Method for improving yield of glucuronic acid in Tempo-enzyme hydrolysis-electrodialysis combined process

Abstract

The invention discloses a method for improving the yield of glucuronic acid in a Tempo-enzyme hydrolysis-electrodialysis combined process, and belongs to the technical field of glucuronic acid preparation. After the Tempo step, oxidized cellulose is subjected to high-temperature water bath treatment, enzyme hydrolysis and electrodialysis, and a glucuronic acid solution is obtained. The glucuronic acid is prepared by utilizing a Tempo-high-temperature hot bath-enzyme hydrolysis-electrodialysis combined process, and the process aim of improving the catalytic preparation of the glucuronic acid can be effectively achieved by combining four stages of modules, so that a final product only contains glucose and the glucuronic acid and does not contain other sugar and sugar acid products at all, and the production cost is reduced. And the quality of the glucuronic acid product is much higher than that of the existing preparation method.

Description

Technical Field

[0001] The present invention belongs to the technical field of glucuronic acid preparation, and particularly relates to a method for improving the yield of glucuronic acid in the combined process of Tempo-enzymatic hydrolysis-electrodialysis. Background Art

[0002] Glucuronic acid (D-Glucuronic acid), abbreviated as glucuronic acid, is a hexose sugar with a carboxyl group at the C-6 position, and its molecular formula is C6H 10 O7, with a molecular weight of 194.14. It widely exists in animals and plants. In plants, it exists in the form of monosaccharide uronic acid in gums such as arabic gum and is an important component of pectin, mucus, and high-level polysaccharides. In animals, glucuronic acid forms a complex with glucuronide and is stored in tissues. It has important biological functions. It can combine with toxic substances containing groups such as hydroxyl, amino, carboxyl, and sulfhydryl groups, enhance the water solubility of toxic substances, and enable them to be quickly excreted from the kidneys, playing a detoxifying role. It is also a common component of proteoglycans and glycosols and is very important for the metabolism of microorganisms, plants, and animals. At the same time, it is also an additive in functional beverages and health foods. With the increasing awareness of people's health concepts and the improvement of the requirements for the quality of life, the demand for glucuronic acid will be increasing.

[0003] Currently, the main methods for preparing glucuronic acid include polysaccharide hydrolysis method, chemical oxidation method, and biological fermentation method, etc. However, there are some problems with the above methods: the yield and purity of glucuronic acid obtained by the chemical oxidation method are low, and a large amount of by-products will be produced, making separation difficult; using the biological fermentation method, the process is complex, and the yield of glucuronic acid obtained is low, and separation is difficult; using the polysaccharide hydrolysis method, due to the high stability of the glycosidic bond connecting the uronic acid, very strong acid-base hydrolysis conditions are required, hydrolysis is difficult, the product yield is low, and a large amount of wastewater is produced, which cannot meet the production needs. Therefore, in view of the above problems, it is necessary to develop a method for preparing glucuronic acid. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for improving the yield of glucuronic acid in the combined process of Tempo-enzymatic hydrolysis-electrodialysis. This method uses the combined process of "Tempo-high temperature water bath-enzymatic hydrolysis-electrodialysis" to prepare glucuronic acid, which is a relatively mild preparation method and provides a new way for the production method of glucuronic acid in industry.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] A method for improving the yield of glucuronic acid in the combined process of Tempo-enzymatic hydrolysis-electrodialysis. After the Tempo step, the oxidized cellulose is treated in a high-temperature water bath, then enzymatically hydrolyzed and electrodialyzed to obtain a glucuronic acid solution.

[0007] Furthermore, the method for improving the yield of glucuronic acid in the combined process of Tempo-enzymatic hydrolysis-electrodialysis includes the following steps:

[0008] 1) Using microcrystalline cellulose as a substrate, preparing oxidized cellulose with an alkaline TEMPO-NaBr-NaClO chemical oxidation system;

[0009] 2) Washing the oxidized cellulose to neutral, centrifuging and drying it, and subjecting the dried oxidized cellulose to a high-temperature water bath treatment;

[0010] 3) Enzymatically hydrolyzing the cellulose obtained in step 2) with cellulase;

[0011] 4) Separating the solid and liquid of the enzymatic hydrolysate in step 3), transferring the supernatant to a bipolar membrane electrodialysis system for separation and purification, and thus obtaining a glucuronic acid solution.

[0012] Furthermore, the dry weight of the microcrystalline cellulose in step 1) is 8 - 9 g.

[0013] Furthermore, the mass of TEMPO in step 1) is 0.128 - 0.16 g.

[0014] Furthermore, the mass of NaBr in step 1) is 0.8 - 1 g.

[0015] Furthermore, the volume of NaClO in step 1) is 18 - 23 mL.

[0016] Furthermore, the temperature of the high-temperature water bath treatment in step 2) is 120 - 180 °C, and the time is 2 h.

[0017] Furthermore, the dosage of cellulase in step 3) is 18 - 20 FPIU / g.

[0018] Furthermore, the temperature of the enzymatic hydrolysis reaction in step 3) is 50 - 55 °C, and the rotation speed is 150 - 160 rpm.

[0019] Furthermore, the voltage of the electrodialysis equipment in step 4) is 20 - 30 V, and the current is below 5 A.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] 1) This application uses microcrystalline cellulose as a raw material and adopts the combined process of "Tempo-high temperature water bath-enzyme hydrolysis-electrodialysis" to prepare glucuronic acid. It is a relatively mild and effective preparation method, providing a new approach for the production method of glucuronic acid in industry.

[0022] 2) This application uses the combined process of "Tempo-high temperature water bath-enzyme hydrolysis-electrodialysis" to prepare glucuronic acid. Through the combination of four-stage modules, it can effectively achieve the process purpose of improving the catalytic preparation of glucuronic acid, making the final product contain only glucose and glucuronic acid, completely free of other sugars and sugar acid products, and the product quality of glucuronic acid is much higher than that of existing preparation methods.

[0023] 3) This application uses the combined process of "Tempo-high temperature water bath-enzyme hydrolysis-electrodialysis" to prepare glucuronic acid with microcrystalline cellulose as the raw material. The raw material is easily soluble, effectively reducing the process cost and achieving the process purpose of sustainable development. Brief Description of the Drawings

[0024] Figure 1 It is the adsorption test effect diagram of microcrystalline cellulose and cellulose after TEMPO oxidation in Example 1 of the present invention;

[0025] Figure 2 It is the high-performance ion chromatogram of the product in Example 3 of the present invention;

[0026] Figure 3 It is the SEM diagram of the product in Example 3 of the present invention; among them, Figure 3 a is the SEM diagram of microcrystalline cellulose; Figure 3 b is the SEM diagram of microcrystalline cellulose after oxidation; Figure 3 c is the SEM diagram of cellulose after hot hydrolysis treatment. Detailed Embodiments

[0027] The following further clarifies the present invention in conjunction with specific embodiments. The embodiments are implemented on the premise of the technical solution of the present invention. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0028] The bipolar membrane electrodialysis system used in the following embodiments is designed by China Kejia High Polymer Materials Technology Co., Ltd. The total membrane area of the polyphenylene ether membrane stack in the device is 21 cm × 9 cm × 15 cm, and a titanium-coated electrode plate is set.

[0029] The main conditions for the high-performance ion chromatography detection used in the following embodiments are: chromatographic column: CarboPacTM PA200, mobile phase: 100 mmol sodium hydroxide and 500 mmol sodium acetate.

[0030] The specific steps of high - efficiency ion chromatography detection used in the following examples are as follows: gradient elution is carried out with the eluent according to the following method: first, elute with eluent A for 7 min, then elute eluent A and eluent B at a volume ratio of 80:20 until 15 min, then elute eluent A and eluent B at a volume ratio of 65:35 until 25 min, and finally elute eluent A and eluent B at a volume ratio of 30:70 until 30 min, and the elution is completed; among them, the elution speed is 0.25 - 0.35 mL / min. The detection sample concentration range is 1 - 50 mg / L, so the sample concentration needs to be diluted to this concentration range, and then the detection is carried out.

[0031] The specific process of the Congo red adsorption experiment in the following examples is as follows: Congo red staining is used to determine the accessibility of the enzyme to cellulose. All samples are fully mixed with Congo red solutions of different concentrations (0, 0.05, 0.1, 0.5, 1, 2, 3, and 4 g / L) with a 1% (w / v) concentration of the substrate. The accessibility experiment is carried out in a 50 mL plastic centrifuge tube. The centrifuge tube is placed in a thermostatic oscillator at a rotation speed of 150 rpm and continuously reacted at 50 °C for 24 hours. Then the sample is centrifuged at 8000 rpm for 5 min, and the absorbance values of the supernatant and the prepared standard solution are measured using a UV / visible spectrophotometer at 498 nm. The amount of material adsorbed by the dye is calculated according to the remaining content of the dye in the solution, and the accessibility of the material is simulated using the Langmuir adsorption isotherm and calculated using the following equation:

[0032]

[0033] Where T is the concentration of the dye adsorbed by the substrate (mg / g); T m is the concentration of the adsorbed dye (mg / g); K is the distribution coefficient, and C is the content of free dye in the supernatant (g / L).

[0034] The specific process of the SEM experiment in the following examples: The microstructure of microcrystalline cellulose before and after pretreatment is observed using a Quanta 200 type scanning electron microscope (SEM) from Thermo Fisher Scientific, USA. Before observation, the microcrystalline cellulose before and after pretreatment needs to be freeze - dried to absolute dryness, pasted on conductive glue, and vacuum sputtered with gold.

[0035] The specific process of the crystallinity experiment in the following examples: The crystallinity of microcrystalline cellulose is detected using an X - ray diffractometer (XRD, Japan). The 2θ range of the X - ray diffraction spectrum is 5 to 35°. The crystallinity index (CrI) of all samples is obtained from the following formula:

[0036]

[0037] Where, I amis the intensity between plane reflections (110) and (200), I 200 is the maximum peak intensity.

[0038] Example 1

[0039] A method for improving the yield of glucuronic acid in the combined process of Tempo-enzymatic hydrolysis-electrodialysis, comprising the following steps:

[0040] (1) Add 0.144 g of TEMPO and 0.9 g of NaBr to 900 mL of distilled water and stir continuously to obtain a TEMPO-NaBr reaction system; add 9 g of dry weight of microcrystalline cellulose and disperse it in the reaction system until TEMPO and NaBr are completely dissolved; in the case of sufficient dispersion of microcrystalline cellulose, add 21 mL of NaClO solution, adjust the pH value of the reaction system to 10, slowly drop 0.5 M NaOH into the reaction system, and the whole process is carried out on a magnetic stirrer equipped with a pH control sensor for 4 h while maintaining the reaction conditions at a pH value of 10; in addition, after the reaction is completed, terminate the reaction with a small amount of ethanol.

[0041] (2) After the reaction is completed, wash the obtained product with distilled water 3-5 times until neutral, and then centrifuge and dry it under the conditions of 8000 rpm and 5 min.

[0042] (3) Subsequently, in a 50 mL centrifuge tube, enzymatic hydrolysis is carried out at a concentration of 5% (w / v) of oxidized cellulose, using 0.05 M citrate as a buffer; in addition, the dosage of cellulase is 20 FPIU / g of cellulose, and 0.2% (w / v) of tetracycline is used as an anti-pollution agent, and the whole process is carried out in a constant temperature oscillator at 50 °C and 150 rpm for 72 hours.

[0043] (4) Separate the solid-liquid mixture of the enzymatic hydrolysate at 8000 rpm, and transfer the supernatant to a bipolar membrane electrodialysis system. Start the bipolar membrane electrodialysis system, control the operating voltage of the electrodialysis system to be 20 V, and the separation process is detected in real time by a conductivity meter. When the conductivity unit level in the feed chamber drops to 100 μs / cm, the separation stops.

[0044] Through high-performance ion chromatography detection, finally 0.313 g / L of glucose and 0.231 g / L of glucuronic acid can be obtained by the combined process of "Tempo-enzymatic hydrolysis-electrodialysis", and the enzymatic hydrolysis rate only reaches 1.09%, among which the yield of glucuronic acid is 0.46%.

[0045] Figure 1It is the adsorption test effect diagram of microcrystalline cellulose and TEMPO-oxidized cellulose. To a certain extent, the accessibility of congo red to cellulose can represent the enzymatic hydrolysis degree. The adsorption tests of microcrystalline cellulose and TEMPO-oxidized cellulose were carried out respectively. From Figure 1 it can be seen that after TEMPO oxidation, congo red no longer shows adsorption ability to cellulose, and the accessibility is less than 0. This is because the degree of polymerization is too low and the relative carboxyl content is too large, resulting in the negative charge repulsion force being greater than the affinity, and then the adsorption ability is 0 or negative. Thus, it can be seen that the accessibility confirms the degradation effect from the side.

[0046] Example 2

[0047] A method for improving the yield of glucuronic acid in the combined process of Tempo-enzymatic hydrolysis-electrodialysis, comprising the following steps:

[0048] (1) Perform a high-temperature water bath of microcrystalline cellulose at 120 °C for 2 h. After completion, wash with distilled water until colorless, leave the precipitate and freeze-dry it.

[0049] (2) Add 0.144 g of TEMPO and 0.9 g of NaBr to 900 mL of distilled water and stir continuously to obtain a TEMPO-NaBr reaction system; add 9 g of microcrystalline cellulose with a dry weight and disperse it in the reaction system until TEMPO and NaBr are completely dissolved; in the case of sufficient dispersion of microcrystalline cellulose, add 21 mL of NaClO solution, adjust the pH value of the reaction system to 10, slowly drip 0.5 M NaOH into the reaction system, and carry out the whole process on a magnetic stirrer equipped with a pH control sensor for 4 h while maintaining the reaction conditions at a pH value of 10; in addition, after the reaction is completed, terminate the reaction with a small amount of ethanol.

[0050] (3) After the reaction is completed, wash the obtained product with distilled water 3-5 times until neutral, and then centrifuge and dry it under the conditions of 8000 rpm and 5 min.

[0051] (4) Subsequently, use 0.05 M citrate as a buffer to carry out enzymatic hydrolysis in a 50 mL centrifuge tube at an oxidized cellulose concentration of 5% (w / v); in addition, the dosage of cellulase is 20 FPIU / g of cellulose, and 0.2% (w / v) of tetracycline is an anti-pollution agent; the whole process is carried out in a constant temperature oscillator at 50 °C and 150 rpm for 72 hours.

[0052] (5) Separate the enzymatic hydrolysate into solid and liquid at 8000 rpm, and transfer the supernatant to a bipolar membrane electrodialysis system. Start the bipolar membrane electrodialysis system, control the operating voltage of the electrodialysis system at 20 V, and monitor the separation process in real-time with a conductivity meter. Stop the separation when the unit series of conductivity in the feed chamber drops to 100 μs / cm.

[0053] After detection by high-performance ion chromatography, through the combined process of "high-temperature water bath - Tempo - enzymatic hydrolysis - electrodialysis", 0.278 g / L of glucose and 0.372 g / L of glucuronic acid can be finally obtained. The enzymatic hydrolysis rate is 1.3%, and the yield of glucuronic acid is 0.74%.

[0054] Through the crystallinity experiment, the experimental results show that the original crystallinity of microcrystalline cellulose is 88.66%, and the crystallinity of microcrystalline cellulose after TEMPO oxidation increases to 92.83%. Therefore, TEMPO oxidation will increase the crystallinity, and the pretreatment process should be placed after TEMPO.

[0055] Example 3

[0056] A method for improving the yield of glucuronic acid in the combined process of Tempo - enzymatic hydrolysis - electrodialysis, comprising the following steps:

[0057] (1) Add 0.144 g of TEMPO and 0.9 g of NaBr to 900 mL of distilled water and stir continuously to obtain a TEMPO - NaBr reaction system; add 9 g of microcrystalline cellulose with a dry weight to disperse in the reaction system until TEMPO and NaBr are completely dissolved; with the microcrystalline cellulose fully dispersed, add 21 mL of NaClO solution, adjust the pH value of the reaction system to 10, and slowly drip 0.5 M NaOH into the reaction system. The whole process is carried out on a magnetic stirrer equipped with a pH control sensor for 4 h while maintaining the reaction conditions at a pH value of 10; in addition, after the reaction is completed, terminate the reaction with a small amount of ethanol.

[0058] (2) After the reaction is completed, wash the obtained product with distilled water 3 - 5 times until neutral, then centrifuge and dry it at 8000 rpm for 5 min; subject the dried oxidized cellulose to a high-temperature water bath at 120 °C for 2 h, and after completion, wash it with distilled water until it is colorless, leaving the precipitate.

[0059] (3) Subsequently, in a 50 mL centrifuge tube, carry out enzymatic hydrolysis at an oxidized cellulose concentration of 5% (w / v), using 0.05 M citrate as a buffer; in addition, the dosage of cellulase is 20 FPIU / g of cellulose, and 0.2% (w / v) of tetracycline is used as an anti-pollution agent. The whole process is carried out in a constant temperature oscillator at 50 °C and 150 rpm for 72 hours.

[0060] (4) Separate the enzymatic hydrolysate into solid and liquid at 8000 rpm, and transfer the supernatant to the bipolar membrane electrodialysis system. Start the bipolar membrane electrodialysis system, control the operating voltage of the electrodialysis system at 20 V, and monitor the separation process in real time with a conductivity meter. Stop the separation when the unit series of conductivity in the feed chamber drops to 100 μs / cm.

[0061] After detection by high-performance ion chromatography, through the combined process of "Tempo - high-temperature water bath - enzymatic hydrolysis - electrodialysis", 17.024 g / L of glucose and 4.617 g / L of glucuronic acid can be finally obtained. The enzymatic hydrolysis rate reaches 43.3%, among which the yield of glucuronic acid is 9.2%, and there are no other sugars and sugar acid products in the product. The chromatogram is as Figure 2 shown.

[0062] Figure 3 a is microcrystalline cellulose, Figure 3 b is oxidized microcrystalline cellulose, and Figure 3 c is the scanning electron micrograph of cellulose after hydrothermal treatment. It can be seen from the SEM image after hydrothermal treatment that obvious erosion effects occur on the surface, indicating that the improvement of enzymatic hydrolysis efficiency is due to the fact that hydrothermal treatment breaks the stubborn crystalline region.

[0063] Example 4

[0064] A method for improving the yield of glucuronic acid in the combined process of Tempo - enzymatic hydrolysis - electrodialysis, comprising the following steps:

[0065] (1) Add 0.144 g of TEMPO and 0.9 g of NaBr to 900 mL of distilled water and stir continuously to obtain a TEMPO - NaBr reaction system; add 9 g of dry weight microcrystalline cellulose and disperse it in the reaction system until TEMPO and NaBr are completely dissolved; with the microcrystalline cellulose fully dispersed, add 21 mL of NaClO solution, adjust the pH value of the reaction system to 10, slowly drip 0.5 M NaOH into the reaction system, and carry out the whole process on a magnetic stirrer equipped with a pH control sensor for 4 h while maintaining the reaction conditions at a pH value of 10; in addition, after the reaction is completed, terminate the reaction with a small amount of ethanol.

[0066] (2) After the reaction is completed, wash the obtained product with distilled water 3 - 5 times until neutral, then centrifuge and dry it at 8000 rpm for 5 min; subject the dried oxidized cellulose to a high-temperature water bath at 150 °C for 2 h, and after completion, wash it with distilled water until it is colorless, leaving the precipitate.

[0067] (3) Subsequently, enzymatic hydrolysis was carried out in a 50 mL centrifuge tube with a 5% (w / v) concentration of oxidized cellulose, using 0.05 M citrate as a buffer; in addition, the dosage of cellulase was 20 FPIU / g cellulose, and 0.2% (w / v) tetracycline was used as an anti-pollution agent; the whole process was carried out in a constant temperature oscillator at 50 °C and 150 rpm for 72 hours.

[0068] (4) The enzymatic hydrolysate was separated into solid and liquid phases at 8000 rpm, and the supernatant was transferred to a bipolar membrane electrodialysis system. The bipolar membrane electrodialysis system was started, and the operating voltage of the electrodialysis system was controlled at 20 V. The separation process was monitored in real time by a conductivity meter, and the separation was stopped when the unit series of conductivity in the feed chamber dropped to 100 μs / cm.

[0069] After detection by high-performance ion chromatography, 18.545 g / L of glucose and 2.564 g / L of glucuronic acid could be finally obtained through the combined process of "Tempo - high-temperature water bath - enzymatic hydrolysis - electrodialysis". The enzymatic hydrolysis rate reached 42.2%, and the yield of glucuronic acid was 5.1%. Moreover, there were no other sugars and sugar acid products in the product.

[0070] Example 5

[0071] A method for improving the yield of glucuronic acid in the combined process of Tempo - enzymatic hydrolysis - electrodialysis, comprising the following steps:

[0072] (1) Add 0.144 g of TEMPO and 0.9 g of NaBr to 900 mL of distilled water and stir continuously to obtain a TEMPO - NaBr reaction system; add 9 g of microcrystalline cellulose with a dry weight and disperse it in the reaction system until TEMPO and NaBr are completely dissolved; in the case of sufficient dispersion of microcrystalline cellulose, add 21 mL of NaClO solution, adjust the pH value of the reaction system to 10, and slowly drip 0.5 M NaOH into the reaction system. The whole process is carried out on a magnetic stirrer equipped with a pH control sensor for 4 h, while maintaining the reaction conditions at a pH value of 10; in addition, after the reaction is completed, terminate the reaction with a small amount of ethanol.

[0073] (2) After the reaction is completed, wash the obtained product with distilled water 3 - 5 times until neutral, then centrifuge and dry it at 8000 rpm for 5 min; the dried oxidized cellulose is subjected to a high-temperature water bath at 180 °C for 2 h, and after completion, wash it with distilled water until colorless and leave the precipitate.

[0074] (3) Subsequently, enzymatic hydrolysis was carried out in a 50 mL centrifuge tube with a 5% (w / v) concentration of oxidized cellulose, using 0.05 M citrate as a buffer; in addition, the dosage of cellulase was 20 FPIU / g cellulose, 0.2% (w / v) tetracycline was used as an anti-pollution agent, and the whole process was carried out in a constant temperature oscillator at 50 °C and 150 rpm for 72 hours.

[0075] (4) The enzymatic hydrolysate was separated into solid and liquid phases at 8000 rpm, and the supernatant was transferred to a bipolar membrane electrodialysis system. The bipolar membrane electrodialysis system was started, and the operating voltage of the electrodialysis system was controlled at 20 V. The separation process was monitored in real-time by a conductivity meter, and the separation was stopped when the unit series of conductivity in the feed chamber decreased to 100 μs / cm.

[0076] After detection by high-performance ion chromatography, 19.987 g / L of glucose and 2.478 g / L of glucuronic acid could be obtained finally through the combined process of "Tempo - high-temperature water bath - enzymatic hydrolysis - electrodialysis". The enzymatic hydrolysis rate reached 44.9%, among which the yield of glucuronic acid was 5.0%, and no other sugars and sugar acid products were contained in the product.

[0077] Example 6

[0078] A method for improving the yield of glucuronic acid in the combined process of Tempo - enzymatic hydrolysis - electrodialysis, comprising the following steps:

[0079] (1) Add 0.144 g of TEMPO and 0.9 g of NaBr to 900 mL of distilled water and stir continuously to obtain a TEMPO-NaBr reaction system; add 8 g of dry weight of microcrystalline cellulose and disperse it in the reaction system until TEMPO and NaBr are completely dissolved; under the condition that the microcrystalline cellulose is fully dispersed, add 21 mL of NaClO solution, adjust the pH value of the reaction system to 10, and slowly drip 0.5 M NaOH into the reaction system. The whole process is carried out on a magnetic stirrer equipped with a pH control sensor for 4 h, and the reaction conditions are maintained at a pH value of 10; in addition, after the reaction is completed, the reaction is terminated with a small amount of ethanol.

[0080] (2) After the reaction is completed, wash the obtained product with distilled water 3 - 5 times until neutral, then centrifuge and dry it under the conditions of 8000 rpm and 5 min; the dried oxidized cellulose is subjected to a high-temperature water bath at 120 °C for 2 h, and after the end, it is washed with distilled water until colorless, and the precipitate is left.

[0081] (3) Subsequently, enzymatic hydrolysis was carried out in a 50 mL centrifuge tube with a 5% (w / v) concentration of oxidized cellulose, using 0.05 M citrate as a buffer; in addition, the dosage of cellulase was 20 FPIU / g cellulose, and 0.2% (w / v) tetracycline was used as an anti-pollution agent. The whole process was carried out in a constant temperature oscillator at 50 °C and 150 rpm for 72 hours.

[0082] (4) The enzymatic hydrolysate was separated into solid and liquid phases at 8000 rpm, and the supernatant was transferred to a bipolar membrane electrodialysis system. The bipolar membrane electrodialysis system was started, and the operating voltage of the electrodialysis system was controlled at 20 V. The separation process was monitored in real time by a conductivity meter, and the separation was stopped when the unit series of conductivity in the feed chamber decreased to 100 μs / cm.

[0083] After detection by high-performance ion chromatography, 14.004 g / L of glucose and 3.047 g / L of glucuronic acid could be finally obtained through the combined process of "Tempo - high-temperature water bath - enzymatic hydrolysis - electrodialysis". The enzymatic hydrolysis rate reached 34.1%, among which the yield of glucuronic acid was 6.1%, and there were no other sugars and sugar acid products in the product.

[0084] Example 7

[0085] A method for improving the yield of glucuronic acid in the combined process of Tempo-enzymatic hydrolysis-electrodialysis, comprising the following steps:

[0086] (1) Add 0.128 g of TEMPO and 0.8 g of NaBr to 900 mL of distilled water and stir continuously to obtain a TEMPO-NaBr reaction system; add 9 g of dry weight of microcrystalline cellulose and disperse it in the reaction system until TEMPO and NaBr are completely dissolved; with the microcrystalline cellulose fully dispersed, add 18 mL of NaClO solution, adjust the pH value of the reaction system to 10, and slowly drip 0.5 M NaOH into the reaction system. The whole process was carried out on a magnetic stirrer equipped with a pH control sensor for 4 h while maintaining the reaction conditions at a pH value of 10; in addition, after the reaction was completed, the reaction was terminated with a small amount of ethanol.

[0087] (2) After the reaction ended, the obtained product was washed with distilled water 3 - 5 times until neutral, and then centrifuged and dried under the conditions of 8000 rpm and 5 min; the dried oxidized cellulose was subjected to a high-temperature water bath at 120 °C for 2 h, and after completion, it was washed with distilled water until colorless, leaving a precipitate.

[0088] (3) Subsequently, enzymatic hydrolysis was carried out in a 50 mL centrifuge tube with a 5% (w / v) concentration of oxidized cellulose, using 0.05 M citrate as a buffer; in addition, the dosage of cellulase was 20 FPIU / g cellulose, and 0.2% (w / v) tetracycline was used as an anti-pollution agent. The whole process was carried out in a constant temperature oscillator at 50 °C and 150 rpm for 72 hours.

[0089] (4) The enzymatic hydrolysate was separated into solid and liquid phases at 8000 rpm, and the supernatant was transferred to a bipolar membrane electrodialysis system. The bipolar membrane electrodialysis system was started, and the operating voltage of the electrodialysis system was controlled at 20 V. The separation process was monitored in real-time by a conductivity meter, and the separation was stopped when the unit series of conductivity in the feed chamber decreased to 100 μs / cm.

[0090] After detection by high-performance ion chromatography, 14.987 g / L of glucose and 3.902 g / L of glucuronic acid could be finally obtained through the combined process of "Tempo - high-temperature water bath - enzymatic hydrolysis - electrodialysis". The enzymatic hydrolysis rate reached 37.8%, and the yield of glucuronic acid was 7.8%. Moreover, there were no other sugars and sugar acid products in the product.

[0091] Example 8

[0092] A method for improving the yield of glucuronic acid in the combined process of Tempo - enzymatic hydrolysis - electrodialysis, comprising the following steps:

[0093] (1) Add 0.16 g of TEMPO and 1 g of NaBr to 900 mL of distilled water and stir continuously to obtain a TEMPO - NaBr reaction system; add 9 g of dry weight of microcrystalline cellulose and disperse it in the reaction system until TEMPO and NaBr are completely dissolved; with the microcrystalline cellulose fully dispersed, add 23 mL of NaClO solution, adjust the pH value of the reaction system to 11, and slowly drip 0.5 M NaOH into the reaction system. The whole process is carried out on a magnetic stirrer equipped with a pH control sensor for 4 h, while maintaining the reaction conditions at a pH value of 11; in addition, after the reaction is completed, terminate the reaction with a small amount of ethanol.

[0094] (2) After the reaction is completed, wash the obtained product with distilled water 3 - 5 times until it is neutral, then centrifuge and dry it at 8000 rpm for 5 min; the dried oxidized cellulose is subjected to a high-temperature water bath at 120 °C for 2 h, and after completion, wash it with distilled water until it is colorless, leaving the precipitate.

[0095] (3) Subsequently, enzymatic hydrolysis was carried out in a 50 mL centrifuge tube with a 5% (w / v) concentration of oxidized cellulose, using 0.05 M citrate as a buffer; in addition, the dosage of cellulase was 20 FPIU / g cellulose, and 0.2% (w / v) tetracycline was used as an anti-pollution agent. The whole process was carried out in a constant temperature oscillator at 50 °C and 150 rpm for 72 hours.

[0096] (4) The enzymatic hydrolysate was separated into solid and liquid phases at 8000 rpm, and the supernatant was transferred to a bipolar membrane electrodialysis system. The bipolar membrane electrodialysis system was started, and the operating voltage of the electrodialysis system was controlled at 20 V. The separation process was monitored in real time by a conductivity meter, and the separation was stopped when the unit series of conductivity in the feed chamber decreased to 100 μs / cm.

[0097] After detection by high-performance ion chromatography, 14.025 g / L of glucose and 2.982 g / L of glucuronic acid could be finally obtained through the combined process of "Tempo - high-temperature water bath - enzymatic hydrolysis - electrodialysis". The enzymatic hydrolysis rate reached 34%, and the yield of glucuronic acid was 6.0%. Moreover, the product did not contain other sugars and sugar acid products.

[0098] Example 9

[0099] A method for increasing the yield of glucuronic acid in the combined process of Tempo - enzymatic hydrolysis - electrodialysis, comprising the following steps:

[0100] (1) Add 0.144 g of TEMPO and 0.9 g of NaBr to 900 mL of distilled water and stir continuously to obtain a TEMPO - NaBr reaction system; add 9 g of dry weight of microcrystalline cellulose and disperse it in the reaction system until TEMPO and NaBr are completely dissolved; with the microcrystalline cellulose fully dispersed, add 21 mL of NaClO solution, adjust the pH value of the reaction system to 10, and slowly drip 0.5 M NaOH into the reaction system. The whole process is carried out on a magnetic stirrer equipped with a pH control sensor for 4 h, while maintaining the reaction conditions at a pH value of 10; in addition, after the reaction is completed, terminate the reaction with a small amount of ethanol.

[0101] (2) After the reaction is completed, wash the obtained product with distilled water 3 - 5 times until neutral, then centrifuge and dry it at 8000 rpm for 5 min; the dried oxidized cellulose is subjected to a high-temperature water bath at 120 °C for 2 h, and after completion, wash it with distilled water until colorless and leave the precipitate.

[0102] (3) Subsequently, enzymatic hydrolysis was carried out in a 50 mL centrifuge tube with a 5% (w / v) concentration of oxidized cellulose, using 0.05 M citrate as a buffer; in addition, the dosage of cellulase was 18 FPIU / g cellulose, and 0.2% (w / v) tetracycline was used as an anti-pollution agent. The whole process was carried out in a constant temperature oscillator at 55 °C and 160 rpm for 72 hours.

[0103] (4) The enzymatic hydrolysate was separated into solid and liquid phases at 8000 rpm, and the supernatant was transferred to a bipolar membrane electrodialysis system. The bipolar membrane electrodialysis system was started, and the operating voltage of the electrodialysis system was controlled at 20 V. The separation process was monitored in real-time by a conductivity meter, and the separation was stopped when the unit series of conductivity in the feed chamber dropped to 100 μs / cm.

[0104] By high-performance ion chromatography detection, 15.024 g / L of glucose and 3.617 g / L of glucuronic acid can be finally obtained through the combined process of "Tempo - high-temperature water bath - enzymatic hydrolysis - electrodialysis". The enzymatic hydrolysis rate reaches 47.3%, and the yield of glucuronic acid is 7.2%. Moreover, there are no other sugars and sugar acid products in the product.

[0105] Example 10

[0106] A method for improving the yield of glucuronic acid in the combined process of Tempo-enzymatic hydrolysis-electrodialysis, comprising the following steps:

[0107] (1) Add 0.144 g of TEMPO and 0.9 g of NaBr to 900 mL of distilled water and stir continuously to obtain a TEMPO-NaBr reaction system; add 9 g of dry weight of microcrystalline cellulose and disperse it in the reaction system until TEMPO and NaBr are completely dissolved; under the condition of sufficient dispersion of microcrystalline cellulose, add 21 mL of NaClO solution, adjust the pH value of the reaction system to 10, and slowly drip 0.5 M NaOH into the reaction system. The whole process is carried out on a magnetic stirrer equipped with a pH control sensor for 4 h, and the reaction conditions are maintained at a pH value of 10; in addition, after the reaction is completed, the reaction is terminated with a small amount of ethanol.

[0108] (2) After the reaction is completed, wash the obtained product with distilled water 3 - 5 times until neutral, then centrifuge and dry it under the conditions of 8000 rpm and 5 min; the dried oxidized cellulose is subjected to a high-temperature water bath at 120 °C for 2 h, and after completion, it is washed with distilled water until colorless, and the precipitate is left.

[0109] (3) Subsequently, enzymatic hydrolysis was carried out in a 50 mL centrifuge tube with a 5% (w / v) concentration of oxidized cellulose, using 0.05 M citrate as a buffer; in addition, the dosage of cellulase was 20 FPIU / g cellulose, and 0.2% (w / v) tetracycline was used as an anti-pollution agent. The whole process was carried out in a constant temperature oscillator at 50 °C and 150 rpm for 72 hours.

[0110] (4) The enzymatic hydrolysate was separated into solid and liquid phases at 8000 rpm, and the supernatant was transferred to a bipolar membrane electrodialysis system. The bipolar membrane electrodialysis system was started, and the operating voltage of the electrodialysis system was controlled at 30 V. The separation process was monitored in real time by a conductivity meter, and the separation was stopped when the unit series of conductivity in the feed chamber decreased to 100 μs / cm.

[0111] After detection by high-performance ion chromatography, 16.354 g / L of glucose and 4.022 g / L of glucuronic acid could be finally obtained through the combined process of "Tempo - high-temperature water bath - enzymatic hydrolysis - electrodialysis". The enzymatic hydrolysis rate reached 40.7%, and the yield of glucuronic acid was 8.0%. Moreover, no other sugars and sugar acid products were contained in the product.

[0112] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for improving the yield of glucuronic acid in a combined process of Tempo-enzymatic hydrolysis-electrodialysis, characterized in that: After the Tempo step, the oxidized cellulose is treated in a high temperature water bath, then enzymatically hydrolyzed and electrodialyzed to obtain a glucuronic acid solution.

2. The method for improving the yield of glucuronic acid in the combined process of Tempo-enzymatic hydrolysis-electrodialysis according to claim 1, characterized in that: The following steps are involved: 1) Using microcrystalline cellulose as substrate, oxidized cellulose was prepared by alkaline TEMPO-NaBr-NaClO chemical oxidation system; 2) The oxidized cellulose is washed to neutrality, centrifuged and dried, and the dried oxidized cellulose is subjected to a high temperature water bath treatment; 3) hydrolyzing the cellulose obtained in step 2) with cellulase; 4) The enzymatic hydrolysate of step 3) is subjected to solid-liquid separation, and the supernatant is transferred to a bipolar membrane electrodialysis system for separation and purification to obtain a glucuronic acid solution.

3. The method for improving the yield of glucuronic acid in the Tempo-enzymatic hydrolysis-electrodialysis combined process according to claim 2, characterized in that: The dry weight of microcrystalline cellulose in step 1) is 8-9 g.

4. The method for improving the yield of glucuronic acid in the combined process of Tempo-enzymatic hydrolysis-electrodialysis according to claim 2, characterized in that: The mass of TEMPO in step 1) is 0.128-0.16 g.

5. The method for improving the yield of glucuronic acid in the Tempo-enzymatic hydrolysis-electrodialysis combined process according to claim 2, characterized in that: The mass of NaBr in step 1) is 0.8-1 g.

6. The method for accurately preparing high-purity glucuronic acid according to claim 2, characterized in that: The volume of NaClO in step 1) is 18-23 mL.

7. The method for improving the yield of glucuronic acid in the combined process of Tempo-enzymatic hydrolysis-electrodialysis according to claim 2, characterized in that: In step 2), the temperature of the high temperature water bath treatment is 120-180°C and the time is 2 h.

8. The method for improving the yield of glucuronic acid in the combined process of Tempo-enzymatic hydrolysis-electrodialysis according to claim 2, characterized in that: The amount of cellulase used in step 3) is 18-20 FPIU / g.

9. The method for improving the yield of glucuronic acid in the combined process of Tempo-enzymatic hydrolysis-electrodialysis according to claim 2, characterized in that: The temperature of the enzymatic hydrolysis reaction in step 3) is 50-55°C and the rotation speed is 150-160 rpm.

10. The method for improving the yield of glucuronic acid in the combined process of Tempo-enzymatic hydrolysis-electrodialysis according to claim 2, characterized in that: In the step 4), the voltage of the electrodialysis equipment is 20-30 V, and the current is below 5 A.

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