阅读说明：本技术 生物酶法利用猪肺制备粗品肝素钠的方法 (Method for preparing crude heparin sodium by utilizing pig lungs through biological enzyme method ) 是由 辛玉昌 黄蓉屏 邓泽胜 于 2019-09-27 设计创作，主要内容包括：本发明公开了生物酶法利用猪肺制备粗品肝素钠的方法。本发明中包括以下步骤：肺糜提取、酶解、分离、降温、吸附、树脂收集以及固化,本发明能够增加油脂与蛋白质之间的分离效果,降低生产成本,技术成熟、实用,肝素收率高,工艺品质稳定。(The invention discloses a method for preparing crude heparin sodium by utilizing pig lungs through a biological enzyme method. The invention comprises the following steps: the invention has the advantages of capability of increasing the separation effect between oil and protein, reduction of production cost, mature and practical technology, high heparin yield and stable process quality.)

1. The method for preparing crude heparin sodium by utilizing pig lungs through the biological enzyme method comprises the following steps:

(1) preparation of pulmonary chyme

Cleaning healthy pig lungs, removing trachea and fat, mincing with a meat mincer, and mixing and stirring with deionized water to obtain a lung paste mixed solution;

(2) enzymolysis

Pumping the minced lung mixed liquor into an enzymolysis tank by using a lift pump, continuously stirring, regulating the salinity of the mixed liquor to 5.5 +/-0.5 ℃ by using saturated concentrated brine, then heating, adding 1kg of trypsin per 350kg of pig lung when the temperature reaches a first preset temperature, adding the trypsin for enzymolysis, then stirring, continuously heating to 60 ℃, preserving heat, and then inactivating at high temperature to obtain an enzyme inactivation solution;

(3) separation of

Separating oil and fat proteins from the enzyme inactivation liquid by a low-speed liquid-solid three-phase separator and a high-speed liquid-solid three-phase separator respectively to obtain a salinization liquid after the proteins and the oil are separated, and pouring the salinization liquid into a buffer tank;

(4) temperature reduction

Cooling the salt solution by using a plate heat exchanger to obtain an adsorption solution;

(5) adsorption

Pumping the adsorption solution into an adsorption tank, adjusting the pH value to 8.2 +/-0.2 and the salinity to 2.9 +/-0.1, adding the treated resin for ion adsorption, and accelerating the adsorption by stirring;

(6) resin collection

Collecting the adsorbed resin by a resin collecting tank, and pumping the resin into a rinsing tank for thorough cleaning to remove impurities such as protein, grease and the like;

(7) curing

And eluting, precipitating and drying the collected resin by using a traditional process to obtain a crude product of heparin sodium.

2. The method for preparing crude heparin sodium by using the pig lung through the biological enzyme method as claimed in claim 1, which is characterized in that: the mass ratio of the deionized water quantity to the pig lung obtained in the step (1) is 1: 1.

3. The method for preparing crude heparin sodium by using the pig lung through the biological enzyme method as claimed in claim 1, which is characterized in that: the first preset temperature in the step (2) is 30 ℃, and the stirring speed is 60 +/-5 rpm.

4. The method for preparing crude heparin sodium by using the pig lung through the biological enzyme method as claimed in claim 1, which is characterized in that: the concentration of the sodium hydroxide solution used in steps (2) and (5) is 0.3 + -0.2 mol/L, and the trypsin used in step (2) is added according to the standard of adding 1Kg of trypsin (20 ten thousand units) per 350Kg of pig lung.

5. The method for preparing crude heparin sodium by using the pig lung through the biological enzyme method as claimed in claim 1, which is characterized in that: the method comprises the following steps:

preparing an enzyme inactivation solution;

(1) heat preservation

Heating the mixed solution to 48 ℃, adjusting the pH value to 8 +/-0.5, and keeping the temperature for 210 +/-30 min;

(2) high temperature inactivation

Raising the temperature to 95 + -2 deg.C, pH 8.5 + -0.5, and salinity 5.5 + -0.5, stopping stirring, and maintaining the temperature for 10min to obtain enzyme inactivation solution.

6. The method for preparing crude heparin sodium by using the pig lung through the biological enzyme method as claimed in claim 1, which is characterized in that: the method comprises the following steps:

preparing a salinization solution;

(1) rough fraction

Separating oil and protein of the enzyme inactivation liquid by using a low-speed liquid-solid three-phase separator with the separation factor of 2500 +/-150 g and the rpm of 3500 +/-100 to obtain a first salinization liquid;

(2) finely dividing

And (3) performing oil and protein fine separation on the coarsely separated salinized solution again by using a high-speed liquid-solid three-phase separator with the separation factor of 12200 +/-200 g and the rpm of 6400 +/-300 to obtain a second salinized solution.

7. The method for preparing crude heparin sodium by using the pig lung through the biological enzyme method as claimed in claim 1, which is characterized in that: the temperature of the step (4) is controlled to be 60 +/-2 ℃.

8. The method for preparing crude heparin sodium by using the pig lung through the biological enzyme method as claimed in claim 1, which is characterized in that: the resin in the step (5) is Bayer resin, and the dosage of the resin is 1Kg of Bayer resin added to every 400Kg of enzymolysis liquid.

9. The method for preparing crude heparin sodium by using the pig lung through the biological enzyme method as claimed in claim 1, which is characterized in that: the stirring speed in the step (5) is 55 +/-5 rpm, and the stirring time is 420 +/-60 min.

Technical Field

The invention belongs to a preparation method of crude heparin sodium, and particularly relates to a method for preparing crude heparin sodium by utilizing pig lungs through a biological enzyme method.

Background

Heparin sodium interferes with many links of the blood coagulation process, has an anticoagulant effect both in vitro and in vivo, has a complex action mechanism, enhances the inhibition effect of the antithrombin III on activated blood coagulation factors II, IX, X, XI and XII mainly through the combination with the antithrombin III, and has the effects of preventing platelet aggregation and destruction, preventing the formation of blood coagulation activating enzyme, preventing prothrombin from being changed into thrombin, inhibiting thrombin, preventing fibrinogen from being changed into fibrin and exerting the anticoagulant effect.

The technical problems in production of extracting heparin sodium from pig lungs mainly include high production cost, low heparin yield and lack of competitive advantages due to the fact that the oil and protein separation technology is not solved for a long time.

Disclosure of Invention

The invention aims to: in order to solve the technical problems in reality, a method for preparing crude heparin sodium by utilizing pig lungs through a biological enzyme method is provided.

The technical scheme adopted by the invention is as follows:

(1) preparation of pulmonary chyme

Cleaning healthy pig lungs, removing trachea and fat, mincing with a meat mincer, and mixing and stirring with deionized water to obtain a lung paste mixed solution;

(2) enzymolysis

Pumping the minced lung mixed liquor into an enzymolysis tank by using a lift pump, continuously stirring, regulating the salinity of the mixed liquor to 5.5 +/-0.5 ℃ by using saturated concentrated brine, then heating, adding 1kg of trypsin per 350kg of pig lung when the temperature reaches a first preset temperature, adding the trypsin for enzymolysis, then stirring, continuously heating to 60 ℃, preserving heat, and then inactivating at high temperature to obtain an enzyme inactivation solution;

(3) separation of

Separating oil and fat proteins from the enzyme inactivation liquid by a low-speed liquid-solid three-phase separator and a high-speed liquid-solid three-phase separator respectively to obtain a salinization liquid after the proteins and the oil are separated, and pouring the salinization liquid into a buffer tank;

(4) temperature reduction

Cooling the salt solution by using a plate heat exchanger to obtain an adsorption solution;

(5) adsorption

Pumping the adsorption solution into an adsorption tank, adjusting the pH value to 8.2 +/-0.2 and the salinity to 2.9 +/-0.1, adding the treated resin for ion adsorption, and accelerating the adsorption by stirring;

(6) resin collection

Collecting the adsorbed resin by a resin collecting tank, and pumping the resin into a rinsing tank for thorough cleaning to remove impurities such as protein, grease and the like;

(7) curing

And eluting, precipitating and drying the collected resin by using a traditional process to obtain a crude product of heparin sodium.

Wherein the mass ratio of the deionized water quantity to the pig lung obtained in the step (1) is 1: 1.

Wherein the first preset temperature in the step (2) is 30 ℃, and the stirring speed used is 60 +/-5 rpm.

Wherein, the concentration of the sodium hydroxide solution used in the steps (2) and (5) is 0.3 +/-0.2 mol/L, and the trypsin used in the step (2) is added according to the standard of adding 1Kg of trypsin (20 ten thousand units) per 350Kg of pig lung.

Wherein the method further comprises the steps of:

preparing an enzyme inactivation solution;

(3) heat preservation

Heating the mixed solution to 48 ℃, adjusting the pH value to 8 +/-0.5, and keeping the temperature for 210 +/-30 min;

(4) high temperature inactivation

Raising the temperature to 95 + -2 deg.C, pH 8.5 + -0.5, and salinity 5.5 + -0.5, stopping stirring, and maintaining the temperature for 10min to obtain enzyme inactivation solution.

Wherein the method further comprises the steps of:

preparing a salinization solution;

(1) rough fraction

Separating oil and protein of the enzyme inactivation liquid by using a low-speed liquid-solid three-phase separator with the separation factor of 2500 +/-150 g and the rpm of 3500 +/-100 to obtain a first salinization liquid;

(2) finely dividing

And (3) performing oil and protein fine separation on the coarsely separated salinized solution again by using a high-speed liquid-solid three-phase separator with the separation factor of 12200 +/-200 g and the rpm of 6400 +/-300 to obtain a second salinized solution.

Wherein the temperature of the step (4) is controlled to be 60 +/-2 ℃.

Wherein the resin in the step (5) is Bayer resin, and the dosage of the resin is 1Kg of Bayer resin added to every 400Kg of enzymolysis solution.

Wherein the stirring speed in the step (5) is 55 +/-5 rpm, and the stirring time is 420 +/-60 min.

In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:

1. in the invention, because deionized water is adopted in the step (1) to extract and mix the porcine lung mucosa, the reaction between ions in water and the effective component heparin sodium is reduced, and the concentration of the heparin sodium in the extracted intestinal mucosa mixed solution is improved.

2. In the invention, the temperature of the mixed liquor is controlled between 35 ℃ and 40 ℃ in the step (2), the salinity of the mixed liquor is adjusted to 5.5 +/-0.5 ℃, the pH is adjusted to 8.5 +/-0.5 ℃, and the mixed liquor is stirred at the speed of 60 +/-5 rpm, so that the enzymolysis reaction effect of the trypsin is improved.

3. In the invention, because the heat preservation and high-temperature inactivation steps are adopted in the step (2), different enzymes can be subjected to enzymolysis and then inactivation treatment on different proteins, so that the decomposition effect on impurities such as the rest proteins is improved

4. In the invention, two centrifuges are respectively adopted in the step (3) to carry out coarse separation and fine separation on the enzyme inactivation liquid, so that protein and grease can be separated, and the purification effect of heparin sodium is improved.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the following embodiments further describe the present invention in detail. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In the description of the present invention, it should be noted that the terms "first", "second", and "third" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance; furthermore, unless expressly stated or limited otherwise, the terms "mounted," "connected," and "connected" are to be construed broadly, as they may be fixedly connected, detachably connected, or integrally connected, for example; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.

Example (b):

a. cleaning healthy fresh pulmonis Sus Domestica, removing trachea and fat, mincing with meat mincer, mixing with deionized water, and stirring to obtain pulmonis Domestica mixture, wherein the mass ratio of pulmonis Sus Domestica to water is 1: 1;

b. pumping the minced lung mixed solution into an enzymolysis tank by using a lift pump, continuously stirring, regulating the salinity of the mixed solution to 5.5 +/-0.5 ℃ by using saturated concentrated brine, heating to 30 ℃, regulating the pH to 8.5 +/-0.5 ℃ by using 0 +/-0.2 mol/L sodium hydroxide solution, continuously heating to 48 +/-2 ℃, adding 1kg of trypsin (20 ten thousand units) according to the dosage of each 350kg of pig lung when the temperature is 48 ℃, adding the trypsin for enzymolysis, stirring at the stirring speed of 60 +/-5 rpm, and continuously heating to 60 ℃;

c. keeping the pH value of the mixed solution at 8 +/-0.5 and the temperature at 60 ℃, and keeping the temperature for 210 +/-30 min;

d. raising the temperature to 95 + -2 deg.C, adjusting pH to 8.5 + -0.5 and salinity to 5.5 + -0.5, stopping stirring, and maintaining the temperature for 10min to obtain enzyme inactivation solution;

e. separating oil and protein of the enzyme inactivation liquid by using a low-speed liquid-solid three-phase separator with separation factors of 2500 +/-150 g and rpm of 3500 +/-100 to obtain a first salinization liquid, and performing oil and protein fine separation on the coarsely separated first salinization liquid again by using a high-speed liquid-solid three-phase separator with separation factors of 12200 +/-200 g and rpm of 6400 +/-300 to obtain a second salinization liquid;

f. cooling the second salinization solution by using a plate heat exchanger, and controlling the temperature at 60 ℃ to obtain an adsorption solution;

g. the adsorption solution is pumped into an adsorption tank, the PH value is adjusted to 8.2 +/-0.2, the salinity is 2.9 +/-0.1, 1Kg of Bayer resin is added into every 400Kg of enzymolysis solution for ion adsorption, and the treated resin is added for ion adsorption, and is stirred and adsorbed for 420 +/-60 min at the speed of 55 +/-5 rpm;

h. collecting the adsorbed resin by a resin collecting tank, pumping into a rinsing tank for thorough cleaning, removing impurities such as protein, grease and the like, discharging the adsorption waste liquid into a sewage treatment center, or performing deep processing to co-produce and extract protein peptide powder.

i. And eluting, precipitating and drying the collected resin by using a traditional process to obtain a crude product of heparin sodium.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.