Multi-physical field online collaborative enzymatic preparation of narrow molecular weight chitooligosaccharide integrated system

Abstract

The application discloses a multi-physical-field online collaborative enzymolysis integrated system for preparing narrow-molecular-weight chitooligosaccharides, which comprises a reaction system, a conveying system, a monitoring system and an intelligent control system. The reaction system comprises a reaction kettle and a multi-physical-field coupling module integrated with a plasma generator, an ultrasonic generator and a microwave generator, and is used for applying physical field pretreatment to materials in the reaction kettle; the conveying system is used for conveying the materials; the monitoring system comprises an online monitoring module and is used for monitoring the absorbance, pH value and temperature of sample liquid in the reaction system in real time; and the intelligent control system comprises an intelligent control unit and is used for adjusting in real time according to the feedback signals of the monitoring system to maintain the preset values of temperature and pH, and the physical field pretreatment time and the enzymolysis time, so as to control the degradation process of chitosan. The application completely avoids chemical residues by means of physical field cooperation, has little environmental pollution, the enzymolysis time is shortened by more than 80%, and the yield of target products is increased by more than 4 times.

Description

Technical Field

[0001] This invention relates to the fields of biochemical engineering and functional oligosaccharide preparation technology, and in particular to an integrated system for the online synergistic enzymatic hydrolysis preparation of narrow molecular weight chitosan oligosaccharides using multiple physical fields. Background Technology

[0002] Chitosan, obtained by deacetylation of chitin, is a basic natural amino polysaccharide composed of N-acetyl-D-glucosamine and D-glucosamine. It is safe and non-toxic, possessing various biological activities such as antibacterial and antioxidant properties, and good biocompatibility. However, chitosan's large molecular weight and poor solubility in aqueous solutions with a pH above 4.5 limit its applications. Further degradation of chitosan to form chitosan oligosaccharides, especially those with lower degrees of polymerization and narrower molecular weight distribution, not only exhibits good water solubility but also stronger biological activity and higher absorption and utilization rates, showing greater potential for applications in immunomodulation, antibacterial, antioxidant, and antitumor activities.

[0003] However, current methods for preparing chitosan oligosaccharides still have many limitations. Methods for preparing water-soluble chitosan oligosaccharides mainly include chemical degradation methods (acid hydrolysis, oxidative degradation, etc.), enzymatic degradation methods (specific enzymes, non-specific enzymes), and physical degradation methods (ultrasound, microwave, etc.). Acid hydrolysis is low-cost and simple to operate, but it consumes a large amount of acid, has a long dissolution time, makes it difficult to control the molecular weight distribution of the obtained chitosan oligosaccharides, and subsequent separation and purification are difficult, and it easily causes environmental pollution. In addition, the harsh chemical environment during acid hydrolysis can easily lead to structural modification of chitosan oligosaccharides, reducing or destabilizing their biological activity, limiting their application in functional foods and biomedicine. Oxidative degradation methods are prone to browning during degradation, affecting the color of the product and subsequent purification effects. Enzymatic hydrolysis has advantages such as mild reaction conditions, green efficiency, and effective preservation of the biological activity of chitosan oligosaccharides, but the degree of hydrolysis of chitosan by a single hydrolytic enzyme is limited, and the process is time-consuming, limiting large-scale production applications. Ultrasonic degradation has the advantages of reducing energy consumption and pollution, while microwave degradation has the advantages of saving raw materials, less pollution, and shorter time consumption. However, it is difficult to obtain chitosan oligosaccharides with a narrow molecular weight distribution by using ultrasonic or microwave degradation methods alone, and they are more suitable as auxiliary degradation methods.

[0004] Plasma technology is a novel technique that utilizes the physicochemical properties of partially or completely ionized gases to achieve material processing and surface modification. It has been reported that active substances (such as hydroxyl radicals and superoxide radicals) generated during plasma treatment of solutions can attack the glycosidic bonds of chitosan, causing them to break and promoting chitosan degradation. While some studies have explored the use of ultrasound-microwave pretreatment combined with enzymatic methods to prepare chitosan oligosaccharides, the enzyme-to-substrate mass ratio (enzyme-to-substrate ratio) is as high as 24%, resulting in high costs. Similarly, the ultrasound-microwave combined method used in patent CN105218701A still requires the addition of 10%-20% oxidant (H2O2) during the degradation process. Therefore, exploring a new, highly efficient, and environmentally friendly process that can significantly reduce enzyme usage and completely eliminate the need for external oxidants has become crucial for driving industrial development.

[0005] Currently, there are no reports, either domestically or internationally, of any dedicated equipment that combines plasma technology with other physical processing technologies (such as ultrasound and microwave) for synergistic enzymatic hydrolysis, nor of any equipment that couples plasma, ultrasound, and microwave physical fields with the enzymatic hydrolysis process in real time and achieves narrow molecular weight distribution oligosaccharide preparation through online multi-parameter feedback. Summary of the Invention

[0006] This invention aims to provide an integrated system for the online synergistic enzymatic hydrolysis of narrow molecular weight chitosan oligosaccharides using multiple physical fields. By applying this dedicated equipment to prepare narrow molecular weight chitosan oligosaccharides, it is possible to pretreat chitosan stepwise or simultaneously using three physical fields: plasma, ultrasound, and microwave, to promote the enzymatic hydrolysis process. During this process, key parameters can be monitored online to achieve dynamic and precise control of the entire degradation process, thereby solving the defects of existing chitosan oligosaccharide preparation technologies, such as wide molecular weight distribution of chitosan oligosaccharides, low efficiency, serious pollution, and crude process control.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention provides an integrated system for the online synergistic enzymatic hydrolysis preparation of narrow molecular weight chitosan oligosaccharides using multiple physical fields, including a reaction system, a delivery system, a monitoring system, and an intelligent control system; The reaction system includes a reaction vessel and a multi-physics coupling module integrating a plasma generator, an ultrasonic generator, and a microwave generator. The reaction vessel is used for mixing, processing, or reacting materials, and the multi-physics coupling module is used to apply physical field pretreatment to the materials in the reaction vessel. The conveying system is used to transport materials; The monitoring system includes an online monitoring module for real-time monitoring of the absorbance, pH value, and temperature of the sample solution within the reaction system. The intelligent control system includes an intelligent control unit that is connected to the reaction system, the delivery system, and the monitoring system. It is used to adjust in real time according to the feedback signal from the monitoring system to maintain the preset values ​​of temperature and pH in the reaction system, and to dynamically adjust the physical field pretreatment time and enzymatic hydrolysis time in the reaction system to control the degradation process of chitosan.

[0008] Preferably, the plasma generator is an atmospheric pressure jet plasma generator with a power range of 300~800W.

[0009] Preferably, the ultrasonic generator is a dual-frequency ultrasonic generator with a frequency range of 20 / 40 kHz and a power range of 25-2500 W.

[0010] Preferably, the microwave generator has a frequency of 2.45 GHz and a power range of 50-4000 W.

[0011] Preferably, the reaction system further includes a temperature control system for assisting in temperature regulation within the reaction vessel; the materials include chitosan or chitosan solution, sample solution, acid-base adjustment solution, and enzyme preparation stock solution, wherein the sample solution is a solution of chitosan solution after physical field pretreatment or enzymatic hydrolysis.

[0012] Preferably, the temperature control system uses a water bath temperature control jacket for programmed heating and utilizes condensate circulation for controllable cooling.

[0013] Preferably, the reaction system further includes an observation window and an intracavitary camera.

[0014] Preferably, the online monitoring module includes an ultraviolet spectrophotometer, a pH meter, and a temperature sensor; the ultraviolet spectrophotometer and pH meter are connected to the reaction vessel through a delivery system to receive sample liquid, so as to realize real-time monitoring of the absorbance and pH value of the sample liquid; the temperature sensor is located inside the reaction vessel to monitor the temperature inside the reaction vessel.

[0015] Preferably, the intelligent control unit controls the temperature control system to adjust in real time to maintain the preset temperature value, controls the delivery system to deliver acid-base adjustment solution to maintain the preset pH value, and dynamically adjusts the physical field pretreatment time and enzymatic hydrolysis time in the reaction system based on the temperature, pH and absorbance monitored in real time by the online monitoring module.

[0016] Preferably, the physical field pretreatment time in the dynamically adjusted reaction system refers to the second pretreatment time of the plasma generator in the dynamically adjusted reaction system.

[0017] This invention also provides a method for preparing low-polymerization-degree narrow-molecular-weight chitosan oligosaccharides using the above-described integrated system, comprising the following steps: S1. Prepare a 1-2% chitosan solution in the reactor or transport the prepared chitosan solution to the reactor through a conveying system. Preparing the chitosan solution in the reactor means adding chitosan and a 1-2% acetic acid solution (v / v) to the reactor and starting the variable speed stirrer through the intelligent control unit to stir and dissolve the chitosan. S2. Set the target process parameters through the intelligent control unit, and use three physical field treatments, namely plasma, dual-frequency ultrasound and microwave, for the first pretreatment. The three physical field treatments include step-by-step start-up, or simultaneous start-up of plasma and dual-frequency ultrasound, or simultaneous start-up of microwave and dual-frequency ultrasound. The step-by-step startup refers to the sequential startup of plasma, dual-frequency ultrasound, and microwave in any order. The synchronous activation of plasma and dual-frequency ultrasound refers to the simultaneous activation of two physical fields, plasma and dual-frequency ultrasound, and microwaves in any order. The synchronous startup of microwave and dual-frequency ultrasound refers to the simultaneous startup of two physical fields, microwave and dual-frequency ultrasound, with plasma in any order. S3. After the initial pretreatment is completed, the sample liquid in the reactor is transported to the online monitoring system through the delivery system. The absorbance of the sample liquid is obtained in real time through the online monitoring system. The intelligent control unit adjusts the second pretreatment time of the plasma generator in real time based on the data until the absorbance is stable within the first target range. S4. The physical field treatment is automatically terminated, and the enzyme preparation stock solution is pumped in through the delivery system to carry out the enzymatic reaction; S5, the intelligent control unit, based on the pH value and temperature obtained by the online monitoring system, pumps acid-base adjustment solution into the temperature control system and delivery system in real time to maintain the preset values ​​of temperature and pH; at the same time, it dynamically adjusts the enzymatic hydrolysis time according to the absorbance data until the absorbance stabilizes within the second target range, which is determined as the reaction endpoint.

[0018] Preferably, the first target range is 1.8 to 2.0, and the second target range is 2.3 to 2.5.

[0019] The present invention also provides a computer device, including a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor executes the computer program to implement the steps of the method described above.

[0020] The present invention also provides a computer-readable storage medium having program instructions stored thereon, which, when executed by a processor, implement the steps of the above-described method.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The method for preparing narrow molecular weight chitosan oligosaccharides using the integrated system of the present invention, compared with traditional enzymatic hydrolysis, increases the yield of polymers with a degree of polymerization of 2-10 (DP2-10) by ≥400%; the enzyme-to-bottom ratio is only 10%, and the enzymatic hydrolysis time is shortened by more than 80%; moreover, the integrated system provided by the present invention can realize the real-time coupling of three physical fields, and achieve continuous production through online multi-parameter feedback dynamic adjustment of the process.

[0022] 2. Existing technologies for preparing narrow molecular weight chitosan oligosaccharides rely on oxidants, leading to difficulties in product purification. In contrast, this invention avoids chemical residues through the synergistic effect of physical fields and does not require the use of oxidants such as hydrogen peroxide, resulting in less environmental pollution. The overall preparation time is also reduced by 70%. Furthermore, this equipment can intelligently monitor changes in pH, temperature, and absorbance during the chitosan degradation process online, facilitating the optimization of the process for quantitative preparation of chitosan oligosaccharides with different degrees of polymerization. Attached Figure Description

[0023] Figure 1 Structural framework diagram of an integrated system for online synergistic enzymatic hydrolysis of narrow molecular weight chitosan oligosaccharides using multiple physics fields; Figure 2 Flowchart of a method for preparing narrow molecular weight chitosan oligosaccharides via enzymatic hydrolysis in an integrated system; Figure 3 A schematic diagram of a small-scale integrated system for preparing narrow molecular weight chitosan oligosaccharides via online synergistic enzymatic hydrolysis using multiple physics fields; The reference numerals in the accompanying drawings include: 1. Reaction system; 101. Reactor; 102. Plasma generator; 103. Ultrasonic generator; 104. Microwave generator; 2. Conveying system; 3. Monitoring system; 301. Ultraviolet spectrophotometer; 302. pH meter; 303. Temperature sensor; 4. Intelligent control system. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] The specific implementation of this invention relies on a specially designed integrated reaction system. This system consists of a core reaction vessel that combines microwave transmission, acid corrosion resistance, and visualization, integrated with a multi-physics field generation module (including a plasma generator, an ultrasonic generator, and a microwave generator), a delivery system, a monitoring system that integrates pH, temperature, and absorbance monitoring modules for online monitoring, and an intelligent control system equipped with an intelligent feedback control module (intelligent control unit).

[0026] In practice, the chitosan solution is first added to the reactor. The target process parameters and preset absorbance values ​​of the product are set via the touchscreen in the intelligent feedback control module. Three physical fields—plasma, dual-frequency ultrasound, and microwave—are used for pretreatment. This includes step-by-step startup, simultaneous startup of plasma and dual-frequency ultrasound, or simultaneous startup of microwave and dual-frequency ultrasound for the first pretreatment. The intelligent control unit adjusts the second pretreatment time in real-time based on the absorbance monitored by the online monitoring module (the second pretreatment uses only a plasma generator) until the absorbance stabilizes in the range of 1.8–2.0, at which point the physical field treatment automatically terminates. During the multi-physical field pretreatment, the active free radicals (such as ·OH) generated by plasma attack glycosidic bonds, achieving "pre-fracture" of the macromolecules. The cavitation effect of ultrasound not only assists in molecular chain breakage but also greatly enhances mass transfer, facilitating subsequent contact between enzymes, active substances, and substrates. Microwaves provide rapid and uniform heating, activating glycosidic bonds through electromagnetic effects.

[0027] Simultaneously with the termination of pretreatment, the pump valve was activated, and the enzyme preparation stock solution was pumped in through the delivery system to initiate the enzymatic hydrolysis reaction. During the hydrolysis process, the intelligent control unit adjusted the pH and temperature in real time based on real-time data monitored by the online monitoring module to maintain their preset values. Simultaneously, it dynamically adjusted the hydrolysis time based on absorbance data until the absorbance stabilized within the range of 2.3–2.5, which was determined as the reaction endpoint. Dynamically adjusting the second pretreatment time and the hydrolysis time in the physical field allows the degradation process to be precisely guided towards a preset narrow molecular weight distribution target. This precise real-time control ensures that the chitosan degradation process proceeds efficiently and controllably towards the preset narrow molecular weight distribution target, resulting in high-quality chitosan oligosaccharides.

[0028] This invention utilizes a comprehensive research approach combining real-time synergy of multiple physical fields and online feedback control. Compared to a single enzymatic hydrolysis method, the reaction time can be shortened by more than 80%; compared to a single physical field-assisted method, the yield of the target product (DP2-10) is increased by more than 82%.

[0029] The present invention provides an integrated system for the online synergistic enzymatic hydrolysis preparation of narrow molecular weight chitosan oligosaccharides using multiple physical fields, such as... Figure 1 , Figure 3 As shown, it includes a reaction system 1, a conveying system 2, a monitoring system 3, and an intelligent control system 4; The reaction system 1 includes a reactor 101 with a built-in variable speed stirrer for mixing reactants and carrying out enzymatic reactions; and a multiphysics coupling module integrating a plasma generator 102, an ultrasonic generator 103 and a microwave generator 104 for applying synergistic effects to the materials in the reactor.

[0030] The plasma generator 102 is an atmospheric pressure jet plasma generator with a power range of 300~800 W. It is installed above the reactor and the distance between it and the liquid surface inside the reactor is adjusted by a lifting mechanism.

[0031] The ultrasonic generator 103 is a dual-frequency ultrasonic generator with a frequency range of 20 / 40 kHz and a power range of 25-2500 W, which is continuously adjustable. It is installed on the upper side wall of the reactor.

[0032] The microwave generator 104 has a frequency of 2.45 GHz, a power range of 50-4000 W, is continuously adjustable, supports pulse or continuous wave modes, and is connected to the outer wall of the reaction tank; microwave leakage complies with the national standard GB10436-89.

[0033] The reaction system 1 also includes a temperature control system for assisting in temperature regulation within the reaction vessel 101, with an accuracy of ±0.1 ℃. The temperature control system uses a water bath temperature control jacket for programmed heating and utilizes condensate circulation for controllable cooling.

[0034] The reaction system 1 is also equipped with an observation window and an internal camera. The reactor 101 has a microwave-permeable stainless steel shell, with an inner surface lined with a PTFE layer resistant to acetic acid corrosion. The variable-speed stirring paddle inside the reactor 101 is connected to a control motor mounted above the reactor 101.

[0035] The conveying system 2 is used to convey materials, including chitosan or chitosan solution, sample solution, acid-base adjustment solution and enzyme preparation stock solution. The sample solution is a solution of chitosan solution after physical field pretreatment or enzymatic hydrolysis.

[0036] The monitoring system 3 includes an online monitoring module for real-time monitoring of the absorbance, pH value and temperature of the reaction system within the reaction system; The online monitoring module includes an ultraviolet spectrophotometer 301, a pH meter 302, and a temperature sensor 303, which are used to monitor the absorbance, pH value, and temperature of the reaction system, respectively.

[0037] The UV spectrophotometer 301 and pH meter 302 in the online monitoring module are connected to the reaction vessel via a peristaltic pump in the connecting pipeline to receive the sample solution, so as to realize real-time monitoring of the pH and absorbance of the sample solution; the absorbance range of the target product chitosan oligosaccharide with a degree of polymerization of 2-10 is 2.3~2.5.

[0038] The absorbance monitoring is based on the principle that degradation products generate ultraviolet absorbing groups. The sample solution is continuously pumped into the flow cell of the ultraviolet detector through a peristaltic pump to realize the dynamic tracking of absorbance during the degradation process. In this invention, the ultraviolet spectrophotometer detects the ultraviolet absorption wavelength of the sample solution at 385 nm.

[0039] In the online monitoring module, temperature sensor 303 is installed inside the reactor to monitor the temperature inside the reactor.

[0040] The intelligent control system 4 includes an intelligent control unit that is connected to the reaction system and the monitoring system. It is used to adjust in real time according to the feedback signal from the monitoring system to maintain the preset values ​​of temperature and pH, and to adjust the multi-physical pretreatment time and enzymatic hydrolysis time in real time to control the degradation process of chitosan.

[0041] Specifically, the intelligent control unit is connected to the multi-physics field coupling module and the online monitoring module. During the multi-physics field (ultrasonic generator 103, microwave generator 104, and plasma generator 102) pretreatment process, either sequentially or collaboratively, the intelligent control unit adjusts the working time of the plasma generator based on the absorbance obtained by the online monitoring module. During the enzymatic hydrolysis process, based on the absorbance, temperature, and pH obtained by the online monitoring module, the intelligent control unit controls the temperature control system and the delivery system to pump in an acid-base regulating solution, thereby maintaining the preset values ​​of temperature and pH in real time, and dynamically adjusting the enzymatic hydrolysis time to obtain chitosan oligosaccharides with a degree of polymerization of 2-10.

[0042] The present invention provides a method for preparing low-polymerization-degree, narrow-molecular-weight chitosan oligosaccharides using the above-described integrated system, such as... Figure 2 As shown, it includes the following steps: S1. Prepare a 1-2% chitosan solution in the reactor or transport the prepared chitosan solution to the reactor through the conveying system 2. Preparing the chitosan solution in the reactor means adding chitosan and a 1-2% acetic acid solution (v / v) to the reactor and starting the variable speed stirrer through the intelligent control unit to stir and dissolve the chitosan. S2. Set the target process parameters through the intelligent control unit, and use three physical field treatments, namely plasma, dual-frequency ultrasound and microwave, for the first pretreatment. The three physical field treatments include step-by-step start-up, or simultaneous start-up of plasma and dual-frequency ultrasound, or simultaneous start-up of microwave and dual-frequency ultrasound. Step-by-step startup refers to the sequential startup of plasma, dual-frequency ultrasound, and microwave in any order. Synchronous activation of plasma and dual-frequency ultrasound refers to the simultaneous activation of two physical fields, plasma and dual-frequency ultrasound, and microwaves in any order. Synchronous activation of microwave and dual-frequency ultrasound refers to the simultaneous activation of two physical fields, microwave and dual-frequency ultrasound, with plasma in any order. S3. After the initial pretreatment is completed, the absorbance of the sample solution is obtained in real time through the online monitoring system. The intelligent control unit adjusts the processing time of the plasma generator in real time based on the data to perform a second pretreatment until the absorbance stabilizes in the range of 1.8~2.0. S4. The physical field treatment is automatically terminated and the pump valve is opened to pump the enzyme preparation stock solution into the delivery system for enzymatic hydrolysis. S5, the intelligent control unit, based on the pH value and temperature obtained by the online monitoring system, pumps in the acid-base adjustment solution in real time through the temperature control system and the delivery system to maintain its preset value; at the same time, it dynamically adjusts the enzymatic hydrolysis time according to the absorbance data until the absorbance stabilizes in the range of 2.3~2.5, which is determined as the reaction endpoint.

[0043] It should be noted that, in production, to save time in preparing the chitosan solution, the prepared chitosan solution can be directly transported to the reactor via a conveying system.

[0044] The present invention provides a computer device, including a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor executes the computer program to implement the steps of the above-described method.

[0045] The present invention provides a computer-readable storage medium storing program instructions that, when executed by a processor, implement the steps of the above-described method.

[0046] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments (laboratory scale of 5000 mL). It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0047] Example 1: (1) Take 60.0 g of chitosan and 3000 mL of acetic acid solution with a volume fraction of 1.0% in the reactor, adjust the temperature to 40 °C, and stir mechanically until the chitosan is completely dissolved.

[0048] (2) Turn on the microwave generator (1500 W) and process for 10 min.

[0049] (3) Turn on the ultrasonic generator (1200 W), set the frequency to dual frequency 20 / 40 kHz, and perform ultrasonic treatment for 20 min.

[0050] (4) Adjust the plasma generator probe nozzle to insert 5 cm into the solution, turn on the plasma generator (800W) and intermittently process the sample solution for 10 min to obtain a uniform and transparent solution system with an absorbance of 1.92 (average of three measurements).

[0051] (5) Add 0.2% chitosanase to the above solution system, adjust the temperature of the enzymatic hydrolysate to 37℃ and the pH to 6.3, and enzymatic hydrolyze for 40 min.

[0052] (6) After the enzymatic hydrolysis is completed, adjust the temperature to 90 °C, inactivate the enzyme for 10 min, adjust the pH of the enzymatic hydrolysate to neutral online, and the absorbance in the enzymatic hydrolysate is 2.34 (average of three measurements).

[0053] (7) Collect and filter the sample solution from step (6), evaporate and concentrate the filtrate to 1 / 5 volume, add ethanol for alcohol precipitation, and freeze dry to obtain the degree of polymerization and average molecular weight of the chitosan oligosaccharide product. See Table 1.

[0054] Example 2: The only difference between this embodiment and Embodiment 1 is that the microwave treatment time is 5 min, the ultrasonic treatment time is 10 min, and the plasma treatment time is 5 min; the absorbance is 1.26 (average of three measurements), which does not fall within the first target range. When the plasma treatment time is dynamically adjusted to 6 min, the absorbance is 1.87 (average of three measurements), and the physical field treatment ends. The enzymatic hydrolysis time was 40 min. After the hydrolysis was completed, the absorbance of the hydrolysate was 2.31 (average of three measurements). The degree of polymerization and average molecular weight of the chitosan oligosaccharide product obtained after alcohol precipitation and freeze-drying are shown in Table 1.

[0055] Example 3: The only difference between this embodiment and Embodiment 1 is that the sample solution is first treated with a microwave generator, and then the plasma generator and the ultrasonic generator are started simultaneously. The enzymatic hydrolysis time was 40 min. After the hydrolysis was completed, the absorbance of the hydrolysate was 2.41 (average of three measurements). The degree of polymerization and average molecular weight of the chitosan oligosaccharide product obtained after alcohol precipitation and freeze-drying are shown in Table 1.

[0056] Comparative Example 1: The only differences between this comparative example and Example 1 are: the plasma generator, ultrasonic generator, and microwave generator were not activated; the enzymatic hydrolysis time was 40 min, and the absorbance in the hydrolysate was 0.78 (average of three measurements), which did not fall within the second target range; when the enzymatic hydrolysis time was dynamically adjusted to 300 min, the absorbance in the hydrolysate was 1.29 (average of three measurements), which still did not fall within the second target range. The degree of polymerization and average molecular weight of the chitosan oligosaccharide product obtained after alcohol precipitation and freeze-drying are shown in Table 1.

[0057] Comparative Example 2: The only difference between this comparative example and Example 1 is that only the plasma generator was activated; after enzymatic hydrolysis, the absorbance in the hydrolysate was 1.95 (average of three measurements). The degree of polymerization and average molecular weight of the chitosan oligosaccharide product obtained after alcohol precipitation and freeze-drying are shown in Table 1.

[0058] Comparative Example 3: The only difference between this comparative example and Example 1 is that only the ultrasonic generator was activated; after enzymatic hydrolysis, the absorbance in the hydrolysate was 1.38 (average of three measurements). The degree of polymerization and average molecular weight of the chitosan oligosaccharide product obtained after alcohol precipitation and freeze-drying are shown in Table 1.

[0059] Comparative Example 4: The only difference between this comparative example and Example 1 is that only the microwave generator was activated; after enzymatic hydrolysis, the absorbance in the hydrolysate was 1.46 (average of three measurements). The degree of polymerization and average molecular weight of the chitosan oligosaccharide product obtained after alcohol precipitation and freeze-drying are shown in Table 1.

[0060] Table 1. Results of degree of polymerization and average molecular weight of chitosan oligosaccharide products in the examples and comparative examples. Note: The degree of polymerization and average relative molecular weight were determined by thin-layer chromatography and acetylacetone method, respectively.

[0061] As shown in Table 1, the yield of DP2-10 was significantly improved after pretreatment with a plasma generator, an ultrasonic generator, and a microwave generator; especially the synergistic pretreatment with these three generators. In Example 1, compared to the traditional single enzymatic hydrolysis group, the yield of DP2-10 increased by 400%, and the enzymatic hydrolysis time and overall preparation time were shortened by 86% and 70%, respectively; compared to the plasma-enzymatic hydrolysis group, the yield of DP2-10 increased by 82%; compared to the ultrasonic-enzymatic hydrolysis group, the yield of DP2-10 increased by 332%; and compared to the microwave-enzymatic hydrolysis group, the yield of DP2-10 increased by 229%.

[0062] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. An integrated system for the online synergistic enzymatic hydrolysis preparation of narrow molecular weight chitosan oligosaccharides using multiple physical fields, characterized in that: This includes a reaction system, a delivery system, a monitoring system, and an intelligent control system; The reaction system includes a reaction vessel and a multiphysics coupling module integrating a plasma generator, an ultrasonic generator, and a microwave generator; the reaction vessel is used for mixing, processing, or reacting materials, and the multiphysics coupling module is used for applying physical field pretreatment to the materials in the reaction vessel; The conveying system is used to transport materials; The monitoring system includes an online monitoring module for real-time monitoring of the absorbance, pH value, and temperature of the sample solution within the reaction system. The intelligent control system includes an intelligent control unit that is connected to the reaction system, the delivery system, and the monitoring system. It is used to adjust in real time according to the feedback signal from the monitoring system to maintain the preset values ​​of temperature and pH in the reaction system, and to dynamically adjust the physical field pretreatment time and enzymatic hydrolysis time in the reaction system to control the degradation process of chitosan.

2. The integrated system for preparing narrow molecular weight chitosan oligosaccharides via online synergistic enzymatic hydrolysis according to claim 1, characterized in that, The plasma generator is an atmospheric pressure jet plasma generator with a power range of 300~800 W.

3. The integrated system for online synergistic enzymatic hydrolysis preparation of narrow molecular weight chitosan oligosaccharides according to claim 1, characterized in that, The ultrasonic generator is a dual-frequency ultrasonic generator with a frequency range of 20 / 40 kHz and a power range of 25-2500 W.

4. The integrated system for preparing narrow molecular weight chitosan oligosaccharides via online synergistic enzymatic hydrolysis according to claim 1, characterized in that, The microwave generator has a frequency of 2.45 GHz and a power range of 50-4000 W.

5. The integrated system for preparing narrow molecular weight chitosan oligosaccharides via online synergistic enzymatic hydrolysis according to claim 1, characterized in that, The reaction system also includes a temperature control system for assisting in temperature regulation within the reactor; the materials include chitosan or chitosan solution, sample solution, acid-base adjustment solution, and enzyme preparation stock solution, wherein the sample solution is a solution of chitosan solution after physical field pretreatment or enzymatic hydrolysis.

6. The integrated system for online synergistic enzymatic hydrolysis preparation of narrow molecular weight chitosan oligosaccharides according to claim 1, characterized in that, The reaction system also includes an observation window and an intracavitary camera.

7. The integrated system for preparing narrow molecular weight chitosan oligosaccharides via online synergistic enzymatic hydrolysis according to claim 5, characterized in that, The online monitoring module includes an ultraviolet spectrophotometer, a pH meter, and a temperature sensor; the ultraviolet spectrophotometer and pH meter are connected to the reaction vessel through a delivery system to monitor the absorbance and pH value of the sample solution in real time; the temperature sensor is located inside the reaction vessel to monitor the temperature inside the reaction vessel.

8. The integrated system for online synergistic enzymatic hydrolysis preparation of narrow molecular weight chitosan oligosaccharides according to claim 7, characterized in that, The intelligent control unit controls the temperature control system to maintain the preset temperature value, controls the delivery system to deliver acid-base adjustment solution to maintain the preset pH value, and dynamically adjusts the physical field pretreatment time and enzymatic hydrolysis time in the reaction system based on the temperature, pH and absorbance monitored in real time by the online monitoring module.

9. The integrated system for preparing narrow molecular weight chitosan oligosaccharides via online synergistic enzymatic hydrolysis according to claim 8, characterized in that, The physical field preprocessing time in the dynamically regulated reaction system refers to the second preprocessing time of the plasma generator in the dynamically regulated reaction system.

10. A method for preparing low-polymerization-degree, narrow-molecular-weight chitosan oligosaccharides using the integrated system according to any one of claims 1-9, characterized in that, Includes the following steps: S1. Prepare a chitosan solution in the reactor or transfer the prepared chitosan solution into the reactor; S2. Set the target process parameters through the intelligent control unit, and use three physical field treatments, namely plasma, dual-frequency ultrasound and microwave, for the first pretreatment. The three physical field treatments include step-by-step start-up, or simultaneous start-up of plasma and dual-frequency ultrasound, or simultaneous start-up of microwave and dual-frequency ultrasound. S3. After the initial pretreatment is completed, the absorbance of the sample liquid is obtained in real time through the online monitoring system. The intelligent control unit adjusts the second pretreatment time of the plasma generator in real time based on the data until the absorbance is stable within the first target range. S4. The physical field treatment is automatically terminated and the pump valve is opened. The enzyme preparation stock solution is pumped in through the delivery system to carry out the enzymatic hydrolysis reaction. S5. Based on the pH value and temperature obtained by the online monitoring system, the intelligent control unit pumps acid-base adjustment solution in real time through the temperature control system and delivery system to maintain the preset values ​​of temperature and pH. At the same time, the enzymatic hydrolysis time is dynamically adjusted according to the absorbance data until the absorbance stabilizes in the second target range, which is determined as the reaction endpoint.

11. The method according to claim 10, characterized in that, The first target range is 1.8 to 2.0, and the second target range is 2.3 to 2.

5.

12. A computer device, comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, characterized in that, When the processor executes the computer program, it implements the steps of the method of claim 10 or 11.

13. A computer-readable storage medium having program instructions stored thereon, characterized in that, When the program instructions are executed by the processor, the steps of the method described in claim 10 or 11 are implemented.

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