Fermentation method for improving titer of lincomycin
By adding B vitamins such as lipoic acid, VB1 or VB12 to the fermentation medium, the fermentation conditions are optimized, and the problem of low lincomycin yield is solved, and the significant improvement of lincomycin titer is achieved.
Patent Information
- Application Number
- CN202410009862.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-01-04
- Publication Date
- 2025-07-11
AI Technical Summary
我国林可霉素生产存在产量低和发酵周期长的问题,现有技术中添加其他物质如金属离子、氨基酸、生物素等未能有效提高林可霉素的效价。
Add B vitamins such as lipoic acid, VB1 or VB12 to the fermentation medium to optimize fermentation conditions such as temperature, pressure and time, and improve the fermentation titer of lincomycin.
The fermentation titer of lincomycin was significantly improved, and the fermentation titer was increased by more than 8%, which improved the fermentation level of lincomycin and laid the foundation for industrial production.
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of antibiotic fermentation, and particularly relates to a fermentation method for improving the titer of lincomycin. Background Art
[0002] Lincomycin (LCM), also known as lincomycin, belongs to the lincosamide antibiotics. It was isolated from an actinomycete in the soil near Lincoln, Nebraska in 1962, and lincomycin was isolated from the culture broth of this bacterium. Lincomycin has strong resistance to Gram-positive bacteria, no cross-infection with existing antibiotics, and low toxicity. Therefore, it is widely used clinically to treat diseases caused by Gram-positive bacteria.
[0003] Researchers have found that adding some substances to the fermentation medium or during the fermentation process can effectively improve the titer of lincomycin. For example, adding metal ions, amino acids, biotin, surfactants, and the key precursor of lincomycin, propylproline, etc. during the fermentation process can significantly improve the titer of lincomycin A. However, compared with foreign countries, the existing technology for producing lincomycin in China still has problems such as low yield and long fermentation cycle. Vitamin B has unique physiological functions and is a biologically active substance that maintains cell growth. It plays a regulatory and controlling role in cell metabolism and is essential for the life activities of microorganisms. Generally, it acts as a coenzyme of enzymes, and can also provide reducing power NADH and NADPH for the growth and metabolism of bacteria, accelerate sugar consumption and cell substance synthesis, promote product synthesis, and increase yield. However, the yield of lincomycin in the existing technology is relatively low. Therefore, a fermentation method capable of improving the titer of lincomycin is needed. Summary of the Invention
[0004] In order to solve the problems existing in the prior art, the present invention provides a fermentation method for improving the titer of lincomycin, which uses a lincomycin-producing bacterium to ferment and produce lincomycin in a fermentation medium, and B vitamins are added to the fermentation medium.
[0005] Among them, the B vitamins are selected from at least one of lipoic acid, VB1 or VB12.
[0006] Among them, the addition time of the B vitamins is 80 - 120 h of fermentation culture.
[0007] Among them, the addition amount of the B vitamins is 0.005 - 0.08% of the mass of the medium used during fermentation.
[0008] Among them, the composition of the fermentation medium is as follows: soybean cake powder 1.2%, soluble starch 3.0%, glucose 3.5%, corn steep liquor 1.4%, NaCl 0.2%, (NH4)2SO4 0.54%, NaNO3 0.4%, NH4NO3 0.15%, MgSO4·7H2O 0.15%, KH2PO4 0.05%, CaCO3 0.5%, antifoam agent 0.03%, sodium hydroxide flakes 0.1%.
[0009] Among them, the pH of the fermentation medium is 7.2.
[0010] Among them, the fermentation culture conditions include: temperature is 30±1°C, pressure is 0.02 - 0.04 MPa, and the fermentation culture period is 160 - 180 h.
[0011] The technical solution of the present invention has at least the following beneficial technical effects:
[0012] The present invention utilizes the influence of B vitamins on microorganisms, adds lipoic acid, VB1, and VB12 during the fermentation process, significantly improves the titer of lincomycin. Under the experimental conditions of the present invention, the fermentation titer is increased to more than 10950 μg / mL, which is more than 8% higher than that of the comparative example, improves the fermentation level of lincomycin, enhances the economic benefits, and lays a foundation for the industrial production of lincomycin. Specific embodiments
[0013] The following will clearly and completely describe the implementation schemes of the present invention in combination with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. The following embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.
[0014] The experimental methods without specific conditions noted in the following embodiments are usually carried out according to conventional conditions, such as the conditions described in "Molecular Cloning: A Laboratory Manual" (New York: Cold Spring Harbor Laboratory PRESS, 1989).
[0015] The titer of lincomycin is determined by high performance liquid chromatography, and the method is as follows:
[0016] Collect 10 mL of the bacterial fermentation broth, place it in a 15 mL centrifuge tube, centrifuge at 3000 rpm for 10 min, take an appropriate amount of the supernatant with a syringe, filter it with an aqueous needle filter membrane with a pore size of 0.20 μm, and then place it in a centrifuge tube and store it in a -20°C refrigerator for future use.
[0017] Chromatographic conditions: Column model: Phenomex C18 (4.6 mm × 250 mm, 5 μm); Mobile phase conditions: 50 mmol / L ammonium acetate: methanol = 4:6; Column temperature: 30 °C; Flow rate: 0.6 mL / min; Injection volume: 20 μL; Detection wavelength: 210 nm.
[0018] In the following examples of the present invention, those skilled in the art know that the strain cultured in the avermectin fermentation medium of the present invention is generally Streptomyces lincolnenisis, preferably Streptomyces lincolnenisis CCTCC M 208064.
[0019] Example 1
[0020] The obtained inoculum was inoculated into a 50 L fermenter medium at an inoculation amount of 15%, and then sterile VB1 was added at 100 h of fermentation. The addition amount of VB1 was 0.01% of the medium, and finally a fermentation broth containing lincomycin was obtained. Fermentation culture conditions: The culture temperature was controlled at 30 ± 1 °C, the filtered and sterilized compressed air inlet was opened throughout the process, the tank pressure was maintained at 0.02 - 0.04 MPa, the stirring was turned on throughout the process, the rotation speed was 120 rpm from 0 to 90 h, and the rotation speed was 80 rpm from 90 h to the end of fermentation. Finally, a fermentation broth containing lincomycin was obtained.
[0021] Final titer detection result: The titer of lincomycin in the fermentation broth at the end of fermentation in Example 1 was 10950 μg / mL, which was 8.53% higher than that in the comparative example.
[0022] Example 2
[0023] The obtained inoculum was inoculated into a 50 L fermenter medium at an inoculation amount of 15%, and then sterile VB12 was added at 110 h of fermentation. The addition amount of VB12 was 0.04% of the medium, and finally a fermentation broth containing lincomycin was obtained. Fermentation culture conditions: The culture temperature was controlled at 30 ± 1 °C, the filtered and sterilized compressed air inlet was opened throughout the process, the tank pressure was maintained at 0.02 - 0.04 MPa, the stirring was turned on throughout the process, the rotation speed was 120 rpm from 0 to 90 h, and the rotation speed was 80 rpm from 90 h to the end of fermentation. Finally, a fermentation broth containing lincomycin was obtained.
[0024] Final titer detection result: The titer of lincomycin in the fermentation broth at the end of fermentation in Example 2 was 11550 μg / mL, which was 14.48% higher than that in the comparative example.
[0025] Example 3
[0026] The obtained inoculum was inoculated into a 50 L fermenter medium at an inoculation amount of 15%, and then sterile lipoic acid was added at 80 h of fermentation. The addition amount of lipoic acid was 0.08% of the medium, and finally a fermentation broth containing lincomycin was obtained. Fermentation culture conditions: The culture temperature was controlled at 30 ± 1°C, the filtered and sterilized compressed air inlet was opened throughout the process, the tank pressure was maintained at 0.02 - 0.04 MPa, stirring was turned on throughout the process, the rotation speed was 120 rpm from 0 to 90 h, and the rotation speed was 80 rpm from 90 h to the end of fermentation. Finally, a fermentation broth containing lincomycin was obtained.
[0027] Final titer detection result: The titer of lincomycin in the fermentation broth at the end of fermentation in Example 3 was 12056 μg / mL, which was 19.50% higher than that in the comparative example.
[0028] Example 4
[0029] The obtained inoculum was inoculated into a 50 L fermenter medium at an inoculation amount of 15%. Then, at 90 h of fermentation, sterile VB1 was added, and the addition amount of VB1 was 0.005% of the medium. At 110 h of fermentation, sterile VB12 was added, and the addition amount of VB12 was 0.01% of the medium. Finally, a fermentation broth containing lincomycin was obtained. Fermentation culture conditions: The culture temperature was controlled at 30 ± 1°C, the filtered and sterilized compressed air inlet was opened throughout the process, the tank pressure was maintained at 0.02 - 0.04 MPa, stirring was turned on throughout the process, the rotation speed was 120 rpm from 0 to 90 h, and the rotation speed was 80 rpm from 90 h to the end of fermentation. Finally, a fermentation broth containing lincomycin was obtained.
[0030] Final titer detection result: The titer of lincomycin in the fermentation broth at the end of fermentation in Example 4 was 12236 μg / mL, which was 21.28% higher than that in the comparative example.
[0031] Example 5
[0032] The obtained inoculum was inoculated into a 50 L fermentor medium at an inoculation amount of 15%. Then, at 80 h of fermentation, sterile lipoic acid was added, and the addition amount of lipoic acid was 0.01% of the medium. At 100 h of fermentation, sterile VB1 was added, and the addition amount of VB1 was 0.005% of the medium. Finally, the lincomycin fermentation broth was obtained. Fermentation culture conditions: The culture temperature was controlled at 30 ± 1 °C. The filtered and sterilized compressed air inlet was opened throughout the process. The tank pressure was maintained at 0.02 - 0.04 MPa. Stirring was turned on throughout the process. The rotation speed was 120 rpm from 0 to 90 h and 80 rpm from 90 h to the end of fermentation. Finally, the fermentation broth containing lincomycin was obtained.
[0033] Final titer detection result: The lincomycin titer in the fermentation broth at the end of fermentation in Example 5 was 12370 μg / mL, which was 22.61% higher than that in the comparative example.
[0034] Example 6
[0035] The obtained inoculum was inoculated into a 50 L fermentor medium at an inoculation amount of 15%. Then, at 80 h of fermentation, sterile lipoic acid was added, and the addition amount of lipoic acid was 0.01% of the medium. At 120 h of fermentation, sterile VB12 was added, and the addition amount of VB12 was 0.01% of the medium. Finally, the lincomycin fermentation broth was obtained. Fermentation culture conditions: The culture temperature was controlled at 30 ± 1 °C. The filtered and sterilized compressed air inlet was opened throughout the process. The tank pressure was maintained at 0.02 - 0.04 MPa. Stirring was turned on throughout the process. The rotation speed was 120 rpm from 0 to 90 h and 80 rpm from 90 h to the end of fermentation. Finally, the fermentation broth containing lincomycin was obtained.
[0036] Final titer detection result: The lincomycin titer in the fermentation broth at the end of fermentation in Example 5 was 12196 μg / mL, which was 20.88% higher than that in the comparative example.
[0037] Example 7
[0038] The obtained inoculum was inoculated into a 50 L fermentor medium at an inoculation amount of 15%. Then, at 80 h of fermentation, sterile lipoic acid was added, and the addition amount of lipoic acid was 0.01% of the medium. At 100 h of fermentation, sterile VB12 was added, and the addition amount of VB12 was 0.01% of the medium. At 120 h of fermentation, sterile VB1 was added, and the addition amount of VB1 was 0.005% of the medium. Finally, the fermentation broth containing lincomycin was obtained. Fermentation culture conditions: The culture temperature was controlled at 30 ± 1 °C. The filtered and sterilized compressed air inlet was opened throughout the process. The tank pressure was maintained at 0.02 - 0.04 MPa. Stirring was turned on throughout the process. The rotation speed was 120 rpm from 0 to 90 h and 80 rpm from 90 h to the end of fermentation. Finally, the fermentation broth containing lincomycin was obtained.
[0039] Final titer detection result: the titer of lincomycin in the fermentation broth at the end of fermentation in Example 7 was 12,539 μg / mL, which was 24.28% higher than that in the comparative example.
[0040] Comparative example
[0041] The inoculum was inoculated into the 50 L fermentation tank medium at an inoculation amount of 15%. The culture temperature was controlled at 30 ± 1 °C. The filtered and sterilized compressed air inlet was opened throughout the process. The tank pressure was maintained at 0.02 - 0.04 MPa. The stirring was turned on throughout the process. The rotation speed was 120 rpm from 0 to 90 h and 80 rpm from 90 h to the end of fermentation, and finally a fermentation broth containing lincomycin was obtained.
[0042] Final titer detection result: the titer of lincomycin in the fermentation broth at the end of fermentation in the comparative example was 10,089 μg / mL.
Claims
1. A fermentation method for improving the titer of lincomycin, characterized in that, Produce lincomycin by fermenting lincomycin-producing bacteria in a fermentation medium, and at least one B vitamin is added to the fermentation medium.
2. The fermentation method according to claim 1, wherein The B vitamin is selected from at least one of lipoic acid, VB1, and VB12.
3. The fermentation method according to claim 1, wherein The addition time of the B vitamin is 80 - 120 h during fermentation culture.
4. The fermentation method according to claim 1, characterized in that The addition amount of the B vitamin is 0.005 - 0.08% of the mass of the medium used during fermentation.
5. The fermentation method according to claim 1, characterized in that The composition of the fermentation medium is as follows: soybean cake powder 1.2%, soluble starch 3.0%, glucose 3.5%, corn steep liquor 1.4%, NaCl 0.2%, (NH4)2SO4 0.54%, NaNO3 0.4%, NH4NO3 0.15%, MgSO4·7H2O 0.15%, KH2PO4 0.05%, CaCO3 0.5%, antifoaming agent 0.03%, sodium hydroxide flakes 0.1%.
6. The fermentation method according to claim 1, wherein The pH of the fermentation medium is 7.
2.
7. The fermentation method according to claim 1, characterized in that The fermentation culture conditions include: temperature is 30 ± 1°C, pressure is 0.02 - 0.04 MPa, and the fermentation culture period is 160 - 180 h.
8. The fermentation method according to claim 1, wherein The lincomycin-producing bacteria is Streptomyces lincolnenisis.