Method for improving fermentation production level of abamectin

Abstract

The invention discloses a method for improving the fermentation production level of abamectin, and belongs to the technical field of pesticide product production. The invention relates to a method for improving the fermentation production level of abamectin, which comprises the following steps: in the fermentation process, monitoring hypha morphology and dissolved oxygen change, placing 5% of the total volume of a main fermentation tank in batches during 90-200 hours, and supplementing an isometric fresh culture medium after placing; transferring the substitute liquid to a substitute tank for continuous culture, and finally combining and extracting in the main fermentation tank and the substitute tank. According to the method for improving the fermentation production level of the abamectin, the mycelium form and the dissolved oxygen value are monitored in real time, and the placing opportunity is dynamically adjusted by adopting weighted comprehensive scoring, so that the mycelium can still keep a relatively good growth state in the later fermentation period, the aging autolysis phenomenon is delayed, and the yield of the abamectin is improved. 5% of the total volume of the fresh culture medium is placed in batches, so that the problem of drastic change of the components of the culture medium due to overlarge single-time placing amount is avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of pesticide product production, and specifically to a method for improving the fermentation production level of avermectin. Background Art

[0002] Currently, the industrial fermentation production of avermectin mainly faces the following problems: imbalance between mycelial growth and metabolism: in the traditional single-batch fermentation process, the mycelium is prone to senescence and autolysis in the later stage of growth, resulting in a decline in the ability to produce antibiotics; dissolved oxygen limitation: during high-density fermentation, the mycelial clumps increase, resulting in insufficient dissolved oxygen (DO), affecting the growth of the mycelium and the synthesis of products; large fluctuations in the titer at the end of fermentation: when the single batch discharge volume is too large, the composition of the culture medium changes violently, affecting the metabolic stability of the mycelium and resulting in a stagnation or even decline in the titer. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for improving the fermentation production level of avermectin. By real-time monitoring of the mycelial morphology and dissolved oxygen value, and dynamically adjusting the discharge time through weighted comprehensive scoring, the mycelium can still maintain a good growth state in the later stage of fermentation, solving the problems raised in the above background art.

[0004] To achieve the above purpose, the present invention provides the following technical solution: A method for improving the fermentation production level of avermectin, including the following steps: During the fermentation process, monitor the changes in mycelial morphology and dissolved oxygen. During the period of 90 - 200 hours in the main fermentation tank, batch-wise discharge 5% of the total volume, and replenish an equal volume of fresh culture medium after discharge; transfer the discharged liquid to a discharge tank for continued cultivation, and finally combine and extract the contents in the main fermentation tank and the discharge tank.

[0005] Preferably, the discharge time is determined by the weighted comprehensive score of the mycelial morphology score and the dissolved oxygen value, where the weight coefficient is dynamically adjusted with the fermentation time.

[0006] Preferably, the mycelial morphology score includes three sub-indicators: aggregation degree, number of branches, and mycelial length. The scoring weight ratio of the three sub-indicators of aggregation degree, number of branches, and mycelial length is 5:3:2.

[0007] Preferably, the aggregation degree is detected online, using a probe-type particle analyzer and a laser scattering instrument to quantify the aggregation level, which is divided into three levels. Level 1: Mycelia are dispersed, no visible clusters, <50μm; Level 2: Small clusters, 50 - 100μm, 3 - 5 mycelia entangled; Level 3: Large clumps, >100μm, >10 mycelia entangled. The scores for levels 1 to 3 are 1.0, 0.6, and 0.2 respectively.

[0008] Preferably, the number of branches is analyzed by AI image analysis. The U-Net model is trained to segment the hyphal network, and more than 500 images are labeled to output the branch point density. If the number of branches per 100 μm ≥ 4, it indicates that the hyphae are growing vigorously and the score is 1.0. If the number of branches per 100 μm is 2 - 3, it indicates that the physiological state of the hyphae is normal, and the score = 0.5 + 0.25×(measured value - 2). If the number of branches per 100 μm < 2, it indicates that the hyphae are aging or nutrient-limited, and the score = 0.1×measured value.

[0009] Preferably, the hyphal length is detected by a Malvern particle size analyzer to measure the volume average length. The judgment criteria for the length of healthy hyphae are as follows: during the logarithmic phase, it is 150 - 300 μm, and during the antibiotic production phase, it is 50 - 150 μm, with an error range not exceeding ±15%. During the antibiotic production phase, according to the deviation degree of the measured length from the target interval, a piecewise linear function is used to calculate the score. The ideal length during the antibiotic production phase is 80 - 120 μm, and the score coefficient decreases in a gradient of 0.1 / 10 μm. When the hyphal length is between 50 - 150 μm, calculate according to , where L is the measured hyphal length, otherwise the score is 0.

[0010] Preferably, the dissolved oxygen monitoring method is as follows: at least 3 optical probes are axially arranged in the main fermenter, and the distance between each probe is not less than 20% of the tank diameter. After the data is filtered by the Kalman filter, multi-probe fusion is performed. The multi-probe fusion adopts the method of weighted comprehensive scoring, and the sum of all weights is 1 to ensure the rationality of the fusion result. The weight coefficient of the i-th probe is equal to the precision weight of the i-th probe divided by the sum of the weights of all probes. Among them, the precision weight of the i-th probe = 1 / σ i 2 , where σ i represents the standard deviation of the measurement data of the i-th probe. If the data of a certain probe exceeds the range of μ ± 3σ for 5 consecutive minutes, its weight is 0, where μ is the mean value of the remaining probes, and the dissolved oxygen value is calculated.

[0011] Preferably, during the period of 90 - 200 hours in the main fermenter, when the fermentation time is 90 - 120 h, the weight ratio of hyphal morphology to dissolved oxygen change is (2 - 3):(7 - 8). The change of dissolved oxygen in the early stage dominates the prevention of premature senescence. When it is 120 - 150 h, the weight ratio of hyphal morphology to dissolved oxygen change is (5 - 6):(4 - 5). During the antibiotic production phase, the health of the hyphae is concerned. When it is 150 - 200 h, the weight ratio of hyphal morphology to dissolved oxygen change is (7 - 8):(2 - 3). In the later stage, delaying autolysis is prioritized.

[0012] Preferably, the substitute discharging tank is a sterile fermentation tank that has been repaired, sterilized, and maintained under pressure. Connect the substitute discharging tank to the main fermentation tank through a pipeline. The conditions for sterilizing the substitute discharging tank are as follows: at a temperature of 120 - 125°C, a tank pressure of 0.1 - 0.14 MP, and maintaining for 15 - 20 minutes. After sterilization, keep the pressure inside the tank at 0.1 - 0.15 MP. The substitute discharging tank uses a differential medium containing sodium butyrate to enable low-activity mycelia to restore their antibiotic-producing ability.

[0013] Preferably, the fresh medium includes nutrients at the following concentrations: 15 g / L glucose, 3 g / L ammonium sulfate, 0.5 g / L sodium propionate, and a trace element solution. The trace element solution includes Co2+ or Mn2+, and 5 mL of the trace element solution is added per liter of fresh medium.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] A method for improving the fermentation production level of avermectin in the present invention, by real-time monitoring the mycelial morphology and dissolved oxygen value, and dynamically adjusting the substitute discharging time by using a weighted comprehensive score, enables the mycelia to still maintain a good growth state in the later stage of fermentation, delays the occurrence of senescence and autolysis phenomena, and batches the substitution of 5% of the total volume of fresh medium, avoiding the problem of drastic changes in the medium composition caused by excessive single-time substitution volume, ensuring the continuity and stability of nutrient supply during the fermentation process, reducing the impact on the metabolism of the bacteria, and dynamically adjusting the weight ratio of mycelial morphology and dissolved oxygen changes according to different stages of fermentation time, paying attention to preventing premature senescence in the early stage, focusing on the health of mycelia in the middle stage, and giving priority to delaying autolysis in the later stage. This refined management method greatly improves the overall efficiency of the fermentation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a flowchart of the method for improving the fermentation production level of avermectin in the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0017] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0018] To solve the problems in the existing traditional single-batch fermentation process, such as the easy senescence and autolysis of mycelia in the later stage of growth, resulting in a decrease in antibiotic-producing ability, insufficient dissolved oxygen, affecting the growth of bacteria and the synthesis of products, large fluctuations in the titer at discharging, and drastic changes in the medium composition when the single-time substitution volume is too large, affecting the metabolic stability of the bacteria and resulting in a stagnant or even decreasing titer, please refer to Figure 1, this embodiment provides the following technical solutions:

[0019] A method for improving the fermentation production level of avermectin, comprising the following steps: during the fermentation process, monitor the mycelial morphology and dissolved oxygen changes. During the period of 90 - 200 hours in the main fermenter, batch-wise discharge 5% of the total volume, and after the discharge, supplement the same volume of fresh medium; transfer the discharged liquid to the discharge tank for continued cultivation, and finally combine and extract the contents in the main fermenter and the discharge tank.

[0020] Determine the discharge timing through the weighted comprehensive score of the mycelial morphology score and the dissolved oxygen value, where the weight coefficient is dynamically adjusted with the fermentation time. The mycelial morphology score includes three sub-indicators: degree of aggregation, number of branches, and mycelial length. The scoring weight ratio of the three sub-indicators of degree of aggregation, number of branches, and mycelial length is 5:3:2.

[0021] The degree of aggregation is detected online. Use a probe-type particle analyzer and a laser scattering instrument to quantify the aggregation level, which is divided into three levels. Level 1: Mycelia are dispersed, no visible clusters, <50μm; Level 2: Small clusters, 50 - 100μm, 3 - 5 mycelia intertwined; Level 3: Large clumps, >100μm, >10 mycelia entangled. The scores for levels 1 to 3 are 1.0, 0.6, and 0.2 respectively.

[0022] The number of branches is analyzed by AI image analysis. Train the U-Net model to segment the mycelial network, annotate more than 500 images, and output the branch point density. If the number of branches per 100μm ≥ 4, it indicates that the mycelia grow vigorously and the score is 1.0. If the number of branches per 100μm is 2 - 3, it indicates that the physiological state of the mycelia is normal, and the score = 0.5 + 0.25×(measured value - 2). If the number of branches per 100μm < 2, it indicates that the mycelia are aging or nutrient-limited, and the score = 0.1×measured value. Very few branches indicate mycelial aging or nutrient deficiency.

[0023]

[0024]

[0025] The mycelial length is detected by a Malvern particle size analyzer to measure the volume average length D[4,3]. The judgment standard for the length of healthy mycelia is: during the logarithmic phase, it is 150 - 300μm, and during the antibiotic production phase, it is 50 - 150μm, with an error range not exceeding ±15%. During the antibiotic production phase, according to the deviation degree of the measured length from the target interval, a piecewise linear function is used to calculate the score. The ideal length during the antibiotic production phase is 80 - 120μm, and the score coefficient decreases at a gradient of 0.1 / 10μm. When the mycelial length is between 50 - 150μm, according to Calculation: L is the measured hyphal length, otherwise the score is 0. For example, when the actual measured value is 110 μm, S = 1.0 - 0.1×1 = 0.9; when the actual measured value is 70 μm, S = 1.0 - 0.1×3 = 0.7.

[0026] The dissolved oxygen monitoring method is as follows: At least 3 optical probes are axially arranged in the main fermenter, and the distance between each probe is not less than 20% of the tank diameter. After the data is filtered by Kalman filter, multi-probe fusion is carried out. The multi-probe fusion adopts the method of weighted comprehensive scoring, and the sum of all weights is 1 to ensure the rationality of the fusion result. The weight coefficient of the i-th probe is equal to the precision weight of the i-th probe divided by the sum of the weights of all probes. Among them, the precision weight of the i-th probe = 1 / σ i 2 , σ i represents the standard deviation of the measurement data of the i-th probe. If the data of a certain probe exceeds the range of μ±3σ continuously for 5 minutes, its weight is 0, μ is the mean value of the remaining probes, and the dissolved oxygen value is calculated.

[0027] During the period of 90 - 200 hours in the main fermenter, when the fermentation time is 90 - 120 h, the weight ratio of hyphal morphology to dissolved oxygen change is (2 - 3):(7 - 8). In the early stage, the change of dissolved oxygen dominates the prevention of premature senescence. When the fermentation time is 120 - 150 h, the weight ratio of hyphal morphology to dissolved oxygen change is (5 - 6):(4 - 5). During the antibiotic production period, the health of hyphae is concerned. When the fermentation time is 150 - 200 h, the weight ratio of hyphal morphology to dissolved oxygen change is (7 - 8):(2 - 3). In the later stage, delaying autolysis is given priority. The threshold for triggering subculture is set as follows: ≥0.8: The system is healthy and no intervention is required; 0.6 - 0.8: Early warning, adjust process parameters, such as increasing agitation, preheating the pipeline, and preparing for subculture.

[0028] The subculture tank is a sterile fermenter that has been repaired, empty sterilized, and kept under pressure. The subculture tank is connected to the main fermenter through a pipeline. The empty sterilization conditions of the subculture tank are at a temperature of 120 - 125 °C, a tank pressure of 0.1 - 0.14 MP, and maintained for 15 - 20 minutes. After the empty sterilization is completed, the pressure in the tank is maintained at 0.1 - 0.15 MP. The subculture tank uses a differential medium containing sodium butyrate to restore the antibiotic production ability of low-activity hyphae.

[0029] The fresh medium includes the following concentrations of nutrients: 15 g / L glucose, 3 g / L ammonium sulfate, 0.5 g / L sodium propionate, and trace element solution. The trace element solution includes Co2+ or Mn2+, and 5 mL of the trace element solution is added to each liter of fresh medium.

[0030] Example 1:

[0031] Adopt 50 m 3Avermectin is fermented in a fermenter. During the fermentation process, the mycelial morphology and dissolved oxygen changes are monitored. In the main fermenter during the period of 90 - 200 hours, the trigger for sub - batch discharge: the comprehensive score < 0.65, and the weight ratio of the mycelial morphology score to the dissolved oxygen value is: 3:7 for 90 - 120h, 6:4 for 120 - 150h, 8:2 for 150 - 200h; 5% of the total volume is discharged in batches, and the total number of discharges is 4 times, at 105h, 135h, 165h, and 190h respectively. After discharge, an equal volume of fresh medium is added; the discharged liquid is transferred to a discharge tank for continuous cultivation, and finally, the contents of the main fermenter and the discharge tank are combined for extraction. In this example, the medium in the discharge tank includes 0.1 mM sodium butyrate + 3 g / L yeast powder.

[0032] Example Two:

[0033] Using a 50m 3 Avermectin is fermented in a fermenter. During the fermentation process, the mycelial morphology and dissolved oxygen changes are monitored. In the main fermenter during the period of 90 - 200 hours, the trigger for sub - batch discharge: the comprehensive score < 0.6, and the weight ratio of the mycelial morphology score to the dissolved oxygen value is: 3:7 for 90 - 120h, 6:4 for 120 - 150h, 8:2 for 150 - 200h; 5% of the total volume is discharged in batches, and the total number of discharges is 6 times, at 95h, 115h, 140h, 160h, 180h, and 195h respectively. After discharge, an equal volume of fresh medium is added; the discharged liquid is transferred to a discharge tank for continuous cultivation, and finally, the contents of the main fermenter and the discharge tank are combined for extraction. In this example, the medium in the discharge tank includes 0.08 mM sodium butyrate + 2 g / L yeast powder.

[0034] Example Three:

[0035] Using a 50m 3 Avermectin is fermented in a fermenter. During the fermentation process, the mycelial morphology and dissolved oxygen changes are monitored. In the main fermenter during the period of 90 - 200 hours, the trigger for sub - batch discharge: the comprehensive score < 0.55, and the weight ratio of the mycelial morphology score to the dissolved oxygen value is: 3:7 for 90 - 120h, 6:4 for 120 - 150h, 8:2 for 150 - 200h; 5% of the total volume is discharged in batches, and the total number of discharges is 3 times, at 110h, 150h, and 185h respectively. After discharge, an equal volume of fresh medium is added; the discharged liquid is transferred to a discharge tank for continuous cultivation, and finally, the contents of the main fermenter and the discharge tank are combined for extraction. In this example, the medium in the discharge tank includes 0.15 mM sodium butyrate + 5 g / L yeast powder.

[0036] Example Four:

[0037] Using a 50m 3Ferment avermectin in a fermenter. During the fermentation process, monitor the mycelial morphology and dissolved oxygen changes. The subculture starts after 120 h. Subculture trigger: comprehensive score < 0.5, delay subculture, subculture 5% of the total volume in batches. The total number of subcultures: 2 times, at 140 h and 175 h respectively. After subculture, add an equal volume of fresh medium; transfer the subculture liquid to the subculture tank for continued culture, and finally combine and extract the contents in the main fermenter and the subculture tank. In this example, the subculture tank medium includes 0.05 mM sodium butyrate + 1 g / L yeast powder.

[0038] Example 5:

[0039] Use a 50 m 3 Ferment avermectin in a fermenter. During the fermentation process, monitor the mycelial morphology and dissolved oxygen changes. In the main fermenter during the period of 90 - 200 hours, subculture trigger: comprehensive score < 0.6, the weight ratio of mycelial morphology score to dissolved oxygen value is: 2:8 for 90 - 120 h, 5:5 for 120 - 150 h, 7:3 for 150 - 200 h; subculture 5% of the total volume in batches. The total number of subcultures: 5 times, at 92 h, 122 h, 145 h, 168 h, and 192 h respectively. After subculture, add an equal volume of fresh medium; transfer the subculture liquid to the subculture tank for continued culture, and finally combine and extract the contents in the main fermenter and the subculture tank. In this example, the subculture tank medium includes 0.12 mM sodium butyrate + 4 g / L yeast powder.

[0040] Comparative Example 1:

[0041] Use a 50 m 3 Fermenter, during the fermentation process, no subculture operation, the same fermentation cycle of 200 h, conventional medium.

[0042] Carry out discharging for the above-mentioned examples and comparative examples to obtain the following data:

[0043]

[0044]

[0045] Compared with the comparative example, the total yield of avermectin is significantly increased through the batch subculture strategy in the examples. This is mainly due to better dissolved oxygen conditions and nutrient supplementation, enabling the mycelium to maintain a good growth state in the later stage of fermentation. The differences between different examples are mainly reflected in the selection of subculture timing, the number of subcultures, and the subculture tank medium formula. These factors jointly affect the final fermentation effect. Example 2 shows the best performance, not only because of its high subculture frequency and reasonable subculture timing selection, but also because of the relatively optimized subculture tank medium formula, which helps to further improve the fermentation efficiency in the subculture tank.

[0046] It should be noted that in this text, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprising", "including" or any other variant thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or elements inherent to such process, method, article or device.

[0047] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention.

Claims

1. A method for improving the fermentation production level of avermectin, characterized in that, It includes the following steps: During the fermentation process, monitor the mycelial morphology and dissolved oxygen changes. During the period of 90 - 200 hours in the main fermenter, batch-wise discharge 5% of the total volume in portions, and after the discharge, supplement with an equal volume of fresh medium; transfer the discharged liquid to the discharge tank for continued cultivation, and finally combine and extract the contents in the main fermenter and the discharge tank.

2. The method for improving the fermentation production level of avermectin according to claim 1, characterized in that: The timing of discharge is determined by the weighted comprehensive score of the mycelial morphology score and the dissolved oxygen value, where the weight coefficient is dynamically adjusted with the fermentation time.

3. The method for improving the fermentation production level of avermectin according to claim 2, characterized in that: The mycelial morphology score includes three sub-indicators: degree of aggregation, number of branches, and mycelial length. The scoring weight ratio of the three sub-indicators of degree of aggregation, number of branches, and mycelial length is 5:3:

2.

4. The method for improving the fermentation production level of avermectin according to claim 3, characterized in that: The degree of aggregation is divided into three grades. Grade 1: Mycelia are dispersed, no visible clusters, <50μm; Grade 2: Small clusters, 50 - 100μm, 3 - 5 mycelia intertwined; Grade 3: Large lumps, >100μm, >10 mycelia entangled. The scores for grades 1 to 3 are 1.0, 0.6, and 0.2 respectively.

5. The method for improving the fermentation production level of avermectin according to claim 3, wherein: The number of branches is classified as follows: If the number of branches per 100μm ≥ 4, it indicates vigorous mycelial growth and the score is 1.0; if the number of branches per 100μm is 2 - 3, it indicates normal mycelial physiological state, and the score = 0.5 + 0.25×(measured value - 2); if the number of branches per 100μm < 2, it indicates mycelial aging or nutrient limitation, and the score = 0.1×measured value.

6. The method for improving the fermentative production level of avermectin according to claim 3, wherein: When the hyphal length is between 50 - 150 μm, calculate according to , otherwise the score is 0, where L is the measured hyphal length.

7. The method for improving the fermentation production level of avermectin according to claim 2, characterized in that: The method for monitoring dissolved oxygen is as follows: Arrange at least 3 optical probes axially in the main fermenter, with the distance between each probe not less than 20% of the tank diameter. After the data is filtered by the Kalman filter, multi-probe fusion is performed, and the multi-probe fusion adopts the method of weighted comprehensive scoring.

8. The method for improving the fermentation production level of avermectin according to claim 2, characterized in that: During the period of 90 - 200 hours in the main fermenter, when the fermentation time is 90 - 120h, the weight ratio of mycelial morphology to dissolved oxygen change is (2 - 3):(7 - 8); when it is 120 - 150h, the weight ratio of mycelial morphology to dissolved oxygen change is (5 - 6):(4 - 5); when it is 150 - 200h, the weight ratio of mycelial morphology to dissolved oxygen change is (7 - 8):(2 - 3).

9. The method for improving the fermentation production level of avermectin according to claim 1, characterized in that: The discharge tank is a sterile fermenter that has been overhauled, empty-sterilized, and maintained under pressure. Connect the discharge tank to the main fermenter through a pipeline. The empty-sterilization conditions of the discharge tank are: at a temperature of 120 - 125°C, a tank pressure of 0.1 - 0.14MP, and maintain for 15 - 20 minutes. After the empty-sterilization is completed, keep the pressure in the tank at 0.1 - 0.15MP. The discharge tank uses a differential medium containing sodium butyrate to restore the anti-producing ability of low-activity mycelia.

10. The method for improving the fermentation production level of avermectin according to claim 1, characterized in that: The fresh medium includes nutrients with the following concentrations: 15g / L glucose, 3g / L ammonium sulfate, 0.5g / L sodium propionate, and trace element solution. The trace element solution includes Co2+ or Mn2+, and 5mL of the trace element solution is added to each liter of fresh medium.

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