阅读说明：本技术 能常温环境贮藏和长途运输的蚓激酶浓缩浆料生产工艺 (Production process of lumbrokinase concentrated slurry capable of being stored in normal temperature environment and transported for long distance ) 是由 倪成 倪宝谦 于 2019-10-21 设计创作，主要内容包括：本发明公开了一种能常温环境贮藏和长途运输的蚓激酶浓缩浆料生产工艺,包括磨浆、除渣、过滤、脱脂、脱盐浓缩、灭菌等步骤,能够剔除水溶性杂质、变性蛋白、蚯蚓油脂等杂质,提高蚓激酶浓缩浆料中蚓激酶丰度,降低生产线分离介质的负荷,提高蚓激酶收率；且生产得到的蚓激酶浓缩浆料物化性质稳定,其中的酶活力保持持久。本发明的生产工艺适应不同蚓激酶工艺路线,制得的蚓激酶浓缩浆料能直接用于蚓激酶精制工程的蚯蚓体原料替代品,且能够常温储藏和运输,具有常年保质的特点,适用于蚓激酶工厂生产的起始原料。(The invention discloses a production process of lumbrokinase concentrated slurry capable of being stored in a normal-temperature environment and transported for a long distance, which comprises the steps of grinding, deslagging, filtering, degreasing, desalting, concentrating, sterilizing and the like, and can remove water-soluble impurities, denatured protein, earthworm oil and other impurities, improve the abundance of lumbrokinase in the lumbrokinase concentrated slurry, reduce the load of a separation medium of a production line, and improve the yield of lumbrokinase; the physical and chemical properties of the produced lumbrokinase concentrated slurry are stable, and the enzyme activity in the slurry is kept lasting. The production process is suitable for different lumbrukinase process routes, and the prepared lumbrukinase concentrated slurry can be directly used as a substitute of earthworm raw materials in lumbrukinase refining engineering, can be stored and transported at normal temperature, has the characteristic of long-term quality guarantee, and is suitable for starting raw materials produced in lumbrukinase factories.)

1. A production process of lumbrukinase concentrated slurry capable of being stored in normal temperature environment and transported for a long distance is characterized by comprising the following steps:

a) collecting earthworm slurry

Emptying impurities in the abdominal cavity of the earthworms, cleaning the impurities, and grinding the impurities into slurry to obtain earthworm ground slurry;

b) continuous centrifugal deslagging

Centrifuging and removing the residue of the earthworm mill slurry obtained in the step a) at 12000r/min to obtain supernatant;

c) clarifying, filtering, retaining residual solid of lumbricus slurry, and releasing biological macromolecules of lumbrokinase

Adding a membrane component into the supernatant obtained in the step b) to clarify the feed liquid;

d) degreasing treatment

Adding sodium chloride into the feed liquid obtained in the step c) until the concentration of the sodium chloride reaches 1M, centrifuging for 30min at 10000r/min, and separating earthworm oil from fat;

e) desalting and concentrating

Introducing the clarified liquid obtained in the step D) into a membrane module with molecular weight cutoff of 5KD to obtain concentrated liquid, adding purified water into the concentrated liquid, and introducing the membrane module with molecular weight cutoff of 5000D to obtain lumbrokinase concentrated slurry;

the whole operation process of the steps a) to e) is operated under ultraviolet continuous irradiation, x-ray cumulative irradiation or gamma-ray cumulative irradiation.

2. The process for producing lumbrukinase concentrated slurry capable of being stored at room temperature and transported for a long distance as claimed in claim 1, wherein the ultraviolet light is emitted from an ultraviolet device with power of 300-500W.

3. The process for producing lumbrokinase concentrated slurry capable of being stored at room temperature and transported over a long distance as claimed in claim 1, wherein the cumulative x-ray irradiation dose is 60 kilorads and the cumulative gamma-ray irradiation dose is 100 kilorads.

4. The process for producing lumbrokinase concentrated slurry capable of being stored at normal temperature and transported for a long distance according to claim 1, wherein the step e) is specifically as follows: and (3) introducing the clarified liquid into a membrane module with the molecular weight cutoff of 5KD, concentrating to 1/5 of the volume of the original clarified liquid, adding purified water into a storage tank, introducing the membrane module with the molecular weight cutoff of 5KD, repeating the operation, and performing 8-10 circulation processes to obtain the lumbrokinase concentrated slurry.

5. The process for producing lumbrokinase concentrated slurry capable of being stored at room temperature and transported for a long distance as claimed in claim 4, wherein the amount of pure water is equal to the volume of the membrane module effluent.

6. The process for producing lumbrokinase concentrated slurry capable of being stored at room temperature and transported for a long distance as claimed in claim 1, wherein the concentration end point of step e) is that the solid content of lumbrokinase concentrated slurry is below 100 mg/ml.

7. The process for producing lumbrokinase concentrated slurry capable of being stored in room temperature environment and transported for a long distance as claimed in claim 1, wherein the molecular weight cut-off of the membrane module in step c) is 50 KD.

8. The process for producing lumbrokinase concentrated slurry capable of being stored at room temperature and transported for a long distance as claimed in claim 1, wherein step e) is followed by freezing or spray drying the lumbrokinase concentrated slurry to obtain powder.

9. The process for producing lumbrokinase concentrated slurry capable of being stored in normal temperature environment and transported for a long distance according to any one of claims 1 to 8, wherein the membrane module in step c) and step e) is a hollow fiber membrane, a roll-type membrane, a folded membrane, a sintered metal membrane or a ceramic membrane.

Technical Field

The invention relates to the field of lumbrukinase, in particular to a production process of lumbrukinase concentrated slurry capable of being stored in a normal-temperature environment and transported for a long distance.

Background

Lumbrukinase is a group of proteolytic enzymes (acid proteins) extracted from special earthworms, and has a molecular weight of 1.6-4.5 ten thousand D. Lumbrokinase is an antithrombotic drug and is mainly used for thrombotic diseases clinically, in particular for patients with increased fibrinogen and platelet aggregation rate; can be used for treating ischemic cardiovascular and cerebrovascular diseases, and improving symptoms and preventing disease development.

However, since lumbrokinase is affected by external environmental factors during processing and storage, when the environment is changed or some groups interact with each other to be weakened, the enzyme structure is easily broken down and the conformation is easily disordered. Once the spatial structure of the enzyme is destroyed, the conformation of the active center is changed, and finally the enzyme is inactivated. The freeze-drying storage method can avoid the problem of enzyme inactivation caused by high temperature, but has high cost and long time consumption, and needs to be carried out at low temperature for sale and application.

Disclosure of Invention

In view of the above, the invention aims to provide a production process of lumbrokinase concentrated slurry capable of being stored and transported in a normal temperature environment for a long distance, which can be stored and transported at a normal temperature and has the characteristic of long-term quality guarantee.

In order to achieve the purpose, the invention provides the following technical scheme:

a production process of lumbrukinase concentrated slurry capable of being stored in normal temperature environment and transported for long distance comprises the following steps:

a) collecting earthworm slurry

Emptying impurities in the abdominal cavity of the earthworms, cleaning the impurities, and grinding the impurities into slurry to obtain earthworm ground slurry;

b) continuous centrifugal deslagging

Centrifuging and removing the residue of the earthworm mill slurry obtained in the step a) at 12000r/min to obtain supernatant;

c) clarifying, filtering, retaining residual solid of lumbricus slurry, and releasing biological macromolecules of lumbrokinase

Adding a membrane component into the supernatant obtained in the step b) to clarify the feed liquid;

d) degreasing treatment

Adding sodium chloride into the feed liquid obtained in the step c) until the concentration of the sodium chloride reaches 1M, centrifuging for 30min at 10000r/min, and separating earthworm oil from fat;

e) desalting and concentrating

Introducing the clarified liquid obtained in the step D) into a membrane module with molecular weight cutoff of 5KD to obtain concentrated liquid, adding purified water into the concentrated liquid, and introducing the membrane module with molecular weight cutoff of 5000D to obtain lumbrokinase concentrated slurry;

the whole operation process of the steps a) to e) is operated under ultraviolet continuous irradiation, x-ray cumulative irradiation or gamma-ray cumulative irradiation.

Further, the ultraviolet rays are emitted by ultraviolet equipment with power of 300-500W.

Further, the cumulative irradiation dose of x-rays is 60 kilorads, and the cumulative irradiation dose of gamma-rays is 100 kilorads.

Further, the step e) is specifically as follows: and (3) introducing the clarified liquid into a membrane module with the molecular weight cutoff of 5KD, concentrating to 1/5 of the volume of the original clarified liquid, adding purified water into a storage tank, introducing the membrane module with the molecular weight cutoff of 5KD, repeating the operation, and performing 8-10 circulation processes to obtain the lumbrokinase concentrated slurry.

Further, the adding amount of the purified water is equal to the volume of the membrane module effluent.

Further, the concentration end point of the step e) is that the solid content of the lumbrokinase concentrated slurry is below 100 mg/ml.

Further, the molecular weight cut-off of the membrane module in step c) is 50 KD.

Further, after the step e), the lumbrokinase concentrated slurry is frozen or spray-dried to prepare powder.

Further, the membrane modules in step c) and step e) are hollow fiber membranes, spiral wound membranes, folded membranes, sintered metal membranes or ceramic membranes.

The invention discloses a production process of lumbrokinase concentrated slurry capable of being stored in a normal-temperature environment and transported for a long distance, which comprises the steps of grinding, deslagging, filtering, degreasing, desalting, concentrating, sterilizing and the like, and can remove water-soluble impurities, denatured protein, earthworm oil and other impurities, improve the abundance of lumbrokinase in the lumbrokinase concentrated slurry, reduce the load of a separation medium of a production line, and improve the yield of lumbrokinase; the physical and chemical properties of the produced lumbrokinase concentrated slurry are stable, and the enzyme activity in the slurry is kept lasting. The production process is suitable for different lumbrokinase process routes, the prepared lumbrokinase concentrated slurry can be directly used for lumbrokinase refining engineering, and can also be prepared into powder form through freezing or spray drying treatment, and the lumbrokinase concentrated slurry is used as a substitute of earthworm body raw materials, has the characteristics of normal-temperature storage and transportation and long-term quality guarantee, and is suitable for starting raw materials of various lumbrokinase production processes.

Drawings

FIG. 1 is a UV spectrum scan of lumbrokinase in experimental process;

FIG. 2 is a UV spectrum scan of lumbrokinase in a comparative process;

FIG. 3 is the diagram of the measurement of lumbrokinase potency obtained from the experimental process and the comparative process.

Detailed Description

For a further understanding of the invention, preferred embodiments of the invention are described below in conjunction with the examples, but it should be understood that these descriptions are included merely to further illustrate the features and advantages of the invention and are not intended to limit the invention to the claims.

The invention provides a production process of lumbrokinase concentrated slurry capable of being stored in a normal-temperature environment and transported for a long distance, which comprises the following steps:

a) collecting earthworm slurry

Emptying impurities in the abdominal cavity of the earthworms, cleaning the impurities, and grinding the impurities into slurry to obtain earthworm ground slurry;

b) continuous centrifugal deslagging

Centrifuging and removing the residue of the earthworm mill slurry obtained in the step a) at 12000r/min to obtain supernatant;

c) clarifying, filtering, retaining residual solid of lumbricus slurry, and releasing biological macromolecules of lumbrokinase

Adding a membrane component into the supernatant obtained in the step b) to clarify the feed liquid;

d) degreasing treatment

Adding sodium chloride into the feed liquid obtained in the step c) until the concentration of the sodium chloride reaches 1M, centrifuging for 30min at 10000r/min, and separating earthworm oil from fat;

e) desalting and concentrating

Introducing the clarified liquid obtained in the step D) into a membrane module with molecular weight cutoff of 5KD to obtain concentrated liquid, adding purified water into the concentrated liquid, and introducing the membrane module with molecular weight cutoff of 5000D to obtain lumbrokinase concentrated slurry;

the whole operation process of the steps a) to e) is operated under ultraviolet continuous irradiation, x-ray cumulative irradiation or gamma-ray cumulative irradiation.

The production process comprises the steps of grinding, deslagging, filtering, degreasing, desalting and concentrating, sterilizing and the like, can remove impurities such as water-soluble impurities, denatured protein, earthworm oil and the like, improves the abundance of lumbrokinase in the lumbrokinase concentrated slurry, reduces the load of a separation medium of a production line, and improves the yield of the lumbrokinase; the physical and chemical properties of the produced lumbrokinase concentrated slurry are stable, and the enzyme activity in the slurry is kept lasting. The production process is suitable for different lumbrukinase process routes, and the prepared lumbrukinase concentrated slurry can be directly used as a substitute of earthworm raw materials in lumbrukinase refining engineering, can be stored and transported at normal temperature, has the characteristic of long-term quality guarantee, and is suitable for starting raw materials produced in lumbrukinase factories.

The invention empties impurities in the abdominal cavity of the earthworms, cleans the impurities, and grinds the impurities into slurry to obtain earthworm ground slurry; in order to ensure the activity of lumbrokinase in earthworms, the temperature in the preparation process of the earthworm slurry is not more than 15 ℃ and the time is not more than 8 hours. In the embodiment of the invention, the device used for grinding into slurry is a grinding wheel mill, a colloid mill, a vibration mill or a ball mill.

And centrifuging the earthworm milled slurry at 12000r/min to remove residues to obtain supernatant.

Adding a membrane component into the supernatant to clarify the feed liquid; the method is used for intercepting residual solid matters in the earthworm cast and releasing the lumbrokinase biomacromolecules. In an embodiment of the invention, the membrane module has a molecular weight cut-off of 50 KD.

Adding sodium chloride into the feed liquid until the concentration of sodium chloride reaches 1M, centrifuging at 10000r/min for 30min, and separating the earthworm oil phase; the steps are used for removing impurities such as earthworm oil and fat and improving the abundance of the lumbrokinase in the lumbrokinase concentrated slurry.

Introducing the clarified solution into a membrane module with molecular weight cutoff of 5KD to obtain a concentrated solution, adding purified water into the concentrated solution, and introducing a membrane module with molecular weight cutoff of 5000D to obtain lumbrokinase concentrated slurry; the ultra-output liquid is small molecular impurities less than 5000D, and the solute of the retentate is mainly lumbrokinase.

In the embodiment of the invention, the step is specifically that the clarified liquid is introduced into a membrane module with the molecular weight cutoff of 5KD, when the clarified liquid is concentrated to 1/5 of the volume of the original clarified liquid, purified water is added into a storage tank, and then the membrane module with the molecular weight cutoff of 5KD is introduced, the operation is repeated, and the lumbrokinase concentrated slurry is obtained through 8-10 circulation processes. Wherein the addition amount of purified water is equal to the volume of the super-extract. The solid content in the lumbrukinase concentrated slurry obtained by the steps is below 100mg/ml, and the solid content is the end point of the concentration step.

The whole operation process of the steps a) to e) is operated under ultraviolet continuous irradiation, x-ray cumulative irradiation or gamma-ray cumulative irradiation; the sterilization steps are beneficial to normal-temperature storage and transportation of the lumbrukinase concentrated slurry, and the quality of the lumbrukinase concentrated slurry is ensured.

Wherein the ultraviolet rays are emitted by ultraviolet equipment with the power of 300-500W; the cumulative irradiation dose of the x rays is 60 kilorads, and the cumulative irradiation dose of the gamma rays is 100 kilorads.

The invention can eliminate 5 x 10³～10×10⁴D water-soluble impurities outside the molecular weight range, and the removal of the impurities can reduce the production line load and improve the lumbrokinase yield.

It should be noted that the membrane module mentioned herein is a hollow fiber membrane, a roll membrane, a folded membrane, a sintered metal membrane, or a ceramic membrane.

In order to further illustrate the present invention, the process for producing concentrated lumbrukinase slurry, which can be stored and transported in a long distance under normal temperature environment, is described in detail with reference to the following examples, which should not be construed as limiting the scope of the present invention.