Method for continuous production of chitooligosaccharide with specific degree of polymerization by immobilized enzyme membrane reactor

Abstract

The application provides a method for continuously producing chitooligosaccharide with specific polymerization degree by using an immobilized enzyme membrane reactor. The method is as follows: 1. preparing a PLGA nanofiber-temperature-sensitive hydrogel composite carrier, fixing the carrier in the inner wall of a ceramic membrane to obtain a composite carrier membrane; 2. assembling the composite carrier membrane into a folding enzyme membrane reactor with temperature control, pressure monitoring and material switching units; 3. introducing enzyme solution to fix chitosanase, and then introducing pretreated chitosan substrate to catalyze the reaction; 4. monitoring the polymerization degree on line to control the reaction process, washing the reactor after the reaction to realize continuous production, and concentrating and spray drying the product to obtain the target product. The application fuses the characteristics of multiple carriers to construct a “rigid support-flexible regulation” structure: the ceramic provides stable mechanical support, the temperature-sensitive hydrogel adjusts the swelling degree with temperature, the enzyme active site is exposed at low temperature to improve the efficiency, and the product is released by shrinking at a later stage, so that the problem of product retention and side reaction is solved.

Description

Technical Field

[0001] This invention relates to the technical field of functional sugar preparation, and specifically to a method for the continuous production of chitosan oligosaccharides with a specific degree of polymerization using an immobilized enzyme membrane reactor. Background Technology

[0002] Chitosan oligosaccharide, an oligosaccharide product with a degree of polymerization between 2 and 20 obtained by the degradation of chitosan through special bio-enzyme technology and a molecular weight ≤3200 Da, plays an indispensable role in many fields. In the pharmaceutical field, thanks to its anti-inflammatory, antioxidant, and immunomodulatory biological activities, chitosan oligosaccharide has a significant effect on improving inflammatory diseases such as arthritis, reducing joint inflammation, relieving joint pain and swelling, and promoting the repair and regeneration of articular cartilage. In addition, it also has medicinal effects such as lowering blood lipids, lowering blood sugar, lowering blood pressure, and improving digestive function, showing great potential in drug development and disease treatment. In the food field, chitosan oligosaccharide has been recognized by the state as a new food raw material and can be used to produce functional foods, beverages, food ingredients, and special diets. It can not only enhance the health functions of food but also improve its texture and flavor. For example, in baked goods such as bread and pastries, it can increase the stability of dough and improve the taste of the final product, while also acting as a prebiotic to promote the growth of beneficial intestinal bacteria. In agriculture, chitosan oligosaccharides can be used as natural pesticides, possessing bactericidal, bacteriostatic, and antiviral properties. They can effectively prevent and control plant diseases, enhance crop disease resistance, improve soil structure and fertility, promote plant growth and development, and increase plant resilience and adaptability. In environmental protection, chitosan oligosaccharides, with their excellent adsorption properties, can effectively remove heavy metal ions and other harmful substances from wastewater, reducing environmental pollution. They can also serve as a raw material for biodegradable plastics, helping to alleviate the pressure of plastic waste on ecosystems.

[0003] Chitosan oligosaccharides with different degrees of polymerization exhibit significant functional differences. Low-polymerization-degree chitosan oligosaccharides often possess better water solubility and higher bioactivity, demonstrating superior performance in antioxidant, blood glucose regulation, and cholesterol regulation. For example, in blood glucose regulation, they can more effectively slow the rate of blood glucose rise and positively impact insulin secretion and sensitivity. High-polymerization-degree chitosan oligosaccharides, on the other hand, may have unique advantages in certain applications, such as playing a role in scenarios requiring stable structures. With the deepening of research and the expansion of applications of chitosan oligosaccharides across various industries, the demand for chitosan oligosaccharides with specific degrees of polymerization is becoming increasingly urgent. Traditional chitosan oligosaccharide production methods have many limitations, making it difficult to meet the demand for large-scale, continuous production of chitosan oligosaccharides with specific degrees of polymerization. Therefore, developing an efficient and stable method for the continuous production of chitosan oligosaccharides with specific degrees of polymerization using an immobilized enzyme membrane reactor has significant practical and economic value, playing a crucial role in promoting the development of chitosan oligosaccharide-related industries. Summary of the Invention

[0004] Technical Problem to be Solved: To address the aforementioned technical problems, the purpose of this invention is to provide a method for the continuous production of chitosan oligosaccharides with a specific degree of polymerization using an immobilized enzyme membrane reactor. This method integrates the properties of a temperature-sensitive hydrogel-ceramic composite carrier and a PLGA nanofiber membrane carrier to construct a "rigid support-flexible regulation" composite carrier structure, achieving dynamic regulation of enzyme activity and precise product release. The ceramic component imparts stable mechanical properties to the carrier, serving as the supporting framework for the PLGA nanofiber membrane. The temperature-sensitive hydrogel component can adjust its own swelling degree according to changes in the reaction system temperature, thereby altering the microenvironment of the enzyme molecules and achieving real-time regulation of enzyme activity—at low temperatures, the hydrogel swelling fully exposes the enzyme's active sites, improving catalytic efficiency; in the later stages of the reaction, the heating and shrinkage promotes the rapid detachment of the product from the carrier, solving the side reaction problem caused by product retention in traditional processes.

[0005] Technical solution: A method for continuous production of chitosan oligosaccharides with a specific degree of polymerization using an immobilized enzyme membrane reactor, comprising the following steps: Step 1: Prepare a PLGA nanofiber-thermosensitive hydrogel composite carrier and fix it on the inner wall of a ceramic membrane to obtain a composite carrier membrane; Step 2: Assemble the composite carrier membrane into a folded enzyme membrane reactor, which is equipped with a temperature control unit, a pressure monitoring unit, and a material switching unit; Step 3: Introduce enzyme solution into the reactor to complete the immobilization of chitosanase. After rinsing with buffer to remove free enzyme, introduce pretreated chitosan substrate solution to carry out the catalytic reaction. Step 4: The reaction process is controlled by online monitoring of the degree of polymerization of the product. After the reaction is completed, the reactor is washed to restore its state and achieve continuous production. The product is concentrated by nanofiltration and spray dried to obtain chitosan oligosaccharides with a specific degree of polymerization.

[0006] Further, the preparation method of the PLGA nanofiber-thermosensitive hydrogel composite carrier in step 1 is as follows: Take 10-20 parts of PLGA particles, add 75-95 parts of a mixed solvent of dichloromethane and N,N-dimethylformamide in a volume ratio of 2-4:1, stir at 20-30℃ and 120-180r / min for 1.5-2.5h to form a spinning solution, and obtain a PLGA nanofiber membrane with a thickness of 0.2mm after electrospinning; immerse the membrane in an aqueous solution containing 8-12 parts of NIPAM monomer, 0.3-0.7 parts of N,N'-methylenebisacrylamide, and 0.2-0.4 parts of ammonium persulfate, deoxygenate with nitrogen for 25-35min, react at 55-65℃ for 2.5-3.5h, rinse and dry to obtain the final product.

[0007] Furthermore, the parameters for electrospinning are as follows: a 40-60mL syringe is used, the needle inner diameter is 0.6-1.0mm, the spinning voltage is 15-21kV, the receiving distance is 12-18cm, the feed rate is 0.6-1.0mL / h, and the receiving roller speed is 40-60r / min.

[0008] Furthermore, the ceramic membrane mentioned in step 1 is a tubular porous ceramic membrane with a pore size of 0.1-0.3 μm, a diameter of 25-35 cm, and a length of 50-70 cm; the composite carrier is fixed by vacuum adsorption, with an adsorption pressure of 0.1-0.15 MPa and an adsorption time of 15-25 min. After fixation, it is rinsed 2-4 times with a 0.01-0.03 mol / L acetate-sodium acetate buffer solution at pH 5.5-6.5, for 8-12 min each time.

[0009] Furthermore, the reactor described in step 2 is made of 316L or 304 stainless steel, with a 4-6cm thick insulation jacket welded to the outer wall and connected to a circulating water bath. One to three temperature sensors with an accuracy of ±0.8℃ are embedded in the jacket. The composite carrier membrane is cut into a rectangle of 0.8-1.2m × 0.4-0.6m and made into an 8-12 layer “Z” structure by folding units of 8-12cm. It is fixed by a polytetrafluoroethylene or polyethylene support. A guide tube with a diameter of 4-6cm is provided in the center of the reactor.

[0010] Furthermore, the enzyme solution mentioned in step 3 is prepared with chitosanase with an enzyme activity of 1000-1400 U / g and an enzyme concentration of 1.2-1.8 mg / mL. During enzyme immobilization, the feed rate is 4-6 mL / min, and the enzyme is kept at a constant temperature of 35-41℃ for 4-6 h. The buffer washing rate is 10-14 mL / min, and the enzyme is washed 3-5 times, each time for 25-35 min. The pressure inside the reactor is controlled at 0.12-0.23 MPa.

[0011] Furthermore, the preparation process of the chitosan substrate solution in step 3 is as follows: Take 20-30 parts of chitosan with a degree of deacetylation ≥ 85%, add 900-1100 parts of acetic acid solution with a mass fraction of 1.0%-1.4%, stir at 50-60℃ and 180-220r / min for 1.5-2.5h, adjust the pH to 5.5-6.5, and then filter through a 0.2-0.25μm microporous membrane to obtain a substrate solution with a mass concentration of 20-30g / L.

[0012] Furthermore, the reaction process described in step 4 is monitored by online GPC. The GPC detection parameters are as follows: mobile phase 0.08-0.12 mol / L sodium nitrate solution, flow rate 0.6-1.0 mL / min, column temperature 32-38℃, and sampling every 15-25 min. When the degree of polymerization of the product stabilizes in the range of 2-7, collection begins. The reaction is terminated after 1.0-1.5 h and the product is washed with buffer at 12-18 mL / min for 12-18 min.

[0013] Furthermore, in step 4, the nanofiltration concentration uses a polyamide nanofiltration membrane with a molecular weight cutoff of 250-350 Da, an operating pressure of 0.25-0.35 MPa, a temperature of 35-45℃, a flow rate of 5-7 mL / min, and is concentrated to a chitosan oligosaccharide mass concentration of 45-55 g / L; the spray drying parameters are an inlet air temperature of 170-190℃, an outlet air temperature of 75-85℃, and a feed rate of 12-18 mL / min.

[0014] The chitosan oligosaccharide with a specific degree of polymerization prepared by the above method is characterized in that the degree of polymerization of the chitosan oligosaccharide is 2-7, the purity is ≥95%, and the moisture content is ≤4%. Beneficial effects

[0015] This invention constructs a three-dimensional network carrier structure with uniform pore size and large specific surface area by controlling the concentration of PLGA spinning solution and the electrospinning voltage (15-21kV), thereby improving enzyme immobilization and catalytic efficiency. This carrier structure not only provides ample attachment sites for enzyme molecules, reducing enzyme aggregation, but also optimizes the mass transfer channels of the reaction system through the network pores, enabling efficient contact between the substrate and the enzyme active site. Simultaneously, it avoids carrier breakage caused by low-concentration spinning solution or pore blockage caused by high-concentration spinning solution, ensuring continuous and stable reaction. On one hand, the optimized immobilized enzyme membrane carrier achieves an enzyme immobilization capacity of 1.4-2.2 U / cm², an increase of 40%-120% compared to comparative examples 1-2; the substrate conversion rate is ≥92%, far exceeding the 55%-80% of the comparative examples; and the single-batch yield can be increased from 18g to 26g, achieving highly efficient production. On the other hand, the carrier structure is stable (no breakage or pore blockage issues), the membrane fouling level is low, the rinsing cycle is long, and the drying efficiency is improved, which greatly reduces equipment maintenance and production time, and lowers the energy consumption and material cost per unit product.

[0016] This invention enhances the binding force between enzyme molecules and the carrier by regulating the hydrophilicity / hydrophobicity of the carrier surface and optimizing its spatial structure, thereby extending the lifespan of the enzyme membrane and improving enzyme activity retention. Compared to traditional carriers, the carrier prepared by this process can increase the enzyme activity retention rate to 68%-72%, increase the number of times the enzyme membrane can be reused, reduce enzyme preparation consumption, lower production costs, and avoid product contamination caused by enzyme molecule shedding.

[0017] This invention utilizes biodegradable polylactic-co-glycolic acid copolymer (PLGA) to prepare nanofiber membrane carriers, combining its excellent biocompatibility and natural degradation characteristics to reduce environmental pollution from waste carriers. Compared to traditional non-degradable carriers, PLGA carriers can naturally degrade into harmless substances after their service life, avoiding the environmental pressure caused by the accumulation of carrier waste. Furthermore, the high specific surface area of ​​its nanofiber structure functionally matches the requirements of enzyme immobilization, achieving a dual improvement in environmental value and process efficiency.

[0018] This invention integrates the properties of a thermosensitive hydrogel-ceramic composite carrier and a PLGA nanofiber membrane carrier to construct a "rigid support-flexible regulation" composite carrier structure, achieving dynamic regulation of enzyme activity and precise product release. The ceramic component endows the carrier with stable mechanical properties, serving as the supporting framework for the PLGA nanofiber membrane; the thermosensitive hydrogel component can adjust its own swelling degree according to changes in the reaction system temperature, thereby altering the microenvironment of enzyme molecules and achieving real-time regulation of enzyme activity—the hydrogel swelling at low temperatures fully exposes the enzyme active sites, improving catalytic efficiency; the contraction due to temperature rise in the later stages of the reaction promotes the rapid detachment of the product from the carrier, solving the side reaction problem caused by product retention in traditional processes.

[0019] This invention develops a folded enzyme membrane reactor by designing a bifunctional composite carrier with a folded structure, achieving the effect of expanding membrane area and improving reaction efficiency within a limited space. The folded structure increases the carrier surface area per unit volume by 3-5 times, significantly increasing the probability of enzyme-substrate contact. Simultaneously, the flow channels formed by the folds optimize the flow state of the reaction solution, avoiding problems of excessively high or low local substrate concentrations and improving reaction uniformity. Compared with traditional flat-plate reactors, this structure can reduce the reactor volume by more than 60% for the same production capacity, reducing production site occupancy costs, and is particularly suitable for space optimization requirements in large-scale production. Detailed Implementation

[0020] This invention proposes a method for the continuous production of chitosan oligosaccharides with a specific degree of polymerization using an immobilized enzyme membrane reactor. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the following will provide a more detailed description of the invention with reference to specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0021] Example 1 A method for continuous production of chitosan oligosaccharides with a specific degree of polymerization using an immobilized enzyme membrane reactor includes the following steps: Step 1: Preparation of composite carrier: 15g of PLGA particles with a number-average molecular weight of 100kDa and a lactic acid to glycolic acid molar ratio of 50:50 were precisely added to 85mL of a mixed solvent of dichloromethane and N,N-dimethylformamide in a volume ratio of 3:1. The mixture was stirred at 25℃ and 150r / min for 2h to form a uniform spinning solution. After standing at room temperature for 30min to remove bubbles, electrospinning was performed using an electrospinning machine equipped with a 0.8mm inner diameter stainless steel needle. The process parameters were set as follows: spinning voltage 18kV, receiving distance 15cm, feed rate 0.8mL / h, and receiving roller speed 50r / min. The spinning was carried out continuously for 4h in a clean environment at room temperature 25℃ and relative humidity 45% to obtain a PLGA nanofiber membrane with a thickness of 0.2mm. The membrane was then cut into 20cm×20cm sheets for later use. Step 2: Weigh 10g of NIPAM monomer, 0.5g of N,N'-methylenebisacrylamide crosslinking agent, and 0.3g of ammonium persulfate initiator, and add them sequentially to 100mL of deionized water. Place the mixture in a constant temperature water bath stirrer and stir at 30℃ and 200r / min until completely dissolved. Control the flow rate of high-purity nitrogen at 50mL / min using a gas flow meter and pass it into the solution to remove oxygen for 30min to obtain a temperature-sensitive hydrogel prepolymer solution. Step 3: Completely immerse the membrane in the thermosensitive hydrogel prepolymer solution, transfer it to a 500mL high-pressure reactor, seal it, continue to purge with nitrogen for 10 minutes to remove residual air from the reactor, close the gas inlet, set the reaction temperature to 60℃, and react at a constant temperature for 3 hours; after the reaction is completed, remove the membrane, rinse it with deionized water by vacuum filtration 5 times, 10 minutes each time; after drying the washed membrane, remove it to obtain the PLGA nanofiber-thermosensitive hydrogel composite carrier; Step 4: Carrier immobilization and composite carrier membrane preparation: Select an α-Al2O3 tubular porous ceramic membrane with a pore size of 0.2μm, an inner diameter of 30cm, and a length of 60cm. Connect it to a deionized water pipeline and rinse the inner wall three times at a flow rate of 10L / h for 5min each time to remove residual impurities from the factory. Cut the PLGA nanofiber-thermosensitive hydrogel composite carrier into strips 5cm wide and 60cm long. Lay the carrier flat on the inner wall of the ceramic membrane using a vacuum adsorption device. Set the adsorption pressure to 0.12MPa and the adsorption time to 20min to ensure that the carrier and the ceramic membrane are tightly bonded without wrinkles or air bubbles. Then prepare a 0.02mol / L acetate-sodium acetate buffer solution with pH 6.0 and rinse the inner wall of the ceramic membrane three times at a flow rate of 5L / h for 10min each time to obtain the composite carrier membrane. Step 5: Assembly of the folded enzyme membrane reactor: Select a 316L stainless steel cylindrical reactor with an inner diameter of 30cm and a height of 60cm. The inner wall is manually sanded with 400-grit sandpaper until the surface roughness Ra≤0.8μm. Wipe with an anhydrous ethanol-soaked lint-free cloth three times for disinfection, and allow to air dry for 30 minutes. Weld a 5cm thick rock wool insulation layer to the outer wall of the reactor. DN20 inlet and outlet ports are pre-drilled at the top and bottom of the insulation layer, respectively. Connect the reactor to an HH-S6 constant temperature water bath as a temperature control unit via food-grade silicone tubing. Embed two PT100 temperature sensors within the insulation layer, located at the upper 1 / 3 and middle 1 / 2 of the reactor, respectively. The sensor signals are connected to the temperature controller to display and record the internal temperature in real time. Completely peel the composite carrier membrane from the ceramic membrane and cut it into 1m×0.5m rectangular sheets. Manually fold these sheets into 10 layers of a "Z" structure using 10cm folding units. Use custom-made polytetrafluoroethylene (PTFE) membranes. After the ethylene support is fixed, it is slowly placed into the reactor. A feeler gauge is used to ensure the gap between the support and the reactor wall is 1.5cm, and that the membrane is free of damage or wrinkles. An inlet with a DN25 stainless steel ball valve is installed at the top of the reactor, with a Y-100 pointer-type pressure gauge connected in parallel next to the valve for real-time monitoring of the reactor pressure. An outlet with a DN25 stainless steel ball valve is installed at the bottom. A 5cm diameter PTFE guide tube is vertically fixed at the center of the reactor, with its tip extending 10cm beyond the top of the reactor. It is connected to a peristaltic pump via a food-grade hose. The inlet of the feed pump is connected to the enzyme solution tank, substrate solution tank, and buffer solution tank via a DN15 three-way solenoid valve. The outlet is connected to the product tank via a hose. An LWGY turbine flow meter is installed on the main feed line to monitor the feed flow rate in real time. The solenoid valve switching function is tested to ensure that the material pipeline switching response time is ≤10s and that there is no pipeline residue. Step 6: Immobilization of chitosanase: Weigh 1.5 g of chitosanase (enzyme activity 1200 U / g), add 1000 mL of 0.02 mol / L acetic acid-sodium acetate buffer solution with a pre-prepared pH of 6.0, place it in a 25 °C constant temperature water bath stirrer, stir at 100 r / min for 30 min until completely dissolved. The obtained enzyme solution is filtered through a 0.22 μm polyethersulfone microporous membrane to remove insoluble impurities, obtaining an enzyme solution, which is stored in an enzyme solution storage tank; Start the constant temperature water bath of the reactor, set the temperature to 38 °C, monitor the temperature inside the reactor through a temperature controller. When the temperature stabilizes at 38 ± 0.5 °C, close the bottom discharge valve of the reactor, start the peristaltic pump, manually adjust the flow rate to 5 mL / min, pump the enzyme solution into the reactor through the central diversion tube, observe through the quartz observation window on the side wall of the reactor. When the enzyme solution completely submerges the "Z"-type composite carrier membrane, stop the feed pump; Open the exhaust valve at the top of the reactor for 1 min to remove the bubbles attached to the pipeline and the membrane surface, then close the exhaust valve, and keep it standing at a constant temperature of 38 °C for 5 h for the enzyme immobilization reaction; During this period, every 1 h, use a sterile syringe to draw a small amount of residual enzyme solution from the sampling port of the reactor, and detect the enzyme activity on a UV-2600 type ultraviolet-visible spectrophotometer (Shimadzu) using the DNS method (3,5-dinitrosalicylic acid colorimetric method). When the enzyme activity does not decrease significantly (the difference < 0.05 U / mL) after two consecutive detections, it is determined that the immobilization reaction is completed; Step 7: Removal of free enzyme and system equilibration: Open the bottom discharge valve of the reactor, drain the residual enzyme solution into the waste liquid collection tank, close the discharge valve, switch to the buffer solution storage tank through a three-way solenoid valve, start the peristaltic pump, adjust the flow rate to 12 mL / min, and pump the acetic acid-sodium acetate buffer solution with a pH of 6.0 into the reactor for flushing 4 times, each time for 30 min; During the flushing process, monitor the pressure inside the reactor in real time through the pressure gauge at the top. When the pressure exceeds 0.25 MPa, manually reduce the flow rate of the peristaltic pump; When the pressure is lower than 0.15 MPa, appropriately increase the flow rate to keep the pressure stable within the range of 0.15 - 0.2 MPa. At the same time, assist in removing the residual bubbles on the membrane surface through the exhaust valve. After each flushing, sample and detect the enzyme activity of the effluent. When the enzyme activity < 0.1 U / mL, stop flushing, complete the preparation of the immobilized enzyme membrane and system equilibration, and the reactor enters the state of waiting for production; Step 8: Pretreatment of chitosan substrate solution: While starting the enzyme immobilization reaction, simultaneously perform substrate pretreatment. Weigh 25 g of chitosan with a deacetylation degree of 90% and a molecular weight of 50 - 100 kDa, add 1000 mL of acetic acid solution with a mass fraction of 1.2%, place it in a 55 °C constant temperature water bath stirrer, stir at 200 r / min for 2 h until completely dissolved; Slowly adjust the pH value of the solution to 6.0 with 0.1 mol / L sodium hydroxide solution, and continuously stir during the adjustment process. The obtained solution is filtered through a 0.22 μm polyethersulfone microporous membrane to obtain a chitosan substrate solution, which is stored in a substrate solution storage tank for standby; Step 9: Catalytic Reaction and Product Collection: Switch the feed line using the three-way solenoid valve, close the buffer tank passage, connect the substrate solution tank, start the peristaltic pump, and adjust the feed flow rate to 15 mL / min to ensure the substrate solution enters the reactor uniformly and contacts the immobilized enzyme membrane; start the gel permeation chromatography (GPC) and set the detection parameters: mobile phase is 0.1 mol / L sodium nitrate solution (filtered through a 0.22 μm filter and degassed by ultrasonication), flow rate is 0.8 mL / min, and the chromatographic column is Ultrahydrogel™ 2000 (300 mm × 7). The reactor outlet solution was sampled every 20 minutes using an automatic sampler at a column temperature of 35℃ and a differential refractive index detector temperature of 35℃. During the reaction, the reactor temperature was maintained at 38±0.5℃ using a temperature controller. If the temperature deviated from the set value, the power of the constant temperature water bath was automatically adjusted. The pressure inside the reactor was maintained at 0.15-0.2MPa through a pressure gauge and flow meter linkage control. When GPC detection showed that the product degree of polymerization was stable within the range of 2-6, the product collection valve was opened to collect the qualified product solution into the No. 1 product storage tank. Step 10: Reaction Termination and Reactor Regeneration: After 1.2 hours of continuous feeding and reaction in the reactor, when GPC monitoring shows that the degree of polymerization of the product is close to the upper limit of 6.0, close the substrate solution feed valve and product collection valve to terminate the reaction; switch to the buffer storage tank via a three-way solenoid valve, maintain the peristaltic pump flow rate at 15 mL / min, and flush the reactor with a pH 6.0 acetate-sodium acetate buffer solution for 15 min to remove residual substrate and product on the membrane surface. The flushing solution is discharged into the waste liquid tank. After flushing, the reactor is restored to the ready-to-react state and can be switched to the substrate solution again for the next batch of reaction to achieve continuous production; after each batch, sample the enzyme activity of the immobilized enzyme membrane to ensure that the enzyme activity is ≥ 65% of the initial immobilized enzyme activity. Step 11: Product Refining and Drying: Combine the product solutions collected from each batch and transfer them to a storage tank. Start the nanofiltration system, using a polyamide nanofiltration membrane with a molecular weight cutoff of 300 Da. Set the operating parameters as follows: pressure 0.3 MPa, temperature 40℃, feed flow rate 6 mL / min. Perform desalting and concentration, taking samples every 1 hour to test the concentration of the concentrate. When the chitosan oligosaccharide mass concentration reaches 50 g / L, stop nanofiltration and collect the concentrate. Transfer the concentrate to spray drying, setting the process parameters as follows: inlet air temperature 180℃, outlet air temperature 80℃, feed rate 15 mL / min, compressed air pressure 0.2 MPa. Perform spray drying and collect the powdery product at the bottom of the drying tower, which is the chitosan oligosaccharide product with a specific degree of polymerization.

[0022] Example 2 (PLGA dosage adjustment) Difference from Example 1: In step 1, the amount of PLGA particles used was 10g, the mixed solvent was 90mL, and the mass concentration of the spinning solution was 10%. Example 3 (PLGA dosage adjustment)

[0023] Difference from Example 1: In step 1, the amount of PLGA particles used is 20g, the amount of mixed solvent is 80mL, and the mass concentration of the spinning solution is 20%. Example 4 (Spinning Voltage Adjustment)

[0024] The difference from Example 1 is that the electrospinning voltage in step 1 is 15kV. Example 5 (Spinning Voltage Adjustment)

[0025] The difference from Example 1 is that the electrospinning voltage in step 1 is 21kV. Example 6 (Enzyme Activity Adjustment)

[0026] Difference from Example 1: In step 4, the chitosanase activity was 1000 U / g and the enzyme concentration was 1.2 mg / mL. Example 7 (Enzyme Activity Adjustment)

[0027] Difference from Example 1: In step 4, the chitosanase activity was 1400 U / g and the enzyme concentration was 1.8 mg / mL. Example 8 (Substrate Concentration Adjustment)

[0028] Difference from Example 1: In step 5, the amount of chitosan used is 20g, and the concentration of the substrate solution is 20g / L. Example 9 (Substrate Concentration Adjustment)

[0029] Difference from Example 1: In step 5, the amount of chitosan used is 30g, and the concentration of the substrate solution is 30g / L. Example 10 (Adjustment of nanofiltration and drying parameters)

[0030] Differences from Example 1: In step 10, the nanofiltration membrane has a molecular weight cutoff of 350 Da and an operating pressure of 0.35 MPa; the spray drying inlet temperature is 190°C and the outlet temperature is 85°C. Comparative Example 1 (PLGA spinning solution concentration too low)

[0031] Difference from Example 1: In step 1, the amount of PLGA particles used is 5g, and the mass concentration of the spinning solution is 5%. Comparative Example 2 (PLGA spinning solution concentration too high)

[0032] Difference from Example 1: In step 1, the amount of PLGA particles used is 25g, and the mass concentration of the spinning solution is 25%. Comparative Example 3 (Electrospinning voltage too low)

[0033] The difference from Example 1 is that the electrospinning voltage in step 1 is 12kV. Comparative Example 4 (Enzyme activity too low)

[0034] Difference from Example 1: In step 4, the chitosanase activity is 800 U / g. Comparative Example 5 (reaction temperature too high)

[0035] The difference from Example 1 is that the enzyme immobilization temperature in step 4 is 45°C, and the reaction temperature in step 10 is 45°C. Comparative Example 6 (substrate with excessively low deacetylation)

[0036] Difference from Example 1: In step 5, the degree of deacetylation of chitosan is 80%.

[0037] Performance testing: 1. Determination of number-average degree of polymerization and degree of polymerization distribution (PDI) A Waters 1525 gel permeation chromatography (GPC) system was used to construct a standard curve with dextran as the standard. Chromatographic conditions: Ultrahydrogel™ 2000 column (300 mm × 7.8 mm), column temperature 35℃; mobile phase: 0.1 mol / L sodium nitrate solution, filtered through a 0.22 μm filter and ultrasonically degassed for 30 min, flow rate 0.8 mL / min; differential refractive index detector temperature 35℃; sample concentration 10 mg / mL, injection volume 20 μL. The number-average degree of polymerization (NMR) was calculated from the standard curve, and PDI was the ratio of weight-average degree of polymerization to number-average degree of polymerization.

[0038] 2. Purity determination An Agilent 1260 high-performance liquid chromatograph (HPLC) equipped with an evaporative light scattering detector (ELSD) was used. Chromatographic conditions: Zorbax SB-C18 column (250 mm × 4.6 mm, 5 μm), column temperature 30 °C; mobile phase acetonitrile-water (30:70 v / v), flow rate 1.0 mL / min; ELSD detector drift tube temperature 80 °C, carrier gas flow rate 2.0 L / min. Sample concentration was 5 mg / mL, injection volume 10 μL. Purity was calculated using chitosan oligosaccharide standards via external standard method.

[0039] 3. Moisture content determination According to GB5009.3-2016 standard, a Mettler Toledo V20 Karl Fischer moisture analyzer was used. Approximately 0.5 g of sample (accurate to 0.0001 g) was weighed and added to the calibrated Karl Fischer reagent. The mixture was stirred until the reaction was complete, and the instrument automatically recorded the moisture content. The results were measured in triplicate, and the average value was taken.

[0040] 4. Enzyme fixation assay The DNS (3,5-dinitrosalicylic acid) colorimetric method was used. The immobilized enzyme membrane was cut into 1cm × 1cm pieces, and 5mL of a pH 6.0 acetate-sodium acetate buffer solution was added. The mixture was shaken and extracted for 30 min, and the supernatant was used as the test solution. 1mL of the test solution was taken, 3mL of DNS reagent was added, and the mixture was boiled in a water bath for 5 min. After cooling, the volume was adjusted to 25mL, and the absorbance was measured at 540nm. A standard curve was plotted using chitosanase standards, and the enzyme immobilization amount was calculated. The results are expressed as U / cm² (1U is defined as the amount of enzyme required to catalyze the production of 1μmol of reducing sugar in 1 min at 38℃).

[0041] 5. Substrate conversion determination After the reaction was completed, 1 mL of the reaction solution was filtered through a 0.22 μm filter membrane, and the residual reducing sugar content was determined using the DNS colorimetric method. The substrate conversion rate was calculated using the following formula: Conversion rate (%) = (Reducing sugar equivalent in the substrate before reaction - Residual reducing sugar content after reaction) / Reducing sugar equivalent in the substrate before reaction × 100%. The reducing sugar equivalent in the substrate before reaction was determined by completely hydrolyzing chitosan.

[0042] The results are shown in Table 1 below: Table 1

[0043] As shown in Table 1 above, the number-average degree of polymerization in the examples remained stable between 2.8 and 4.2, with a PDI ≤ 1.5. Higher enzyme activity resulted in lower polymerization degree; higher spinning voltage and larger carrier surface area led to better enzyme immobilization and consequently lower polymerization degree. The product purity was ≥ 96.8%, and the moisture content was ≤ 2.4%, meeting food-grade or pharmaceutical-grade chitosan oligosaccharide standards. Example 7, with its high enzyme activity and thorough catalysis, achieved a purity of 98.5%, making it the optimal choice. Example 10 optimized nanofiltration and drying parameters, reducing the moisture content to 1.8% and improving storage stability. Enzyme immobilization capacity was 1.4-2.2 U / cm³. 2 The substrate conversion rate was ≥92%, and the yield per batch increased with increasing substrate concentration (18g in Example 8 and 26g in Example 9). Furthermore, the membrane fouling was low, making it suitable for continuous production. In Comparative Examples 1 and 2, due to improper PLGA dosage, the carrier structure was defective, resulting in enzyme immobilization at only 0.7-1.0 U / cm³. 2 The substrate conversion rate plummeted to 55%-75%, while the degree of polymerization increased (4.8-5.2), PDI ≥ 1.8, and product uniformity was poor. Comparative Examples 4 and 5 both resulted in a severe decrease in enzyme catalytic efficiency; the former achieved a substrate conversion rate of 60% and a product degree of polymerization as high as 8.2; the latter had an enzyme inactivation rate of 50%, a conversion rate of only 58%, and the product was prone to clumping. Comparative Example 6, due to its low enzyme-substrate binding efficiency, extended the reaction period to 2 hours, achieving a conversion rate of 70%.

[0044] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for continuous production of chitosan oligosaccharides with a specific degree of polymerization using an immobilized enzyme membrane reactor, characterized in that, Includes the following steps: Step 1: Prepare a PLGA nanofiber-thermosensitive hydrogel composite carrier and fix it on the inner wall of a ceramic membrane to obtain a composite carrier membrane; Step 2: Assemble the composite carrier membrane into a folded enzyme membrane reactor, which is equipped with a temperature control unit, a pressure monitoring unit, and a material switching unit; Step 3: Introduce enzyme solution into the reactor to complete the immobilization of chitosanase. After rinsing with buffer to remove free enzyme, introduce pretreated chitosan substrate solution to carry out the catalytic reaction. Step 4: The reaction process is controlled by online monitoring of the degree of polymerization of the product. After the reaction is completed, the reactor is washed to restore its state and achieve continuous production. The product is concentrated by nanofiltration and spray dried to obtain chitosan oligosaccharides with a specific degree of polymerization.

2. The method according to claim 1, characterized in that, The preparation method of the PLGA nanofiber-thermosensitive hydrogel composite carrier in step 1 is as follows: Take 10-20 parts of PLGA particles, add 75-95 parts of a mixed solvent of dichloromethane and N,N-dimethylformamide, stir at 20-30℃ and 120-180r / min for 1.5-2.5h to form a spinning solution, and obtain a PLGA nanofiber membrane with a thickness of 0.2mm after electrospinning; immerse the membrane in an aqueous solution containing 8-12 parts of NIPAM monomer, 0.3-0.7 parts of N,N'-methylenebisacrylamide, and 0.2-0.4 parts of ammonium persulfate, deoxygenate with nitrogen for 25-35min, react at 55-65℃ for 2.5-3.5h, rinse and dry to obtain the final product.

3. The method according to claim 2, characterized in that, The electrospinning parameters are as follows: a 40-60mL syringe is used, the needle inner diameter is 0.6-1.0mm, the spinning voltage is 15-21kV, the receiving distance is 12-18cm, the feed rate is 0.6-1.0mL / h, and the receiving roller speed is 40-60r / min.

4. The method according to claim 1, characterized in that, The ceramic membrane mentioned in step 1 is a tubular porous ceramic membrane with a pore size of 0.1-0.3 μm, a diameter of 25-35 cm, and a length of 50-70 cm. The composite carrier is fixed by vacuum adsorption at an adsorption pressure of 0.1-0.15 MPa and an adsorption time of 15-25 min. After fixation, it is rinsed 2-4 times with a 0.01-0.03 mol / L acetate-sodium acetate buffer solution at pH 5.5-6.5 for 8-12 min each time.

5. The method according to claim 1, characterized in that, The reactor described in step 2 is made of 316L or 304 stainless steel, with a 4-6cm thick insulation jacket welded to the outer wall and connected to a circulating water bath. One to three temperature sensors with an accuracy of ±0.8℃ are embedded in the jacket. The composite carrier membrane is cut into a rectangle of 0.8-1.2m × 0.4-0.6m and made into an 8-12 layer "Z" structure by folding units of 8-12cm. It is fixed by a polytetrafluoroethylene or polyethylene support. A guide tube with a diameter of 4-6cm is provided in the center of the reactor.

6. The method according to claim 1, characterized in that, The enzyme solution mentioned in step 3 is prepared with chitosanase with an enzyme activity of 1000-1400 U / g and an enzyme concentration of 1.2-1.8 mg / mL. During enzyme immobilization, the feed rate is 4-6 mL / min, and the enzyme is kept at a constant temperature of 35-41℃ for 4-6 h. The buffer washing rate is 10-14 mL / min, and the enzyme is washed 3-5 times for a total of 25-35 min each time. The pressure inside the reactor is controlled at 0.12-0.23 MPa.

7. The method according to claim 1, characterized in that, The preparation process of the chitosan substrate solution in step 3 is as follows: Take 20-30 parts of chitosan with a degree of deacetylation ≥85%, add 900-1100 parts of acetic acid solution with a mass fraction of 1.0%-1.4%, stir at 50-60℃ and 180-220r / min for 1.5-2.5h, adjust the pH to 5.5-6.5, and then filter through a 0.2-0.25μm microporous membrane to obtain a substrate solution with a mass concentration of 20-30g / L.

8. The method according to claim 1, characterized in that, The reaction process described in step 4 was monitored by online GPC. The GPC detection parameters were as follows: mobile phase 0.08-0.12 mol / L sodium nitrate solution, flow rate 0.6-1.0 mL / min, column temperature 32-38℃, and sampling every 15-25 min. When the degree of polymerization of the product stabilized in the range of 2-7, collection began. The reaction was terminated after 1.0-1.5 h and the product was washed with buffer at 12-18 mL / min for 12-18 min.

9. The method according to claim 1, characterized in that, The nanofiltration concentration in step 4 uses a polyamide nanofiltration membrane with a molecular weight cutoff of 250-350 Da, with an operating pressure of 0.25-0.35 MPa, a temperature of 35-45℃, a flow rate of 5-7 mL / min, and concentration to a chitosan oligosaccharide mass concentration of 45-55 g / L; the spray drying parameters are an inlet air temperature of 170-190℃, an outlet air temperature of 75-85℃, and a feed rate of 12-18 mL / min.

10. The chitosan oligosaccharide with a specific degree of polymerization prepared by the method according to any one of claims 1-9, characterized in that, The chitosan oligosaccharide has a degree of polymerization of 2-7, a purity of ≥95%, and a moisture content of ≤4%.

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