A method for efficiently purifying nattokinase

Abstract

This invention relates to the field of compound purification technology, and provides a method for efficiently purifying nattokinase. The method includes preparing a crude nattokinase extract; adding a surfactant to the crude nattokinase extract and mixing thoroughly; adding an aqueous two-phase system to the nattokinase mixture; mixing and stirring to form the aqueous two-phase system; allowing the mixed aqueous two-phase system to stand and separate into layers, collecting the upper phase solution rich in nattokinase; adsorbing the upper phase solution onto activated carbon; and ultrafiltration the upper phase solution after activated carbon adsorption to obtain a high-purity nattokinase solution. This invention improves the separation rate of the upper and lower phases by treating the crude nattokinase extract with a surfactant followed by aqueous two-phase affinity purification, with minimal impact on enzyme activity. Subsequent activated carbon adsorption and ultrafiltration remove residual impurities while reducing the impact of the surfactant on the ultrafiltration membrane, thus improving the purity of the nattokinase solution.

Description

Technical Field

[0001] This invention relates to the field of compound purification technology, specifically a method for efficiently purifying nattokinase. Background Technology

[0002] With increasing emphasis on health and in-depth research into natural bioactive substances, nattokinase, as an enzyme with important physiological functions, has received widespread attention. Nattokinase has good thrombolytic effects and potential application value in the prevention and treatment of cardiovascular diseases. However, the efficient purification of nattokinase from natto has always been a technical challenge. Purification methods mainly include chromatography, magnetic microsphere separation, and integrated separation technologies, including aqueous two-phase affinity partitioning, expanded bed adsorption, and mixed-mode expanded bed adsorption. Among these, aqueous two-phase affinity partitioning is often used for the purification of nattokinase. An aqueous 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 utilizes the differences in the distribution of different substances in an aqueous two-phase system to achieve the separation and purification of the target substance.

[0003] In traditional aqueous two-phase affinity partitioning purification of nattokinase, the crude nattokinase extract is usually directly mixed with an aqueous two-phase system, and then phase separation is achieved through stirring and settling. However, this method has some drawbacks. First, the phase separation process may not be rapid or complete enough, resulting in unsatisfactory separation and prolonged purification time. Second, due to the lack of effective surfactants, the interfacial tension is high, affecting the speed and efficiency of phase separation. In addition, traditional methods have limited effectiveness in removing impurities, making it difficult to obtain high-purity nattokinase products. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a highly efficient method for purifying nattokinase, solving the problem of slow purification speed in existing water-dependent affinity partitioning techniques.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for efficiently purifying nattokinase, comprising the following steps:

[0006] S1. Prepare a crude extract of nattokinase, which is obtained by extracting natto. Add a surfactant to the crude extract of nattokinase and mix well.

[0007] S2. Add an aqueous two-phase system to the nattokinase mixture, wherein the aqueous two-phase system consists of a polymer and a phosphate, wherein the polymer has a mass fraction of 10%-20% and the phosphate has a mass fraction of 8%-12%;

[0008] S3. Mix and stir the crude extract of nattokinase containing the aqueous two-phase system to ensure thorough mixing and formation of the aqueous two-phase system;

[0009] S4. Allow the mixed aqueous two-phase system to stand and separate into layers;

[0010] S5. Separate the upper and lower phases and collect the upper phase solution rich in nattokinase;

[0011] S6. Activated carbon adsorption is performed on the upper phase solution;

[0012] S7. The superphase solution after activated carbon adsorption is subjected to ultrafiltration to obtain a high-purity nattokinase solution.

[0013] Preferably, the method for preparing the crude nattokinase extract is as follows: after pulverizing natto, add the extract at a mass ratio of natto to extract at 1:5, extract at 30°C for 4 hours, then filter to remove residue to obtain the crude nattokinase extract, wherein the extract is a phosphate buffer solution with pH 7.0.

[0014] Preferably, the surfactant in S1 is selected from Tween 20 or Tween 80, with a mass fraction of 0.1%-0.5%.

[0015] Preferably, the aqueous two-phase 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.

[0016] Preferably, the stirring temperature in step S3 is 20-30℃, and the stirring time is 1-2 hours.

[0017] Preferably, the settling temperature in step S4 is 15-25°C and the settling time is 1-1.5 hours.

[0018] Preferably, the activated carbon in S6 is selected from coconut shell activated carbon.

[0019] Preferably, the ultrafiltration membrane in S7 is made of polyethersulfone, has a molecular weight cutoff of 10 kDa, an ultrafiltration pressure of 0.15-0.25 MPa, and an ultrafiltration temperature of 20-30°C.

[0020] This invention provides a highly efficient method for purifying nattokinase. It has the following beneficial effects:

[0021] This invention reduces interfacial tension by adding a surfactant with good emulsifying and dispersing properties. The specific ratio of polymer and phosphate in the aqueous two-phase system, combined with appropriate stirring temperature and time, enables the system to mix rapidly to form an aqueous two-phase system, accelerating the separation of the upper and lower phases. The overall process is compact and efficient, significantly shortening the purification time.

[0022] In preparing the crude extract, this invention uses a phosphate buffer solution to avoid damage to the enzyme under extreme conditions. Tween 20 or Tween 80 is selected as the surfactant, which has little impact on enzyme activity. The ultrafiltration process uses a polyethersulfone ultrafiltration membrane, whose surface can form hydrogen bonds with water, reducing non-specific binding with nattokinase, thereby reducing adsorption loss and ensuring that the activity of nattokinase is well preserved during the purification process.

[0023] This invention utilizes the differences in substance distribution in a two-phase aqueous system to enrich nattokinase in the upper phase solution, initially separating impurities. The activated carbon adsorption step further removes residual impurities while reducing the impact of surfactants on the ultrafiltration membrane. The polyethersulfone ultrafiltration membrane precisely retains nattokinase, removing small molecule impurities and impurities of similar molecular weight, thereby obtaining a high-purity nattokinase solution and greatly improving product quality. Detailed Implementation

[0024] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0025] Example 1:

[0026] This invention provides a method for efficiently purifying nattokinase, comprising the following steps:

[0027] S1. Prepare crude nattokinase extract. After crushing natto, add phosphate buffer solution with pH 7.0 at a mass ratio of natto to extract solution of 1:5. Extract at 30℃ for 4 hours, filter to remove residue, add 0.3% Tween 20 by mass to crude extract solution and mix well.

[0028] S2. Add a two-phase system consisting of polyethylene glycol 6000 and dextran T500 in a volume ratio of 1:2, wherein the molecular weight of polyethylene glycol 6000 is 6000 and the molecular weight of dextran T500 is 500000, and potassium dihydrogen phosphate and dipotassium hydrogen phosphate in a volume ratio of 1:1, wherein the mass fraction of the polymer is 15% and the mass fraction of the phosphate is 10%.

[0029] S3. Stir at 25°C for 1.5 hours;

[0030] S4. Allow to stand at 20℃ for 1.2 hours to separate into layers;

[0031] S5. Separate the upper and lower phases, and collect the upper phase solution;

[0032] S6. Coconut shell activated carbon adsorption is performed on the upper phase solution. After the separation of the aqueous two-phase system is completed and an upper phase solution rich in nattokinase is obtained, there may still be some surfactants that have not been completely removed and other impurities that may have been introduced during the aqueous two-phase separation process. By adsorption with activated carbon, these impurities, especially surfactants, can be further removed, reducing their pollution to the subsequent ultrafiltration membrane and their adverse effects on the ultrafiltration effect. Then, ultrafiltration treatment can be performed to better ensure the efficiency of the ultrafiltration process and the purity of the nattokinase solution.

[0033] S7. High-purity nattokinase solution is obtained by ultrafiltration using polyethersulfone (PES) material, with a molecular weight cutoff of 10 kDa, an ultrafiltration pressure of 0.2 MPa, and an ultrafiltration temperature of 25°C. The PES ultrafiltration membrane can form hydrogen bonds with water on its surface, reducing non-specific binding with nattokinase and thus reducing adsorption loss. This ensures that the activity of nattokinase is well preserved during the purification process. The PES ultrafiltration membrane precisely retains nattokinase and removes small molecule impurities and impurities with similar molecular weights, thereby achieving the acquisition of a high-purity nattokinase solution.

[0034] Example 2:

[0035] This invention provides a method for efficiently purifying nattokinase, comprising the following steps:

[0036] S1. Prepare crude nattokinase extract as in Example 1, add 0.4% Tween 80 by mass to the crude extract and mix well;

[0037] S2. Added to the aqueous two-phase system, the polymer mass fraction is 18%, and the phosphate mass fraction is 11%;

[0038] S3. Stir at 28°C for 1.8 hours;

[0039] S4. Allow to stand at 22℃ for 1.4 hours to separate into layers;

[0040] S5. Collect the upper phase solution;

[0041] S6. Perform adsorption with coconut shell activated carbon;

[0042] S7. Ultrafiltration is performed using an ultrafiltration membrane at an ultrafiltration pressure of 0.22 MPa and an ultrafiltration temperature of 28°C. The other parameters are the same as in Example 1.

[0043] Example 3:

[0044] This invention provides a method for efficiently purifying nattokinase, comprising the following steps:

[0045] S1. Prepare crude nattokinase extract as in Example 1, add 0.1% Tween 20 by mass, and mix well;

[0046] S2. Added to the aqueous two-phase system, the polymer mass fraction is 12%, and the phosphate mass fraction is 9%;

[0047] S3. Stir at 22°C for 1 hour;

[0048] S4. Let stand at 18℃ for 1 hour to allow the layers to separate.

[0049] S5. Collect the upper phase solution.

[0050] S6. Adsorption by coconut shell activated carbon.

[0051] 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.

[0052] Comparative Example 1:

[0053] This invention provides a comparative example of a method for efficiently purifying nattokinase, comprising the following steps:

[0054] S1. Prepare crude extract of nattokinase without adding surfactants;

[0055] S2. Add to the aqueous two-phase system, with a polymer mass fraction of 15% and a phosphate mass fraction of 10%;

[0056] S3. The steps of stirring, settling, and separating are the same as in Example 1;

[0057] S4. Without activated carbon adsorption, perform ultrafiltration directly, with the same parameters as in Example 1.

[0058] Comparative Example 2:

[0059] This invention provides a comparative example of a method for efficiently purifying nattokinase, comprising the following steps:

[0060] S1. Prepare crude nattokinase extract by adding 0.3% by mass of a non-Tween series common surfactant, specifically 0.3% by mass of Span 80;

[0061] S2. Add to the aqueous two-phase system, with a polymer mass fraction of 15% and a phosphate mass fraction of 10%;

[0062] S3. The steps of stirring, settling, and separating are the same as in Example 1;

[0063] S4. Perform ordinary activated carbon adsorption;

[0064] S5. The ultrafiltration treatment parameters are the same as in Example 1.

[0065] The separation rate, enzyme activity, impurities, and specific activity of the above-mentioned examples and comparative examples were determined, and the results are shown in the table below:

[0066]

[0067] Through the above examples and comparative examples, it is evident that adding Tween series surfactants in the examples can reduce interfacial tension, accelerate the phase separation process, and increase the separation speed. In contrast, Comparative Example 1, due to the absence of surfactants, exhibits a slower phase separation speed. Comparative Example 2, using Span 80, may not be as effective as Tween series surfactants, resulting in a generally lower separation speed and purification effect. The coconut shell activated carbon adsorption in the examples can further remove residual impurities and surfactants, reducing contamination of the ultrafiltration membrane and improving ultrafiltration efficiency and the purity of the nattokinase solution. Comparative Example 1, lacking activated carbon adsorption, may allow impurities to directly enter the ultrafiltration membrane, affecting the ultrafiltration effect and product purity. Comparative Example 2, using ordinary activated carbon, may not be as effective as coconut shell activated carbon, leading to incomplete impurity removal and relatively low product purity. The series of operational steps in the examples, including surfactant selection, optimization of the aqueous two-phase system, activated carbon adsorption, and ultrafiltration treatment, work together to effectively protect the activity of nattokinase while removing most impurities, improving product purity and quality. In contrast, the comparative examples, due to deficiencies in certain steps, resulted in low enzyme activity retention, high impurity content, and relatively poor product quality.

[0068] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for efficiently purifying nattokinase, characterized in that, Includes the following steps: S1. Prepare a crude extract of nattokinase, which is obtained by extracting natto. Add a surfactant to the crude extract of nattokinase and mix evenly to obtain a nattokinase mixture. The surfactant is selected from Tween 20 or Tween 80, with a mass fraction of 0.1%-0.5%. S2. Add an aqueous two-phase system to the nattokinase mixture. The aqueous two-phase system consists of a polymer and a phosphate, wherein the polymer has a mass fraction of 10%-20% and the phosphate has a mass fraction of 8%-12%. The polymer in the aqueous two-phase system consists of polyethylene glycol 6000 and dextran T500 in a volume ratio of 1:2, wherein the molecular weight of polyethylene glycol 6000 is 6000 and the molecular weight of dextran T500 is 500000. The phosphate is a mixture of potassium dihydrogen phosphate and dipotassium hydrogen phosphate in a volume ratio of 1:

1. S3. Mix and stir the crude nattokinase extract containing the aqueous two-phase system to form a mixed aqueous two-phase system; S4. Allow the mixed aqueous two-phase system to stand and separate into layers; S5. Separate the upper and lower phases and collect the upper phase solution rich in nattokinase; S6. The upper phase solution is subjected to activated carbon adsorption, wherein the activated carbon is selected from coconut shell activated carbon; S7. The superphase solution after activated carbon adsorption is subjected to ultrafiltration to obtain a high-purity nattokinase solution.

2. The method for efficiently purifying nattokinase according to claim 1, characterized in that, The method for preparing the crude nattokinase extract is as follows: after pulverizing natto, add the extract at a mass ratio of natto to extract at 1:5, extract at 30°C for 4 hours, then filter to remove residue to obtain the crude nattokinase extract. The extract is a phosphate buffer solution with pH 7.

0.

3. The method for efficiently purifying nattokinase according to claim 1, characterized in that, The stirring temperature in S3 is 20-30℃, and the stirring time is 1-2 hours.

4. The method for efficiently purifying nattokinase according to claim 1, characterized in that, The settling temperature in S4 is 15-25℃, and the settling time is 1-1.5 hours.

5. The method for efficiently purifying nattokinase according to claim 1, characterized in that, The ultrafiltration membrane in S7 is made of polyethersulfone, has a molecular weight cutoff of 10 kDa, an ultrafiltration pressure of 0.15-0.25 MPa, and an ultrafiltration temperature of 20-30℃.

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