glucose content is reduced from 10% to 5%, and the sodium metabisulfite is increased to 45%. The results show that the activity titer of heparin sodium in the enzymatic hydrolyzate is 12.3 USPU / ml (equivalent to a yield of 98,000 USPU / root), a decrease of 16.3% compared to 114,000 USPU / root in Example 1. Furthermore, 1173 grams of dried crude heparin sodium light yellow solid was obtained, with a titer of 84.0 USPU / mg and a crude heparin sodium yield of 78,826 USPU / root. Compared with the crude heparin sodium yield of 88,843 USPU / root in Example 1, it is a decrease of 12.7%.

[0088] The results showed that when the content of glucose, the activating component of the bioactive enzyme, was reduced and the content of sodium metabisulfite, which controls excessive fermentation and corruption, was increased, the yield of crude sodium heparin would decrease significantly.

[0089] Comparative Example 10: Adjusting the Type and Ratio of Auxiliary Agents to Produce Crude Heparin Sodium

[0090] The specific embodiment refers to Example 1. The adjuvant in Example 1 was a homemade mixture containing 30% sodium bicarbonate, 10% glucose, 20% citric acid, and 40% sodium metabisulfite. The difference in Example 10 was that the type and ratio of the adjuvants were adjusted. Under the condition of maintaining the 20% citric acid, the glucose content was increased from 10% to 50%, the sodium metabisulfite was reduced to 15%, and the sodium bicarbonate was reduced to 15%. The results showed that the activity titer of heparin sodium in the enzymatic hydrolyzate was 10.2 USPU / ml (equivalent to a yield of 82,000 USPU / root), a decrease of 39.0% compared to 114,000 USPU / root in Example 1. Furthermore, 1023 grams of dry crude heparin sodium gray-black solid was obtained, with a titer of 76.0 USPU / mg and a crude heparin sodium yield of 62,198 USPU / root. Compared with the crude heparin sodium yield of 88,843 USPU / root in Example 1, a decrease of 42.8%.

[0091] The results showed that when the content of glucose, the activating component of the bioactive enzyme, was increased and the content of sodium bicarbonate and sodium metabisulfite, which control excessive fermentation and spoilage, was reduced, the yield of crude sodium heparin decreased significantly, reaching 42.8%. Moreover, due to excessive fermentation and spoilage, the enzymatic hydrolysis solution turned gray-black, resulting in the crude sodium heparin appearing as a gray-black solid.

[0092] The results showed that when the content of glucose, the activating component of the bioactive enzyme, was reduced and the content of sodium metabisulfite, which controls excessive fermentation and corruption, was increased, the yield of crude sodium heparin would decrease significantly.

[0093] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for extracting crude heparin sodium from pig small intestinal mucosa, characterized in that: The following steps are involved: (1) Preparation of porcine small intestinal mucosal solution: dilute the porcine small intestinal mucosal solution and add sodium chloride to a final concentration of 1.2-1.6% to adjust the pH to 6-8. The dilution is to add water to 6-12 L of each porcine small intestinal mucosal solution; (2) Biofermentation: adding bioactive enzymes and adjuvants to the pig small intestinal mucosal solution obtained in step (1), heating the solution to 25-50° C., and keeping the temperature for 45-120 minutes to obtain fermented small intestinal mucosal solution; the amount of the bioactive enzyme added is 0.05-0.30% of the mass of the pig small intestinal mucosal solution; the amount of the adjuvant added is 0.05-0.30% of the mass of the pig small intestinal mucosal solution; (3) Enzymatic hydrolysis: the pH of the fermented small intestinal mucosal fluid obtained in step (2) is adjusted to 6-8, pancreatic enzyme is added at 0.01-0.10% of the mass of the pig small intestinal mucosal fluid, the temperature is raised to 50-55°C, and the temperature is kept for 120-300 minutes to obtain an enzymatic hydrolyzate; (4) Adsorption: The enzymatic hydrolysate obtained in step (3) is heated to 70-95°C, kept warm for 10-60 minutes, then filtered and cooled to 56-60°C, subjected to resin adsorption and elution, and a crude heparin sodium solution is obtained; the mass of the resin accounts for 0.10-0.50% of the mass of the pig small intestinal mucosal fluid; (5) Collecting crude heparin sodium: subjecting the crude heparin sodium solution obtained in step (4) to alcohol precipitation and drying to obtain crude heparin sodium; The biologically active enzyme in step (2) includes alkaline protease of ≥30000 U / g; including 1×10 9 CFU / g of Bifidobacterium lactis; the amount of the bioactive enzyme added is 0.05-0.15% of the mass of the pig small intestine mucosal solution; The auxiliary agent in step (2) includes, by mass fraction, 20-30% sodium bicarbonate, 10-30% glucose, 15-20% citric acid, and 30-40% sodium metabisulfite; the amount of the auxiliary agent added is 0.15-0.25% of the mass of the porcine small intestinal mucosal solution.

2. The method according to claim 1, wherein The pancreatic enzymes in step (3) include ≥3000 U / g of trypsin, ≥30000 U / g of pancreatic lipase, and ≥60000 U / g of pancreatic amylase. The amount of the pancreatic enzymes added is 0.01-0.04% of the mass of the mucosal fluid of the pig small intestine.

3. The method according to claim 1, wherein: The activity of the alkaline protease is 30,000-60,000 U / g.

4. The method according to claim 1, wherein: The eluent used in step (4) is a sodium chloride solution with a mass fraction of 18 to 25%.

5. Heparin sodium prepared by the method according to any one of claims 1 to 4.

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

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