A fermentation medium for producing mupirocin and a method for producing mupirocin

Abstract

The present application relates to a kind of fermentation medium of mupirocin and the production method of mupirocin.The present application improves fermentation medium and the production method of mupirocin, significantly improves the fermentation level of mupirocin, and when fermenting 5-7 days, the fermentation titer reaches 12000mg / L or more.The whole production process is cheap and easy to obtain, and the fermentation process is simple and easy to operate, which is beneficial to industrial production.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to a fermentation culture medium for producing mupirocin and a method for producing mupirocin. Background Technology

[0002] Mupirocin, also known as Pseudomonas aeruginosa acid A, is a topical antibacterial drug produced by the fermentation of Pseudomonas fluorescens. It can inhibit and kill a variety of bacteria that cause skin diseases and is suitable for skin diseases such as folliculitis, boils, and impetigo. It has the advantages of being easy to use, inexpensive, and having few side effects.

[0003] Currently, mupirocin is mainly produced through microbial fermentation. While there are numerous reports on the purification and preparation processes of mupirocin, reports on its fermentation production process are scarce. CN108277243A reports a method for producing pseudomonadic acid A by supplementing a mixture of linoleic acid and leucine during fermentation, along with the addition of ammonia and sodium citrate to control the fermentation pH to 5.5-5.8. Using this method, the final fermentation potency of pseudomonadic acid A reached 3462 mg / L. Although this method can improve the fermentation potency, the feeding and fermentation process control are complex and costly, and the final fermentation potency is still not ideal. CN108949845A reports a fermentation medium for preparing mupirocin and a method for preparing mupirocin from the fermentation medium. The fermentation medium in this report has complex materials and cumbersome operation, achieving a fermentation potency of approximately 6000 mg / L. CN111996136A reports a method for producing mupirocin by adding sodium molybdate and urea to a fermentation medium, achieving a fermentation potency of approximately 6600 mg / L. Although the fermentation potency is improved compared to the control group, considering that sodium molybdate may cause molybdenum poisoning and the operation process is relatively cumbersome, the fermentation potency of this method is still not ideal.

[0004] In view of the above situation, this invention is proposed. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a fermentation medium for producing mupirocin and a method for producing mupirocin. By improving the fermentation medium and the mupirocin production process, this invention significantly enhances the fermentation level of mupirocin, achieving a fermentation potency of over 12000 mg / L after 5-7 days of fermentation. Furthermore, the fermentation materials are inexpensive and readily available, and the fermentation process is simple and easy to operate, which is beneficial for industrial production.

[0006] The technical solution of the present invention is as follows:

[0007] This invention provides a fermentation medium for producing mupirocin, comprising: 80-120 g / L glucose, 20-60 g / L soybean meal, 10-30 g / L cottonseed meal, and 1-3 g / L ammonium sulfate; preferably, the fermentation medium comprises: 100 g / L glucose, 40 g / L soybean meal, 20 g / L cottonseed meal, and 1 g / L ammonium sulfate, and the pH value of the fermentation medium is 7.5 ± 0.1.

[0008] This invention also provides a method for producing mupirocin, comprising: using *Pseudomonas fluuorescens* HS-PF20-62 (accession number CGMCC No. 25307) as the production strain, and fermenting it using the fermentation medium described above. Preferably, the fermentation temperature is 21℃-23℃. Preferably, during the 48th-96th hour of fermentation, a feedstock is added in batches, characterized in that the feedstock is a glucose solution; preferably, the glucose solution is added in batches at the 48th and 96th hours of fermentation, with each addition of glucose solution being 4% of the original fermentation medium volume, and the glucose solution having a mass percentage concentration of 50%.

[0009] The microbial strain of *Pseudomonas fluorescens* HS-PF20-62 of this invention was deposited on July 15, 2022, at the China General Microbiological Culture Collection Center (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with accession number CGMCC No. 25307, classified and named *Pseudomonas fluorescens*, and registered, proving its viability.

[0010] As one embodiment of the present invention, the method for producing mupirocin is as follows:

[0011] Step (1) Activation of bacterial strain: Pseudomonas fluorescens HS-PF20-62, which was preserved at low temperature with the preservation number CGMCC No.25307, was inoculated onto a plate medium and cultured at 26±1℃ for 24±4 hours to obtain a single colony.

[0012] Step (2) Colony culture: Transfer the single colonies obtained in step (1) to a new plate medium by spot inoculation, with 10-12 single colonies on each plate; incubate at 26±1℃ for 3-6 days to obtain spot-inoculated single colonies;

[0013] The plate culture medium is composed of the following: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, agar 15 g / L, and the pH value of the plate culture medium is 7.5 ± 0.1.

[0014] Step (3) Preparation of seed culture in shake flask: Pick one single colony of the seed culture prepared in step (2) and inoculate it into the seed culture medium. Place the seed shake flask at 26±1℃ and shake at 250±10r / r / min for 28±4 hours.

[0015] The shake flask seed culture medium is composed of the following: 20 g / L glucose, 20 g / L soybean meal, 10 g / L yeast powder, and 2 g / L calcium carbonate. The pH value of the seed culture medium is 7.5 ± 0.1.

[0016] Shake flask seed culture medium volume: 20ml / 250ml;

[0017] Step (4) Shake flask fermentation culture: The seed liquid prepared in step (3) is transferred into the fermentation shake flask at a volume ratio of 5%. The volume of the fermentation medium for shake flask fermentation culture is 20ml / 250ml, the temperature of shake flask fermentation culture is 22±1℃, the shaking speed of fermentation shake flask is 250±10r / min, and the fermentation culture cycle is 5-7 days.

[0018] The fermentation shake flask culture medium is the fermentation culture medium for producing mupirocin as described above in this invention.

[0019] As a specific embodiment of the present invention, the method for producing mupirocin further includes:

[0020] In step (4) shake flask fermentation culture, the feed is added in batches from the 48th to the 96th hour of the fermentation culture. The feed is a glucose solution. Preferably, the glucose solution is added in batches at the 48th and 96th hours of the fermentation culture. The volume of the glucose solution added each time is 4% of the original fermentation culture medium volume, and the mass percentage concentration of the glucose solution is 50%.

[0021] The beneficial effects achieved by this invention are as follows:

[0022] (1). This invention optimizes and improves the existing mupirocin fermentation medium. By screening the components and optimizing the concentration of the medium, the fermentation medium material used is inexpensive and readily available, which greatly reduces the cost of fermentation raw materials.

[0023] (2). By controlling the culture temperature at each stage, not only was the fermentation potency improved, but the energy consumption cost in the production process was also reduced.

[0024] (3) Improve the existing fermentation method by adopting a feeding method and controlling the timing of feeding to further improve the fermentation level. The fermentation process is simple and easy to operate.

[0025] (4). The production method described in this invention is simple, low-cost, and can greatly improve the fermentation level of mupirocin. Detailed Implementation

[0026] The present invention will be clearly and completely described and illustrated below with reference to specific comparative examples and embodiments. The comparative examples and embodiments described are only some examples of the present invention, and not all examples. The following embodiments of the present invention are only used to illustrate the present invention, and are not intended to limit the scope of the present invention.

[0027] In this embodiment, the HPLC detection conditions for mupirocin are as follows:

[0028] Column: Agilent ZORBAX SB-C84.6 * 250 mm 5 μm;

[0029] Buffer solution: Dissolve 10.5g ammonium acetate in 1L of deionized water, and adjust the pH to 5.7 with acetic acid;

[0030] Mobile phase: Buffer solution: Tetrahydrofuran: Water = 500: 300: 200;

[0031] Flow rate: 1.0 ml / min;

[0032] Column temperature: 25℃;

[0033] Detection wavelength: 230nm;

[0034] Injection volume: 5 μl.

[0035] This invention discloses a fermentation culture medium for preparing mupirocin, comprising: 80-120 g / L glucose, 20-60 g / L soybean meal, 10-30 g / L cottonseed meal, and 1-3 g / L ammonium sulfate.

[0036] Comparative Example 1: Fermentation medium

[0037] The fermentation medium for this comparative example includes: 50 g / L glucose, 65 g / L soybean meal powder, 10 g / L peanut meal powder, 6 g / L corn steep liquor powder, 10 g / L glycerol, 2 g / L urea, 6 g / L sodium chloride, 4 g / L calcium chloride, 5 g / L calcium carbonate, and 10 g / L soybean oil. The pH value of the fermentation medium is 7.0 ± 0.1.

[0038] Comparative Example 2: Fermentation medium

[0039] The fermentation medium for this comparative example includes: 10 g / L glucose, 50 g / L glycerol, 20 g / L soybean meal powder, 10 g / L peanut meal powder, 5 g / L corn steep liquor powder, 0.5 g / L ammonium sulfate, 0.5 g / L ammonium chloride, 5 g / L sodium chloride, and 5 g / L calcium carbonate. The pH value of the fermentation medium is 7.0 ± 0.1.

[0040] Comparative Example 3: Fermentation Medium

[0041] The fermentation medium for this comparative example includes: 70 g / L glucose, 60 g / L soybean meal powder, 6 g / L corn steep liquor powder, 2 g / L urea, 5 g / L ammonium sulfate, and 10 g / L soybean oil. The pH value of the fermentation medium is 7.5 ± 0.1.

[0042] Example 1: Fermentation medium

[0043] The fermentation medium in this embodiment includes: 100 g / L glucose, 40 g / L soybean meal, 20 g / L cottonseed meal, and 1 g / L ammonium sulfate. The pH value of the fermentation medium is 7.5 ± 0.1.

[0044] Example 2: Method for preparing mupirocin from fermentation medium

[0045] This invention also discloses a method for preparing mupirocin using the aforementioned fermentation medium. First, the bacterial strain is cultured, and finally, *Pseudomonas fluorescens* is inoculated into the fermentation medium for fermentation to produce mupirocin. The specific method is as follows:

[0046] Step (1) Activation of bacterial strain: Pseudomonas fluorescens HS-PF20-62, which was preserved at low temperature with the preservation number CGMCC No.25307, was inoculated onto a plate medium and cultured at 26±1℃ for 24±4 hours to obtain a single colony.

[0047] Step (2) Colony culture: Transfer the single colonies obtained in step (1) to a new plate medium by spot inoculation, with 10-12 single colonies on each plate; incubate at 26±1℃ for 3-6 days to obtain spot-inoculated single colonies;

[0048] The plate culture medium is composed of the following: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, agar 15 g / L, and the pH value of the plate culture medium is 7.5 ± 0.1.

[0049] Step (3) Preparation of seed culture in shake flask: Pick one single colony of the cultured in step (2) and inoculate it into the seed culture medium. Place the seed shake flask at 26±1℃ and shake at 250±10r / min for 28±4 hours to obtain the seed culture in shake flask.

[0050] The shake flask seed culture medium is composed of the following: 20 g / L glucose, 20 g / L soybean meal, 10 g / L yeast powder, and 2 g / L calcium carbonate. The pH value of the seed culture medium is 7.5 ± 0.1.

[0051] Shake flask seed culture medium volume: 20ml / 250ml;

[0052] Step (4) Shake-flask fermentation: The seed culture prepared in step (3) is transferred into a fermentation shake flask at a volume ratio of 5%. The volume of the fermentation shake flask is 20 / 250 ml, the fermentation shake flask culture temperature is 22±1℃, the fermentation shake flask rotation speed is 250±10 r / min, and the culture period is 5 days.

[0053] The fermentation medium used was the fermentation medium from Comparative Examples 1-3 and Example 1, respectively. After cultivation, a fermentation broth containing mupirocin was obtained. After pretreatment, HPLC analysis was performed, and the specific results are as follows:

[0054] Table 1: Effects of different fermentation medium formulations on the fermentation potency of mupirocin

[0055]

[0056] As can be seen from the results in Table 1, the formulation of the fermentation medium has a significant impact on the fermentation potency of mupirocin. Obviously, the formulation of the fermentation medium in Example 1 is the optimal formulation.

[0057] Example 3: Method for preparing mupirocin from fermentation medium

[0058] Based on the results of Example 2, the fermentation medium of Example 1 was selected as the fermentation shake flask medium, and the effect of culture temperature on fermentation titer was investigated in shake flask fermentation.

[0059] The process of strain activation, colony culture, shake flask seed liquid preparation and shake flask fermentation culture is the same as in Example 2. In this example, the shake flask fermentation culture temperatures are 20±1℃, 22±1℃, 24±1℃, 26±1℃ and 28±1℃, respectively.

[0060] Table 2: Effect of different fermentation temperatures on the fermentation potency of mupirocin

[0061]

[0062] As shown in Table 2, fermentation temperature has a significant impact on the potency of mupirocin. Within the experimental temperature range, lower temperatures are beneficial for increasing the potency, but excessively low temperatures (20±1℃) inhibit the potency. Overall, a fermentation temperature of 22±1℃ is the most suitable.

[0063] Example 4: Method for preparing mupirocin from fermentation medium

[0064] Based on the results of Examples 2 and 3, the fermentation medium of Example 1 was selected as the shake flask fermentation medium, and the shake flask fermentation temperature was selected as 22±1℃.

[0065] The activation of the strain, colony culture, preparation of seed culture in shake flasks, and shake flask fermentation were performed in the same manner as in Example 2. Different substances were added at 72 hours of fermentation, and the flasks were removed from the fermentation flasks on day 7. The added substances, concentrations of the added solutions, volumes of added solutions, and titers at the end of the fermentation flasks are shown in Table 3.

[0066] Table 3: Effects of different substances added during fermentation on the fermentation potency of mupirocin

[0067]

[0068] As can be seen from the results in Table 3, the addition of substances such as leucine, isoleucine, phenylalanine and soybean oil during the fermentation process did not help improve the fermentation titer, while the addition of glucose solution could increase the fermentation yield of mupirocin.

[0069] Example 5: Method for preparing mupirocin from fermentation medium

[0070] Based on the results of Examples 2 and 3, the fermentation medium of Example 1 was selected as the fermentation shake flask medium, and the shake flask fermentation temperature was selected as 22±1℃.

[0071] The activation of the strain, colony culture, preparation of the seed culture in shake flask, and shake flask fermentation process were the same as in Example 2. During fermentation, different volumes of glucose solution were added, with a mass percentage concentration of 50% (m%). The flasks were removed from the fermentation vessel on day 7. The timing, volume, and titer of the added material are shown in Table 4.

[0072] Table 4: Effect of glucose supplementation during fermentation on the fermentation potency of mupirocin

[0073]

[0074] As shown in Table 4, adding glucose solution in batches during fermentation does indeed help improve fermentation titer. Adding 4% (50% by mass) of the original fermentation medium at 48 and 96 hours of fermentation, respectively, had the greatest effect on improving titer.

[0075] The fermentation results from the above examples clearly demonstrate that the fermentation medium and fermentation production process of this invention can greatly improve the fermentation level of mupirocin and ultimately significantly reduce the cost of industrial production.

[0076] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. A method for producing mupirocin, characterized in that... The production strain is Pseudomonas fluorescens HS-PF20-62 with accession number CGMCC No. 25307, and fermentation is carried out using a fermentation medium. The fermentation medium includes: 80-120 g / L glucose, 20-60 g / L soybean meal, 10-30 g / L cottonseed meal, and 1-3 g / L ammonium sulfate.

2. The production method according to claim 1, characterized in that, The fermentation culture comprises: 100 g / L glucose, 40 g / L soybean meal, 20 g / L cottonseed meal, and 1 g / L ammonium sulfate, with a pH of 7.

5. + 0.

1.

3. The production method according to claim 1, characterized in that, The fermentation culture temperature is 21℃-23℃.

4. The production method according to claim 1, wherein the feed is added in batches during the 48th-96th hour of fermentation, characterized in that... The feed material is a glucose solution.

5. The production method according to claim 4, wherein glucose solution is added in batches at the 48th and 96th hours of the fermentation culture, and the volume of glucose solution added each time is 4% of the original fermentation culture medium volume, and the mass percentage concentration of the glucose solution is 50%.

6. The production method according to any one of claims 1-5, characterized in that... include: Step (1) Activation of bacterial strain: Pseudomonas fluorescens HS-PF20-62, which has been preserved at low temperature with the preservation number CGMCC No. 25307, was inoculated onto a plate culture medium and incubated at 26°C. + Cultured at 1℃ for 24 hours + After 4 hours, single colonies were obtained from the coating. Step (2) Colony culture: Transfer the single colonies obtained in step (1) to new agar plates by spot inoculation, with 10-12 single colonies on each plate; incubate at 26°C. + Incubate at 1℃ for 3-6 days to obtain single colonies. The plate culture medium has the following composition: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, and agar 15 g / L. The pH of the plate culture medium is 7.

5. + 0.1; Step (3) Seed culture preparation in shake flasks: Pick one single colony cultured in step (2) and inoculate it into the seed culture medium. Place the seed shake flask at 26°C. + 1℃, shaking speed 250 + Cultured at 10 r / min for 28 days + 4 hours; The shake-flask seed culture medium consists of the following components: 20 g / L glucose, 20 g / L soybean meal, 10 g / L yeast extract, and 2 g / L calcium carbonate. The pH of the seed culture medium is 7.

5. + 0.1; Shake flask seed culture medium volume: 20ml / 250ml; Step (4) Shake-flask fermentation: The seed culture prepared in step (3) is transferred into a shake-flask at a volume ratio of 5%. The volume of the fermentation medium for shake-flask fermentation is 20 ml / 250 ml, and the fermentation temperature is 22°C. + 1℃, fermentation shaker speed 250 + 10 r / min, fermentation culture period 5-7 days; The fermentation medium for shake-flask fermentation is the fermentation medium described in any one of claims 1-2.

7. The production method according to claim 6, wherein in step (4) shake-flask fermentation culture, the feed is added in batches during the 48th-96th hour of the fermentation culture, characterized in that... The feed material is a glucose solution.

8. In the production method according to claim 7, glucose solution is added in batches at the 48th and 96th hours of the fermentation culture, with the volume of glucose solution added each time being 4% of the original fermentation culture medium volume, and the mass percentage concentration of the glucose solution being 50%.

Images