阅读说明：本技术 一种玉米秸秆的预处理及酶解的方法 (Pretreatment and enzymolysis method of corn straw ) 是由 徐建 顾帅令 吴海军 左宗涛 魏无忌 胡世杰 张楷 徐霞 于 2020-12-24 设计创作，主要内容包括：本发明公开了一种玉米秸秆的预处理及酶解的方法,包括以下步骤：包括以下步骤：将玉米秸秆切断,依次进行粉碎、水洗和烘干,得到颗粒物；颗粒物加入稀硫酸溶液中,在超临界二氧化碳条件下预处理一段时间后,经抽滤后收集滤液和滤渣,滤渣放入烘箱干燥一段时间去除水分,得到固形物；固形物作为酶水解底物,按一定固液比添加木聚糖酶和柠檬酸三钠缓冲液,酶水解一段时间后,获得木糖。本发明方法设计科学合理,在低温下利用稀酸强化超临界二氧化碳预处理效果,进而更好的打破木质纤维素本身复杂的交联结构,破坏木质素和半纤维之间的化学键,脱除木质素,并且使半纤维素更多的暴露出来,提升酶水解效率。(The invention discloses a method for pretreatment and enzymolysis of corn straws, which comprises the following steps: the method comprises the following steps: cutting corn stalks, and sequentially crushing, washing and drying to obtain particles; adding the particles into a dilute sulfuric acid solution, pretreating for a period of time under the condition of supercritical carbon dioxide, filtering, collecting filtrate and filter residues, and drying the filter residues in an oven for a period of time to remove moisture to obtain a solid matter; and (3) taking the solid as an enzyme hydrolysis substrate, adding xylanase and trisodium citrate buffer solution according to a certain solid-to-liquid ratio, and performing enzyme hydrolysis for a period of time to obtain xylose. The method disclosed by the invention is scientific and reasonable in design, and the effect of the supercritical carbon dioxide pretreatment is enhanced by using the dilute acid at a low temperature, so that the complex cross-linked structure of the lignocellulose is broken better, the chemical bond between the lignin and the hemicellulose is broken, the lignin is removed, more hemicellulose is exposed, and the enzymatic hydrolysis efficiency is improved.)

1. A method for pretreatment and enzymolysis of corn straws is characterized by comprising the following steps:

(1) cutting corn stalks, and sequentially crushing, washing and drying to obtain particles;

(2) adding the particles obtained in the step (1) into a dilute sulfuric acid solution according to a certain solid-to-liquid ratio, pretreating for a period of time under the condition of supercritical carbon dioxide, filtering, collecting filtrate and filter residues, and drying the filter residues in an oven for a period of time to remove moisture to obtain a solid matter;

(3) and (3) taking the solid obtained in the step (2) as an enzyme hydrolysis substrate, adding xylanase and trisodium citrate buffer solution according to a certain solid-to-liquid ratio, and performing enzyme hydrolysis for a period of time to obtain xylose.

2. The method for pretreating and enzymolyzing corn stalks according to claim 1, wherein the method comprises the following steps: in the step (1), the corn straws are cut off and crushed into the granularity of 30-50 meshes.

3. The method for pretreating and enzymolyzing corn stalks according to claim 1, wherein the method comprises the following steps: in the step (2), the particles are added into a dilute sulfuric acid solution according to the solid-to-liquid ratio of 1:8-12, and H in the dilute sulfuric acid solution₂SO₄The mass percentage of the components is 1-2%.

4. The method for pretreating and enzymolyzing corn stalks according to claim 1, wherein the method comprises the following steps: in the step (2), the pretreatment time under the condition of supercritical carbon dioxide is 30-240 min.

5. The method for pretreating and enzymolyzing corn stalks according to claim 1, wherein the method comprises the following steps: in the step (2), the temperature of the pretreatment under the condition of supercritical carbon dioxide is 35-70 ℃.

6. The method for pretreating and enzymolyzing corn stalks according to claim 1, wherein the method comprises the following steps: in the step (2), the drying temperature of the oven is 30-60 ℃, and the drying time is 10-14 h.

7. The method for pretreating and enzymolyzing corn stalks according to claim 1, wherein the method comprises the following steps: in the step (2), the xylanase is acid xylanase, and the enzyme activity is 200000U/g.

8. The method for pretreating and enzymolyzing corn stalks according to claim 1, wherein the method comprises the following steps: in the step (2), the dosage of the xylanase is 28000 and 33000U/g substrate, preferably 31250U/g substrate.

9. The method for pretreating and enzymolyzing corn stalks according to claim 1, wherein the method comprises the following steps: in the step (3), the pH value of the trisodium citrate buffer solution is 5, and the xylanase and the trisodium citrate buffer solution are added according to the solid-to-liquid ratio of 1:40-60, preferably 1: 50.

10. The method for pretreating and enzymolyzing corn stalks according to claim 1, wherein the method comprises the following steps: in the step (3), the time for enzyme hydrolysis is 20-28h, preferably 24 h.

Technical Field

The invention relates to the technical field of agricultural waste conversion and utilization, in particular to a method for pretreatment and enzymolysis of corn straws.

Background

At present, agricultural and forestry wastes such as corn straws and the like are directly discarded, so that the environment is greatly polluted while resources are wasted; the small amount of the feed is used as animal feed, returned to the field or burnt to generate heat, and the utilization efficiency is low. Therefore, it is very necessary to improve the utilization efficiency of agricultural and forestry wastes such as corn stalks, and taking corn stalks as an example, the annual output is as high as 3.5 hundred million tons, which accounts for about 60% of the total straw output (wheat straws, rice straws, bean straws, cotton straws, corn straws and the like), and the development of the technology for processing and utilizing the corn stalks and other derived products to produce hemicellulose, xylose and the like has a far-reaching significance.

The conventional acid treatment method for preparing xylose uses common acid as a hydrolysis catalyst. The sulfuric acid is cheap, meets the requirement of industrial production, and has good hydrolysis effect, so the sulfuric acid is generally used in a large amount. When lignocellulose is hydrolyzed by acid, the glycosidic bond in the hemicellulose or cellulose is broken, so that the hemicellulose or cellulose is changed as follows: the average polymerization degree of the hemicellulose or cellulose is obviously reduced; the internal surface area of hemicellulose or cellulose increases and the crystallinity decreases; the protective layer of lignin is also destroyed, causing degradation of hemicellulose or cellulose. In lignocellulose acidolysis, hemicellulose is first broken down due to easier separation.

However, the traditional acid hydrolysis method requires a series of processes such as deacidification and neutralization in the production process, and has great pollution to the environment; meanwhile, the production process causes corrosion to equipment, and the economic investment is also large.

Disclosure of Invention

The invention aims to overcome the problems in the prior art and provides a method for pretreatment and enzymolysis of corn straws.

In order to achieve the technical purpose and achieve the technical effect, the invention is realized by the following technical scheme:

a method for pretreatment and enzymolysis of corn straws comprises the following steps:

(1) cutting corn stalks, and sequentially crushing, washing and drying to obtain particles;

(2) adding the particles obtained in the step (1) into a dilute sulfuric acid solution according to a certain solid-to-liquid ratio, pretreating for a period of time under the condition of supercritical carbon dioxide, filtering, collecting filtrate and filter residues, and drying the filter residues in an oven for a period of time to remove moisture to obtain a solid matter;

(3) and (3) taking the solid obtained in the step (2) as an enzyme hydrolysis substrate, adding xylanase and trisodium citrate buffer solution according to a certain solid-to-liquid ratio, and performing enzyme hydrolysis for a period of time to obtain xylose.

Further, according to the method for pretreating and enzymolyzing the corn stalks, in the step (1), the corn stalks are cut off and crushed into the granularity of 30-50 meshes.

Further, according to the method for pretreating and enzymolyzing the corn straws, in the step (2), the particles are added into a dilute sulfuric acid solution according to the solid-to-liquid ratio of 1:8-12, and H in the dilute sulfuric acid solution₂SO₄The mass percentage of the components is 1-2%.

Further, according to the method for the pretreatment and enzymolysis of the corn stalks, in the step (2), the pretreatment time is 30-240min under the condition of supercritical carbon dioxide.

Further, according to the method for pretreating and enzymolyzing the corn stalks, in the step (2), the temperature of pretreatment is 35-70 ℃ under the condition of supercritical carbon dioxide.

Further, according to the method for the pretreatment and the enzymolysis of the corn stalks, in the step (2), the drying temperature of the oven is 30-60 ℃, and the drying time is 10-14 hours.

Further, according to the method for pretreating and hydrolyzing the corn straws, in the step (2), the xylanase is acidic xylanase, and the enzyme activity is 200000U/g.

Further, in the method for pretreating and hydrolyzing corn stalks with enzyme, in the step (2), the dosage of the xylanase is 28000 and 33000U/g substrate, preferably 31250U/g substrate.

Further, according to the method for pretreating and hydrolyzing corn stalks, in the step (3), the pH of the trisodium citrate buffer solution is 5, and the xylanase and the trisodium citrate buffer solution are added according to the solid-to-liquid ratio of 1:40-60, preferably 1: 50.

Further, in the method for pretreating and hydrolyzing the corn stalks by the enzyme, in the step (3), the time for hydrolyzing the enzyme is 20 to 28 hours, preferably 24 hours.

The invention has the beneficial effects that:

the method of the invention has the advantages of simple operation,

the method disclosed by the invention is scientific and reasonable in design, and the effect of the supercritical carbon dioxide pretreatment is enhanced by using the dilute acid at a low temperature, so that the complex cross-linked structure of the lignocellulose is broken better, the chemical bond between the lignin and the hemicellulose is broken, the lignin is removed, more hemicellulose is exposed, and the enzymatic hydrolysis efficiency is improved. Compared with the traditional acid hydrolysis method, the method is environment-friendly and has low economic investment.

Of course, it is not necessary for any one product that embodies the invention to achieve all of the above advantages simultaneously.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The following embodiments are relevant to the present invention:

example 1

Crushing corn straws, sieving the corn straws by using a 2mm sieve, taking 50g of the corn straws as a raw material for biomass pretreatment, adding the raw material into 500ml of 1% dilute sulfuric acid solution according to a solid-to-liquid ratio of 10%, reacting at 35 ℃ for 60min, after pretreatment, carrying out suction filtration by using a non-woven fabric, collecting filtrate and filter residue, washing the filter residue by using 1000m1 deionized water, and then carrying out HPLC analysis on the filtrate to evaluate the pretreatment effect; and drying the filter residue in an oven at 45 ℃ for 12 hours to remove water.

Taking 2g of the pretreated solid as an enzyme hydrolysis substrate, and carrying out enzyme hydrolysis by using acid xylanase (200000U/g of enzyme activity, Shandong Scotto Han Biotech Co., Ltd.), wherein the enzyme dosage is 0.625g (31250U/g substrate); 100ml of trisodium citrate buffer (pH 5) is added, the solid-to-liquid ratio is 1:50, and the enzymatic hydrolysis time is 24 h. Extracting 0.5ml of enzyme hydrolysate at 0, 1, 4, 8, 12, 24h, immediately putting into boiling water for 10min for inactivation after extraction, and centrifuging with a high speed centrifuge at 10000rpm for 10min after inactivation. After centrifugation, taking the supernatant for HPLC analysis, and quantifying the xylose content in the enzyme water solution. The final xylose yield per unit was calculated to be 15.43%, which is a large improvement compared to the 9.40% yield of the feedstock.

Example 2

Crushing corn straws, sieving the corn straws by using a 2mm sieve, taking 50g of the corn straws as a raw material for biomass pretreatment, adding the raw material into 500ml of 1% dilute sulfuric acid solution according to a solid-to-liquid ratio of 10%, reacting at 50 ℃ for 60min, after pretreatment, carrying out suction filtration by using a non-woven fabric, collecting filtrate and filter residue, washing the filter residue by using 1000m1 deionized water, and then carrying out HPLC analysis on the filtrate to evaluate the pretreatment effect; and drying the filter residue in an oven at 45 ℃ for 12 hours to remove water.

Taking 2g of the pretreated solid as an enzyme hydrolysis substrate, and carrying out enzyme hydrolysis by using acid xylanase (200000U/g of enzyme activity, Shandong Scotto Han Biotech Co., Ltd.), wherein the enzyme dosage is 0.625g (31250U/g substrate); 100ml of trisodium citrate buffer (pH 5) is added, the solid-to-liquid ratio is 1:50, and the enzymatic hydrolysis time is 24 h. Extracting 0.5ml of enzyme hydrolysate at 0, 1, 4, 8, 12, 24h, immediately putting into boiling water for 10min for inactivation after extraction, and centrifuging with a high speed centrifuge at 10000rpm for 10min after inactivation. After centrifugation, taking the supernatant for HPLC analysis, and quantifying the xylose content in the enzyme water solution. The final xylose yield was calculated to be 15.74% with a large improvement compared to the 9.40% yield of the feedstock.

Example 3

Crushing corn straws, sieving the corn straws by using a 2mm sieve, taking 50g of the corn straws as a raw material for biomass pretreatment, adding the raw material into 500ml of 1% dilute sulfuric acid solution according to a solid-to-liquid ratio of 10%, reacting at 70 ℃ for 60min, after pretreatment, carrying out suction filtration by using a non-woven fabric, collecting filtrate and filter residue, washing the filter residue by using 1000m1 deionized water, and then carrying out HPLC analysis on the filtrate to evaluate the pretreatment effect; and drying the filter residue in an oven at 45 ℃ for 12 hours to remove water.

Taking 2g of the pretreated solid as an enzyme hydrolysis substrate, and carrying out enzyme hydrolysis by using acid xylanase (200000U/g of enzyme activity, Shandong Scotto Han Biotech Co., Ltd.), wherein the enzyme dosage is 0.625g (31250U/g substrate); 100ml of trisodium citrate buffer (pH 5) is added, the solid-to-liquid ratio is 1:50, and the enzymatic hydrolysis time is 24 h. Extracting 0.5ml of enzyme hydrolysate at 0, 1, 4, 8, 12, 24h, immediately putting into boiling water for 10min for inactivation after extraction, and centrifuging with a high speed centrifuge at 10000rpm for 10min after inactivation. After centrifugation, taking the supernatant for HPLC analysis, and quantifying the xylose content in the enzyme water solution. The final xylose yield was calculated to be 28.51% with a large improvement compared to the 9.40% yield of the feedstock.

Example 4

Crushing corn straws, sieving the corn straws by using a 2mm sieve, taking 50g of the corn straws as a raw material for biomass pretreatment, adding the raw material into 500ml of 2% dilute sulfuric acid solution according to a solid-to-liquid ratio of 10%, reacting at 70 ℃ for 30min, after pretreatment, carrying out suction filtration by using a non-woven fabric, collecting filtrate and filter residue, washing the filter residue by using 1000m1 deionized water, and then carrying out HPLC analysis on the filtrate to evaluate the pretreatment effect; and drying the filter residue in an oven at 45 ℃ for 12 hours to remove water.

Taking 2g of the pretreated solid as an enzyme hydrolysis substrate, and carrying out enzyme hydrolysis by using acid xylanase (200000U/g of enzyme activity, Shandong Scotto Han Biotech Co., Ltd.), wherein the enzyme dosage is 0.625g (31250U/g substrate); 100ml of trisodium citrate buffer (pH 5) is added, the solid-to-liquid ratio is 1:50, and the enzymatic hydrolysis time is 24 h. Extracting 0.5ml of enzyme hydrolysate at 0, 1, 4, 8, 12, 24h, immediately putting into boiling water for 10min for inactivation after extraction, and centrifuging with a high speed centrifuge at 10000rpm for 10min after inactivation. After centrifugation, taking the supernatant for HPLC analysis, and quantifying the xylose content in the enzyme water solution. The final xylose yield per unit was calculated to be 24.82%, which is a large improvement compared to the 9.40% yield of the feedstock.

Example 5

Crushing corn straws, sieving the corn straws by using a 2mm sieve, taking 50g of the corn straws as a raw material for biomass pretreatment, adding the raw material into 500ml of 2% dilute sulfuric acid solution according to a solid-to-liquid ratio of 10%, reacting at 70 ℃ for 60min, after pretreatment, carrying out suction filtration by using a non-woven fabric, collecting filtrate and filter residue, washing the filter residue by using 1000m1 deionized water, and then carrying out HPLC analysis on the filtrate to evaluate the pretreatment effect; and drying the filter residue in an oven at 45 ℃ for 12 hours to remove water.

Taking 2g of the pretreated solid as an enzyme hydrolysis substrate, and carrying out enzyme hydrolysis by using acid xylanase (200000U/g of enzyme activity, Shandong Scotto Han Biotech Co., Ltd.), wherein the enzyme dosage is 0.625g (31250U/g substrate); 100ml of trisodium citrate buffer (pH 5) is added, the solid-to-liquid ratio is 1:50, and the enzymatic hydrolysis time is 24 h. Extracting 0.5ml of enzyme hydrolysate at 0, 1, 4, 8, 12, 24h, immediately putting into boiling water for 10min for inactivation after extraction, and centrifuging with a high speed centrifuge at 10000rpm for 10min after inactivation. After centrifugation, taking the supernatant for HPLC analysis, and quantifying the xylose content in the enzyme water solution. The final xylose yield was calculated to be 25.64% with a large improvement compared to the 9.40% yield of the feedstock.

Example 6

Crushing corn straws, sieving the corn straws by using a 2mm sieve, taking 50g of the corn straws as a raw material for biomass pretreatment, adding the raw material into 500ml of 2% dilute sulfuric acid solution according to a solid-to-liquid ratio of 10%, reacting at 70 ℃ for 120min, after pretreatment, carrying out suction filtration by using a non-woven fabric, collecting filtrate and filter residue, washing the filter residue by using 1000m1 deionized water, and then carrying out HPLC analysis on the filtrate to evaluate the pretreatment effect; and drying the filter residue in an oven at 45 ℃ for 12 hours to remove water.

Taking 2g of the pretreated solid as an enzyme hydrolysis substrate, and carrying out enzyme hydrolysis by using acid xylanase (200000U/g of enzyme activity, Shandong Scotto Han Biotech Co., Ltd.), wherein the enzyme dosage is 0.625g (31250U/g substrate); 100ml of trisodium citrate buffer (pH 5) is added, the solid-to-liquid ratio is 1:50, and the enzymatic hydrolysis time is 24 h. Extracting 0.5ml of enzyme hydrolysate at 0, 1, 4, 8, 12, 24h, immediately putting into boiling water for 10min for inactivation after extraction, and centrifuging with a high speed centrifuge at 10000rpm for 10min after inactivation. After centrifugation, taking the supernatant for HPLC analysis, and quantifying the xylose content in the enzyme water solution. The final xylose yield per unit was calculated to be 26.21%, which is a large improvement compared to the 9.40% yield of the feedstock.

Example 7

Crushing corn straws, sieving the corn straws by using a 2mm sieve, taking 50g of the corn straws as a raw material for biomass pretreatment, adding the raw material into 500ml of 2% dilute sulfuric acid solution according to a solid-to-liquid ratio of 10%, reacting at 70 ℃ for 240min, after pretreatment, carrying out suction filtration by using a non-woven fabric, collecting filtrate and filter residue, washing the filter residue by using 1000m1 deionized water, and then carrying out HPLC analysis on the filtrate to evaluate the pretreatment effect; and drying the filter residue in an oven at 45 ℃ for 12 hours to remove water.

Taking 2g of the pretreated solid as an enzyme hydrolysis substrate, and carrying out enzyme hydrolysis by using acid xylanase (200000U/g of enzyme activity, Shandong Scotto Han Biotech Co., Ltd.), wherein the enzyme dosage is 0.625g (31250U/g substrate); 100ml of trisodium citrate buffer (pH 5) is added, the solid-to-liquid ratio is 1:50, and the enzymatic hydrolysis time is 24 h. Extracting 0.5ml of enzyme hydrolysate at 0, 1, 4, 8, 12, 24h, immediately putting into boiling water for 10min for inactivation after extraction, and centrifuging with a high speed centrifuge at 10000rpm for 10min after inactivation. After centrifugation, taking the supernatant for HPLC analysis, and quantifying the xylose content in the enzyme water solution. The final xylose yield was calculated to be 30.85% with a large improvement compared to the 9.40% yield of the feedstock.

The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.