Feeding method for producing mupirocin through fermentation of pseudomonas fluorescens

Abstract

The invention provides a material supplementing method for producing mupirocin through fermentation of pseudomonas fluorescens. The method specifically comprises the following steps: S1, preparing a material supplementing system: preparing material supplementing liquid, and adjusting the pressure difference between a material supplementing tank and a fermentation tank; s2, monitoring material supplementing trigger conditions: monitoring the dissolved oxygen content and the pH value in the fermentation tank in real time through a PLC control system; s3, executing a feeding program: starting a peristaltic pump to feed a feeding liquid in a quantitative feeding mode, and feeding ammonia water; and S4, dynamic feedback adjustment: controlling the flow of glucose supplement by adjusting the rotating speed of a peristaltic pump, controlling specific dissolved oxygen saturation in different fermentation stages by adjusting the ventilation quantity and the stirring rotating speed, and controlling specific pH values in different fermentation stages by supplementing ammonia water. According to the present invention, by designing the material supplementing liquid with the specific raw materials and the specific ratio, adopting the peristaltic pump to quantitatively supplement the material, and detecting and controlling the glucose content, the dissolved oxygen and the pH value during the fermentation process, the strain can grow and metabolize in the stable environment, the improvement of the mupirocin yield is promoted, the fermentation period is shortened, and the energy consumption is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation production, and particularly relates to a feeding method for producing mupirocin by fermentation with Pseudomonas fluorescens. Background Art

[0002] Skin infections are a common and recurring disease in current medicine. They are caused by a wide variety of pathogens, with Gram-positive cocci being particularly prevalent, significantly impacting patients' health and quality of life. Mupirocin, a topical antibiotic, demonstrates significant advantages in treating these infections.

[0003] Mupirocin is highly sensitive to various Gram-positive cocci associated with skin infections, particularly Staphylococci and Streptococci, and is effective even against drug-resistant Staphylococcus aureus. It also exhibits some antibacterial activity against certain Gram-negative bacteria. Importantly, mupirocin exhibits no cross-resistance with other antibiotics, effectively preventing treatment failures due to drug resistance in clinical applications.

[0004] From a mechanism of action perspective, mupirocin is an inhibitor of aminoacyl-tRNA synthetase (AaRS) and is currently the only marketed bacterial AaRS inhibitor. It selectively inactivates bacterial isoleucyl-tRNA synthetase (AaRS). These enzymes play a vital role in protein biosynthesis by catalyzing the synthesis of aminoacyl-tRNA (aa-rRNA). Once inhibited, protein biosynthesis is forced to terminate, leading to slower bacterial growth. Therefore, these enzymes are targets for antibacterial drugs.

[0005] In clinical applications, mupirocin is suitable for a variety of bacterial skin infections, primarily for the treatment of primary skin infections caused by Gram-positive cocci, such as impetigo, furuncle, and folliculitis. Mupirocin is also effective in treating secondary skin infections such as eczema, ulcers, and wounds. It can also prevent the aggravation of the primary disease, creating favorable conditions for its treatment. Reports have demonstrated that mupirocin is highly effective in reducing suppuration in wounds following dermatologic surgery, whether administered prophylactically or therapeutically.

[0006] It can be seen that mupirocin is of great significance in clinical treatment. In the existing technology, mupirocin is mainly produced by fermentation of genetically engineered Pseudomonas fluorescens. However, the existing fermentation process has the following two major technical bottlenecks: (1) The control of the fermentation process conditions is complex and harsh, and a small fluctuation can lead to a significant decrease in yield; (2) Feeding is a key link in maintaining efficient fermentation, but the existing feeding process is imperfect and lacks a precise control strategy, resulting in a mismatch between nutrient supply and bacterial growth requirements, which in turn affects the stability of fermentation and is difficult to meet the needs of industrial production.

[0007] Therefore, developing a new mupirocin fermentation method that is suitable for industrial large-scale production, simple to control, and easy to execute is an important issue that needs to be solved urgently by those skilled in the art. Summary of the Invention

[0008] The present invention aims to provide a fed-batch method for the fermentation production of mupirocin by Pseudomonas fluorescens. Through a precise control strategy and a simple, easy-to-implement operation method, a high-quality fermentation environment is provided for Pseudomonas fluorescens, maximizing the yield and quality of mupirocin and ensuring the stability of the fermentation process.

[0009] To achieve the above object, the present invention provides a fed-batch method for producing mupirocin by fermentation with Pseudomonas fluorescens, comprising the following steps:

[0010] S1 feeding system preparation, including: preparing feeding solution, adjusting the pressure difference between feeding tank and fermentation tank;

[0011] S2 Feeding trigger condition monitoring: The PLC control system monitors the dissolved oxygen content and pH value in the fermentation tank in real time. When conditions A and B are met at the same time, feeding is started;

[0012] Condition A is that the dissolved oxygen content increases by more than 20% within 10 minutes and reaches 70% saturation;

[0013] The condition B is that the pH value rises by more than 0.1 within 10 minutes;

[0014] S3 feeding program execution includes: starting the peristaltic pump to feed the feeding liquid in a quantitative feeding mode, and adding ammonia water with a mass concentration of 5-25% to adjust the pH;

[0015] S4 dynamic feedback regulation, including: feed control, dissolved oxygen control and pH control;

[0016] The feeding control method specifically includes: controlling the flow rate of glucose feeding by adjusting the rotation speed of the peristaltic pump so that the glucose concentration in the tank is maintained at 1-5g / L throughout the fermentation process;

[0017] The dissolved oxygen control method specifically includes: controlling different fermentation stages by adjusting the ventilation volume and the speed of the peristaltic pump to maintain a specific dissolved oxygen saturation in the tank;

[0018] The pH control method specifically includes: controlling different fermentation stages by adding ammonia water to maintain a specific pH value in the tank.

[0019] In a preferred embodiment, in step S1, the components and mass concentrations of the prepared feed solution include: 40-70% glucose, 0.05-0.07% magnesium sulfate, 0.03-0.08% potassium dihydrogen phosphate, 0.03-0.08% zinc sulfate, 0.001-0.004% cobalt chloride, and 0.2-0.4% folate.

[0020] In the feed solution provided by the present invention, glucose is used as the core carbon source, magnesium sulfate provides Mg 2+ , potassium dihydrogen phosphate provides phosphorus source and K + , zinc sulfate provides Zn 2+ , cobalt chloride provides Co 2+ , foaming enemy as a defoaming agent, and the balance is water. In terms of ratio, the feed liquid of the present invention limits the ultra-high concentration of glucose, which can reduce the feed volume and avoid excessive dilution of the culture solution in the fermentation tank. At the same time, it reduces the operational burden and risk of contamination due to frequent feeding. Trace inorganic salts achieve precise guidance of microbial metabolism and avoid nutritional imbalance. In addition, the addition of a high proportion of foaming enemy can avoid the problem of foam surge caused by high-sugar feeding, and can also avoid the risk of liquid escape, dissolved oxygen decrease and contamination caused by foam. Therefore, the present invention limits the aforementioned components and proportions.

[0021] In a preferred embodiment, in step S1, after the preparation of the feed solution is completed, sterilization treatment is further included. The sterilization treatment method can adopt the common knowledge mastered by those skilled in the art, such as steam moist heat sterilization at 0.1 MPa and 121° C. for 20 minutes.

[0022] In a preferred embodiment, in step S1, after adjusting the pressure difference between the feeding tank and the fermentation tank, the tank pressure of the feeding tank is 0.05-0.15 MPa, and the tank pressure of the fermentation tank is 0.03-0.05 MPa; preferably, after adjusting the pressure difference between the feeding tank and the fermentation tank, the tank pressure of the feeding tank is 0.1 MPa, and the tank pressure of the fermentation tank is 0.03 MPa.

[0023] In the present invention, by adjusting the pressure difference between the feed tank and the fermentation tank, a unidirectional pressure gradient is established, thereby achieving the following objectives: (1) the pressure difference forms a physical barrier to isolate external contamination: the feed liquid can only flow from the high-pressure feed tank to the low-pressure fermentation tank, completely blocking the reverse entry of microorganisms or gases in the fermentation tank into the feed tank; (2) ensuring the precise controllability of the feed flow rate; and (3) reducing dissolved oxygen fluctuations to avoid fermentation tank decompression. As can be seen, in step S1 of the present invention, by optimizing the composition and ratio and the tank pressure gradient, the basic conditions for efficient feeding are established in a coordinated manner.

[0024] The feeding method designed by the present invention for producing mupirocin by fluorescing pseudomonas determines the feeding start time according to the change of dissolved oxygen, and starts feeding when the dissolved oxygen quickly rises to 70% and the pH starts to rise, thereby avoiding delaying feeding due to the inability to quickly detect the glucose concentration in the tank, resulting in sugar deficiency in fermentation metabolism and foam generation; at the same time, a peristaltic pump is used for quantitative feeding, which controls the feeding flow dosage more accurately, allowing the bacteria to grow and metabolize in a more stable environment, thereby achieving high bacteria conversion rates and mupirocin conversion rates, fast bacteria metabolism, shortened fermentation cycle, and reduced energy consumption.

[0025] In a preferred embodiment, in step S4, during the feeding control, the glucose concentration in the tank is measured every 1-5 hours, and the speed of the peristaltic pump is adjusted according to the measurement result.

[0026] Preferably, in step S4, during the feeding control,

[0027] When the glucose concentration in the measuring tank is below 1g / L, increase the speed of the peristaltic pump to make the glucose concentration in the tank reach 1.5-3.0g / L;

[0028] When the glucose concentration in the measuring tank is between 1.5-3.0 g / L, maintain the current peristaltic pump speed;

[0029] When the glucose concentration in the measuring tank is between 3.0-5.0 g / L, reduce the peristaltic pump speed;

[0030] When the glucose concentration in the measuring tank is above 5.0 g / L, stop feeding.

[0031] In a preferred embodiment, in step S4, controlling different fermentation stages to maintain a specific dissolved oxygen saturation in the tank specifically includes: from the 0th to the 24th hour of fermentation, the dissolved oxygen in the tank is 30-50%; from the 24th hour of fermentation to the time of tank release, the dissolved oxygen in the tank is 20-40%.

[0032] Preferably, in step S4, the dissolved oxygen in the tank is 35% from the 0th to the 24th hour of fermentation; and the dissolved oxygen in the tank is 25% from the 24th hour of fermentation to the time of tank release.

[0033] In a preferred embodiment, in step S4, the control of different fermentation stages to maintain a specific pH value in the tank specifically includes: natural fermentation from 0 to 10 hours of fermentation without adjusting the pH in the tank; adding ammonia water at a uniform rate from 10 to 14 hours of fermentation so that the pH in the tank reaches 5.8 at the 14th hour of fermentation; and from the 14th hour of fermentation to the time of tank release, the pH in the tank is 5.6-6.0.

[0034] Another object of the present invention is to provide mupirocin produced by any one of the above-mentioned Pseudomonas fluorescens fermentation fed-batch methods.

[0035] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0036] 1. The present invention designs a feeding method for the fermentation production of mupirocin by Pseudomonas fluorescens. When monitoring the feeding trigger conditions, the dissolved oxygen content and pH in the fermentation tank are monitored in real time by a PLC control system. When the following conditions are met simultaneously: the dissolved oxygen content increases by more than 20% within 10 minutes and reaches 70% saturation, and the pH value increases by more than 0.1 within 10 minutes, feeding is immediately started. This avoids delays in feeding due to the inability to quickly detect the glucose concentration in the tank, resulting in sugar deficiency in fermentation metabolism and the generation of foam.

[0037] 2. The present invention adopts a peristaltic pump for quantitative feeding, which can more accurately control the amount of feeding flow. The bacteria grow and metabolize in a more stable environment. A lower glucose feeding solution can promote the improvement of the bacteria conversion rate and mupirocin conversion rate. The bacteria metabolism rate is fast, the fermentation cycle is shortened, and the energy consumption and raw material costs are reduced.

[0038] 3. The present invention controls the glucose concentration in the fermentation tank at 1-5 g / L (preferably 1.5-3.0 g / L) and stably controls the pH at 5.8, which can greatly reduce the occurrence of fermentation side reactions, improve the quality of the fermentation liquid, reduce the difficulty of post-extraction, and increase the extraction yield.

[0039] 4. The present invention improves the efficiency of hormone production by adding trace elements to the fermentation feed solution, and promotes the secretion of the secondary metabolite mupirocin by controlling the pH in a timely manner, thereby synergistically maintaining the potency growth and improving the final level. DETAILED DESCRIPTION

[0040] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in detail below in conjunction with specific embodiments. However, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0041] The technical solution of this application is described in detail below through specific embodiments:

[0042] Unless otherwise specified, the technical means used in the present invention are conventional means well known to those skilled in the art. The various raw materials, reagents, instruments, and equipment used in the present invention can be purchased commercially or prepared by existing methods. Unless otherwise specified, all reagents used in the present invention are of analytical grade. The Pseudomonas fluorescens used in the present invention is obtained from Henan Tianfang Pharmaceutical Co., Ltd., and the bubo used in the present invention is obtained from Wuhan Hengqiang Chemical Co., Ltd.

[0043] In the present invention, parts by weight may be weight units known in the art such as μg, mg, g, kg, etc., or multiples thereof, such as 1 / 10, 1 / 100, 10 times, 100 times, etc.

[0044] Example 1

[0045] A fed-batch method for producing mupirocin by fermentation with Pseudomonas fluorescens comprises the following steps:

[0046] (1) In a feeding tank, prepare a feeding solution with a specific mass concentration of 50% glucose, 0.05% magnesium sulfate, 0.03% potassium dihydrogen phosphate, 0.03% zinc sulfate, 0.002% cobalt chloride, 0.2% folate, and the balance water. The prepared feeding solution needs to be steam sterilized at 0.1 MPa and 121°C for 20 minutes;

[0047] (2) Adjust the tank pressure of the feed tank to 0.05 MPa and maintain the tank pressure of the fermentation tank at 0.03 MPa;

[0048] (3) Feeding trigger condition monitoring: The dissolved oxygen content and pH value in the fermentation tank are monitored in real time through the PLC control system. When conditions A and B are met at the same time, feeding is started;

[0049] Condition A is that the dissolved oxygen content increases by more than 20% within 10 minutes and reaches 70% saturation;

[0050] The condition B is that the pH value rises by more than 0.1 within 10 minutes;

[0051] (4) Execute the feeding program: start the peristaltic pump to feed the feeding liquid in a quantitative feeding mode, and add ammonia water with a mass concentration of 5% to adjust the pH. At the same time, the feeding program is dynamically feedback-regulated by feeding control, dissolved oxygen control, and pH control, specifically including:

[0052] Measure the glucose concentration in the tank every 4 hours:

[0053] When the glucose concentration in the measuring tank is below 1g / L, increase the speed of the peristaltic pump to make the glucose concentration in the tank reach 2.0g / L;

[0054] When the glucose concentration in the measuring tank is 2.0 g / L, maintain the current peristaltic pump speed;

[0055] When the glucose concentration in the measuring tank is 4.0 g / L, reduce the peristaltic pump speed;

[0056] When the glucose concentration in the measuring tank is above 5.0 g / L, stop feeding.

[0057] When the feeding speed is increased, the dissolved oxygen in the tank will decrease. At this time, the dissolved oxygen in the tank can be adjusted by increasing the ventilation volume and stirring speed.

[0058] Control different fermentation stages and maintain a specific dissolved oxygen saturation in the tank, specifically: from 0 to 24 hours of fermentation, the dissolved oxygen in the tank is 30%; from the 24th hour of fermentation to the time of tank release, the dissolved oxygen in the tank is 20%.

[0059] Control the different fermentation stages to maintain a specific pH value in the tank. Specifically, during fermentation hours 0-10, natural fermentation occurs without adjusting the pH in the tank. The initial pH of the system is around 6.6, which gradually decreases after fermentation begins. During fermentation hours 10-14, ammonia is added at a constant rate to achieve a pH of 5.8 in the tank at the 14th hour. From the 14th hour of fermentation until the tank is released, the pH in the tank is 5.8.

[0060] After the fermentation is completed, the fermentation liquid is tested and the results are as follows:

[0061] By using the feeding method provided in this example, the mupirocin content in the fermentation broth of Pseudomonas fluorescens reached 7.0 g / L after 65 hours of fermentation.

[0062] Example 2

[0063] A fed-batch method for producing mupirocin by fermentation with Pseudomonas fluorescens comprises the following steps:

[0064] (1) In the feeding tank, prepare the feeding solution with a specific mass concentration of 55% glucose, 0.06% magnesium sulfate, 0.05% potassium dihydrogen phosphate, 0.05% zinc sulfate, 0.002% cobalt chloride, 0.3% folate, and the balance water. The prepared feeding solution needs to be steam sterilized at 0.1 MPa and 121°C for 20 minutes;

[0065] (2) Adjust the tank pressure of the feed tank to 0.1 MPa and maintain the tank pressure of the fermentation tank at 0.03 MPa;

[0066] (3) Feeding trigger condition monitoring: The dissolved oxygen content and pH value in the fermentation tank are monitored in real time through the PLC control system. When conditions A and B are met at the same time, feeding is started;

[0067] Condition A is that the dissolved oxygen content increases by more than 20% within 10 minutes and reaches 70% saturation;

[0068] The condition B is that the pH value rises by more than 0.1 within 10 minutes;

[0069] (4) Execute the feeding program: start the peristaltic pump to feed the feeding liquid in a quantitative feeding mode, and add ammonia water with a mass concentration of 20% to adjust the pH. At the same time, the feeding program is dynamically feedback-regulated by feeding control, dissolved oxygen control, and pH control, specifically including:

[0070] Measure the glucose concentration in the tank every 4 hours:

[0071] When the glucose concentration in the measuring tank is below 1g / L, increase the speed of the peristaltic pump to make the glucose concentration in the tank reach 1.5g / L;

[0072] When the glucose concentration in the measuring tank is 1.5 g / L, maintain the current peristaltic pump speed;

[0073] When the glucose concentration in the measuring tank is 3.0 g / L, reduce the speed of the peristaltic pump;

[0074] When the glucose concentration in the measuring tank is above 5.0 g / L, stop feeding.

[0075] When the feeding speed is increased, the dissolved oxygen in the tank will decrease. At this time, the dissolved oxygen in the tank can be adjusted by increasing the ventilation volume and stirring speed.

[0076] Control different fermentation stages and maintain a specific dissolved oxygen saturation in the tank, specifically: from 0 to 24 hours of fermentation, the dissolved oxygen in the tank is 40%; from the 24th hour of fermentation to the time of tank release, the dissolved oxygen in the tank is 25%.

[0077] Control different fermentation stages and maintain a specific pH value in the tank, specifically including: from 0 to 10 hours of fermentation, natural fermentation, no need to adjust the pH in the tank; from 10 to 14 hours of fermentation, uniform flow of ammonia water so that the pH in the tank reaches 5.8 at the 14th hour of fermentation; from the 14th hour of fermentation to the time of tank release, the pH in the tank is 5.8.

[0078] After the fermentation is completed, the fermentation liquid is tested and the results are as follows:

[0079] By using the feeding method provided in this example, the mupirocin content in the fermentation broth of Pseudomonas fluorescens reached 7.5 g / L after 65 hours of fermentation.

[0080] Example 3

[0081] A fed-batch method for producing mupirocin by fermentation with Pseudomonas fluorescens comprises the following steps:

[0082] (1) In a feeding tank, prepare a feeding solution with a specific mass concentration of 45% glucose, 0.05% magnesium sulfate, 0.04% potassium dihydrogen phosphate, 0.03% zinc sulfate, 0.002% cobalt chloride, 0.2% folate, and the balance water. The prepared feeding solution needs to be steam sterilized at 0.1 MPa and 121°C for 20 minutes;

[0083] (2) Adjust the tank pressure of the feed tank to 0.15 MPa and maintain the tank pressure of the fermentation tank at 0.05 MPa;

[0084] (3) Feeding trigger condition monitoring: The dissolved oxygen content and pH value in the fermentation tank are monitored in real time through the PLC control system. When conditions A and B are met at the same time, feeding is started;

[0085] Condition A is that the dissolved oxygen content increases by more than 20% within 10 minutes and reaches 70% saturation;

[0086] The condition B is that the pH value rises by more than 0.1 within 10 minutes;

[0087] (4) Execute the feeding program: start the peristaltic pump to feed the feeding liquid in a quantitative feeding mode, and add ammonia water with a mass concentration of 25% to adjust the pH. At the same time, the feeding program is dynamically feedback-regulated by feeding control, dissolved oxygen control, and pH control, specifically including:

[0088] Measure the glucose concentration in the tank every 4 hours:

[0089] When the glucose concentration in the measuring tank is below 1g / L, increase the speed of the peristaltic pump to make the glucose concentration in the tank reach 2.5g / L;

[0090] When the glucose concentration in the measuring tank is 2.5 g / L, maintain the current peristaltic pump speed;

[0091] When the glucose concentration in the measuring tank is 4.0 g / L, reduce the peristaltic pump speed;

[0092] When the glucose concentration in the measuring tank is above 5.0 g / L, stop feeding.

[0093] When the feeding speed is increased, the dissolved oxygen in the tank will decrease. At this time, the dissolved oxygen in the tank can be adjusted by increasing the ventilation volume and stirring speed.

[0094] Control different fermentation stages and maintain a specific dissolved oxygen saturation in the tank, specifically: from the 0th to the 24th hour of fermentation, the dissolved oxygen in the tank is 40%; from the 24th hour of fermentation to the time of tank release, the dissolved oxygen in the tank is 30%.

[0095] Control different fermentation stages and maintain a specific pH value in the tank, specifically including: from 0 to 10 hours of fermentation, natural fermentation, no need to adjust the pH in the tank; from 10 to 14 hours of fermentation, uniform flow of ammonia water so that the pH in the tank reaches 5.8 at the 14th hour of fermentation; from the 14th hour of fermentation to the time of tank release, the pH in the tank is 5.8.

[0096] After the fermentation is completed, the fermentation liquid is tested and the results are as follows:

[0097] By using the feeding method provided in this example, the mupirocin content in the fermentation broth of Pseudomonas fluorescens reached 8.0 g / L after 65 hours of fermentation.

[0098] Comparative Example 1

[0099] A fed-batch method for producing mupirocin by fermentation with Pseudomonas fluorescens comprises the following steps:

[0100] (1) In the feeding tank, prepare the feeding solution with a specific mass concentration of 45% glucose and the balance water. The prepared feeding solution needs to be steam sterilized at 0.1 MPa and 121°C for 20 minutes;

[0101] (2) Adjust the tank pressure of the feed tank to 0.15 MPa and maintain the tank pressure of the fermentation tank at 0.05 MPa;

[0102] (3) Feed trigger monitoring: When the reducing sugar concentration in the fermentation broth is below 15 g / L, sugar feeding is initiated. The reducing sugar concentration in the fermentation broth is measured every 4 hours. Based on the glucose consumption, glucose solution is fed once to maintain the glucose concentration in the fermentation broth within the range of 5-15 g / L. When the pH is below 5.5, 25% ammonia water is added to adjust the pH to 5.5-6.0.

[0103] When the feeding speed is increased, the dissolved oxygen in the tank will decrease. At this time, the dissolved oxygen in the tank can be adjusted by increasing the ventilation volume and stirring speed to control the dissolved oxygen to not less than 30%;

[0104] After the fermentation is completed, the fermentation liquid is tested and the results are as follows:

[0105] By using the feeding method provided in this example, the mupirocin content in the fermentation broth of Pseudomonas fluorescens reached 5.5 g / L after 65 hours of fermentation.

[0106] This comparative example uses a traditional intermittent feeding scheme, which is relatively extensive and causes large fluctuations in the fermentation process, which is not conducive to bacterial growth and metabolism, affecting yield. In addition, the fermentation liquid contains a high content of glucose, resulting in a waste of raw materials.

[0107] Comparative Example 2

[0108] A fed-batch method for producing mupirocin by fermentation with Pseudomonas fluorescens comprises the following steps:

[0109] (1) In the feeding tank, prepare the feeding solution with a specific mass concentration of 55% glucose and the balance being water. The prepared feeding solution needs to be steam sterilized at 0.1 MPa and 121°C for 20 minutes.

[0110] (2) Adjust the tank pressure of the feed tank to 0.1 MPa and maintain the tank pressure of the fermentation tank at 0.03 MPa;

[0111] (3) Feeding trigger condition monitoring: The dissolved oxygen content and pH value in the fermentation tank are monitored in real time through the PLC control system. When conditions A and B are met at the same time, feeding is started;

[0112] Condition A is that the dissolved oxygen content increases by more than 20% within 10 minutes and reaches 70% saturation;

[0113] The condition B is that the pH value rises by more than 0.1 within 10 minutes;

[0114] (4) Execute the feeding program: start the peristaltic pump to feed the feeding liquid in a quantitative feeding mode, and add ammonia water with a mass concentration of 20% to adjust the pH. At the same time, the feeding program is dynamically feedback-regulated by feeding control, dissolved oxygen control, and pH control, specifically including:

[0115] Measure the glucose concentration in the tank every 4 hours:

[0116] When the glucose concentration in the measuring tank is below 1g / L, increase the speed of the peristaltic pump to make the glucose concentration in the tank reach 1.5g / L;

[0117] When the glucose concentration in the measuring tank is 1.5 g / L, maintain the current peristaltic pump speed;

[0118] When the glucose concentration in the measuring tank is 3.0 g / L, reduce the speed of the peristaltic pump;

[0119] When the glucose concentration in the measuring tank is above 5.0 g / L, stop feeding.

[0120] When the feeding speed is increased, the dissolved oxygen in the tank will decrease. At this time, the dissolved oxygen in the tank can be adjusted by increasing the ventilation volume and stirring speed.

[0121] Control different fermentation stages and maintain a specific dissolved oxygen saturation in the tank, specifically: from 0 to 24 hours of fermentation, the dissolved oxygen in the tank is 40%; from the 24th hour of fermentation to the time of tank release, the dissolved oxygen in the tank is 25%.

[0122] Control different fermentation stages and maintain a specific pH value in the tank, specifically including: from 0 to 10 hours of fermentation, natural fermentation, no need to adjust the pH in the tank; from 10 to 14 hours of fermentation, uniform flow of ammonia water so that the pH in the tank reaches 5.8 at the 14th hour of fermentation; from the 14th hour of fermentation to the time of tank release, the pH in the tank is 5.8.

[0123] After the fermentation is completed, the fermentation liquid is tested and the results are as follows:

[0124] By using the feeding method provided in this example, the mupirocin content in the fermentation broth of Pseudomonas fluorescens reached 6.5 g / L after 65 hours of fermentation.

[0125] Compared with Comparative Example 1, this comparative example enables timely feeding, avoiding delays in feeding due to the inability to quickly detect glucose concentration in the tank, which can lead to sugar deficiency during fermentation and foaming. The use of a peristaltic pump for quantitative feeding allows for more precise control of feed flow, allowing the bacterial strain to grow and metabolize in a more stable environment. This results in high bacterial and mupirocin conversion rates, rapid bacterial metabolism, a shortened fermentation cycle, and reduced energy consumption. However, the lack of trace element addition compromises mupirocin production efficiency.

[0126] Comparative Example 3

[0127] A fed-batch method for producing mupirocin by fermentation with Pseudomonas fluorescens comprises the following steps:

[0128] (1) In a feeding tank, prepare a feeding solution with a specific mass concentration of 45% glucose, 0.05% magnesium sulfate, 0.04% potassium dihydrogen phosphate, 0.03% zinc sulfate, 0.002% cobalt chloride, 0.2% folate, and the balance water. The prepared feeding solution needs to be steam sterilized at 0.1 MPa and 121°C for 20 minutes;

[0129] (2) Adjust the tank pressure of the feed tank to 0.15 MPa and maintain the tank pressure of the fermentation tank at 0.05 MPa;

[0130] (3) Feed trigger monitoring: Sugar feeding is initiated when the reducing sugar concentration in the fermentation broth is below 15 g / L. The reducing sugar concentration in the fermentation broth is measured every 4 hours. Based on the glucose consumption, glucose solution is fed once to maintain the glucose concentration in the fermentation broth within the range of 5-15 g / L. When the pH is below 5.5, 25% ammonia water is fed to adjust the pH to 5.5-6.0.

[0131] When the feeding speed is increased, the dissolved oxygen in the tank will decrease. At this time, the dissolved oxygen in the tank can be adjusted by increasing the ventilation volume and stirring speed to control the dissolved oxygen to not less than 30%;

[0132] After the fermentation is completed, the fermentation liquid is tested and the results are as follows:

[0133] By using the feeding method provided in this example, the mupirocin content in the fermentation broth of Pseudomonas fluorescens reached 6.7 g / L after 65 hours of fermentation.

[0134] Compared with Example 3, the feeding method of this comparative example is rough and not conducive to bacterial growth and metabolism, resulting in a 16% decrease in the mupirocin content in the fermentation broth, which seriously affects the production efficiency for pharmaceutical manufacturers.

[0135] In summary, the present invention reduces frequent feeding by designing a feeding solution with specific raw materials and a specific ratio, while achieving precise guidance of microbial metabolism and avoiding nutrient loss. The use of a peristaltic pump for quantitative feeding allows for more precise control of the feed flow rate. Simultaneously, glucose content, dissolved oxygen, and pH are monitored and controlled during the fermentation process, allowing the bacteria to grow and metabolize in a more stable environment. This promotes high mupirocin yields, accelerates bacterial metabolism, shortens the fermentation cycle, and reduces energy consumption.

[0136] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A fed-batch method for producing mupirocin by fermentation with Pseudomonas fluorescens, characterized in that: The following steps are involved: S1 feeding system preparation, including: preparing feeding solution, adjusting the pressure difference between feeding tank and fermentation tank; S2 Feeding trigger condition monitoring: The PLC control system monitors the dissolved oxygen content and pH value in the fermentation tank in real time. When conditions A and B are met at the same time, feeding is started; Condition A is that the dissolved oxygen content increases by more than 20% within 10 minutes and reaches 70% saturation; The condition B is that the pH value rises by more than 0.1 within 10 minutes; S3 feeding program execution includes: starting the peristaltic pump to feed the feeding liquid in a quantitative feeding mode, and adding ammonia water with a mass concentration of 5-25% to adjust the pH; S4 dynamic feedback regulation, including: feed control, dissolved oxygen control and pH control; The feeding control method specifically includes: controlling the flow rate of glucose feeding by adjusting the rotation speed of the peristaltic pump so that the glucose concentration in the tank is maintained at 1-5g / L throughout the fermentation process; The dissolved oxygen control method specifically includes: controlling different fermentation stages by adjusting the ventilation volume and stirring speed to maintain a specific dissolved oxygen saturation in the tank; The pH control method specifically includes: controlling different fermentation stages by adding ammonia water to maintain a specific pH value in the tank.

2. The fed-batch method for producing mupirocin by fermentation with Pseudomonas fluorescens according to claim 1, wherein: In step S1, the components and mass concentrations of the prepared feeding solution include: 40-70% glucose, 0.05-0.07% magnesium sulfate, 0.03-0.08% potassium dihydrogen phosphate, 0.03-0.08% zinc sulfate, 0.001-0.004% cobalt chloride, and 0.2-0.4% folate.

3. The fed-batch method for producing mupirocin by fermentation with Pseudomonas fluorescens according to claim 1, wherein: In step S1, after adjusting the pressure difference between the feeding tank and the fermentation tank, the tank pressure of the feeding tank is 0.05-0.15 MPa, and the tank pressure of the fermentation tank is 0.03-0.05 MPa.

4. The fed-batch method for producing mupirocin by fermentation with Pseudomonas fluorescens according to claim 1, wherein: In step S4, during the feeding control, the glucose concentration in the tank is measured every 1-5 hours, and the rotation speed of the peristaltic pump is adjusted according to the measurement result.

5. The fed-batch method for producing mupirocin by fermentation with Pseudomonas fluorescens according to claim 4, wherein: In step S4, during the feeding control, When the glucose concentration in the measuring tank is below 1g / L, increase the speed of the peristaltic pump to make the glucose concentration in the tank reach 1.5-3.0g / L; When the glucose concentration in the measuring tank is between 1.5-3.0 g / L, maintain the current peristaltic pump speed; When the glucose concentration in the measuring tank is between 3.0-5.0 g / L, reduce the peristaltic pump speed; When the glucose concentration in the measuring tank is above 5.0 g / L, stop feeding.

6. The fed-batch method for producing mupirocin by fermentation with Pseudomonas fluorescens according to claim 1, wherein: In step S4, the different fermentation stages are controlled to maintain a specific dissolved oxygen saturation in the tank, specifically including: from the 0th to the 24th hour of fermentation, the dissolved oxygen in the tank is 30-50%; from the 24th hour of fermentation to the time of tank release, the dissolved oxygen in the tank is 20-40%.

7. The fed-batch method for producing mupirocin by fermentation with Pseudomonas fluorescens according to claim 1, wherein: In step S4, the control of different fermentation stages to maintain a specific pH value in the tank specifically includes: natural fermentation from 0 to 10 hours of fermentation without adjusting the pH in the tank; adding ammonia water at a uniform rate from 10 to 14 hours of fermentation so that the pH in the tank reaches 5.8 at the 14th hour of fermentation; and from the 14th hour of fermentation to the time of tank discharge, the pH in the tank is 5.6-6.

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8. Mupirocin produced by the Pseudomonas fluorescens fed-batch fermentation method according to any one of claims 1 to 7.