CN115094053A - Method for efficiently separating and purifying nattokinase and application of nattokinase in preparation of nattokinase powder - Google Patents

Method for efficiently separating and purifying nattokinase and application of nattokinase in preparation of nattokinase powder Download PDF

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CN115094053A
CN115094053A CN202210708168.2A CN202210708168A CN115094053A CN 115094053 A CN115094053 A CN 115094053A CN 202210708168 A CN202210708168 A CN 202210708168A CN 115094053 A CN115094053 A CN 115094053A
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nattokinase
membrane
purifying
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杨茂华
邢建民
穆廷桢
苗得露
赵胥浩
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Abstract

The invention relates to a method for efficiently separating and purifying nattokinase and application thereof in preparing nattokinase powder, wherein the method comprises the following steps: taking nattokinase fermentation liquor or nattokinase crude product solution to sequentially carry out I-grade microfiltration treatment, II-grade ultrafiltration treatment and III-grade ultrafiltration treatment, and obtaining purified nattokinase. The method for separating and purifying the nattokinase is simple and easy to operate, does not need large instruments or equipment, does not need chemical reagents, and is low in cost and short in time consumption of the whole process; the purified nattokinase can be effectively stored for a long time, has higher purity and enzyme activity, and is very suitable for large-scale industrial production.

Description

Method for efficiently separating and purifying nattokinase and application of nattokinase in preparation of nattokinase powder
Technical Field
The invention belongs to the technical field of biochemical separation and purification, relates to a method for efficiently separating and purifying nattokinase and application thereof in preparing nattokinase powder, and particularly relates to a method for efficiently separating and purifying nattokinase by utilizing a membrane technology and application thereof in preparing nattokinase powder with high enzyme activity.
Background
Nattokinase (NK) is an enzyme having strong fibrinolytic activity isolated from fermentation of natto. Nattokinase belongs to serine protease, consists of 275 amino acids and has the molecular weight of 27.7 kDa. Compared with other thrombolytic drugs, the nattokinase has the characteristics of high safety, long action time, good thrombolytic activity, low price and the like, and is expected to become a thrombolytic drug with stronger market competitiveness in the future.
The separation and purification of nattokinase is always the key for restricting the development and application of nattokinase, so more researches are carried out on the separation and purification of nattokinase. The separation and purification methods commonly used at present comprise centrifugation, membrane separation, column chromatography, chromatographic purification and the like.
For example, CN101979530A discloses a method for separating and purifying by ethanol precipitation crude extraction, anion and cation exchange resin chromatography and HPD-400 macroporous adsorption resin refining to obtain nattokinase pure product, column chromatography filling material suitable for industrial production is adopted to replace commonly used expensive gel and other materials, the yield can reach 59 percent, the purity is more than 95 percent, and the method has the characteristics of high yield, low cost and large-scale production. But the whole process takes a long time and the operation is relatively complex.
For example, ammonium sulfate fractional precipitation and gel column filtration are adopted, and although a good separation effect is obtained, the operation capacity is limited and the production cost is high. The separation and purification are carried out by an ammonium sulfate fractional salting-out method and phenyl Sepharose hydrophobic column chromatography, the purification multiple is 14.82, and the recovery rate is 44.03%. However, because the ammonium sulfate precipitation results in the increase of the ionic strength of the whole enzyme solution, dialysis desalination is usually performed first when an ion exchange column is used, which otherwise would cause great interference to the subsequent purification steps, thereby often making the whole process time-consuming. In short, the existing separation and purification methods have the defects of difficult industrial production, high cost and the like.
Therefore, it is very significant to develop a method for separating and purifying nattokinase, which is simple and easy to operate, and the purified nattokinase is easy to effectively preserve for a long time and has higher enzyme activity.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for efficiently separating and purifying nattokinase and application thereof in preparing nattokinase powder, in particular to a method for efficiently separating and purifying nattokinase by utilizing a membrane technology and application thereof in preparing nattokinase powder with high enzyme activity.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a method for efficiently separating and purifying nattokinase, which comprises the following steps: taking nattokinase fermentation liquor or nattokinase crude product solution to sequentially carry out I-grade microfiltration treatment, II-grade ultrafiltration treatment and III-grade ultrafiltration treatment, and obtaining purified nattokinase.
The method for separating and purifying the nattokinase is simple and easy to operate, does not need large instruments or equipment, does not need chemical reagents, and is low in cost and short in time consumption of the whole process; the purified nattokinase can be effectively stored for a long time, has higher purity and enzyme activity, and is very suitable for large-scale industrial production.
Preferably, the membrane material used in the stage I microfiltration treatment comprises a flat membrane, a tubular membrane, a roll membrane or a hollow fiber membrane.
Preferably, the material of the membrane material used in the stage i microfiltration treatment comprises any one or a combination of at least two of cellulose acetate, cellulose nitrate, mixed ester, regenerated cellulose or polyether sulfone.
Preferably, the pore diameter of the membrane material used in the stage i microfiltration treatment is 0.1-0.3 μm, such as 0.1 μm, 0.12 μm, 0.15 μm, 0.18 μm, 0.2 μm, 0.22 μm, 0.25 μm, 0.28 μm, 0.3 μm, and the like, and other specific values in the numerical range can be selected, which is not described in detail herein.
Preferably, the operating differential pressure in the stage i microfiltration treatment is 0.01-0.2MPa, such as 0.01MPa, 0.05MPa, 0.08MPa, 0.1MPa, 0.12MPa, 0.15MPa, 0.18MPa, 0.2MPa, etc., and other specific values in the value range can be selected, which is not described in detail herein.
The grade I microfiltration mainly removes thalli to obtain grade I microfiltration permeating liquid, the aperture of the used membrane material is specially selected to be 0.1-0.3 mu m and is matched with the operation pressure difference of 0.01-0.2MPa, the influence on the enzyme activity of the nattokinase is small on the basis of ensuring better purification effect, and the final obtained nattokinase can be effectively stored for a long time by being matched with subsequent grade II ultrafiltration treatment and grade III ultrafiltration treatment, and has higher purity and enzyme activity.
Preferably, the membrane material used in the II-stage ultrafiltration treatment comprises a flat membrane, a tubular membrane, a spiral membrane or a hollow fiber membrane.
Preferably, the material of the membrane material used in the second-stage ultrafiltration treatment comprises any one or a combination of at least two of cellulose acetate, polyethylene, polyamide and polysulfone.
Preferably, the pore diameter of the membrane material used in the second-stage ultrafiltration treatment is 0.03-0.05 μm, such as 0.03 μm, 0.032 μm, 0.035 μm, 0.038 μm, 0.04 μm, 0.042 μm, 0.045 μm, 0.048 μm, 0.05 μm, and the like, and other specific values in the numerical value range can be selected, which is not described in detail herein.
Preferably, the operating pressure difference in the II-stage ultrafiltration treatment is 100-1000kPa, such as 100kPa, 200kPa, 400kPa, 500kPa, 600kPa, 700kPa, 800kPa, 1000kPa, etc., and other specific values in the value range can be selected, which is not described in detail herein.
The II-level ultrafiltration mainly removes high molecular weight protein to obtain II-level ultrafiltration permeate, the aperture of the used membrane material is specially selected to be 0.03-0.05 mu m and is matched with the operation pressure difference of 100 plus 1000kPa, the influence on the enzyme activity of the nattokinase is small on the basis of ensuring better purification effect, and the nattokinase can be effectively stored for a long time by being matched with the subsequent III-level ultrafiltration, and has higher purity and enzyme activity.
Preferably, the membrane material used in the grade III ultrafiltration treatment comprises a flat membrane, a tubular membrane, a spiral membrane or a hollow fiber membrane.
Preferably, the material of the membrane material used in the stage iii ultrafiltration treatment includes any one of cellulose acetate, polyethylene, polyamide or polysulfone or a combination of at least two of them.
Preferably, the pore diameter of the membrane material used in the stage iii ultrafiltration treatment is less than 0.02 μm, such as 0.02 μm, 0.018 μm, 0.015 μm, 0.012 μm, 0.01 μm, 0.005 μm, and the like, and other specific values in the numerical range can be selected, which are not described in detail herein.
Preferably, the operating pressure difference in the III-stage ultrafiltration treatment is 100-1000kPa, such as 100kPa, 200kPa, 400kPa, 500kPa, 600kPa, 700kPa, 800kPa, 1000kPa, etc., and other specific values in the value range can be selected, which is not described in detail herein.
The III-level ultrafiltration treatment mainly removes substances with small molecular weight to obtain III-level ultrafiltration permeate, the specific selection of the aperture of the used membrane material is less than 0.02 mu m, and the operation pressure difference of 100-1000kPa is matched, so that the influence on the enzyme activity of the nattokinase is small on the basis of ensuring better purification effect, the finally obtained nattokinase can be effectively stored for a long time, and the purity and the enzyme activity are higher.
Preferably, the nattokinase fermentation liquid is a microorganism culture liquid for synthesizing nattokinase.
Preferably, the microorganism comprises bacillus subtilis, yeast or escherichia coli.
The separation and purification method has wide application range and is suitable for almost all forms of nattokinase fermentation liquor and nattokinase crude product solution.
Illustratively, the preparation method of the nattokinase fermentation liquor comprises the following steps:
(1) inoculating the strain in a culture medium for culturing to form a seed solution;
(2) inoculating the seed liquid into a fermentation culture medium for culture to obtain a fermentation liquid;
the fermentation medium comprises: 1.0-3.0g of natto powder, 1.0-3.0g of cane sugar, 0.2-0.6g of glycerol, 0.1-0.3g of monopotassium phosphate, 0.7-2.0g of dipotassium phosphate, 0.01-0.03g of calcium chloride and 0-0.1 g of magnesium sulfate
g, and the pH value of the fermentation medium is 7.0-7.5.
Preferably, the temperature of the culture in the step (2) is 25-40 ℃ and the time is 24-72 h.
In a second aspect, the invention provides nattokinase powder with high enzyme activity, which is obtained by freeze-drying a grade-III ultrafiltration concentrated solution obtained by the method for efficiently separating and purifying nattokinase in the first aspect.
Preferably, the temperature of the freeze-drying treatment is-70 to-30 ℃, such as-70 ℃, -60 ℃, -50 ℃, -40 ℃, -30 ℃ and the like; the time is 8-72h, such as 8h, 12h, 16h, 20h, 24h, 36h, 42h, 56h, 72h and the like; other specific point values within the above numerical range can be selected, and are not described in detail herein.
Preferably, the water content of the nattokinase powder is less than 5%, such as 5%, 4%, 3%, 2%, 1%, 0% and the like, and other specific values in the numerical range can be selected, so that the description is omitted.
The special freeze drying process conditions ensure that the obtained natto kinase powder has high enzyme activity and can be effectively stored for a long time.
Preferably, a protective agent is further added into the grade III ultrafiltration concentrated solution before the freeze drying treatment, wherein the protective agent comprises any one or the combination of at least two of glucose, sucrose, lactose, maltose, fructose, sorbitol, mannitol, xylitol, dextran, polyethylene glycol, povidone or glycine.
Compared with the prior art, the invention has the following beneficial effects:
the method for separating and purifying the nattokinase is simple and easy to operate, does not need large instruments or equipment, does not need chemical reagents, and is low in cost and short in time consumption of the whole process; the purified nattokinase can be effectively stored for a long time, has higher purity and enzyme activity, and is very suitable for large-scale industrial production.
Detailed Description
To further illustrate the technical means and effects of the present invention, the following further describes the technical solution of the present invention with reference to the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.
The sources of the nattokinase fermentation liquor used in the following examples or comparative examples are as follows:
(1) inoculating bacillus natto into a culture medium for culture to form a bacterial colony;
(2) selecting a single colony from the colonies, and carrying out amplification culture to form a seed solution;
(3) inoculating the seed solution into a fermentation culture medium by an inoculation amount of 5% for culture to obtain a fermentation liquid; wherein the formula of the fermentation medium comprises: 2.0g of natto powder, 2.0g of cane sugar, 0.4g of glycerol, 0.2g of monopotassium phosphate, 1.0g of dipotassium phosphate, 0.02g of calcium chloride and 0.05g of magnesium sulfate, wherein the pH value of the fermentation medium is 7.4; the temperature of the culture is 37 ℃, and the time is 72 h.
Example 1
The embodiment provides a method for efficiently separating and purifying nattokinase and nattokinase powder, and the method comprises the following steps:
(1) treating the nattokinase fermentation liquor in a hollow fiber membrane microfiltration membrane device, and collecting permeate, wherein the microfiltration membrane is made of a nitrocellulose membrane, the filtering pore diameter is 0.2 mu m, and the operating pressure is about 0.1 MPa;
(2) treating the permeate with a flat ultrafiltration membrane device, and collecting the ultrafiltration permeate, wherein the ultrafiltration membrane is made of cellulose acetate membrane, the filtration pore diameter of the ultrafiltration membrane is 0.04 μm, and the operation pressure is 400 kPa;
(3) performing ultrafiltration treatment on the ultrafiltration permeate by using a flat membrane again, and collecting ultrafiltration concentrate, wherein the ultrafiltration membrane is made of a cellulose acetate membrane, the filtration pore diameter is 0.01 mu m, and the operating pressure is about 800 kPa;
(4) and (3) freeze-drying the ultrafiltration concentrated solution to obtain the natto kinase powder, wherein the freeze-drying temperature is-40 ℃, and the freeze-drying time is 30 hours. The water content was 4% as measured by loss on drying.
Example 2
The embodiment provides a method for efficiently separating and purifying nattokinase and nattokinase powder, and the method comprises the following steps:
(1) treating the nattokinase fermentation liquor in a tubular microfiltration membrane device, and collecting permeate, wherein the microfiltration membrane is made of a cellulose acetate membrane, the filtering pore diameter is 0.3 mu m, and the operating pressure is about 0.08 MPa;
(2) treating the permeate by a roll-type ultrafiltration membrane device, and collecting the ultrafiltration permeate, wherein the ultrafiltration membrane is made of a polyethylene membrane, the filtration pore diameter of the ultrafiltration membrane is 0.05 μm, and the operating pressure is 300 kPa;
(3) performing ultrafiltration treatment on the ultrafiltration permeate by using a flat membrane again, and collecting ultrafiltration concentrate, wherein the ultrafiltration membrane is a polysulfone membrane, the filtration pore diameter is 0.02 mu m, and the operation pressure is about 500 kPa;
(4) freeze-drying the ultrafiltration concentrated solution to obtain natto kinase powder, wherein the freeze-drying temperature is-60 ℃, and the freeze-drying time is 24 h. The water content was 3% as measured by loss on drying method.
Example 3
The embodiment provides a method for efficiently separating and purifying nattokinase and nattokinase powder, and the method comprises the following steps:
(1) treating the nattokinase fermentation liquor in a hollow fiber membrane microfiltration membrane device, and collecting permeate, wherein the microfiltration membrane is made of a nitrocellulose membrane, the filtering pore diameter is 0.1 mu m, and the operating pressure is about 0.2 MPa;
(2) treating the permeate by using a flat ultrafiltration membrane device, and collecting the ultrafiltration permeate, wherein the ultrafiltration membrane is made of a cellulose acetate membrane, the filtration pore diameter of the ultrafiltration membrane is 0.03 mu m, and the operating pressure is 800 kPa;
(3) performing ultrafiltration treatment on the ultrafiltration permeate by using a flat membrane again, and collecting ultrafiltration concentrate, wherein the ultrafiltration membrane is made of a polyamide membrane, the filtration pore diameter is 0.01 mu m, and the operation pressure is about 800 kPa;
(4) freeze-drying the ultrafiltration concentrated solution to obtain nattokinase powder, wherein the freeze-drying temperature is-50 ℃, and the freeze-drying time is 36 h. The water content was 2% as measured by loss on drying method.
Example 4
This example provides a method for efficiently separating and purifying nattokinase and a nattokinase powder, which is different from example 1 only in that the operation pressure in step (1) is about 0.3MPa, and other operations are kept unchanged.
Example 5
This example provides a method for efficiently separating and purifying nattokinase and a nattokinase powder, which is different from example 1 only in that the operation pressure in step (2) is about 1500kPa, and other operations are kept unchanged.
Example 6
This example provides a method for efficiently separating and purifying nattokinase and a nattokinase powder, which is different from example 1 only in that the operation pressure in step (3) is about 1500kPa, and the other operations are kept unchanged.
Comparative example 1
This comparative example provides a method for efficiently separating and purifying nattokinase and a nattokinase powder, which is different from example 1 only in that the ultrafiltration treatment of step (3) is not performed, and the ultrafiltration concentrated solution obtained in step (2) is directly subjected to freeze drying in the same manner, and other operations are consistent with example 1.
Comparative example 2
This comparative example provides a method for efficiently separating and purifying nattokinase and a nattokinase powder, which is different from example 1 only in that the ultrafiltration treatment of step (2) is not performed, the microfiltration concentrate obtained in step (1) is directly subjected to the ultrafiltration treatment of step (3), and other operations are the same as those of example 1.
Test example
(1) The enzyme activities of the nattokinase powders prepared in examples 1 to 6 and comparative examples 1 to 2 were measured according to the enzyme activity measuring method established by the Japan nattokinase Association (5 parallel measurements per group), and the average results were as follows:
Figure BDA0003706193820000081
Figure BDA0003706193820000091
(2) the natto kinase powders prepared in examples 1 to 6 and comparative examples 1 to 2 were left at 4 ℃ for 2 weeks, and the enzyme activities of the respective groups of samples were measured again (5 times for each group), and the average results were as follows:
group of Enzyme activity (IU/mg)
Example 1 28330
Example 2 25370
Example 3 25180
Example 4 19600
Example 5 17900
Example 6 15500
Comparative example 1 9360
Comparative example 2 8320
According to the data, the nattokinase purified by the method for efficiently separating and purifying the nattokinase can be effectively stored for a long time, and has high enzyme activity.
The applicant states that the present invention is illustrated by the above examples, but the present invention is not limited to the above examples, i.e. it is not meant to be dependent on the above examples to practice the present invention. It should be understood by those skilled in the art that any modifications of the present invention, equivalent substitutions of the raw materials of the product of the present invention, and the addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that, in the above embodiments, the various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, the present invention does not separately describe various possible combinations.

Claims (10)

1. A method for efficiently separating and purifying nattokinase is characterized by comprising the following steps: taking nattokinase fermentation liquor or nattokinase crude product solution to sequentially carry out I-level microfiltration treatment, II-level ultrafiltration treatment and III-level ultrafiltration treatment, thus obtaining the purified nattokinase.
2. The method for efficiently separating and purifying nattokinase according to claim 1, wherein the membrane material used in the I-stage microfiltration treatment comprises a flat membrane, a tubular membrane, a roll membrane or a hollow fiber membrane;
preferably, the material of the membrane material used in the stage i microfiltration treatment comprises any one or a combination of at least two of cellulose acetate, cellulose nitrate, mixed ester, regenerated cellulose or polyether sulfone.
3. The method for efficiently separating and purifying nattokinase according to claim 1 or 2, wherein the membrane material used in the I-stage microfiltration treatment has a pore size of 0.1-0.3 μm;
preferably, the operation pressure difference in the I-stage microfiltration treatment is 0.01-0.2 MPa.
4. The method for efficiently separating and purifying nattokinase according to any one of claims 1 to 3, wherein the membrane material used in the II-stage ultrafiltration treatment comprises a flat membrane, a tubular membrane, a roll-type membrane or a hollow fiber membrane;
preferably, the material of the membrane material used in the second-stage ultrafiltration treatment comprises any one or a combination of at least two of cellulose acetate, polyethylene, polyamide and polysulfone.
5. The method for efficiently separating and purifying nattokinase according to any one of claims 1 to 4, wherein the membrane material used in the II-stage ultrafiltration treatment has a pore size of 0.03 to 0.05 μm;
preferably, the operating pressure difference in the II-stage ultrafiltration treatment is 100-1000 kPa.
6. The method for efficiently separating and purifying nattokinase according to any one of claims 1 to 5, wherein the membrane material used in the III-grade ultrafiltration treatment comprises a flat membrane, a tubular membrane, a roll membrane or a hollow fiber membrane;
preferably, the material of the membrane material used in the stage iii ultrafiltration treatment includes any one of cellulose acetate, polyethylene, polyamide or polysulfone or a combination of at least two of them.
7. The method for efficiently separating and purifying nattokinase according to any one of claims 1 to 6, wherein the pore diameter of the membrane material used in the III-stage ultrafiltration treatment is less than 0.02 μm;
preferably, the operating pressure difference in the III-stage ultrafiltration treatment is 100-1000 kPa.
8. The method for efficiently separating and purifying nattokinase according to any one of claims 1 to 7, wherein the nattokinase fermentation liquid is a microorganism culture liquid for synthesizing nattokinase;
preferably, the microorganism comprises bacillus subtilis, yeast or escherichia coli.
9. A nattokinase powder with high enzyme activity, which is characterized in that the nattokinase powder with high enzyme activity is obtained by freeze-drying a grade-III ultrafiltration concentrated solution obtained by the method for efficiently separating and purifying nattokinase according to any one of claims 1 to 8.
10. The nattokinase powder having high enzymatic activity according to claim 9, wherein the temperature of the freeze-drying treatment is-70 to-30 ℃ and the time is 8 to 72 hours;
preferably, the water content of the nattokinase powder is less than 5%.
CN202210708168.2A 2022-06-21 2022-06-21 Method for efficiently separating and purifying nattokinase and application of nattokinase in preparation of nattokinase powder Pending CN115094053A (en)

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