Method for preparing enteromorpha oligosaccharide by composite enzymolysis gradient alcohol precipitation

Abstract

The present application relates to the technical field of biological catalysis engineering, and discloses a method for preparing Enteromorpha oligosaccharides by composite enzymolysis gradient alcohol precipitation, which comprises the following steps: in the process of enzymolysis reaction, a negative feedback coupling between stirring power and the increase operation of ethanol concentration is established, the stirring power is used to represent the consumption degree of non-liquid substrate in real time, and the increase of ethanol is triggered on the basis, so that the phase equilibrium is dynamically regulated, and the selective precipitation and degradation of products are realized. By establishing a strong coupling relationship between chemical reaction and physical phase change, the present application changes the random collision process of enzymolysis into a deterministic and orderly evolution process dominated by physical phase equilibrium, and improves the ability of the system to adapt to the differences in raw materials, thereby solving the technical problems of reaction disorder and poor product selectivity in the existing enzymatic degradation.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for preparing Enteromorpha oligosaccharides by composite enzymatic degradation gradient alcohol precipitation, and belongs to the technical field of biological catalysis engineering. BACKGROUND

[0002] Currently, the enzymatic degradation of natural polysaccharide substrates is a common technical means, which is usually carried out in a homogeneous or near-homogeneous liquid environment, and the enzyme and the substrate molecules are fully contacted by mechanical stirring to achieve a high reaction conversion rate. This method is effective when the total conversion rate of polysaccharide is the goal. However, when the industrial application demand shifts to the output of specific oligosaccharides with a narrow molecular weight distribution range, an inherent limitation of this technical method becomes apparent.

[0003] The reason is that a fully mixed homogeneous reaction system provides equal contact opportunities for enzyme molecules and all sugar chain molecules dissolved therein, whether they are original long-chain substrates or generated target intermediate oligosaccharides. They will all follow their respective kinetic probabilities to become the objects of enzyme action. This means that a target oligosaccharide molecule is generated at the same time, and it immediately becomes a new substrate for further degradation. In a system where the reaction process and the separation process are separated, the target product cannot be protected in situ in time, and the continuous reaction will lead to the continuous degradation of the product to smaller molecular weights. Ultimately, a complex mixture is obtained.

[0004] To improve the selectivity of the product, the common improvement method in the art includes adjusting the reaction conditions such as temperature or pH value, or using multi-stage organic solvent precipitation and chromatography separation technology after the reaction is completed; but the former can only adjust the average molecular weight of the product, and cannot change the random nature of the reaction process, and the latter is a purification step after the reaction is completed, which cannot make up for the material loss caused by excessive degradation of the target product in the reaction stage. Specifically, the existing technology mainly has the following technical problems: 1. In a homogeneous reaction environment, the action of the enzyme lacks selectivity, the generation of the target intermediate product and the re-degradation occur at the same time, and excessive degradation is difficult to avoid; 2. The generation process of the product and the separation and protection process thereof are independent of each other in time and space, which leads to difficult process control and insufficient batch stability of the final product quality; for example, the Chinese invention patent with the authorization announcement number CN108003199B discloses a kind of enteromorpha oligosaccharide with hypoglycemic function and its preparation method and application, and finally a one-time precipitation is carried out by using a fixed concentration of ethanol to prepare enteromorpha oligosaccharide. The essence of this method is to execute the enzymatic reaction and the product separation as two completely independent steps in sequence. In the long fixed reaction time, the target oligosaccharide product generated in the early stage cannot be immediately separated and protected, and will be further degraded as a substrate. This inevitably leads to a wide molecular weight distribution and poor selectivity of the final product. The technical solution fails to effectively solve the core problems of excessive degradation of the target product and disorder of the reaction process in the enzymatic process. Therefore, how to establish a new reaction mechanism under which the generation process of a specific oligosaccharide product can directly trigger its immediate separation, and the reaction path of its excessive degradation is terminated through separation, so as to change the entire enzymatic process from a random reaction to an orderly molecular weight controllable directional reaction, becomes a technical problem to be solved by the present application. SUMMARY

[0005] The present application provides a method for preparing enteromorpha oligosaccharide by complex enzymatic gradient alcohol precipitation, which aims to solve the problem of poor product selectivity caused by the independence of the reaction process and the product separation process in the existing enzymatic degradation process.

[0006] To achieve the above-mentioned purpose, the present application provides a method for preparing enteromorpha oligosaccharide by complex enzymatic gradient alcohol precipitation, which comprises the following steps:

[0007] Step a: providing an aqueous solution containing enteromorpha polysaccharide substrate in a reactor with a stirring device, and adding ethanol to form a heterogeneous system containing a liquid phase and a non-liquid phase composed of enteromorpha polysaccharide substrate in the reactor;

[0008] Step b: adding a complex enzyme to the heterogeneous system to perform an enzymatic reaction on the interface between the liquid phase and the non-liquid phase, so that the enteromorpha polysaccharide substrate in the non-liquid phase is degraded into oligosaccharide products that are soluble in the liquid phase;

[0009] c. establishing a negative feedback coupling between the stirring power in the reactor and the operation of increasing ethanol during the enzymatic reaction, which is achieved by monitoring the stirring power in real time and triggering the operation of increasing the concentration of ethanol in the system when the stirring power drops to a stable threshold due to the consumption of non-liquid phase in the enzymatic reaction, which selectively causes the intermediate products in the molecular weight range determined by the current concentration of ethanol to precipitate from the liquid phase and become new substrates for the enzymatic reaction until the concentration of ethanol reaches a preset endpoint;

[0010] d. separating and collecting the liquid phase after the concentration of ethanol reaches the preset endpoint to obtain the oligosaccharide product.

[0011] Preferably, the determination of the stable threshold in step c and the triggering of the operation of increasing the concentration of ethanol constitute an adaptive cycle, the running mechanism of which is that after one operation of increasing ethanol causes intermediate products to precipitate and form new non-liquid phase, the stirring power rises to a peak value; as the enzymes in the new non-liquid phase interface react and consume, the stirring power decreases accordingly, and when it is monitored that the stirring power decreases to a preset proportion of the peak value, it is determined that the stable threshold is reached, and the next operation of increasing the concentration of ethanol is triggered immediately, so that the increasing rate of the concentration of ethanol directly depends on the reaction characteristics of the Enteromorpha polysaccharide substrate itself.

[0012] Preferably, the complex enzyme added in step b is a first enzyme composition; and the method further comprises: during step c, supplementing a second enzyme composition different from the components or proportions of the first enzyme composition into the reactor, the enzymes in the second enzyme composition having higher ethanol tolerance than the enzymes in the first enzyme composition, so as to superimpose a biological catalytic gradient composed of changes in enzyme components on the physical environmental gradient composed of changes in the concentration of ethanol.

[0013] Preferably, the determination rule of the stable threshold in step c is defined by the following inequality: P_current≤(1-α)·P_peak, wherein P_current is the current stirring power monitored in real time; P_peak is the stirring power peak value that occurs after one operation of increasing ethanol in this adaptive cycle; and α is a preset power drop proportion factor ranging from 0.05 to 0.2, and when the inequality is established, it is determined that the stable threshold is reached.

[0014] Preferably, the method further comprises: monitoring an acoustic signature of the reactor after each increase of the ethanol concentration in step c; and determining a morphological property of the precipitated non-liquid phase based on the acoustic signature, and when the non-liquid phase is determined to be in the form of viscous gel-like clumps with low specific surface area, performing a short interface remodeling agitation procedure comprising high-low speed rapid alternation or reverse rotation before resuming normal agitation to break the viscous gel-like clumps into loose flocs with high specific surface area.

[0015] Preferably, the method further comprises: continuously monitoring a temperature difference between a reaction temperature inside the reactor and a reference temperature outside the reactor during the whole process; and issuing a termination instruction to stop increasing the ethanol concentration and enter step d when the temperature difference is stably below a preset termination threshold close to zero due to the disappearance of the heat of enzymatic reaction, thereby providing a reaction nature-based end point arbitration for the whole self-adaptive running process.

[0016] Preferably, the non-liquid phase in step a is a gel phase or a solid phase; the initial concentration of ethanol in the system in step c is 25% to 35% by volume, and the preset end point concentration is 55% to 75% by volume; the enzymatic reaction is controlled at a temperature of 40 to 60 degrees Celsius and a pH of 4.0 to 6.0.

[0017] Preferably, the first enzyme composition mainly comprises endoglucanases responsible for rapidly reducing the molecular weight of the substrate; the second enzyme composition mainly comprises exoglucanases or debranching enzymes responsible for modifying the ends of oligosaccharides or removing branches; the operation of supplementing the second enzyme composition is performed in a continuous dropwise manner after the ethanol concentration in the system reaches an intermediate concentration point.

[0018] Preferably, the acoustic signature is a vibration signal collected by a vibration sensor installed on the outer wall of the reactor kettle body; the operation of determining the morphological property specifically comprises: temporarily changing the speed of the stirring paddle to apply an active acoustic probing excitation after increasing the ethanol concentration, and performing a fast Fourier transform on the transient response of the vibration signal induced by the excitation to determine whether viscous gel-like clumps are formed according to whether a characteristic low-frequency high-amplitude resonance peak appears in the response signal.

[0019] Preferably, after step d, the method further comprises performing nanofiltration concentration and freeze-drying treatment on the collected liquid phase to recover the solid oligosaccharide product.

[0020] Compared with the prior art, the present application has the following beneficial effects:

[0021] 1. During the enzymatic hydrolysis reaction, the concentration of organic solvent in the system is continuously controlled, establishing a strong coupling relationship between chemical reaction and physical phase transition. The degradation of the solid substrate by the enzyme directly causes the product to dissolve into the liquid phase, while the continuously increasing solvent concentration in the liquid phase selectively precipitates intermediate products of specific molecular weights as new solid-phase reaction interfaces. This process transforms the entire degradation reaction from disordered random collisions in a traditional homogeneous system into a deterministic evolutionary process continuously guided by physical phase equilibrium, proceeding sequentially from high molecular weight to low molecular weight.

[0022] 2. This invention further uses the stirring power in the reactor as a real-time physical quantity characterizing the total amount of non-liquid phase in the system, and uses the change of this physical quantity to trigger the increase of organic solvent; when the interfacial reaction causes the non-liquid phase to consume less stirring power, the system replenishes solvent to cause some intermediate products to precipitate and the stirring power to recover. This constructs a closed-loop feedback regulation based on the actual reaction process, so that the rate of increase of solvent concentration no longer depends on a fixed time program, but directly depends on the reaction characteristics of the substrate itself, thereby enabling the entire process to obtain the inherent adaptability to the differences in the physicochemical properties of different batches of raw materials.

[0023] 3. This invention further divides the complex enzyme system according to the differences in function and environmental tolerance of each component, and performs programmed supplementation at different stages of the reaction process; on top of the physical environmental gradient dominated by changes in solvent concentration, a biocatalytic gradient composed of changes in enzyme components is superimposed, so that in the early stage of the reaction, highly efficient but intolerant enzymes complete the main degradation, while in the later stage of the reaction, enzymes with better tolerance modify the product. This synergy between the physical gradient and the biological gradient allows the regulation of the final product to go beyond the single dimension of molecular weight, but extends to the control of its fine structure; and the acoustic properties of the outer wall of the reaction vessel are also considered. The resonance characteristic signal is used to determine the microscopic physical form of the newly precipitated non-liquid phase after each increase in solvent concentration, and the stirring mode is adjusted instantaneously based on the judgment result to reshape the reaction interface. This adds a mechanism to ensure the effectiveness of the microscopic reaction interface to the macroscopic process control based on stirring power. At the same time, by monitoring the differential heat flow signal inside and outside the reactor, the final disappearance of the heat of biochemical reaction is determined, providing an independent endpoint arbitration based on the nature of the reaction for the entire adaptive operation process. The introduction of these two mechanisms enables the system to get rid of dependence on indirect parameters when dealing with random disturbances in the process and determining the final reaction completion. Attached Figure Description

[0024] Fig. 1 This is a schematic diagram of the process flow for the integrated multi-module feedback control of the present invention;

[0025] Fig. 2 This is a dynamic relationship diagram of gradient alcohol precipitation triggered by negative feedback of stirring power according to the present invention;

[0026] Fig. 3 The adaptive regulation closed-loop control system architecture diagram is used for realizing the adaptive regulation of the application. DETAILED DESCRIPTION

[0027] In order to make the technical scheme and advantages of the application more clear, the technical scheme of the application will be described in detail below. Obviously, the described embodiments are only some of the embodiments of the application, but not all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0028] The application provides a method for preparing Enteromorpha oligosaccharides by gradient alcohol precipitation of complex enzymatic hydrolysis, which establishes the correlation between enzymatic hydrolysis chemical reaction and product physical phase separation, and uses the physical quantity of stirring power to feedback regulate the reaction process, so that the degradation process of polysaccharides is sequentially performed from high molecular weight to low molecular weight. The method mainly includes the construction of an initial heterogeneous system, the dynamic regulation of enzymatic hydrolysis and alcohol precipitation process based on stirring power feedback, and the reaction end point determination based on temperature difference signal.

[0029] In a specific implementation process, first, a heterogeneous reaction system needs to be constructed. This step is to add ethanol to an aqueous solution containing Enteromorpha polysaccharide substrate in a reactor with stirring device, and control the initial concentration of ethanol in the system to be in the range of 25% to 35% by volume. If the ethanol concentration is less than 25%, the precipitation of polysaccharide substrate with the largest molecular weight is not sufficient, the area of non-liquid phase interface formed is insufficient, and the initial reaction rate is affected. If the concentration is higher than 35%, a part of the intermediate product that should be degraded in the subsequent stage may be precipitated prematurely, which will interfere with the order of degradation. Through this step, a non-liquid phase of liquid phase and gel phase or solid phase composed of high molecular weight Enteromorpha polysaccharide is formed in the reactor, and the temperature of the reaction system is controlled at 40-60℃, and the pH value is controlled at 4.0-6.0, thereby forming a reaction interface in which the enzyme is distributed in the liquid phase and the substrate exists in the non-liquid phase. Subsequently, a complex enzyme is added to the heterogeneous system to start the interfacial enzymatic reaction. In the initial stage of the reaction, the first enzyme composition is added first, which mainly contains endoglucanases responsible for cutting the polysaccharide backbone. These enzymes act on the interface between the liquid phase and the non-liquid phase, and degrade the long-chain polysaccharide in the non-liquid phase into oligosaccharide products that are soluble in the current ethanol concentration liquid phase. As the reaction proceeds, after the ethanol concentration rises to an intermediate point according to the gradient program, for example, reaches 45% by volume, the second enzyme composition is added to the reactor. The second enzyme composition mainly contains exoglucanases or debranching enzymes responsible for modifying the ends of oligosaccharides or removing branches, and such enzymes have higher ethanol tolerance. Through this arrangement of supplementing different functional enzyme systems on the physical environment gradient constituted by the change of ethanol concentration, the main degradation is completed by endoglucanases in the early stage of the reaction, and the product structure is modified by ethanol-tolerant special enzymes in the later stage of the reaction.

[0030] In the process of the whole enzymatic reaction, in order to make the process adapt to the reaction characteristics difference of different batches of raw materials, the method establishes a negative feedback coupling relationship between stirring power and ethanol increasing operation; Its operation mode is: the stirring power is used as a physical quantity to represent the total amount of non-liquid phase in the system in real time, when the interface reaction consumes non-liquid phase, the viscosity of the system decreases, and the stirring power required to maintain constant speed decreases; The system monitors the stirring power P_current in real time, and judges according to the inequality P_current≤(1-α)·P_peak; Wherein, P_peak is the stirring power peak that appears after one ethanol increasing operation in this adaptive cycle, and α is a power reduction factor in the range of 0.05 to 0.2; The value of α is determined according to the calibration experiment, for example, in a standard system, when the non-liquid phase substrate is consumed by 80alpha, the central value is 0.1; If α is less than 0.05, the system is too sensitive to power changes and is easily disturbed by signal noise; If α is greater than 0.2, the response is sluggish, which may cause excessive degradation; In a numerical deduction example, assume that the power rises to a peak P_peak=150W after one ethanol increasing operation, and α is set to 0.1, then the stable threshold is calculated as(1-0.1)150W=135W; When the enzymatic reaction makes P_current drop to 135W, the inequality is established, the controller triggers the next operation to increase the ethanol concentration, so that the intermediate product selectively precipitates to form new non-liquid phase, the stirring power rises, and the next cycle begins; In this way, the increasing rate of ethanol concentration depends directly on the reaction rate of the substrate itself.

[0031] After the initial heterogeneous system is constructed and stabilized in step a, and after each ethanol increase operation in step c, the peak stirring power P_peak is determined according to a set of standardized procedures. After the initial ethanol is added, the system is continuously stirred at the preset stirring rate until the monitored stirring power value fluctuates within ±2% of the current average value for 3 consecutive minutes. The stable average value is taken as the initial peak power P_peak for the first enzymatic hydrolysis cycle. For each subsequent cycle triggered by an ethanol increase operation, the control system collects power data at a frequency of no less than 5 Hz within a 60-second time window after the ethanol feeding instruction is issued. The maximum value of the 5-second moving average of the power readings within this window is determined as the peak power P_peak for the new cycle. This procedure filters out the hydrodynamic noise caused by the ethanol injection instant using the moving average algorithm, so that the value of P_peak directly corresponds to the state of the system after the newly precipitated non-liquid phase reaches dispersion equilibrium. In addition, to deal with the possibility of forming low specific surface area viscous gel-like clumps during the alcohol precipitation process, the method also includes an interface morphology control step based on acoustic characteristic signals. A vibration sensor is installed on the outer wall of the reactor kettle body to collect the kettle body vibration signals. After each increase in ethanol concentration, the stirring paddle speed is briefly changed to apply a proactive acoustic detection excitation, and the transient response of the vibration signal induced by the excitation is analyzed by fast Fourier transform. If viscous gel-like clumps are formed, a characteristic low-frequency high-amplitude resonance peak will appear in the response signal. Once the system recognizes this characteristic peak, a short interface remodeling stirring program containing high-low speed rapid alternation or reverse rotation is executed before the normal stirring is restored, and the clumps are dispersed into loose flocs by shear force to maintain the effective area of the reaction interface.

[0032] The interface morphology control based on acoustic characteristic signal is performed in coordination with the stirring of the conventional process to avoid interference of the detection behavior on the natural precipitation morphology of the material. The coordination mechanism includes: after the completion of an ethanol increasing operation, the conventional stirring program is paused, and the system enters a precipitation window of 5 to 10 seconds; after the end of the window period, an active acoustic detection excitation is performed, i.e. the stirring paddle is driven to rotate at a low speed of 50 rpm for 2 seconds, and the tank vibration signal is collected synchronously; the control system performs fast Fourier transform on the collected signal, and if the characteristic frequency and amplitude are within the normal loose flocculent morphology range obtained by calibration, the conventional process stirring is directly restored; if the signal characteristics meet the calibration model of viscous gel-like lumps, the interface remodeling stirring program is first executed, and then the conventional process stirring is restored after the end of the program, thereby forming a closed loop operation sequence of detection, judgment and execution; finally, in order to provide an end point determination basis for the entire process based on whether the biochemical reaction itself is completed, the method further includes an independent termination mechanism; the mechanism works by continuously monitoring the temperature difference between the reaction temperature in the reactor and the reference temperature outside the reactor; since the enzymatic reaction is exothermic, as long as the reaction is ongoing, the temperature in the reactor will be slightly higher than the external reference temperature, forming a small positive temperature difference; when the substrate is depleted and the reaction heat disappears, the temperature difference will be stably below a preset termination threshold, for example 0.02°C; when this phenomenon is monitored, the system issues a termination instruction to stop increasing ethanol and enter step d, i.e. separate and collect the liquid phase; the collected liquid phase can be further nanofiltered, concentrated and freeze-dried to recover the solid oligosaccharide product; this end point determination method based on the disappearance of reaction heat makes the termination decision of the process directly related to the completion of the biochemical reaction.

[0033] In a specific embodiment, the complex enzyme is further defined as comprising a first enzyme composition and a second enzyme composition, the first enzyme composition having as a major active component an endo-enzyme responsible for random cleavage within the polysaccharide backbone, which acts to rapidly depolymerize the initial non-liquid phase interface macromolecular polysaccharide substrate into a series of water-soluble intermediate products, and the second enzyme composition having as a major active component an exo-enzyme responsible for cleaving monosaccharide or oligosaccharide residues from the end of the sugar chain, or a debranching enzyme responsible for removing branched structures, which acts to modify and finely degrade the intermediate products that have been dissolved in the liquid phase to obtain oligosaccharides of a target molecular weight range; in a specific example, the ratio of the endo-enzyme in the first enzyme composition to the exo-enzyme or debranching enzyme in the second enzyme composition, in terms of enzyme activity units (U), is in the range of (2:1) to (5:1); when the ratio is lower than 2:1, the relative content of the endo-enzyme in the system is insufficient, resulting in a too low degradation rate of the initial non-liquid phase, a slow decrease in stirring power, and thus a sluggish response of the ethanol concentration increase based on power feedback, prolonging the total process time; when the ratio is higher than 5:1, the endo-enzyme is in relative excess, which rapidly generates a large amount of intermediate products with a wide molecular weight distribution in the early stage of the reaction, which exceeds the fine modification capacity of the subsequently supplemented second enzyme composition, ultimately leading to an increase in the polydispersity index (PDI) of the product, therefore, controlling the enzyme activity ratio in the range of (2:1) to (5:1) can synergistically ensure the efficiency of the initial interface reaction and the uniformity of the molecular weight of the subsequent product; to ensure the stability of the process in long-term operation, the control system can introduce a periodic recalibration procedure, which is automatically triggered at a preset production batch interval or when the polydispersity index (PDI) of the final product deviates from the target value by more than 5% for three consecutive batches, to recalibrate the stirring power decrease ratio factor a, the acoustic feature judgment threshold, and the reaction endpoint temperature difference threshold, and use the updated parameters as the control benchmark for subsequent production.

[0034] Example 1: In an industrial production of a specific molecular weight range of Enteromorpha oligosaccharides, the raw material of a batch of Enteromorpha polysaccharides processed due to different sources showed the characteristics of high average molecular weight, complex branched structure and easy to form viscous gel. If the enzyme degradation process based on the fixed time program and increasing ethanol concentration is used, the product with the required molecular weight distribution cannot be obtained. In order to process this batch of raw materials, the Enteromorpha polysaccharide substrate aqueous solution of the raw material was placed in a reactor equipped with a power sensor, an outer wall vibration sensor and an inner and outer temperature difference monitoring probe. Ethanol was added to make the initial concentration 30% by volume percentage, and after forming a heterogeneous system, the first enzyme composition was added to start the reaction. Since the degradation rate of this batch of raw material is lower than that of the standard raw material, the stirring power P_current monitored by the system decreases accordingly, and based on the negative feedback coupling relationship between stirring power and ethanol increasing operation, the trigger frequency of the ethanol metering pump is automatically reduced, as a result, the increasing rate of ethanol concentration is directly determined by the degradation rate of the substrate itself. This kind of self-adaptive rate adjustment provides a longer degradation time window for the substrate with lower reaction activity, avoiding the problem of embedding and solidifying the insufficiently degraded substrate due to the rapid increase of ethanol concentration.

[0035] When the reaction proceeds to about 48% of the ethanol concentration of the system, the vibration sensor on the outer wall of the reactor identifies a characteristic low-frequency high-amplitude resonance peak in the vibration signal collected by the active acoustic detection excitation after one ethanol increasing operation, and the system judges that low specific surface area viscous gel-like clumps are precipitated in the reactor. At this time, the control system suspends the regular stirring and executes the interface remodeling stirring program. Through several times of rapid alternation of high and low speed, the clumps are dispersed into loose floc by shear force until the acoustic characteristic signal returns to the normal range. Under this working condition, the feedback regulation of stirring power determines the overall rate of the process, and the monitoring of acoustic characteristic signal corrects the physical form of the reaction interface after each phase separation in time, so that the reaction progress and interface effectiveness are considered; as the reaction continues, when the ethanol concentration exceeds 45%, the system adds the second enzyme composition which is resistant to ethanol according to the program, and modifies the branched structure of the newly generated oligosaccharides; as the reaction enters the end stage, the stirring power changes tend to be flat, and the end point judgment basis of the system switches to the inner and outer temperature difference signal; when the temperature difference between the inside and outside of the reactor is monitored to be stable below the termination threshold of 0.02℃ due to the disappearance of enzyme reaction heat, the system issues a termination instruction to stop increasing ethanol and enters the product separation step; the collected liquid product is analyzed, and the oligosaccharide molecular weight distribution is concentrated, the polydispersity coefficient is less than 1.2, and the branched structure content is lower than the preset requirement. This result is obtained under the condition that the raw material used is a batch with high variability.

[0036] Example 2: To objectively verify the technical effect of the method of the present application in improving the molecular weight uniformity of the product, the following comparative test was designed and carried out; the test used a jacketed 5L standard reaction kettle, which was equipped with a temperature control unit with a temperature control accuracy of ±0.1℃, a stirring device connected with a power meter, and a high-performance gel chromatograph for measuring the molecular weight distribution of the product; the test raw material was the same batch of crude Enteromorpha polysaccharide, with an initial weight average molecular weight of 2.5 x 10 Da and a polydispersity coefficient PDI of 3.8; the enzyme preparation used was a composite cellulase, and the total enzyme activity addition amount was kept the same in each test group; the test set one sample group of the present application and two control groups; the sample group of the present application was executed according to the technical scheme of the specific embodiment, in which the initial ethanol concentration was set to 30% by volume, the stirring power reduction ratio factor was set to 0.1, the preset end point ethanol concentration was 60% by volume, the reaction temperature was 50℃, and the pH was 5.0; control group A used homogeneous enzyme hydrolysis, and the reaction was carried out in a buffer without adding ethanol for 4 hours, after which ethanol was added to reach a final concentration of 60%, and the supernatant was collected; control group B used a fixed time program gradient alcohol precipitation enzyme hydrolysis, and the initial conditions were the same as those of the sample group of the present application, but after the start of the reaction, the ethanol concentration of the system was linearly increased to 60% within 3 hours at a constant rate through a metering pump.

[0037] After the test was completed, the liquid phase products obtained in each group were analyzed, and the results showed that different preparation methods had an effect on the yield and molecular weight distribution of the product; control group A using homogeneous enzyme hydrolysis, the target oligosaccharide, i.e. the component with a degree of polymerization DP of 5 to 15, had a yield of only 18.2%, and the PDI value of the final product was 2.95, indicating that the product components were complex and had poor uniformity; in comparison, control group B using fixed time gradient alcohol precipitation, the target oligosaccharide yield was increased to 35.7%, and the PDI value was reduced to 1.81, showing that the gradient alcohol precipitation operation itself had a certain effect on improving selectivity; in the sample group of the present application, the target oligosaccharide yield reached 62.5% within a total process time of 3.2 hours driven by the stirring power feedback, and the PDI value of the final product was 1.18; this data showed that, compared with the fixed time program used in control group B, the adaptive feedback adjustment mechanism based on stirring power in the sample group of the present application coupled the ethanol concentration increase rate with the actual reaction progress of the substrate, which was a key factor in further improving the yield and molecular weight uniformity of the target product.

[0038] Example 3: This example describes a method for preparing Enteromorpha oligosaccharides by composite enzyme hydrolysis gradient alcohol precipitation, as follows: Figs. 1 to 3 , wherein the initial ethanol concentration is 30% by volume, the stirring power reduction ratio factor is 0.1, the preset end point ethanol concentration is 60% by volume, the reaction temperature is 50℃, and the pH is 5.0. Fig. 1As shown, the process begins with the *Ulva prolifera* polysaccharide substrate as input. First, initial ethanol is added to construct an initial heterogeneous system containing a solid-liquid interface. Then, a complex enzyme is added to initiate interfacial enzymatic hydrolysis, degrading the polysaccharide. The core of the process is an adaptive enzymatic hydrolysis and gradient ethanol precipitation core cycle. In this cycle, the selective precipitation and orderly degradation of the product are achieved by continuously controlling the ethanol concentration. The operation of this core cycle is monitored and regulated in real time by three parallel control modules: one is a stirring power negative feedback control module, which judges the degree of consumption of the non-liquid phase by real-time monitoring of the stirring power, and accordingly... The system consists of three main components: a trigger for ethanol addition; an acoustic characteristic morphology control module, which monitors the acoustic signals of the reactor to determine whether the newly precipitated non-liquid phase is gel-like and optimizes the stirring mode accordingly to execute the interface remodeling stirring program; a temperature difference signal endpoint arbitration module, which continuously monitors the temperature difference inside and outside the reactor to determine whether the heat of reaction has disappeared and issues a termination command accordingly. Upon receiving the termination command, the core cycle ends and enters the product separation and collection step to collect the liquid phase containing the target oligosaccharide. Finally, through post-processing steps such as nanofiltration concentration and freeze drying, high-purity final product Ulva oligosaccharide is obtained.

[0039] like Fig. 2 As shown in the figure, the horizontal axis represents time in minutes (min), the left vertical axis represents stirring power in watts (W), and the right vertical axis represents ethanol concentration in volume percentage (%). The solid line represents the real-time monitored stirring power, which decreases periodically due to the consumption of non-liquid phases. The dashed line represents the ethanol concentration in the system, which increases in a stepwise manner under the trigger of control commands. The dotted line represents the stability threshold calculated based on the peak stirring power. As can be seen from the figure, whenever the stirring power shown by the solid line drops to the same level as the stability threshold shown by the dotted line, the system immediately triggers an ethanol increase operation, causing the ethanol concentration shown by the dashed line to jump to the next plateau. This operation then leads to the precipitation of intermediate products and causes the stirring power to rise to a new peak, thus starting the next adaptive cycle of consumption monitoring triggering precipitation.

[0040] like Fig. 3As shown, the system takes a main reactor as the core, the state of which is collected by a series of sensors, including a power sensor for obtaining stirring power feedback, a reactor body vibration sensor for analyzing non-liquid phase form, an internal and external temperature difference probe for end point arbitration, and a temperature control / acid-base degree probe for monitoring conventional process parameters. All sensor data are transmitted to a central control server, and the process control software running in the server analyzes the data in real time. The software contains core modules corresponding to each control function, namely, a stirring power feedback module, an acoustic form analysis module, and an end point arbitration module. According to the analysis results, the software issues control instructions to a series of actuators, including a stirring device for adjusting the reaction interface and the mixing state of the system, an ethanol feeding pump for performing gradient alcohol precipitation operation, and a complex enzyme feeding system for supplementing special enzymes at specific stages of the process. The state monitoring and manual operation of the entire system are completed through a human-machine interface, i.e., an operator terminal, thereby forming a complete automatic control loop.

[0041] Example 4: To determine the key control parameters of the method of the application in a specific reaction device, a standardized calibration procedure needs to be performed before the process is run. The procedure is performed in a reactor of the same size as that used in actual production, which contains an ethanol aqueous solution with the same initial concentration as the process, and an inert polymer microsphere with stable physicochemical properties and similar characteristics to the target non-liquid phase is used as a non-liquid phase simulator to establish the relationship between process physical quantities and control parameters without biochemical reaction interference. First, when only the ethanol aqueous solution is in the reactor, the process is run at the preset process stirring rate, and the stirring power in the stable state is recorded as the baseline power P_liquid. Then, inert polymer microspheres with a total mass of M_total are added to the reactor, which corresponds to the mass of the initial precipitated non-liquid phase in the actual process. After the microspheres are fully dispersed and stable, the stirring power peak P_peak is recorded. To calibrate the stirring power reduction ratio factor, the solid-liquid mixture is removed from the reactor in batches to gradually reduce the mass of the remaining polymer microspheres, and the stirring power P_current is recorded at each mass point after stabilization. Thus, the power-solid content relationship curve of the system is drawn. According to the relationship curve, the stirring power value corresponding to the remaining microsphere mass of 0.2M_total is found, which is defined as the stable threshold P_threshold when the non-liquid phase is consumed by 80%. Finally, the value of a is calculated by the formula a=(P_peak-P_threshold)P_peak. In this calibration, if P_peak is 150 W and P_threshold is 132 W, then the value of a is calculated to be 0.12, which is set as the control parameter for subsequent process operation.

[0042] To calibrate the judgment threshold of acoustic characteristic signal, a small amount of high viscosity sodium alginate solution is added to the system containing M_total polymer microspheres in the kettle to simulate the formation of viscous gel-like mass; the vibration signal of the kettle outer wall of the gel state system is recorded, and fast Fourier transform is performed on it to obtain the gel acoustic resonance spectrum; compared with the previously recorded loose flocculent normal spectrum containing only polymer microspheres, it is found that the signal amplitude of the gel state jumps in the frequency band of 50Hz to 150Hz; accordingly, the judgment threshold is determined: if the integral power of the vibration signal induced by the active acoustic detection excitation in the frequency band of 50Hz to 150Hz during the process operation exceeds 3 times the integral power of the same frequency band in the loose flocculent state during the calibration stage, it is determined that viscous gel-like mass is formed, and the interface remodeling stirring program is triggered; the program is set to: forward rotation at 300rpm for 5 seconds, and reverse rotation at 100rpm for 3 seconds, and the cycle is executed three times; through the above procedure, the control parameters suitable for the system are obtained.

[0043] In one process operation, due to the failure of external equipment, the valve of the ethanol feeding pump was stuck, resulting in the injection of a large amount of non-pre-set ethanol into the reactor in a short time; such sudden solvent concentration jump makes a large amount of dissolved intermediate product in the liquid phase precipitate instantaneously and quickly aggregate around the stirring paddle, forming a high-density semi-solid gel mass that partially wraps the stirring paddle, which will cause the failure of conventional process control methods.

[0044] Under this sudden condition, the coordination mechanism of the method of the application is triggered, the stirring power monitoring system detects that the power value rises in a short time and exceeds the normal stirring power peak P_peak in the process cycle, and the system judges that mechanical overload occurs accordingly; at the same time, the acoustic characteristic signal collected by the vibration sensor on the outer wall of the kettle changes suddenly, and the vibration amplitude in the low frequency band exceeds the gel state judgment threshold set in the calibration stage, indicating that a large volume of solid obstacle is formed in the reactor; based on the comprehensive judgment of the two physical signals, the control system confirms that a large volume of rapid gel event has occurred, and then automatically enters the fault recovery program, which first forcibly closes the ethanol feeding pump and suspends the conventional adaptive cycle, and then starts an interface remodeling stirring program in high torque mode, which mechanically breaks the semi-solid gel mass through alternating positive and negative rotation pulses until the stirring power falls within the equipment safety threshold and the acoustic characteristic signal returns to the characteristic spectrum range of loose flocculent material, then the system exits the fault recovery program and restores the stable state of the system as the new baseline, and continues to execute the adaptive enzyme hydrolysis and alcohol precipitation process.

[0045] Example 6: To establish a baseline reference model for the determination of the end point of enzymatic reaction under the specific thermodynamic environment of the reactor, a pre-standardized thermodynamic characterization procedure is required to be performed once the process is applied to a new equipment or after the replacement of key heat exchange components; this procedure aims to quantify the background thermal noise level of the specific system and to set a statistically significant threshold for the determination of the end of reaction, while establishing a set of fault-tolerant mechanisms for the stagnation conditions at the end of reaction; this procedure first loads the reactor with a solution similar to the actual end point state of the process, i.e. with a concentration of ethanol at the target end point, containing the concentration of inactivated enzymes and end products, but in the absence of active substrate, and starts the stirring and constant temperature control, recording the temperature difference signal AT between the inside and outside of the reactor for at least 1 hour; the fluctuations of the AT signal collected during this period are considered as the background thermal noise signal of the system; subsequently, a statistical analysis of this noise signal data is performed, calculating its mean μ_noise and standard deviation σ_noise, and the final threshold for the end of reaction T_threshold is set as T_threshold = μ_noise + 3σ_noise; this threshold is stored in the control system as a reference for the determination of the disappearance of the heat of biochemical reaction in all subsequent production batches.

[0046] Further, to cope with the stagnation state of the temperature difference signal slightly above T_threshold but no longer significantly decreasing due to trace amounts of substrate or slow side reactions, the procedure also sets a set of supplementary logical judgment conditions; in the control system, a stagnation judgment timer is set, which is used to measure the time t_elapsed elapsed since the last ethanol addition operation triggered by the decrease in stirring power; if t_elapsed exceeds a pre-set maximum reaction period, for example 60 minutes, and during this period the temperature difference signal AT is always less than a low activity state threshold set as 2T_threshold, the system will determine that the reaction has entered an invalid stagnation state and will forcibly issue a termination instruction; through the construction of the above baseline model and the setting of supplementary logic, a basis is provided for the determination of the end point of the reaction, and the risk of prolonged process abnormalities is avoided.

[0047] It is obvious to those skilled in the art that the present application is not limited to the details of the above exemplary embodiments, and can be implemented in other specific forms without departing from the spirit or essential characteristics of the present application.

[0048] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and are not limiting, and although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the present application.

Claims

1. A method for preparing *Ulva prolifera* oligosaccharides via complex enzymatic hydrolysis and gradient alcohol precipitation, characterized in that, Includes the following steps: Step a, in a reactor equipped with a stirring device, an aqueous solution containing Ulva prolifera polysaccharide substrate is provided, and ethanol is added, so that a heterogeneous system containing a liquid phase and a non-liquid phase composed of Ulva prolifera polysaccharide substrate is formed in the reactor; Step b: Add a complex enzyme to the heterogeneous system and carry out an enzymatic hydrolysis reaction at the interface between the liquid phase and the non-liquid phase, so that the Ulva polysaccharide substrate in the non-liquid phase is degraded into oligosaccharide products that are soluble in the liquid phase. Step c: During the enzymatic hydrolysis reaction, a negative feedback coupling relationship is established between the stirring power in the reactor and the ethanol increase operation. This negative feedback coupling relationship is specifically achieved through the following operation: real-time monitoring of stirring power. When the stirring power decreases to a stable threshold due to the consumption of non-liquid phase in the enzymatic hydrolysis reaction, the operation of increasing the concentration of ethanol in the system is triggered. This increase operation selectively causes intermediate products with a molecular weight range determined by the current ethanol concentration to precipitate from the liquid phase and become new substrates for the enzymatic hydrolysis reaction until the ethanol concentration reaches the preset endpoint. In step d, after the concentration of ethanol reaches the preset endpoint, the liquid phase is separated and collected to obtain the oligosaccharide product.

2. The method for preparing *Ulva prolifera* oligosaccharides by complex enzymatic hydrolysis and gradient alcohol precipitation according to claim 1, characterized in that, The determination of the stability threshold in step c, and the operation of triggering the increase of ethanol concentration, constitute an adaptive loop. The operating mechanism of this adaptive loop is as follows: after an ethanol increase operation causes the intermediate product to precipitate and form a new non-liquid phase, the stirring power rises to a peak value. As the enzyme is consumed in the reaction at the new non-liquid interface, the stirring power decreases accordingly. When the stirring power drops to a preset percentage of the peak value, it is determined that a stable threshold has been reached, and the next operation to increase the ethanol concentration is immediately triggered.

3. The method for preparing *Ulva prolifera* oligosaccharides by complex enzymatic hydrolysis and gradient alcohol precipitation according to claim 1, characterized in that, The complex enzyme added in step b is the first enzyme composition; Furthermore, the method also includes: during step c, adding a second enzyme composition to the reactor that has a different composition or ratio than the first enzyme composition, wherein the enzyme in the second enzyme composition has higher ethanol tolerance than the enzyme in the first enzyme composition, so as to superimpose a biocatalytic gradient composed of changes in enzyme composition on top of the physical environmental gradient composed of changes in ethanol concentration.

4. The method for preparing *Ulva prolifera* oligosaccharides by complex enzymatic hydrolysis and gradient alcohol precipitation according to claim 2, characterized in that, The determination rule for the stability threshold in step c is limited by the following inequality: P_current≤(1-α)·P_peak, where P_current is the current stirring power monitored in real time; P_peak is the peak stirring power that appears immediately after an ethanol increase operation in this adaptive cycle; α is a preset power decrease ratio factor in the range of 0.05 to 0.

2. When the inequality is true, the stability threshold is determined to have been reached.

5. The method for preparing *Ulva prolifera* oligosaccharides by complex enzymatic hydrolysis and gradient alcohol precipitation according to claim 1, characterized in that, Also includes: In step c, after each increase in ethanol concentration, an acoustic characteristic signal of the reactor is monitored. Furthermore, based on acoustic characteristic signals, a morphological characteristic of the precipitated non-liquid phase is determined. When the non-liquid phase is determined to be a viscous gel-like mass with a low specific surface area, a brief interface remodeling stirring program is executed before resuming normal stirring, which involves rapid alternation of high and low speeds or reverse rotation, in order to break up the viscous gel-like mass into loose flocs with a high specific surface area.

6. The method for preparing *Ulva prolifera* oligosaccharides by complex enzymatic hydrolysis and gradient alcohol precipitation according to claim 1, characterized in that, Also includes: Throughout the entire process, the temperature difference between a reaction temperature inside the reactor and a reference temperature outside the reactor is continuously monitored. Furthermore, when the temperature difference steadily falls below a preset termination threshold close to zero due to the disappearance of the heat of enzymatic hydrolysis, a termination command is issued to forcibly stop the operation of increasing the ethanol concentration and proceed to step d.

7. The method for preparing *Ulva prolifera* oligosaccharides by complex enzymatic hydrolysis and gradient alcohol precipitation according to claim 1, characterized in that, In step a, the non-liquid phase is either a gel phase or a solid phase; in step c, the initial concentration of ethanol in the system is 25% to 35% by volume, and the preset endpoint concentration is 55% to 75% by volume; the temperature of the enzymatic hydrolysis reaction is controlled at 40°C to 60°C, and the pH value is controlled at 4.0 to 6.

0.

8. The method for preparing *Ulva prolifera* oligosaccharides by complex enzymatic hydrolysis and gradient alcohol precipitation according to claim 3, characterized in that, The first enzyme composition mainly contains an endonuclease responsible for rapidly reducing the molecular weight of the substrate; the second enzyme composition mainly contains an exonuclease or debranching enzyme responsible for modifying the ends of oligosaccharides or removing branches; the addition of the second enzyme composition is carried out by continuous dropwise addition after the ethanol concentration of the system reaches an intermediate concentration point.

9. The method for preparing *Ulva prolifera* oligosaccharides by complex enzymatic hydrolysis and gradient alcohol precipitation according to claim 5, characterized in that, The acoustic characteristic signal is the vibration signal collected by a vibration sensor installed on the outer wall of the reactor vessel; the operation to determine the morphological characteristics is as follows: after increasing the ethanol concentration, the rotation speed of the stirring paddle is briefly changed to apply an active acoustic detection excitation, and a fast Fourier transform is performed on the transient response of the vibration signal induced by the excitation. Whether a viscous gel-like mass is formed is determined based on whether a characteristic low-frequency high-amplitude resonance peak appears in the response signal.

10. The method for preparing *Ulva prolifera* oligosaccharides by complex enzymatic hydrolysis and gradient alcohol precipitation according to claim 1, characterized in that, Following step d, the collected liquid phase is further subjected to nanofiltration concentration and freeze-drying.

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