Morphological and physiological parameter-based bulk drug production strain culture method

Abstract

The invention belongs to the technical field of industrial microbial fermentation process optimization, and discloses a bulk drug production strain culture method based on morphology and physiological parameters. According to the method, through dual verification of physiological trend and form peak, the misjudgment risk of single parameter judgment is avoided, meanwhile, the trend is captured as soon as possible through change rate analysis, and transplanting can be conducted at the golden moment when the thallus activity reaches the peak but does not decline. By quantifying morphology and setting a clear threshold value, human subjective factors are eliminated, the process reproducibility is extremely high, the principle of the method is suitable for production of raw material medicines fermented by various filamentous bacteria, such as acarbose, moxidectin and tobramycin, and finally accurate seed transfer is realized, so that the fermentation period is effectively shortened, the yield of the raw material medicines is improved, and the method is suitable for industrial production. And remarkable economic benefits are brought.

Description

Technical Field

[0001] This invention belongs to the field of industrial microbial fermentation process optimization technology, specifically relating to a method for culturing active pharmaceutical ingredient (API) strains based on morphological and physiological parameters. More specifically, it relates to a method for determining the optimal transplanting time for API production strains, particularly actinomycetes and filamentous fungi, during the seed culture stage. Background Technology

[0002] In the industrial fermentation production of active pharmaceutical ingredients, this is a crucial step that determines the success and efficiency of fermentation. Transplanting too early results in insufficient cell biomass and weak physiological activity, leading to an excessively long lag period in the early stages of fermentation; transplanting too late causes the cells to age and lose vitality, and may even lead to cell autolysis, severely affecting the yield and efficiency of subsequent fermentation.

[0003] Currently, commonly used judgment methods in the industry have significant limitations: 1) Single time parameter method: This method relies solely on a fixed culture time. Because it cannot dynamically adapt to batch-to-batch differences in culture medium, fluctuations in inoculum size, and changes in the microenvironment, batch-to-batch consistency in the fermentation process is poor. 2) Single physiological parameter method: For example, relying solely on the rebound point of pH "from low to high" or the "sharp rise / fall" of dissolved oxygen (DO) as the basis for judgment. These macroscopic parameters are easily affected by engineering conditions such as stirring and aeration, and have significant lag. By the time significant parameter changes are observed, the optimal window for cell physiological state may have already been missed. 3) Experience-based judgment method: Relying on operators' sensory observation of bacterial viscosity, color, or mycelial morphology. This method is highly subjective, makes it difficult to establish quantitative standards, and severely hinders the standardized control and linear scale-up of the fermentation process.

[0004] Moreover, the growth and metabolic characteristics of filamentous fungi such as actinomycetes and streptomyces during the seed culture stage are fundamentally different from those of common single-celled microorganisms such as Escherichia coli and yeast, making the determination of the optimal transplanting period more complex and critical.

[0005] The complexity of morphological development: Unlike the simple growth cycle of single-celled bacteria, filamentous fungi undergo a complex differentiation process: spores → substrate hyphae → aerial hyphae / spores. The optimal transplanting period is a brief window that must occur during the peak of hyphal vigor, well before the formation of aerial hyphae or spores. The formation of aerial hyphae or spores marks an irreversible change in the physiological state of the fungus, which will severely weaken its subsequent yield. Usually, a single parameter change cannot accurately correspond to this critical morphological node.

[0006] Specific signals of metabolic transition: During their rapid growth phase, filamentous bacteria consume large amounts of oxygen and produce acidic metabolites, leading to a continuous decrease in dissolved oxygen and pH. When growth slows and metabolism shifts, the "double inflection point"—where dissolved oxygen rises from a low level and pH rises from a low level and then stabilizes—is a key signal that they are ending their rapid proliferation and preparing to enter the production phase. This signal is subtle and transient, and relying on a single parameter for judgment is easily misinterpreted by noise.

[0007] Early establishment of production potential: The physiological state of the mycelium during the seed stage directly affects its secondary metabolic efficiency in the fermenter. The optimal transplanting period corresponds not only to sufficient biomass, but also to a specific physiological state in which the mycelium is highly active and possesses the potential to efficiently initiate the synthesis of the target product.

[0008] In summary, due to the unique morphological differentiation, metabolic complexity, and deep correlation with production performance of filamentous fungi such as actinomycetes and Streptomyces, the use of single-parameter judgment methods is inevitably insufficient. Therefore, there is an urgent need in this field for a judgment method that can deeply integrate online physiological parameter trends reflecting internal metabolic state with quantitative indicators of cell morphology reflecting external developmental stage, in order to scientifically and accurately capture the optimal transplantation period of filamentous fungi for active pharmaceutical ingredients (APIs), and ensure the high efficiency and stability of API fermentation production. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of existing technologies that rely on a single parameter to determine the optimal transplanting period, and to provide a method for culturing raw material drug production strains based on morphological and physiological parameters.

[0010] The technical solution adopted in this invention is:

[0011] In a first aspect, the present invention provides a method for determining the optimal transplantation time of a raw material drug production strain based on morphological and physiological parameters, characterized in that the determination method is as follows:

[0012] S1: During the seed tank culture stage of the active pharmaceutical ingredient production strain, physiological parameters were continuously monitored, and samples were taken periodically for morphological analysis of the bacterial cells. The physiological parameters were dissolved oxygen concentration and pH value, and the morphological indicators included average hyphal length, hyphal branching frequency, and fullness.

[0013] S2: Perform trend analysis on the physiological parameters extracted in step S1, and extract the first physiological signal point T_p and the second physiological signal point T_pH;

[0014] S3: Quantify and synthesize the morphological indicators extracted in step S1 to obtain the morphological composite index, thereby determining the morphological signal point T_m;

[0015] S4: Statistically analyze the percentage of vacuoles and autolytic hyphae in the bacterial strains used to produce the active pharmaceutical ingredient. The ratio of the percentage of vacuoles to the percentage of autolytic hyphae is the health status of the bacterial cells.

[0016] S5: The optimal transplantation time point is determined when all of the following conditions are met: the first physiological signal point T_p and the second physiological signal point T_pH have both appeared; the morphological comprehensive index is in the peak plateau phase; the health of the bacterial cells is lower than the health threshold γ; and the three time points T_p, T_pH and T_m are within a reasonable time interval Δt.

[0017] In this invention, the hyphal branching frequency is defined as the number of effective branches generated on a unit length of main hyphae, which is obtained by observing under a microscope and calculating "the total number of observed branches / the total length of observed main hyphae", with units of branches / micrometer or branches / millimeter. Effective branches refer to lateral branches whose length exceeds twice the diameter of the main hyphae.

[0018] In this invention, fullness is defined as an index characterizing the fullness and uniformity of protoplasm within hyphal cells. It is obtained by analyzing hyphal segments through microscopic image analysis, assigning a grade, and calculating the average value. The grade is set according to the state of cytoplasmic filling. For example, 3 points are given for completely filled and uniformly dense hyphal cytoplasm; 2 points are given for basically filled but with slight local shrinkage; and 1 point is given for significant cytoplasmic shrinkage, the presence of vacuoles, or large areas of unevenness. Fullness is ultimately expressed as a dimensionless average score.

[0019] Among them, the peak plateau period is a flat phase in which the curve of the morphological composite index grows to near its maximum value and remains at a high level for a certain period of time without significant fluctuations.

[0020] The vacuolar percentage refers to the percentage of transparent vacuolar areas within a single bacterial cell that, under instrumental observation, occupy the total projected area of ​​the cell. This can be observed using an optical microscope or electron microscope, and the average value of the vacuolar area within a selected region can be calculated using image analysis software.

[0021] The autolytic hyphae ratio refers to the proportion of hyphae that undergo autolysis within a specific field of view relative to the total number of hyphae. This is typically achieved using random sampling to calculate the ratio of autolytic hyphae to healthy hyphae across multiple fields of view.

[0022] Wherein, the first physiological signal point T_p is the starting moment when the dissolved oxygen concentration deviates from its original trajectory and enters a stable upward channel;

[0023] The second physiological signal point T_pH is the starting moment when the pH value deviates from its original change trajectory and enters a stable reverse change channel;

[0024] The morphological signal point T_m is the starting time point when the morphological composite index enters the peak plateau period;

[0025] The hyphal branching frequency is the number of effective branches produced per unit length of main hyphae, expressed as branches per micrometer or per millimeter. Effective branches refer to lateral branches whose length exceeds twice the diameter of the main hyphae.

[0026] The fullness is obtained by scoring the hyphal segments using image analysis and calculating the average value.

[0027] In some embodiments, the morphological composite index is obtained by calculating the weighted sum of various morphological indicators, the weighted product, the decision tree analysis, or the neural network decision.

[0028] In some embodiments, the morphological composite index is obtained by calculating a weighted sum of the individual morphological indicators.

[0029] In some embodiments, the weights of each morphological indicator, the health threshold γ, and Δt are obtained through data analysis of past production batches of the active pharmaceutical ingredient strain.

[0030] In some embodiments, the weights, health thresholds γ and Δt of each morphological indicator are obtained by regression analysis or machine learning analysis using data from past production batches of the active pharmaceutical ingredient strain.

[0031] In some embodiments, the active pharmaceutical ingredient is an antibiotic or a secondary metabolite produced by fermentation of filamentous fungi.

[0032] In some embodiments, the active pharmaceutical ingredient is selected from acarbose, moxicritin, or tobramycin.

[0033] Secondly, the present invention provides a method for culturing a strain for producing active pharmaceutical ingredients, comprising the judgment method described in the first aspect.

[0034] Thirdly, the present invention provides a system for determining the optimal transplantation time of a strain used in the production of active pharmaceutical ingredients, employing the determination method described in the first aspect, and comprising the following modules:

[0035] Data acquisition module: used to acquire dissolved oxygen concentration, pH value, average hyphal length, hyphal branching frequency, fullness, and cell health of the active pharmaceutical ingredient (API) production strain. At the same time, it calculates the weighted sum of average hyphal length, hyphal branching frequency, and fullness to obtain the morphological comprehensive index.

[0036] Data processing module: Processes the curve relationships between dissolved oxygen concentration, pH value, morphological comprehensive index and time for the raw material production strains;

[0037] Analysis module: Based on the curve relationship obtained by the data processing module, output the first physiological signal point T_p, the second physiological signal point T_pH, and the morphological signal point T_m;

[0038] Early warning module: When the first physiological signal point T_p and the second physiological signal point T_pH have both appeared, the morphological comprehensive index is in the peak plateau period, the health of the bacteria is lower than the system preset health threshold γ, and the three time points T_p, T_pH and T_m are within the reasonable time interval Δt, an early warning will be issued indicating that the optimal transplanting time point has been entered.

[0039] Fourthly, the present invention provides an apparatus comprising the system described in the third aspect for determining the optimal transplantation time of the active pharmaceutical ingredient strain.

[0040] The beneficial effects of this invention are:

[0041] Precise decision-making: The dual verification of physiological trends and peak morphology avoids the risk of misjudgment based on a single parameter.

[0042] High predictability: By analyzing the rate of change, trends can be captured early, allowing for transplantation at the "golden moment" when bacterial activity reaches its peak but has not yet declined.

[0043] Objective quantification: By quantifying morphology and setting clear thresholds, subjective human factors are eliminated, resulting in extremely high process reproducibility.

[0044] High applicability: The principle of this method is applicable to the production of raw materials for various filamentous bacteria fermentation, such as acarbose, moxifloxacin, and tobramycin.

[0045] Improved efficiency: Precision transplanting can shorten the fermentation cycle, increase the yield of raw materials, and bring significant economic benefits. Attached Figure Description

[0046] Figure 1 This is a diagram of the mycelial state of the batch transplanted at 19 hours in Example 1.

[0047] Figure 2 This is a diagram of the mycelial state of the batch transplanted at 22 hours in Example 1.

[0048] Figure 3 This is a diagram of the mycelial state of the batch transplanted at 15 hours in Example 1. Detailed Implementation

[0049] The following disclosure provides many different implementations or examples for different ways of implementing the present invention.

[0050] To achieve the scientific and precise capture of the optimal inoculation period for filamentous bacteria used in active pharmaceutical ingredients (API) production, and to ensure the high efficiency and stability of API fermentation production, this invention adopts the following technical solution:

[0051] A method for determining the optimal transplanting time of active pharmaceutical ingredient (API) production strains based on a combination of morphological and physiological parameters includes the following steps:

[0052] S1: Data Acquisition: During the seed tank culture process, online physiological parameters (DO, pH) and bacterial cell morphology images obtained by microscopic imaging were acquired in parallel.

[0053] S2: Physiological parameter trend feature identification

[0054] This stage aims to identify the metabolic characteristics of bacteria transitioning from the early to the middle and late stages of logarithmic growth, as detailed below:

[0055] Dissolved oxygen (DO) trend feature identification:

[0056] Case A (classic mode): If the dissolved oxygen concentration has a clear process of decreasing to a minimum point and then rising again, then when it rises continuously and steadily from the minimum point, and the absolute value of its rate of change exceeds the preset threshold α, it is recorded as the physiological characteristic point T_p.

[0057] Scenario B (Non-classical model): If the dissolved oxygen concentration does not show a clear minimum point, but rather hovers at a low level or slowly decreases, then when it deviates from its original trajectory and first enters and maintains a clear, stable upward trend (e.g., its rate of change turns from negative to positive and continuously exceeds the threshold α, or mathematical analysis determines that its slope has undergone a significant change), this is recorded as the physiological characteristic point T_p. This signifies that the bacterial cell's oxygen consumption rate begins to fall below its oxygen supply rate, reflecting a shift in basal metabolism.

[0058] pH trend feature identification:

[0059] Case A (classic mode): If the pH value has a clear process of decreasing to the lowest point (or rising to the highest point) and then rising (or falling back), then when it starts to change in the opposite direction continuously and stably from the extreme point, and its rate of change exceeds the preset threshold β, it is recorded as the physiological characteristic point T_pH.

[0060] Scenario B (Non-classical model): If the pH value does not show a clear extreme point, but is in a plateau or slow-changing phase, then when it deviates from its original trajectory and first enters and maintains a clear, stable reverse change channel (e.g., from steady to continuous increase, or from slow decrease to rapid increase, and its rate of change exceeds the threshold β), it is recorded as the physiological characteristic point T_pH. This signifies a fundamental shift in the metabolic pattern of bacterial acid / alkali production.

[0061] S3: Quantitative analysis of morphological parameters: Quantify hyphal length, branching frequency, and fullness from the image, calculate the comprehensive morphological index, and record the signal point T_m when it enters the peak plateau phase; at the same time, assess cell health (vacuolation / autolysis ratio).

[0062] S4: Joint judgment: When T_p and T_pH have appeared, the morphological comprehensive index is at its peak plateau, the cell health meets the standard (vacuolation / autolysis ratio < γ), and the time points of T_p, T_pH, and T_m are close (within the Δt time period), then it is determined that the optimal transplanting window has been entered.

[0063] Those skilled in the art can obtain the optimal values ​​for the aforementioned key thresholds α, β, γ, and Δt by performing regression analysis or machine learning analysis using relevant data from past high-yield batches of this strain.

[0064] In this invention, the hyphal branching frequency is defined as the number of effective branches produced on a unit length of main hyphae. It is obtained by observing under a microscope and calculating "the total number of observed branches / the total length of observed main hyphae", with units of branches / micrometer or branches / millimeter. Effective branches refer to lateral branches whose length exceeds twice the diameter of the main hyphae.

[0065] In this invention, fullness is defined as an index characterizing the fullness and uniformity of protoplasm within hyphal cells. It is obtained by analyzing hyphal segments through microscopic image analysis, assigning a grade, and calculating the average value. The grade is set according to the state of cytoplasmic filling. For example, 3 points are given for completely filled and uniformly dense hyphal cytoplasm; 2 points are given for basically filled but with slight local shrinkage; and 1 point is given for significant cytoplasmic shrinkage, the presence of vacuoles, or large areas of unevenness. Fullness is ultimately expressed as a dimensionless average score.

[0066] Due to the complex internal environment of the fermenter, stirring, aeration, and bubble impact on the electrodes may cause high-frequency noise interference in the raw physiological parameter data (DO and pH) collected by the DCS system. To prevent false triggering of judgments due to instantaneous fluctuations, the raw data needs to be smoothed and denoised before performing trend feature identification in step S2.

[0067] Specifically, a moving average filtering method or a Savitzky-Gore filtering method can be used. For example, within a set time window, the arithmetic mean of the data within that window is calculated as the effective value at the current time.

[0068] In addition, an outlier removal mechanism can be set up: when the rate of change at a certain moment instantly exceeds the physical limit (for example, the DO value changes by 50% within 1 minute), the system determines that the signal is abnormal (such as bubbles sticking to the wall), automatically removes the point and keeps the value of the previous moment until the signal returns to the normal range.

[0069] Example 1: Acarbose-producing bacteria (Actinoplanes)

[0070] 1) The culture conditions for the acarbose-producing bacterium (Actinoplanes) in this embodiment are as follows:

[0071] Temperature: 28.0±2.0℃, pH: 7.2±0.5, Airflow: 200~400 m³ / h 3 The stirring speed is 70~150 rpm, and the tank pressure is 0.05±0.01 MPa. The specific ingredients for the seed culture tank are shown in Table 1 below.

[0072] Table 1. Ingredients for 1.5 kL Seed Culture Tank

[0073]

[0074] 2) Data collection is as follows:

[0075] Physiological parameters: pH and dissolved oxygen (DO) data in the seed tank were recorded in real time at a frequency of once per minute through a distributed control system (DCS system).

[0076] Morphological parameters: Aseptic samples were taken every 3 hours and observed using a microscope equipped with a 100x objective lens and image analysis software. The average hyphal length, number of branches, and fullness index were calculated, and a comprehensive morphological index (weighted sum of the three) was generated. The operator also assessed the proportion of vacuolated hyphae.

[0077] 3) The key thresholds α, β, γ, and Δt are set as follows:

[0078] In this embodiment, the dissolved oxygen trend change threshold α = 0.05% / min, the pH trend change threshold β = 0.008 / min, the mycelial health threshold γ = 3%, and the multi-signal synchronization window Δt = 3 hours are set.

[0079] 4) The determination process is as follows:

[0080] Before 18 hours of culture, the instantaneous slope of DO remained negative (in a downward trend). At 18.3 hours, the instantaneous slope first turned positive and remained positive for the next 20 minutes, with the mean exceeding the threshold α. The system determined that a clear metabolic trend shift had occurred, and T_p was recorded at 18.5 hours.

[0081] The pH value started to decrease slowly and continuously from the initial value of 7.2. After about 16.5 hours of incubation, the rate of decrease in the pH curve slowed down significantly and tended to stabilize. The rate of change exceeded the threshold β, triggering T_pH.

[0082] Between 17 and 22 hours, the morphological composite index reached its peak and stabilized at a plateau. Microscopic observation showed robust hyphae with dense branching and a vacuolar percentage of <3%. T_m was at its peak plateau and met the health criteria. Simultaneously, the time difference between the three signal points was within 3 hours, meeting the preset conditions.

[0083] Decisions and Results:

[0084] The system issued an alarm after 19 hours of cultivation, indicating that the optimal inoculation window had been reached. The operator then performed inoculation on a portion of the mycelium. At this point, the mycelial state was as shown in the attached image. Figure 1 As shown;

[0085] Meanwhile, another portion was transplanted based on conventional experience (i.e., after 22 hours, when the inoculum concentration reached 28% and the pH was 6.8). The mycelial state at this time is shown in the attached image. Figure 2 As shown;

[0086] In addition, for another part, based on subjective judgment, after 15 hours of cultivation, the on-site operators observed that the dissolved oxygen (DO) curve changed from a continuous decline to slight fluctuation. At the same time, based on experience, they believed that the viscosity of the bacterial solution had begun to increase. To avoid "transplanting too late," they subjectively decided to transplant. At this time, the mycelial state is shown in the attached figure. Figure 3 As shown.

[0087] The batch subsequent fermentation period for the 19-hour group was 113 hours, with a fermentation potency of approximately 6402 mg / L; the batch subsequent fermentation period for the conventional empirical transfer (22-hour group) was 121 hours, with a final fermentation potency of approximately 5820 mg / L; and the batch subsequent fermentation period for the subjective judgment transfer (15-hour group) reached 128 hours, with a final fermentation potency of approximately 5360 mg / L.

[0088] The subsequent fermentation titers of the 19-hour transplanted batches were on average 10% higher than those transplanted based on experience, and on average 19% higher than those transplanted based on subjective judgment. The fermentation cycles were also shortened by approximately 8 hours and approximately 15 hours, respectively.

[0089] Example 2: Moxicidin producing bacteria (Streptomyces cyaneogriseus)

[0090] 1) The culture conditions for the moxicidin-producing bacterium (Streptomyces cyaneogriseus) in this embodiment are as follows:

[0091] A 3 kL seed culture vessel was used, with a temperature of 28.0±2.0℃, a pH of 6.5±1.5, and an air flow rate of 50~160 m³ / h. 3The stirring speed is 100~250 rpm, and the tank pressure is 0.01~0.08 MPa. The specific ingredients for the seed culture tank are shown in Table 2 below.

[0092] Table 2. Ingredients for 3 kL Seed Tank Culture Medium

[0093]

[0094] 2) Data collection is as follows:

[0095] Physiological parameters: pH and dissolved oxygen (DO) data in the seed tank were recorded in real time at a frequency of once per minute through a distributed control system (DCS system).

[0096] Morphological parameters: Aseptic samples were taken every 3 hours and observed using a microscope equipped with a 100x objective lens and image analysis software. The average hyphal length, number of branches, and fullness index were calculated, and a comprehensive morphological index (weighted sum of the three) was generated. The operator also assessed the proportion of vacuolated hyphae.

[0097] 3) The key thresholds α, β, γ, and Δt are set as follows:

[0098] The dissolved oxygen trend transition threshold was set to α = 0.15 % / min, the pH trend transition threshold to β = 0.012 pH / min, the mycelial health threshold to γ ​​= 3%, and the multi-signal synchronization window to Δt = 3 hours.

[0099] 4) The determination process is as follows:

[0100] After approximately 16 hours of incubation, dissolved oxygen (DO) stopped its continuous decline and began to turn positive, indicating a clear shift in metabolic trend. T_p was recorded at 16.5 hours.

[0101] After a period of rapid decline and stabilization, the pH began to rise after about 17 hours of incubation, with the rate of change exceeding the threshold β. T_pH was recorded as 17.5 hours.

[0102] From 16.5 hours onwards, the morphological composite index continuously increased. It reached its peak at 18-20 hours and stabilized at a plateau. During this stage, the average hyphal length and branching frequency were the highest, but spore differentiation had not yet begun in large quantities. This was recorded as the morphological characteristic point T_m=18 hours.

[0103] The time difference between the three signal points is within 3 hours, which meets the preset conditions.

[0104] The system issued an optimal transfer alert after 18 hours of cultivation. After the transfer was performed, the fermentation units of this batch of moxicidin increased by an average of 8% compared to batches using traditional concentration-based transfer methods, and batch-to-batch production stability was significantly enhanced.

[0105] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. A method for determining the optimal transplantation time of a raw material drug production strain based on morphological and physiological parameters, characterized in that, The determination method is as follows: S1: During the seed tank culture stage of the active pharmaceutical ingredient production strain, physiological parameters were continuously monitored, and samples were taken periodically for morphological analysis of the bacterial cells. The physiological parameters were dissolved oxygen concentration and pH value, and the morphological indicators included average hyphal length, hyphal branching frequency, and fullness. S2: Perform trend analysis on the physiological parameters extracted in step S1, and extract the first physiological signal point T_p and the second physiological signal point T_pH; S3: Quantify and synthesize the morphological indicators extracted in step S1 to obtain the morphological composite index, thereby determining the morphological signal point T_m; S4: Statistically analyze the percentage of vacuoles and autolytic hyphae in the bacterial strains used to produce the active pharmaceutical ingredient. The ratio of the percentage of vacuoles to the percentage of autolytic hyphae is the health status of the bacterial cells. S5: The optimal transplantation time point is determined when all of the following conditions are met: the first physiological signal point T_p and the second physiological signal point T_pH have both appeared; the morphological comprehensive index is in the peak plateau phase; the health of the bacterial cells is lower than the health threshold γ; and the three time points T_p, T_pH and T_m are within a reasonable time interval Δt. Wherein, the first physiological signal point T_p is the starting moment when the dissolved oxygen concentration deviates from its original trajectory and enters a stable upward channel; The second physiological signal point T_pH is the starting moment when the pH value deviates from its original change trajectory and enters a stable reverse change channel; The morphological signal point T_m is the starting time point when the morphological composite index enters the peak plateau period; The hyphal branching frequency is the number of effective branches produced per unit length of main hyphae, expressed as branches per micrometer or per millimeter. Effective branches refer to lateral branches whose length exceeds twice the diameter of the main hyphae. The fullness is obtained by scoring the hyphal segments using image analysis and calculating the average value.

2. The judgment method according to claim 1, characterized in that, The morphological composite index is obtained by calculating the weighted sum, product, decision tree output, or neural network determination of various morphological indicators.

3. The judgment method according to claim 2, characterized in that, The morphological composite index is obtained by calculating the weighted sum of various morphological indicators.

4. The determination method according to claim 1 or 3, characterized in that, The weights of each morphological indicator, the health threshold γ, and Δt were obtained through data analysis of past production batches of the active pharmaceutical ingredient strain.

5. The method for culturing the active pharmaceutical ingredient strain according to claim 4, characterized in that, The weights, health thresholds γ and Δt of each morphological indicator were obtained through regression analysis or machine learning analysis using data from past production batches of the active pharmaceutical ingredient strain.

6. The determination method according to claim 1, characterized in that, The active pharmaceutical ingredient is an antibiotic or secondary metabolite produced by fermentation of filamentous fungi.

7. The determination method according to claim 1, characterized in that, The active pharmaceutical ingredient is selected from acarbose, moxifloxacin, or tobramycin.

8. A method for culturing a strain used in the production of active pharmaceutical ingredients, characterized in that, It includes the determination method described in claim 1.

9. A system for determining the optimal transplantation time of a bacterial strain used in the production of active pharmaceutical ingredients, characterized in that, The determination method described in claim 1 includes the following modules: Data acquisition module: used to acquire dissolved oxygen concentration, pH value, average hyphal length, hyphal branching frequency, fullness, and cell health of the active pharmaceutical ingredient (API) production strain. At the same time, it calculates the weighted sum of average hyphal length, hyphal branching frequency, and fullness to obtain the morphological comprehensive index. Data processing module: Processes the curve relationships between dissolved oxygen concentration, pH value, morphological comprehensive index and time for the raw material production strains; Analysis module: Based on the curve relationship obtained by the data processing module, output the first physiological signal point T_p, the second physiological signal point T_pH, and the morphological signal point T_m; Early warning module: When the first physiological signal point T_p and the second physiological signal point T_pH have both appeared, the morphological comprehensive index is in the peak plateau period, the health of the bacteria is lower than the system preset health threshold γ, and the three time points T_p, T_pH and T_m are within the reasonable time interval Δt, an early warning will be issued indicating that the optimal transplanting time point has been entered.

10. An apparatus, characterized in that, The system comprising, as described in claim 9, for determining the optimal transplantation time of the active pharmaceutical ingredient strain.

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