CN111118081A - Method for improving lignocellulose enzymolysis saccharification efficiency through glucose-assisted ball milling and application of method - Google Patents
Method for improving lignocellulose enzymolysis saccharification efficiency through glucose-assisted ball milling and application of method Download PDFInfo
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Abstract
The invention discloses a method for improving lignocellulose enzymolysis saccharification efficiency by glucose-assisted ball milling and application thereof. The method comprises the following steps: (1) crushing a wood fiber raw material to obtain wood fiber powder; (2) extracting wood fiber powder with an organic solvent, and drying to obtain a degreased wood fiber raw material; (3) uniformly mixing the degreased wood fiber raw material with a sodium hydroxide solution, reacting at 110-180 ℃, cooling after the reaction is finished, performing suction filtration, taking filter residues, washing, and drying to obtain a solid product; (4) mixing the solid product with glucose and then carrying out ball milling to obtain a ball-milled product; (5) and adding the ball-milling product into a cellulase solution for carrying out an enzymolysis reaction to obtain reducing sugar. The method has the characteristics of mild process conditions, simple operation, high conversion rate and the like, and can be used for preparing reducing sugar such as glucose and xylose.
Description
Technical Field
The invention belongs to the field of biomass utilization, and particularly relates to a method for improving lignocellulose enzymolysis saccharification efficiency by glucose-assisted ball milling and application thereof.
Background
Because the excessive use of petrochemical resources by human beings causes a series of environmental problems while causing global petrochemical resource exhaustion, finding a new energy source which can meet the increasing energy consumption requirement and keep ecological balance has become a common target of scientific researchers of various countries. Among many new energy sources, biomass is considered as an ideal material for replacing petrochemical resources due to its advantages of wide source, low price, environmental friendliness, and the like. Among them, the production of ethanol, a clean fuel, from lignocellulose as a raw material is receiving more and more attention. However, since the lignocellulosic raw material has a compact structure and is highly resistant to enzymes, enzymatic saccharification efficiency is low. Therefore, how to effectively improve the saccharification efficiency of lignocellulose plays an important role in further research on high-value utilization of lignin.
Eucalyptus grows very fast and is a good raw material for wood boards. The eucalyptus has developed roots, has low requirements on the growth environment, contains rich lignocellulose and is a good biomass resource. Therefore, the method for improving the enzymolysis saccharification efficiency of the eucalyptus by the aid of the glucose ball milling can provide important theoretical and technical guidance for lignocellulose enzymolysis saccharification.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a method for improving the enzymolysis and saccharification efficiency of lignocellulose by glucose-assisted ball milling.
The invention also aims to provide application of the method for improving the enzymolysis and saccharification efficiency of the lignocellulose by glucose-assisted ball milling.
The purpose of the invention is realized by the following technical scheme: a method for improving lignocellulose enzymolysis saccharification efficiency by glucose-assisted ball milling comprises the following steps:
(1) crushing: crushing a wood fiber raw material to obtain wood fiber powder;
(2) degreasing treatment: wrapping the wood fiber powder obtained in the step (1) with gauze, adding an organic solvent for extraction, and drying to obtain a degreased wood fiber raw material;
(3) dilute alkali pretreatment: uniformly mixing the degreased wood fiber raw material obtained in the step (2) with a sodium hydroxide solution, reacting at 110-180 ℃, cooling after the reaction is finished, performing suction filtration, taking filter residue, washing and drying the filter residue to obtain a solid product;
(4) and (3) glucose ball milling treatment: mixing the solid product obtained in the step (3) with glucose, and then carrying out ball milling to obtain a ball-milled product;
(5) and (3) enzymolysis treatment: and (3) adding the ball-milled product obtained in the step (4) into a cellulase solution, carrying out enzymolysis reaction at 40-80 ℃, soaking in boiling water to inactivate enzyme after the reaction is finished, centrifuging, taking supernatant, concentrating, and crystallizing to obtain reducing sugar.
The wood fiber raw material in the step (1) is a biomass raw material rich in lignocellulose; preferably at least one of eucalyptus, poplar and pine; more preferably eucalyptus.
The size of the wood fiber powder in the step (1) is 40-80 meshes.
The organic solvent in the step (2) is at least one of acetone and ethanol; preferably a mixed solvent obtained by mixing acetone and ethanol; more preferably acetone and ethanol in a volume ratio of 2: 0.6-1.5, and mixing to obtain a mixed solvent; most preferably acetone and ethanol in a volume ratio of 2:1 to 1.5 to obtain a mixed solvent.
The solid-liquid ratio of the wood fiber powder to the organic solvent in the step (2) is 1-2: 10 (g/ml); preferably 1.2-1.5: 10 (g/ml).
The gauze in the step (2) is 150-250 meshes of gauze; preferably 200-250 mesh gauze.
The extraction conditions in the step (2) are as follows: extracting for 4-12 h at 75-90 ℃; preferably: extracting for 4-8 h at 75-90 ℃.
The drying temperature in the step (2) is 50-70 ℃.
The solid-to-liquid ratio of the wood fiber raw material to the sodium hydroxide in the step (3) is 1: 8-15 (g/mL); preferably 1:10 to 15 (g/mL).
The concentration of the sodium hydroxide solution in the step (3) is 0.5-3% by mass; preferably 1 to 2.5 percent by mass.
The reaction temperature in the step (3) is preferably 120-150 ℃.
The reaction time in the step (3) is 40-120 min; preferably 45-100 min.
And (4) washing in the step (3) is washing by adopting ultrapure water.
The drying temperature in the step (3) is 50-70 ℃; preferably 60 deg.c.
The mass ratio of the solid product to the glucose in the step (4) is 10: 2-10; preferably 10: 4-8.
The ball milling conditions in the step (4) are as follows: adding 50-70 ball milling media into every 5-10 g of solid product (preferably adding 60-70 ball milling media into every 6-8 g of solid product); the rotation speed is 300-500 rpm (preferably 400-450 rpm); the ball milling time is 2-8 h (preferably 3-6 h); because high temperature is generated in the ball milling process, the ball milling needs to be stopped for 5min and then is cooled to the room temperature, namely the total time is 4-16 hours.
The ball milling medium is zirconia beads.
The cellulase in the step (5) is preferably a cellulose complex enzyme.
The concentration of the cellulase solution in the step (5) is 0.1 to 0.5 percent by mass; preferably 0.1 to 0.3 percent by mass.
The dosage of the cellulase in the step (5) is calculated according to the proportion of 0.03-0.15 mg of cellulase in each milligram (mg) of ball-milling product.
The enzymolysis reaction in the step (5) is preferably carried out on a shaking table at the rotating speed of 100-300 rpm (preferably 150-200 rpm).
The temperature of the enzymolysis reaction in the step (5) is preferably 45-60 ℃.
The time of the enzymolysis reaction in the step (5) is 40-80 h; preferably 48 to 60 hours.
Soaking in boiling water in the step (5) for 5-15 min; preferably 8-12 min.
The centrifugation conditions in the step (5) are as follows: centrifuging at 1000-1600 rpm for 5-15 min; preferably: centrifuging at 1200rpm for 10-12 min.
And (5) concentrating by adopting a rotary evaporator.
The concentration conditions in the step (5) are as follows: concentrating for 20-60 min at 40-80 ℃; preferably: concentrating at 60-80 deg.C for 30-40 min.
And (5) crystallizing by using a crystallizing tank.
The crystallization conditions in the step (5) are as follows: the crystallization temperature is 40-80 ℃ (preferably 60-80 ℃), and the crystallization time is 6-12 h (preferably 8-12 h).
The reducing sugar in the step (5) is at least one of glucose and xylose.
The method for improving the lignocellulose enzymolysis saccharification efficiency by glucose-assisted ball milling is applied to the preparation of reducing sugar.
The reducing sugar is at least one of glucose and xylose; glucose is preferred.
Compared with the prior art, the invention has the following advantages and effects:
(1) the preparation process of the invention is as follows: the method comprises the steps of eucalyptus wood powder preparation, degreasing treatment, diluted alkali pretreatment, glucose ball milling assisting treatment, enzymolysis treatment and concentration crystallization, wherein the diluted alkali pretreatment has higher reaction activity under the glucose ball milling assisting condition, and the technological process of lignocellulose enzymolysis and saccharification is shortened.
(2) The preparation method has the characteristics of mild process conditions, simple operation, high conversion rate and the like, and no acid is added in the reaction process, so that the corrosion of equipment is avoided, and the preparation method is environment-friendly.
Drawings
FIG. 1 is an infrared spectrum of a pretreated sample, eucalyptus material and enzymatic residue.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available.
The eucalyptus sapwood related to the embodiment of the invention is the xylem part of the eucalyptus; the eucalyptus wood chips are obtained by cutting the xylem of eucalyptus into sheets (convenient for crushing).
The extraction involved in the embodiment of the invention can be performed by an extraction device such as a Soxhlet extractor (a condenser tube, an extraction bottle and a heating device from top to bottom). During extraction, the eucalyptus tree powder is wrapped in gauze and placed in an extraction tube; adding organic solvent into the extraction bottle, heating the extraction bottle, gasifying the organic solvent, feeding the gasified organic solvent into a condenser, circularly cooling tap water in the condenser to condense the gasified organic solvent into liquid, and dripping the liquid into the extraction tube to extract lipid substances in the eucalyptus wood powder; when the liquid level of the organic solvent in the extraction tube reaches a certain height, the organic solvent dissolved with the crude fat flows into the extraction bottle through a siphon tube; the organic solvent flowing into the extraction bottle is continuously heated, gasified, ascended and condensed, and is dripped into the extraction tube, and the process is repeated in a circulating way until the extraction is complete. Heating at 75-90 ℃; the extraction time is 4-12 h (preferably 4-8 h). The heating device can use a water bath for heating.
Example 1
A method for improving enzymolysis saccharification efficiency of eucalyptus by using glucose-assisted ball milling comprises the following specific steps:
(1) preparing eucalyptus wood powder: after the eucalyptus sapwood chips are crushed by the medicine crusher, the eucalyptus sapwood chips are sieved and screened by a 40-60-mesh eucalyptus powder.
(2) Degreasing treatment: wrapping the ground eucalyptus wood powder with 200-mesh gauze, placing the gauze in an extraction tube, adding acetone and ethanol into the extraction tube according to the volume ratio of 2:1, completely covering the eucalyptus wood powder (the solid-to-liquid ratio is 1.2:10, g/ml) with mixed liquid, extracting the eucalyptus wood powder by circularly heating and cooling extraction liquid after the eucalyptus wood powder is completely soaked (the heating temperature is 75 ℃, the time is 6 hours), taking out the eucalyptus raw material in the gauze, and drying at 50-70 ℃ to obtain the degreased eucalyptus raw material.
(3) Dilute alkali pretreatment: adding NaOH solution with the mass fraction of 1% into the extracted eucalyptus raw material, adding the material and the NaOH solution according to the proportion of 1:10(g/mL), reacting in a reaction kettle at 120 ℃ for 45min, cooling to normal temperature after the reaction is finished, performing suction filtration, washing reaction filter residues for multiple times by ultrapure water, and drying at 60 ℃ to obtain a solid product.
As can be seen from the infrared spectrum of FIG. 1, 898cm-1The characteristic peak of the cellulose β -D-glucoside is shown, compared with the peak of the eucalyptus raw material, the absorption intensity of the characteristic peak of the cellulose β -D-glucoside is basically consistent after the eucalyptus raw material is subjected to dilute alkali pretreatment, which indicates that the dilute alkali pretreatment has much retention to the cellulose and is not degraded basically, 1510cm-1The characteristic peak of lignin is shown, and the value of the characteristic peak is lower after the dilute alkali pretreatment, which shows that the dilute alkali pretreatment has a promoting effect on lignin degradation, so that the lignin is dissolved in the hydrolysate.
(4) And (3) glucose ball milling treatment: mixing the pretreated solid product with glucose according to the mass ratio of 10:4, placing the mixture in a ball mill (manufacturer: Germany; model: DECO-PBM-V-2L; ball milling medium: zirconia beads; ratio: 6g solid product can be added with 60 zirconia beads) (note: because high temperature is generated in the ball milling process, the ball milling is stopped for 5min, the ball milling is stopped, the temperature is reduced to the room temperature, and the stopped ball milling is stopped for 5min, the ball milling time is not counted in the ball milling time), and mixing and ball milling at 400rpm for 3h to obtain a ball-milled product.
(5) And (3) enzymolysis treatment: 50mg of ball-milling product is weighed and added into 1.5mL of cellulose complex enzyme (purchased from Heshi wall chemical industry) solution with the mass fraction of 0.1 percent, and the shaking is carried out by a shaking table for 48 hours at the temperature of 45 ℃ and the rpm of 150. After the reaction is finished, soaking (soaking the shake flask filled with the reaction product into boiling water) into boiling water for 10min, during which time the enzyme is inactivated. Then centrifuging (1200rpm, 10min), and collecting supernatant to obtain an enzymolysis product; then, the enzymolysis product is subjected to sugar analysis by using HPLC (high performance liquid chromatography); wherein, the chromatographic conditions are as follows: a chromatographic column: daidan PA 20; column temperature: 30 ℃; flow rate: 4 ml/min; sample introduction amount: 20 mu L of the solution; the ampere detector detects a change in current.
(6) Concentration and crystallization: and (3) placing the enzymolysis product in a rotary evaporator at 60 ℃ for 30min for evaporation concentration, and then inputting the enzymolysis product into a crystallization tank for crystallization at 80 ℃ for 8h to obtain reducing sugar crystals.
The glucose yield (%) (glucose content in the enzymatic hydrolysate-added glucose amount)/glucose content in the raw material.
Xylose yield (%) -, xylose content in the enzymatic hydrolysate/xylose content in the raw material.
Wherein the glucose content and the xylose content in the raw material are respectively determined by hydrolyzing the extracted material in the step (2) with 72% concentrated sulfuric acid for 1h, adding water to dilute into 4% dilute sulfuric acid, continuously hydrolyzing at high temperature (121 ℃) for 1h, and measuring the glucose content and the xylose content of the filtered hydrolysate by HPLC (the same chromatographic conditions as in the step (5)).
Through calculation, the glucose yield of the example 1 is 58.03%, and the xylose yield is 57.23%.
Example 2
A method for improving enzymolysis saccharification efficiency of eucalyptus by using glucose-assisted ball milling comprises the following specific steps:
(1) preparing eucalyptus wood powder: after the eucalyptus sapwood chips are crushed by the medicine crusher, the eucalyptus sapwood chips are sieved and screened by a 40-60-mesh eucalyptus powder.
(2) Degreasing treatment: wrapping the eucalyptus wood powder obtained by crushing with 250-mesh gauze, placing the gauze in an extraction tube, adding acetone and ethanol into the extraction tube according to the volume ratio of 2:1.2, completely covering the wood powder (the solid-to-liquid ratio is 1.5:10, g/ml) with the mixed solution, extracting the eucalyptus wood powder by circularly heating, cooling and extracting the extract (80 ℃ for 8 hours), taking out the eucalyptus raw material in the gauze, and drying (50-70 ℃) to obtain the degreased eucalyptus raw material.
(3) Dilute alkali pretreatment: adding NaOH solution with the mass fraction of 1.5% into the extracted eucalyptus raw material, adding the materials and the NaOH solution according to the proportion of 1:12(g/mL), respectively reacting in a reaction kettle for 60min at 140 ℃, cooling to normal temperature after the reaction is finished, carrying out suction filtration, washing reaction filter residues for multiple times by using ultrapure water, and drying at 60 ℃ to obtain a solid product.
(4) And (3) glucose ball milling treatment: mixing the pretreated solid product and glucose according to the mass ratio of 10:5, placing the mixture in a ball mill (manufacturer: Germany; model: DECO-PBM-V-2L; ball milling medium: zirconia beads; ratio: 6g solid product can be added with 65 zirconia beads (note: because high temperature is generated in the ball milling process, the ball milling is stopped for 5min, the temperature is reduced to the room temperature, and the stopped ball milling is stopped for 5min, the ball milling time is not counted in the ball milling time), and mixing and ball milling for 4h at 400rpm to obtain a ball milling product.
(5) And (3) enzymolysis treatment: weighing 50mg of ball-milling product, adding the ball-milling product into 1.5mL of cellulose complex enzyme solution with the mass fraction of 0.15%, and shaking for 50h by using a shaking table under the shaking conditions of 50 ℃ and 150 rpm. After the reaction is finished, soaking the mixture into boiling water for 8min, and inactivating the enzyme in the course of soaking. Then, the mixture was centrifuged (1200rpm, 12min), and the supernatant was collected and analyzed for sugar by HPLC.
(6) Concentration and crystallization: and (3) placing the enzymolysis product in a rotary evaporator at 70 ℃ for 35min for evaporation concentration, and then inputting the enzymolysis product into a crystallization tank for crystallization at 70 ℃ for 10h to obtain reducing sugar crystals.
Through calculation (the method is the same as the example 1), the glucose yield of the example 2 is 59.93%, and the xylose yield is 60.02%.
Example 3
A method for improving enzymolysis saccharification efficiency of eucalyptus by using glucose-assisted ball milling comprises the following specific steps:
(1) preparing eucalyptus wood powder: after the eucalyptus sapwood chips are crushed by the medicine crusher, the eucalyptus sapwood chips are sieved and screened by 60-80 meshes of eucalyptus wood powder.
(2) Degreasing treatment: wrapping eucalyptus wood powder obtained by crushing with 220-mesh gauze, placing the gauze in an extraction tube, adding acetone and ethanol into the extraction tube according to the volume ratio of 2:1.5 (the solid-liquid ratio is 1.5:10, g/ml), completely covering the wood powder with a mixed solution, completely soaking the wood powder, extracting the eucalyptus wood powder by circularly heating, cooling and extracting the extract (85 ℃ for 10 hours), taking out eucalyptus raw materials in the gauze, and drying (50-70 ℃) to obtain the degreased eucalyptus raw materials.
(3) Dilute alkali pretreatment: adding NaOH solution with the mass fraction of 2.5% into the extracted eucalyptus raw material, adding the materials and the NaOH solution according to the proportion of 1:15(g/mL), respectively reacting in a reaction kettle for 100min at 150 ℃, cooling to normal temperature after the reaction is finished, carrying out suction filtration, washing reaction filter residues for multiple times by using ultrapure water, and drying at 60 ℃ to obtain a solid product.
(4) And (3) glucose ball milling treatment: mixing the pretreated solid product and glucose according to the mass ratio of 10:8, placing the mixture in a ball mill (manufacturer: Germany; model: DECO-PBM-V-2L; ball milling medium: zirconia beads; ratio: 8g solid product can be added with 70 zirconia beads (note: because high temperature is generated in the ball milling process, the ball milling is stopped for 5min, the temperature is reduced to the room temperature, the ball milling time is not counted for 5min, the ball milling is stopped), mixing and ball milling is carried out for 6h at 450rpm, and thus the ball milling product is obtained.
(5) And (3) enzymolysis treatment: weighing 50mg of ball-milling product, adding the ball-milling product into 1.5mL of cellulose complex enzyme solution with the mass fraction of 0.3%, and shaking by a shaker for 60 hours at the temperature of 200 rpm. After the reaction is finished, soaking the mixture into boiling water for 12min, and inactivating the enzyme in the course of reaction. Then centrifuged (1200rpm, 10min) and the supernatant was collected for sugar analysis by HPLC.
(6) Concentration and crystallization: and (3) placing the enzymolysis product in a rotary evaporator at 80 ℃ for 40min for evaporation concentration, and then inputting the enzymolysis product into a crystallization tank for crystallization at 60 ℃ for 12h to obtain reducing sugar crystals.
Through calculation (the method is the same as the example 1), the glucose yield of the example 2 is 56.8 percent, and the xylose yield is 55.24 percent.
Comparative example
(1) Preparing eucalyptus wood powder: after the eucalyptus sapwood chips are crushed by the medicine crusher, the eucalyptus sapwood chips are sieved and screened by 60-80 meshes of eucalyptus wood powder.
(2) Degreasing treatment: wrapping eucalyptus powder obtained by crushing with 200-mesh gauze, placing the gauze in an extraction tube, adding acetone and ethanol into the extraction tube according to the volume ratio of 2:1.2 (the solid-liquid ratio is 2:10, g/ml), completely covering the wood powder with mixed liquid, extracting the eucalyptus powder by circularly heating and cooling extraction liquid after the wood powder is completely soaked (85 ℃) for 8 hours, taking out eucalyptus raw materials in the gauze, and drying (50-70 ℃) to obtain degreased eucalyptus raw materials.
(3) Dilute alkali pretreatment: adding NaOH solution with the mass fraction of 1% into the extracted eucalyptus raw material, adding the materials and the NaOH solution according to the proportion of 1:12(g/mL), respectively reacting in a reaction kettle for 90min at 120 ℃, cooling to normal temperature after the reaction is finished, performing suction filtration, washing reaction filter residues for multiple times by ultrapure water, and drying at 65 ℃ to obtain a solid product.
(4) Ball milling treatment: 500mg of the pretreated solid product is put into a ball mill, mixed and then put into the ball mill (manufacturer: Germany; model: DECO-PBM-V-2L; ball milling medium: zirconia beads; ratio: 8g of the solid product can be added with 65 zirconia beads (note: because high temperature is generated in the ball milling process, the ball milling is stopped for 5min and then is cooled to the room temperature, and the ball milling is stopped for 5min and is not counted for ball milling time), and mixed and ball milled for 3h at 500rpm to obtain the ball-milled product.
(5) And (3) enzymolysis treatment: weighing 50mg of ball-milling product, adding the ball-milling product into 1.5mL of cellulose complex enzyme solution with the mass fraction of 0.2%, and shaking for 45h by using a shaking table under the shaking conditions of 55 ℃ and 150 rpm. After the reaction is finished, soaking the mixture into boiling water for 8min, and inactivating the enzyme in the course of soaking. Then centrifuged (1200rpm, 10min) and the supernatant was collected for sugar analysis by HPLC.
(6) Concentration and crystallization: and (3) placing the enzymolysis product in a rotary evaporator at 80 ℃ for 35min for evaporation concentration, and then inputting the enzymolysis product into a crystallization tank for crystallization at 60 ℃ for 12h to obtain reducing sugar crystals.
Through calculation (the method is the same as that of example 1), the glucose yield of the comparative example is 17.21%, and the xylose yield is 15.14%.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A method for improving lignocellulose enzymolysis saccharification efficiency by glucose-assisted ball milling is characterized by comprising the following steps:
(1) crushing: crushing a wood fiber raw material to obtain wood fiber powder;
(2) degreasing treatment: wrapping the wood fiber powder obtained in the step (1) with gauze, adding an organic solvent for extraction, and drying to obtain a degreased wood fiber raw material;
(3) dilute alkali pretreatment: uniformly mixing the degreased wood fiber raw material obtained in the step (2) with a sodium hydroxide solution, reacting at 110-180 ℃, cooling after the reaction is finished, performing suction filtration, taking filter residue, washing and drying the filter residue to obtain a solid product;
(4) and (3) glucose ball milling treatment: mixing the solid product obtained in the step (3) with glucose, and then carrying out ball milling to obtain a ball-milled product;
(5) and (3) enzymolysis treatment: and (3) adding the ball-milled product obtained in the step (4) into a cellulase solution, carrying out enzymolysis reaction at 40-80 ℃, soaking in boiling water to inactivate enzyme after the reaction is finished, centrifuging, taking supernatant, concentrating, and crystallizing to obtain reducing sugar.
2. The method for improving the enzymatic saccharification efficiency of lignocellulose by glucose assisted ball milling as recited in claim 1, wherein:
the wood fiber raw material in the step (1) is at least one of eucalyptus, poplar and pine;
the mass ratio of the solid product to the glucose in the step (4) is 10: 2-10.
3. The method for improving the enzymatic saccharification efficiency of lignocellulose by glucose assisted ball milling as recited in claim 2, wherein:
the wood fiber raw material in the step (1) is eucalyptus;
the mass ratio of the solid product to the glucose in the step (4) is 10: 4-8.
4. The method for improving the enzymatic saccharification efficiency of lignocellulose by glucose assisted ball milling as recited in claim 1, wherein:
the ball milling conditions in the step (4) are as follows: adding 50-70 ball milling media into every 5-10 g of solid product; the rotating speed is 300-500 rpm; ball milling time is 2-8 h;
the ball milling medium is zirconia beads.
5. The method for improving the enzymatic saccharification efficiency of lignocellulose by glucose assisted ball milling as recited in claim 1, wherein:
the solid-liquid ratio of the wood fiber powder to the organic solvent in the step (2) is 1-2: 10, g/ml;
the solid-to-liquid ratio of the wood fiber raw material to the sodium hydroxide in the step (3) is 1: 8-15 g/mL;
the concentration of the sodium hydroxide solution in the step (3) is 0.5-3% by mass;
the concentration of the cellulose solution in the step (5) is 0.1-0.5% by mass percent;
the dosage of the cellulase in the step (5) is calculated according to the proportion of 0.03-0.15 mg of cellulase in each mg of ball-milling product.
6. The method for improving the enzymatic saccharification efficiency of lignocellulose by glucose assisted ball milling as recited in claim 1, wherein:
the organic solvent in the step (2) is at least one of acetone and ethanol;
the gauze in the step (2) is 150-250 meshes of gauze;
the extraction conditions in the step (2) are as follows: extracting for 4-12 h at 75-90 ℃;
the cellulase in the step (5) is a cellulose complex enzyme.
7. The method for improving the enzymatic saccharification efficiency of lignocellulose by glucose assisted ball milling of claim 6, wherein:
the organic solvent in the step (2) is acetone and ethanol according to a volume ratio of 2: 0.6-1.5, and mixing to obtain a mixed solvent;
the gauze in the step (2) is 200-250 meshes of gauze;
the extraction conditions in the step (2) are as follows: extracting for 4-8 h at 75-90 ℃.
8. The method for improving the enzymatic saccharification efficiency of lignocellulose by glucose assisted ball milling as recited in claim 1, wherein:
the size of the wood fiber powder in the step (1) is 40-80 meshes;
the drying temperature in the step (2) is 50-70 ℃;
the reaction temperature in the step (3) is 120-150 ℃;
the reaction time in the step (3) is 40-120 min;
the washing in the step (3) is carried out by adopting ultrapure water;
the drying temperature in the step (3) is 50-70 ℃;
the enzymolysis reaction in the step (5) is carried out on a shaking table at the rotating speed of 100-300 rpm;
the temperature of the enzymolysis reaction in the step (5) is 45-60 ℃;
the time of the enzymolysis reaction in the step (5) is 40-80 h;
soaking in boiling water in the step (5) for 5-15 min;
the centrifugation conditions in the step (5) are as follows: centrifuging at 1000-1600 rpm for 5-15 min;
the concentration conditions in the step (5) are as follows: concentrating for 20-60 min at 40-80 ℃;
the crystallization conditions in the step (5) are as follows: the crystallization temperature is 40-80 ℃, and the crystallization time is 6-12 h;
the reducing sugar in the step (5) is at least one of glucose and xylose.
9. Use of the method for improving the enzymatic saccharification efficiency of lignocellulose by glucose-assisted ball milling as recited in any one of claims 1 to 8 in the preparation of reducing sugars.
10. Use according to claim 9, characterized in that: the reducing sugar is at least one of glucose and xylose.
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