Tissue culture control method for improving polysaccharide content of protocorm of dendrobium huoshanense

Abstract

The invention relates to the technical field of tissue culture, in particular to a tissue culture control method for increasing the content of polysaccharide in protocorm of dendrobium huoshanense, the method is carried out in a bioreactor provided with an online monitoring system, and the online monitoring system is at least used for monitoring a culture characteristic parameter set of a culture solution in real time; the method comprises the following steps: culturing dendrobium huoshanense protocorm under a first culture condition suitable for cell proliferation; judging whether cell growth enters a proliferation saturation state or not based on a culture characteristic parameter set monitored by an online monitoring system in real time; if yes, triggering a polysaccharide induction stage, and taking the culture characteristic parameter set for triggering the polysaccharide induction stage as a reference culture characteristic parameter set; after a time interval is set, collecting the real-time culture characteristic parameter set again, and calculating a culture characteristic deviation vector based on the real-time culture characteristic parameter set and the reference culture characteristic parameter set; and on the basis of the culture characteristic deviation vector, judging the metabolic state characteristic of the current dendrobium protocorm cell.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tissue culture, and in particular to a tissue culture control method for improving polysaccharide content of protocorm of Dendrobium huoshanense. BACKGROUND

[0002] As a rare medicinal plant, the polysaccharide in the protocorm of Dendrobium huoshanense is an important active ingredient. Currently, to improve the polysaccharide content of the protocorm of Dendrobium huoshanense, a tissue culture method is usually adopted, in which an inducer is added or the culture condition is changed at a specific time point to stimulate the biosynthesis of polysaccharide.

[0003] However, most of the prior art methods adopt fixed culture time sequence and static culture condition adjustment strategy, that is, the inducement pressure is uniformly applied or the culture medium is replaced at a predetermined time point. The existing method cannot perceive and respond to the real-time physiological state changes of the protocorm cells of Dendrobium huoshanense during the culture process. Due to the differences in growth rate and metabolic activity of different batches of cells, the fixed culture scheme often cannot accurately match the best polysaccharide synthesis opportunity of the cells, resulting in low inducement efficiency, unstable polysaccharide yield and difficulty in maximizing. SUMMARY

[0004] The present application provides a tissue culture control method for improving polysaccharide content of protocorm of Dendrobium huoshanense, which can effectively solve the problems in the background art.

[0005] In order to achieve the above-mentioned purpose, the present application provides a tissue culture control method for improving polysaccharide content of protocorm of Dendrobium huoshanense, comprising:

[0006] culturing the protocorm of Dendrobium huoshanense under first culture conditions suitable for cell proliferation;

[0007] judging whether the cell growth enters a proliferation saturation state based on the set of culture characteristic parameters monitored in real time by the online monitoring system;

[0008] when the judgment is yes, triggering a polysaccharide induction phase, and taking the set of culture characteristic parameters triggering the polysaccharide induction phase as a reference set of culture characteristic parameters;

[0009] after a set time interval, collecting a real-time set of culture characteristic parameters again, and calculating a culture characteristic deviation vector based on the real-time set of culture characteristic parameters and the reference set of culture characteristic parameters;

[0010] judging the metabolic state characteristics of the current protocorm cells of Dendrobium huoshanense based on the culture characteristic deviation vector;

[0011] adjusting or maintaining the current culture condition to a second culture condition corresponding to the metabolic state characteristics based on the metabolic state characteristics;

[0012] The process of repeatedly collecting real-time culture characteristic parameter sets, calculating culture characteristic deviation vectors, judging metabolic state characteristics, and adjusting or maintaining the second culture conditions continues until the culture endpoint is reached.

[0013] In one possible design, the first culture conditions include the control of the culture medium and environmental conditions.

[0014] In one possible design, the set of culture characteristic parameters includes pH, dissolved oxygen concentration, turbidity, and sugar content.

[0015] In one possible design, determining whether cell growth has entered a state of proliferation saturation includes: calculating the biomass growth rate derived from the turbidity value; when the biomass growth rate is lower than a preset threshold, it is determined that the cell has entered a state of proliferation saturation.

[0016] In one possible design, the culture characteristic deviation vector includes a proton flux change index, a respiratory metabolic intensity index, a carbon source utilization efficiency index, and a biomass accumulation rate.

[0017] In one possible design, the metabolic state characteristics include an efficient synthetic state, a proliferative state, and a metabolically inhibited state.

[0018] In one possible design, the culture condition adjustment strategy for the highly efficient synthesis state includes maintaining the current pH value, reducing the dissolved oxygen concentration, and maintaining the current sugar content.

[0019] In one possible design, the culture condition adjustment strategy for the proliferative tendency state includes adjusting the pH value to be higher than the baseline pH value, reducing the dissolved oxygen concentration, and reducing the sugar content.

[0020] In one possible design, the culture condition adjustment strategy for the metabolically inhibited state includes rapidly restoring the pH to a baseline pH, increasing dissolved oxygen concentration, increasing sugar content, and removing metabolic waste accumulated in the culture medium.

[0021] In one possible design, the criteria for determining the reaching of the culture endpoint include the peak polysaccharide content and the carbon source consumption saturation.

[0022] The technical solution of this invention achieves the following technical effects: By equipping the system with an online monitoring system, multiple parameters in the culture medium can be monitored in real time, allowing for timely assessment of cell growth status and avoiding the inflexibility of fixed time sequences. The culture strategy can be dynamically adjusted based on actual growth conditions. By real-time monitoring and calculation of the culture characteristic deviation vector, different metabolic states can be identified, allowing for adjustment of culture conditions under different metabolic states. This ensures that each batch of cells can be induced under optimal physiological conditions, maximizing polysaccharide synthesis. Culture conditions can be precisely adjusted according to the cell's metabolic state. If cells enter a proliferation saturation state, the polysaccharide induction phase can be triggered and adjusted to the corresponding second culture conditions, ensuring that cells synthesize polysaccharides under optimal metabolic conditions, improving induction efficiency, and stabilizing yield. Real-time data acquisition and analysis allow for precise control of the optimal induction timing for each batch of cells, and dynamic adjustment of culture conditions reduces induction errors, ensuring stable polysaccharide yield. Real-time monitoring and dynamic adjustment make the culture process more flexible and precise. Adjusting culture conditions based on the real-time physiological state of the cells improves polysaccharide induction efficiency and yield stability. Attached Figure Description

[0023] Figure 1 A flowchart of a tissue culture control method for increasing the polysaccharide content of Dendrobium huoshanense protocorms. Detailed Implementation

[0024] This application will now be described with reference to the accompanying drawings.

[0025] like Figure 1 As shown, the tissue culture control method for increasing the polysaccharide content of Dendrobium huoshanense protocorms according to the present invention specifically includes the following steps:

[0026] S1. Cultivate the protocorms of Dendrobium huoshanense under the first culture conditions suitable for cell proliferation;

[0027] S2. Based on the set of culture characteristic parameters monitored in real time by the online monitoring system, determine whether cell growth has entered a state of proliferation saturation;

[0028] S3. When the determination is yes, the polysaccharide induction phase is triggered, and the culture characteristic parameter set that triggers the polysaccharide induction phase is used as the baseline culture characteristic parameter set.

[0029] S4. After a set time interval, collect the real-time culture characteristic parameter set again, and calculate the culture characteristic deviation vector based on the real-time culture characteristic parameter set and the benchmark culture characteristic parameter set.

[0030] S5. Based on the culture feature deviation vector, determine the metabolic state characteristics of the current Dendrobium protocorm cells;

[0031] S6. Based on the metabolic state characteristics, adjust or maintain the current culture conditions to a second culture condition corresponding to the metabolic state characteristics;

[0032] S7. Repeat the process of collecting real-time culture characteristic parameter set, calculating culture characteristic deviation vector, judging metabolic state characteristics, and adjusting or maintaining the second culture conditions until the culture endpoint is reached.

[0033] In this embodiment, by equipping the system with an online monitoring system, multiple parameters in the culture medium can be monitored in real time, allowing for timely assessment of cell growth status. This avoids the inflexibility of fixed time sequences and enables dynamic adjustment of the culture strategy based on actual growth conditions. By monitoring and calculating the culture characteristic deviation vector in real time, different metabolic states can be identified, allowing for adjustment of culture conditions under different metabolic states. This ensures that each batch of cells can be induced under optimal physiological conditions, maximizing polysaccharide synthesis. The system can precisely adjust culture conditions based on the cell's metabolic state. If cells enter a proliferation saturation state, a polysaccharide induction phase can be triggered, and the corresponding second culture conditions can be adjusted to ensure that cells synthesize polysaccharides under optimal metabolic conditions, improving induction efficiency and stabilizing yield. By collecting and analyzing data in real time, the optimal induction timing for each batch of cells can be accurately grasped, and dynamic adjustment of culture conditions can reduce induction errors, ensuring the stability of polysaccharide yield. Real-time monitoring and dynamic adjustment make the culture process more flexible and precise. The system can adjust culture conditions based on the real-time physiological state of the cells, thereby improving polysaccharide induction efficiency and yield stability.

[0034] In some embodiments of the present invention, for step S1, the protocorms of Dendrobium huoshanense are cultured under first culture conditions suitable for cell proliferation;

[0035] The first culture conditions include the control of culture medium and environmental conditions;

[0036] The culture medium is the nutrient source for the proliferation of protocorms and needs to meet the requirements of cell division for carbon, nitrogen, phosphorus, trace elements and growth regulators. MS medium is used for the proliferation of protocorms of Dendrobium huoshanense.

[0037] Sucrose is used as the main carbon source, with the concentration controlled at 20-30 g / L. Sucrose not only provides energy but also regulates the osmotic pressure of the culture medium. Too low a concentration can cause cells to absorb water and swell, while too high a concentration can inhibit cell division.

[0038] The nitrogen source is a mixture of nitrate nitrogen and ammonium nitrogen. The amount of ammonium nitrogen should be appropriate, as an excess will cause the pH of the culture medium to drop rapidly and inhibit cell growth.

[0039] Add a combination of cytokinins and auxins as growth regulators;

[0040] 6-Benzylaminopurine was selected as a cytokinin to promote cell division and induce the formation of clustered bud-like structures in protocorms;

[0041] Naphthaleneacetic acid was selected as an auxin to help regulate cell elongation and prevent the protocorm from becoming too dense, which could lead to internal hypoxia.

[0042] The trace elements and vitamins are prepared according to the standard MS medium formula, without any additional additions or subtractions.

[0043] After the culture medium is prepared, the pH needs to be adjusted to 5.6-5.8 with hydrochloric acid or sodium hydroxide. The pH will drop slightly after sterilization, so a buffer space needs to be reserved in advance.

[0044] The original corms of Dendrobium huoshanense are heterotrophically cultured and have no photosynthetic capacity. Environmental conditions need to be precisely controlled through a bioreactor to avoid factors such as light, temperature, and humidity affecting cell proliferation efficiency.

[0045] A constant temperature control was adopted, set at 23-25℃. Temperatures below 20℃ would cause a decrease in the division rate, while temperatures above 28℃ would trigger oxidative stress in the cells, resulting in browning.

[0046] The proliferation phase does not require light. The bioreactor should be placed in a dark environment or wrapped with a light shield, and only briefly exposed to weak light during sampling to avoid light signals interfering with the cell cycle.

[0047] Bioreactors need to maintain a suitable dissolved oxygen concentration; during the proliferation period of Dendrobium huoshanense protocorms, the air saturation needs to be controlled at 30%-50%.

[0048] Sterile air bubble ventilation is used to avoid mechanical damage to the protocorm caused by excessive ventilation.

[0049] The inoculation amount of protocorms is calculated based on the effective volume of the bioreactor. Too low an inoculation amount will lead to insufficient cell density in the early stage and a prolonged proliferation cycle; too high an inoculation amount will rapidly consume nutrients and lead to inhibition of cell competition in the later stage.

[0050] Select protocorms with a diameter of 2-3mm, a light yellow-green color, no browning, and no contamination by bacteria. They should be rinsed with sterile water in advance to remove any old culture medium residue and avoid introducing impurities that could affect the stability of the new culture medium.

[0051] Inoculation should be performed in a sterile environment under a clean bench. After inoculation, the bioreactor should be sealed and sterile air should be introduced for 30 minutes to ensure that the internal environment is stable before starting the temperature, stirring and other control programs.

[0052] In this embodiment, a suitable combination of carbon source, nitrogen source, and growth regulators provided adequate nutritional support, promoting cell division and growth of the protocorms. Precise control of temperature, humidity, and oxygen concentration provided an optimal environment for cell proliferation. A constant temperature range ensured the normal cell division rate. Strict control of dissolved oxygen concentration within the bioreactor prevented mechanical damage to cells from excessive aeration. A light-free environment avoided interference from light signals on the cell cycle, ensuring efficient cell proliferation. Aseptic operation and strict inoculation conditions effectively reduced the risk of exogenous contamination during culture, ensuring the stability of the culture medium. The use of a bioreactor for aeration and dissolved oxygen control ensured that cells were not subjected to unnecessary damage during proliferation. An appropriate inoculation amount ensured a balanced cell density in the early stages, avoiding excessive nutrient consumption or cell competition inhibition, thereby stabilizing the proliferation cycle and ultimately improving the efficiency and stability of Dendrobium huoshanense protocorm proliferation.

[0053] In some embodiments of the present invention, for step S2, based on the set of culture characteristic parameters monitored in real time by the online monitoring system, it is determined whether cell growth has entered a proliferation saturation state;

[0054] The set of culture characteristic parameters includes pH value, dissolved oxygen concentration, turbidity, and sugar content;

[0055] pH value reflects the acidity or alkalinity of the culture medium and indirectly indicates the intensity of cellular metabolic activity;

[0056] Dissolved oxygen concentration is used to measure the intensity of cellular respiration; a hypoxic environment may inhibit proliferation or trigger metabolic shifts.

[0057] Turbidity is directly related to cell density through the principle of light scattering and is a direct indicator of biomass growth;

[0058] Sugar content is used to monitor the rate of carbon source consumption in the culture medium, reflecting the degree of cellular metabolic activity;

[0059] Determining whether cell growth has entered a state of proliferation saturation includes: calculating the biomass growth rate derived from turbidity value; when the biomass growth rate is lower than a preset threshold, it is determined that the cell has entered a state of proliferation saturation.

[0060] A quantitative relationship between turbidity value and cell dry weight or fresh weight was established in advance through offline experiments, and real-time turbidity data was converted into absolute biomass values.

[0061] The biomass growth rate, i.e. the change in biomass per unit time, is calculated using the sliding window method.

[0062] The critical growth rate for proliferation saturation is determined based on historical data; when the growth rate is detected to be below the threshold twice consecutively, a saturation state judgment is triggered.

[0063] In this embodiment, based on the online monitoring system, dynamic data of cell culture can be acquired in real time, reflecting the cell growth status in a timely manner, thereby helping operators accurately grasp the stage and changes of cell growth. By comprehensively considering multiple culture characteristic parameters such as pH value, dissolved oxygen concentration, turbidity, and sugar content, the system can comprehensively reflect the cell's metabolic activity, respiration intensity, and carbon source consumption rate, providing more accurate cell growth information. By calculating the biomass growth rate using the sliding window method and setting a reasonable growth rate threshold, it is possible to accurately determine whether cells have entered a state of proliferation saturation, avoiding over-culture or waste of resources. By establishing a quantitative relationship between turbidity and cell biomass through offline experiments, the real-time monitored turbidity data can be directly converted into the absolute value of biomass, thereby providing more intuitive and reliable cell growth data. This step can automatically determine whether cell growth has reached saturation, reducing human intervention and errors, and improving the accuracy and efficiency of the production process.

[0064] In some embodiments of the present invention, for step S3, when it is determined that the polysaccharide induction stage is triggered, and the culture characteristic parameter set that triggers the polysaccharide induction stage is used as the benchmark culture characteristic parameter set.

[0065] During the proliferation saturation phase, cells shift from primary metabolism, which is mainly based on DNA replication and cell division, to secondary metabolism, which is mainly based on the synthesis of secondary metabolites. Induction at this time can maximize the utilization of cellular metabolic resources. If induction is performed during the proliferation phase, cells will still preferentially allocate energy to growth, resulting in low polysaccharide synthesis efficiency. If induction is delayed, cells will enter the senescence phase due to nutrient depletion or accumulation of metabolic waste, and their synthetic capacity will decline.

[0066] After induction is triggered, the system records the current set of culture characteristic parameters as the baseline culture characteristic parameter set. The role of the baseline culture characteristic parameter set is to serve as a reference value for subsequent bioreactor control, helping to monitor cell growth, metabolism and polysaccharide synthesis processes in real time.

[0067] In this embodiment, after cell growth reaches proliferation saturation, the cell's metabolic focus shifts from primary metabolism, primarily DNA replication and cell division, to secondary metabolism, primarily the synthesis of secondary metabolites. At this point, inducing polysaccharide synthesis can fully utilize the cell's metabolic resources, thereby maximizing polysaccharide synthesis efficiency. If induction occurs during the proliferation phase, the cell will preferentially use energy for growth rather than polysaccharide synthesis, resulting in poor induction effects. If induction is delayed, the cell will enter senescence, and its synthetic capacity will decrease. By accurately triggering induction during proliferation saturation, the yield and quality of polysaccharides can be improved. After triggering induction, the system records the current set of culture characteristic parameters as a baseline set of culture characteristic parameters, providing a stable reference for subsequent bioreactor control and helping the system monitor cell growth, metabolic status, and polysaccharide synthesis process in real time.

[0068] In some embodiments of the present invention, for step S4, after a set time interval, the real-time culture characteristic parameter set is collected again, and the culture characteristic deviation vector is calculated based on the real-time culture characteristic parameter set and the benchmark culture characteristic parameter set;

[0069] After the polysaccharide induction phase is triggered, samples are taken at fixed time intervals to refer to the cycle of changes in metabolic parameters during plant cell suspension culture. Too short a time will increase the system load, while too long a time will miss the transition of metabolic state.

[0070] The culture characteristic deviation vector includes the proton flux change index, respiratory metabolic intensity index, carbon source utilization efficiency index, and biomass accumulation rate;

[0071] The proton flux change index can reflect the activity of cell membrane ATPase and the rate of extracellular acidification, and indirectly indicate the intensity of organic acid secretion during polysaccharide synthesis. If the index is negative, it indicates that the cell's metabolic acid production is increased; if it is positive, it may be biased towards nitrogen metabolism.

[0072] The respiratory metabolic intensity index is calculated based on changes in dissolved oxygen concentration and characterizes the activity of the mitochondrial respiratory chain; the higher the index, the stronger the cellular respiration and the more abundant the energy supply; a low index may indicate decreased metabolic activity.

[0073] The carbon source utilization efficiency index is calculated by the ratio of sugar consumption rate to biomass accumulation rate, reflecting the rate at which cells consume sugar in the culture medium. Polysaccharide synthesis depends on carbon source supply; too high a carbon source supply will lead to premature carbon source depletion, while too low a carbon source supply indicates that the carbon source is not being effectively used for polysaccharide synthesis.

[0074] The biomass accumulation rate is derived from turbidity and directly reflects cell division activity. Since the cell has entered the proliferation saturation phase, this rate should be close to 0. If a significant positive value is found, it indicates that the cell is still biased towards proliferation and subsequent conditions need to be adjusted to inhibit division.

[0075] In this embodiment, the real-time collected parameters accurately reflect the metabolic activities of cells at different culture stages, helping to assess the health status of cell metabolism. Sampling at fixed time intervals avoids the drawbacks of excessively short or long sampling periods, ensuring effective data is obtained at critical moments of metabolic transition, thus enabling appropriate regulatory decisions. The various indices provided by the culture characteristic deviation vector can provide early warnings before metabolic problems arise, helping to adjust culture conditions in a timely manner. By monitoring the biomass accumulation rate, measures can be taken promptly when cells approach saturation, avoiding ineffective proliferation and ensuring that resources are prioritized for polysaccharide synthesis, thereby improving culture efficiency. This step, through a multi-dimensional, real-time data monitoring and feedback mechanism, enhances the accuracy, stability, and production efficiency of the plant cell suspension culture process.

[0076] In some embodiments of the present invention, for step S5, the metabolic state characteristics of the current Dendrobium protocorm cells are determined based on the culture feature deviation vector;

[0077] Metabolic state characteristics include highly efficient anabolic state, proliferative state, and metabolically inhibited state;

[0078] A highly efficient synthetic state indicates that cellular metabolic activity is concentrated on synthesizing important metabolites, particularly polysaccharides. In this state, the cell's energy and resources are preferentially used for the production of secondary metabolites, while proliferation is inhibited or slowed. The corresponding deviation vector is characterized by:

[0079] A high positive value for the proton flux change index indicates active secretion of organic acids;

[0080] A moderate or slightly high respiratory metabolic intensity index indicates that the cell has sufficient energy supply and supports efficient metabolic activities.

[0081] A medium or high carbon source utilization efficiency index indicates that the cells are relatively efficient in utilizing glycogen sources, meaning that glycogen sources are effectively converted into polysaccharides.

[0082] The rate of biomass accumulation is low or close to zero at this time, indicating that the growth and proliferation of cells have slowed down and the focus is mainly on the synthesis of polysaccharides.

[0083] The proliferation tendency state indicates that the cell's metabolic activities are mainly concentrated on cell growth and proliferation; the efficiency of polysaccharide synthesis is low, and cellular resources are allocated more to division and expansion; the characteristics of the corresponding deviation vector are:

[0084] A negative proton flux change index indicates that the cell is undergoing more nitrogen metabolism or acid metabolism.

[0085] A high respiratory metabolic intensity index indicates that cells are consuming a large amount of energy for division and growth;

[0086] The low carbon source utilization efficiency index indicates that the cells mainly consume sugar sources in the culture medium for proliferation rather than polysaccharide synthesis.

[0087] A high rate of biomass accumulation indicates rapid cell proliferation and an increase in biomass.

[0088] Metabolic inhibition indicates a slowdown in cellular metabolic activity, with cells entering a state of senescence or stress, growth almost halting, and a decreased ability to synthesize polysaccharides; the corresponding bias vector characteristics are:

[0089] A significantly negative proton flux change index indicates that the cell has entered a state of metabolic inhibition, resulting in a greater accumulation of metabolic waste.

[0090] A low respiratory metabolic intensity index indicates decreased respiratory chain activity and insufficient energy supply in cells.

[0091] A carbon source utilization efficiency index that is too low indicates that the consumption efficiency of sugar sources is extremely low, and the cells have entered a state of inefficient sugar utilization, indicating that the cells have entered the senescence stage.

[0092] A biomass accumulation rate close to zero indicates that cells have almost stopped proliferating, and resources are being diverted to maintaining life activities rather than growth.

[0093] In this embodiment, by analyzing characteristic parameters such as proton flux change index, respiratory metabolic intensity index, carbon source utilization efficiency index, and biomass accumulation rate, it is possible to accurately determine whether cells are in different metabolic states, such as high-efficiency synthesis, proliferation tendency, or metabolic inhibition. By identifying cells in different metabolic states, culture conditions can be optimized according to specific needs, promoting polysaccharide production in the high-efficiency synthesis state and supporting rapid growth in the proliferation tendency state. In the high-efficiency synthesis state, cells concentrate resources on polysaccharide synthesis, which can improve the synthesis efficiency of target metabolites. In the proliferation tendency state, biomass accumulation is increased by accelerating cell division and proliferation. By identifying the metabolic inhibition state, it is possible to detect cells entering a state of senescence or stress in a timely manner, avoiding reduced product synthesis efficiency and growth stagnation, and prolonging the cell's active phase. This step can effectively improve the production efficiency of Dendrobium protocorm cells, optimize their growth and metabolic state, and thus improve the yield and quality of target products.

[0094] In some embodiments of the present invention, for step S6, based on the metabolic state characteristics, the current culture conditions are adjusted or maintained to a second culture condition corresponding to the metabolic state characteristics.

[0095] When in a state of high-efficiency synthesis, carbon metabolism mainly flows to the polysaccharide synthesis pathway rather than cell proliferation, and metabolic activities are stable and efficient. At this time, the core of the culture conditions is to maintain a stable microenvironment, prolong the high-efficiency synthesis window, and avoid parameter fluctuations from interrupting the synthesis process.

[0096] Strategies for adjusting culture conditions to achieve efficient synthesis include:

[0097] Maintain the current pH value by adding a small amount of acid-base regulator in real time through an online monitoring system to control pH fluctuations;

[0098] Dissolved oxygen concentration is reduced to 80%-90% of the baseline value, inhibiting excessive activity of the mitochondrial respiratory chain;

[0099] Maintain the current carbon source concentration by slowly replenishing the carbon source through the bioreactor's feeding system to avoid excessive carbon source causing cells to revert to proliferation, or insufficient carbon source causing a shortage of synthetic raw materials.

[0100] No new inducers will be added for the time being; the concentration of the inducers added during the polysaccharide induction phase will be maintained to avoid metabolic disorders caused by repeated stress.

[0101] When a cell is in a proliferative state, its metabolic flow is biased towards cell division, and the polysaccharide synthesis pathway is inhibited. At this time, the key to culture conditions is to apply appropriate stress signals to guide the metabolic flow from proliferation to polysaccharide synthesis.

[0102] Strategies for adjusting culture conditions in a proliferative state include:

[0103] Fine-tuning the pH value slightly above the optimal proliferation pH; a slightly alkaline environment can inhibit the activity of cell cycle-related enzymes and slow down the proliferation rate, while not significantly affecting the activity of key enzymes in polysaccharide synthesis;

[0104] Dissolved oxygen concentration is reduced to 25%-30% of saturated dissolved oxygen; moderate hypoxia stress can activate the cell's secondary metabolic defense mechanism, promote the synthesis of protective substances such as polysaccharides, and inhibit the cell division process that requires strong oxygen.

[0105] By moderately reducing the carbon source concentration, the supply of raw materials for cell proliferation is reduced, forcing cells to prioritize the use of limited carbon sources for polysaccharide synthesis.

[0106] Supplement with a low concentration of elicitor once; the elicitor can act as a signaling molecule to activate the expression of polysaccharide synthesis-related genes and further guide the shift of metabolic flux.

[0107] When in a state of metabolic inhibition, overall metabolic activity decreases significantly and polysaccharide synthesis almost stops. At this time, the core of the culture conditions is to alleviate the inhibitory pressure and restore the basal metabolic activity of cells, rather than directly pursuing polysaccharide synthesis.

[0108] Strategies for adjusting culture conditions in a state of metabolic inhibition include:

[0109] Quickly adjust the pH back to the optimal basal metabolic pH; if the pH deviates too much, it needs to be adjusted slowly through gradient feeding to avoid further damage to cells from sudden pH changes;

[0110] Dissolved oxygen concentration is increased to 50%-60% of saturated dissolved oxygen; sufficient oxygen can restore the respiratory chain function of cells, provide adenosine triphosphate for metabolic activities, and alleviate inhibition caused by insufficient energy.

[0111] Replenishing fresh carbon sources provides cells with sufficient energy resources, helping them restore their basal metabolic cycle;

[0112] Initiate a partial culture medium change procedure in the bioreactor; remove accumulated metabolic waste from the culture medium, as this waste inhibits enzyme activity and exacerbates metabolic stagnation.

[0113] In this embodiment, by adjusting culture conditions for different metabolic states, the direction of cellular metabolism can be effectively guided, thereby achieving efficient production of target products such as polysaccharide synthesis, thus improving production efficiency and avoiding metabolic disorders. In the efficient synthesis state, maintaining stable culture conditions can extend the window of efficient synthesis, avoiding fluctuations and interruptions in the synthesis process, thereby improving polysaccharide synthesis efficiency. In the proliferation-prone state, applying appropriate stress signals can effectively inhibit cell proliferation and promote polysaccharide synthesis. In the metabolic inhibition state, strategies such as rapidly adjusting pH, increasing dissolved oxygen concentration, supplementing carbon sources, and initiating partial medium replacement can effectively restore the basal metabolic activity of cells, alleviate metabolic stagnation caused by inhibitory stress, and thus restore production capacity. By finely adjusting the elicitor concentration and avoiding repeated stress, excessive metabolic stress can be avoided, thus ensuring that cells synthesize polysaccharides under stable conditions.

[0114] In some embodiments of the present invention, for step S7, the process of collecting real-time culture characteristic parameter set, calculating culture characteristic deviation vector, judging metabolic state characteristics, and adjusting or maintaining the second culture conditions is repeated until the culture endpoint is reached.

[0115] The criteria for determining whether the culture endpoint has been reached include:

[0116] To determine the peak polysaccharide content, original corm samples were taken at regular intervals, and the polysaccharide content was determined using the phenol-sulfuric acid method. When the polysaccharide content growth rate showed its first decline in two or three consecutive test results, it was preliminarily determined that the endpoint was approaching.

[0117] Carbon source consumption saturation: When the remaining carbon source concentration in the culture medium is less than 10%-15% of the initial concentration, and the carbon source consumption rate is less than 0.1 g / (L·h) for three consecutive monitoring cycles, it indicates that the carbon source supply for cell polysaccharide synthesis has reached its limit, and polysaccharide accumulation is difficult to continue to increase.

[0118] In this embodiment, by real-time acquisition of culture characteristic parameter sets, calculation of culture characteristic deviation vectors, and judgment of metabolic state characteristics, the second culture conditions can be dynamically adjusted or maintained, improving the stability of cell metabolic state during culture and optimizing the polysaccharide accumulation process. Regular monitoring of polysaccharide content, combined with continuous monitoring results, allows for more accurate determination of the culture endpoint. When the polysaccharide growth rate first declines, it can be promptly determined that the culture is nearing its endpoint, avoiding resource waste caused by over-culture or improper culture conditions. By monitoring the remaining carbon source concentration and carbon source consumption rate in the culture medium, the carbon source supply required for cell polysaccharide synthesis can be accurately determined. When the carbon source supply approaches its limit, culture conditions can be adjusted early to prevent the cessation of cell polysaccharide accumulation, thereby increasing the final polysaccharide yield. This step, through precise real-time monitoring, timely adjustment of culture conditions, and precise control of polysaccharide content and carbon source consumption, optimizes the polysaccharide production process, improving culture efficiency and final yield.

[0119] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A tissue culture control method for increasing the polysaccharide content of Dendrobium huoshanense protocorms, characterized by, The method is performed in a bioreactor equipped with an online monitoring system for at least monitoring a set of culture characteristic parameters of the culture solution in real time; the method comprises: culturing the Dendrobium huoshanense protocorm under first culture conditions suitable for cell proliferation; judging whether the cell growth enters a proliferation saturation state based on the set of culture characteristic parameters monitored in real time by the online monitoring system; when the judgment is yes, triggering a polysaccharide induction phase, and taking the set of culture characteristic parameters triggering the polysaccharide induction phase as a reference set of culture characteristic parameters; after a set time interval, collecting a real-time set of culture characteristic parameters again, and calculating a culture characteristic deviation vector based on the real-time set of culture characteristic parameters and the reference set of culture characteristic parameters; judging a metabolic state feature of the current Dendrobium huoshanense protocorm cell based on the culture characteristic deviation vector; adjusting or maintaining the current culture conditions to second culture conditions corresponding to the metabolic state feature based on the metabolic state feature; repeating the processes of collecting a real-time set of culture characteristic parameters, calculating a culture characteristic deviation vector, judging a metabolic state feature, and adjusting or maintaining second culture conditions until a culture endpoint is reached.

2. The tissue culture control method for improving the polysaccharide content of Dendrobium huoshanense protocorms according to claim 1, characterized in that, The first culture conditions include culture medium and environmental condition control.

3. The method of claim 1, wherein the method is characterized by, The set of culture characteristic parameters includes pH value, dissolved oxygen concentration, turbidity, and sugar content.

4. The method of claim 3, wherein the method is characterized by, The judgment of whether the cell growth enters a proliferation saturation state comprises calculating a biomass growth rate derived based on the turbidity value, and when the biomass growth rate is lower than a preset threshold value, it is judged that the proliferation saturation state is entered.

5. The method of claim 1, wherein the method is characterized by, The culture characteristic deviation vector includes proton flux change index, respiratory metabolic intensity index, carbon source utilization efficiency index, and biomass accumulation rate.

6. The tissue culture control method for increasing the polysaccharide content of Dendrobium huoshanense protocorms according to claim 5, characterized in that, The metabolic state feature includes high-efficiency synthesis state, proliferation tendency state, and metabolic inhibition state.

7. The method of claim 6, wherein the method is characterized by, The culture condition adjustment strategy for the high-efficiency synthesis state includes maintaining the current pH value, reducing the dissolved oxygen concentration, and maintaining the current sugar content. 8.The method of claim 6, wherein the method is characterized by, The culture condition adjustment strategy for the proliferation tendency state includes adjusting the pH value to be higher than the reference pH value, reducing the dissolved oxygen concentration, and reducing the sugar content. 9.The method of claim 6, wherein the method is characterized by, The culture condition adjustment strategy for the metabolic inhibition state includes quickly adjusting the pH value to the reference pH value, increasing the dissolved oxygen concentration, increasing the sugar content, and removing accumulated metabolic waste in the culture medium.

10. The method of claim 1, wherein the method is characterized by, The judgment basis for reaching the culture endpoint includes polysaccharide content peak value and carbon source consumption saturation degree.

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