Isolation method of curcuma phaeocaulis protoplast

Abstract

The present application relates to a kind of methods for separating Curcuma zedoary protoplast, comprising: Curcuma zedoary tender leaf is cut into filament, is placed in the mixed enzyme liquid containing specific concentration;Carry out two-stage vacuum pretreatment of combined vacuumizing, transient low temperature stimulation, mechanical vibration and the sterile CO2 of being passed into;Enzymolysis is under constant temperature condition, and regularly detects the concentration of glucose, cellobiose or galacturonic acid in enzyme solution;According to concentration data, the pre-established enzymolysis kinetics model is fitted, and the best time window of protoplast starting to release in large quantities is predicted;When enzymolysis enters the window, according to the rising rate of real-time monitored metabolite concentration, near-infrared light intermittent irradiation is automatically triggered;Finally, the quantity dynamics of intact and broken protoplast is observed by microscope until meeting stop condition.The method is mainly used for efficiently, stably preparing high yield, high integrity and high activity Curcuma zedoary protoplast, and provides high-quality material for subsequent genetic transformation, somatic hybridization and other biological technology research.

Description

Technical Field

[0001] This invention relates to the field of protoplast separation technology, specifically to a method for separating Curcuma zedoaria protoplasts. Background Technology

[0002] Curcuma zedoaria, an important medicinal plant, provides ideal materials for genetic transformation, somatic cell hybridization, and physiological and biochemical research using its protoplasts. Obtaining efficient and highly viable protoplasts is a prerequisite for related downstream biotechnological operations. Traditional methods for isolating plant protoplasts typically rely on prolonged immersion of plant tissue cell walls using mixed enzyme solutions such as cellulase and pectinase. However, for medicinal plant materials like Curcuma zedoaria, which have a unique cell wall structure, conventional enzymatic hydrolysis methods face several technical bottlenecks that urgently need to be addressed.

[0003] First, enzymatic hydrolysis is a complex and dynamic biochemical reaction, and its efficiency is directly related to the degree of cell wall degradation. However, traditional methods lack real-time and precise monitoring of the hydrolysis process. Hydrolysis time is generally set empirically, leading to significant uncertainty. Too short a time results in insufficient cell wall degradation, leading to low protoplast release and yield; too long a time causes a significant decrease in the viability of released protoplasts due to prolonged exposure to the enzyme solution, and the cell membrane is more prone to breakage due to the continuous action of enzymes and osmotic pressure, resulting in a lower proportion of intact protoplasts. This difficulty in accurately determining the "optimal harvest window" is one of the main obstacles to obtaining large quantities of highly viable protoplasts.

[0004] Secondly, vacuum infiltration is often used to promote enzyme penetration, but negative pressure treatment alone can have uneven effects on tissues like Curcuma zedoaria and may cause physical damage to cells. During enzymatic hydrolysis, factors such as local pH fluctuations and accumulation of metabolites can also affect enzyme activity and stability, leading to decreased hydrolysis efficiency and uncontrollable reaction progress, resulting in poor reproducibility between different batches of experiments.

[0005] Furthermore, how to further promote the mild and efficient degradation of the cell wall without damaging the protoplast is a technical challenge. Simple chemical enzymatic hydrolysis is sometimes insufficient to completely overcome the resistance of certain regions of the cell wall, and if the application of external physical fields (such as light, ultrasound, etc.) is not properly controlled, it is easy to generate overheating or mechanical shearing forces, which may exacerbate the damage to the protoplast.

[0006] Therefore, existing technologies for the isolation of Curcuma zedoaria protoplasts urgently require a method that can sense the enzymatic hydrolysis process in real time, dynamically predict the optimal termination time, and intelligently intervene in this critical stage in a non-invasive manner to improve yield and quality. This method needs to overcome the "black box" nature of the enzymatic hydrolysis process, achieving a shift from experience-dependent to process-controllable processes, thereby stably and efficiently preparing large quantities of Curcuma zedoaria protoplasts with good integrity and high viability. Summary of the Invention

[0007] One object of the present invention is to address at least the aforementioned deficiencies and to provide at least the advantages described below.

[0008] To achieve these and other advantages of the present invention, a method for isolating Curcuma zedoaria protoplasts is now provided, comprising the following steps: S1. Take tender leaves of Curcuma zedoaria, wash them with sterile water, use absorbent material to dry the surface moisture of the leaves, and then cut them into thin strips of 0.5~1.0 mm.

[0009] S2. Place the filaments into a container containing a mixed enzyme solution. The mixed enzyme solution includes: cellulase at a concentration of 1.5%–2.0% (w / v), pectinase at a concentration of 0.5%–1.0% (w / v), cleavage enzyme at a concentration of 0.2%–0.5% (w / v), and MES buffer. The MES buffer is added either directly as a 0.2M stock solution at pH 5.6–5.8 or via a pH-responsive carrier. The final concentration of MES in the mixed enzyme solution is 0.05M–0.1M. The method of adding the MES buffer can be selected according to the required precision of pH stability control: if continuous and precise buffering is required, a pH-responsive sustained-release microcapsule form can be used; if direct control is possible, it can be added directly as a stock solution.

[0010] S3. Wrap the container with light-proof material and perform a two-stage vacuum pretreatment: Use a vacuum pump to evacuate the inside of the container. The vacuum pretreatment is carried out in two stages: The first stage maintains a pressure of 25℃~28℃ and -0.07~-0.08MPa for 20 minutes. Under this negative pressure condition, mechanical vibration at a frequency of 100 times / minute for 5 seconds is applied every 10 minutes, along with a brief low-temperature stimulus of -2~0℃ for 2 seconds. The second stage stops the vacuum and introduces sterile CO2 gas at a flow rate of 5 mL / min into the container for 10 minutes. The first stage, by simultaneously applying mechanical vibration and brief low temperature while maintaining negative pressure, aims to initially promote enzyme penetration using negative pressure, further loosen the tissue through mechanical force, and combine this with thermal shock to help weaken the cell wall structure. The second stage immediately introduces sterile CO2 gas to stabilize the microenvironment of the pretreatment using gas replacement and to provide uniform and mild starting conditions for subsequent enzymatic hydrolysis. Each step is executed sequentially and complements the others, together forming a coherent operational unit aimed at maximizing the preprocessing effect and minimizing physical damage.

[0011] S4. Place the container in a constant temperature shaker and carry out the enzymatic hydrolysis reaction at 25℃~28℃ and 40~60 rpm. During the enzymatic hydrolysis, take a sample every 20~30 minutes. After taking the sample, add an equal volume of sterile mixed enzyme solution to maintain a constant system volume. Use high performance liquid chromatography to detect the concentration of key metabolites in the enzymatic hydrolysate. The key metabolites are one or more of glucose, cellobiose, and galacturonic acid.

[0012] S5. Based on the concentration change data of key metabolites over time obtained in step S4, by fitting the characteristic inflection point of the key metabolite release curve, and based on the pre-established enzymatic hydrolysis kinetic model that correlates the inflection point with the protoplast release mass, predict the start and end points of the optimal time window for the large-scale release of protoplasts in this enzymatic hydrolysis reaction.

[0013] S6. When the enzymatic hydrolysis time reaches the start of the predicted time window, intermittent near-infrared irradiation is applied to the enzymatic hydrolysis vessel. The triggering and stopping of irradiation are controlled by the real-time monitoring of the concentration rise rate of the key metabolites. The trigger threshold and irradiation duration of near-infrared irradiation need to be dynamically adjusted according to the following progressive logic: first, basic dynamic fine-tuning is performed, then advanced synergistic adjustment is performed, and finally the result of the advanced synergistic adjustment is used as the execution standard for the current irradiation parameters. If, after entering the predicted time window, the metabolite concentration rise rate does not reach the trigger threshold for three consecutive monitoring cycles, the system can automatically execute a low-intensity, short-duration default irradiation program to maintain a mild auxiliary effect.

[0014] S7. Continue the enzymatic hydrolysis and irradiation process of step S6, and observe the number of intact spherical protoplasts and broken protoplasts in the hydrolysate under a microscope at 20-60 minute intervals. Stop enzymatic hydrolysis and irradiation when the following core stopping conditions are met: when the number of intact protoplasts reaches a plateau and the number of broken protoplasts remains below 15% of the number of intact protoplasts. In addition, the system can automatically terminate the reaction based on a synergistic stopping condition, which follows the principle of choosing the applicable condition first and prioritizing the one met, in accordance with the aforementioned core conditions: the system continuously calculates the moving average EA of the rate-effectiveness coefficient E from the most recent N consecutive irradiations and records its peak value EM; when EA decreases for at least two consecutive near-infrared irradiation monitoring cycles (Δt = 10 minutes) from EM, and the value drops below 30% of EM, the system also automatically executes a stop command, simultaneously terminating sampling detection, near-infrared irradiation, and constant-temperature shaking incubator operation. In the above scheme, it should be noted that the reliability of the pre-established enzymatic hydrolysis kinetic model is based on the following biological foundation: For young leaves of Curcuma zedoaria, sufficient degradation of the cell wall is a necessary prerequisite for the release of protoplasts. Monitoring the concentration of key metabolites is essentially an indirect but highly sensitive monitoring of the overall progress of the rate-limiting step of cell wall degradation. The mathematical model established through numerous parallel experiments in the early stages has already included the possible transient physiological delay between the "completeness of cell wall degradation" and the "initiation of actual protoplast release" in the statistical correlation. Therefore, the prediction of the "optimal time window" is for the window at which protoplasts begin to be released in large quantities, and its starting point naturally includes the necessary process time from the degradation of key structures to the smooth expulsion of the plasma membrane, thus ensuring the biological rationality and operational guidance value of the prediction.

[0015] Preferably, in step S2 of the present invention, the MES buffer is added in the form of pH-responsive sustained-release microcapsules, the particle size of which ranges from 10 to 200 μm; the pH-responsive sustained-release microcapsules have a double-layer structure: the core is 0.2M, pH 5.7 MES buffer crystals, and the outer layer is a hydroxypropyl methylcellulose phthalate coating that swells and degrades at pH above 5.9; the sustained-release rate of the microcapsules in the enzymatic hydrolysate is 0.01 to 0.02 mol / (L•h) per unit volume of enzymatic hydrolysate.

[0016] Preferably, the pre-established enzymatic hydrolysis kinetic model in step S5 of the present invention is obtained through the following method: Multiple parallel experiments are conducted under standard enzymatic hydrolysis conditions, the concentration of the key metabolites in the hydrolysate is monitored periodically, and protoplast yield and viability are recorded simultaneously; a curve is plotted with the concentration of the key metabolites as the vertical axis and the hydrolysis time as the horizontal axis, and the curve is fitted to a piecewise function containing an exponential growth phase and a plateau phase using a nonlinear regression method; the optimal time point when the number of protoplasts released in each experiment enters the plateau phase and the breakage rate is less than 15% is correlated with the first significant inflection point on the corresponding curve to establish a mathematical model that predicts the optimal enzymatic hydrolysis time window using the inflection point characteristic value. It should be noted that the kinetic model established by nonlinear regression fitting described above is a general method for predicting the optimal time window. In practical applications, to facilitate rapid operation, simplified empirical prediction formulas can be summarized from the above general model based on a large number of prior calibration experiments. For example, the start and end points (TE) of the optimal time window can be expressed as linear functions of the inflection point time (TP) of the key metabolite release curve, i.e., TS = a×TP, TE = b×TP, where the coefficients a and b are determined through the calibration experiments. The specific coefficients given in the examples (e.g., a=0.95, b=1.25) are merely examples and should not be construed as the sole limitation of the model of the present invention.

[0017] Preferably, the specific control method of intermittent irradiation in step S6 of the present invention is as follows: use a near-infrared light source, the spot area of which should cover the entire surface of the enzymatic hydrolysate or the light-transmitting cross-section of the reaction vessel to ensure uniform irradiation; the distance from the light-emitting surface of the light source to the surface of the enzymatic hydrolysate is fixed at 10-20 cm; at this distance, adjust the output power of the light source so that the irradiation intensity received by the surface of the enzymatic hydrolysate is 50-100 mW / cm 2 ; define the monitoring period Δt as 10 minutes, and calculate the average concentration increase rate V of key metabolites within each period; after the enzymatic hydrolysis enters the prediction time window, take the V value measured in the first monitoring period as the reference rate V0; when V>1.5V0 is measured in any monitoring period, trigger an irradiation automatically; for a single irradiation executed after triggering, its wavelength is 800-1000 nm and the duration is 5-10 seconds; after each irradiation ends, the system enters a pause and recovery period, which lasts at least 5 minutes (to avoid cumulative damage to the protoplast cell membrane caused by local thermal effects of irradiation), and no new trigger judgment is made during this period; after the pause and recovery period ends, the system resumes monitoring and rate judgment; if V does not exceed 1.5V0 in three consecutive monitoring periods after entering the time window, automatically execute a default irradiation program with an intensity of 20 mW / cm 2 and a duration of 5 seconds, and also follow the above intermittent logic.

[0018] Preferably, polymer nanoparticles are uniformly embedded in the hydroxypropyl methylcellulose phthalate coating of the present invention; the polymer nanoparticles are prepared by an imprinting technique with cellobiose or galacturonic acid dimer as the template molecule, and carboxyl group-containing pH-sensitive groups are grafted onto the polymer network of the polymer nanoparticles, and the pH-sensitive groups respond to the same pH as the degradation pH of the HPMCP coating, and the pH is >5.9; the mass fraction of the polymer nanoparticles in the HPMCP coating is 0.1% - 5.0%.

[0019] Preferably, the basic dynamic fine-tuning rule of the near-infrared irradiation of the present invention is as follows: in the next monitoring period after the first trigger of irradiation, calculate the growth rate R1 of the number of intact protoplasts in this period; compare R1 with the average growth rate R0 corresponding to the same monitoring period ordinal number after entering the prediction time window, which is statistically obtained based on multiple sets of historical experimental data under the same experimental conditions; according to the ratio k = R1 / R0, fine-tune the next trigger threshold and irradiation duration: if k>1.1, the next trigger threshold is increased to 1.7V0 and the irradiation duration is reduced by 20%; if 0.9<k ≤ 1.1, the parameters remain unchanged; if k ≤ 0.9, the next trigger threshold is reduced to 1.3V0 and the irradiation duration is increased by 20%.

[0020] Preferably, the advanced coordinated adjustment rule for near-infrared irradiation of the present invention is as follows: The system records the V value measured in the monitoring cycle before each triggered irradiation as the pre-trigger rate VA, and records the V value measured in the first monitoring cycle after the irradiation ends and recovers as the post-response rate VB; calculates the rate-efficiency coefficient E = (VB – VA) / VA for a single irradiation; calculates its moving average EA based on the rate-efficiency coefficient E of the most recent N consecutive irradiations, where N ≥ 3; sets an adjustment threshold Δ, where Δ ranges from 0.05 to 0.2; if the increase in the current EA compared to the previous EA is greater than Δ, the system automatically increases the trigger threshold by 5% to 15% and reduces the irradiation duration by 10% to 20%; if the decrease is greater than Δ, the system automatically decreases the trigger threshold by 5% to 15% and increases the irradiation duration by 10% to 20%.

[0021] Preferably, the dynamic adjustment of the present invention also incorporates real-time monitoring of the pH value of the enzymatic hydrolysate as a collaborative control parameter: within the predicted time window, the pH value of the enzymatic hydrolysate is continuously monitored; when the pH value is detected to rise to the range of 5.85~5.95, the system automatically halve the adjustment range of the trigger threshold (originally 5%~15%) and doubles the adjustment range of the irradiation duration (originally 10%~20%).

[0022] Preferably, the system of the present invention automatically generates and executes a stop command when the following conditions are met, so as to simultaneously terminate the sampling detection in step S4, the near-infrared irradiation in step S6, and the operation of the constant temperature shaker; the triggering of the stop command follows the principle of choosing one of the core condition and the synergistic condition, and prioritizing the one that is met first; wherein, the core condition is that the number of intact protoplasts enters a plateau period, and the number of broken protoplasts is always less than 15% of the number of intact protoplasts; the synergistic condition is that the system continuously calculates the moving average value EA and records its peak value EM; when it is detected that EA decreases for at least two consecutive near-infrared irradiation monitoring cycles (Δt=10 minutes) from EM, and its value drops to less than 30% of EM; when either of the above two conditions is met, the system immediately executes the stop command.

[0023] The present invention has at least the following beneficial effects: 1. This invention constructs a complete and highly controllable protoplast separation system by integrating physical pretreatment, real-time biochemical monitoring, kinetic model prediction, and near-infrared intelligent irradiation based on metabolic rate. It solves the problems of the "black box" nature and reliance on experience in the enzymatic hydrolysis process, achieving precise prediction and intervention of the optimal harvest time window through data-driven approaches. This significantly improves the yield and integrity of Curcuma zedoaria protoplasts, while also significantly enhancing batch-to-batch stability and experimental reproducibility.

[0024] 2. This invention solves the problem of pH fluctuations during enzymatic hydrolysis by introducing pH-responsive sustained-release microcapsules as buffer carriers. When the pH of the hydrolysate rises above 5.9 due to the reaction, the outer coating of the microcapsule degrades, intelligently releasing the MES buffer in the core, thereby stabilizing the pH back to the optimal range. This achieves automated and stable control of the pH of the reaction microenvironment, avoiding the decrease in enzyme activity caused by pH drift, and ensuring that the entire enzymatic hydrolysis reaction proceeds stably in an optimal chemical environment without the need for manual adjustments during the process.

[0025] 3. This invention solves the problem of the inability to scientifically predict the end point of enzymatic hydrolysis by establishing and applying a pre-trained enzymatic hydrolysis kinetic model. This model mathematically correlates the inflection point of the metabolite concentration curve, which is easily monitored online, with the optimal protoplast release mass point confirmed offline. This allows operators to quickly and accurately predict the optimal enzymatic hydrolysis time window for the current batch based on real-time collected concentration data, shifting the separation process from experience-driven to model-predictive, improving the scientific rigor of the process and the predictability of the results, and reducing the waste of materials and time caused by blind experimentation.

[0026] 4. This invention designs an intelligent irradiation triggering mechanism based on real-time metabolic rate feedback, solving the problem that fixed-mode irradiation may not be suitable for specific reaction states. By dynamically binding the irradiation action to the rate of increase in metabolite concentration V (e.g., triggering when V>1.5V0), it ensures that near-infrared light assistance is applied only when enzymatic activity is high and a "bottleneck" may be encountered, achieving "on-demand stimulation." This avoids the risk of thermal damage that may be caused by ineffective or excessive irradiation, and can effectively promote cell wall loosening and protoplast release at critical moments, improving the safety and efficiency of irradiation.

[0027] 5. This invention further upgrades the precise release logic of pH buffering by embedding molecularly imprinted polymer nanoparticles into sustained-release microcapsule coatings. These nanoparticles can specifically recognize specific oligosaccharide fragments, such as cellobiose, produced by cell wall degradation, and after recognition, synergistically promote the early or accelerated degradation of the coating locally through their pH-sensitive groups. This allows the release of the buffer to respond not only to the overall pH but also to the "signals" of the enzymatic hydrolysis process, achieving earlier and more precise pH stabilization intervention, thereby better maintaining enzyme activity and improving the consistency of enzymatic hydrolysis efficiency.

[0028] 6. Building upon intelligent triggering, this invention introduces a dynamic parameter fine-tuning mechanism based on the actual growth effect of protoplasts, solving the problem that static parameters cannot adapt to individual differences. The system adaptively adjusts the trigger threshold and duration of the next irradiation by comparing the protoplast growth rate after irradiation with the historical average. This allows the irradiation strategy to be optimized based on the real biological feedback from each intervention, forming a preliminary adaptive learning capability. It achieves personalized optimal assistance for different material batches, further enhancing the potential for maximizing protoplast yield.

[0029] 7. This invention establishes a more agile, dynamic adjustment mechanism based on direct feedback signals by defining and tracking the "rapid-effect coefficient E" and its moving average EA. It solves the lag problem of relying on adjustments made with a delay in protoplast growth. By analyzing the immediate effect of recent irradiation on metabolic rate, the system can more quickly and directly determine the effectiveness of current parameter settings and proactively optimize subsequent trigger thresholds and irradiation durations accordingly. This achieves more precise and faster closed-loop process control, ensuring that irradiation intervention remains within a high-efficiency range.

[0030] 8. This invention addresses the issue of potential conflicts between irradiation parameter adjustments and pH buffering effects when the pH of the enzymatic hydrolysate approaches the release threshold of the buffer microcapsules, by introducing pH as a co-control parameter. When the pH enters the 5.85–5.95 range, the system automatically adjusts its strategy to minimize interference with potential pH buffering events, utilizing a gentler but slightly longer irradiation period. This achieves intelligent synergy between physical assistance and chemical regulation, ensuring the stable and harmonious operation of the entire control system.

[0031] 9. This invention solves the problem of comprehensively determining the endpoint of enzymatic hydrolysis by setting an automatic stop rule based on the peak decay of the moving average value EA (rapid-effect coefficient). When EA continuously decreases from its peak to below 30%, it indicates that the effect of near-infrared irradiation, the core regulatory method, on promoting the reaction has significantly weakened, meaning that protoplast release is nearing its limit, and further enzymatic hydrolysis yields very little benefit while increasing the risk of breakage. At this point, the system automatically and safely shuts down all processes, achieving the effect of timely termination of the reaction at the optimal time point, maximizing the preservation of protoplast viability, and realizing fully automated endpoint determination and execution of the experimental process. Detailed Implementation

[0032] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0033] Example 1 The method for isolating Curcuma zedoaria protoplasts includes the following steps: Step 1: Prepare materials and equipment: Tender new leaves of Curcuma zedoaria seedlings that have grown for 8 days, free from diseases and pests, with a fresh weight of 5g / group, and set up 6 parallel samples.

[0034] Cellulase (enzyme activity ≥1000U / g), pectinase (enzyme activity ≥800U / g), analyte (enzyme activity ≥500U / g), MES buffer (purity ≥99%), glucose (purity ≥99%), cellobiose (purity ≥98%), galacturonic acid (purity ≥98%), FDA (fluorescein diacetate, purity ≥97%), mannitol (purity ≥99%), sterile CO2 gas (purity ≥99.99%), sterile CPW medium (containing 0.5M mannitol, pH 5.7).

[0035] Vacuum pump (ultimate pressure ≤ -0.1MPa), constant temperature shaker (temperature control accuracy ±0.5℃, speed range 0~200rpm), high performance liquid chromatograph (equipped with ultraviolet detector), near-infrared irradiation system (wavelength adjustable 800~1000nm, intensity adjustable 0-200mW / cm²). 2 ), biological microscope (400x magnification), high-speed refrigerated centrifuge (speed range 0~15000rpm), sterile operating table, analytical balance (accuracy 0.1mg), ultrapure water system (resistivity 18.2MΩ・cm), sterile Erlenmeyer flasks, sterile filter paper, sharp blades, 200-mesh sterile filter screen.

[0036] Step 2: Take 5g of tender leaves of Curcuma zedoaria, rinse 4 times with sterile water, dry the surface with sterile filter paper, and cut them into 0.8mm fine shreds on a sterile operating table with a sharp blade, ensuring that the cut is flat and undamaged.

[0037] Step 3: Prepare 100 mL of mixed enzyme solution with the following components: 1.8% (w / v) cellulase, 0.8% (w / v) pectinase, 0.3% (w / v) cleavage enzyme, and add 0.2 M MES buffer stock solution at pH 5.7 to make the final MES concentration in the mixed enzyme solution 0.08 M. Place the leaf filaments into a sterile Erlenmeyer flask, pour in the prepared mixed enzyme solution, and gently shake to ensure that all filaments are completely immersed in the enzyme solution.

[0038] Step 4: Completely wrap the Erlenmeyer flask with aluminum foil (to protect it from light) and perform a two-stage vacuum pretreatment: In the first stage, place the Erlenmeyer flask in a constant temperature environment of 26°C, start the vacuum pump to evacuate to -0.075MPa, and maintain it for 20 minutes; under this negative pressure condition, every 10 minutes, simultaneously perform the following: a small semiconductor cooling plate based on the Peltier effect, triggered by a cooling unit controlled by the control system, is tightly attached to the bottom of the container for 2 seconds, causing the enzymatic hydrolysate to generate an instantaneous low temperature stimulus of -2°C, while applying mechanical vibration at a frequency of 100 times / minute for 5 seconds; In the second stage, turn off the vacuum pump, stop the vacuum state, and immediately introduce sterile CO2 gas into the Erlenmeyer flask at a flow rate of 5 mL / min for 10 minutes.

[0039] Step 5: Place the pre-treated Erlenmeyer flask in a constant temperature shaker, set the temperature to 26℃ and the rotation speed to 50 rpm, and start the enzymatic hydrolysis reaction. During the enzymatic hydrolysis, take a 0.5 mL sample every 25 minutes (immediately after sampling, add an equal volume of sterile mixed enzyme solution to the Erlenmeyer flask to maintain the stability of the system volume). Use HPLC to detect the concentrations of glucose, cellobiose, and galacturonic acid in the sample solution. The HPLC detection conditions are: C18 column (4.6 mm × 250 mm, 5 μm), mobile phase is methanol-0.1% phosphoric acid aqueous solution (volume ratio 20:80), flow rate is 1.0 mL / min, column temperature is 30℃, detection wavelength is 210 nm, and injection volume is 20 μL.

[0040] Step 6: Substitute the concentration data of the three key metabolites obtained in real time in Step 5 into the pre-established enzymatic hydrolysis kinetic model (this model was constructed through 10 sets of parallel experiments under standard conditions, fitted as a piecewise function including the exponential growth period and the plateau period, and the inflection point of the correlation curve with the optimal enzymatic hydrolysis time window). The fitted result shows that the optimal time window for this enzymatic hydrolysis reaction is 4.2-6.8h.

[0041] Step 7: When the enzymatic hydrolysis time reaches 4.2 hours, start the near-infrared irradiation system; in this embodiment, a basic trigger control method is used. A monitoring cycle is defined as 10 minutes. The average concentration increase rate V of the three key metabolites within each cycle is calculated. The V value of the first monitoring cycle after entering the time window is set as the baseline rate V0 (in this experiment, V0 = 0.048 mg / (mL•h)). When the measured V > 1.5V0 (i.e., > 0.072 mg / (mL•h)) within any monitoring cycle, near-infrared irradiation is automatically triggered, with irradiation parameters of wavelength 900 nm and intensity 80 mW / cm². 2 The irradiation lasts for 8 seconds; after each irradiation, there is a 5-minute pause until the temperature of the enzyme digest returns to 26°C, then the rate judgment and triggering restart; if V does not exceed 0.072 mg / (mL•h) for 3 consecutive monitoring cycles, then an intensity of 20 mW / cm² will be automatically executed once.2 The default irradiation program lasts for 5 seconds.

[0042] Step 8: During the continuous enzymatic digestion and irradiation process, every 30 minutes, 10 fields of view are randomly selected under a 400x microscope to observe and count the number of intact spherical protoplasts and broken protoplasts. After 6.0 hours of enzymatic digestion, the change rate of the number of intact protoplasts at two consecutive observation points is 3.2%, which means that the plateau period has begun. The number of broken protoplasts accounts for 10.5% of the number of intact protoplasts. At this point, the enzymatic digestion reaction and near-infrared irradiation are stopped.

[0043] Step 9: Filter the enzymatic hydrolysate through a 200-mesh sterile filter to remove undegraded leaf tissue fragments; collect the filtrate and place it in a sterile centrifuge tube, centrifuge at 4℃ and 800 rpm for 5 minutes; discard the supernatant and wash the precipitate twice with sterile CPW medium containing 0.5M mannitol and pH 5.7 to finally obtain purified Curcuma zedoaria protoplasts.

[0044] Example 2 Based on Example 1, the following reagent was added: hydroxypropyl methylcellulose phthalate (HPMCP, viscosity 50-100 mPa•s); the remaining materials and equipment are the same as in Example 1.

[0045] Replace step 3 with: Prepare 100 mL of mixed enzyme solution with the following components: 1.8% (w / v) cellulase, 0.8% (w / v) pectinase, and 0.3% (w / v) cleavage enzyme; add MES buffer in the form of pH-responsive slow-release microcapsules with an average particle size of 100 μm. The microcapsules have a double-layer structure: the core is 0.2 M, pH 5.7 MES buffer crystals, and the outer layer is HPMCP coating that swells and degrades at pH above 5.9. The slow-release rate of the double-layer structure is 0.015 mol / (L·h), and the amount added ensures that the final concentration of MES in the mixed enzyme solution is 0.08 M; place the leaf filaments into a sterile Erlenmeyer flask, pour in the prepared mixed enzyme solution, and gently shake to ensure that all filaments are completely immersed in the enzyme solution.

[0046] Example 3 Based on Example 2, the following reagents were added: cellobiose (purity ≥98%) and carboxyl pH-sensitive monomers (such as methacrylic acid); the remaining materials and equipment were the same as in Example 2.

[0047] Replace step 3 with: preparing 100 mL of mixed enzyme solution, with the same components as in Example 2; uniformly embedding polymer nanoparticles in the HPMCP coating, with an addition amount of 1.0% (w / w) of the HPMCP mass. These particles are prepared using molecular imprinting technology with cellobiose as a template molecule, and carboxyl pH-sensitive groups are grafted onto the polymer network, with a response pH consistent with the degradation pH of the HPMCP coating (>5.9); place the leaf filaments into a sterile Erlenmeyer flask, pour in the prepared mixed enzyme solution, and gently shake to ensure that all filaments are completely immersed in the enzyme solution.

[0048] Example 4 Based on Example 1, step seven is replaced as follows: when the enzymatic hydrolysis time reaches 4.2 h (the start of the predicted time window), the near-infrared irradiation system is activated; the monitoring period Δt is defined as 10 minutes, and the average concentration increase rate V of the key metabolite in each period is calculated; the V value measured in the first monitoring period after entering the time window is used as the baseline rate V0 (V0 = 0.048 mg / (mL•h) in this experiment); when V > 1.5V0 (i.e. > 0.072 mg / (mL•h)) is measured in any monitoring period, a wavelength of 900 nm and an intensity of 80 mW / cm² are automatically triggered. 2 Near-infrared irradiation lasts for 8 seconds; after each irradiation, pause for 5 minutes until the enzyme digestion solution temperature returns to 26℃, then restart rate judgment and triggering; after entering the time window, if V does not exceed 0.072 mg / (mL•h) for 3 consecutive monitoring cycles, then automatically execute a 20mW / cm intensity test. 2 The default irradiation program lasts for 5 seconds.

[0049] Example 5 Based on Example 3, the following equipment is added: an online pH sensor (accuracy ±0.01), whose real-time data is connected to the irradiation control system; the remaining materials and equipment are the same as in Example 3.

[0050] Step 3 is replaced with: Prepare 100 mL of mixed enzyme solution, with the same components as in Example 3 (pH-responsive sustained-release MES microcapsules containing molecularly imprinted nanoparticles).

[0051] Step six is ​​replaced by: inputting the key metabolite concentration-time data obtained in real-time from step five into a pre-established enzymatic hydrolysis kinetic model for fitting. This model has the following built-in relationships: the optimal harvest time window start point TS = 0.95 × TP, and the end point TE = 1.25 × TP (this coefficient was determined based on 20 previous calibration experiments). The system calculates the fitting inflection point time TP of the current metabolite concentration curve in real-time. When enzymatic hydrolysis has progressed to approximately 3.5 hours, the real-time fitting determines TP = 4.42 hours, and the model predicts the optimal time window for this reaction to be 4.2 hours (TS) to 5.5 hours (TE).

[0052] Step seven is replaced with: When the enzymatic hydrolysis time reaches 4.2 hours (the start of the predicted time window), start the near-infrared irradiation system. The specific control and dynamic adjustment methods are as follows: 1. Basic Control Logic: Define the monitoring period Δt as 10 minutes, and calculate the average concentration rise rate V of the key metabolite within each period; use the V value measured in the first monitoring period after entering the time window as the baseline rate V0 (V0 = 0.05 mg / (mL•h) in this experiment); when V > 1.5V0 (i.e. > 0.075 mg / (mL•h)) is measured in any monitoring period, automatically trigger a wavelength of 900 nm and an intensity of 80 mW / cm². 2 Near-infrared irradiation lasts for 8 seconds; after each irradiation, pause for 5 minutes until the enzyme digest temperature returns to 26℃ (pre-irradiation temperature) before restarting rate judgment and triggering; if V does not exceed 0.075 mg / (mL•h) for 3 consecutive monitoring cycles after entering the time window, then automatically execute a 20 mW / cm² intensity test. 2 The default irradiation program lasts for 5 seconds.

[0053] 2. Dynamic adjustment based on protoplast growth rate: Calculate the growth rate R1 of intact protoplasts during the next near-infrared irradiation monitoring cycle (Δt = 10 minutes) after the initial irradiation trigger (R1 = (N...). n -N n-1 ) / N n-1 ×100%, N n N represents the number of complete protoplasts in the current cycle. n-1(the quantity in the previous cycle); compare R1 with the average growth rate R0 at this time point obtained from historical experimental data (R0 = 12.5% in this experiment), and calculate the ratio k = R1 / R0; in this experiment, k = 1.08, which is in the range of 0.9 < k ≤ 1.1, so maintain the trigger threshold (1.5V0) and irradiation duration (8 seconds) unchanged; if k > 1.1, then increase the next trigger threshold to 1.7V0 and reduce the irradiation duration by 20%; if k ≤ 0.9, then reduce the next trigger threshold to 1.3V0 and increase the irradiation duration by 20%.

[0054] 3. Dynamic adjustment based on the rapid effect coefficient: The system records the V value measured in the previous monitoring cycle before each irradiation trigger as the pre-trigger rate VA, and records the V value measured in the first monitoring cycle after the irradiation ends and returns to normal as the post-response rate VB; calculate the rapid effect coefficient E of a single irradiation as E = (VB - VA) / VA; take the moving average EA of the rapid effect coefficients of the last three consecutive irradiations, and set the adjustment threshold Δ = 0.1; in this experiment, the E values for three consecutive times are 0.28, 0.31, and 0.29 respectively, and the EA values are 0.28, 0.295, and 0.293 in sequence. The decrease of the current EA compared with the previous EA is 0.68% < Δ, so maintain the parameters unchanged; if the increase of the current EA compared with the previous EA > Δ, then increase the trigger threshold by 10% and reduce the irradiation duration by 15%; if the decrease > Δ, then reduce the trigger threshold by 10% and increase the irradiation duration by 15%.

[0055] 4. pH co-control: During the predicted time window, continuously monitor the pH value of the enzymatic hydrolysate through an online pH sensor; when it is monitored that the pH rises to 5.9, the system automatically halves the adjustment range of the trigger threshold (the original adjustment range is ±10%, and after adjustment it is ±5%), and doubles the adjustment range of the irradiation duration (the original adjustment range is ±15%, and after adjustment it is ±30%).

[0056] Replace step eight with: Continuously carry out the enzymatic hydrolysis and irradiation process, and at the same time execute the automatic stop condition: The system continuously calculates EA, and records the peak value EM = 0.32; when it is monitored that EA continuously decreases starting from EM and drops to 28% (0.09) of EM, the system automatically generates a stop instruction to terminate the sampling detection, near-infrared irradiation, and operation of the constant temperature shaker (at this time, the enzymatic hydrolysis time is 5.2h).

[0057] Comparative Example 1 Based on Example 1, the only difference is: Exclude the operation process of step seven, and stop the enzymatic hydrolysis reaction until the number of intact protoplasts enters the plateau phase and the fragmentation rate is lower than 15%.

[0058] Experiment and Analysis 1. Set up a control group (traditional enzymatic hydrolysis method), comparative example 1 and examples 1-5, and perform the corresponding operation steps of the above examples and comparative examples. Each group has 6 parallel samples, and the protoplast yield, integrity rate, viability, batch-to-batch RSD and enzymatic hydrolysis time are uniformly detected.

[0059] The materials and equipment in the control group were exactly the same as those in Example 1, and the specific operating steps included: S1: Take 5g of tender leaves of Curcuma zedoaria, rinse 3 times with sterile water, blot dry with sterile filter paper, and cut into 1.0~1.5mm fine shreds.

[0060] S2: Prepare 100 mL of mixed enzyme solution containing only 1.8% (w / v) cellulase and 0.8% (w / v) pectinase, with 0.08M ordinary MES buffer (non-slow-release, non-microencapsulated form, pH 5.7) added, without enzyme separation; the material-to-liquid ratio is 1 g fresh weight: 12 mL enzyme solution, with the leaf filaments immersed in the enzyme solution.

[0061] S3: Single vacuum pretreatment: Wrap the Erlenmeyer flask with aluminum foil, evacuate to -0.075MPa at 25℃, maintain for 20 minutes, without instantaneous low temperature or mechanical vibration, and do not introduce CO2 gas after vacuuming.

[0062] S4: Enzymatic hydrolysis: Enzymatic hydrolysis at 25℃ and 50 rpm in a constant temperature shaker. No sampling or detection is performed during the enzymatic hydrolysis process, and there is no monitoring of metabolite concentration.

[0063] S5: No enzymatic hydrolysis kinetic model fitting, no optimal time window prediction, the enzymatic hydrolysis time is set to 8h based only on experience.

[0064] S6: After 8 hours of enzymatic hydrolysis, observe the protoplast state under a microscope and manually stop the enzymatic hydrolysis; the post-treatment is the same as in Example 1 (filtering through a 200-mesh screen, centrifuging at 4°C and 800 rpm for 5 minutes, and washing twice with CPW medium).

[0065] 2. The results of the comparative experiment are shown in Table 1: Table 1: 3. Experimental Analysis A. The yield of Example 1 was 166.7% higher than that of the control group, and the integrity rate and viability were increased by 17.5% and 18.5% respectively, proving that the separation method of the present invention can solve the core pain points of the traditional method.

[0066] B. Compared with Example 1, Example 2 showed a 9.4% increase in yield and a 20.6% decrease in RSD, demonstrating that pH stabilization can improve enzymatic hydrolysis efficiency and batch stability.

[0067] C. Compared with Example 1, Example 5 showed a 6.6% increase in protoplast integrity and a 6.7% reduction in enzymatic hydrolysis time, demonstrating that precise irradiation control can further optimize protoplast quality and separation efficiency.

[0068] D. Compared with Example 2, Example 3 showed an 11.4% increase in yield and a 3.7% increase in integrity, demonstrating that the specific recognition mechanism can enhance the accuracy of pH buffering.

[0069] E. Example 5 showed the best performance across all indicators, with a yield increase of 31.2% compared to Example 1, an RSD decrease of 3.2%, and a 13.3% reduction in enzymatic hydrolysis time, demonstrating the synergistic effect of multiple technical features.

[0070] F. The yield of Comparative Example 1 was reduced by 21.9% compared to Example 1, demonstrating that near-infrared intelligent irradiation is one of the key technologies for improving protoplast release efficiency.

[0071] 4. Vitality Sustainability and Functional Validation To evaluate the effects of near-infrared irradiation on the long-term viability and function of protoplasts, protoplasts obtained from Example 5 (representing the optimal solution of the present invention) and Comparative Example 1 (without irradiation) were selected for subsequent culture experiments.

[0072] Method: The purified protoplasts were subjected to a 1×10⁻⁶ ppm... 5 Cells were suspended at a density of 1 cell / mL in CPW-13M liquid medium and incubated statically at 25°C under low light. Samples were taken on day 0 (the day of isolation), day 3, and day 7 after culture. Viability was determined using the FDA staining method, and the percentage of first cell divisions was counted under a microscope.

[0073] The results are shown in Table 2: Table 2: As shown in Table 2, the protoplasts isolated by the method of this invention not only exhibited higher viability immediately after isolation but also showed a slower decline in viability during the one-week culture period, maintaining a higher survival rate. More importantly, their cell division rate was significantly higher than that of the control group. This directly proves that the protoplasts prepared by this invention not only have good integrity but also are metabolically active, maintaining vigorous division and proliferation capabilities, fully meeting the stringent requirements for protoplast regeneration capabilities in downstream applications such as somatic cell hybridization and genetic transformation.

[0074] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Further modifications can be readily implemented by those skilled in the art.

Claims

1. A method for isolating Curcuma zedoaria protoplasts, characterized in that, Includes the following steps: S1. Take tender leaves of Curcuma zedoaria, wash them with sterile water, use absorbent material to dry the surface moisture of the leaves, and then cut them into thin strips of 0.5~1.0mm. S2. Place the filaments into a container containing a mixed enzyme solution, which includes: cellulase at a concentration of 1.5%–2.0% (w / v), pectinase at a concentration of 0.5%–1.0% (w / v), cleavage enzyme at a concentration of 0.2%–0.5% (w / v), and MES buffer. The MES buffer is added either directly as a 0.2M stock solution with a pH of 5.6–5.8 or via a pH-responsive carrier. The final concentration of MES in the mixed enzyme solution is 0.05M–0.1M. S3. Wrap the container with light-proof material and perform a two-stage vacuum pretreatment: Use a vacuum pump to evacuate the inside of the container. The vacuum pretreatment is carried out in two stages: The first stage is maintained at 25℃~28℃ and -0.07~-0.08MPa pressure for 20 minutes; under this negative pressure condition, mechanical vibration at a frequency of 100 times / minute for 5 seconds is applied every 10 minutes, as well as a momentary low temperature stimulus of -2~0℃ for 2 seconds; The second stage is to stop the vacuum and introduce sterile CO2 gas at a flow rate of 5 mL / min into the container for 10 minutes. S4. Place the container in a constant temperature shaker and carry out the enzymatic hydrolysis reaction at 25℃~28℃ and 40~60 rpm. During the enzymatic hydrolysis, take a sample every 20~30 minutes. After each sample, add an equal volume of sterile mixed enzyme solution to maintain a constant system volume. Use high performance liquid chromatography to detect the concentration of key metabolites in the enzymatic hydrolysate. The key metabolites are one or more of glucose, cellobiose, and galacturonic acid. S5. Based on the concentration change data of key metabolites over time obtained in step S4, by fitting the characteristic inflection point of the key metabolite release curve, and based on the pre-established enzymatic hydrolysis kinetic model that correlates the inflection point with the protoplast release mass, predict the start and end points of the optimal time window for the large-scale release of protoplasts in this enzymatic hydrolysis reaction. S6. When the enzymatic hydrolysis time reaches the start of the predicted time window, intermittent near-infrared light irradiation is applied to the enzymatic hydrolysis container. The triggering and stopping of irradiation are controlled by the real-time monitoring of the concentration rise rate of the key metabolites. The triggering threshold and irradiation duration of near-infrared irradiation need to be dynamically adjusted according to the following progressive logic: first, basic dynamic fine-tuning is performed, then advanced collaborative adjustment is performed, and finally the result of advanced collaborative adjustment is used as the execution standard for the current irradiation parameters. S7. Continue the enzymatic hydrolysis and irradiation process of step S6, and observe the number of intact spherical protoplasts and broken protoplasts in the hydrolysate under a microscope in 20-60 minute intervals. Stop the enzymatic hydrolysis and irradiation when the following core stopping conditions are met: when the number of intact protoplasts enters a plateau phase, and the number of broken protoplasts is always less than 15% of the number of intact protoplasts.

2. The method for isolating Curcuma zedoaria protoplasts as described in claim 1, characterized in that, In step S2, the MES buffer is added in the form of pH-responsive sustained-release microcapsules with a particle size range of 10 ~ 200 μm. The pH-responsive sustained-release microcapsules have a bilayer structure: the core is a 0.2M, pH 5.7 MES buffer crystal, and the outer layer is a hydroxypropyl methylcellulose phthalate coating that swells and degrades at pH above 5.9; the sustained-release rate of the microcapsules in the enzymatic hydrolysate is 0.01~0.02 mol / (L•h) per unit volume of enzymatic hydrolysate.

3. The method for isolating Curcuma zedoaria protoplasts as described in claim 1, characterized in that, The pre-established enzymatic hydrolysis kinetic model in step S5 was obtained in the following way: Multiple parallel experiments were conducted under standard enzymatic hydrolysis conditions. The concentrations of the key metabolites in the hydrolysate were monitored periodically, and protoplast yield and viability were recorded simultaneously. A curve was plotted with the concentration of key metabolites on the vertical axis and the enzymatic hydrolysis time on the horizontal axis. The curve was fitted to a piecewise function containing the exponential growth period and the plateau period using a nonlinear regression method. The optimal time point when the number of protoplasts released in each group of experiments reaches a plateau and the breakage rate is less than 15% is correlated with the first significant inflection point on the corresponding curve to establish a mathematical model for predicting the optimal enzymatic hydrolysis time window based on the inflection point characteristic value.

4. The method for isolating Curcuma zedoaria protoplasts as described in claim 1, characterized in that, The specific control method for intermittent irradiation in step S6 is as follows: Using a near-infrared light source, the light spot area should cover the entire surface of the enzyme hydrolysate or the light-transmitting section of the reaction vessel to ensure uniform irradiation. The distance from the emitting surface of the light source to the surface of the enzyme hydrolysate should be fixed at 10-20 cm. At this distance, the output power of the light source should be adjusted so that the irradiation intensity received by the surface of the enzyme hydrolysate is 50-100 mW / cm². 2 ; Define the monitoring period Δt as 10 minutes, and calculate the average concentration rise rate V of key metabolites within each period; After the enzymatic hydrolysis enters the predicted time window, the V value measured in the first monitoring cycle is used as the baseline rate V0. When V > 1.5V0 is measured within any monitoring cycle, an irradiation is automatically triggered. The single irradiation, triggered, has a wavelength of 800-1000 nm and a duration of 5-10 seconds. After each irradiation, the system enters a pause and recovery period, which lasts for at least 5 minutes, during which no new trigger judgment is performed; after the pause and recovery period ends, the system resumes monitoring and rate judgment. If, after entering the time window, V does not exceed 1.5V0 for three consecutive monitoring cycles, an intensity of 20 mW / cm² will be automatically applied. 2 The default irradiation procedure lasts for 5 seconds and also follows the intermittent logic described above.

5. The method for isolating Curcuma zedoaria protoplasts as described in claim 2, characterized in that, Hydroxypropyl methylcellulose phthalate coating uniformly embeds polymer nanoparticles; Polymer nanoparticles are prepared using cellobiose or galacturonic acid dimers as template molecules and molecular imprinting technology. The polymer nanoparticles have carboxyl pH-sensitive groups grafted onto their polymer network, and the pH response of these groups is consistent with the degradation pH of the HPMCP coating, both being >5.

9. The mass fraction of the polymer nanoparticles in the HPMCP coating is 0.1% to 5.0%.

6. The method for isolating Curcuma zedoaria protoplasts as described in claim 4, characterized in that, The basic dynamic fine-tuning rules for near-infrared irradiation are as follows: In the next monitoring cycle following the first triggered irradiation, calculate the growth rate R1 of the number of intact protoplasts during that cycle; Compare R1 with R0, which is the average growth rate of the same monitoring period after entering the prediction time window, obtained by statistical analysis of multiple sets of historical experimental data under the same experimental conditions. Based on the ratio k = R1 / R0, the trigger threshold and irradiation duration for the next time are fine-tuned: if k > 1.1, the trigger threshold for the next time is increased to 1.7V0, and the irradiation duration is reduced by 20%; if 0.9 < k ≤ 1.1, the parameters remain unchanged; if k ≤ 0.9, the trigger threshold for the next time is reduced to 1.3V0, and the irradiation duration is increased by 20%.

7. The method for isolating Curcuma zedoaria protoplasts as described in claim 4, characterized in that, The advanced coordinated adjustment rules for near-infrared irradiation are as follows: The system records the V value measured in the monitoring cycle before each irradiation trigger as the pre-trigger rate VA, and records the V value measured in the first monitoring cycle after the irradiation ends and recovery as the post-response rate VB. Calculate the efficiency coefficient of a single irradiation, E = (VB – VA) / VA; Calculate the moving average EA based on the rate-effectiveness coefficient E of the most recent N consecutive irradiations, where N≥3; Set an adjustment threshold Δ, with a value ranging from 0.05 to 0.

2. If the increase of the current EA compared to the previous EA is greater than Δ, the system will automatically increase the trigger threshold by 5% to 15% and decrease the irradiation duration by 10% to 20%. If the decrease is greater than Δ, the system will automatically decrease the trigger threshold by 5% to 15% and increase the irradiation duration by 10% to 20%.

8. The method for isolating Curcuma zedoaria protoplasts as described in claim 7, characterized in that, Dynamic adjustment also incorporates real-time monitoring of the enzyme hydrolysate pH value as a synergistic control parameter: Within the predicted time window, continuously monitor the pH value of the enzymatic hydrolysate; When the pH value is detected to rise to the range of 5.85 to 5.95, the system automatically halve the adjustment range of the trigger threshold and double the adjustment range of the irradiation duration.

9. The method for isolating Curcuma zedoaria protoplasts as described in claim 7 or 8, characterized in that, When the following conditions are met, the system automatically generates and executes a stop command to simultaneously terminate the sampling detection in step S4, the near-infrared irradiation in step S6, and the operation of the constant temperature shaker. The triggering of the stop command follows the principle of applying either the core condition or the coordination condition, with the one that is satisfied first taking precedence. The core condition is that "the number of intact protoplasts enters a plateau period, and the number of broken protoplasts is always less than 15% of the number of intact protoplasts" as specified in step S7 of claim 1; the synergistic condition is that the system continuously calculates the moving average value EA as described in claim 7 and records its peak value EM; when it is detected that EA decreases for at least two consecutive near-infrared irradiation monitoring cycles from EM, and its value drops to less than 30% of EM; When either of the above two conditions is met, the system immediately executes the stop command.

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