Device for removing impure protein in traditional Chinese medicine crude polysaccharide by utilizing immobilized protease

Abstract

The utility model relates to a device for removing impure protein in crude polysaccharide of traditional Chinese medicine by utilizing immobilized protease, which comprises a protein removal reactor loaded with the immobilized protease, a dissolved oxygen tester and a constant-temperature magnetic stirrer, and a dissolved oxygen electrode of the dissolved oxygen tester is connected to the protein removal reactor. The papain immobilized on the microcrystalline cellulose is used for carrying out enzymolysis reaction on impure protein in the traditional Chinese medicine crude polysaccharide, and the device has the characteristics of simplicity in operation, mild conditions and environment friendliness. The immobilized papain is limited between the two layers of screens, so that the problem of separation of the immobilized papain in the traditional Chinese medicine crude polysaccharide solution can be avoided, and the method has the characteristics of rapidness and convenience. The immobilized papain in the device can be expanded to immobilize other proteases, such as bromelain, subtilisin, protease K and the like, on cellulose according to protein types in different traditional Chinese medicine polysaccharide solutions.

Description

Technical Field

[0001] This utility model relates to a device for removing impurities from crude polysaccharides of traditional Chinese medicine using immobilized protease, and relates to the field of impurity removal technology for crude polysaccharides of traditional Chinese medicine. Background Technology

[0002] Polysaccharides are natural biological macromolecules composed of 10 or more monosaccharides linked by glycosidic bonds. They are important components of plant cell walls and animal and microbial cell membranes. Traditional Chinese medicine polysaccharides refer to the collective term for active polysaccharide components isolated from animal and plant medicinal materials. They possess pharmacological activities such as immunomodulation, hypoglycemic agents, and antitumor activity, and are one of the important material bases for the unique therapeutic effects of traditional Chinese medicine.

[0003] In the purification of polysaccharides from traditional Chinese medicine (TCM), protein removal is a crucial step in improving polysaccharide quality and purity. Denaturing and precipitating proteins using chemical reagents is the most common method for removing impurities from TCM polysaccharides, primarily including the Sevage method (chloroform: n-butanol = 4:1), the TCA method (trichloroacetic acid), and the hydrochloric acid method. The Sevage method is widely used due to its mild conditions and high protein removal rate; however, it requires large amounts of chloroform, posing serious health risks and causing environmental pollution. Furthermore, this method is cumbersome and time-consuming. In recent years, the use of free proteases to enzymatically hydrolyze polysaccharides to remove impurities has been developed, offering advantages such as specificity, high efficiency, environmental friendliness, and low polysaccharide loss. However, free enzymes are difficult to recover and reuse, and the enzymes themselves, being proteins, can actually increase the content of impurities in the polysaccharide. Currently, some researchers are using a combination of enzymatic hydrolysis and the Sevage method to reduce polysaccharide loss while improving protein removal efficiency, but the risks associated with the use of chloroform in the Sevage method still need to be addressed. Therefore, finding a method that can utilize the advantages of protease's specificity and efficiency while also rapidly separating and recovering it is of great research significance.

[0004] Enzyme immobilization is a method of confining water-soluble free enzymes to a specific region using physical or chemical methods, forming an enzyme that is insoluble in water but still retains its enzymatic activity. Compared to free enzymes, immobilized enzymes are generally insoluble in the reaction system, and the enzyme and product can be separated by standing or filtration. Immobilized enzymes also have good stability, can be reused to reduce production costs, and are easily implemented in continuous industrial production. The main methods for protease immobilization include adsorption, encapsulation, covalent bonding, and cross-linking, and their advantages and disadvantages are shown in Table 1. Research reports on the removal of impurities from polysaccharides of traditional Chinese medicine using immobilized proteases are still relatively few. Most studies focus on directly extracting polysaccharides or purifying crude polysaccharides using encapsulation or cross-linking methods. For example, Gao Mingxia et al. immobilized papain using chitosan as a carrier and glutaraldehyde as a cross-linking agent to extract polysaccharides from burdock; Liao Qiyuan et al. encapsulated papain in calcium alginate particles to remove impurities from crude polysaccharides of cat's claw. However, plant-based polysaccharides are mainly found in cell walls, and adding immobilized proteases does not promote the dissolution of polysaccharides. Immobilized proteases formed by encapsulation reduce the probability of enzyme-substrate binding, resulting in longer polysaccharide purification time. Furthermore, the encapsulation material is prone to dissolution at higher temperatures, causing the protease to be released.

[0005] Table 1. Main methods of protease immobilization and their advantages and disadvantages

[0006] advantage shortcoming Adsorption method Mild conditions, low cost, and reusable Sensitive to factors such as pH, ionic strength, and temperature, the enzyme is prone to detachment and has a small enzyme loading capacity. Embedding method The conditions are relatively mild, there is a wide range of choices in embedding materials and methods, and immobilized enzymes have a broad range of applications. It can only be used with low molecular weight substrates and often has diffusion limitation problems. Covalent bonding method The vector and coupling method offer high selectivity, strong enzyme binding affinity, and are very stable. The coupling conditions are harsh, easily causing enzyme inactivation, and are costly; some couplings are actually toxic. Crosslinking method The cross-linking reagents are plentiful, the technology is simple, the enzyme binding force is strong, and the stability is high. The cross-linking conditions are intense, resulting in poor mechanical properties.

[0007] To address the aforementioned issues, our research group initially selected microcrystalline cellulose as the immobilization carrier. We activated the microcrystalline cellulose using a strong oxidant (sodium periodate-sulfuric acid solution) and then prepared immobilized papain by immobilizing it onto the surface of the activated microcrystalline cellulose via a self-assembly method. Water-insoluble carbohydrates such as microcrystalline cellulose have small particles and large specific surface areas, allowing for greater binding of the target protease and thus improving the enzymatic hydrolysis efficiency of impurities in polysaccharide solutions. Furthermore, water-insoluble carbohydrates such as microcrystalline cellulose and chitin are byproducts of agricultural processing, possessing the advantages of low price, easy availability, and recyclability. Immobilized papain was used to remove impurities from crude polysaccharide solutions of Astragalus membranaceus and Pueraria lobata. Results showed that the immobilized protease effectively removed impurities from the polysaccharides of traditional Chinese medicine with a low polysaccharide loss rate, and maintained 80% enzyme activity even after 4-5 reuses. Filtration or low-speed centrifugation can completely separate and recover the immobilized protease, providing a prerequisite for reuse. However, the high viscosity of the polysaccharide solution prevents the use of these methods in large-scale polysaccharide purification processes, directly limiting the application of immobilized protease in actual production. Utility Model Content

[0008] In view of the shortcomings of the prior art, the technical problem to be solved by this utility model is to provide a device for removing impurities from crude polysaccharides of traditional Chinese medicine using immobilized protease.

[0009] To solve the above-mentioned technical problems, the technical solution of this utility model is: an apparatus for removing impurities from crude polysaccharides of traditional Chinese medicine using immobilized protease, comprising a protein removal reactor loaded with immobilized protease, a dissolved oxygen meter and a constant temperature magnetic stirrer, wherein the protein removal reactor is placed in the constant temperature magnetic stirrer, and the dissolved oxygen electrode of the dissolved oxygen meter is connected to the protein removal reactor.

[0010] Preferably, the protein removal reactor includes upper and lower cylindrical containers. The top of the upper cylindrical container has three interfaces from left to right: an inlet / outlet of the crude polysaccharide solution from traditional Chinese medicine, a temperature sensor interface, and a dissolved oxygen electrode interface, respectively. The upper cylindrical container has a screen in the middle and a threaded interface and a gasket at the bottom. The lower cylindrical container has a threaded interface at the top, a screen at the bottom, and a magnetic stir bar at the bottom. The upper and lower cylindrical containers are rotated and tightened at the threaded interface.

[0011] Preferably, a temperature sensor is installed on the temperature sensor interface, and a dissolved oxygen electrode is installed on the dissolved oxygen electrode interface.

[0012] Preferably, the screen is a stainless steel screen with a particle size ≥400 mesh, and immobilized papain is loaded between the two screen layers.

[0013] Preferably, the length of the magnetic stir bar is 40~70 mm.

[0014] Compared with existing technologies, this invention has the following advantages: The papain immobilized on microcrystalline cellulose performs enzymatic hydrolysis of impurities in crude polysaccharides from traditional Chinese medicine, characterized by simple operation, mild conditions, and environmental friendliness. Confining the immobilized protease between two layers of sieves not only ensures sufficient contact between the immobilized protease and the polysaccharide solution, thereby improving hydrolysis efficiency, but also avoids the separation problem of the immobilized protease in the crude polysaccharide solution, simplifying the operation steps and providing a rapid and convenient process.

[0015] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the structure of an embodiment of the present utility model.

[0017] Figure 2 The images show the FT-IR spectra of microcrystalline cellulose before and after activation.

[0018] Figure 3 Transmission electron microscopy images of activated microcrystalline cellulose (A) and activated microcrystalline cellulose-papain (B).

[0019] Figure 4The histograms show the protein removal rate and polysaccharide loss rate before and after microcrystalline cellulose-papain treatment of Astragalus membranaceus crude polysaccharide solution and Pueraria lobata crude polysaccharide solution. Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0021] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0022] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0023] like Figures 1-4 As shown, this embodiment provides an apparatus for removing impurities from crude polysaccharides of traditional Chinese medicine using immobilized protease, including a protein removal reactor 2 loaded with immobilized protease 1, a dissolved oxygen meter 3, and a constant temperature magnetic stirrer 4. The protein removal reactor is placed in the constant temperature magnetic stirrer, and the dissolved oxygen electrode 5 of the dissolved oxygen meter is connected to the protein removal reactor.

[0024] In this embodiment of the invention, the protein removal reactor includes two cylindrical containers 6, an upper one and a lower one. The top of the upper cylindrical container has three interfaces from left to right: an inlet / outlet 7 for the crude polysaccharide solution of traditional Chinese medicine, a temperature sensor interface, and a dissolved oxygen electrode interface, respectively. The upper cylindrical container has a screen 8 in the middle and a threaded interface 9 and a gasket at the bottom. The lower cylindrical container has a threaded interface at the top, a screen at the bottom, and a magnetic stir bar 10 at the bottom. The upper and lower cylindrical containers are rotated and tightened at the threaded interface.

[0025] In this embodiment of the invention, the immobilized protease loaded in the protein removal reactor is papain immobilized on activated microcrystalline cellulose, with an enzyme activity of 500~950 U / g.

[0026] In this embodiment of the invention, the papain immobilization matrix is ​​activated microcrystalline cellulose with a particle size of 100-300 mesh.

[0027] In this embodiment of the invention, a temperature sensor 11 is installed on the temperature sensor interface, and a dissolved oxygen electrode is installed on the dissolved oxygen electrode interface.

[0028] In this embodiment of the invention, the screen is a stainless steel screen with a particle size ≥ 400 mesh, and immobilized papain is loaded between the two screen layers.

[0029] In this embodiment of the invention, the length of the magnetic stir bar is 40~70 mm.

[0030] In this embodiment of the invention, the method for preparing immobilized papain is as follows:

[0031] (1) Microcrystalline cellulose (particle size 100-300 mesh) was placed in a mixed solution containing sodium periodate and sulfuric acid and stirred at room temperature for 24 h to activate it. The molar ratio of sodium periodate to sulfuric acid was 3:1 to 1:2. After the activated microcrystalline cellulose was thoroughly washed, it was dried for later use.

[0032] (2) Disperse the activated cellulose obtained in step (1) in Tris-HCl buffer solution (50 mM, pH 7.6), add papain, vortex mix, and shake at room temperature for 24 h to immobilize the enzyme. The mass ratio of activated cellulose to papain is 1:1 to 1:4.

[0033] (3) Centrifuge the suspension from step (2) at 500-1500 rpm for 2-10 min to remove free papain, and redisperse the lower precipitate in Tris-HCl buffer solution. Repeat 3-4 times to obtain a dispersion of microcrystalline cellulose-papain complex. The enzyme activity of microcrystalline cellulose-papain was calculated to be 500-950 U / g.

[0034] The temperature of the constant temperature magnetic stirrer is 50~60 °C, the rotation speed is 15~40 r / min, and the stirring time is 30~90 min.

[0035] Specific implementation process:

[0036] Example 1

[0037] A magnetic stir bar (50 mm in length) was placed at the bottom of the cylindrical container below the protein removal reactor. A 400-mesh stainless steel sieve was fixed to both the upper and lower cylindrical containers of the reactor. 0.2 g of immobilized papain was weighed and placed on the lower sieve in the lower cylindrical container. The two cylindrical containers were then tightened by rotation. Astragalus membranaceus crude polysaccharide solution was added through the inlet. The temperature sensor of the thermostatic magnetic stirrer and the dissolved oxygen electrode of the dissolved oxygen meter were placed in their respective ports. The reactor was then placed in the thermostatic magnetic stirrer at 55 °C, a rotation speed of 30 r / min, and a stirring time of 60 min. The immobilized papain was prepared as follows: 2.0 g of 100-mesh microcrystalline cellulose was weighed and activated for 24 h in a mixed solution of 200 mL sodium periodate (1.2 g) and concentrated sulfuric acid (2 mL). Subsequently, the washed activated cellulose (0.1 g) and papain (0.32 g) were dispersed in Tris-HCl solution (50 mM, pH 7.6), shaken for 24 h, and then centrifuged and washed to obtain immobilized papain.

[0038] Example 2

[0039] A magnetic stir bar (50 mm in length) was placed at the bottom of the cylindrical container below the protein removal reactor. A 400-mesh stainless steel sieve was fixed to both the upper and lower cylindrical containers of the reactor. 0.6 g of immobilized papain was weighed and placed on the lower sieve in the lower cylindrical container. The two cylindrical containers were then tightened by rotation. Astragalus membranaceus crude polysaccharide solution was added through the inlet. The temperature sensor of the thermostatic magnetic stirrer and the dissolved oxygen electrode of the dissolved oxygen meter were placed in their respective ports. The reactor was then placed in the thermostatic magnetic stirrer at 55 °C, a rotation speed of 45 r / min, and a stirring time of 60 min. The immobilized papain was prepared as follows: 200-mesh microcrystalline cellulose (2.0 g) was weighed and activated for 24 h in a mixed solution of 200 mL sodium periodate (1.2 g) and concentrated sulfuric acid (2 mL). Subsequently, the washed activated cellulose (0.1 g) and papain (0.16 g) were dispersed in Tris-HCl solution (50 mM, pH 7.6), shaken for 24 h, and then centrifuged and washed to obtain immobilized papain.

[0040] Example 3

[0041] A magnetic stir bar (70 mm in length) was placed at the bottom of the cylindrical container below the protein removal reactor. A 400-mesh stainless steel sieve was fixed to both the upper and lower cylindrical containers of the reactor. 0.48 g of immobilized papain was weighed and placed on the lower sieve in the lower cylindrical container. The two cylindrical containers were then tightened by rotation. Pueraria lobata crude polysaccharide solution was added through the inlet. The temperature sensor of the thermostatic magnetic stirrer and the dissolved oxygen electrode of the dissolved oxygen meter were placed in their respective ports. The reactor was then placed in the thermostatic magnetic stirrer at 55 °C, a rotation speed of 45 r / min, and a stirring time of 90 min. The immobilized papain was prepared as follows: 200-mesh microcrystalline cellulose (2.0 g) was weighed and activated for 24 h in a mixed solution of 200 mL sodium periodate (1.2 g) and concentrated sulfuric acid (2 mL). Subsequently, the washed activated cellulose (0.1 g) and papain (0.32 g) were dispersed in Tris-HCl solution (50 mM, pH 7.6), shaken for 24 h, and then centrifuged and washed to obtain immobilized papain.

[0042] Example 4

[0043] A magnetic stir bar (50 mm in length) was placed at the bottom of the cylindrical container below the protein removal reactor. A 600-mesh stainless steel sieve was fixed to both the upper and lower cylindrical containers of the reactor. 1.6 g of immobilized papain was weighed and placed on the lower sieve in the lower cylindrical container. The two cylindrical containers were then tightened by rotation. Pueraria lobata crude polysaccharide solution was added through the inlet. The temperature sensor of the thermostatic magnetic stirrer and the dissolved oxygen electrode of the dissolved oxygen meter were placed in their respective ports. The reactor was then placed in the thermostatic magnetic stirrer at 60 °C, a rotation speed of 30 r / min, and a stirring time of 120 min. The immobilized papain was prepared as follows: 200-mesh microcrystalline cellulose (2.0 g) was weighed and activated for 24 h in a mixed solution of 200 mL sodium periodate (1.2 g) and concentrated sulfuric acid (2 mL). Subsequently, the washed activated cellulose (0.1 g) and papain (0.32 g) were dispersed in Tris-HCl solution (50 mM, pH 7.6), shaken for 24 h, and then centrifuged and washed to obtain immobilized papain.

[0044] Example 5

[0045] A magnetic stir bar (70 mm in length) was placed at the bottom of the cylindrical container below the protein removal reactor. A 500-mesh stainless steel sieve was fixed to both the upper and lower cylindrical containers of the reactor. 2.0 g of immobilized papain was weighed and placed on the lower sieve in the lower cylindrical container. The two cylindrical containers were then tightened by rotation. Pueraria lobata crude polysaccharide solution was added through the inlet. The temperature sensor of the thermostatic magnetic stirrer and the dissolved oxygen electrode of the dissolved oxygen meter were placed in their respective ports. The reactor was then placed in the thermostatic magnetic stirrer at 60 °C, a rotation speed of 45 r / min, and a stirring time of 120 min. The immobilized papain was prepared as follows: 2.0 g of 100-mesh microcrystalline cellulose was weighed and activated for 24 h in a mixed solution of 200 mL sodium periodate (1.2 g) and concentrated sulfuric acid (2 mL). Subsequently, the washed activated cellulose (0.1 g) and papain (0.64 g) were dispersed in Tris-HCl solution (50 mM, pH 7.6), shaken for 24 h, and then centrifuged and washed to obtain immobilized papain.

[0046] The above description is merely a preferred embodiment of this utility model and is not intended to limit the utility model in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this utility model without departing from its technical solution shall still fall within the protection scope of this utility model.

Claims

1. An apparatus for removing impurities from crude polysaccharides of traditional Chinese medicine using immobilized protease, characterized in that: The protein removal reactor loaded with immobilized protease is placed in the constant temperature magnetic stirrer, and the dissolved oxygen electrode of the dissolved oxygen meter is connected to the protein removal reactor.

2. The device for removing impurities from traditional Chinese medicine crude polysaccharides by using immobilized protease according to claim 1, characterized in that: The protein removal reactor comprises an upper columnar container and a lower columnar container, the top end of the upper columnar container is provided with three interfaces from left to right, which are a traditional Chinese medicine crude polysaccharide solution inlet and outlet, a temperature sensor interface and a dissolved oxygen electrode interface in sequence, the upper columnar container is provided with a layer of screen mesh in the middle, and the lower end is provided with a threaded interface and a gasket; the top end of the lower columnar container is provided with a threaded interface, the lower layer is provided with a layer of screen mesh, and a magnetic stirring rod is placed at the bottom end; the upper columnar container and the lower columnar container are screwed at the threaded interfaces. 3.The device for removing impurities from traditional Chinese medicine crude polysaccharides by using immobilized protease according to claim 2, characterized in that: A temperature sensor is installed on the temperature sensor interface, and a dissolved oxygen electrode is installed on the dissolved oxygen electrode interface.

4. The device for removing impurities from traditional Chinese medicine crude polysaccharides by using immobilized protease according to claim 2, characterized in that: The screen mesh is a stainless steel screen mesh with a particle size of greater than or equal to 400 meshes, and immobilized papain is loaded between the two layers of screen mesh.

5. The device for removing impurities from traditional Chinese medicine crude polysaccharides by using immobilized protease according to claim 2, characterized in that: The length of the magnetic stirring rod is 40-70 mm.

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