Lucid ganoderma extraction method based on reusable magnetic enzyme

Abstract

The invention discloses a ganoderma lucidum extraction method based on a reusable magnetic enzyme. The ganoderma lucidum extraction method comprises the following steps: S1, preparing a nanoscale amination modified Fe3O4-SiO2 magnetic composite carrier; s2, on the basis of the amination modified Fe3O4-SiO2 magnetic composite carrier, cellulase is added, and a magnetic immobilized enzyme buffer solution is obtained; s3, the ganoderma lucidum sporocarp is put into a magnetic immobilized enzyme buffer solution to be subjected to enzymolysis, extraction and filtration, effective components of ganoderma lucidum are obtained, and magnetic immobilized enzyme is recycled for repeated utilization; according to the method, the magnetic nanoparticles are modified, so that the surfaces of the magnetic nanoparticles have specific active groups, meanwhile, the magnetic response advantage of an inorganic magnetic material is reserved, the nanoscale amination-modified Fe3O4-SiO2 magnetic composite carrier is prepared, enzyme molecules can be directly anchored on the surfaces of the magnetic nanoparticles, and enzyme activity is improved. The immobilized enzyme can be conveniently recycled from a reaction system, so that the cost of an enzyme preparation can be reduced, and the enzyme activity stability can be improved.

Description

Technical Field

[0001] The invention relates to the technical field of ganoderma lucidum extraction, and in particular to a ganoderma lucidum extraction method based on reusable magnetic enzyme. Background Art

[0002] Ganoderma lucidum is a fungus of the class Basidiomycetes, the family Polyporaceae, and the genus Ganoderma. It is a medicinal fungus, and its fruiting bodies are often used. Ganoderma lucidum contains a variety of active substances such as polysaccharides, triterpenes, organic germanium, nucleosides, amino acids, proteins, alkaloids, oils, sterols, etc. Ganoderma lucidum triterpenes have a variety of biological activities, such as improving the body's immunity, anti-tumor, anti-inflammatory, antioxidant, lowering blood lipids, lowering blood sugar, and protecting the liver. Studies have shown that the pharmacological effects of Ganoderma lucidum are closely related to the content of Ganoderma lucidum triterpenes, and Ganoderma lucidum triterpenes have become one of the important indicators for evaluating the quality of Ganoderma lucidum products. The cell wall of Ganoderma lucidum fruiting bodies is mainly composed of chitin, cellulose, hemicellulose and other substances, with a complex structure, hardness, and acid and alkali resistance. These reasons lead to a relatively low yield of Ganoderma lucidum triterpenes in the conventional reflux extraction method, which is difficult to meet the actual use needs. At present, the extraction of Ganoderma lucidum mainly adopts solvent extraction, ultrasonic assisted extraction and other methods. Although these methods can extract the effective ingredients in Ganoderma lucidum to a certain extent, they often have disadvantages such as solvent residue, high energy consumption, low extraction efficiency and environmental unfriendliness. In recent years, with the development of biotechnology, enzymatic hydrolysis has gradually attracted attention due to its mild, efficient and environmentally friendly characteristics. However, the existing enzymatic hydrolysis method in Ganoderma lucidum extraction still faces problems such as high cost of enzyme preparations, difficulty in recycling and poor enzyme activity stability, which limits its large-scale application. Summary of the invention

[0003] In order to solve the above problems, the present invention proposes a method for extracting Ganoderma lucidum based on a reusable magnetic enzyme.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] A method for extracting ganoderma lucidum based on a reusable magnetic enzyme comprises the following steps:

[0006] S1, preparing nano-scale amino-modified Fe3O4-SiO2 magnetic composite carrier;

[0007] S2, based on the amino-modified Fe3O4-SiO2 magnetic composite carrier, adding cellulase to obtain a magnetic immobilized enzyme buffer;

[0008] S3, placing the ganoderma lucidum fruiting body into a magnetic immobilized enzyme buffer for enzymolysis, extraction and filtration to obtain the effective components of ganoderma lucidum, and recovering the magnetic immobilized enzyme for repeated use.

[0009] Preferably, the specific process of step S1 is:

[0010] S11, weigh 2mmol of triacetylacetonate iron, add 10ml of benzyl alcohol to a high-pressure reactor, add 2ml of oleylamine and 2ml of oleic acid in sequence for ultrasonic dissolution; after mixing evenly, react at 200°C in a muffle furnace for 10h, pour the black solution obtained after the reaction into a beaker, and remove the supernatant by magnet adsorption; then redisperse the black precipitate in cyclohexane to obtain a Fe3O4 cyclohexane solution;

[0011] S12, weigh 5 ml of Trinaton X-100, 25 ml of cyclohexane and 30 mg of Fe3O4 cyclohexane solution and dissolve them in a three-necked flask by ultrasonic; then add 6 ml of n-hexanol and dissolve them in ultrasonic for 10 minutes until they are evenly dispersed;

[0012] S13, transfer the three-necked flask to a mechanical stirrer, add 2 ml of H2O and 2 ml of ammonia water dropwise during stirring to form a stable microemulsion, and stir at room temperature for 1 hour; then slowly add 3 ml of TEOS, react at room temperature for 24 hours, centrifuge at 12000 rpm for 10 minutes, collect the precipitate, wash it with ethanol and distilled water for several times, and disperse it in 10 ml of distilled water to obtain a HMFSs aqueous solution;

[0013] S14. Centrifuge the HMFSs aqueous solution to remove the supernatant and wash it twice with ethanol. Transfer it to a three-necked flask and sonicate for 15 minutes. Add 2 ml of distilled water. Add 2 ml of ammonia water under stirring and continue stirring for 30 minutes. Add 1 ml of APTES at 80°C and react for 2 hours. After the reaction, centrifuge at 12000 rpm for 10 minutes to collect the precipitate, wash it with ethanol and distilled water for 3 times each, and dry it in vacuum at room temperature for 24 hours to obtain an amino-modified Fe3O4-SiO2 magnetic composite carrier containing amino functional groups.

[0014] Preferably, the specific process of step S2 is:

[0015] S21, place the amino-modified Fe3O4-SiO2 magnetic composite carrier in a 50 ml conical flask, add purified water, shake and sonicate for 10 seconds, add glutaraldehyde by volume to make the final concentration of the solution reach 5%, and activate at 30°C and 180 r / min for 2 hours;

[0016] S22, after activation, wash with ultrapure water and pH 4.8 citric acid buffer, then add pH 4.8 citric acid buffer and cellulase, and immobilize at 20°C and 180 r / min for 3 h;

[0017] S23, after the immobilization reaction is completed, the cellulase is separated by a magnet and repeatedly washed with a pH 4.8 citric acid buffer until no protein is detected in the supernatant, thereby obtaining an immobilized cellulase as a cross-linked composite carrier;

[0018] S24, adding a citric acid buffer solution of pH 4.8 to obtain a magnetic immobilized enzyme buffer solution, and storing it in a 4°C refrigerator for later use.

[0019] Preferably, the specific process of step S3 is:

[0020] S31, weigh the Ganoderma lucidum fruiting body and place it in a 500 mL distillation flask, add the magnetic immobilized enzyme buffer, and perform enzymolysis in a 37° C. constant temperature water bath for 2 h;

[0021] S32, after the enzymatic hydrolysis is completed, the magnetic immobilized enzyme is separated by adsorption with a magnet, taken out and cooled, and ethanol is added, and refluxed in a water bath at 80°C for 3h;

[0022] S33, after the extraction is completed, the filtrate is filtered, and the filtrate is the effective component extracted from Ganoderma lucidum. The magnetic immobilized enzyme is recovered through the magnetic field for repeated use.

[0023] Preferably, the Ganoderma lucidum fruiting bodies in step S31 are pre-treated by removing impurities and crushing before enzymatic hydrolysis to obtain Ganoderma lucidum particles suitable for enzymatic hydrolysis.

[0024] After adopting the above technical scheme, the present invention has the following beneficial effects: although the unmodified magnetic Fe3O4 nanoparticles have the advantages of high purity, stable magnetism, large specific surface area, etc., as metal particles, they are easily combined with hydrogen ions in the solution so that their surface has a strong positive charge, and are more likely to agglomerate than other organic materials; therefore, the present invention can modify the magnetic nanoparticles, not only so that the surface of the particles has specific active groups, but also retain the magnetic response advantages of inorganic magnetic materials, thereby obtaining nano-scale amino-modified Fe3O4-SiO2 magnetic composite carriers; in the amino-modified Fe3O4-SiO2 magnetic composite carriers, enzyme molecules can be directly anchored on the surface of the magnetic nanoparticles, and the magnetic Fe3O4 nanoparticles have a large specific surface area and superparamagnetism, that is, they have magnetism in an external magnetic field, and their magnetism will disappear when the external magnetic field disappears. The immobilized enzyme can be easily recovered and reused from the reaction system to form a reusable magnetic enzyme, which can not only reduce the cost of enzyme preparations, but also improve the enzyme activity stability, and can be applied on a large scale. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is an electron microscope image of the exposed Fe3O4 nanoparticles of the present invention;

[0026] Figure 2 This is an electron microscope image of the Fe3O4-SiO2 magnetic composite carrier of the present invention that has not been modified by amino;

[0027] Figure 3 This is an electron microscope image of the Fe3O4-SiO2 magnetic composite carrier modified by amino group of the present invention;

[0028] Figure 4 This is a diagram of the Zeta potential measurement results of the present invention;

[0029] Figure 5 It is the absorbance test diagram of different glucose concentrations of the present invention;

[0030] Figure 6 This is a diagram showing the enzyme activity test results of the magnetic immobilized enzyme during the six-times reuse process of the present invention. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0032] like Figures 1 to 6 As shown, a method for extracting Ganoderma lucidum based on a reusable magnetic enzyme comprises the following steps:

[0033] S1, preparing nano-scale amino-modified Fe3O4-SiO2 magnetic composite carrier;

[0034] The specific process of step S1 is:

[0035] S11, weigh 2mmol of triacetylacetonate iron, add 10ml of benzyl alcohol to a high-pressure reactor, add 2ml of oleylamine and 2ml of oleic acid in sequence for ultrasonic dissolution; after mixing evenly, react at 200°C in a muffle furnace for 10h, pour the black solution obtained after the reaction into a beaker, and remove the supernatant by magnet adsorption; then redisperse the black precipitate in cyclohexane to obtain a Fe3O4 cyclohexane solution;

[0036] S12, weigh 5 ml of Trinaton X-100, 25 ml of cyclohexane and 30 mg of Fe3O4 cyclohexane solution and dissolve them in a three-necked flask by ultrasonic; then add 6 ml of n-hexanol and dissolve them in ultrasonic for 10 minutes until they are evenly dispersed;

[0037] S13, transfer the three-necked flask to a mechanical stirrer, add 2 ml of H2O and 2 ml of ammonia water dropwise during stirring to form a stable microemulsion, and stir at room temperature for 1 hour; then slowly add 3 ml of TEOS, react at room temperature for 24 hours, centrifuge at 12000 rpm for 10 minutes, collect the precipitate, wash it with ethanol and distilled water for several times, and disperse it in 10 ml of distilled water to obtain a HMFSs aqueous solution;

[0038] S14, centrifuge the HMFSs aqueous solution to remove the supernatant and wash it with ethanol twice, transfer it to a three-necked flask and perform ultrasonic treatment for 15 minutes, add 2 ml of distilled water, add 2 ml of ammonia water under stirring conditions, and continue stirring for 30 minutes; add 1 ml of APTES at 80°C for 2 hours, and after the reaction, centrifuge at 12000 rpm for 10 minutes to collect the precipitate, wash it with ethanol and distilled water three times each, and vacuum dry it at room temperature for 24 hours to obtain an amino-modified Fe3O4-SiO2 magnetic composite carrier containing amino functional groups;

[0039] Among them, the electron microscope image of Fe3O4 nanoparticles is as follows Figure 1 As shown, the electron microscope image of the Fe3O4-SiO2 magnetic composite carrier without amino modification is as follows Figure 2 As shown, the electron microscope image of the Fe3O4-SiO2 magnetic composite carrier modified by amino is as follows Figure 3 As shown. Figures 1 to 3 It can be seen that the morphology of the bare Fe3O4 nanoparticles is a regular sphere, and the particle size is relatively uniform. After being coated with a silica shell and an amino-modified monomer, the prepared FS and HMFS-NH2 are both core-shell structures, and the shell thickness increases to varying degrees. Compared with the bare Fe3O4 nanoparticles, the particle dispersion properties of FS and HMFS-NH2 are improved to varying degrees.

[0040] Furthermore, the Zeta potential of bare Fe3O4 nanoparticles, FS and HMFS-NH2 was measured respectively, and the relevant tests were carried out under the same conditions. Zeta potential can indirectly reflect the success of amino-modified monomer grafting, and can also be used to evaluate the particle dispersion performance. The Zeta potential measurement results are shown in Figure 2. Figure 4 As shown in the figure, before amino modification, the Zeta potential of the bare Fe3O4 nanoparticles and FS surface was negative. When the amino group was successfully grafted onto the material surface, the Zeta potential became positive.

[0041] S2, based on the amino-modified Fe3O4-SiO2 magnetic composite carrier, adding cellulase to obtain a magnetic immobilized enzyme buffer;

[0042] The specific process of step S2 is:

[0043] S21, place the amino-modified Fe3O4-SiO2 magnetic composite carrier in a 50 ml conical flask, add purified water, shake and sonicate for 10 seconds, add glutaraldehyde by volume to make the final concentration of the solution reach 5%, and activate at 30°C and 180 r / min for 2 hours;

[0044] S22, after activation, wash with ultrapure water and pH 4.8 citric acid buffer, then add pH 4.8 citric acid buffer and cellulase, and immobilize at 20°C and 180 r / min for 3 h;

[0045] S23, after the immobilization reaction is completed, the cellulase is separated by a magnet and repeatedly washed with a pH 4.8 citric acid buffer until no protein is detected in the supernatant, thereby obtaining an immobilized cellulase as a cross-linked composite carrier;

[0046] S24, adding a citric acid buffer solution of pH 4.8 to obtain a magnetic immobilized enzyme buffer solution, and storing it in a refrigerator at 4°C for later use;

[0047] The activity test of the obtained magnetic immobilized enzyme was carried out:

[0048] The activity of free and magnetically immobilized enzymes was measured at 540 nm using the CMCA-DNS method. The specific activity of free or immobilized cellulase is defined as: 1g of solid enzyme (or liquid enzyme) hydrolyzes the substrate CMC-Na at 50°C and a certain pH for 1h to produce reducing sugar equivalent to 1mg of glucose, which is 1 enzyme activity unit, expressed as u / mL or u / g. The enzyme activity measured by the CMCA-DNS method can be converted to the international enzyme activity OU / ML or IU by multiplying it by 0.093;

[0049] Determination of magnetic immobilized enzyme activity: Take 4 25mL test tubes, one of which is used as a blank (1 tube), and the other is used as the sample to be tested, and make 3 tubes in parallel. Add 2.0mL CMC-Na solution to the 4 test tubes, add 0.5mL of magnetic immobilized enzyme diluted a certain multiple to the 3 test tubes of the sample group to be tested, and add 0.5mL of citric acid buffer solution with pH 4.8 to the blank tube. Then place it in a 50℃ constant temperature water bath to react for 30min. After the reaction is completed, adsorb the magnetic immobilized enzyme with a magnet, take the supernatant, quickly add 3.0mL of DNS reagent, and put the 4 test tubes into a boiling water bath at the same time and heat for 10min. After the reaction, take it out, quickly cool it to room temperature with running water, and add ultrapure water to make it 25mL. Use the blank tube to zero the instrument and measure the absorbance at a wavelength of 540nm.

[0050] The enzyme activity of magnetically immobilized enzyme was calculated using the following formula:

[0051]

[0052] Where: X1 is the enzyme activity of cellulase u / mL or u / g; A is the amount of reducing sugar (mg) calculated from the absorbance on the standard curve; is converted into 1 ml enzyme solution; n is the dilution multiple of the enzyme sample; 2 is the time conversion factor;

[0053] The substances added to different test tubes are shown in Table 1.

[0054] Table 1: Substances added to different test tubes

[0055]

[0056]

[0057] According to GA concentration of 5%, activation for 2h, 2mg cellulase, immobilization temperature of 20℃, and immobilization time of 3h, the enzyme activity of immobilized cellulase was measured to be 2457u / g, which is higher and more stable than the enzyme activity of free enzyme; the absorbance of different glucose concentrations is as follows Figure 5 shown.

[0058] S3, placing the ganoderma lucidum fruiting body into a magnetic immobilized enzyme buffer for enzymolysis, extraction and filtration to obtain the effective components of ganoderma lucidum, and recovering the magnetic immobilized enzyme for repeated use;

[0059] The specific process of step S3 is:

[0060] S31, weigh the Ganoderma lucidum fruiting body and place it in a 500 mL distillation flask, add the magnetic immobilized enzyme buffer, and perform enzymolysis in a 37° C. constant temperature water bath for 2 h;

[0061] S32, after the enzymatic hydrolysis is completed, the magnetic immobilized enzyme is separated by adsorption with a magnet, taken out and cooled, and ethanol is added, and refluxed in a water bath at 80°C for 3h;

[0062] S33, after the extraction is completed, the extraction is performed, and the filtrate is the effective component extracted from Ganoderma lucidum, and the magnetic immobilized enzyme is recovered through a magnetic field for repeated use;

[0063] In step S31, the ganoderma lucidum fruiting bodies are pre-treated by removing impurities and crushing before enzymatic hydrolysis to obtain ganoderma lucidum particles suitable for enzymatic hydrolysis.

[0064] Enzyme activity test of the magnetic immobilized enzyme of the present invention for repeated use:

[0065] Place the Ganoderma lucidum fruiting body in a 500 mL distillation flask, add the magnetic immobilized enzyme buffer, and perform enzymolysis in a 37°C constant temperature water bath for 2 hours. After the enzymolysis is completed, separate the magnetic immobilized enzyme with a magnet, remove it and cool it. Add the Ganoderma lucidum fruiting body and the recovered magnetic immobilized enzyme again, repeat the above operation six times to measure the enzyme activity, and the test results are as follows: Figure 6 shown.

[0066] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for extracting Ganoderma lucidum based on a reusable magnetic enzyme, characterized in that: The following steps are involved: S1, preparing nano-scale amino-modified Fe3O4-SiO2 magnetic composite carrier; S2, based on the amino-modified Fe3O4-SiO2 magnetic composite carrier, adding cellulase to obtain a magnetic immobilized enzyme buffer; S3, placing the ganoderma lucidum fruiting body into a magnetic immobilized enzyme buffer for enzymolysis, extraction and filtration to obtain the effective components of ganoderma lucidum, and recovering the magnetic immobilized enzyme for repeated use.

2. A method for extracting Ganoderma lucidum based on reusable magnetic enzyme according to claim 1, characterized in that: The specific process of step S1 is: S11, weigh 2mmol of triacetylacetonate iron, add 10ml of benzyl alcohol to a high-pressure reactor, add 2ml of oleylamine and 2ml of oleic acid in sequence for ultrasonic dissolution; after mixing evenly, react at 200°C in a muffle furnace for 10h, pour the black solution obtained after the reaction into a beaker, and remove the supernatant by magnet adsorption; then redisperse the black precipitate in cyclohexane to obtain a Fe3O4 cyclohexane solution; S12, weigh 5 ml of Trinaton X-100, 25 ml of cyclohexane and 30 mg of Fe3O4 cyclohexane solution and dissolve them in a three-necked flask by ultrasonic; then add 6 ml of n-hexanol and dissolve them in ultrasonic for 10 minutes until they are evenly dispersed; S13, transfer the three-necked flask to a mechanical stirrer, add 2 ml of H2O and 2 ml of ammonia water dropwise during stirring to form a stable microemulsion, and stir at room temperature for 1 hour; then slowly add 3 ml of TEOS, react at room temperature for 24 hours, centrifuge at 12000 rpm for 10 minutes, collect the precipitate, wash it with ethanol and distilled water for several times, and disperse it in 10 ml of distilled water to obtain a HMFSs aqueous solution; S14. Centrifuge the HMFSs aqueous solution to remove the supernatant and wash it twice with ethanol. Transfer it to a three-necked flask and sonicate for 15 minutes. Add 2 ml of distilled water. Add 2 ml of ammonia water under stirring and continue stirring for 30 minutes. Add 1 ml of APTES at 80°C and react for 2 hours. After the reaction, centrifuge at 12000 rpm for 10 minutes to collect the precipitate, wash it with ethanol and distilled water for 3 times each, and dry it in vacuum at room temperature for 24 hours to obtain an amino-modified Fe3O4-SiO2 magnetic composite carrier containing amino functional groups.

3. The method for extracting Ganoderma lucidum based on reusable magnetic enzyme according to claim 1, characterized in that: The specific process of step S2 is: S21, place the amino-modified Fe3O4-SiO2 magnetic composite carrier in a 50 ml conical flask, add purified water, shake and sonicate for 10 seconds, add glutaraldehyde by volume to make the final concentration of the solution reach 5%, and activate at 30°C and 180 r / min for 2 hours; S22, after activation, wash with ultrapure water and pH 4.8 citric acid buffer, then add pH 4.8 citric acid buffer and cellulase, and immobilize at 20°C and 180 r / min for 3 h; S23, after the immobilization reaction is completed, the cellulase is separated by a magnet and repeatedly washed with a pH 4.8 citric acid buffer until no protein is detected in the supernatant, thereby obtaining an immobilized cellulase as a cross-linked composite carrier; S24, adding a citric acid buffer solution of pH 4.8 to obtain a magnetic immobilized enzyme buffer solution, and storing it in a 4°C refrigerator for later use.

4. The method for extracting Ganoderma lucidum based on a reusable magnetic enzyme according to claim 1, characterized in that: The specific process of step S3 is: S31, weigh the Ganoderma lucidum fruiting body and place it in a 500 mL distillation flask, add the magnetic immobilized enzyme buffer, and perform enzymolysis in a 37° C. constant temperature water bath for 2 h; S32, after the enzymatic hydrolysis is completed, the magnetic immobilized enzyme is separated by adsorption with a magnet, taken out and cooled, and ethanol is added, and refluxed in a water bath at 80°C for 3h; S33, after the extraction is completed, the filtrate is filtered, and the filtrate is the effective component extracted from Ganoderma lucidum. The magnetic immobilized enzyme is recovered through the magnetic field for repeated use.

5. The method for extracting Ganoderma lucidum based on reusable magnetic enzyme according to claim 4, characterized in that: In step S31, the ganoderma lucidum fruiting bodies are pre-treated by removing impurities and crushing before enzymatic hydrolysis to obtain ganoderma lucidum particles suitable for enzymatic hydrolysis.

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