Method for electrophoretic migration enrichment separation of pectin and other plant polysaccharides

Abstract

The present application belongs to the technical field of plant extraction and separation, and discloses a method for electrophoretic migration enrichment and separation of pectin and other plant polysaccharides. The electrophoretic separation device is divided into multiple compartments, and the crude pectin extract is filtered and then put into the electrophoretic separation device. After the solution is adjusted to a suitable temperature and pH, the pectin / polysaccharide, which is electronegative, starts to migrate from the cathode to the anode under the action of the electric field. The solid content of the pectin / polysaccharide extract can be increased by 10-30 times, which greatly saves the amount of alcohol used for alcohol precipitation, significantly shortens the pectin deesterification time, and increases the deesterification rate by three times compared with the traditional deesterification method, with a recovery rate of more than 80%. The product has good emulsifying and gelling properties. Compared with the existing pectin or polysaccharide extraction and separation technology, the method is simple in process, high in separation efficiency, energy-saving, environmentally friendly and safe, can reduce the production cost of pectin and polysaccharide, and can further broaden the application of polysaccharide in the fields of food, medicine and chemical industry.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant extraction and separation, and particularly relates to a method for electrophoretic migration enrichment and separation of pectin and other plant polysaccharides. BACKGROUND

[0002] Natural pectin / polysaccharide substances exist in the form of protopectin, pectin and pectic acid in the fruits, roots, stems and leaves of plants, and are a component of cell walls, forming a gel-like matrix in the cell walls of plants, and are often used as thickening agents, colloidal stabilizers, gelling agents, emulsifiers and drug delivery carriers in the food industry. The main structural domains of pectin include homogalacturonan (HG), rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II), and in addition, there are a small amount of substituted galacturonan. Commercial pectin, such as citrus peel and apple pomace pectin, must have a galacturonic acid (GalA) content of more than 65%. According to the degree of methoxylation (DM) of pectin, pectin can be divided into high-methoxy pectin (HMP, DM>50%) and low-methoxy pectin (LMP, DM<50%), and the size of DM also determines the gelling mode of pectin. Since most of the carboxyl groups in HMP are esterified by methoxyl groups, they cannot interact with each other, and the gelation of HMP requires an acidic condition (for example, pH 3.5) and a high concentration of sucrose as a co-solvent (usually 65% sucrose), while the gelation of LMP relies on the formation of a so-called “egg-box” model gel by divalent cations (such as Ca 2+ , Zn 2+ and Mg 2+ , etc.

[0003] With the improvement of people's living standards, the number of patients with diabetes, high blood lipids, high blood pressure and their complications is increasing year by year, and low-sugar, low-calorie health foods are increasingly favored. Since LMP has a wider application range in terms of pH and solid content, and can be used to produce low-sugar, low-calorie, low-sweetness and other nutrient-rich foods, its demand is increasing. In addition, pectin is also a kind of excellent natural water-soluble dietary fiber, which has certain physiological functional properties and biological activities, such as antioxidant activity, immune regulation, anti-inflammatory effect, cholesterol regulation, bowel movement, blood lipid reduction, heavy metal removal in the human body and anticancer effect, and pectin can also be applied to health foods.

[0004] Most of the natural pectin on the market is HMP, and the proportion of LMP is small and the price is high. At present, acid, alkali and enzyme methods are often used to produce commercial LMP by deesterification of HMP. The ammonia method for deesterification of HMP is mainly the deesterification system of ammonia-ethanol, in addition, hydroxylamine and other ammonia derivatives are also used for the deesterification of HMP; the acid method for deesterification of HMP mainly relies on strong acids such as HCl, and the alkali method for deesterification mainly relies on strong alkalis such as NaOH. However, the above two methods have their own disadvantages. The acid deesterification reaction not only has pH dependence, but also has low efficiency, and the pectin molecules will undergo hydrolysis reaction at low pH. The object of pectin acid hydrolysis reaction is mainly the neutral sugar side chain of pectin, followed by some proteins and phenolic acids. Under alkaline conditions, deesterification of pectin will cause β-elimination reaction of pectin molecular chain, affecting the quality of pectin. In addition to acid and alkali methods, enzyme deesterification of HMP is welcomed due to its high efficiency and environmental protection. Enzymatic deesterification is beneficial to produce random or regional distribution of unesterified GalA units due to its relative specificity. The most commonly used HMP deesterification enzyme is pectin methyl esterase. However, the cost of enzyme deesterification is high, and it is difficult to carry out large-scale commercial production.

[0005] Therefore, the defects of the prior art are: high cost, difficult to carry out large-scale commercial production; low efficiency; complex process; low purity and activity; not environmentally friendly.

[0006] On September 19, 2024, a search was conducted in the China Patent Publication Database with "Pectin and electrophoresis and membrane and collection" as the abstract keyword, and the synonym expansion was checked. No relevant literature was found.

[0007] On September 19, 2024, an abstract search was conducted on China's CNKI with "Pectin and electrophoresis and membrane and collection and platinum". It was found that Dong Zhangyong, Luo Mei, and Wang Zhong published "Purification and identification of banana wilt fungus 4th strain β-1, 6-galactanase"; Li Anqi published "Effect of soil chenopodium zygophylla tissue culture root exudates on soybean root cells".

[0008] On September 19, 2024, a search was conducted on the website of the United States Patent and Trademark Office (https: / / ppubs.uspto.gov / pubwebapp / ) with "Pectin with electrophoresis with membrane with collection". No relevant literature was found.

[0009] September 19, 2024, a search was made on the Korean Patent Office with "Pectin and electrophoresis and membrane and collection" in the search box; search website http: / / eng.kipris.or.kr / enghome / main.jsp. Five documents were found, none of which are related to the present patent: 1020127002889 (2010.07.26) Withdrawn [1] METHODS AND COMPOSITIONS FOR IMPROVING SUGAR TRANSPORT, MIXED SUGAR FERMENTATION, AND PRODUCTION OF BIOFUELS;

[0010] 1020177023785 (2016.01.13) GLYCAN THERAPEUTICS AND RELATED METHODS THEREOF;

[0011] 1020100049639 (2010.05.27) COMPOSITION FOR PREVENTING OR TREATING STROKE CONTAINING SALVIA MILTIORRHIZA EXTRACT OR CRYSTOL

[0012] 1020170184796 (2017.12.29) An anticancer agent for treating cancers resistant to PI3K / mTOR inhibitor;

[0013] 1020120010787 (2012.02.02) Method for Screening of Pathogenic Factors Against Rice Blast Disease.

[0014] September 19, 2024, a search was made on the Korean Patent Office with "Pectin and electrophoresis and membrane and collection" in the search box; search website http: / / eng.kipris.or.kr / enghome / main.jsp. Five documents were found, none of which are related to the present patent: 1020127002889 (2010.07.26) Withdrawn [1] METHODS AND COMPOSITIONS FOR IMPROVING SUGAR TRANSPORT, MIXED SUGAR FERMENTATION, AND PRODUCTION OF BIOFUELS;

[0015] 202010601311.9 Use of a metapolymer extract for preventing and / or treating arthritis and joint degeneration; 201310003302.X Variants of glycoside hydrolases; 200480031516.7 Variants of glycoside hydrolases.

[0016] On September 19, 2024, an application titled "Pectin and electrophoresis and membrane and collection" was published on the website of the Japan Patent Office.

[0017] A search on https: / / www.j-platpat.inpit.go.jp / yielded no relevant literature.

[0018] This concept is completely different from that of this patent. Summary of the Invention

[0019] The purpose of the invention is to provide a more effective method for electrophoretic migration enrichment and separation of pectin and other plant polysaccharides. Specific objectives are detailed in the specific implementation section, which outlines several substantial technical effects.

[0020] To achieve the above objectives, the present invention adopts the following technical solution:

[0021] A method for electrophoretic migration enrichment and separation of pectin and other plant polysaccharides, characterized in that the method comprises the following steps:

[0022] The raw material from which pectin or polysaccharides can be extracted is initially extracted using hot acid / alkali or enzymatic methods to obtain an initial pectin extract. The extract is then filtered through a filter to obtain a clear extract.

[0023] The prepared extract was poured into an electrophoretic separation device. Several membrane modules were placed at intervals in the tank of the device. NaCl solution was placed in dialysis bags at both ends of the separation device. Platinum-plated electrodes were placed in the dialysis bags.

[0024] By selecting voltage and current, under the influence of an electric field, negatively charged pectin / polysaccharide molecules migrate directionally from the cathode to the anode via electrophoresis.

[0025] During the migration process, the membrane inside the separation device acts as a barrier, causing pectin / polysaccharide to gradually accumulate and precipitate on the membrane;

[0026] Collect the aggregates enriched on each membrane module to obtain pectin / polysaccharide with high solids content;

[0027] After alcohol precipitation, washing and drying, pectin / polysaccharide enriched and separated by electrophoretic migration method was obtained.

[0028] A further technical solution of the present invention is that the separation tank is divided into multiple chambers by a number of membrane plates, and the chambers are interconnected; a dialysis bag containing NaCl solution is placed at each end of the electrophoretic separation device, and a platinum electrode is implanted into the dialysis bag; under the action of an electric field, pectin / polysaccharide molecules move directionally toward the anode and are collected and separated by each membrane plate in sequence according to their molecular weight and charge strength.

[0029] A further technical solution of the present invention is that the voltage during the electrolysis process is 150-20000V, the current is 15-2000mA, and the reaction time is 0.5-3h.

[0030] A further technical solution of the present invention is that the electrolyte is sodium chloride or potassium chloride; the pectin / polysaccharide mass: sodium chloride or potassium chloride mass = 1000:1 to 10:1; the concentration of the pectin solution is 0.1 to 1 w / w%; the molecular weight cutoff of the membrane is 100 Da to 2000 kDa; and both the anode electrode and the cathode electrode are made of platinum sheets.

[0031] A further technical solution of the present invention is that the products on the membrane are adjusted to pH 3-5 with 1-3M NaOH / HCl, and then 3 times the volume of 75%-95% ethanol is added to each product and stirred. The mixture is then centrifuged at 1000-20000 rpm for 10-30 min. After that, the mixture is washed with 75%-95% ethanol and dried at 40-60℃ to obtain the pectin product.

[0032] A further technical solution of the present invention is that the electrophoretic separation device is a dedicated electrophoretic separation device;

[0033] The device has the following structure: it includes an electrophoresis container 1, an upper support portion 2 is arranged on the electrophoresis container 1, and a membrane frame 4. The membrane frame 4 has side support portions 8 on both sides, and the side support portions 8 have grooves.

[0034] The groove of the side support part 8 faces downward and can overlap the upper support part 2;

[0035] Multiple membrane holes 3 are arranged on the membrane frame 4;

[0036] A liftable membrane unit can be placed on the membrane aperture 3. The membrane unit includes a lower membrane body and an upper support bar 6. A handle 7 is arranged on the upper support bar 6.

[0037] The number and spacing of the membranes can be adjusted by adjusting the arrangement of the membranes in membrane pores 3.

[0038] The electrophoresis container 1 is connected to an external cold water container via a fixed shaft 11. The cold water container is connected to an inlet 9 and an outlet 12. There is a cold water space 10 between the cold water container and the electrophoresis container 1. By pumping flowing cold water into the cold water space, the electrophoresis container 1 can be cooled. The electrophoresis container 1 is suspended, that is, except for the top surface of the electrophoresis container 1, the other surfaces are placed in cold water, and the bottom surface of the electrophoresis container 1 is immersed in the flowing cold water.

[0039] A further technical solution of the present invention is that, after electrophoresis is completed, pulling the handle 7 upwards allows the membrane pores 3 to clean the deposits on the membrane; when the handle 7 is pulled upwards, a container is placed below, and the deposits cleaned from the membrane by the membrane pores 3 will fall into the container.

[0040] A further technical solution of the present invention is that, before electrophoresis begins, the number of membranes on the membrane holder 4 is adjusted;

[0041] The membrane frame is fixed by the groove of the side support portion 8 facing downwards and overlapping the upper support portion 2;

[0042] Electrophoresis;

[0043] After electrophoresis is completed;

[0044] The entire membrane frame is held upright.

[0045] A container is placed below. Pulling the handle 7 upwards will cause the cleaning residue on the membrane pores 3 to fall into the container.

[0046] A further technical solution of the present invention is that the electrophoretic separation device includes an outer jacket, which is connected to a cold water circulation machine; the temperature of the coolant is kept as low as possible without freezing.

[0047] A further technical solution of the present invention is that,

[0048] For any of the following schemes:

[0049] Option 1:

[0050] 20g of beet pulp was dispersed in 400g of distilled water and 0.2g of sodium chloride was placed in a bipolar dialysis bag. The pH of the solution was adjusted to 1.5 and the solution was placed in an 85℃ water bath for 2 hours. After cooling the crude extract solution from the hot acid water bath, it was coarsely filtered through a 400-mesh filter cloth. The crude extract was centrifuged at 8000r / min for 20min, and the supernatant was collected and the pH was adjusted to 3.5 to obtain the initial beet pectin electrolyte.

[0051] (2) The self-made electrophoretic separation device is divided into several reaction chambers using a 500Da regenerated cellulose membrane. The electrophoretic separation device is placed in a container slightly larger than itself. Both ends of the container are connected to a low-temperature constant temperature bath through interfaces. Platinum anode electrodes are placed in the anode area and platinum cathode electrodes are placed in the cathode area. Two transparent PVC pipes are connected through the low-temperature constant temperature bath to allow low-temperature cooling water to be introduced into the container and contact the outer wall of the electrophoretic separation device to reduce the temperature of the pectin solution during the electromigration enrichment of pectin.

[0052] (3) Add 400 mL of the electrolyte from step (1) to the electrophoretic separation device from step (2), and stir at 800 rpm; adjust the water temperature in the low-temperature constant temperature bath to 8°C so that the temperature of the electrolyte in each reaction chamber is maintained at 15-30°C.

[0053] (4) When the water temperature of the low temperature constant temperature bath in step (3) reaches the set temperature, turn on the circulation pump of the low temperature constant temperature bath with a flow rate of 3L / min.

[0054] (5) Turn on the power, adjust the voltage to 200V, and the reaction time is 1 hour;

[0055] (6) After the reaction is complete, adjust the pH of the products on the dialysis membrane in each reaction chamber described in step (5) to 3.8 using 1M NaOH / HCl.

[0056] (7) Add 3 times the volume of 95% v / v ethanol to the product in step (6) and centrifuge at 25°C and 12000 rpm for 10 min; then remove the precipitated pectin and wash it with 75% v / v and 95% v / v ethanol.

[0057] (8) Finally, place the pectin washed in step (7) in an electric heating drying oven, set the temperature to 40°C and the drying time to 12 hours.

[0058] Option 2:

[0059] (1) Disperse 25g of beet pulp into 500g of distilled water and place 0.2g of sodium chloride in a bipolar dialysis bag; adjust the pH of the solution to 1.5 and bathe it in an 85℃ water bath for 2h; cool the crude extract solution after the hot acid water bath and filter it through a 400-mesh filter cloth; centrifuge the crude extract at 8000r / min for 20min, take the supernatant and adjust the pH to 3.5 to obtain the initial beet pectin electrolyte;

[0060] (2) The self-made electrochemical separation device is divided into several reaction chambers using a 500Da regenerated cellulose membrane. The electrophoretic separation device is placed in a container slightly larger than itself. Both ends of the container are connected to a low-temperature constant temperature bath through interfaces. Platinum anode electrodes are placed in the anode area and platinum cathode electrodes are placed in the cathode area. Two transparent PVC pipes are connected through the low-temperature constant temperature bath to introduce low-temperature cooling water into the container, which is in contact with the outer wall of the electrophoretic separation device to reduce the temperature of the pectin solution during the electromigration enrichment of pectin. The ideal reaction temperature is 20-30 degrees Celsius, and the cooling water is set to 10-12 degrees Celsius, but the reaction temperature (i.e., the electrophoretic solution temperature) will rise to 40-50 degrees Celsius.

[0061] (3) Add 500 mL of the electrolyte from step (1) to the electrophoretic separation device from step (2), with the magnetic stirrer rotating at 800 rpm; adjust the water temperature in the low-temperature constant temperature bath to 12°C so that the temperature of the electrolyte in each reaction chamber is maintained at 30-45°C.

[0062] (4) When the water temperature of the low temperature constant temperature bath in step (3) reaches the set temperature, turn on the circulation pump of the low temperature constant temperature bath with a flow rate of 4L / min.

[0063] (5) Turn on the power supply connecting the anode and cathode, adjust the voltage to 220V, and the reaction time to 1h;

[0064] (6) After the reaction is complete, pour the products on the dialysis membrane in each reaction chamber described in step (5) into beakers, and then adjust the pH to 3.8 with 1M NaOH / HCl.

[0065] (7) Add 3 times the volume of 95% v / v ethanol to the product in step (6) and centrifuge at 25°C and 12000 rpm for 12 min; then remove the precipitated pectin and wash it twice with 75% v / v and 95% v / v ethanol.

[0066] (8) Finally, place the pectin washed in step (7) in an electric heating drying oven, set the temperature to 40°C and the drying time to 12 hours.

[0067] Option 3:

[0068] Same as Scheme 2, the only difference being the reaction time is 1.5 hours;

[0069] Option 4

[0070] Same as Scheme 2, the only difference being the reaction time is 2 hours;

[0071] Option 5

[0072] Similar to Option 2, the only difference is that the reaction time is 2.5 hours.

[0073] The present invention, which adopts the above technical solution, has the following beneficial effects compared with the prior art: During the electrolysis process, a series of electrochemical reactions will occur in the sodium chloride or potassium chloride solution: acidic oxidizing water will be generated in the anode chamber, and alkaline reducing water will be generated in the cathode chamber.

[0074] Higher temperatures can improve the deesterification efficiency of the pectin solution in the anolyte region under acidic conditions, and also accelerate the oxidation of methoxy groups in high-ester pectin by free radicals in the oxidizing water, thus jointly improving the efficiency of obtaining low-methoxy pectin under acidic conditions. Outside the electrophoretic separation device, there is a larger, uncovered container (i.e., a cooling water tank) in which the electrophoretic separation device is placed. Low-temperature cooling water is introduced into this container to cool the solution inside the electrophoretic separation device. Because the resistance is low when energized, the current is high, resulting in significant heat generation in the solution. Therefore, the solution inside the electrophoretic separation device is at a relatively high temperature. The low-temperature cooling water in the outer container is used to cool the solution inside the electrophoretic separation device. The "relatively high temperature" in this section refers to a reaction temperature of 40–50 degrees Celsius.

[0075] High efficiency: Electrochemical reactions can improve reaction rates and product recovery rates. The method provided by this invention can significantly shorten pectin deesterification time, achieve a deesterification rate three times that of traditional deesterification methods, and maintain a recovery rate of over 80%.

[0076] Simplified process: Compared with the complex processes of traditional enzymatic, acidic or alkaline methods for preparing pectin, this invention uses an electrochemical reaction, which is simple in steps, easy to operate, and reduces the process flow and equipment requirements.

[0077] Excellent product quality: The low-methoxyl pectin obtained under electrochemical reaction conditions has high purity and activity, which can meet the requirements of pectin in different fields, such as food, medicine, cosmetics, etc.

[0078] Environmentally friendly: This invention uses an electrochemical method to prepare low-methoxyl pectin, which eliminates the need for harmful chemical reagents, reduces environmental pollution, and improves food safety, in line with the concepts of sustainable development and green chemistry.

[0079] In summary, the electrophoretic migration enrichment and separation technique for pectin and other plant polysaccharides provided by this invention has the advantages of high efficiency, simplified process, high-quality products, and environmental friendliness, and is suitable for industrial production and wide application. Attached Figure Description

[0080] To further illustrate the present invention, the following description is provided in conjunction with the accompanying drawings:

[0081] Figure 1 Schematic diagram of the technical process for electrophoretic migration enrichment and separation of pectin and other plant polysaccharides;

[0082] Figure 2 Molecular weight distribution diagrams of Examples 1, 2 and Comparative Example 1;

[0083] Figure 3 Scanning electron microscope images of cathode pectin aerogels from Example 1, Example 2, and Comparative Example 1;

[0084] Figure 4 Structural diagram of the auxiliary equipment of the invention;

[0085] Figure 5 for Figure 4 A partial structural diagram;

[0086] Figure 6 for Figure 4 A partial structural diagram;

[0087] Figure 7 The physicochemical parameters of the pectin of the invention and the physicochemical parameters of the comparative example are shown.

[0088] Figure 8 Design structure diagram for a dedicated electrolytic cell;

[0089] The components are: 1. Electrophoresis container; 2. Upper support part; 3. Membrane pores; 4. Membrane frame; 5. Membrane; 6. Upper support bar; 7. Handle; 8. Side support part; 9. Inlet; 10. Cold water space; 11. Fixed shaft; 12. Outlet. Detailed Implementation

[0090] This patent provides multiple parallel solutions. The different descriptions indicate that they are either improved versions of the basic solution or parallel solutions. Each solution has its own unique characteristics.

[0091] The purpose of this invention is to provide a technical method for electrophoretic migration enrichment and separation of pectin and other plant polysaccharides.

[0092] During the electrolysis of sodium chloride or potassium chloride solutions, a series of electrochemical reactions occur: acidic oxidizing water is produced in the anode region, and alkaline reducing water is produced in the cathode region.

[0093] At higher temperatures, not only can the deesterification efficiency of the pectin solution in the anode chamber be improved under acidic conditions, but the efficiency of free radical oxidation of methoxy groups in high-ester pectin can also be accelerated, thereby jointly improving the efficiency of preparing low-methoxy pectin under acidic conditions.

[0094] The electrophoretic separation device is divided into several reaction chambers by a membrane. Dialysis bags containing a certain amount of NaCl solution are placed at both ends of the cathode and anode. Platinum electrodes connected to the cathode and anode are inserted into the bags for conduction. Under the action of the electric field, pectin molecules carrying negative charges migrate directionally from the cathode to the anode and will gradually accumulate on the membrane, thereby completing the separation and purification process of pectin.

[0095] To achieve the above objectives, the present invention provides a technical method for electrophoretic migration enrichment and separation of pectin and other plant polysaccharides, comprising the following steps:

[0096] (1) Stir and disperse 30g of raw pectin meal into 600g of distilled water, adjust the pH of the solution to 1.5, and place it in an 85℃ water bath for 2 hours;

[0097] (2) After cooling the hot acid water bath crude extract solution obtained in step (1), filter it coarsely with a 400-mesh filter cloth; centrifuge the crude extract at 8000 r / min for 20 min and take the supernatant.

[0098] (3) Adjust the pH of the supernatant obtained in step (2) to 3.5 to obtain the initial pectin electrolyte;

[0099] (4) Select an electrophoretic separation device of appropriate size, install several slots on both sides of the separation device to fix the clips; then hollow out the middle of the double-layer clips and clamp the single-layer 5kDa membrane in the middle; the whole clip can be fixed in the slots.

[0100] (5) The electrophoretic separation device is divided into several reaction chambers by a membrane. Dialysis bags containing a certain amount of NaCl solution are placed at both ends of the cathode and anode. Platinum electrodes connected to the cathode and anode are inserted into the bags for conduction. Under the action of the electric field, pectin molecules carrying negative charges migrate directionally from the cathode to the anode and will gradually accumulate on the membrane.

[0101] (6) Turn on the power supply connecting the anode and cathode, adjust the voltage to 220V, and the reaction time to 1h;

[0102] (7) After the reaction is complete, pour the product from the electrophoresis tank described in step (6) into a beaker, and then adjust the pH to 3.0 with 1M NaOH / HCl.

[0103] (8) Add 3 times the volume of 75% to 95% ethanol to the product in step (7) and centrifuge at 25°C and 12000 rpm for 10 min; then remove the precipitated pectin and wash it with 75% to 95% ethanol.

[0104] (9) Finally, place the pectin washed in step (8) in an electric heating drying oven, set the temperature to 40°C and the drying time to 12 hours.

[0105] Furthermore, in step (5), the electrolyte is sodium chloride or potassium chloride.

[0106] Further, in step (5), the ratio of pectin mass to sodium chloride or potassium chloride mass is 1000:1 to 10:1.

[0107] Furthermore, the concentration of the pectin solution in step (3) is 0.1–1 w / w%.

[0108] Furthermore, the molecular weight cutoff of the membrane in step (4) is 100 Da to 2000 kDa.

[0109] Furthermore, in step (5), both the anode electrode and the cathode electrode are made of platinum-plated metal sheets.

[0110] Furthermore, the electrophoretic separation device of step (5) is placed in another container slightly larger than itself. The container has interfaces at both ends, namely the water inlet and the water outlet, which are connected to the low temperature constant temperature bath through a hose. The low temperature constant temperature bath can introduce low temperature cooling water into the container and then discharge it from the water outlet through the hose. By introducing and discharging low temperature cooling water, the heat generated in the electrophoretic tank due to electrophoretic migration caused by the power supply can be discharged, thereby realizing the temperature control of the pectin solution.

[0111] Furthermore, in step (5), the volume of electrolyte in each reaction chamber is the same, which is 100-1000 mL, and the speed of the magnetic stirrer is 500-1000 rpm.

[0112] Furthermore, in step (5), the temperature of each reaction chamber is 10-50°C, the flow rate of the cooling water circulator is 6-20 L / min, and the flow rate of the circulation pump is 1-8 L / min.

[0113] Furthermore, in step (6), the power supply voltage is 150–20000V and the reaction time is 0.5–3h.

[0114] Further, in step (6), the products in each reaction chamber after removal are adjusted to pH 3-5 with 1-3M NaOH / HCl, and then 3 times the volume of 75%-95% ethanol is added. The mixture is then centrifuged at room temperature and 1000-20000 rpm for 10-30 min. Afterward, the mixture is washed with 75%-95% ethanol.

[0115] Furthermore, the pectin is dried at 40–60°C until the moisture content meets national standards.

[0116] The beneficial effects of this invention are:

[0117] Electrophoretic migration: Electrochemical reactions are carried out using electrophoretic migration. The electrophoretic separation device is divided into different reaction chambers by membranes. Under the action of an electric field, pectin molecules carrying negative charges undergo directional electrophoretic migration from the cathode to the anode. During the migration process, the membrane in the electrophoresis tank acts as a barrier, which can gradually enrich and precipitate pectin on the membrane, thereby obtaining electrophoretically migrated and enriched pectin, ensuring the quality and activity of the product.

[0118] High efficiency: The method provided by this invention can significantly shorten the pectin deesterification time, the deesterification rate can reach three times that of traditional deesterification, and the recovery rate is over 80%.

[0119] Simplified process: Compared with the complex process of separating and purifying pectin by traditional ethanol precipitation, this invention uses an electrochemical reaction, which is simple in steps and easy to operate, reducing the process flow and equipment requirements, and greatly reducing the production cost and energy consumption.

[0120] Excellent product quality: The low-methoxyl pectin prepared under electrochemical reaction conditions has high purity and activity, which can meet the requirements of pectin in different fields, such as food, medicine, cosmetics, etc.

[0121] Environmentally Friendly / Environmentally Friendly: This invention uses an electrochemical method to prepare low-methoxyl pectin, eliminating the need for harmful chemical reagents, reducing environmental pollution, and improving food safety, aligning with the principles of sustainable development and green chemistry. Using an electrochemical method instead of ethanol precipitation saves approximately 70% of ethanol usage, reduces energy consumption during ethanol recovery, and minimizes ethanol wastewater treatment / pollution.

[0122] In summary, the electrophoretic migration enrichment and separation technique for pectin and other plant polysaccharides provided by this invention has the advantages of high efficiency, simplified process, high-quality products, and environmental friendliness, and is suitable for industrial production and wide application.

[0123] Example 1

[0124] (1) Disperse 20g of beet pulp into 400g of distilled water and place 0.2g of sodium chloride in a bipolar dialysis bag; adjust the pH of the solution to 1.5 and bathe it in an 85℃ water bath for 2h; cool the crude extract solution after the hot acid water bath and filter it through a 400-mesh filter cloth; centrifuge the crude extract at 8000r / min for 20min, take the supernatant and adjust the pH to 3.5 to obtain the initial beet pectin electrolyte;

[0125] (2) The self-made electrophoretic separation device (1000 mL) was divided into several reaction chambers using a 500 Da regenerated cellulose membrane. The electrophoretic separation device was placed in a container slightly larger than itself, and both ends of the container were connected to a cryogenic bath via interfaces. A anolyte platinum electrode (length × width = 10 mm × 10 mm) was placed in the anolyte region, and a cathode platinum electrode (length × width = 10 mm × 10 mm) was placed in the cathode region. Two transparent PVC pipes (outer diameter: 6 mm, inner diameter: 4 mm) were connected to the cryogenic bath to allow low-temperature cooling water to be introduced into the container and contact the outer wall of the electrophoretic separation device to reduce the temperature of the pectin solution during the electromigration enrichment of pectin.

[0126] (3) Add 400 mL of the electrolyte from step (1) to the electrophoretic separation device described in (2), with the magnetic stirrer rotating at 800 rpm. Adjust the water temperature in the low-temperature constant temperature bath to 8°C, so that the temperature of the electrolyte in each reaction chamber is maintained between 15 and 30°C.

[0127] (4) When the water temperature of the low temperature constant temperature bath in step (3) reaches the set temperature, turn on the circulation pump of the low temperature constant temperature bath with a flow rate of 3L / min.

[0128] (5) Turn on the power, adjust the voltage to 200V, and the reaction time is 1h.

[0129] (6) After the reaction is complete, adjust the pH of the products on the dialysis membrane in each reaction chamber described in step (5) to 3.8 using 1M NaOH / HCl.

[0130] (7) Add 3 volumes of 95% v / v ethanol to the product from step (6) and centrifuge at 25°C and 12000 rpm for 10 min. Then remove the precipitated pectin and wash it with 75% v / v and 95% v / v ethanol.

[0131] (8) Finally, place the pectin washed in step (7) in an electric heating drying oven, set the temperature to 40°C and the drying time to 12h.

[0132] Example 2

[0133] (1) Disperse 25g of beet pulp into 500g of distilled water and place 0.2g of sodium chloride in a bipolar dialysis bag; adjust the pH of the solution to 1.5 and bathe it in an 85℃ water bath for 2h; cool the crude extract solution after the hot acid water bath and filter it through a 400-mesh filter cloth; centrifuge the crude extract at 8000r / min for 20min, take the supernatant and adjust the pH to 3.5 to obtain the initial beet pectin electrolyte;

[0134] (2) The self-made electrochemical separation device (1000 mL) was divided into several reaction chambers using a 500 Da regenerated cellulose membrane. The electrophoretic separation device was placed in a container slightly larger than itself, and the two ends of the container were connected to a low-temperature thermostat via interfaces. A platinum anode electrode was placed in the anode region.

[0135] (Length × Width = 10mm × 10mm), a cathode platinum electrode is placed in the cathode area.

[0136] (Length × Width = 10mm × 10mm). Two transparent PVC pipes (outer diameter: 6mm, inner diameter: 4mm) are connected to a low-temperature constant temperature bath, through which low-temperature cooling water can be introduced into the container and contact the outer wall of the electrophoretic separation device to reduce the temperature of the pectin solution during the electromigration enrichment of pectin.

[0137] (3) Add 500 mL of the electrolyte from step (1) to the electrophoretic separation device described in (2), with the magnetic stirrer rotating at 800 rpm. Adjust the water temperature in the low-temperature constant temperature bath to 12°C, so that the temperature of the electrolyte in each reaction chamber is maintained at 30–45°C.

[0138] (4) When the water temperature of the low temperature constant temperature bath in step (3) reaches the set temperature, turn on the circulation pump of the low temperature constant temperature bath with a flow rate of 4L / min.

[0139] (5) Turn on the power supply connecting the anode and cathode, adjust the voltage to 220V, and the reaction time to 1h.

[0140] (6) After the reaction is complete, pour the products on the dialysis membrane in each reaction chamber described in step (5) into beakers, and then adjust the pH to 3.8 with 1M NaOH / HCl.

[0141] (7) Add 3 volumes of 95% v / v ethanol to the product from step (6) and centrifuge at 25°C and 12000 rpm for 12 min. Then remove the precipitated pectin and wash it twice with 75% v / v and 95% v / v ethanol.

[0142] (8) Finally, place the pectin washed in step (7) in an electric heating drying oven, set the temperature to 40°C and the drying time to 12h.

[0143] Example 3

[0144] Same as Example 2, except that the reaction time is 1.5h.

[0145] Example 4

[0146] Same as Example 2, except that the reaction time is 2 hours.

[0147] Example 5

[0148] Same as Example 2, except that the reaction time is 2.5h.

[0149] Comparative Example 1

[0150] Similar to Example 1, except that a low-temperature thermostat is not used to control the temperature of the electrolyte in the electrophoretic separation device, and the reaction is carried out at room temperature (25±2℃).

[0151] Experimental Example 1 – Determination of Weight-Average Molecular Weight (Mw) and its Distribution

[0152] The molecular weight (Mw) of pectin was analyzed and calculated using an HPGPC system. The mobile phase was 100 mmol / L sodium nitrate (containing 0.04% sodium azide), the flow rate was 0.6 mL / min, and the column temperature was 35 °C for isocratic elution. Pectin samples were prepared into 1 mg / mL solutions using the mobile phase. Separation was performed using an Ultrahydrogel Guard guard column in series with Ultrahydrogel 2000 and 1000 columns, and the samples were detected using a 2414 differential refractive index detector. Eight dextran standards (Mw: 1 × 10⁻⁶) were used.3 ~1×10 7 Da) was dissolved in the mobile phase to prepare a 2 mg / mL solution, filtered through a 0.45 μm filter membrane, and then introduced into a liquid chromatography system for detection. The obtained data were processed using Breeze data processing software to establish a standard curve. Subsequently, the differential elution curve of the obtained pectin sample was integrated based on the standard curve to obtain the molecular weight of the corresponding pectin.

[0153] Experimental Example 2 – Determination of Galacturonic Acid (GalA) Content

[0154] The galacturonic acid (GalA) content in pectin was determined colorimetrically using 3-phenylphenol as the colorimetric reagent. After color development, the maximum absorbance at 520 nm was measured using a UV-Vis spectrophotometer. A standard curve was established using D-galacturonic acid as a standard, and the GalA content in pectin was calculated using the standard curve.

[0155] Experimental Example 3 – Determination of Degree of Methyl Esterification (DM) and Degree of Acetylation (DA)

[0156] The degree of methyl esterification and acetylation of pectin was determined by HPLC. 30 mg of pectin sample was saponified in 1 mL of a saponification solution containing 0.4 M NaOH (isopropanol:water 1:1, v / v) at 25 °C for 2 h. The resulting saponified solution was then filtered through a 0.45 μm filter and analyzed by HPLC. The chromatographic system used 5 mmol / L H₂SO₄ as the mobile phase with isocratic elution at a flow rate of 0.6 mL / min. The column temperature was 45 °C, and the injection volume was 25 μL. A 2414 differential refractive index detector was used for sample detection. The DM and DA values ​​in this experiment were obtained by calculating the molar ratio of methanol to acetic acid to GalA after saponification.

[0157] Experimental Example 4 – Preparation and Characterization of Pectin Gel

[0158] A certain amount of glucono-delta-lactone (GDL) was added to a fully hydrated pectin solution, and after stirring until homogeneous, a certain amount of calcium carbonate (CaCO3) was added, resulting in final concentrations of pectin, GDL, and CaCO3 of 1.25 wt%, 45 mM, and 37.5 mM, respectively. The solution was then placed in a 4°C refrigerator for 24 hours to form a homogeneous pectin gel. The morphology of the freeze-dried pectin aerogel was photographed using a scanning electron microscope.

[0159] Table 1 Physicochemical parameters of pectin

[0160]

[0161]

[0162] In summary, as shown in Table 1, the method for preparing low-methoxylated beet pectin disclosed in this invention not only increases the galacturonic acid content but also minimizes damage to pectin molecules while achieving deesterification, preserving the integrity of the pectin structure and realizing the controllable preparation of low-methoxylated beet pectin. Figure 2 It can be seen that the low-methoxyl beet pectin prepared by this method has good uniformity and high purity; from Figure 3 It can be seen that the low-methoxyl beet pectin-based hydrogel prepared by this method has a uniform and stable structure, which helps to enhance the application potential of pectin in food and medicine.

[0163] It is important to note that this patent provides a dedicated device:

[0164] It has the following special effects: 1. It can directly lift the membrane module; 2. When the membrane module is extracted, pectin on the membrane surface is removed; 3. The spacing between the membrane modules can be flexibly adjusted; 4. It has its own cooling space, which is a five-sided water-adjacent cooling space, increasing the contact area. This greatly facilitates related research.

[0165] The device includes an electrophoresis container 1, an upper support portion 2 arranged on the electrophoresis container 1, and a membrane frame 4. The membrane frame 4 has side support portions 8 on both sides, and the side support portions 8 have grooves.

[0166] The groove of the side support part 8 faces downward and can overlap the upper support part 2;

[0167] Multiple membrane holes 3 are arranged on the membrane frame 4;

[0168] A liftable membrane unit can be placed on the membrane aperture 3. The membrane unit includes a lower membrane body and an upper support bar 6. A handle 7 is arranged on the upper support bar 6.

[0169] The number and spacing of the membranes can be adjusted by adjusting the arrangement of the membranes in membrane pores 3.

[0170] The electrophoresis container 1 is connected to an external cold water container via a fixed shaft 11. The cold water container is connected to an inlet 9 and an outlet 12. There is a cold water space 10 between the cold water container and the electrophoresis container 1. By pumping flowing cold water into the cold water space, the electrophoresis container 1 can be cooled. The electrophoresis container 1 is suspended, that is, except for the top surface of the electrophoresis container 1, the other surfaces are placed in cold water, and the bottom surface of the electrophoresis container 1 is immersed in the flowing cold water.

[0171] In the field of food processing, a method for electrophoretic migration-induced enrichment and separation of pectin is disclosed. The method involves pouring the initially extracted beet pectin electrolyte into an electrophoretic separation device, adjusting to a suitable temperature, turning on the power, and after the reaction is complete, adjusting the obtained beet pectin electrolyte to a suitable pH value. The pectin electrolyte is then subjected to alcohol precipitation, washing, and drying to obtain low-methoxylated beet pectin. This invention provides a method capable of simultaneously preparing low-methoxylated beet pectin with different molecular weights and degrees of methyl esterification. The prepared pectin not only has a high galacturonic acid content but also exhibits good gelling properties, which helps to enhance the application potential of pectin in food, medicine, and environmental protection fields. Compared with existing technologies for preparing low-methoxylated pectin, this method is simpler, more efficient, produces higher quality products, and is safer and more environmentally friendly.

[0172] In summary, this invention belongs to the field of plant extraction and separation technology, and discloses a technical method for electrophoretic migration enrichment and separation of pectin and other plant polysaccharides. The method involves dividing an electrophoretic separation device into multiple compartments using membranes of the same or different pore sizes. The crude pectin extract is filtered and placed into the electrophoretic separation device. After adjusting the solution to a suitable temperature and pH, the electronegativity of pectin / polysaccharides is utilized, causing them to migrate from the cathode to the anode under the influence of an electric field. After the reaction, the aggregates on the membranes are collected, and after alcohol precipitation, washing, and drying, the pectin / polysaccharide product is obtained. The method provided by this invention can increase the solids content of the pectin / polysaccharide extract by 10 to 30 times, significantly reduce the amount of alcohol used in alcohol precipitation, and significantly shorten the pectin deesterification time. The deesterification rate is three times that of traditional deesterification methods, and the recovery rate reaches over 80%. The main indicators of the separated pectin, such as molecular weight, degree of esterification, and galacturonic acid content, are no different from those of pectin obtained by traditional alcohol precipitation methods, and it exhibits good emulsifying and gelling properties. Compared with existing pectin or polysaccharide extraction and separation technologies, this method is simple, efficient, energy-saving, environmentally friendly, and safe. It can reduce the production cost of pectin and polysaccharides and further broaden the application of polysaccharides in food, medicine, and chemical industries.

[0173] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claims.

Claims

1. A method for electrophoretic migration enrichment and separation of pectin, characterized in that, The method includes the following steps: The raw material from which pectin or polysaccharides can be extracted is initially extracted using hot acid / alkali or enzymatic methods to obtain an initial pectin extract. The extract is then filtered through a filter to obtain a clear extract. The prepared extract was poured into an electrophoretic separation device. Several membrane modules were placed at intervals in the tank of the device. Dialysis bags were placed at both ends of the separation device, and NaCl solution was placed in the bags. Platinum-plated electrodes were placed in the dialysis bags. By selecting voltage and current, under the influence of an electric field, negatively charged pectin migrates directionally from the cathode to the anode via electrophoresis. During the migration process, the membrane inside the separation device acts as a barrier, causing pectin to gradually accumulate and precipitate on the membrane. Collect the aggregates enriched on each membrane module to obtain pectin with high solids content; Pectin was obtained by electrophoretic migration after alcohol precipitation, washing and drying.

2. The method for electrophoretic migration enrichment and separation of pectin as described in claim 1, characterized in that, The separation tank is divided into multiple chambers by several membrane plates, and the chambers are interconnected. A dialysis bag containing NaCl solution is placed at each end of the electrophoretic separation device, and a platinum electrode is implanted into the dialysis bag. Under the action of an electric field, as the pectin moves directionally toward the anode, it is collected and separated by each membrane plate in sequence according to its molecular weight and charge strength.

3. The method for electrophoretic migration enrichment and separation of pectin as described in claim 1, characterized in that, The voltage during the electrolysis process is 150–20000 V, the current is 15–2000 mA, and the reaction time is 0.5–3 h.

4. The method for electrophoretic migration enrichment and separation of pectin as described in claim 1, characterized in that, The electrolyte is sodium chloride or potassium chloride; the ratio of pectin mass to sodium chloride or potassium chloride mass is 1000:1 to 10:1; the concentration of the pectin solution is 0.1 to 1 w / w%; the molecular weight cutoff of the membrane is 100 Da to 2000 kDa; both the anode and cathode electrodes are made of platinum sheets.

5. The method for electrophoretic migration enrichment and separation of pectin as described in claim 1, characterized in that, The pectin on the membrane was adjusted to pH 3-5 with 1-3 M NaOH / HCl, and then 3 times the volume of 75%-95% ethanol was added to each product and stirred. The mixture was then centrifuged at 1000-20000 rpm for 10-30 min. After washing with 75%-95% ethanol, the product was dried at 40-60 °C to obtain the pectin product.

6. The method for electrophoretic migration enrichment and separation of pectin as described in claim 1, characterized in that, The electrophoretic separation device is a dedicated electrophoretic separation device; The device has the following structure: the device includes an electrophoresis container (1), an upper support part (2) is arranged on the electrophoresis container (1), and a membrane frame (4). The membrane frame (4) has side support parts (8) on both sides, and the side support parts (8) have grooves. The groove of the side support part (8) faces downward and can overlap the upper support part (2); Multiple membrane holes (3) are arranged on the membrane frame (4); A liftable membrane unit can be placed on the membrane aperture (3). The membrane unit includes a lower membrane body and an upper support bar (6); a handle (7) is arranged on the upper support bar (6). The number and distance of membranes can be adjusted by adjusting the arrangement of membranes in the membrane pores (3); The electrophoresis container (1) is connected to an external cold water container via a fixed shaft (11). The cold water container is connected to an inlet (9) and an outlet (12). There is a cold water space (10) between the cold water container and the electrophoresis container (1). The electrophoresis container (1) can be cooled by pumping in flowing cold water into the cold water space. The electrophoresis container (1) is suspended, that is, except for the top surface of the electrophoresis container (1), the other surfaces are placed in cold water, and the bottom surface of the electrophoresis container (1) is immersed in flowing cold water.

7. The method for electrophoretic migration enrichment and separation of pectin as described in claim 6, characterized in that, After electrophoresis is completed, pull up the handle (7) to clean the deposits on the membrane using the membrane pores (3); when the handle (7) is pulled up, a container is placed below, and the deposits cleaned from the membrane by the membrane pores (3) will fall into the container.

8. The method for electrophoretic migration enrichment and separation of pectin as described in claim 6, characterized in that, Before electrophoresis begins, adjust the number of membranes on the membrane rack (4); The membrane frame is fixed by the groove of the side support part (8) facing downwards and overlapping the upper support part (2); Electrophoresis; After electrophoresis is completed; The entire membrane frame is held upright. A container is placed below. When the handle (7) is pulled upwards, the residue on the membrane that was cleaned by the membrane pores (3) will fall into the container.

9. The method for electrophoretic migration enrichment and separation of pectin as described in claim 6, characterized in that, The electrophoretic separation device includes an outer jacket connected to a chilled water circulator; the coolant temperature is kept as low as possible without freezing.

10. A method for electrophoretic migration enrichment and separation of pectin, characterized in that, For any of the following schemes: Option 1: 20 g of beet pulp was dispersed in 400 g of distilled water and 0.2 g of sodium chloride was placed in a bipolar dialysis bag. The pH of the solution was adjusted to 1.5 and the solution was placed in an 85 ℃ water bath for 2 h. After cooling the crude extract solution from the hot acid water bath, it was coarsely filtered through a 400-mesh filter cloth. The crude extract was centrifuged at 8000 r / min for 20 min, and the supernatant was collected and the pH was adjusted to 3.5 to obtain the initial beet pectin electrolyte. (2) Divide the self-made electrophoretic separation device into several reaction chambers using a 500 Da regenerated cellulose membrane. Place the electrophoretic separation device in a container slightly larger than itself. Connect the two ends of the container to the low-temperature constant temperature bath through interfaces. Place the anode platinum sheet electrode in the anode area and the cathode platinum sheet electrode in the cathode area. Connect two transparent PVC pipes through the low-temperature constant temperature bath to allow low-temperature cooling water to be introduced into the container and contact the outer wall of the electrophoretic separation device to reduce the temperature of the pectin solution during the electromigration enrichment of pectin. (3) Add 400 mL of the electrolyte from step (1) to the electrophoretic separation device described in (2), with the magnetic stirrer rotating at 800 rpm; adjust the water temperature in the low-temperature constant temperature bath to 8 ℃, so that the temperature of the electrolyte in each reaction chamber is maintained at 15-30 ℃. (4) When the water temperature of the low temperature constant temperature bath in step (3) reaches the set temperature, turn on the circulation pump of the low temperature constant temperature bath with a flow rate of 3 L / min; (5) Turn on the power, adjust the voltage to 200 V, and the reaction time to 1 h; (6) After the reaction is complete, adjust the pH of the products on the dialysis membrane in each reaction chamber described in step (5) to 3.8 using 1M NaOH / HCl; (7) Add 3 times the volume of 95% v / v ethanol to the product in step (6) and centrifuge at 25°C and 12000 rpm for 10 min; then remove the precipitated pectin and wash it with 75% v / v and 95% v / v ethanol. (8) Finally, place the pectin washed in step (7) in an electric heating drying oven, set the temperature to 40 ℃, and dry for 12 h. Option 2: (1) Disperse 25 g of beet pulp into 500 g of distilled water and place 0.2 g of sodium chloride in the bipolar dialysis bag; Adjust the pH of the solution to 1.5 and bathe it in an 85 ℃ water bath for 2 h; after cooling the crude extract solution from the hot acid water bath, filter it coarsely with a 400-mesh filter cloth; centrifuge the crude extract at 8000 r / min for 20 min, take the supernatant and adjust the pH to 3.5 to obtain the initial beet pectin electrolyte. (2) The self-made electrochemical separation device is divided into several reaction chambers using a 500 Da regenerated cellulose membrane. The electrophoretic separation device is placed in a container slightly larger than itself. Both ends of the container are connected to a low-temperature constant temperature bath through interfaces. Platinum anode electrodes are placed in the anode area and platinum cathode electrodes are placed in the cathode area. Two transparent PVC pipes are connected through the low-temperature constant temperature bath to allow low-temperature cooling water to be introduced into the container and contact the outer wall of the electrophoretic separation device to reduce the temperature of the pectin solution during the electromigration enrichment of pectin. (3) Add 500 mL of the electrolyte from step (1) to the electrophoretic separation device described in (2), with the magnetic stirrer rotating at 800 rpm; adjust the water temperature in the low-temperature constant temperature bath to 12 ℃, so that the temperature of the electrolyte in each reaction chamber is maintained at 30-45 ℃. (4) When the water temperature of the low temperature constant temperature bath in step (3) reaches the set temperature, turn on the circulation pump of the low temperature constant temperature bath with a flow rate of 4 L / min. (5) Turn on the power supply connecting the anode and cathode, adjust the voltage to 220 V, and the reaction time to 1 h; (6) After the reaction is complete, pour the products on the dialysis membrane in each reaction chamber described in step (5) into beakers, and then adjust the pH to 3.8 with 1M NaOH / HCl. (7) Add 3 times the volume of 95% v / v ethanol to the product in step (6) and centrifuge at 25°C and 12000 rpm for 12 min; then remove the precipitated pectin and wash it twice with 75% v / v and 95% v / v ethanol. (8) Finally, place the pectin washed in step (7) in an electric heating drying oven, set the temperature to 40 ℃, and dry for 12 h. Option 3: Same as Option 2, except that the reaction time is 1.5 hours; Option 4: Same as Option 2, except that the reaction time is 2 hours; Option 5: Same as Option 2, except that the reaction time is 2.5 h.

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