Method for extracting lignin from traditional Chinese medicine residues

Abstract

The invention relates to the technical field of lignin extraction, and provides a method for extracting lignin from traditional Chinese medicine residues. Comprising the following steps: medicine residue crushing, medicine residue dissolving, residual enzyme inactivation, enzymolysis and oscillation reaction, lignin extraction with a choline chloride solution, ultrasonic reaction, centrifugal extraction of lignin-containing liquid, lignin precipitation, and washing and drying of the precipitate. By means of the technical scheme, the problems that an existing system for extracting lignin from traditional Chinese medicine residues is complex, and the lignin yield is low are solved.

Description

Technical Field

[0001] The invention relates to the technical field of lignin extraction, and in particular to a method for extracting lignin from traditional Chinese medicine residues. Background Art

[0002] Lignin is a natural biodegradable material, mainly composed of cellobiose, and is widely used in the industrial field. It is used in the textile industry to produce environmentally friendly materials and can replace traditional polymer fibers. Lignin is also used to make biodegradable packaging materials, food additives and new composite materials. Its renewability and environmental friendliness make it widely used in the environmental protection field and become a key material to promote sustainable development.

[0003] Traditional Chinese medicine residues are rich in lignin, and their extraction and utilization have important scientific and economic value. First, from a scientific point of view, the lignin in traditional Chinese medicine residues can be used as a natural biodegradable material with good renewability and stability, which can provide a new resource transformation pathway for the industry. Secondly, from an environmental protection point of view, traditional Chinese medicine residues usually contain harmful substances such as heavy metals and heavy metal salts. In the process of extracting lignin, it can be converted into a more easily handled biodegradable form, thereby reducing pollution to the environment. In addition, the extraction of lignin can also provide potential raw materials for industrial applications, promoting green industry and sustainable development. Therefore, the extraction of lignin from traditional Chinese medicine residues not only has important academic research significance, but also has broad social value.

[0004] However, although lignin can be extracted from the existing traditional Chinese medicine residues, the extraction equipment is numerous, the system is complex, the maintenance cost is high, and the lignin yield is low.

[0005] Therefore, a new method for extracting lignin is urgently needed. Summary of the invention

[0006] The invention provides a method for extracting lignin from traditional Chinese medicine residues, which solves the problems of complex system and low lignin yield in the existing extraction of lignin from traditional Chinese medicine residues.

[0007] The technical solution of the present invention is as follows: A method for extracting lignin from traditional Chinese medicine residues, comprising the following steps:

[0008] Step 1: Dry the Chinese medicine residue to be used, crush it into 50-100 mesh after drying, and weigh it after crushing;

[0009] Step 2: Dissolve the crushed Chinese medicine residues into pure water, dissolving 100g of Chinese medicine residues into each liter of pure water;

[0010] Step 3: Adjust the pH value to 4.5-6.0 and heat to 90-100°C to inactivate the residual enzyme for 10-20 minutes;

[0011] Step 4: Add the complex enzyme and perform an oscillation reaction. The conditions required for the oscillation reaction are: a temperature of 30-45°C, a stirring speed of 150 rpm, and a time of 4-5 hours; wherein, 0.5 ml of the complex enzyme solution corresponds to 1 g of the Chinese medicine residue;

[0012] Step 5: After the oscillation reaction is completed, the mixture is heated to 75-80°C to inactivate the enzyme for 10-15 minutes; then centrifuged to obtain a separation liquid and a solid residue for 10-15 minutes at a centrifugal speed of 8000-8500 rpm;

[0013] Step 6: adding choline chloride solution into the separation liquid, the mass ratio of the separation liquid to the choline chloride solution is 1:15, and the concentration of the choline chloride solution is 0.2%;

[0014] Step 7, adding the mixture in step 6 into the ultrasonic reactor, intermittently starting the ultrasonic reactor, the intermittent time is 1min, the ultrasonic working time is 2min, the reaction time is 1.5-2h, the reaction temperature is 90-95°C, and the working power of the ultrasonic reactor is 180-200W;

[0015] Step 8: Place the reaction mixture into a centrifuge for 10-15 min at a centrifugal speed of 10000-11000 rpm;

[0016] Step 9: After centrifugation, take the upper solution, add deionized water to the upper solution, and take out the precipitate after standing for 30-40 minutes;

[0017] Step 10: Wash the precipitate in step 9 with 70% ethanol and obtain lignin powder after drying.

[0018] As a further technical solution, the complex enzyme comprises, by weight: 30-50 parts of lignin peroxidase, 20-35 parts of laccase, 25-55 parts of cellulase, 10-15 parts of glycerophospholipids, and 5-15 parts of chitosan.

[0019] As a further technical solution, the complex enzyme comprises, by weight: 40 parts of lignin peroxidase, 25 parts of laccase, 35 parts of cellulase, 13 parts of glycerophospholipids, and 10 parts of chitosan.

[0020] As a further technical solution, in step 2, stirring is performed while the Chinese medicine residue is dissolved.

[0021] As a further technical solution, in step three, a citric acid buffer solution is used to adjust the pH value.

[0022] As a further technical solution, in step six, the solid residue is washed with pure water and then put into the choline chloride solution.

[0023] As a further technical solution, in step nine, the ratio of the chaotropic liquid to deionized water is 1:2.

[0024] As a further technical solution, in step ten, the precipitate is washed three times with ethanol and then dried.

[0025] As a further technical solution, in step ten, the washed precipitate is treated by hot air drying.

[0026] As a further technical solution, in step five, the solid residue needs to be rendered harmless before being discharged.

[0027] The method for extracting lignin from traditional Chinese medicine residues provided by the present invention has the following beneficial effects:

[0028] 1. Using conventional equipment, adjusting parameters and the amount of reactants to extract lignin, a large amount of lignin was obtained at a lower cost;

[0029] Second, the new compound enzyme is used to extract the lignin in the drug residue, which not only quickly degrades the lignin, but also ensures the stability and activity of the compound enzyme during the degradation process, and prevents the compound enzyme from becoming ineffective when entering the liquid environment;

[0030] 3. The presence of liposome-chitosan complex system in the complex enzyme can not only further enhance the stability and activity of the enzyme, but also provide an immobilized carrier and protection for the enzyme molecules;

[0031] 4. Extracting lignin from Chinese medicine residues can not only realize the recycling of resources and increase the high added value of Chinese medicine, but also reduce the pressure of waste disposal and reduce pollution to the environment. DETAILED DESCRIPTION

[0032] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] The present invention provides a method for extracting lignin from traditional Chinese medicine residues, comprising the following steps:

[0034] Step 1: Dry the Chinese medicinal residues to be used, crush them into 50-100 meshes after drying, and weigh them after crushing.

[0035] In this step, first take a certain weight of Chinese medicine residue, preferably the Chinese medicine residue that is naturally dried after boiling the medicine, and dry it naturally for about a week. Then put the Chinese medicine residue into an oven to dry. After drying, take out the Chinese medicine residue and put it into a grinder for crushing. The crushed Chinese medicine residue is between 50-100 meshes. Collect the Chinese medicine residue sieved out by the grinder and then weigh it. Avoid impurities when weighing, and record the weight after weighing.

[0036] Step 2: Dissolve the crushed Chinese medicine residues fully in pure water, dissolving 100g of Chinese medicine residues in every liter of pure water.

[0037] In this step, take the crushed Chinese herbal medicine residue and pure water, with 100g of Chinese herbal medicine residue corresponding to each liter of pure water. In the process of dissolving the Chinese herbal medicine residue into pure water, stirring is required. Stirring can not only speed up the dissolution rate of the Chinese herbal medicine residue, but also prevent the residue from forming "lump"-like substances when entering the water.

[0038] Step 3: Adjust the pH value to 4.5-6.0 and heat to 90-100°C to inactivate the residual enzyme for 10-20 minutes.

[0039] In this step, there will be certain enzymes in the Chinese medicine residues after crushing. These enzymes need to be inactivated, otherwise they will affect the activity of the complex enzyme in the next step. The pH value of the solution is adjusted to 4.5-6.0 using citric acid buffer, and then heated to 90-100°C. Maintaining this state for 10-20 minutes can complete the activity of the enzymes remaining in the Chinese medicine residues.

[0040] Step 4: Add complex enzyme and perform oscillation reaction. The conditions required for the oscillation reaction are: temperature of 30-45°C, stirring speed of 150rpm, and time of 4-5h; wherein, 0.5ml of complex enzyme solution corresponds to every 1g of Chinese medicine residue.

[0041] In this step, a compound enzyme is added to the mixed solution, with 0.5 ml of compound enzyme solution corresponding to 1 g of Chinese medicine residue; after the corresponding amount of compound enzyme is added, the mixed solution is subjected to an oscillation reaction, the temperature of the oscillation reaction is 30-45° C., and the speed of the oscillator during the oscillation reaction is 150 rpm. The oscillation reaction can accelerate the decomposition of the cell wall by the compound enzyme, thereby causing the plant cells to precipitate lignin.

[0042] The components of the complex enzyme include lignin peroxidase, laccase, cellulase, glycerophospholipid and chitosan. Lignin peroxidase can efficiently oxidize and degrade non-phenolic structures in lignin; laccase can oxidize phenolic lignin and produce free radicals to promote the degradation of lignin; cellulase can degrade cellulose, thereby causing cells to release lignin. Glycerol phospholipids can form micelles or liposomes, which can encapsulate enzyme molecules and protect them from the influence of the external environment, thereby improving the stability of the complex enzyme; in addition, glycerophospholipids can also be used as surfactants to improve the contact efficiency between drug residues and enzymes, and increase the catalytic activity of enzymes in the degradation of lignin. Chitosan has high biocompatibility and degradability. As an enzyme immobilization carrier, enzyme molecules are fixed on the surface of chitosan by physical adsorption, thereby improving the stability of the enzyme. Moreover, since chitosan is usually a porous structure, when enzyme molecules adhere to the surface of chitosan, the contact area between enzyme molecules and lignin can be increased, thereby improving the catalytic efficiency of the enzyme. Glycerol phospholipids and chitosan can work synergistically to form a stable composite system (such as liposome-chitosan complex), further enhancing the stability and activity of the enzyme. Glycerol phospholipids improve the interfacial properties, and chitosan provides an immobilization carrier and protection. The combination of the two can significantly improve the performance of the composite enzyme.

[0043] Step 5: After the oscillation reaction is completed, heat to 75-80°C to inactivate the enzyme for 10-15 minutes; then centrifuge to obtain a separation liquid and a solid residue, the centrifugation time is 10-15 minutes, and the centrifugal speed is 8000-8500rpm.

[0044] After the oscillation reaction is completed, the liquid in the oscillator is removed and then heated to 75-80°C to inactivate the enzyme in the liquid. The inactivation is mainly to remove the complex enzyme. After the complex enzyme is extinguished, the liquid is centrifuged for 10-15 minutes at a speed of 8000-8500rpm. After centrifugation in the centrifuge, the mixed liquid is separated into a separation liquid and a solid residue. At this time, the lignin is dissolved in the separation liquid, and the solid residue needs to be harmlessly treated before it can be thrown away.

[0045] Step 6: Add the choline chloride solution into the separation liquid, the mass ratio of the separation liquid to the choline chloride solution is 1:15, and the concentration of the choline chloride solution is 0.2%.

[0046] In this step, choline chloride is a component of a deep eutectic solvent (DES), which is usually mixed with a hydrogen bond donor (such as urea, lactic acid, organic acid, etc.) to form a deep eutectic solvent system. It has strong dissolving power, high polarity and adjustability. After mixing with the separation liquid, the choline chloride solution can destroy the chemical bonds (such as ether bonds, ester bonds, etc.) between lignin and cellulose and hemicellulose, thereby releasing lignin while ensuring the integrity of lignin. In addition, due to the low concentration of choline chloride, a more complete lignin structure can be obtained.

[0047] Step 7: Add the mixture in step 6 into the ultrasonic reactor, start the ultrasonic reactor intermittently, the intermittent time is 1 min, the ultrasonic working time is 2 min, the reaction time is 1.5-2 h, the reaction temperature is 90-95° C., and the working power of the ultrasonic reactor is 180-200 W.

[0048] In this step, the cavitation effect generated by ultrasound can significantly improve the mixing and mass transfer efficiency between liquids, promote the contact between choline chloride and lignin, and accelerate the dissolution and separation of lignin. In addition, intermittent ultrasonic reaction can not only avoid local overheating caused by long-term ultrasound, protect heat-sensitive components (such as certain lignin derivatives) from being destroyed, but also facilitate real-time monitoring of the reaction process and sampling and analysis of the extraction effect.

[0049] Step 8: Place the reaction mixture into a centrifuge for 10-15 minutes at a centrifugal speed of 10000-11000 rpm.

[0050] In this step, after the choline chloride solution and the separation liquid are fully mixed and reacted, the mixture enters a centrifuge for separation. Since lignin is dissolved in the liquid and impurities such as complex enzymes are present in the mixture, centrifugation can be used to separate lignin from these impurities.

[0051] Step 9. After centrifugation, take the upper solution, then add deionized water to the upper solution, and take out the precipitate after standing for 30-40 minutes.

[0052] In this step, since the upper liquid contains a large amount of lignin, after adding deionized water, the solubility of lignin will be greatly reduced due to the hydrophobicity of lignin itself, and it will precipitate and form a precipitate in the upper liquid. After standing for a period of time, you can further wait for more precipitation. When standing, it is best to choose room temperature, that is, the temperature is between 25-27°C.

[0053] Step 10: Wash the precipitate in step 9 with 70% ethanol and obtain lignin powder after drying.

[0054] In this step, washing the precipitate with ethanol can not only remove impurities, but also reduce the loss of lignin, because ethanol is a polar organic solvent, which will not cause a large amount of lignin to dissolve during the washing process, and the residual ethanol will evaporate quickly during the drying process, leaving a lignin powder with higher purity. The volatilization of ethanol can be further improved by using the form of thermal drying.

[0055] The method for extracting lignin from traditional Chinese medicine residues provided by the present invention has the following beneficial effects:

[0056] 1. Using conventional equipment, adjusting parameters and the amount of reactants to extract lignin, a large amount of lignin was obtained at a lower cost;

[0057] Second, the new compound enzyme is used to extract the lignin in the drug residue, which not only quickly degrades the lignin, but also ensures the stability and activity of the compound enzyme during the degradation process, and prevents the compound enzyme from becoming ineffective when entering the liquid environment;

[0058] 3. The presence of liposome-chitosan complex system in the complex enzyme can not only further enhance the stability and activity of the enzyme, but also provide an immobilized carrier and protection for the enzyme molecules;

[0059] 4. Extracting lignin from Chinese medicine residues can not only realize the recycling of resources and increase the high added value of Chinese medicine, but also reduce the pressure of waste disposal and reduce pollution to the environment.

[0060] In order to verify that the composite enzyme involved in the present invention can efficiently degrade lignin in traditional Chinese medicine residues, 3 examples and 4 comparative examples are provided.

[0061] Embodiment 1,

[0062] The composite enzyme involved in the present invention comprises, by weight: 40 parts of lignin peroxidase, 25 parts of laccase, 35 parts of cellulase, 13 parts of glycerophospholipid and 10 parts of chitosan.

[0063] The preparation method of the complex enzyme involved in the present invention comprises the following steps:

[0064] Step S1, using a balance to accurately weigh 40 parts of lignin peroxidase, 25 parts of laccase, 35 parts of cellulase, 13 parts of glycerophospholipids, and 10 parts of chitosan;

[0065] Step S2, adding chitosan to an appropriate amount of deionized water, stirring until completely dissolved, and heating during stirring, the heating temperature not exceeding 40° C.;

[0066] Step S3, adding glycerophospholipids to the mixed solution in step S2, stirring until completely dissolved, and heating during stirring, the heating temperature not exceeding 40° C.;

[0067] Step S4, adding lignin peroxidase, laccase and cellulase into the mixed solution in step S3 in sequence, stirring until completely dissolved, and controlling the pH value of the solution to 5.0-7.0 during the dissolution process;

[0068] Step S5, placing the mixed solution in step S4 on a magnetic stirrer and stirring for 0.5-1h at a stirring speed of 100-300rpm;

[0069] Step S6: After sufficient stirring, the complex enzyme solution is filled into a sterile container, and then placed in a refrigerator for refrigerated storage at a storage temperature of 4-5°C.

[0070] Embodiment 2,

[0071] The composite enzyme involved in the present invention comprises, by weight: 30 parts of lignin peroxidase, 20 parts of laccase, 25 parts of cellulase, 10 parts of glycerophospholipid and 5 parts of chitosan.

[0072] The preparation method of the complex enzyme in this example is the same as the method disclosed in Example 1.

[0073] Embodiment 3,

[0074] The composite enzyme involved in the present invention comprises, by weight: 50 parts of lignin peroxidase, 35 parts of laccase, 55 parts of cellulase, 15 parts of glycerophospholipid and 15 parts of chitosan.

[0075] The preparation method of the complex enzyme in this example is the same as the method disclosed in Example 1.

[0076] Comparative Example 1

[0077] The composite enzyme involved in the present invention comprises, by weight, 25 parts of laccase, 35 parts of cellulase, 13 parts of glycerophospholipid and 10 parts of chitosan.

[0078] Comparative Example 2

[0079] The complex enzyme involved in the present invention comprises, by weight: 40 parts of lignin peroxidase, 35 parts of cellulase, 13 parts of glycerophospholipids and 10 parts of chitosan.

[0080] Comparative Example 3

[0081] The composite enzyme involved in the present invention comprises, by weight: 40 parts of lignin peroxidase, 25 parts of laccase, 13 parts of glycerophospholipids and 10 parts of chitosan.

[0082] Comparative Example 4

[0083] The composite enzyme involved in the present invention comprises, by weight: 40 parts of lignin peroxidase, 25 parts of laccase, 35 parts of cellulase and 10 parts of chitosan.

[0084] Comparative Example 5

[0085] The composite enzyme involved in the present invention comprises, by weight: 40 parts of lignin peroxidase, 25 parts of laccase, 35 parts of cellulase and 13 parts of glycerophospholipid.

[0086] Take a portion of Chinese medicine residue, and then use steps 1 to 3 provided by the present invention to obtain a residue mixture, and then add the complex enzyme solution in Examples 1-3 and Comparative Examples 1-5 in sequence. Since the pH value is adjusted to 4.5-6.0 in step 3, the lignin extracted from the residue is acid-soluble lignin, and the following table is obtained according to the standard NB / T 34057.5-2017.

[0087] Acid-soluble lignin content of standard sample (%) Example 1 7.8 Example 2 6.6 Example 3 6.9 Comparative Example 1 2.8 Comparative Example 2 3.3 Comparative Example 3 2.6 Comparative Example 4 2.9 Comparative Example 5 3.5

[0088] By comparison of Examples 1-3, it can be seen that a composite enzyme with a suitable ratio can extract lignin more efficiently; by comparison of Example 1, Comparative Example 1 and Comparative Example 2, it can be seen that both lignin peroxidase and laccase can improve the degradation efficiency and degradation amount of lignin. If lignin peroxidase or laccase is lacking, a composite system cannot be formed between lignin peroxidase and laccase, and the connection structure of lignin and cellulose cannot be degraded; by Example 1, Comparative Example 4 and Comparative Example 5, it can be seen that glycerophospholipids can also be used as surfactants to improve the contact efficiency between drug residues and enzymes, and increase the catalytic activity of enzymes in the degradation of lignin; chitosan has high biocompatibility and degradability. As an immobilized carrier of the enzyme, the enzyme molecules are fixed on the surface of chitosan by physical adsorption, thereby improving the stability of the enzyme.

[0089] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for extracting lignin from traditional Chinese medicine residues, characterized in that: The following steps are involved: Step 1: Dry the Chinese medicine residue to be used, crush it into 50-100 mesh after drying, and weigh it after crushing; Step 2: Dissolve the crushed Chinese medicine residues into pure water, dissolving 100g of Chinese medicine residues into each liter of pure water; Step 3: Adjust the pH value to 4.5-6.0 and heat to 90-100°C to inactivate the residual enzyme for 10-20 minutes; Step 4: Add the complex enzyme solution and perform an oscillation reaction. The conditions required for the oscillation reaction are: a temperature of 30-45°C, a stirring speed of 150 rpm, and a time of 4-5 hours; wherein, 0.5 ml of the complex enzyme solution corresponds to 1 g of the Chinese medicinal residue; Step 5: After the oscillation reaction is completed, the mixture is heated to 75-80°C to inactivate the enzyme for 10-15 minutes; then centrifuged to obtain a separation liquid and a solid residue for 10-15 minutes at a centrifugal speed of 8000-8500 rpm; Step 6: adding choline chloride solution into the separation liquid, the mass ratio of the separation liquid to the choline chloride solution is 1:15, and the concentration of the choline chloride solution is 0.2%; Step 7, adding the mixture in step 6 into the ultrasonic reactor, intermittently starting the ultrasonic reactor, the intermittent time is 1min, the ultrasonic working time is 2min, the reaction time is 1.5-2h, the reaction temperature is 90-95°C, and the working power of the ultrasonic reactor is 180-200W; Step 8: Place the reaction mixture into a centrifuge for 10-15 min at a centrifugal speed of 10000-11000 rpm; Step 9: After centrifugation, take the upper solution, add deionized water to the upper solution, and take out the precipitate after standing for 30-40 minutes; Step 10: Wash the precipitate in step 9 with 70% ethanol and obtain lignin powder after drying.

2. A method for extracting lignin from traditional Chinese medicine residues according to claim 1, characterized in that: The complex enzyme comprises, by weight: 30-50 parts of lignin peroxidase, 20-35 parts of laccase, 25-55 parts of cellulase, 10-15 parts of glycerophospholipids and 5-15 parts of chitosan.

3. A method for extracting lignin from traditional Chinese medicine residues according to claim 2, characterized in that: The complex enzyme comprises, by weight: 40 parts of lignin peroxidase, 25 parts of laccase, 35 parts of cellulase, 13 parts of glycerophospholipids and 10 parts of chitosan.

4. A method for extracting lignin from traditional Chinese medicine residues according to any one of claims 1 or 2, characterized in that: In the step 2, stirring is performed while the Chinese medicinal residues are dissolved.

5. A method for extracting lignin from traditional Chinese medicine residues according to any one of claims 1 or 2, characterized in that: In the step three, a citric acid buffer solution is used to adjust the pH value.

6. A method for extracting lignin from Chinese medicinal residues according to any one of claims 1 or 2, characterized in that: In step six, the solid residue is washed with pure water and then put into the choline chloride solution.

7. A method for extracting lignin from Chinese medicinal residues according to any one of claims 1 or 2, characterized in that: In step nine, the ratio of the chaotropic liquid to deionized water is 1:

2.

8. A method for extracting lignin from Chinese medicinal residues according to any one of claims 1 or 2, characterized in that: In the step 10, the precipitate is washed three times with ethanol and then dried.

9. A method for extracting lignin from Chinese medicinal residues according to any one of claims 1 or 2, characterized in that: In the step 10, the washed precipitate is treated by hot air drying.

10. A method for extracting lignin from traditional Chinese medicine residues according to any one of claims 1 or 2, characterized in that: In step five, the solid residue needs to be rendered harmless before being discharged.