A method for preparing xylose or xylooligosaccharides using corn cobs
The corn cob was hydrolyzed by enzymatic method, combined with deacetylation and delignin treatment, and enzymatically lysed by composite enzymes, solving the problems of high acid and alkali consumption and large wastewater discharge in the traditional xylose and oligosaccharide production processes, achieving mild process and environmentally friendly and efficient production.
Patent Information
- Application Number
- CN202211576208.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-12-08
- Publication Date
- 2025-04-18
- Estimated Expiration
- 2042-12-08
AI Technical Summary
The existing xylose and oligosaccharide production processes have problems such as high acid and alkali consumption, large sewage discharge, long production cycle, high production costs, and unstable product quality.
Xylose or oligoxylose is prepared by enzymatic hydrolysis of corn cobs. After deacetylation and delignin treatment, complex plant hydrolase, cellulase and xylanase are added for enzymatic decomposition to obtain xylose or oligoxylose product solution.
The traditional acid-removal process has avoided the problems of high acid and alkali consumption and large sewage discharge, and achieved the effect of mild process conditions, low energy consumption, low environmental pollution, and safer and more reliable product quality.
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Figure CN115896205B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biochemical engineering, and in particular relates to a method for preparing xylose or xylooligosaccharides by utilizing corn cobs. Background Art
[0002] Xylose is a pentose sugar that appears as colorless to white crystals or powder, has a sweet taste, is easily soluble in water, and is difficult to dissolve in ethanol. In nature, xylose is the most abundant pentose sugar. In addition to the free xylose in bamboo shoots, most natural xylose is widely present in the hemicellulose of plants in the form of polypentose, especially in the waste parts of agricultural products.
[0003] Xylo-oligosaccharide is a functional oligosaccharide, which is composed of 2 to 7 xyloses connected by β-1,4 glycosidic bonds, with xylobiose and xylotriose as the main components. Xylo-oligosaccharide not only has a highly selective proliferation effect on intestinal probiotics such as bifidobacteria, lactobacilli and other probiotics, but also has an inhibitory effect on harmful bacteria such as Escherichia coli, so it is also called a super prebiotic.
[0004] At present, the industrial production process of xylose and xylooligosaccharides mainly uses corn cobs, bagasse, cottonseed hulls and other agricultural and forestry wastes rich in hemicellulose as raw materials. Among them, corn cobs have the highest content of pentose, are easy to store, have a relatively loose structure, and are easy to extract. Therefore, they are widely used in the production of xylose and xylooligosaccharides. Corn is a food and feed crop with abundant resources. In recent years, with the advancement of science and technology, my country's annual corn production has exceeded 100 million tons, ranking second in the world. According to statistics, my country's annual corn production in 2021 is about 273 million tons. Calculated based on the production of 1 kg of corn, 1 / 3 kg of corn cobs can be obtained at the same time, and my country's annual corn cob production is about 91 million tons, which is an extremely considerable output.
[0005] The traditional xylose and oligoxylose production process is a chemical method, which uses acid as a catalyst to hydrolyze the raw materials under high temperature conditions, and then removes harmful impurities such as acid, pigment, colloid, ash, etc. contained in the hydrolyzate through multiple processes such as neutralization, activated carbon decolorization, ion exchange, concentration, crystallization, etc., before obtaining xylose and oligoxylose products. According to statistics, 1 ton of finished xylose requires nearly 10 tons of corn cobs, 3 tons of acid, 2 tons of liquid alkali, 120-150 kilograms of activated carbon, 30-50 tons of steam, and more than 100m3 of water.
[0006] The existing xylose and xylooligosaccharide production process has many problems, such as complex process, high acid and alkali consumption, large sewage discharge, long production cycle, high production cost, unstable product quality, etc. In addition, the product yield is low and the loss is large during the crystallization process of xylose and xylooligosaccharide preparation, which also greatly increases the production cost of xylose and xylooligosaccharides. Summary of the invention
[0007] The invention aims to avoid the problems of high acid and alkali consumption, large sewage discharge, long production cycle, high production cost, unstable product quality, etc. in the traditional acid hydrolysis process for obtaining xylose and xylooligosaccharides.
[0008] The present invention provides a method for preparing xylose or xylooligosaccharides by using corn cobs, comprising the following steps: deacetylation and delignification treatment of the corn cobs, adding composite plant hydrolase Viscozyme L, enzymolysis, and then adding xylanase and cellulase for secondary enzymolysis to obtain a xylose product solution;
[0009] Alternatively, after the corn cob is subjected to deacetylation and delignification treatment, two enzymes, cellulase and xylanase, are added for enzymatic hydrolysis or two enzymes, Pentopan Mono BG and xylanase, are added for enzymatic hydrolysis to obtain a xylo-oligosaccharide product solution.
[0010] It is further defined that the steps of the deacetylation treatment are as follows: distilled water and alkali are added to the corn cob raw material, and the mixture is stirred at 600 rpm / min in a 70° C. water bath for 3 hours, and centrifuged after the reaction is completed to obtain a deacetylated raw material.
[0011] It is further defined that the mass ratio of corn cob to distilled water is 1:8; the alkali used is Ca(OH)2, and the dosage is 0.8 mmol / g corn cob.
[0012] It is further defined that the delignification treatment is as follows: distilled water and Na2SO3 are added to the deacetylated raw material, reacting at a high temperature of 120°C for 30 minutes, and centrifuging after the reaction to obtain a delignified raw material.
[0013] It is further defined that the mass ratio of the deacetylated raw material to distilled water is 1:10; and the amount of Na2SO3 used is 12% of the mass of the corn cob.
[0014] It is further defined that the conditions of the composite plant hydrolase Viscozyme L are as follows: the temperature is controlled at 50°C and the pH is 3.5, the enzyme is inactivated after 12 hours of enzymolysis, and the addition amount of the composite plant hydrolase Viscozyme L is 0.33% of the mass of the corn cob raw material; the conditions of the secondary enzymolysis are as follows: the temperature is controlled at 50°C and the pH is 4.8, the enzyme is inactivated after 36 to 48 hours of enzymolysis, the amount of xylanase and cellulase is each 0.33% of the mass of the corn cob raw material, and the usage ratio of the composite plant hydrolase Viscozyme L: xylanase: cellulase is 1:1:1; the total amount of enzymes is 0.99%.
[0015] It is further defined that the conditions for the combined enzymatic hydrolysis of cellulase and xylanase or the enzymatic hydrolysis of pentosanase Pentopan Mono BG and xylanase are as follows: the temperature is controlled at 50°C and the pH is 4.8, and the enzymes are inactivated after 24 to 48 hours of enzymatic hydrolysis; the dosage of xylanase and cellulase is 0.5% of the mass of the corn cob raw material, the dosage ratio of the two enzymes is 1:1, and the total dosage of xylanase and cellulase is 1%; the dosage of pentosanase Pentopan Mono BG and xylanase is 0.5% of the mass of the corn cob raw material, the dosage ratio of the two enzymes is 1:1, and the total dosage of pentosanase Pentopan Mono BG and xylanase is 1%.
[0016] It is further defined that the enzyme inactivation condition is 85° C. for 15 min.
[0017] The present invention provides xylose or xylooligosaccharide obtained by the above method.
[0018] The present invention provides application of the above method in improving the yield and concentration of xylose or xylooligosaccharides.
[0019] Beneficial effects:
[0020] 1. The present invention adopts enzymatic hydrolysis of corn cobs to produce xylose or xylooligosaccharides, avoiding the problems of high acid and alkali consumption, large sewage discharge, long production cycle, high production cost, unstable product quality, etc. in the traditional acid hydrolysis process, and has the advantages of mild process conditions, low energy consumption, low environmental pollution, and safer and more reliable product quality.
[0021] 2. The present invention performs deacetylation and delignification treatment on corn cobs, and its beneficial effects are: ① Acetyl groups attach to hemicellulose to form steric hindrance, which hinders the enzyme from entering the raw material system. After deacetylation, the enzyme can smoothly contact the target, thereby improving the enzymatic hydrolysis rate. ② Delignification destroys the dense tissue formed by cellulose, hemicellulose and lignin in the corn cob, and the structure becomes loose, which is more conducive to the enzyme acting on hemicellulose, thereby improving the enzymatic hydrolysis rate.
[0022] 3. Hemicellulose is difficult to be hydrolyzed by a single enzyme. The present invention uses multiple enzymes to perform composite enzymolysis on corn cobs. By changing the combination of different enzymes, the content of xylose or oligoxylose in the product can be regulated. When composite plant hydrolyzing enzymes, cellulase and xylanase are used for composite enzymolysis, hemicellulose is hydrolyzed more thoroughly, and most of the products are xylose. The hydrolysis rate of hemicellulose is above 95%, the yield of xylose can reach up to 81.6%, and the concentration of xylose solution can reach up to 38.8g / L. When composite enzymolysis of cellulase and xylanase, or composite enzymolysis of pentosanase and xylanase, most of the products are oligoxylose, the hydrolysis rate of hemicellulose is above 90%, the yield of oligoxylose can reach up to 86.7%, and the concentration of oligoxylose in the product solution can reach up to 41.2g / L.
[0023] Purpose of pretreatment: The cellulose, hemicellulose and lignin components in corn cobs are discontinuous layered structures, which hinder the hydrolysis of enzymes. In order to fully hydrolyze hemicellulose, the corn cobs must be properly pretreated to destroy their original dense structure to facilitate further hydrolysis by enzymes.
[0024] Pretreatment process: ① Deacetylation: Acetyl groups attach to hemicellulose, forming steric hindrance, which hinders the enzyme from entering the raw material system. After deacetylation, the enzyme can smoothly contact the target. ② Delignification: Remove lignin from the raw material, the raw material structure becomes loose, making it easier for the enzyme to act on xylan.
[0025] Principle of enzymatic hydrolysis process: Hemicellulose is difficult to be hydrolyzed by a single enzyme, and the expected production effect can be achieved only when multiple enzymes work together. The present invention utilizes plant composite hydrolases, xylanases and cellulases for combined hydrolysis. Composite plant hydrolases have the ability to release restraints and degrade non-starch polysaccharides, and can be used to decompose plant cell walls to release effective ingredients from plant tissues. Cellulase has the function of removing cellulose from plant cell walls, and can release sugars in cells. Xylanase can degrade xylan in hemicellulose into xylose or xylooligosaccharides. The present invention uses a combination of different enzymes to enzymatically hydrolyze corn cobs, and can regulate the content of xylose or xylooligosaccharides in the enzymatic hydrolyzate, thereby achieving efficient production of xylose or xylooligosaccharides. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 This is the SEM photo of the untreated raw material;
[0027] Figure 2 This is the SEM photo of the deacetylated raw material;
[0028] Figure 3 This is the SEM photo of delignified raw material. DETAILED DESCRIPTION
[0029] Example 1.
[0030] A method for preparing xylose by enzymatic hydrolysis of corn cobs comprises the following steps:
[0031] (1) Weigh 100 g corn cobs, crush them, pass them through an 80-mesh sieve, and wash the resulting corn cob powder with clean water to remove dust from the raw material;
[0032] (2) Deacetylation: Add 800 mL of distilled water and 5.92 g of Ca(OH)2 to the corncob raw material, stir and react at 600 rpm / min in a 70°C water bath for 3 h, and centrifuge after the reaction to obtain a deacetylated raw material; the amount of Ca(OH)2 used is 80 mmol Ca(OH)2 / 100 g corncob = 0.8 mmol / g corncob;
[0033] (3) Delignification: Add 1L of distilled water and 12g of Na2SO3 to the deacetylated raw material, and react at 120°C for 30min. Centrifuge after the reaction to obtain a delignified raw material; the amount of Na2SO3 used is 12gNa2SO3 / 100g corn cob = 12% of the mass of the corn cob;
[0034] (4) Enzymatic hydrolysis: After the pretreatment, 800 L of distilled water was added to the corn cob raw material, and 0.33 g of composite plant hydrolase Viscozyme L (Novozymes) was added. The temperature was controlled at 50° C. and pH 3.5. The enzyme was inactivated after 12 h of enzymatic hydrolysis. Then, 0.33 g of xylanase (Jiangsu Ruiyang Biotechnology Co., Ltd.) and 0.33 g of cellulase (Jiangsu Ruiyang Biotechnology Co., Ltd.) were added for enzymatic hydrolysis. The temperature was controlled at 50° C. and pH 4.8. The enzymatic hydrolysis time was 48 h. The dosage of composite plant hydrolase Viscozyme L was 0.33 g enzyme / 100 g corn cob = 0.33% of the mass of the corn cob raw material.
[0035] The dosage of xylanase is 0.33g enzyme / 100g corncob = 0.33% of the mass of corncob raw material, the dosage of cellulase is 0.33g enzyme / 100g corncob = 0.33% of the mass of corncob raw material, and the total dosage of enzyme is 0.99g enzyme
[0036] / 100g corncob raw material = 0.99% of the mass of corncob raw material;
[0037] (5) After the enzymatic hydrolysis is completed, the enzyme is inactivated at 85°C for 15 min and the solution is centrifuged to obtain a xylose product solution.
[0038] Example 2
[0039] A method for preparing xylose by enzymatic hydrolysis of corn cobs comprises the following steps:
[0040] (1) Weigh 500 g corn cobs, crush them, pass them through a 90-mesh sieve, and wash the resulting corn cob powder with clean water to remove dust from the raw material;
[0041] (2) Deacetylation: Add 4 L of distilled water to the corncob raw material, add 29.6 g of Ca(OH)2, stir and react at 600 rpm / min in a 70°C water bath for 3 h, centrifuge after the reaction to obtain a deacetylated raw material; the amount of Ca(OH)2 used is 0.4 mol Ca(OH)2 / 500 g corncob = 0.8 mmol / g corncob;
[0042] (3) Delignification: Add 5L of distilled water and 60g of Na2SO3 to the deacetylated raw material, and react at 120°C for 30min. Centrifuge after the reaction to obtain a delignified raw material; the amount of Na2SO3 used is 60gNa2SO3 / 500g corn cob = 12% of the mass of the corn cob;
[0043] (4) Enzymatic hydrolysis: After the pretreatment, 5 L of distilled water was added to the corn cob raw material, and 1.67 g of composite plant hydrolase Viscozyme L (Novozymes) was added. The temperature was controlled at 50° C. and pH 3.5. The enzyme was inactivated after 12 h of enzymatic hydrolysis. Then, 1.67 g of xylanase (Jiangsu Ruiyang Biotechnology Co., Ltd.) and 1.67 g of cellulase (Jiangsu Ruiyang Biotechnology Co., Ltd.) were added for enzymatic hydrolysis. The temperature was controlled at 50° C. and pH 4.8. The enzymatic hydrolysis time was 36 h. The dosage of composite plant hydrolase Viscozyme L was 1.67 g enzyme / 500 g corn cob = 0.33% of the mass of the corn cob raw material.
[0044] The dosage of xylanase is 1.67 g enzyme / 500 g corncob = 0.33% of the mass of corncob raw material, the dosage of cellulase is 1.67 g enzyme / 500 g corncob = 0.33% of the mass of corncob raw material, and the total dosage of enzyme is 5.01 g enzyme
[0045] / 500g corn cob raw material = 0.99% of the mass of corn cob raw material;
[0046] (5) After the enzymatic hydrolysis is completed, the enzyme is inactivated at 85°C for 15 min and the solution is centrifuged to obtain a xylose product solution.
[0047] Example 3
[0048] A method for preparing xylose by enzymatic hydrolysis of corn cobs comprises the following steps:
[0049] (1) Weigh 1 kg of corn cobs, crush them, pass them through a 100-mesh sieve, and wash the resulting corn cob powder with clean water to remove dust from the raw material;
[0050] (2) Deacetylation: Add 8 L of distilled water to the corn cob raw material, add 59.2 g of Ca(OH)2, stir and react at 600 rpm / min in a 70°C water bath for 3 h, centrifuge after the reaction to obtain a deacetylated raw material; the amount of Ca(OH)2 used is 0.8 mol Ca(OH)2 / 1000 g corn cob = 0.8 mmol / g corn cob;
[0051] (3) Delignification: Add 10L of distilled water and 120g of Na2SO3 to the deacetylated raw material, and react at 120°C for 30min. Centrifuge after the reaction to obtain a delignified raw material; the amount of Na2SO3 used is 120gNa2SO3 / 1000g corn cob = 12% of the mass of the corn cob;
[0052] (4) Enzymatic hydrolysis: After the pretreatment, 9 L of distilled water was added to the corn cob raw material, and 3.33 g of the composite plant hydrolase Viscozyme L (Novozymes) was added. The temperature was controlled at 50 ° C and pH 3.5. The enzyme was inactivated after 12 h of enzymatic hydrolysis. Then, 3.33 g of xylanase (Jiangsu Ruiyang Biotechnology Co., Ltd.) and 3.33 g of cellulase (Jiangsu Ruiyang Biotechnology Co., Ltd.) were added for enzymatic hydrolysis. The temperature was controlled at 50 ° C and pH 4.8. The enzymatic hydrolysis time was 42 h. The composite plant hydrolase Viscozyme L was added. The total dosage of L is 3.33g enzyme / 1000g corn cob = 0.33% of the mass of corn cob raw material; the dosage of xylanase is 3.33g enzyme / 1000g corn cob = 0.33% of the mass of corn cob raw material, the dosage of cellulase is 3.33g enzyme / 1000g corn cob = 0.33% of the mass of corn cob raw material, and the total dosage of enzymes is 9.99g enzyme / 1000g corn cob raw material = 0.999% of the mass of corn cob raw material;
[0053] (5) After the enzymatic hydrolysis is completed, the enzyme is inactivated at 85°C for 15 min and the solution is centrifuged to obtain a xylose product solution.
[0054] Example 4
[0055] A method for preparing xylo-oligosaccharides by enzymatic hydrolysis of corncobs comprises the following steps:
[0056] (1) Weigh 100 g corn cobs, crush them, pass them through an 80-mesh sieve, and wash the resulting corn cob powder with clean water to remove dust from the raw material;
[0057] (2) Deacetylation: Add 800 mL of distilled water and 5.92 g of Ca(OH)2 to the corncob raw material, stir and react at 600 rpm / min in a 70°C water bath for 3 h, and centrifuge after the reaction to obtain a deacetylated raw material; the amount of Ca(OH)2 used is 80 mmol Ca(OH)2 / 100 g corncob = 0.8 mmol / g corncob;
[0058] (3) Delignification: Add 1L of distilled water and 12g of Na2SO3 to the deacetylated raw material, and react at 120°C for 30min. Centrifuge after the reaction to obtain a delignified raw material; the amount of Na2SO3 used is 12gNa2SO3 / 100g corn cob = 12% of the mass of the corn cob;
[0059] (4) Enzymolysis: After the pretreatment, 800 L of distilled water was added to the corn cob raw material, and 0.5 g of xylanase (Jiangsu Ruiyang Biotechnology Co., Ltd.) and 0.5 g of cellulase (Jiangsu Ruiyang Biotechnology Co., Ltd.) were added for enzymolysis. The temperature was controlled at 50° C. and pH 4.8, and the enzymolysis time was 36 h. The amount of xylanase was 0.5 g enzyme / 100 g corn cob = 0.5% of the mass of the corn cob raw material, the amount of cellulase was 0.5 g enzyme / 100 g corn cob = 0.5% of the mass of the corn cob raw material, and the total amount of xylanase and cellulase was 1 g enzyme / 100 g corn cob raw material = 1% of the mass of the raw material;
[0060] (5) After the enzymatic hydrolysis is completed, the enzyme is inactivated at 85°C for 15 minutes and the solution is centrifuged to obtain a xylo-oligosaccharide product solution.
[0061] Example 5
[0062] A method for preparing xylo-oligosaccharides by enzymatic hydrolysis of corncobs comprises the following steps:
[0063] (1) Weigh 500 g corn cobs, crush them, pass them through a 90-mesh sieve, and wash the resulting corn cob powder with clean water to remove dust from the raw material;
[0064] (2) Deacetylation: Add 4 L of distilled water to the corncob raw material, add 29.6 g of Ca(OH)2, stir and react at 600 rpm / min in a 70°C water bath for 3 h, centrifuge after the reaction to obtain a deacetylated raw material; the amount of Ca(OH)2 used is 0.4 mol Ca(OH)2 / 500 g corncob = 0.8 mmol / g corncob;
[0065] (3) Delignification: Add 5L of distilled water and 60g of Na2SO3 to the deacetylated raw material, and react at 120°C for 30min. Centrifuge after the reaction to obtain a delignified raw material; the amount of Na2SO3 used is 60gNa2SO3 / 500g corn cob = 12% of the mass of the corn cob;
[0066] (4) Enzymolysis: After the pretreatment, 5 L of distilled water was added to the corn cob raw material, and 2.5 g of xylanase (Jiangsu Ruiyang Biotechnology Co., Ltd.) and 2.5 g of cellulase (Jiangsu Ruiyang Biotechnology Co., Ltd.) were added for enzymolysis. The temperature was controlled at 50° C. and pH 4.8, and the enzymolysis time was 24 h. The amount of xylanase was 2.5 g enzyme / 500 g corn cob = 0.5% of the mass of the corn cob raw material, the amount of cellulase was 2.5 g enzyme / 500 g corn cob = 0.5% of the mass of the corn cob raw material, and the total amount of xylanase and cellulase was 5 g enzyme / 500 g corn cob raw material = 1% of the mass of the raw material;
[0067] (5) After the enzymatic hydrolysis is completed, the enzyme is inactivated at 85°C for 15 minutes and the solution is centrifuged to obtain a xylo-oligosaccharide product solution.
[0068] Example 6
[0069] A method for preparing xylo-oligosaccharides by enzymatic hydrolysis of corncobs comprises the following steps:
[0070] (1) Weigh 1 kg of corn cobs, crush them, pass them through a 100-mesh sieve, and wash the resulting corn cob powder with clean water to remove dust from the raw material;
[0071] (2) Deacetylation: Add 8 L of distilled water to the corn cob raw material, add 59.2 g of Ca(OH)2, stir and react at 600 rpm / min in a 70°C water bath for 3 h, centrifuge after the reaction to obtain a deacetylated raw material; the amount of Ca(OH)2 used is 0.8 mol Ca(OH)2 / 1000 g corn cob = 0.8 mmol / g corn cob;
[0072] (3) Delignification: Add 10L of distilled water and 120g of Na2SO3 to the deacetylated raw material, and react at 120°C for 30min. Centrifuge after the reaction to obtain a delignified raw material; the amount of Na2SO3 used is 120gNa2SO3 / 1000g corn cob = 12% of the mass of the corn cob;
[0073] (4) Enzymolysis: After the pretreatment, the corn cob raw material was added with 9 L of distilled water, and 5 g of xylanase (Jiangsu Ruiyang Biotechnology Co., Ltd.) and 5 g of cellulase (Jiangsu Ruiyang Biotechnology Co., Ltd.) were added for enzymolysis. The temperature was controlled at 50° C. and pH 4.8, and the enzymolysis time was 48 h. The amount of xylanase was 5 g enzyme / 1000 g corn cob = 0.5% of the mass of the corn cob raw material, the amount of cellulase was 5 g enzyme / 1000 g corn cob = 0.5% of the mass of the corn cob raw material, and the total amount of xylanase and cellulase was 10 g enzyme / 1000 g corn cob raw material = 1% of the mass of the raw material.
[0074] (5) After the enzymatic hydrolysis is completed, the enzyme is inactivated at 85°C for 15 minutes and the solution is centrifuged to obtain a xylo-oligosaccharide product solution.
[0075] Example 7
[0076] A method for preparing xylo-oligosaccharides by enzymatic hydrolysis of corncobs comprises the following steps:
[0077] (1) Weigh 100 g corn cobs, crush them, pass them through an 80-mesh sieve, and wash the resulting corn cob powder with clean water to remove dust from the raw material;
[0078] (2) Deacetylation: Add 800 mL of distilled water to the corncob raw material, add 5.92 g of Ca(OH)2, and stir in a 70°C water bath at 600 rpm / min for 3 h. After the reaction, centrifuge to obtain a deacetylated raw material; the amount of Ca(OH)2 used is 80 mmol Ca(OH)2 / 100 g corncob = 0.8 mmol / g corncob.
[0079] (3) Delignification: Add 1L of distilled water and 12g of Na2SO3 to the deacetylated raw material, and react at 120°C for 30min. Centrifuge after the reaction to obtain a delignified raw material; the amount of Na2SO3 used is 12gNa2SO3 / 100g corn cob = 12% of the mass of the corn cob;
[0080] (4) Enzymatic hydrolysis: After the pretreatment, the corn cob raw material was added with 800 L of distilled water, and 0.5 g of xylanase (Jiangsu Ruiyang Biotechnology Co., Ltd.) and 0.5 g of pentosanase Pentopan Mono BG (Novozymes) were added for enzymatic hydrolysis. The temperature was controlled at 50° C. and pH 4.8. The enzymatic hydrolysis time was 36 h. The amount of xylanase was 0.5 g enzyme / 100 g corn cob = 0.5% of the mass of the corn cob raw material, and the amount of pentosanase Pentopan Mono BG was 0.5 g enzyme / 100 g corn cob = 0.5% of the mass of the corn cob raw material.
[0081] / 100g corncob = 0.5% of the mass of corncob raw material, the total amount of pentosanase Pentopan Mono BG and xylanase is 1g enzyme / 100g corncob raw material = 1% of the mass of raw material;
[0082] (5) After the enzymatic hydrolysis is completed, the enzyme is inactivated at 85°C for 15 minutes and the solution is centrifuged to obtain a xylo-oligosaccharide product solution.
[0083] Example 8
[0084] A method for preparing xylo-oligosaccharides by enzymatic hydrolysis of corncobs comprises the following steps:
[0085] (1) Weigh 500 g corn cobs, crush them, pass them through a 90-mesh sieve, and wash the resulting corn cob powder with clean water to remove dust from the raw material;
[0086] (2) Deacetylation: Add 4 L of distilled water to the corncob raw material, add 29.6 g of Ca(OH)2, stir and react at 600 rpm / min in a 70°C water bath for 3 h, centrifuge after the reaction to obtain a deacetylated raw material; the amount of Ca(OH)2 used is 0.4 mol Ca(OH)2 / 500 g corncob = 0.8 mmol / g corncob;
[0087] (3) Delignification: Add 5L of distilled water and 60g of Na2SO3 to the deacetylated raw material, and react at 120°C for 30min. Centrifuge after the reaction to obtain a delignified raw material; the amount of Na2SO3 used is 60gNa2SO3 / 500g corn cob = 12% of the mass of the corn cob;
[0088] (4) Enzymolysis: After the pretreatment, 5 L of distilled water was added to the corn cob raw material, and 2.5 g of xylanase (Jiangsu Ruiyang Biotechnology Co., Ltd.) and 2.5 g of pentosanase Pentopan Mono BG (Novozymes) were added for enzymolysis. The temperature was controlled at 50° C. and pH 4.8, and the enzymolysis time was 24 h. The amount of xylanase was 2.5 g enzyme / 500 g corn cob = 0.5% of the mass of the corn cob raw material, the amount of pentosanase Pentopan Mono BG was 2.5 g enzyme / 500 g corn cob = 0.5% of the mass of the corn cob raw material, and the total amount of pentosanase Pentopan Mono BG and xylanase was 5 g enzyme / 500 g corn cob raw material = 1% of the mass of the raw material;
[0089] (5) After the enzymatic hydrolysis is completed, the enzyme is inactivated at 85°C for 15 minutes and the solution is centrifuged to obtain a xylo-oligosaccharide product solution.
[0090] Example 9
[0091] A method for preparing xylo-oligosaccharides by enzymatic hydrolysis of corncobs comprises the following steps:
[0092] (1) Weigh 1 kg of corn cobs, crush them, pass them through a 100-mesh sieve, and wash the resulting corn cob powder with clean water to remove dust from the raw material;
[0093] (2) Deacetylation: Add 8 L of distilled water to the corn cob raw material, add 59.2 g of Ca(OH)2, stir and react at 600 rpm / min in a 70°C water bath for 3 h, centrifuge after the reaction to obtain a deacetylated raw material; the amount of Ca(OH)2 used is 0.8 mol Ca(OH)2 / 1000 g corn cob = 0.8 mmol / g corn cob;
[0094] (3) Delignification: Add 10L of distilled water and 120g of Na2SO3 to the deacetylated raw material, and react at 120°C for 30min. Centrifuge after the reaction to obtain a delignified raw material; the amount of Na2SO3 used is 120gNa2SO3 / 1000g corn cob = 12% of the mass of the corn cob;
[0095] (4) Enzymatic hydrolysis: After the pretreatment, the corn cob raw material was added with 9 L of distilled water, 5 g of xylanase (Jiangsu Ruiyang Biotechnology Co., Ltd.) and 5 g of pentosanase Pentopan Mono BG (Novozymes) for enzymatic hydrolysis. The temperature was controlled at 50 °C and pH 4.8. The enzymatic hydrolysis time was 48 h. The amount of xylanase used was 5 g enzyme / 1000 g corn cob.
[0096] = 0.5% of the mass of corn cob raw material, the amount of pentosanase Pentopan Mono BG is 5g enzyme / 1000g corn cob = 0.5% of the mass of corn cob raw material, the total amount of pentosanase Pentopan Mono BG and xylanase is 10g enzyme / 1000g corn cob raw material = 1% of the mass of raw material;
[0097] (5) After the enzymatic hydrolysis is completed, the enzyme is inactivated at 85°C for 15 minutes and the solution is centrifuged to obtain a xylo-oligosaccharide product solution.
[0098] Example 10
[0099] The microstructural characterization of the unprocessed corncob raw material includes the following steps:
[0100] (1) Weigh 10 g corn cob, crush it, pass it through a 100-mesh sieve, wash the resulting corn cob powder with clean water to remove dust from the raw material, and dry it;
[0101] (2) Take a small amount of untreated raw material for SEM characterization. The results are as follows Figure 1 shown.
[0102] The structure of the untreated raw material is regular and dense, and the active sites of hemicellulase are wrapped in the dense structure.
[0103] Embodiment 11
[0104] The microstructural characterization of the deacetylated corncob raw material includes the following steps:
[0105] (1) Weigh 10 g corn cob, crush it, pass it through a 100-mesh sieve, and wash the resulting corn cob powder with clean water to remove dust from the raw material;
[0106] (2) Deacetylation: Add 80 mL of distilled water and 0.592 g of Ca(OH)2 to the corncob raw material, stir at 600 rpm / min in a 70°C water bath for 3 h, centrifuge after the reaction, and dry to obtain the deacetylated raw material;
[0107] (3) Take a small amount of deacetylated raw material for SEM characterization. The results are as follows Figure 2 shown.
[0108] The raw material structure after deacetylation is loose and broken into microscopic flake structures.
[0109] Example 12
[0110] The microstructural characterization of delignified corncob raw materials includes the following steps:
[0111] (1) Weigh 10 g corn cob, crush it, pass it through a 100-mesh sieve, and wash the resulting corn cob powder with clean water to remove dust from the raw material;
[0112] (2) Deacetylation: Add 80 mL of distilled water and 0.592 g of Ca(OH)2 to the corncob raw material, stir at 600 rpm / min in a 70°C water bath for 3 h, and centrifuge after the reaction to obtain a deacetylated raw material;
[0113] (3) Delignification: Add 100 mL of distilled water and 1.2 g of Na2SO3 to the deacetylated raw material, and react at 120°C for 30 min. After the reaction, centrifuge and dry to obtain a delignified raw material.
[0114] (4) Take a small amount of delignified raw material for SEM characterization, the results are as follows Figure 3 shown.
[0115] After the removal of lignin, the raw materials have a large number of porous gaps in the structure, which completely destroys the original dense structure of the raw materials, fully exposes the cellulose and hemicellulose structures in the raw materials, further increases the contact area between the enzyme and the raw materials, and is conducive to enzymatic hydrolysis and saccharification.
[0116] Comparative Example 1
[0117] Referring to the method for preparing xylose in Example 1, the order of deacetylation and delignification was changed.
[0118] Comparative Example 2
[0119] Referring to the method for preparing xylose in Example 1, the hydrolysis step of the composite plant hydrolase Viscozyme L was omitted.
[0120] Comparative Example 3
[0121] Referring to the method for preparing xylose in Example 1, the total amount of enzyme added remained unchanged, and the ratio of enzyme addition was replaced by composite plant hydrolase Viscozyme L: xylanase: cellulase = 2:1:1.
[0122] Comparative Example 4
[0123] Referring to the method for preparing xylose in Example 1, the total amount of enzyme added remained unchanged, and the ratio of enzyme added was replaced by composite plant hydrolase Viscozyme L: xylanase: cellulase = 1:2:1.
[0124] Comparative Example 5
[0125] Referring to the method for preparing xylose in Example 1, the total amount of enzyme added remained unchanged, and the ratio of enzyme addition was replaced by composite plant hydrolase Viscozyme L: xylanase: cellulase = 1:1:2.
[0126] Comparative Example 6
[0127] Referring to the method for preparing xylose in Example 1, the xylanase (Jiangsu Ruiyang Biotechnology Co., Ltd.) was replaced with xylanase from another manufacturer (Manufacturer A).
[0128] Comparative Example 7
[0129] Referring to the method for preparing xylose in Example 1, the xylanase (Jiangsu Ruiyang Biotechnology Co., Ltd.) was replaced with xylanase from another manufacturer (Manufacturer B).
[0130] Comparative Example 8
[0131] Referring to the method for preparing xylose in Example 1, the cellulase (Jiangsu Ruiyang Biotechnology Co., Ltd.) was replaced with cellulase from another manufacturer (Manufacturer C).
[0132] Comparative Example 9
[0133] Referring to the method for preparing xylose in Example 1, the cellulase (Jiangsu Ruiyang Biotechnology Co., Ltd.) was replaced with cellulase from another manufacturer (Manufacturer D).
[0134] Comparative Example 10
[0135] Referring to the method for preparing xylo-oligosaccharides in Example 4, the total amount of enzyme added remained unchanged, and the ratio of enzyme added was replaced by xylanase:cellulase=2:1.
[0136] Comparative Example 11
[0137] Referring to the method for preparing xylo-oligosaccharides in Example 4, the total amount of enzyme added remained unchanged, and the ratio of enzyme added was replaced by xylanase:cellulase=1:2.
[0138] Comparative Example 12
[0139] Referring to the method for preparing xylo-oligosaccharides in Example 7, the total amount of enzyme added remained unchanged, and the ratio of enzyme added was replaced by xylanase:pentosanase=2:1.
[0140] Comparative Example 13
[0141] Referring to the method for preparing xylo-oligosaccharides in Example 7, the total amount of enzyme added remained unchanged, and the ratio of enzyme addition was replaced by xylanase:pentosanase=1:2.
[0142] Describe the experimental verification of the invention effect:
[0143] 1. Composition analysis of the xylose product solutions obtained in Examples 1-3 and Comparative Examples 1-9
[0144] Test method: Use high performance liquid chromatography to analyze the components in the product solution and calculate the main indicators, including hemicellulose hydrolysis rate, xylose yield, and xylose concentration.
[0145] Hemicellulose hydrolysis rate formula = (hemicellulose content of raw material - hemicellulose content of enzymatic residue) / hemicellulose content of raw material;
[0146] Xylose yield formula = xylose yield / raw material hemicellulose content;
[0147] Table 1 Xylose product solution data
[0148]
[0149]
[0150] 1. By comparing the data of Example 1 with that of Comparative Example 1, it can be found that after exchanging the order of deacetylation and delignification in the pretreatment, the yield of xylose decreased by nearly 40%. Removing lignin first will make the structure of the corn cob raw material loose, resulting in a large amount of hemicellulose loss in the subsequent deacetylation process, thereby reducing the yield. Therefore, deacetylation first and then delignification is a necessary pretreatment order.
[0151] 2. By comparing the data of Example 1 with that of Comparative Example 2, it can be found that the yield of xylose is greatly reduced after the enzymatic hydrolysis with the composite plant hydrolase is omitted, indicating that the enzymatic hydrolysis with the composite plant hydrolase is an essential process.
[0152] 3. By comparing the data of Example 1 with Comparative Examples 3, 4 and 5, it can be found that when the enzyme addition ratio is changed, the xylose yield decreases to varying degrees, indicating that when the enzyme addition ratio is 1:1:1, the raw material can be hydrolyzed to the greatest extent and the xylose yield is also the highest.
[0153] 4. By comparing the data of Example 1 with Comparative Examples 6, 7, 8 and 9, it can be found that after replacing the original xylanase (Jiangsu Ruiyang Biotechnology Co., Ltd.) or cellulase (Jiangsu Ruiyang Biotechnology Co., Ltd.), the xylose production decreased to varying degrees, indicating that the enzymatic hydrolysis effect of the original xylanase and cellulase was better and the xylose production was also higher.
[0154] 2. Composition Analysis of the Xylo-oligosaccharide Product Solutions Obtained from Examples 4-9 and Comparative Examples 10-13
[0155] Test method: Use high performance liquid chromatography to analyze the components in the product solution and calculate the main indicators, including hemicellulose hydrolysis rate, xylooligosaccharide yield, and xylooligosaccharide concentration.
[0156] Hemicellulose hydrolysis rate formula = (hemicellulose content of raw material - hemicellulose content of enzymatic residue) / hemicellulose content of raw material;
[0157] The formula of xylooligosaccharide yield = xylooligosaccharide yield / hemicellulose content of raw materials;
[0158] Table 2 Xylo-oligosaccharide product solution data
[0159]
[0160]
[0161] 1. By comparing the data of Example 4 with those of Comparative Examples 10 and 11, it can be found that when the addition ratio of xylanase to cellulase is changed, the yield of xylo-oligosaccharides decreases to varying degrees, indicating that when the enzyme addition ratio is 1:1, the raw material can be hydrolyzed to the greatest extent and the yield of xylo-oligosaccharides is also the highest.
[0162] 2. By comparing the data of Example 7 with Comparative Examples 12 and 13, it can be found that when the addition ratio of xylanase to pentosanase is changed, the yield of xylooligosaccharides decreases to varying degrees, indicating that when the enzyme addition ratio is 1:1, the raw material can be hydrolyzed to the greatest extent and the yield of xylooligosaccharides is also the highest.
Claims
1. A method for preparing xylose or xylooligosaccharides using corncobs, characterized in that: The method comprises the following steps: after deacetylation and delignification treatment of corn cobs, adding composite plant hydrolase Viscozyme L, after enzymolysis, adding xylanase and cellulase for secondary enzymolysis to obtain a xylose product solution; Alternatively, after deacetylation and delignification treatment of corn cobs, two enzymes of cellulase and xylanase are added for enzymatic hydrolysis or two enzymes of pentopan Mono BG and xylanase are added for enzymatic hydrolysis to obtain a xylo-oligosaccharide product solution; The deacetylation treatment is as follows: distilled water and alkali are added to the corncob raw material, and the mixture is stirred at 600 rpm in a 70° C. water bath for 3 h, and centrifuged after the reaction to obtain a deacetylated raw material; The delignification treatment is as follows: adding distilled water and Na2SO3 to the deacetylated raw material, reacting at a high temperature of 120°C for 30 minutes, and centrifuging after the reaction to obtain a delignified raw material; The conditions for the enzymatic hydrolysis of the composite plant hydrolase Viscozyme L are as follows: the temperature is controlled at 50° C. and pH 3.5, the enzyme is inactivated after 12 hours of enzymatic hydrolysis, and the addition amount of the composite plant hydrolase Viscozyme L is 0.33% of the mass of the corn cob raw material; The conditions of the secondary enzymolysis are as follows: the temperature is controlled at 50°C and the pH is 4.8, the enzyme is inactivated after 36-48 hours of enzymolysis, the dosage of xylanase and cellulase is 0.33% of the mass of the corn cob raw material, the dosage ratio of composite plant hydrolase Viscozyme L: xylanase: cellulase is 1:1:1; the total dosage of the enzyme is 0.99%; The conditions for enzymolysis of the cellulase and xylanase or enzymolysis of the pentosanase Pentopan Mono BG and xylanase are as follows: the temperature is controlled at 50°C and the pH is 4.8, and the enzymes are inactivated after enzymolysis for 24 to 48 hours; the dosage of the xylanase and the cellulase is 0.5% of the mass of the corn cob raw material, the dosage ratio of the two enzymes is 1:1, and the total dosage of the xylanase and the cellulase is 1%; the dosage of the pentosanase Pentopan Mono BG and the xylanase is 0.5% of the mass of the corn cob raw material, the dosage ratio of the two enzymes is 1:1, and the total dosage of the pentosanase Pentopan Mono BG and the xylanase is 1%.
2. The method according to claim 1, characterized in that The mass ratio of corn cob to distilled water during the deacetylation process was 1:8; the alkali used was Ca(OH)2, and the dosage was 0.8 mmol / g corn cob.
3. The method according to claim 1, characterized in that The mass ratio of the deacetylated raw material to distilled water is 1:10; the amount of Na2SO3 used is 12% of the mass of the corn cob.
4. The method according to claim 1, characterized in that: The enzyme inactivation conditions were all 85° C. for 15 min.
5. Use of the method according to any one of claims 1 to 4 in improving the yield and concentration of xylose or xylooligosaccharides.