CN113117128A - Homogeneous multifunctional bacterial cellulose-based skin dressing and preparation method thereof - Google Patents
Homogeneous multifunctional bacterial cellulose-based skin dressing and preparation method thereof Download PDFInfo
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- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
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Abstract
The invention relates to a homogeneous multifunctional bacterial cellulose-based skin dressing and a preparation method thereof.
Description
Technical Field
The invention relates to a homogeneous multifunctional bacterial cellulose-based skin dressing and a preparation method thereof, in particular to a multifunctional bacterial cellulose-based skin dressing which is prepared by a layer-by-layer in-situ composite method and has good moisture absorption, adhesion prevention, infection resistance and healing promotion capability, belonging to the field of biomedical materials.
Background
The skin caused by burn, scald and chronic wound is seriously damaged, difficult to repair and easy to cause complications such as infection, and is one of the diseases which are difficult to treat in the current orthopedic surgery and dermatology department. The development of the skin dressing with multiple functions of resisting infection, promoting healing and the like has positive economic and social values for promoting the recovery of patients, relieving the pain of the patients and reducing the expenditure of medical and health expenses. The Bacterial Cellulose (BC) has a three-dimensional nano network structure, can prevent external pathogenic bacteria from invading, has good water absorption, water retention, air permeability, anti-adhesion performance, biocompatibility, immunogenicity and certain hemostatic function, can maintain the moist environment of a wound surface, promotes granulation growth, and is one of ideal skin dressing raw materials. However, the BC nanofiber dressing does not have a sterilization function and has no effect on invaded pathogenic bacteria and infected wound surfaces; on the other hand, the BC has poor cell compatibility, and the ideal healing promoting effect is difficult to achieve for large-area wound repair.
The active ingredients are introduced into the three-dimensional network structure of the BC support to prepare the BC-based composite material, which is an effective way for improving the performance of the BC-based dressing. One introduces Silver Nanoparticles (Wu CN. TEMPO-Oxidized Bacterial Cellulose with Silver Nanoparticles for round dropping. biomacromolecules.2018; 19:544-54.), quaternary ammonium salt compound(s) (II) (III)
Modification of Bacterial cell with Quaternary Ammonium Compounds Based on Amino Acids and the Effect on Antimicrobial activity, biomacromolecules, 2018; 1528-38.), an antibacterial peptide (furnatz M. function of bacterial cellulose bottom delivery with the antibacterial peptide epsilon-poly-L-lysine. biological materials.2018; 13:025014.), Amoxicillin (Ye S. Flexible Amoxicillin-modified Bacterial cells sites for Wound addressing: In Vitro and In Vivo evaluation&Interfaces.2018; 5862-70.) and the like to obtain the BC-based dressing with good antibacterial performance. Collagen type I (Moraes PRFdS. bacterial cell/Collagen Hydrogel for round health materials research. 2016; 19:106-16.), hyaluronic acid (Li Y. bacterial cell-hyaluronic acid nanocomposites as a ground addressing for a driver skin in therapy repair. journal of Materials Chemistry B.2015; 3498-; 184:323-32.) and the like with BC to obtain the BC-based composite material which can better promote the cell growth. However, the above methods only seek single performance in one aspect, and the effect is not ideal in a complex clinical environment.
Although the prior art also exists for the simultaneous introduction of both of these materials into the BC. However, in terms of the preparation method, the conventional BC-based composite material is prepared by synthesizing BC with bacteria such as acetobacter xylinum and then compounding the BC with additives. Since the BC has a nano-scale network structure, macromolecular additives cannot enter the BC, and homogeneous materials are difficult to obtain. For example, a patent (chinese publication No. CN102888027A) discloses a bacterial cellulose/collagen-chitosan composite material and a method for preparing the same, which uses a positive pressure method or a negative pressure method to make collagen enter BC, but the collagen distribution in the composite material obtained by the method is not uniform.
Disclosure of Invention
In view of the above problems, the present invention aims to provide a homogeneous multifunctional bacterial cellulose-based skin dressing and a preparation method thereof.
On one hand, the invention provides a homogeneous multifunctional bacterial cellulose-based skin dressing, which is prepared by introducing type I collagen and chitosan quaternary ammonium salt into a three-dimensional network structure of bacterial cellulose through a layer-by-layer in-situ compounding method.
Preferably, the chitosan quaternary ammonium salt is a chitosan derivative synthesized by reacting chitosan and a quaternary ammonium salt, and the quaternary ammonium salt is at least one of 2, 3-epoxypropyltrimethylammonium chloride, dodecyl dimethyl benzyl ammonium chloride, chloropropylamine trimethyl ammonium chloride and hydroxypropyl trimethyl ammonium chloride. Wherein, the chitosan quaternary ammonium salt endows the skin dressing with anti-infection performance.
Preferably, the content of the bacterial cellulose in the multifunctional bacterial cellulose-based skin dressing is 40-60 wt%.
Preferably, the multifunctional bacterial cellulose-based skin dressing has a thickness of 0.5-3 mm.
In another aspect, the present invention also provides a method for preparing a multifunctional bacterial cellulose-based skin dressing, comprising:
(1) preparing a culture medium containing type I collagen and chitosan quaternary ammonium salt;
(2) obtaining a bacterial cellulose base membrane by adopting a static culture method;
(3) dropwise adding a culture medium containing the type I collagen and the chitosan quaternary ammonium salt on the surface of the bacterial cellulose base membrane until the culture medium containing the type I collagen and the chitosan quaternary ammonium salt is completely consumed;
(4) and (4) after repeating the step (3) for multiple times, removing the bacterial cellulose base membrane, and then purifying and freeze-drying to obtain the multifunctional bacterial cellulose base skin dressing.
In the present disclosure, a static culture method is used to obtain a bacterial cellulose base membrane to obtain a strain suitable for biosynthesis of bacterial cellulose. Then, a culture medium containing the type I collagen and the chitosan quaternary ammonium salt is dripped on the surface of the bacterial cellulose base membrane, and bacteria on the surface layer of the bacterial cellulose base membrane are biologically synthesized into bacterial cellulose and fully compounded with the type I collagen and the chitosan quaternary ammonium salt on a micrometer scale, so that the technical problem that the collagen and the chitosan quaternary ammonium salt are not easy to introduce is solved, and the distribution of the collagen and the chitosan quaternary ammonium salt is more uniform. On the other hand, the additive is added into the culture medium, and the additive is compounded layer by layer while the bacterial cellulose layer grows, so that a very uniform compound is formed. Specifically, bacterial cellulose is secreted by bacteria in an aerobic environment, and only the layer of bacteria at the membrane-liquid interface can secrete the bacterial cellulose in a culture solution containing the bacteria. The invention utilizes the characteristic, the culture medium containing the type I collagen and the chitosan quaternary ammonium salt is reduced to a very small amount, namely, a very thin layer (preferably reaching the thickness of micron order) is covered on the bacterial cellulose base membrane and the grown bacterial cellulose, so that the bacterial cellulose growing before and after is continuous, and the continuous growth of the bacterial cellulose is realized. And finally removing the bacterial cellulose-based basement membrane, and then purifying and freeze-drying to obtain the homogeneous multifunctional bacterial cellulose-based skin dressing.
Preferably, the concentration of the chitosan quaternary ammonium salt in the culture medium containing the type I collagen and the chitosan quaternary ammonium salt is 1-5 mg/mL, and the concentration of the type I collagen is 0.5-2 mg/mL.
Preferably, the thickness of the bacterial cellulose base membrane is 0.5-2 mm.
Preferably, the thickness of the culture medium containing the type I collagen and the chitosan quaternary ammonium salt dripped on the surface of the bacterial cellulose base membrane every time is 30-80 mu m.
Preferably, the purification comprises: firstly, carrying out ultrasonic treatment in 0.3-0.7 mol/L NaOH solution for 1-3 hours; placing the mixture into pure water, performing ultrasonic treatment for 1 hour, and repeating the ultrasonic treatment for 3-5 times; and finally, washing the mixture to be neutral by adopting pure water.
Preferably, the temperature of the freeze drying is-30 to-50 ℃, and the time is 48 to 96 hours.
Has the advantages that:
according to the invention, the bacterial cellulose is grown in situ in the culture medium containing the type I collagen and the chitosan quaternary ammonium salt for the first time by a layer-by-layer in-situ compounding method, so that the uniform distribution of the type I collagen and the chitosan quaternary ammonium salt is directly realized, and finally the homogeneous multifunctional bacterial cellulose-based skin dressing is obtained;
in the invention, the preparation process of the obtained I type collagen/chitosan quaternary ammonium salt/bacterial cellulose functional dressing with anti-infection and tissue repair promoting properties is simple, high in efficiency and good in repeatability;
according to the invention, the prepared type I collagen/chitosan quaternary ammonium salt/bacterial cellulose functional dressing integrates the advantages of three materials, namely type I collagen, chitosan quaternary ammonium salt and bacterial cellulose, has the basic performance required by skin dressings, and has good antibacterial performance and cell compatibility.
Drawings
FIG. 1 is a scanning electron micrograph of Bacterial Cellulose (BC) prepared in example 1;
FIG. 2 is an SEM image of a collagen I/chitosan hyamine/bacterial cellulose (CHBC) functional dressing prepared in example 1;
FIG. 3 is a scanning electron microscope image of a type I collagen/chitosan quaternary ammonium salt/bacterial cellulose (CHBC-2) functional dressing prepared in example 2;
FIG. 4 is a bacterial experimental drawing of the Bacterial Cellulose (BC) and type I collagen/chitosan quaternary ammonium salt/bacterial cellulose (CHBC) functional dressing of example 3;
FIG. 5 is a schematic representation of the cell experiments of the Bacterial Cellulose (BC) and type I collagen/chitosan quaternary ammonium salt/bacterial cellulose (CHBC) functional dressing of example 4;
fig. 6 is the attached diagram of animal experiments of the Bacterial Cellulose (BC) and type I collagen/chitosan quaternary ammonium salt/bacterial cellulose (CHBC) functional dressing in example 5.
Detailed Description
The present invention is further illustrated by the following examples, which are to be understood as merely illustrative and not restrictive.
In the method, the I-type collagen and the chitosan quaternary ammonium salt are introduced into a three-dimensional network structure of the bacterial cellulose by a layer-by-layer in-situ compounding method for the first time, and finally the homogeneous multifunctional bacterial cellulose-based skin dressing, namely the I-type collagen/chitosan quaternary ammonium salt/bacterial cellulose functional dressing, is obtained. The preparation process of the multifunctional bacterial cellulose-based skin dressing is exemplarily described below.
Preparing a common culture medium. And preparing a culture medium, placing the culture medium in a sterilization pot, and sterilizing the culture medium for 30-60 min at high temperature and high pressure to obtain a common culture medium. Specifically, glucose, peptone, disodium hydrogen phosphate and yeast powder are dissolved in deionized water to prepare a culture medium, the pH of the culture medium is 3.5-5.5, and the culture medium is placed in a sterilization pot to be sterilized for 20-40 min at 110-120 ℃ and 0.1MPa for later use, so that a common culture medium is obtained. As an example of preparing a common medium, 25g of glucose, 10g of peptone, 10g of disodium hydrogen phosphate and 7.5g of yeast powder are dissolved in 1L of deionized water to prepare a medium, the pH of the medium is controlled to be 4-5, and the medium is placed in a sterilization pot to be sterilized at 115 ℃ and 0.1MPa for 30min for later use, so as to obtain the common medium.
Preparing a culture medium containing type I collagen and chitosan quaternary ammonium salt. Dissolving chitosan quaternary ammonium salt in common culture medium, and sterilizing at high temperature and high pressure. Wherein, the concentration of the chitosan quaternary ammonium salt is controlled to be 1-5 mg/mL. Then, continuously dissolving the type I collagen in an aseptic environment to obtain a culture medium containing the type I collagen and the chitosan quaternary ammonium salt. Wherein the concentration of the type I collagen is controlled to be 0.5-2 mg/mL.
In an alternative embodiment, the chitosan quaternary ammonium salt is a chitosan derivative synthesized by reacting chitosan and a quaternary ammonium salt. The quaternary ammonium salt can be at least one of 2, 3-epoxypropyltrimethyl ammonium chloride, dodecyl dimethyl benzyl ammonium chloride, chloropropylamine trimethyl ammonium chloride, hydroxypropyl trimethyl ammonium chloride and the like.
And (3) obtaining the bacterial cellulose base membrane by adopting a static culture method. The thickness of the obtained bacterial cellulose base membrane can be 0.5-2 mm.
And (3) dropwise adding a culture medium containing type I collagen and chitosan quaternary ammonium salt to cover the obtained bacterial cellulose base membrane still containing the strain. And (3) when the culture medium containing the type I collagen and the chitosan quaternary ammonium salt which is dripped last time is just consumed, dripping again and repeatedly for many times until the required thickness is reached. Preferably, the timing device is set to control the dropping time and frequency of the culture medium containing the type I collagen and the chitosan quaternary ammonium salt by controlling the thickness thereof. In addition, when a culture medium containing type I collagen and chitosan quaternary ammonium salt is dripped to cover the bacterial cellulose base membrane, the thickness of the coated specific culture medium is controlled to be 30-80 mu m, and the aim is to compound the bacterial cellulose, the type I collagen and the chitosan quaternary ammonium salt layer by layer on a micrometer scale.
And after the multifunctional bacterial cellulose-based skin dressing reaches the required thickness, removing the bacterial cellulose-based basement membrane, and then purifying and freeze-drying to obtain the multifunctional bacterial cellulose-based skin dressing. Wherein the purification step comprises: firstly, putting the mixture into 0.3-0.7 mol/L NaOH solution, and carrying out ultrasonic treatment for 1-3 hours; placing the mixture into pure water, performing ultrasonic treatment for 1 hour, and repeating the ultrasonic treatment for 3-5 times; finally, washing the mixture to be neutral by pure water. The temperature of freeze drying is-30 to-50 ℃, and the time is 48 to 96 hours.
In the invention, the prepared I type collagen/chitosan quaternary ammonium salt/bacterial cellulose functional dressing integrates the advantages of the I type collagen, the chitosan quaternary ammonium salt and the bacterial cellulose, and has a plurality of functions of adhesion prevention, moisture absorption, infection resistance, healing promotion and the like.
In an alternative embodiment, the content of the bacterial cellulose in the multifunctional bacterial cellulose-based skin dressing may be 40-60 wt%. The thickness of the obtained multifunctional bacterial cellulose-based skin dressing can be 0.5-3 mm.
The present invention will be described in further detail with reference to examples. It is also to be understood that the following examples are illustrative of the present invention and are not to be construed as limiting the scope of the invention, and that certain insubstantial modifications and adaptations of the invention by those skilled in the art in light of the foregoing description are intended to be included within the scope of the invention. The specific process parameters and the like of the following examples are also only one example of suitable ranges, i.e., those skilled in the art can select the appropriate ranges through the description herein, and are not limited to the specific values exemplified below. Unless otherwise specified, the quaternary ammonium salt of chitosan used in the following examples is 2, 3-epoxypropyltrimethylammonium chloride.
Example 1
The preparation method of the I type collagen/chitosan quaternary ammonium salt/bacterial cellulose functional dressing 1 comprises the following steps:
s1: dissolving 25g of glucose, 10g of peptone, 10g of disodium hydrogen phosphate and 7.5g of yeast powder in 1L of deionized water to prepare a culture medium, wherein the pH value of the culture medium is 4-5, and placing the culture medium in a sterilization pot to sterilize for 30min at 115 ℃ and 0.1MPa for later use to obtain a common culture medium;
s2: the chitosan quaternary ammonium salt is dissolved in a common culture medium, and the concentration is 3 mg/mL. After high-temperature sterilization, dissolving type I collagen with the concentration of 1mg/mL in an aseptic environment to obtain a culture medium containing the type I collagen and chitosan quaternary ammonium salt;
s3: inoculating a bacterial liquid into a culture medium in an aseptic environment, wherein the strain of the bacterial liquid is acetobacter xylinum, inoculating 200 mu L of the culture medium inoculated with the acetobacter xylinum into each hole of a 24-hole plate, and statically culturing for 2d at the temperature of 30 ℃ to obtain a bacterial cellulose base basement membrane (BC) with the thickness of 1.5 mm;
s4: dripping a culture medium containing type I collagen and chitosan quaternary ammonium salt to cover the basement membrane, wherein the thickness is about 50 mu m, and culturing for 2 hours; when the bacterial cellulose culture medium which is dripped last time is just consumed, dripping again and repeatedly for many times until the required thickness is reached, wherein the thickness is about 1 mm;
s5: after removing the basement membrane, placing the basement membrane in 0.5mol/L NaOH solution, and carrying out ultrasonic treatment for 1-3 hours; placing the mixture into pure water, performing ultrasonic treatment for 1 hour, and repeating the ultrasonic treatment for 3-5 times; washing with pure water to neutral. The sample is then frozen in a refrigerator at-20 ℃ for 12h, and finally freeze-dried for 72h to obtain the type I collagen/chitosan quaternary ammonium salt/bacterial cellulose functional dressing (CHBC).
Observing by using a scanning electron microscope:
FIG. 1 is an SEM image of a BC showing that pure BC has a relatively uniform and smooth fiber surface and relatively large pores between fibers. FIG. 2 is an SEM image of CHBC with the interstitial pores between the bacterial cellulose of the type I collagen/chitosan quat/bacterial cellulose functional dressing (CHBC) sample filled with type I collagen and chitosan quat. The content of the bacterial cellulose in the obtained multifunctional bacterial cellulose-based skin dressing is 50 wt%.
Example 2
The preparation method of the I type collagen/chitosan quaternary ammonium salt/bacterial cellulose functional dressing 2 comprises the following steps:
preparing a common culture medium and a culture medium containing type I collagen and chitosan quaternary ammonium salt according to the method of example 1, and culturing to obtain a bacterial cellulose base membrane;
s1: dripping culture medium containing type I collagen and chitosan quaternary ammonium salt to cover the basement membrane, wherein the thickness is about 80 mu m, and culturing for 2.5 hours; when the bacterial cellulose culture medium which is dripped last time is just consumed, dripping again and repeatedly for many times until the required thickness is reached, wherein the thickness is about 1.5 mm;
s2: after removing the basement membrane, placing the basement membrane in 0.5mol/L NaOH solution, and carrying out ultrasonic treatment for 1-3 hours; placing the mixture into pure water, carrying out ultrasonic treatment for 1 hour, and repeating for 3-5 times; washing with pure water to neutral. The sample is then frozen in a refrigerator at-20 ℃ for 12h, and finally freeze-dried for 72h, so as to obtain the type I collagen/chitosan quaternary ammonium salt/bacterial cellulose functional dressing 2 (CHBC-2).
Observing by using a scanning electron microscope:
FIG. 3 is an SEM image of CHBC-2, wherein the pores between the bacterial cellulose of the type I collagen/chitosan hyamine/bacterial cellulose functional dressing 2(CHBC-2) sample are filled with type I collagen and chitosan hyamine. The content of the bacterial cellulose in the obtained multifunctional bacterial cellulose-based skin dressing is 45 wt%.
Example 3
The antibacterial performance test of the I type collagen/chitosan quaternary ammonium salt/bacterial cellulose functional dressing CHBC comprises the following steps:
the bacterial cellulose BC prepared in the example 1 and the type I collagen/chitosan quaternary ammonium salt/bacterial cellulose functional dressing CHBC are respectively cultured together with the pathogenic bacteria Staphylococcus aureus ATCC 25923 in a 37 ℃ incubator for 6 hours and 24 hours. And observing the adhesion and growth conditions of bacteria on the surface of the material by a scanning electron microscope, and observing the killing conditions of the bacteria by laser confocal observation.
In FIG. 4, A is an SEM picture of the material after co-culturing with bacteria for a certain period of time. Compared with the bacterial cellulose BC, the type I collagen/chitosan quaternary ammonium salt/bacterial cellulose functional dressing CHBC can well inhibit the colonization of bacteria and the formation of bacterial biofilms; in FIG. 4, B is a laser confocal photograph of dead and live bacteria, wherein green is live bacteria and red is dead bacteria.
As can be seen from the figure, the type I collagen/chitosan quaternary ammonium salt/bacterial cellulose functional dressing is able to kill bacteria in contact.
Example 4
The cell biology performance test of the I type collagen/chitosan quaternary ammonium salt/bacterial cellulose functional dressing CHBC comprises the following steps:
the biological performance of the collagen I/chitosan quaternary ammonium salt/bacterial cellulose functional dressing CHBC prepared in example 1 was examined with fibroblasts (NIH3T3) and endothelial cells (HUVEC): the proliferation behavior of cells on the surface of the material is inspected by a CCK-8 method; and observing the adhesion morphology of the cells on the surface of the material by using a scanning electron microscope.
In FIG. 5, A is the result of CCK-8 experiment of NIH3T3 proliferation on the surface of the material, and in FIG. 5, B is the result of CCK-8 experiment of HUVEC proliferation on the surface of the material. As can be seen from the figure, the type I collagen/chitosan quaternary ammonium salt/bacterial cellulose functional dressing is more capable of promoting the proliferation of two types of cells. The result of the scanning electron microscope C in fig. 5 shows that the two cells spread better on the surface of the type I collagen/chitosan quaternary ammonium salt/bacterial cellulose functional dressing, indicating that the bacterial cellulose-based composite material has better cell compatibility.
Example 5
An animal experiment of the I type collagen/chitosan quaternary ammonium salt/bacterial cellulose functional dressing comprises the following steps:
referring to the method of example 1, type I collagen/chitosan quaternary ammonium salt/bacterial cellulose functional dressing was prepared and ethylene oxide sterilized. Several mice of 8 weeks old were selected, anesthetized, and sterilized to perform an operation. After a wound of a certain size is cut with a scalpel, 10 is injected4And (5) coating materials on the cfu staphylococcus aureus, and fixing the materials by using a medical adhesive tape. The dressing was changed every two days, the healing of the wound was observed, and a photograph was taken.
Fig. 6 is a rough photograph of wound healing, wherein the blank group (Control group) is the BC group without added bacteria. As can be seen from the figure, the BC group had significant infection after 2 days, and no foci of infection were found in the Control combination CHBC group; the healing profile of BC was expected to be poor compared to control group, indicating that infection affected tissue healing; the healing was significantly better in the CHBC group than in the BC group.
Claims (9)
1. The homogeneous multifunctional bacterial cellulose-based skin dressing is characterized in that a layer-by-layer in-situ compounding method is adopted to introduce type I collagen and chitosan quaternary ammonium salt into a three-dimensional network structure of bacterial cellulose, so that the homogeneous multifunctional bacterial cellulose-based skin dressing is obtained.
2. The multifunctional bacterial cellulose-based skin dressing according to claim 1, wherein the chitosan quaternary ammonium salt is a chitosan derivative synthesized by reacting chitosan and a quaternary ammonium salt, and the quaternary ammonium salt is at least one of 2, 3-epoxypropyltrimethylammonium chloride, dodecyl dimethyl benzyl ammonium chloride, chloropropylamine trimethyl ammonium chloride and hydroxypropyl trimethyl ammonium chloride.
3. The multifunctional bacterial cellulose-based skin dressing according to claim 1 or 2, wherein the content of bacterial cellulose in the multifunctional bacterial cellulose-based skin dressing is 40-60 wt%.
4. The multifunctional bacterial cellulose-based skin dressing according to any one of claims 1-3, wherein the multifunctional bacterial cellulose-based skin dressing has a thickness of 0.5-3 mm.
5. A method of preparing the multifunctional bacterial cellulose-based skin dressing according to any one of claims 1-4, comprising:
(1) preparing a culture medium containing type I collagen and chitosan quaternary ammonium salt;
(2) obtaining a bacterial cellulose base membrane by adopting a static culture method;
(3) dropwise adding a culture medium containing the type I collagen and the chitosan quaternary ammonium salt on the surface of the bacterial cellulose base membrane until the culture medium containing the type I collagen and the chitosan quaternary ammonium salt is completely consumed;
(4) and (4) after repeating the step (3) for multiple times, removing the bacterial cellulose base membrane, and then purifying and freeze-drying to obtain the multifunctional bacterial cellulose base skin dressing.
6. The method according to claim 5, wherein the concentration of the quaternary ammonium salt of chitosan in the culture medium containing type I collagen and the quaternary ammonium salt of chitosan is 1 to 5mg/mL, and the concentration of the type I collagen is 0.5 to 2 mg/mL.
7. The method according to claim 5 or 6, wherein the thickness of the culture medium containing the type I collagen and the chitosan quaternary ammonium salt dripped on the surface of the bacterial cellulose base membrane each time is 30-80 μm.
8. The method according to any one of claims 5 to 7, wherein the bacterial cellulose base film has a thickness of 0.5 to 2 mm.
9. The production method according to any one of claims 5 to 8, wherein the purification comprises: firstly, carrying out ultrasonic treatment in 0.3-0.7 mol/L NaOH solution for 1-3 hours; placing the mixture into pure water, performing ultrasonic treatment for 1 hour, and repeating the ultrasonic treatment for 3-5 times; finally, washing the mixture to be neutral by pure water; the temperature of the freeze drying is-20 to-50 ℃, and the time is 48 to 96 hours.
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