阅读说明：本技术 生物酶法利用母猪肠制备粗品肝素钠的方法 (Method for preparing crude heparin sodium by using sow intestines through biological enzyme method ) 是由 辛玉昌 黄蓉屏 邓泽胜 范时兵 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母猪肠制备粗品肝素钠的方法。本发明中包括以下步骤：肠粘膜提取、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 sow intestines through a biological enzyme method. The invention comprises the following steps: the invention can increase the separation effect between the grease and the protein, reduce the production cost, has mature and practical technology, high heparin yield and stable process quality.)

1. The method for preparing crude heparin sodium by using sow intestines through a biological enzyme method comprises the following steps:

(1) intestinal mucosa extraction

Rolling healthy fresh sow intestines with deionized water by using a rolling machine for multiple times, then feeding the rolled intestines into an intestine scraping machine for scraping, and collecting intestinal mucosa mixed liquor prepared in the rolling and scraping processes;

(2) enzymolysis

Pumping the intestinal mucosa mixed solution into an enzymolysis tank by using a lift pump, stirring at normal temperature, adjusting the salinity of the mixed solution to be more than 4.5 by using saturated strong brine, adjusting the pH to 9.0 by using a sodium hydroxide solution, keeping the temperature of the mixed solution at 35-10 ℃, then adding trypsin, and carrying out primary heat preservation, secondary heat preservation and high-temperature inactivation 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

The adsorption solution is pumped into an adsorption tank, the pH value is adjusted to be more than 8.0, the salinity is more than 3.0, the treated resin is added for ion adsorption, and the adsorption is accelerated 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 the biological enzyme method as claimed in claim 1, which comprises the following steps: the water quantity of the deionized water used in the step (1) is controlled to be 5 +/-1 Kg per sow intestine.

3. The method for preparing crude heparin sodium by the biological enzyme method as claimed in claim 1, which comprises the following steps: the stirring speed used in step (2) was 60. + -. 5 rpm.

4. The method for preparing crude heparin sodium by the biological enzyme method as claimed in claim 1, which comprises the following steps: the concentration of the sodium hydroxide solution used in steps (2) and (5) was 0.3. + -. 0.2mol/L, and the trypsin used in step (2) was added as 1Kg of trypsin (20 ten thousand units) per 750Kg of sow intestine.

5. The method for preparing crude heparin sodium by the biological enzyme method as claimed in claim 1, which comprises the following steps: the method comprises the following steps:

preparing an enzyme inactivation solution;

one-time heat preservation

Heating the mixed solution to 48 ℃, adjusting the pH value to 8.5, adjusting the salinity to be more than 3.6, and keeping the temperature for 50 +/-10 min;

secondary heat preservation

Heating the mixed solution to 58 deg.C, adjusting pH to 8.5, salinity >3.2, and maintaining the temperature for 120 min;

high temperature inactivation

Raising the temperature to 80 ℃, keeping the pH value more than 8.0 and the salinity more than 3.0, and keeping the temperature for 10min to obtain the enzyme inactivation solution.

6. The method for preparing crude heparin sodium by the biological enzyme method as claimed in claim 1, which comprises the following steps: 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 the biological enzyme method as claimed in claim 1, which comprises the following steps: the temperature of the step (4) is controlled to be 60 +/-2 ℃.

8. The method for preparing crude heparin sodium by the biological enzyme method as claimed in claim 1, which comprises the following steps: the resin in the step (5) is Bayer resin, and the dosage of the resin is 1Kg of Bayer resin added to every 60Kg of sow intestines.

9. The method for preparing crude heparin sodium by the biological enzyme method as claimed in claim 1, which comprises the following steps: 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 method for preparing crude heparin sodium, in particular to a method for preparing crude heparin sodium by utilizing sow intestines 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 in a sow farm 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, the method for preparing the crude heparin sodium by the sow intestines through the biological enzyme method is provided.

The technical scheme adopted by the invention is as follows:

(1) intestinal mucosa extraction

Rolling healthy fresh sow intestines with deionized water by using a rolling machine for multiple times, then feeding the rolled intestines into an intestine scraping machine for scraping, and collecting intestinal mucosa mixed liquor prepared in the rolling and scraping processes;

(2) enzymolysis

Pumping the intestinal mucosa mixed solution into an enzymolysis tank by using a lift pump, stirring at normal temperature, adjusting the salinity of the mixed solution to be higher than 4.5 ℃ by using saturated strong brine, adjusting the pH to 9.0 by using a sodium hydroxide solution, adjusting the temperature of the mixed solution to be 35-10 ℃, then adding trypsin, and carrying out primary heat preservation, secondary heat preservation and high-temperature inactivation 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

The adsorption solution is pumped into an adsorption tank, the pH value is adjusted to be more than 8.0, the salinity is adjusted to be more than 3.0 ℃, the treated resin is added for ion adsorption, and the adsorption is accelerated 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 water amount of the deionized water used in the step (1) is controlled to be 5 +/-1 Kg per sow intestine.

Wherein the stirring speed used in the step (2) is a speed of 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 that 1Kg of trypsin (20 ten thousand units) is added to every 750Kg of sow intestines.

Wherein the method comprises the steps of:

preparing an enzyme inactivation solution;

(1) one-time heat preservation

Heating the mixed solution to 48 ℃, adjusting the pH value to 8.5, adjusting the salinity to be more than 3.6 ℃, and keeping the temperature for 50 +/-10 min;

(2) secondary heat preservation

Heating the mixed solution to 58 deg.C, adjusting pH to 8.5, salinity >3.2 deg.C, and maintaining for 120 min;

(3) high temperature inactivation

Raising the temperature to 80 ℃, keeping the pH value more than 8.0 and the salinity more than 3.0, and keeping the temperature for 10min to obtain the enzyme inactivation solution.

Wherein the method 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 more than 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 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 intestinal mucosa of the sow, 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 be 4.5 ℃, the pH is adjusted to be 9.0, 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 steps of primary heat preservation, secondary heat preservation and high-temperature inactivation are adopted in the step (2), different enzymes can carry out enzymolysis and then inactivation treatment on different proteins, so that the decomposition effect on impurities such as other 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 "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. indicate orientations or positional relationships, and are only used for convenience in describing the present invention and simplifying the description, but do not indicate or imply that the device or element referred to must have a particular orientation, be constructed in a particular orientation, and be operated, and thus should not be construed as limiting the present invention; 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; either directly or indirectly through intervening media, or 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. rolling healthy fresh sow intestines with deionized water by using a rolling machine for multiple times, then feeding the rolled intestines into a intestine scraping machine for scraping, and collecting intestinal mucosa mixed liquor prepared in the rolling and scraping processes, wherein the water amount is controlled to be 5 +/-1 Kg per sow intestine;

b. pumping the mixed liquor of the intestinal mucosa into an enzymolysis tank by using a lift pump, stirring at the speed of 60 +/-5 rpm at normal temperature, adjusting the salinity of the mixed liquor to be higher than 4.5 ℃ by using saturated concentrated brine, adjusting the pH to 9.0 by using 0.3 +/-0.2 mol/L sodium hydroxide solution, controlling the temperature of the mixed liquor to be 35-40 ℃, and adding trypsin according to the standard of adding 1Kg of trypsin (20 ten thousand units) to every 750Kg of sow intestine;

c. heating the mixed solution to 48 ℃, adjusting the pH value to 8.5 and the salinity to be more than 3.6 ℃, preserving heat for 50 +/-10 min, then heating the mixed solution to 58 ℃, adjusting the pH value to 8.5 and the salinity to be more than 3.2 ℃, preserving heat for 120min, then heating to 80 ℃, adjusting the pH value to be more than 8.0 and the salinity to be more than 3.0 ℃, preserving heat for 10min, and obtaining an enzyme inactivation solution;

d. 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;

e. cooling the second salinization solution by using a plate heat exchanger, and controlling the temperature to be 60 +/-2 ℃ to obtain an adsorption solution;

f. pumping the adsorption solution into an adsorption tank, adjusting the pH to be more than 8.0 by using 0.3 +/-0.2 mol/L sodium hydroxide solution, adjusting the salinity of the mixed solution to be more than 3.0 ℃ by using saturated concentrated brine, adding 1Kg of Bayer resin into each 60Kg of sow intestines for ion adsorption, and stirring and adsorbing at the speed of 55 +/-5 rpm for 420 +/-60 min;

g. collecting the adsorbed resin by a resin collecting tank, pumping the resin into a rinsing tank for thorough cleaning, removing impurities such as protein, grease and the like, and discharging the adsorption waste liquid into sewage treatment;

h. 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.