CN112159486B - Method for preparing crude heparin sodium by utilizing low salt of small intestine mucosa of pig - Google Patents

Abstract

The invention provides a method for preparing crude heparin sodium by utilizing low salt of a small intestine mucosa of a pig, which comprises the steps of carrying out low salt compound enzymolysis to obtain an enzymolysis solution, concentrating the enzymolysis solution to obtain a concentrated solution, carrying out ion exchange to obtain a crude heparin sodium eluent, and drying alcohol precipitation to obtain the crude heparin sodium: in the low-salt compound enzymolysis, saturated casing saline is added, and the salt concentration of the mucosal solution is regulated to be 0.8-1.0% by mass. The method for preparing the crude heparin sodium by utilizing the low salt of the pig intestinal mucosa can realize enzymolysis and adsorption processes of the crude heparin sodium under the low salt condition, can ensure the yield of the crude heparin sodium, simultaneously reduce the salt concentration in discharged wastewater, reduce the wastewater treatment cost, reduce environmental pollution and increase comprehensive economic benefits.

Description

Method for preparing crude heparin sodium by utilizing low salt of small intestine mucosa of pig

Technical Field

The invention relates to the technical field of extraction processes of crude heparin sodium of animal casings, in particular to a method for preparing crude heparin sodium by utilizing low salt of small intestinal mucosa of pigs.

Background

Heparin is a mucopolysaccharide widely existing in mammalian tissues, mainly existing in mast cells, has an anticoagulant effect, and is widely used for surgery and treatment of cerebral thrombosis, myocardial infarction and the like. Heparin has a molecular weight of about 3000-37500 and studies on commercial heparin suggest at least 21 molecular individuals. Heparin sodium is sodium salt of heparin, is the most practical anticoagulation medicine in clinical medicine field, is mucopolysaccharide sulfate composed of D-glucosamine, N-acetylglucosamine, D-glucuronic acid and L-iduronic acid alternately, and is widely used for preventing and treating cerebral thrombosis. Heparin sodium is mostly present in protein-heparin complexes in combination with proteins, and is mostly present in the small intestinal mucosa and lung with the highest content. Heparin sodium is mainly used for anticoagulation, and is one of the most important anticoagulation and antithrombotic biochemical drugs at present. The crude heparin sodium product is a traditional export product in China and plays an important role internationally.

At present, heparin sodium cannot be artificially synthesized and can only be extracted from animal tissues. Heparin sodium is mainly extracted from small intestine mucous membranes of pigs and sheep, beef lung, saline casing saline and the like. The pig small intestine mucosa is the main raw material for extracting heparin sodium at present. The raw material for producing the crude heparin sodium is pig small intestine, the pig small intestine is processed by an intestine scraping machine, the casing is separated from intestinal mucosa, the casing is salted by saturated saline, sterilization and preservation are carried out, and the saturated saline of the casing is soaked with heparin components. The crude heparin sodium is sodium salt of heparin extracted from pig small intestine mucosa, and is raw material of refined heparin sodium. The crude heparin sodium product contains more impurities and is not pure in sample, so that the crude heparin sodium product can not be directly used as a clinical medicine. The quality of crude heparin sodium determines the quality of refined heparin sodium to a certain extent, so that the preparation of the crude heparin sodium is important.

The production methods of crude heparin are mainly high-salt hydrolysis method, enzymolysis salt hydrolysis compound method and enzymolysis method. Because the sausage casing salting process can produce a large amount of saturated sausage casing salt water, the treatment of high-concentration salt water is very difficult, enterprises usually adopt a high-salt salting method and an enzymolysis salt hydrolysis compounding method, and the sausage casing salt water is added in the reaction process, so that the inner part is naturally consumed, and the sausage casing salt water is discharged along with production wastewater. However, the salt concentration of the production wastewater in the industry is generally 3% or more, and the wastewater treatment is difficult and has high cost.

For example, CN105218705a relates to a method for preparing heparin sodium by using double enzymolysis method. The method for preparing heparin sodium by using the double enzymolysis method comprises the following steps: and the heparin sodium in the heparin sodium-protein complex is dissociated by adopting a double enzymolysis method, and then the heparin sodium is completely extracted through the steps of heating, resin adsorption, elution, alcohol precipitation, drying and the like, so that the heparin sodium yield is improved. The adoption of double enzymolysis can properly reduce salt concentration, reduce pollution, and reduce steam consumption and cooling water consumption. The pig pancreas can be used after being activated in mass production, and the cost of the pig pancreas per 6 tons of reaction liquid is about 100 yuan. Pig pancreas costs and steam costs can be offset. The salt concentration in this patent application is 3.0-4.0%.

In another example, CN108456262a provides a process for preparing high-purity heparin sodium, which mainly comprises the steps of 3.0% (w/v) sodium chloride salt hydrolysis, 0.1% -0.2% (w/v) trypsin enzymolysis, hydrogen peroxide-ozone high-efficiency oxidation, absolute ethanol precipitation, acetone dehydration, vacuum drying and the like, wherein the hydrogen peroxide-ozone high-efficiency oxidation system can generate HO2-, which is an inducer generated by free radical oh, and once the free radical oh is generated, chain reaction can be initiated, so that the oxidation speed is increased, and the oxidation effect is improved. Compared with the traditional multistage hydrogen peroxide oxidation treatment, the process for purifying heparin sodium saves at least 40 percent of time, improves the potency by 9U/mg, improves the potency recovery rate by 8 percent, and has high purity and good color of the prepared heparin sodium.

Along with the increasing severity of domestic environmental protection situation, many small and medium enterprises are gradually eliminated due to serious pollution of high-salt wastewater. Therefore, developing a low-salt or even salt-free crude heparin sodium preparation method becomes a problem to be solved urgently by those skilled in the art.

Disclosure of Invention

In view of the above, the invention aims to provide a method for preparing crude heparin sodium by utilizing low salt of the mucosa of the small intestine of a pig, so as to solve the problem of high-salt wastewater pollution on the premise of ensuring the quality of the crude heparin sodium.

In order to achieve the above purpose, the technical scheme of the invention is realized as follows:

a method for preparing crude heparin sodium by using low salt of small intestine mucosa of pig comprises the following steps

a. Low-salt composite enzymolysis: preparing a mucous membrane solution of pig small intestine, adding saturated enteric-coated saline, regulating the salt concentration of the mucous membrane solution to 0.8-1.0%, and regulating the pH of the mucous membrane solution to 7.0-8.0; heating to 50-55 ℃, adding compound enzyme for enzymolysis to obtain enzymolysis liquid;

b. concentrating the enzymolysis liquid: concentrating the enzymolysis liquid to obtain concentrated liquid, wherein the volume of the concentrated liquid is 1/10 of that of the enzymolysis liquid;

c. ion exchange: ion exchange is carried out on the concentrated solution to obtain crude heparin sodium eluent;

d. alcohol precipitation and drying: and (3) carrying out alcohol precipitation on the crude heparin sodium eluent, and drying the precipitate of the alcohol precipitation to obtain the crude heparin sodium.

Further, in the step a, the enzymolysis conditions are as follows: firstly preserving heat for 2-3 h, secondly heating to 80-85 ℃, preserving heat for 20-30 min again, and finally cooling to 50-55 ℃.

Further, in step a, the complex enzymes are 2709 alkaline protease and pancreatin.

Further, the addition amount of the complex enzyme is 0.1 to 0.2 percent of the mass of the mucosa solution of the small intestine of the pig.

Further, the mass ratio of 2709 alkaline protease to pancreatin is 2:1.

Further, in the step a, saturated casing saline is added, and the salt concentration of the mucosal solution is adjusted to be 0.88-0.92% by mass.

Further, in the step b, the step of concentrating the enzymolysis liquid comprises the following steps:

b1, centrifuging the enzymolysis liquid, wherein the rotation speed of the centrifuge is 5000r/min, the flow is 3-6 m < 3 >/h, and the continuous operation is carried out;

b2, filtering the supernatant after centrifugation through a 500nm ceramic membrane;

b3, concentrating the filtered supernatant through a nanofiltration membrane, wherein the molecular weight cut-off of the nanofiltration membrane is more than 300-400 daltons, the operating pressure is 1.5Mpa, and the operating temperature is 35 ℃.

Further, in step c, the step of ion exchanging includes:

c1, transferring the concentrated solution into an ion exchange column filled with anion exchange resin to adsorb heparin, wherein the adsorption time is 10 hours;

c2, washing the ion exchange column with a NaCl solution with the mass fraction of 2%;

and c3, eluting the ion exchange column by using 20% NaCl solution by mass fraction to obtain crude heparin sodium eluent.

Further, in the step d, the step of drying the alcohol starch includes:

d1, adding 90-95 DEG food grade alcohol into the crude heparin sodium eluent, and adjusting the alcohol content to 40-50 DEG for alcohol precipitation, wherein the alcohol precipitation time is 8-12 h;

d2, separating out an alcohol precipitation;

d3, drying the precipitate of the alcohol precipitation at 80 ℃ for 8 hours to obtain crude heparin sodium.

Compared with the prior art, the invention has the following advantages:

1. the method for preparing the crude heparin sodium by utilizing the low-salt porcine intestinal mucosa can realize enzymolysis and adsorption processes of the heparin sodium under the low-salt condition, can reduce the salt concentration in discharged wastewater, reduce the wastewater treatment cost, reduce environmental pollution and increase comprehensive economic benefits while ensuring the yield of the heparin sodium.

2. The invention uses the composite enzymolysis technology to separate heparin from protein more thoroughly, and the heparin sodium yield is further improved.

3. The invention has the advantages of low reaction temperature, low consumption of precipitated alcohol, energy conservation, consumption reduction, simple and convenient process steps and strong operability, and can be applied to mass production.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained from these drawings without inventive effort for a person skilled in the art. In the drawings:

FIG. 1 is a graph showing the enzyme activity of 2709 alkaline protease of example 1 of the present invention at various salt concentrations;

FIG. 2 is a graph showing enzymatic hydrolysis efficiencies of 2709 alkaline protease and pancreatin at different salt concentrations in example 1 of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The invention will be described in detail below with reference to the drawings in connection with embodiments.

Example 1

The embodiment relates to a method for preparing crude heparin sodium by utilizing low-salt of a small intestinal mucosa of a pig, which comprises four steps of low-salt compound enzymolysis to obtain an enzymolysis solution, concentration of the enzymolysis solution to obtain a concentrated solution, ion exchange to obtain a crude heparin sodium eluent and alcohol precipitation drying to obtain heparin sodium.

Step one, low-salt compound enzymolysis: preparing a mucous membrane solution of pig small intestine, adding saturated enteric-coated saline, regulating the salt concentration of the mucous membrane solution to 0.8-1.0% by mass, and regulating the pH of the mucous membrane solution to 7.0-8.0; heating to 50-55 ℃, adding compound enzyme for enzymolysis, and obtaining enzymolysis liquid. Specifically, firstly, fresh small intestine mucosa solution of pigs is put into an enzymolysis reaction tank, preferably, the mass ratio of mucosa to water in the mucosa solution of the small intestine of pigs is 1:6-1:8, so as to ensure that enzyme has good diffusivity in the solution and is fully contacted with a reaction substrate. And then, adding saturated casing saline into the enzymolysis reaction tank, and adjusting the salt concentration of the mucous membrane solution to 0.8-1.0% by mass. And regulating the pH value of the mucous membrane liquid to 7.0-8.0 by using a NaOH solution with the mass fraction of 25%. Finally, heating to 50-55 ℃, and adding complex enzyme for enzymolysis to obtain enzymolysis liquid. More preferably, the conditions for enzymolysis are: preserving heat for 2-3 h, heating to 80-85 ℃, preserving heat for 20-30 min, and cooling to 50-55 ℃.

The design idea of the invention is mainly as follows: the high concentration brine can inhibit the growth and activity of microorganisms, which is unfavorable for the maximum efficacy of biological enzymes. Therefore, the physiological environment similar to the animal body, namely, the NaCl physiological saline concentration of 0.9 percent is designed to be adopted, so as to improve the activity of the biological enzyme, and further improve the decomposition rate of the biological enzyme on the protein-heparin conjugate in animal tissues.

Physiological saline in animals is 0.9% NaCl, under the environment, biological enzyme activity can exert the maximum effect, and various biological activity tests are carried out by taking physiological saline as a basic solution. Under the condition that a plurality of proteases exist in the mucosa internal environment of the small intestine of the pig, the heparin sodium combined on the protein can be effectively separated by using the complex enzyme for enzymolysis, and the product yield of the heparin sodium is further improved. The pancreatin is mainly extracted by animal pancreas tissue processing, and the pancreatin used in the invention belongs to enzyme produced by pig pancreas processing, is a native endogenous enzyme of pigs, and has highest natural activity in a physiological saline environment.

First, the enzyme activity of 2709 alkaline protease was measured at different salt concentrations (mass fractions of 0.45%, 0.9%, 1.8%, 2.7% and 3.6%) and was examined by the Furline method (since this method is a common method for enzyme activity measurement, the steps of the measuring method are not repeated here), and the measurement results are shown in Table 1 and FIG. 1. As can be seen from the analysis of the data in Table 1 and FIG. 1, 2709 alkaline protease had the highest enzymatic activity at salt concentrations of 0.8 to 1.0%.

TABLE 1 alkaline protease Activity at 2709 under different salt concentration conditions

Salt concentration (%)	0.45	0.9	1.8	2.7	3.6
						Enzyme activity (Wan U/g)	20.808	21.6495	21.114	20.349	19.69875

In order to further increase the performance of the method for preparing the crude heparin sodium by utilizing the low salt of the small intestinal mucosa of the pig, in one specific embodiment of the invention, the complex enzyme is 2709 alkaline protease and pancreatin, the pancreatin is added based on the environment in the pig intestine, the components of the small intestinal mucosa of the pig are complex, the protein content is higher, the variety is more, and the 2709 alkaline protease is added to supplement the pancreatin and decompose the protein. Preferably, the adding amount of the complex enzyme is 0.1-0.2% of the mass of the mucosa solution of the small intestine of the pig, so as to prevent the enzymolysis reaction from influencing the yield of crude heparin sodium. More preferably, the mass ratio of 2709 alkaline protease to pancreatin is 2:1, various macromolecular substances are decomposed, impurities in the adsorption of crude heparin sodium are reduced, and the product quality is improved.

Next, the enzymatic hydrolysis efficiency of the complex enzyme composed of pancreatin and 2709 alkaline protease was examined at different salt concentrations (0.45%, 0.9%, 1.8%, 2.7% and 3.6%), wherein the added mass of the porcine small intestine mucosa liquid, 2709 alkaline protease and pancreatin was 150g, 0.5g and 0.25g, respectively, and under this condition, a porcine intestine mucosa decomposition experiment was performed to verify the salt concentration of the complex enzyme with the optimal decomposition efficiency. The specific experimental steps are as follows:

1. equal amounts of 150g of pig small intestine mucosa solution are taken respectively, and the volume is fixed to 400ml.

2. The enzymolysis is carried out by adding reagent with normal equivalent of 400ml. Wherein the added reagent is 2709 alkaline protease and pancreatin complex enzyme, and the addition amounts of the two are 0.5g and 0.25g respectively. Meanwhile, the pig small intestine mucosa liquid without any reagent is used as blank contrast, and other conditions are the same. The enzymolysis reaction condition is pH8.0 and 52 deg.c for 2 hr.

3. After the enzymolysis reaction is finished, 100ml of mixed solution is taken and transferred to a large test tube, and naturally settled for 48 hours.

4. The remaining undissolved precipitate was compared.

Wherein, the blank sediment height is 3.2cm, and then the decomposition height of the pig small intestine mucosa liquid = blank sediment height-insoluble sediment height. The data comparison results are shown in table 2 and fig. 2. As can be seen from the analysis of the data in Table 2 and FIG. 2, the complex enzyme has the highest decomposition efficiency at a salt concentration of 0.8 to 1.0%.

TABLE 2 enzymolysis efficiency of complex enzyme under different salt concentration environments

Salt concentration (%)	0.45	0.9	1.8	2.7	3.6
						Insoluble sediment height (cm)	1.5	1.2	1.4	1.6	1.7
Decomposition height (cm)	1.7	2	1.8	1.6	1.5

Step two, concentrating enzymolysis liquid: concentrating the enzymolysis liquid to obtain concentrated liquid, wherein the volume of the concentrated liquid is 1/10 of that of the enzymolysis liquid. Specifically, firstly, separating the enzymolysis liquid by a disc type centrifugal machine, wherein the rotating speed is 5000r/min, the flow is 3-6 m < 3 >/h, so as to remove visible insoluble matters, and continuously running. And filtering the supernatant after centrifugation through a 500nm ceramic membrane to remove the impurity proteins. And finally, concentrating the filtered supernatant through a nanofiltration membrane, wherein the molecular weight cut-off of the nanofiltration membrane is more than 300-400 daltons, the operating pressure is 1.5Mpa, the operating temperature is 35 ℃, and finally the volume of the enzymolysis liquid is reduced to 1/10 of the original volume.

Step three, ion exchange: and (3) carrying out ion exchange on the concentrated solution to obtain crude heparin sodium eluent. Specifically, first, the concentrated solution is transferred to an ion exchange column filled with anion exchange resin to adsorb heparin for 10 hours. Next, the resin was washed with a 2% mass fraction of NaCl solution to remove impurities such as proteins, nucleic acids, etc. And finally, eluting the crude heparin by using a NaCl solution with the mass fraction of 20% to obtain a crude heparin sodium eluting solution.

Step four, alcohol precipitation and drying: and (3) carrying out alcohol precipitation on the crude heparin sodium eluent, and drying the precipitate of the alcohol precipitation to obtain the crude heparin sodium. Specifically, firstly, 90-95 degrees of food grade alcohol is added into the heparin sodium eluent, alcohol precipitation is carried out by adjusting the alcohol content to 40-50 degrees, and the alcohol precipitation time is 8-12 hours. Next, an alcohol precipitate was isolated. And finally, drying the precipitate of the alcohol precipitation at 80 ℃ for 8 hours to obtain crude heparin sodium.

Example 2

The present example relates to a method for preparing crude heparin sodium with low salt from pig small intestine mucosa, which is a specific application under example 1.

Example 2.1

1.1 The mucous membrane scraped from the small intestines of pigs is put into an enzymolysis reaction tank, the number of the small intestines is 1450, and the volume is 10700L.

1.2 Stirring is started, 400L of saturated casing saline is added into the enzymolysis reaction tank, 12050L of the saturated casing saline is fixed, and the concentration of the mucous membrane liquid salt is regulated to be 0.91 percent.

1.3 37L of 25% NaOH solution is added into an enzymolysis reaction tank, and the PH is measured to be 7.5 by using a PH 5.5-9.0 precision test paper.

1.4 Steam was turned on and the temperature was raised to 52 ℃. 2.7kg of compound protease is added and the temperature is kept for 2 hours. Heating to 83 ℃, and preserving heat for 25min. Cooling to 55 ℃ by cooling water.

1.5 40kg of anionic resin is added into an enzymolysis reaction tank, heparin is adsorbed for 8 hours by stirring, and the resin is collected by a 80-mesh filter bag.

1.6 The resin is washed by 72L of NaCl solution with the mass fraction of 2 percent to remove impurities such as protein, nucleic acid and the like, then the heparin is eluted by 72L of NaCl solution with the mass fraction of 20 percent, and the eluent is injected into a precipitation tank.

1.7 Starting a precipitation tank for stirring, regulating the alcoholic strength of heparin eluent to 46 DEG by using food-grade alcohol with the alcoholic strength of 90-95 DEG, closing stirring, precipitating for 12 hours, and distinguishing the solution from top to bottom.

1.8, extracting supernatant, opening a tank bottom valve to discharge sediment, then entering a hot air circulation drying box, drying at 80 ℃ and packaging to obtain crude heparin sodium. Wherein, the yield of the crude heparin sodium is 1380 roots/hundred million units, and the salt concentration in the discharged wastewater is 1.1 percent.

Example 2.2

2.1 The mucous membrane scraped from the small intestine of the pig is put into an enzymolysis reaction tank, the quantitative amount is 1500, and the volume is 10450L.

2.2 Stirring is started, 380L of saturated casing saline is added into the enzymolysis reaction tank, 12030L of the saturated casing saline is fixed in volume, and the concentration of the mucous membrane liquid salt is regulated to be 0.88%.

2.3 Adding 39L of 25% NaOH solution into an enzymolysis reaction tank, and measuring the pH value to be 8.0 by using a precision test paper with the pH value of 5.5-9.0.

2.4 Steam was turned on and the temperature was raised to 54 ℃. 3.8kg of compound protease is added and the temperature is kept for 2 hours. Heating to 82 ℃, and preserving heat for 28min. Cooling to 52 ℃ by cooling water.

2.5 Separating the enzymolysis liquid by a disc type centrifugal machine, wherein the rotating speed is 5000r/min, the flow is 4m < 3 >/h, and removing visible insoluble substances. The ceramic membrane is used for removing the foreign proteins, the nanofiltration membrane is used for concentrating, and the volume of the liquid is reduced to 1/10 of the original volume.

2.6 The nanofiltration membrane concentrate of the enzymolysis liquid enters an ion exchange column filled with anion exchange resin to carry out heparin adsorption, the resin is washed by 72L of NaCl solution with the mass fraction of 2% concentration to remove impurities such as protein, nucleic acid and the like, the heparin is eluted by 72L of NaCl solution with the mass fraction of 20% concentration, and the eluent is injected into a precipitation tank.

2.7 Starting a precipitation tank for stirring, regulating the alcoholic strength of heparin eluent to 47 DEG by using food-grade alcohol with the alcoholic strength of 90-95 DEG, closing stirring, precipitating for 12 hours, and distinguishing the solution from top to bottom.

2.8, extracting supernatant, opening a tank bottom valve to discharge sediment, then entering a hot air circulation drying box, drying at 80 ℃ and packaging to obtain crude heparin sodium. Wherein, the yield of the crude heparin sodium is 1335 roots/hundred million units, and the salt concentration in the discharged wastewater is 1.0 percent.

Example 2.3

3.1 The mucous membrane scraped from the small intestine of the pig is put into an enzymolysis reaction tank, the amount of the mucous membrane is 1550, and the volume is 10600L.

3.2 Stirring is started, 410L of saturated casing saline is added into the enzymolysis reaction tank, 12040L of the saturated casing saline is fixed in volume, and the concentration of the mucosal fluid salt is regulated to be 0.93 percent.

3.3 37L of 25% NaOH solution is added into an enzymolysis reaction tank, and the PH is measured to be 7.5 by using a PH 5.5-9.0 precision test paper.

3.4 Steam was turned on and the temperature was raised to 53 ℃. 3kg of compound protease is added, and the temperature is kept for 2.5h. Heating to 84 ℃, and preserving heat for 26min. Cooling to 54 ℃ by cooling water.

3.5 Separating the enzymolysis liquid by a disc type centrifugal machine, wherein the rotating speed is 5000r/min, the flow is 4m < 3 >/h, and removing visible insoluble substances. The ceramic membrane is used for removing the foreign proteins, the nanofiltration membrane is used for concentrating, and the volume of the liquid is reduced to 1/10 of the original volume.

3.6 The nanofiltration membrane concentrate of the enzymolysis liquid enters an ion exchange column filled with anion exchange resin to carry out heparin adsorption, the resin is washed by 72L of NaCl solution with the mass fraction of 2% concentration to remove impurities such as protein, nucleic acid and the like, the heparin is eluted by 72L of NaCl solution with the mass fraction of 20% concentration, and the eluent is injected into a precipitation tank.

3.7 Starting a precipitation tank for stirring, regulating the alcoholic strength of heparin eluent to 46 DEG by using food-grade alcohol with the alcoholic strength of 90-95 DEG, closing stirring, precipitating for 12 hours, and distinguishing the solution from top to bottom.

And 3.8, extracting supernatant, opening a tank bottom valve to discharge sediment, then entering a hot air circulation drying box, drying at 80 ℃ and packaging to obtain crude heparin sodium. Wherein, the yield of the crude heparin sodium is 1320 roots/hundred million units, and the salt concentration in the discharged wastewater is 1.1 percent.

Example 2.4

4.1 The mucous membrane scraped from the small intestine of the pig is put into an enzymolysis reaction tank, the amount of the mucous membrane is 1520, and the volume is 10550L.

4.2 Stirring is started, 370L of saturated casing saline is added into the enzymolysis reaction tank, 12020L of the saturated casing saline is fixed in volume, and the concentration of the mucosal fluid salt is regulated to be 0.8%.

4.3 38L of 25% NaOH solution is added into an enzymolysis reaction tank, and the PH value is measured to be 8.0 by using a PH 5.5-9.0 precision test paper.

4.4 Steam was turned on and the temperature was raised to 53 ℃. 3.2kg of compound protease is added and the temperature is kept for 2 hours. Heating to 84 deg.C, and maintaining for 21min. Cooling to 54 ℃ by cooling water.

4.5 Separating the enzymolysis liquid by a disc type centrifugal machine, wherein the rotating speed is 5000r/min, the flow is 4.3m < 3 >/h, and removing visible insoluble substances. The ceramic membrane is used for removing the foreign proteins, the nanofiltration membrane is used for concentrating, and the volume of the liquid is reduced to 1/10 of the original volume.

4.6 The nanofiltration membrane concentrate of the enzymolysis liquid enters an ion exchange column filled with anion exchange resin to carry out heparin adsorption, the resin is washed by 72L of NaCl solution with the mass fraction of 2% concentration to remove impurities such as protein, nucleic acid and the like, the heparin is eluted by 72L of NaCl solution with the mass fraction of 20% concentration, and the eluent is injected into a precipitation tank.

4.7 Starting a precipitation tank for stirring, regulating the alcoholic strength of heparin eluent to 46 DEG by using food-grade alcohol with the alcoholic strength of 90-95 DEG, closing stirring, precipitating for 12 hours, and distinguishing the solution from top to bottom.

4.8, extracting supernatant, opening a tank bottom valve to discharge sediment, then entering a hot air circulation drying box, drying at 80 ℃ and packaging to obtain crude heparin sodium. Wherein, the yield of the crude heparin sodium is 1350 roots/hundred million units, and the salt concentration in the discharged wastewater is 1.0 percent.

Example 2.5

5.1 The mucous membrane scraped from the small intestine of the pig is put into an enzymolysis reaction tank, the quantitative amount is 1490, and the volume is 10580L.

5.2 Stirring is started, 460L of saturated casing saline is added into the enzymolysis reaction tank, 12010L of the saturated casing saline is fixed, and the concentration of the mucosal fluid salt is regulated to be 1.0%.

5.3 36L of 25% NaOH solution is added into an enzymolysis reaction tank, and the PH is measured to be 7.0 by using a PH 5.5-9.0 precision test paper.

5.4 Steam was turned on and the temperature was raised to 53 ℃. 3.4kg of compound protease is added, and the temperature is kept for 2.5 hours. Heating to 84 deg.C, and maintaining for 25min. Cooling to 54 ℃ by cooling water.

5.5 40kg of anionic resin is added into an enzymolysis reaction tank, heparin is adsorbed for 8 hours by stirring, and the resin is collected by a 80-mesh filter bag.

5.6 The resin is washed by 72L of NaCl solution with the mass fraction of 2 percent to remove impurities such as protein, nucleic acid and the like, then the heparin is eluted by 72L of NaCl solution with the mass fraction of 20 percent, and the eluent is injected into a precipitation tank.

5.7 Starting a precipitation tank for stirring, regulating the alcoholic strength of heparin eluent to 46 DEG by using food-grade alcohol with the alcoholic strength of 90-95 DEG, closing stirring, precipitating for 12 hours, and distinguishing the solution from top to bottom.

And 5.8, extracting supernatant, opening a tank bottom valve to discharge sediment, then entering a hot air circulation drying box, drying at 80 ℃ and packaging to obtain crude heparin sodium. Wherein, the yield of the crude heparin sodium is 1360 roots/hundred million units, and the salt concentration in the discharged wastewater is 1.2 percent.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims (4)

1. A method for preparing crude heparin sodium by utilizing low salt of small intestine mucosa of a pig is characterized by comprising the following steps: comprises the following steps:

a. low-salt composite enzymolysis: preparing a mucous membrane solution of pig small intestine, adding saturated enteric-coated saline, regulating the salt concentration of the mucous membrane solution to 0.88-0.92% by mass, and regulating the pH of the mucous membrane solution to 7.0-8.0; heating to 50-55 ℃, adding compound enzyme for enzymolysis to obtain enzymolysis liquid; the complex enzyme is 2709 alkaline protease and pancreatin; the enzymolysis conditions are as follows: firstly preserving heat for 2-3 h, secondly heating to 80-85 ℃, preserving heat for 20-30 min again, and finally cooling to 50-55 ℃; the addition amount of the complex enzyme is 0.1-0.2% of the mass of the mucosa solution of the small intestine of the pig;

b. concentrating the enzymolysis liquid: concentrating the enzymolysis liquid to obtain concentrated liquid, wherein the volume of the concentrated liquid is 1/10 of that of the enzymolysis liquid;

c. ion exchange: ion exchange is carried out on the concentrated solution to obtain crude heparin sodium eluent;

d. alcohol precipitation and drying: carrying out alcohol precipitation on the crude heparin sodium eluent, and drying precipitate of the alcohol precipitation to obtain crude heparin sodium;

the mass ratio of 2709 alkaline protease to pancreatin is 2:1.

2. The method for preparing crude heparin sodium by low-salt preparation of pig small intestine mucosa according to claim 1, wherein the method comprises the following steps: in the step b, the step of concentrating the enzymolysis liquid comprises the following steps:

b1, centrifuging the enzymolysis liquid, wherein the rotation speed of the centrifuge is 5000r/min, and the flow is 3-6 m ³ And/h, continuously operating;

b2, filtering the supernatant after centrifugation through a 500nm ceramic membrane;

b3, concentrating the filtered supernatant through a nanofiltration membrane, wherein the molecular weight cut-off of the nanofiltration membrane is more than 300-400 daltons, the operating pressure is 1.5Mpa, and the operating temperature is 35 ℃.

3. The method for preparing crude heparin sodium by low-salt preparation of pig small intestine mucosa according to claim 1, wherein the method comprises the following steps: in step c, the step of ion exchange includes:

c1, transferring the concentrated solution into an ion exchange column filled with anion exchange resin to adsorb heparin, wherein the adsorption time is 10 hours;

c2, washing the ion exchange column with a NaCl solution with the mass fraction of 2%;

and c3, eluting the ion exchange column by using 20% NaCl solution by mass fraction to obtain crude heparin sodium eluent.

4. The method for preparing crude heparin sodium by low-salt preparation of pig small intestine mucosa according to claim 1, wherein the method comprises the following steps: in the step d, the alcohol precipitation drying step comprises the following steps:

d1, adding 90-95 DEG food grade alcohol into the crude heparin sodium eluent, and adjusting the alcohol content to 40-50 DEG for alcohol precipitation, wherein the alcohol precipitation time is 8-12 h;

d2, separating out an alcohol precipitation;

d3, drying the precipitate of the alcohol precipitation at 80 ℃ for 8 hours to obtain crude heparin sodium.

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