Method for extracting lignin from litter

Abstract

The invention belongs to the technical field of lignin extraction, and particularly relates to a method for extracting lignin from litter. The method comprises the following steps: mixing litter and a sodium chloride solution, and refrigerating to obtain a pretreated raw material; sequentially performing first dipping treatment, second dipping treatment, third dipping treatment and fourth dipping treatment on the pretreated raw material in a first organic solvent, a second organic solvent, a third organic solvent and a fourth organic solvent to obtain an intermediate extract; carrying out starch removal treatment on the intermediate extract to obtain lignin; the first organic solvent is ethanol; the second organic solvent is acetone or ethyl acetate; the third organic solvent is a mixed solvent of methane chloride and methanol; the fourth organic solvent is acetone or ethyl acetate. The method provided by the invention can be used for effectively separating and extracting lignin, and has a relatively high extraction rate. The adopted raw materials are low in cost, and the method is simple and suitable for industrial production.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of lignin extraction, and particularly relates to a method for extracting lignin from litter. BACKGROUND

[0002] As a renewable resource, lignin has the advantages of rich reserves, low cost, high carbon, high aromaticity, biodegradability and low density. Adding lignin to soil can improve soil nutrient utilization efficiency, improve soil aggregate structure, and improve soil enzyme activity, thereby improving degraded soil and complexing and leaching pollutants in soil, or inducing microbial degradation of organic pollutants, thereby repairing contaminated soil, and having high potential value and application prospect in soil improvement and repair.

[0003] Existing lignin extraction methods mainly include sulfite method / alkali method, organic solvent method and ionic liquid extraction method. The sulfite method / alkali method has the problems of serious pollution and structural damage. The organic solvent method has the defects of low extraction rate, difficult solvent recovery and high cost. The ionic liquid extraction method has the problems of high reagent price and complex purification steps.

[0004] Therefore, there is a need for a lignin extraction method with low cost and high extraction efficiency. SUMMARY

[0005] The application aims to provide a method for extracting lignin from litter, which has low cost and high lignin extraction efficiency.

[0006] To achieve the above-mentioned purpose, the application provides the following technical scheme:

[0007] The application provides a method for extracting lignin from litter, comprising the following steps:

[0008] Mixing litter and a sodium chloride solution, and performing cold storage treatment to obtain pretreated raw materials;

[0009] Performing first impregnation treatment, second impregnation treatment, third impregnation treatment and fourth impregnation treatment on the pretreated raw materials in a first organic solvent, a second organic solvent, a third organic solvent and a fourth organic solvent in sequence to obtain intermediate extract;

[0010] Performing starch removal treatment on the intermediate extract to obtain lignin;

[0011] The first organic solvent is ethanol;

[0012] The second organic solvent is acetone or ethyl acetate;

[0013] The third organic solvent is a mixed solvent of methane chlorides and methanol, and the methane chlorides are at least one of chloroform and dichloromethane.

[0014] The fourth organic solvent is acetone or ethyl acetate.

[0015] Preferably, the litter includes leaf litter; the leaf litter includes at least one of leaf litter of woody plants and leaf litter of herbaceous plants.

[0016] The woody plants include at least one of Populus euphratica and Populus canescens;

[0017] The herbaceous plants include at least one of Caragana jubata and Alhagi sparsifolia.

[0018] Preferably, before the mixing, the litter is dried and ground.

[0019] The concentration of the sodium chloride solution is 45-55 mmol / L; the ratio of the use amount of the litter to the sodium chloride solution is 1 g: 30-40 mL; the temperature of the refrigeration treatment is 4-8℃, and the time is 8-24 h.

[0020] Preferably, the first immersion treatment includes: mixing the pretreated litter and the first organic solvent, sequentially performing ultrasonic treatment and centrifugal treatment, and repeating the above process twice.

[0021] The volume concentration of the ethanol is 70-90%; the ratio of the use amount of the litter to the first organic solvent is 1 g: 30-40 mL.

[0022] The time of the ultrasonic treatment is 20-25 min.

[0023] The rotation speed of the centrifugal treatment is 3000-4000 rpm, the time is 8-15 min, and the temperature is 1-4℃.

[0024] Preferably, the second immersion treatment includes: mixing the product obtained by the first immersion treatment and the second organic solvent, sequentially performing ultrasonic treatment and centrifugal treatment.

[0025] The volume concentration of the acetone is 70-100%; the ratio of the use amount of the litter to the second organic solvent is 1 g: 30-40 mL.

[0026] The time of the ultrasonic treatment is 20-25 min.

[0027] The rotation speed of the centrifugal treatment is 3000-4000 rpm, the time is 8-15 min, and the temperature is 1-4℃.

[0028] Preferably, the third immersion treatment includes: mixing the product obtained by the second immersion treatment and the third organic solvent, sequentially performing ultrasonic treatment and centrifugal treatment.

[0029] The volume ratio of the methane chloroform and the methanol is 1-2:1; the use amount ratio of the litter and the third organic solvent is 1g:30-40mL;

[0030] The ultrasonic time is 20-25min;

[0031] The centrifugal rotation speed is 3000-4000rpm, the time is 8-15min, and the temperature is 1-4℃.

[0032] Preferably, the fourth dipping treatment comprises: mixing the product obtained by the third dipping treatment and the fourth organic solvent, and sequentially performing ultrasonic and centrifugal treatment;

[0033] The volume concentration of the acetone is 70-100%; the use amount ratio of the litter and the fourth organic solvent is 1g:30-40mL;

[0034] The ultrasonic time is 20-25min;

[0035] The centrifugal rotation speed is 3000-4000rpm, the time is 8-15min, and the temperature is 1-4℃.

[0036] Preferably, the starch removal treatment comprises: first mixing the intermediate extract and the Tris-acetic acid buffer, performing liquid phase reaction to obtain a product system;

[0037] Second mixing the Tris-acetic acid buffer and the amylase to obtain an enzyme preparation;

[0038] Third mixing the product system and the enzyme preparation to perform enzymolysis reaction.

[0039] Preferably, the concentration of the Tris-acetic acid buffer is 10-15mmol / L;

[0040] In the first mixing process, the use amount ratio of the litter and the Tris-acetic acid buffer is 1g:25-30mL;

[0041] The liquid phase reaction temperature is 80-100℃, and the time is 1-4h;

[0042] The amylase comprises Bacillus licheniformis alpha-amylase and amylase glucosidase;

[0043] In the enzyme preparation, the content of the Bacillus licheniformis alpha-amylase is 15-25 units / 5-6mL, and the content of the amylase glucosidase is 30-50 units / 5-6mL;

[0044] The volume ratio of the product system and the enzyme preparation is 5:1;

[0045] The enzymatic reaction is carried out at a temperature of 55-60℃ for 1.5-2.5h.

[0046] Preferably, after the enzymatic reaction, the obtained system is sequentially subjected to centrifugation, water washing and acetone washing.

[0047] The centrifugation is carried out at a speed of 3000-4000rpm for 8-15min at a temperature of 1-4℃.

[0048] The water washing process comprises mixing the obtained precipitate after centrifugation with pure water, sequentially performing ultrasonic treatment and centrifugation, and repeating the above steps twice.

[0049] The acetone washing process comprises mixing the product after water washing with acetone, sequentially performing ultrasonic treatment and centrifugation, and repeating the above steps twice.

[0050] The present application provides a method for extracting lignin from litter, comprising the following steps: mixing litter and sodium chloride solution, carrying out cold storage treatment to obtain pretreated raw material; sequentially carrying out first impregnation treatment, second impregnation treatment, third impregnation treatment and fourth impregnation treatment on the pretreated raw material in a first organic solvent, a second organic solvent, a third organic solvent and a fourth organic solvent to obtain intermediate extract; carrying out starch removal treatment on the intermediate extract to obtain lignin; the first organic solvent is ethanol; the second organic solvent is acetone or ethyl acetate; the third organic solvent is a mixed solvent of methane chlorides and methanol, and the methane chlorides are at least one of trichloromethane and dichloromethane; the fourth organic solvent is acetone or ethyl acetate. The method provided by the present application can effectively separate and extract lignin by gradually removing different polar impurities in different organic solvents, and has a high extraction rate. The raw material cost of the present application is low, the method is simple, and is suitable for industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 is the test process diagram provided by the present application;

[0052] Figure 2 is the H spectrum of lignin components of different species obtained by the examples and comparative examples of the present application;

[0053] Figure 3 is the HSQC spectrum of lignin components of different species obtained by the examples of the present application;

[0054] Figure 4 is the flowchart of the extraction method adopted by the examples of the present application. DETAILED DESCRIPTION

[0055] The application provides a method for extracting lignin from litter, comprising the following steps:

[0056] Mixing the litter and a sodium chloride solution, and performing refrigeration treatment to obtain pretreated raw materials;

[0057] Performing first, second, third and fourth impregnation treatments on the pretreated raw materials in a first, a second, a third and a fourth organic solvent in sequence to obtain intermediate extracts;

[0058] Performing starch removal treatment on the intermediate extracts to obtain lignin;

[0059] The first organic solvent is ethanol;

[0060] The second organic solvent is acetone or ethyl acetate;

[0061] The third organic solvent is a mixed solvent of methane chlorides and methanol, and the methane chlorides are at least one of trichloromethane and dichloromethane;

[0062] The fourth organic solvent is acetone or ethyl acetate.

[0063] The application mixes the litter and a sodium chloride solution, and performs refrigeration treatment to obtain pretreated raw materials.

[0064] In the application, the litter preferably comprises leaf litter; the leaf litter preferably comprises at least one of leaf litter of woody plants and leaf litter of herbaceous plants; the woody plants preferably comprise at least one of Populus euphratica and Populus pruinosa; and the herbaceous plants preferably comprise at least one of Hedysarum polybotrys and Alhagi sparsifolia. In specific embodiments of the application, the litter is leaf litter of Populus euphratica, leaf litter of Alhagi sparsifolia or leaf litter of Hedysarum polybotrys collected from the southern edge of the Taklimakan Desert.

[0065] In the application, before the mixing, the litter is preferably dried and ground; the drying and grinding process is not particularly limited in the application, and any process known to those skilled in the art can be used.

[0066] In the present application, the concentration of the sodium chloride solution is preferably 45-50 mmol / L, and further preferably 50 mmol / L; the ratio of the use amount of the litter and the sodium chloride solution is preferably 1 g: 30-40 mL. In the present application, the temperature of the refrigeration treatment is preferably 4-8℃, and the time is preferably 8-24 h. In the present application, after the refrigeration treatment, it is further preferred to centrifuge the obtained system; the rotation speed of the centrifugation is preferably 3000-4000 rpm, and further preferably 3480 rpm, the time is preferably 8-15 min, and further preferably 10 min; and the temperature is preferably 1-4℃. In the present application, the effects of the refrigeration treatment in the sodium chloride solution include: (1) destroying the cell osmotic pressure and promoting the release of lignin: the sodium chloride solution dehydrates the cells through osmotic action, destroys the cell membrane structure, and thus releases the lignin and other components in the cells, which is especially suitable for the pretreatment of plant cell walls, and helps to more efficiently separate lignin in the subsequent step, and at the same time, the refrigeration method can further promote the diffusion of water-soluble components; (2) removing water-soluble impurities: the supernatant removed after centrifugation may contain a large amount of water-soluble impurities (such as sugars, proteins, inorganic salts, etc.), which will interfere with the purification of lignin; (3) the addition of NaCl can promote the precipitation of part of the water-soluble impurities through salting-out effect, but more importantly, it helps to separate the lignin that is insoluble in salt solution; (4) promoting the precipitation or aggregation of lignin: lignin may aggregate in salt solution due to the charge shielding effect (Na + neutralizing the negative charge on the surface of lignin), forming larger particles, which is convenient for separation from the supernatant by centrifugation; after removing the supernatant, the remaining precipitate may contain lignin, cellulose, hemicellulose, etc., which is convenient for further extraction of lignin with organic solvents; (5) sodium chloride pretreatment can reduce the interference of impurities in subsequent solvent extraction.

[0067] After obtaining the pretreated raw material, the pretreated raw material is sequentially subjected to first, second, third and fourth impregnation treatments in a first, second, third and fourth organic solvent, respectively, to obtain an intermediate extract.

[0068] In the present application, the first organic solvent is ethanol. In the present application, the first impregnation treatment preferably comprises: mixing the pretreated raw material and the first organic solvent, sequentially performing ultrasonic and centrifugation, repeating the above process twice; the volume concentration of the ethanol is preferably 70-90%; the ratio of the use amount of the litter and the first organic solvent is preferably 1g: 30-40mL; the time of the ultrasonic is preferably 20-25min; the speed of the centrifugation is preferably 3000-4000rpm, further preferably 3480rpm, the time is preferably 8-15min, further preferably 10min; the temperature is preferably 1-4℃. In the present application, ethanol is a polar organic solvent, and after the first impregnation treatment, ethanol-soluble components (such as low-molecular-weight phenols, pigments, part of lipids, residual sugars) can be removed to reduce the interference in the subsequent steps; lignin has low solubility in ethanol and remains in the residue. At the same time, ultrasonic assistance can destroy the cell wall and improve the permeability of the solvent.

[0069] In the present application, the second organic solvent is acetone or ethyl acetate; the volume concentration of the acetone is preferably 70-100%. In the present application, the second impregnation treatment preferably comprises: mixing the product obtained by the first impregnation treatment and the second organic solvent, sequentially performing ultrasonic and centrifugation; the ratio of the use amount of the litter and the second organic solvent is preferably 1g: 30-40mL; the time of the ultrasonic is preferably 20-25min; the speed of the centrifugation is preferably 3000-4000rpm, further preferably 3480rpm, the time is preferably 10min, and the temperature is preferably 1-4℃. In the present application, the second organic solvent is a nonpolar solvent, and after the second impregnation treatment, lipids, pigments and residual organic solvent-soluble substances can be further removed, and ethanol is more effective in removing hydrophobic impurities; lignin is almost insoluble in the second organic solvent.

[0070] In the present application, the third organic solvent is a mixed solvent of methane chlorides and methanol, and the methane chlorides are at least one of trichloromethane and dichloromethane. In the present application, the volume ratio of the methane chlorides and methanol is preferably 1-2:1. In the present application, the third impregnation treatment preferably comprises mixing the product obtained by the second impregnation treatment and the third organic solvent, and sequentially performing ultrasonic treatment and centrifugation; the ratio of the use amount of the litter and the third organic solvent is preferably 1g:30-40mL; the time of the ultrasonic treatment is preferably 20-25min; the rotation speed of the centrifugation is preferably 3000-4000rpm, and further preferably 3480rpm, and the time is preferably 8-15min, and further preferably 10min; and the temperature is preferably 1-4℃. In the present application, the third organic solvent used is a mixed solvent of medium polarity, and after the third impregnation treatment, the liposoluble substances (such as waxes, resins, and part of membrane lipids) can be removed, the methane chlorides can destroy the membrane lipid bilayer, and the methanol can enhance the dissolution of the polar components. The third organic solvent is more efficient than a single solvent (such as pure trichloromethane) and is suitable for the extraction of stubborn lipids; and lignin is insoluble in the third organic solvent.

[0071] In the present application, the fourth organic solvent is acetone or ethyl acetate. In the present application, the volume concentration of the acetone is preferably 70-100%. In the present application, the fourth impregnation treatment preferably comprises mixing the product obtained by the third impregnation treatment and the fourth organic solvent, and sequentially performing ultrasonic treatment and centrifugation; the ratio of the use amount of the litter and the fourth organic solvent is preferably 1g:30-40mL; the time of the ultrasonic treatment is preferably 20-25min; the rotation speed of the centrifugation is preferably 3000-4000rpm, and further preferably 3480rpm, and the time is preferably 8-15min, and further preferably 10min; and the temperature is preferably 1-4℃. In the present application, after the fourth impregnation treatment, the residual water-soluble impurities can be removed, and the product is easy to dry.

[0072] After obtaining the intermediate extract, the present application removes starch from the intermediate extract to obtain lignin.

[0073] In the present application, the starch removal treatment preferably comprises: first mixing the intermediate extract and a Tris-acetic acid buffer, performing a liquid phase reaction to obtain a product system; second mixing the Tris-acetic acid buffer and amylase to obtain an enzyme preparation; and third mixing the product system and the enzyme preparation to perform an enzymatic reaction.

[0074] In the present application, the concentration of the Tris-acetic acid buffer is preferably 10-15 mmol / L, and the pH value of the Tris-acetic acid buffer is preferably 6. In the present application, the ratio of the use amount of the litter and the Tris-acetic acid buffer is preferably 1 g: 25-30 mL during the mixing of the litter and the Tris-acetic acid buffer; the temperature of the liquid phase reaction is preferably 80-100 ℃, and the time is preferably 1-4 h. In the present application, the liquid phase reaction with the Tris-acetic acid buffer can destroy the cross-linking of hemicellulose-lignin and further release lignin; at high temperature, hemicellulose is hydrolyzed, but lignin is still not dissolved; by controlling the pH value of the Tris-acetic acid buffer, the acidic condensation of lignin can be avoided; and by the above treatment, 10-30% of the hemicellulose can be removed.

[0075] In the present application, the amylase preferably includes Bacillus licheniformis alpha-amylase and amyloglucosidase (derived from Aspergillus niger); the content of the Bacillus licheniformis alpha-amylase in the enzyme preparation is preferably 15-25 units / 5-6 mL, and specifically can be 20 units / 5 mL; and the content of the amyloglucosidase is preferably 30-50 units / 5-6 mL, and specifically can be 40 units / 5 mL. In the present application, the volume ratio of the product system and the enzyme preparation is preferably 5:1; the temperature of the enzymatic reaction is preferably 55-60 ℃, and the time is preferably 1.5-2.5 h, and further preferably 2 h. In the present application, the residual starch can be degraded by enzyme treatment to avoid interfering with subsequent lignin analysis and further improve the purity of lignin; and the starch enzyme only acts on starch without affecting the structure of lignin.

[0076] In the present application, after the enzymatic reaction, the obtained system is further preferably sequentially subjected to centrifugation, water washing and acetone washing; the rotation speed of the centrifugation is preferably 3000-4000 rpm, and further preferably 3480 rpm, and the time is preferably 8-15 min, and further preferably 10 min; and the temperature is preferably 1-4 ℃.

[0077] In the present application, the process of water washing preferably includes mixing the precipitate obtained after centrifugation and pure water, sequentially performing ultrasonic and centrifugation, and repeating the above steps twice; and the ratio of the use amount of the litter and the pure water is preferably 1 g: 40 mL. In the present application, the time of the ultrasonic is preferably 20-25 min; the rotation speed of the centrifugation is preferably 3000-4000 rpm, and further preferably 3480 rpm, and the time is preferably 8-15 min, and further preferably 10 min; and the temperature is preferably 1-4 ℃. In the present application, the enzymatic products (such as glucose) and the buffer residues can be removed by water washing.

[0078] In the present application, the acetone washing process comprises mixing the water-washed product with acetone, sequentially performing ultrasonic treatment and centrifugation, and repeating the above steps twice; the ratio of the amount of the litter to the amount of acetone is preferably 1 g:40 mL, and the volume concentration of the acetone is preferably 100%. In the present application, the ultrasonic treatment time is preferably 20-25 min; the centrifugation speed is preferably 3000-4000 rpm, further preferably 3480 rpm, the centrifugation time is preferably 10 min, and the centrifugation temperature is preferably 1-4℃. In the present application, the purpose of the acetone washing is to remove water and residual water-soluble impurities, facilitating drying.

[0079] In the present application, after the starch removal treatment, the obtained system is preferably further subjected to drying and grinding. In the present application, the drying temperature is preferably 50℃, and the drying time is preferably 24 h. In the present application, the grinding speed is preferably 600 rpm, and the grinding time is 10 min; the above grinding process is repeated 5 times, and the interval between adjacent two times is preferably 5 min.

[0080] Unless otherwise specified, the materials and devices used in the present application are commercially available in the art.

[0081] The technical solutions in the present application will be clearly and completely described below in combination with the embodiments in the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0082] Embodiment 1

[0083] The litter of Populus euphratica leaves collected from the southern edge of the Taklimakan Desert was used as the raw material, and the extraction was performed according to the process shown in the figure; Figure 4

[0084] After drying and grinding the raw material, 1 g of the raw material was added into a 50-mL conical centrifuge tube, 30 mL of a 50-mM sodium chloride solution was added, and the mixture was uniformly mixed and then placed in a refrigerator for cold storage at 4℃ for 12 h; the cold-stored system was centrifuged at 3480 rpm for 10 min at 1℃ to obtain the pretreated raw material;

[0085] 40 mL of ethanol with a volume concentration of 80% was added to the pretreated raw material, and ultrasonic treatment was performed for 20 min; the system was centrifuged at 3480 rpm for 10 min at 1℃ to remove the solvent; the above process was repeated twice;

[0086] 40 mL of acetone with a volume concentration of 100% was added to the product after the first immersion treatment, and ultrasonic treatment was performed for 20 min; the system was centrifuged at 3480 rpm for 10 min at 1℃; ​

[0087] After the second impregnation treatment, 40 mL of chloroform / methanol (volume ratio of 1:1) was added to the product, which was ultrasonically treated for 20 min, centrifuged at 1°C at a speed of 3480 rpm for 10 min;

[0088] After the third impregnation treatment, 40 mL of acetone with a volume concentration of 100% was added to the product, which was ultrasonically treated for 20 min, centrifuged at 1°C at a speed of 3480 rpm for 10 min to obtain an intermediate extract;

[0089] 25 mL of Tris-acetic acid buffer with a concentration of 10 mM (pH 6.0) was added to the intermediate extract obtained above, which was mixed thoroughly, placed in a water bath at 90°C for 2 h, and then cooled to 55-60°C to obtain a product system;

[0090] 20 and 40 units of Bacillus licheniformis α-amylase and starch glucosidase (derived from Aspergillus niger) were added to 5 mL of Tris-acetic acid buffer with a concentration of 10 mM, respectively, to obtain an enzyme preparation;

[0091] The 5 mL of enzyme preparation obtained above was added to the product system, and an enzymatic reaction was carried out in a 55°C water bath for 2 h; the system after the enzymatic reaction was centrifuged at 1°C at a speed of 3480 rpm for 10 min to remove the solvent; 40 mL of pure water was added, ultrasonically treated for 20 min, centrifuged at 1°C at a speed of 3480 rpm for 10 min to remove the solvent, and the above water washing process was repeated twice; then 40 mL of acetone with a volume concentration of 100% was added, ultrasonically treated for 20 min, centrifuged at 1°C at a speed of 3480 rpm for 10 min to remove the solvent, and the above acetone washing was repeated twice;

[0092] The product obtained above was dried at 50°C for 24 h, and then ground at a speed of 600 rpm for 5 times, 10 min each time, with an interval of 5 min between adjacent two times, to obtain lignin.

[0093] Example 2

[0094] The leaves of Alhagi sparsifolia collected from the southern edge of the Taklimakan Desert were used as raw materials, and lignin was obtained by extraction according to the manner of Example 1.

[0095] Example 3

[0096] The leaves of Caragana floridus collected from the southern edge of the Taklimakan Desert were used as raw materials, and lignin was obtained by extraction according to the manner of Example 1.

[0097] Comparative Example 1

[0098] The leaf litter of Populus euphratica, Calligonum rubicundum or Caragana pygmaea collected from the south edge of Taklimakan Desert was used as raw material;

[0099] 5g of the dried and ground raw material was weighed and added to 50mL of 95% ethanol solution, soaked for 72h, and then filtered to obtain the lignin solution dissolved in ethanol. Most of the ethanol solvent was removed by rotary evaporation to obtain a viscous solution. The solution was added to 30mL of 7M hydrochloric acid aqueous solution, and after standing, the lignin was precipitated and filtered to obtain the purified lignin. After drying, the precipitate was obtained.

[0100] Comparative Example 2

[0101] The leaf litter of Populus euphratica, Calligonum rubicundum or Caragana pygmaea collected from the south edge of Taklimakan Desert was used as raw material;

[0102] 5g of the dried and ground raw material was weighed and added to 50mL of 95% ethanol solution, soaked for 72h, and then filtered to obtain the lignin solution dissolved in ethanol. Most of the ethanol solvent was removed by rotary evaporation to obtain a viscous solution. The solution was added to 30mL of 7M hydrochloric acid aqueous solution, and after standing, the lignin was precipitated and filtered to obtain the purified lignin. After drying, the precipitate was obtained.

[0103] Performance test

[0104] The lignin extracted from the examples and comparative examples was characterized;

[0105] 70mg of the obtained lignin was transferred to a 5mm NMR tube, 1mL of deuterated dimethyl sulfoxide / deuterated pyridine (volume ratio 4:1) was added; the NMR tube was shaken in an ultrasonic bath for 4h until the gel became uniform, and the final sample height should be about 4-5cm in the NMR tube.

[0106] H spectrum and HSQC spectrum were obtained using Bruker pulse program, and the spectrum was analyzed by MestReNova software.

[0107] Figure 1 is the test process diagram provided by the present application, Figure 2 is the H spectrum of lignin components of different species obtained by the examples and comparative examples of the present application; Figure 3 is the HSQC spectrum of lignin components of different species obtained by the examples of the present application, wherein method one represents examples 1-3, method two represents comparative example 1, and method three represents comparative example 2.

[0108] Table 1 is the purity of lignin obtained by the examples and comparative examples (the purity is the mass percentage of lignin in the final extract);

[0109]

[0110] Table 2 is the extraction rate of lignin obtained by the examples and comparative examples (the extraction rate is the mass percentage of lignin in the final extract relative to the original lignin of the raw material);

[0111]

[0112] Table 3 is the proportion of three different types of lignin extracted in Examples 1-3;

[0113] In Tables 1-2, Method 1 represents Examples 1-3, Method 2 represents Comparative Example 1, and Method 3 represents Comparative Example 2.

[0114] Table 1 is the purity of lignin obtained by the examples and comparative examples

[0115] Species Method One (%) Method Two (%) Method Three (%) Populus euphratica 79.2±0.4a 70.1±0.4b 61.9±0.4c Alhagi maurorum 67.3±0.5a 65.5±0.4b 59.9±0.4c Caragana pygmaea 65.4±1.9a 56.3±1.7b 48.1±1.7c

[0116] Note: The values are the average values of each species (±SE, n=3), and different letters in the same row represent significant differences between different methods.

[0117] Table 2 is the extraction rate of lignin obtained by the examples and comparative examples

[0118] Species Method One (%) Method Two (%) Method Three (%) Populus euphratica 84.5±0.3a 64.6±0.1b 37.1±0.2c Alhagi maurorum 73.2±0.2a 31.3±0.2b 21.6±0.3c Caragana pygmaea 89.1±0.4a 80.6±0.2b 53.1±0.1c

[0119] Note: The values are the average values of each species (±SE, n=3), and different letters in the same row represent significant differences between different methods.

[0120] Table 3 is the proportion of three different types of lignin extracted in Examples 1-3;

[0121]

[0122] The lignin content in Populus euphratica leaf litter collected from the southern edge of the Taklimakan Desert was measured to be 9.0%, the lignin content in Alhagi sparsifolia leaf litter was measured to be 16.9%, and the lignin content in Caragana jubata leaf litter was measured to be 5.8% using the method of GB / T 20805-2006. The above results were used as the theoretical value of lignin content to calculate the results in Tables 1 and 2. It can be seen from the comparison that the lignin purity and extraction rate of Method 1 are significantly higher than those of Methods 2 and 3 in all species. Therefore, Method 1 (i.e., the extraction method provided by the present application) is the optimal lignin extraction method.

[0123] In combination with Table 3, the proportion of different groups in the lignin HSQC spectrum extracted according to Method I proves that the proportion of S and G groups in the leaves of dicotyledonous plants (Populus euphratica, Alhagi sparsifolia and Karelinia caspia) is high. In addition, it can be seen that the leaves of desert vegetation have a unique drought-resistant function, and the proportion of G groups in general lignin is high.

[0124] Although the above embodiments have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained under the premise of no creativity according to the present embodiments, which all belong to the protection scope of the present application.

Claims

1. A method for extracting lignin from litter, characterized in that: The following steps are involved: mixing the litter and sodium chloride solution, and performing a cold storage treatment to obtain a pretreated raw material; performing a first immersion treatment, a second immersion treatment, a third immersion treatment, and a fourth immersion treatment on the pretreated raw material in a first organic solvent, a second organic solvent, a third organic solvent, and a fourth organic solvent in sequence to obtain an intermediate extract; The intermediate extract is subjected to a starch removal treatment to obtain lignin; The first organic solvent is ethanol; The second organic solvent is acetone or ethyl acetate; The third organic solvent is a mixed solvent of methane chloride and methanol, and the methane chloride is at least one of chloroform and dichloromethane; The fourth organic solvent is acetone or ethyl acetate.

2. The extraction method according to claim 1, wherein The litter includes leaf litter; the leaf litter includes at least one of leaf litter of woody plants and leaf litter of herbaceous plants; The woody plant includes at least one of Populus euphratica and Populus gracile; The herbaceous plant includes at least one of ophiopogon wiltii and camel thorn.

3. The extraction method according to claim 1, wherein Before the mixing, the litter is dried and ground; The concentration of the sodium chloride solution is 45 to 55 mmol / L; the usage ratio of the litter and the sodium chloride solution is 1 g: 30 to 40 mL; the temperature of the cold storage treatment is 4 to 8° C., and the time is 8 to 24 hours.

4. The extraction method according to claim 1, wherein The first immersion treatment comprises: mixing the pretreated litter with a first organic solvent, performing ultrasound and centrifugation in sequence, and repeating the above process twice; The volume concentration of the ethanol is 70-90%; the usage ratio of the litter and the first organic solvent is 1g:30-40mL; The ultrasound time is 20 to 25 minutes; The centrifugal speed is 3000-4000 rpm, the time is 8-15 minutes, and the temperature is 1-4°C.

5. The extraction method according to claim 1, wherein The second impregnation treatment comprises: mixing the product obtained from the first impregnation treatment with a second organic solvent, and sequentially performing ultrasonication and centrifugation; The volume concentration of the acetone is 70-100%; the usage ratio of the litter and the second organic solvent is 1g:30-40mL; The ultrasound time is 20 to 25 minutes; The centrifugal speed is 3000-4000 rpm, the time is 8-15 minutes, and the temperature is 1-4°C.

6. The extraction method according to claim 1, characterized in that The third impregnation treatment comprises: mixing the product obtained by the second impregnation treatment with a third organic solvent, and sequentially performing ultrasonication and centrifugation; The volume ratio of the methane chloride and methanol is 1 to 2:1; the amount ratio of the litter and the third organic solvent is 1 g: 30 to 40 mL; The ultrasound time is 20 to 25 minutes; The centrifugal speed is 3000-4000 rpm, the time is 8-15 minutes, and the temperature is 1-4°C.

7. The extraction method according to claim 1, characterized in that The fourth immersion treatment comprises: mixing the product obtained by the third immersion treatment with a fourth organic solvent, and sequentially performing ultrasonication and centrifugation; The volume concentration of the acetone is 70-100%; the usage ratio of the litter and the fourth organic solvent is 1g:30-40mL; The ultrasound time is 20 to 25 minutes; The centrifugal speed is 3000-4000 rpm, the time is 8-15 minutes, and the temperature is 1-4°C.

8. The extraction method according to claim 1, characterized in that The starch removal treatment comprises: first mixing the intermediate extract and Tris-acetate buffer, performing a liquid phase reaction, and obtaining a product system; a second mixing of Tris-acetate buffer and amylase to obtain an enzyme preparation; The product system and the enzyme preparation are mixed for a third time to carry out an enzymatic hydrolysis reaction.

9. The extraction method according to claim 8, characterized in that The concentration of the Tris-acetate buffer is 10-15 mmol / L; During the first mixing process, the ratio of the litter to Tris-acetate buffer is 1 g: 25-30 mL; The temperature of the liquid phase reaction is 80-100°C and the time is 1-4 hours; The amylase includes Bacillus licheniformis α-amylase and amyloglucosidase; In the enzyme preparation, the content of Bacillus licheniformis α-amylase is 15-25 units / 5-6 mL, and the content of amyloglucosidase is 30-50 units / 5-6 mL; The volume ratio of the product system to the enzyme preparation is 5:1; The temperature of the enzymatic hydrolysis reaction is 55-60° C., and the time is 1.5-2.5 hours.

10. The extraction method according to claim 8, characterized in that After the enzymatic hydrolysis reaction, the obtained system is sequentially centrifuged, washed with water and washed with acetone; The centrifugal speed is 3000-4000 rpm, the time is 8-15 min, and the temperature is 1-4°C; The water washing process comprises: mixing the precipitate obtained after centrifugation with pure water, performing ultrasonication and centrifugation in sequence, and repeating the above steps twice; The acetone washing process comprises: mixing the product after water washing with acetone, performing ultrasonication and centrifugation in sequence, and repeating the above steps twice.

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