Method for efficiently purifying nattokinase

Abstract

The invention relates to the technical field of compound purification, and provides a method for efficiently purifying nattokinase, which comprises the following steps: preparing a nattokinase crude extract, adding a surfactant into the nattokinase crude extract, and uniformly mixing; adding the aqueous two-phase system into the nattokinase mixed solution; mixing and stirring to form an aqueous two-phase system; standing and layering the mixed aqueous two-phase system, and collecting an upper phase solution rich in nattokinase; performing activated carbon adsorption on the upper-phase solution; and carrying out ultrafiltration treatment on the upper-phase solution adsorbed by the activated carbon to obtain a high-purity nattokinase solution. According to the method disclosed by the invention, the nattokinase crude extract is treated by the surfactant and then is subjected to double-water-phase affinity purification, so that the separation speed of an upper phase and a lower phase is increased, and the influence on enzyme activity is small; the residual impurities can be removed through activated carbon adsorption and ultrafiltration treatment subsequently, and the purity of the nattokinase solution is improved while the influence of the surfactant on the ultrafiltration membrane is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of compound purification, in particular to a method for efficiently purifying nattokinase. Background Art

[0002] With people's increasing attention to health and in-depth research on natural bioactive substances, nattokinase, as an enzyme substance with important physiological functions, has received widespread attention. Nattokinase has good thrombolytic effect and has potential application value in the prevention and treatment of cardiovascular diseases. However, the efficient purification of nattokinase from natto has always been a technical problem. The main purification methods are chromatography, magnetic microsphere separation, and integrated separation technology, including two-phase affinity distribution technology, expanded bed adsorption technology, mixed mode expanded bed adsorption technology, etc. Among them, two-phase affinity distribution technology is often used for the purification of nattokinase. The two-phase system is a two-phase system formed by two different water-soluble polymers or a polymer and a salt under certain conditions. This technology uses the distribution difference of different substances in the two-phase system to achieve the separation and purification of the target substance.

[0003] In the traditional method of purifying nattokinase by affinity partitioning of two-phase aqueous solution, the crude nattokinase extract is usually directly mixed with the two-phase aqueous system, and then phase separation is achieved through stirring, standing and other steps. However, this method has some shortcomings. First, the phase separation process may not be rapid and complete enough, resulting in unsatisfactory separation effect and prolonged purification time. Second, due to the lack of effective surfactants, the interfacial tension is high, which affects the speed and efficiency of phase separation. In addition, the traditional method has limited effect in removing impurities, and it is difficult to obtain high-purity nattokinase products. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the present invention provides a method for efficiently purifying nattokinase, which solves the problem of slow purification speed in the existing two-phase affinity partitioning technology.

[0005] To achieve the above object, the present invention is achieved by the following technical scheme: a method for efficiently purifying nattokinase, comprising the following steps: S1. Prepare a crude extract of nattokinase, the crude extract is obtained by extracting natto, adding a surfactant to the crude extract of nattokinase and mixing; S2. A two-phase aqueous system is added to the nattokinase mixture, wherein the two-phase aqueous system consists of a polymer and a phosphate, wherein the mass fraction of the polymer is 10%-20% and the mass fraction of the phosphate is 8%-12%; S3. The crude extract of nattokinase containing the aqueous two-phase system was mixed and stirred to form a two-phase aqueous system; S4. The mixed two-phase aqueous system is allowed to stand and separate; S5. Separate the upper and lower phases and collect the upper phase solution rich in nattokinase; S6. performing activated carbon adsorption on the upper phase solution; S7. The upper phase solution after activated carbon adsorption is subjected to ultrafiltration to obtain a high-purity nattokinase solution.

[0006] Preferably, the preparation method of the crude extract of nattokinase is: after crushing the natto, adding the extract at a mass ratio of natto to the extract of 1:5, extracting at 30°C for 4 hours, and then filtering to remove the residue to obtain a crude extract of nattokinase, wherein the extract is a phosphate buffer solution with a pH of 7.0.

[0007] Preferably, the surfactant in S1 is selected from Tween 20 or Tween 80, with a mass fraction of 0.1%-0.5%.

[0008] Preferably, the aqueous two-phase system polymer in S2 is composed of polyethylene glycol 6000 and dextran T500 in a volume ratio of 1:2, wherein the molecular weight of polyethylene glycol 6000 is 6000; the molecular weight of dextran T500 is 500000, and the phosphate is a mixture of potassium dihydrogen phosphate and dipotassium hydrogen phosphate in a volume ratio of 1:1.

[0009] Preferably, the stirring temperature in S3 is 20-30° C., and the stirring time is 1-2 hours.

[0010] Preferably, the standing temperature in S4 is 15-25° C., and the standing time is 1-1.5 hours.

[0011] Preferably, the activated carbon in S6 is coconut shell activated carbon.

[0012] Preferably, the material of the ultrafiltration membrane in S7 is polyethersulfone, the molecular weight cut-off of the ultrafiltration membrane is 10 kDa, the ultrafiltration pressure is 0.15-0.25 MPa, and the ultrafiltration temperature is 20-30°C.

[0013] The present invention provides a method for efficiently purifying nattokinase. It has the following beneficial effects: The present invention reduces interfacial tension by adding a surfactant with good emulsifying and dispersing properties. The specific ratio of polymer and phosphate in the two-phase aqueous system, combined with appropriate stirring temperature and time, allows the system to quickly mix to form a two-phase aqueous system, accelerating the separation of the upper and lower phases. The overall process is compact and efficient, and the purification time is greatly shortened.

[0014] When preparing the crude extract, the present invention uses a phosphate buffer to avoid damage to the enzyme under extreme conditions. Tween 20 or Tween 80 is selected as a surfactant, which has little effect on the enzyme activity. Ultrafiltration treatment uses a polyethersulfone ultrafiltration membrane, the surface of which can form hydrogen bonds with water, reducing nonspecific binding with nattokinase, thereby reducing adsorption losses and ensuring that the activity of nattokinase is better retained during the purification process.

[0015] The present invention utilizes the difference in material distribution through a two-phase aqueous system to enrich nattokinase in the upper phase solution, preliminarily separate impurities, and further removes residual impurities in the activated carbon adsorption step while reducing the influence of surfactants on the ultrafiltration membrane. Nattokinase is accurately intercepted by the polyethersulfone ultrafiltration membrane, small molecule impurities and impurities with similar molecular weight are removed, thereby achieving the acquisition of a high-purity nattokinase solution and greatly improving product quality. DETAILED DESCRIPTION

[0016] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0017] Example 1:

[0018] The embodiment of the present invention provides a method for efficiently purifying nattokinase, comprising the following steps: S1. Prepare a crude nattokinase extract by grinding natto and adding pH 7.0 phosphate buffer at a 1:5 mass ratio of natto to extract. Extract at 30°C for 4 hours, filter to remove residue, and add 0.3% Tween 20 to the crude extract and mix thoroughly. S2. Add a two-phase aqueous system consisting of polyethylene glycol 6000 (MW 6000) and dextran T500 (MW 500,000) in a volume ratio of 1:2, and potassium dihydrogen phosphate and dipotassium hydrogen phosphate in a volume ratio of 1:1, with a polymer content of 15% and a phosphate content of 10%. S3. Stir at 25°C for 1.5 hours; S4. Let stand at 20°C for 1.2 hours; S5. Separate the upper and lower phases and collect the upper phase solution; S6. carry out coconut shell activated carbon adsorption with upper phase solution, after completing the separation of two-phase aqueous system and obtaining being rich in the upper phase solution of nattokinase, now, may still exist some surfactants that are not completely removed and other impurities that may be introduced in the two-phase aqueous separation process in the solution, by activated carbon adsorption, these impurities can be further removed, especially surfactant, reduce its pollution to subsequent ultrafiltration membrane and to the adverse effect of ultrafiltration effect, and then carry out ultrafiltration process, can better ensure the efficiency of ultrafiltration process and the purity of nattokinase solution; S7. Ultrafiltration treatment using polyethersulfone material, a molecular weight cutoff of 10kDa, an ultrafiltration pressure of 0.2MPa, and an ultrafiltration temperature of 25°C was used to obtain a high-purity nattokinase solution. The polyethersulfone ultrafiltration membrane used had a surface that could form hydrogen bonds with water, reducing nonspecific binding with nattokinase and thus reducing adsorption losses, ensuring that the activity of nattokinase was better retained during the purification process. The polyethersulfone ultrafiltration membrane was used to accurately retain nattokinase, remove small molecule impurities and impurities with similar molecular weight, and achieve the acquisition of a high-purity nattokinase solution.

[0019] Example 2: The embodiment of the present invention provides a method for efficiently purifying nattokinase, comprising the following steps: S1. A crude extract of nattokinase was prepared as in Example 1, and 0.4% Tween 80 was added to the crude extract and mixed well; S2. Adding a two-phase aqueous system with a polymer mass fraction of 18% and a phosphate mass fraction of 11%; S3. Stir at 28°C for 1.8 hours; S4. Let stand at 22°C for 1.4 hours; S5. Collect the upper phase solution; S6. performing coconut shell activated carbon adsorption; S7. Ultrafiltration was performed using an ultrafiltration membrane. The ultrafiltration pressure was 0.22 MPa, the ultrafiltration temperature was 28° C., and the remaining parameters were the same as in Example 1.

[0020] Example 3: The embodiment of the present invention provides a method for efficiently purifying nattokinase, comprising the following steps: S1. Prepared the same crude extract of nattokinase as in Example 1, added 0.1% by mass fraction of Tween 20 and mixed well; S2. Adding a two-phase aqueous system with a polymer mass fraction of 12% and a phosphate mass fraction of 9%; S3. Stir at 22°C for 1 hour; S4. Let stand at 18°C ​​for 1 hour; S5. Collect the upper phase solution.

[0021] S6. Coconut shell activated carbon adsorption.

[0022] S7. The ultrafiltration pressure is 0.18 MPa, the ultrafiltration temperature is 22°C, and the other parameters are the same as in Example 1.

[0023] Comparative Example 1: The comparative example of the present invention provides a method for efficiently purifying nattokinase, comprising the following steps: S1. Preparation of crude nattokinase extract without adding surfactant; S2. Adding a two-phase aqueous system with a polymer mass fraction of 15% and a phosphate mass fraction of 10%; S3. Stirring, standing, separation and other steps are the same as in Example 1; S4. Direct ultrafiltration was performed without activated carbon adsorption, using the same parameters as in Example 1.

[0024] Comparative Example 2: The comparative example of the present invention provides a method for efficiently purifying nattokinase, comprising the following steps: S1. Preparation of crude nattokinase extract, adding 0.3% by mass fraction of a non-Tween series of ordinary surfactants, specifically 0.3% by mass fraction of Span 80; S2. Adding a two-phase aqueous system with a polymer mass fraction of 15% and a phosphate mass fraction of 10%; S3. Stirring, standing, separation and other steps are the same as in Example 1; S4. Perform ordinary activated carbon adsorption; S5. Ultrafiltration treatment parameters are the same as those in Example 1.

[0025] The above examples and comparative examples were tested for separation speed, enzyme activity, impurities and specific activity content, and the test results are shown in the following table:

[0026] By the above-mentioned embodiment and comparative example, adding Tween series surfactant in the embodiment can reduce interfacial tension, accelerate the phase separation process, and improve separation speed. By contrast, comparative example one is owing to not adding surfactant, and phase separation speed is slow. Comparative example two uses Span 80, and its effect may not be as good as Tween series surfactant, resulting in general separation speed and purification effect; Coconut shell activated carbon adsorption in the embodiment can further remove residual impurities and surfactant, reduce the pollution to ultrafiltration membrane, improve ultrafiltration efficiency and the purity of nattokinase solution, comparative example one does not carry out activated carbon adsorption, may cause impurities to directly enter the ultrafiltration membrane, affect ultrafiltration effect and product purity, comparative example two uses ordinary activated carbon, and its adsorption effect may not be as good as coconut shell activated carbon, resulting in incomplete impurity removal, and product purity is relatively low; a series of operating steps in the embodiment, including the selection of surfactant, optimization of two-phase aqueous system, activated carbon adsorption and ultrafiltration treatment, etc., work together, effectively protect the activity of nattokinase, remove most of impurities, improve product purity and quality, and comparative example, due to the deficiency in some steps, causes enzyme activity retention rate low, impurity content is high, and product quality is relatively poor.

[0027] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for efficiently purifying nattokinase, characterized in that, The following steps are involved: S1. Prepare a crude extract of nattokinase, the crude extract is obtained by extracting natto, adding a surfactant to the crude extract of nattokinase and mixing to obtain a nattokinase mixture; S2. A two-phase aqueous system is added to the nattokinase mixture, wherein the two-phase aqueous system consists of a polymer and a phosphate, wherein the mass fraction of the polymer is 10%-20% and the mass fraction of the phosphate is 8%-12%; S3. The crude extract of nattokinase containing the aqueous two-phase system was mixed and stirred to form a mixed two-phase aqueous system; S4. The mixed two-phase aqueous system is allowed to stand and separate; S5. Separate the upper and lower phases and collect the upper phase solution rich in nattokinase; S6. performing activated carbon adsorption on the upper phase solution; S7. The upper phase solution after activated carbon adsorption is subjected to ultrafiltration to obtain a high-purity nattokinase solution.

2. a kind of method of efficient purification of nattokinase according to claim 1, is characterized in that, The preparation method of the crude nattokinase extract comprises the following steps: crushing natto, adding the extract at a mass ratio of natto to the extract of 1:5, extracting at 30° C. for 4 hours, and then filtering to remove the residue to obtain the crude nattokinase extract, wherein the extract is a phosphate buffer solution with a pH of 7.

0.

3. a kind of method of efficient purification of nattokinase according to claim 1, is characterized in that, The surfactant in S1 is selected from Tween 20 or Tween 80, with a mass fraction of 0.1%-0.5%.

4. a kind of method of efficient purification of nattokinase according to claim 1, is characterized in that, The polymer in the aqueous two-phase system in S2 is composed of polyethylene glycol 6000 and dextran T500 in a volume ratio of 1:2, wherein the molecular weight of polyethylene glycol 6000 is 6000; the molecular weight of dextran T500 is 500000, and the phosphate is a mixture of potassium dihydrogen phosphate and dipotassium hydrogen phosphate in a volume ratio of 1:

1.

5. a kind of method of efficient purification of nattokinase according to claim 1, is characterized in that, The stirring temperature in S3 is 20-30° C., and the stirring time is 1-2 hours.

6. a method for efficiently purifying nattokinase according to claim 1, is characterized in that, The standing temperature in S4 is 15-25° C., and the standing time is 1-1.5 hours.

7. a kind of method of efficient purification of nattokinase according to claim 1, is characterized in that, The activated carbon in S6 is coconut shell activated carbon.

8. a kind of method of efficient purification of nattokinase according to claim 1, is characterized in that, The ultrafiltration membrane in S7 is made of polyethersulfone, has a molecular weight cut-off of 10 kDa, an ultrafiltration pressure of 0.15-0.25 MPa, and an ultrafiltration temperature of 20-30°C.

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