Method for increasing fermentation titer of acetyl-isovaleryl tylosin

Abstract

A method for increasing the fermentation titer of Acetyl-isovaleryl tylosin. An oxygen carrier and a surfactant are added in the fermentation process of Acetyl-isovaleryl tylosin, and Streptomyces thermotolerans is used for fermentation in a fermentation culture medium to produce the Acetyl-isovaleryl tylosin. The level of dissolved oxygen of a fermentation liquid in different fermentation periods is effectively regulated and controlled, so that the oxygen dissolving capacity of the Streptomyces thermotolerans is improved, and synthesis of the Acetyl-isovaleryl tylosin is promoted, thereby remarkably increasing the fermentation titer of the Acetyl-isovaleryl tylosin.

Description

A method for increasing the fermentation unit of tyvalosin

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application number 202410006592.1 filed with the State Intellectual Property Office of China on January 3, 2024, entitled “A Method for Improving Tylvalosin Fermentation Units,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The invention relates to a method for improving the fermentation unit of tyvalosin, and belongs to the technical field of antibiotic fermentation. Background Art

[0004] Acetyl-isovaleryl tylosin (AIV) is an important derivative of tylosin. It was first discovered by Japanese scholar Okamoto in 1979 through the ATCC 11416 is obtained by acetylation at the 3-position and isovalerylation at the 4"-position of the tylosin A structure. Like tylosin, tylosin belongs to the 16-membered macrolide antibiotic class and has a similar antimicrobial spectrum. It is effective against porcine pancreatitis, blue ear virus, proliferative enteritis, and mycoplasma infections in chickens. Furthermore, tylosin can enhance the chemotaxis and phagocytic capacity of phagocytes, increase cell number and activation, and improve the body's nonspecific immunity. It is a new antibiotic specifically for livestock and poultry. Its efficacy against mycoplasmas is 4-5 times that of tylosin, making it an ideal treatment for mycoplasma infections in poultry and livestock. Tylvalosin exhibits no cross-resistance with other antibiotics, exhibits low post-administration residues, is low in toxicity, and can be used long-term, thus possessing broad market prospects.

[0005] The closest existing technology (a method for modifying dissolved oxygen during the tylosin fermentation process to reduce the 4"-O-isovaleryl tylosin component, CN 114606282A) regulates the pH of the fermentation broth during the initial fermentation phase, subsequently adding the substrate tylosin dry powder and L-leucine, and removing the axial flow impeller in the upper layer of the fermentor. This method uses a fermentor equipped with a stirring device to control the dissolved oxygen content during fermentation to reduce the 4"-O-isovaleryl tylosin component. However, this prior art utilizes a mechanically stirred fermentor. During fermentation, faster stirring speeds increase dissolved oxygen levels. However, excessively high stirring speeds generate shear forces that damage the bacteria, easily breaking the hyphae and forming short branches, hindering fermentation. Therefore, to address the issues of significant tylosin autolysis, excessive foaming in the fermentation broth, and low fermentation unit, it is necessary to regulate the dissolved oxygen level of the fermentation broth during different fermentation cycles to improve the dissolved oxygen capacity of thermotolerant Streptomyces, thereby increasing the fermentation unit. Summary of the Invention

[0006] In response to the problems in the prior art of the shear force generated by the stirring speed causing damage to the bacterial body, easy breakage of the hyphae, and low fermentation unit, the present invention provides a method for improving the fermentation unit of tylvalosin, belonging to the field of antibiotic fermentation technology. The method uses thermotolerant Streptomyces as the production strain, and adopts the staged addition of oxygen carriers and surfactants during the fermentation process to control the dissolved oxygen level of the fermentation liquid in different fermentation cycles, thereby improving the oxygen dissolving capacity of the thermotolerant Streptomyces, promoting the synthesis of tylvalosin, and increasing the fermentation unit of tylvalosin.

[0007] To achieve the above object, the present invention provides a method for increasing the fermentation unit of tylvalosin, wherein tylvalosin-producing bacteria are used to ferment and produce tylvalosin in a fermentation medium, wherein an oxygen carrier and a surfactant are added to the fermentation medium.

[0008] The oxygen carrier is first added during 0-30 hours of fermentation, and the oxygen carrier is second added during 60-100 hours of fermentation.

[0009] Wherein, the added oxygen carrier is n-hexadecane.

[0010] Wherein, the concentration of the added oxygen carrier is 1.5-2.5%.

[0011] Wherein, the added amount of the oxygen carrier is 0.1-0.3% of the volume of the fermentation liquid.

[0012] Wherein, the surfactant is added for 30-60 hours during the fermentation culture.

[0013] Wherein, the added surfactant is span-20.

[0014] Wherein, the added concentration of the surfactant is 0.1-0.3%.

[0015] Wherein, the added amount of the surfactant is 0.2-0.3% of the volume of the fermentation liquid.

[0016] The dissolved oxygen content during the fermentation culture for 0-30 hours is above 45%, the dissolved oxygen content during the fermentation culture for 30-60 hours is 25-40%, and the dissolved oxygen content during the fermentation culture for 60-100 hours is above 65%.

[0017] The technical solution of the present invention has at least the following beneficial technical effects:

[0018] The present invention regulates the dissolved oxygen content in different fermentation cycles by adding oxygen carriers and surfactants to the thermotolerant Streptomyces fermentation broth in stages, reduces the oxygen transfer resistance between the gas and liquid phases, thereby improving the oxygen dissolving capacity of the thermotolerant Streptomyces, promoting the synthesis of tylvalosin, and increasing the tylvalosin fermentation unit. DETAILED DESCRIPTION

[0019] In the following embodiments of the present invention, those skilled in the art know that the bacterial species cultured in the tyvalosin fermentation medium of the present invention is generally Streptomyces thermotolirans, preferably Streptomyces thermotolirans ATCC 25500.

[0020] Potency detection method (HPLC method): C18 (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile (0.15 mol / L), ammonium acetate solution, acetic acid = 45:45:10; flow rate: 1.0 mL / min; column temperature: 30°C; injection volume: 10 μL; UV detector wavelength: 280 nm.

[0021] Example 1

[0022] After the thermotolerant Streptomyces is matured through seed culture, it is transferred to a fermentation tank for fermentation culture. 3 The fermentation volume is used as the unit. When the fermenter is running at 0h, 20ml of 1.5% n-hexadecane is added to the fermentation liquid, and the dissolved oxygen content of the fermentation liquid is detected to be 58%. 40ml of 0.1% span-20 is added at 30h, and the dissolved oxygen content of the fermentation liquid is detected to be 30%. 18ml of 1.5% n-hexadecane is added at 60h, and the dissolved oxygen content of the fermentation liquid is detected to be 78%. At the end of fermentation, the fermentation unit of tyvalosin is detected to be 39274μg / mL.

[0023] Example 2

[0024] After the thermotolerant Streptomyces is matured through seed culture, it is transferred to a fermentation tank for fermentation culture. 3 The fermentation volume is taken as the unit. When the fermenter has been running for 15 hours, 40 ml of 2.0% n-hexadecane is added to the fermentation liquid, and the dissolved oxygen content of the fermentation liquid is detected to be 62%. After 45 hours, 50 ml of 0.2% span-20 is added to the fermentation liquid, and the dissolved oxygen content of the fermentation liquid is detected to be 36%. After 80 hours, 36 ml of 2.0% n-hexadecane is added to the fermentation liquid, and the dissolved oxygen content of the fermentation liquid is detected to be 83%. At the end of fermentation, the fermentation unit of tyvalosin is detected to be 39685 μg / mL.

[0025] Example 3:

[0026] After the thermotolerant Streptomyces is matured through seed culture, it is transferred to a fermentation tank for fermentation culture. 3The fermentation volume is taken as the unit. When the fermenter has been running for 30 hours, 60 ml of 2.5% n-hexadecane is added to the fermentation liquid, and the dissolved oxygen content of the fermentation liquid is detected to be 68%. After 60 hours, 60 ml of 0.3% span-20 is added to the fermentation liquid, and the dissolved oxygen content of the fermentation liquid is detected to be 39%. After 100 hours, 54 ml of 2.5% n-hexadecane is added to the fermentation liquid, and the dissolved oxygen content of the fermentation liquid is detected to be 85%. At the end of fermentation, the fermentation unit of tyvalosin is detected to be 39210 μg / mL.

[0027] Comparative Example 1:

[0028] During the fermentation of tyvalosin, no substance was added, and the remaining culture conditions were the same as those in Example 1. At the end of the fermentation, the tyvalosin fermentation unit was 37410 μg / mL.

[0029] Comparative Example 2:

[0030] After the thermotolerant Streptomyces is matured through seed culture, it is transferred to a fermentation tank for fermentation culture. 3 The fermentation volume is used as the unit. When the fermenter is running for 45 hours, 40 ml of 2.0% n-hexadecane is added to the fermentation liquid, and the dissolved oxygen content of the fermentation liquid is detected to be 60%. After 80 hours, 36 ml of 2.0% n-hexadecane is added to the fermentation liquid, and the dissolved oxygen content of the fermentation liquid is detected to be 82%. At the end of fermentation, the fermentation unit of tyvalosin is detected to be 38225 μg / mL.

[0031] Comparative Example 3:

[0032] After the thermotolerant Streptomyces is matured through seed culture, it is transferred to a fermentation tank for fermentation culture. 3 The fermentation volume was used as the unit. When the fermenter was running for 45 hours, 50 ml of 0.2% span-20 was added. The dissolved oxygen content of the fermentation liquid was detected to be 70%. At the end of fermentation, the fermentation unit was detected to be 38220 μg / mL.

Claims

1. A method for increasing the fermentation unit of tylvalosin, characterized in that, Tylvalosin is fermented and produced by tylvalosin-producing bacteria in a fermentation medium, and an oxygen carrier and a surfactant are added to the fermentation medium.

2. The method according to claim 1, characterized in that, The first addition time of the oxygen carrier is 0 - 30 h of fermentation culture, and the second addition time of the oxygen carrier is 60 - 100 h of fermentation culture.

3. The method according to claim 1, characterized in that The added oxygen carrier is n-hexadecane.

4. The method according to claim 1, characterized in that, The concentration of the added oxygen carrier is 1.5 - 2.5%.

5. The method according to claim 1, characterized in that, The addition amount of the oxygen carrier is 0.1 - 0.3% of the volume of the fermentation broth.

6. The method for increasing the fermentation unit of tylvalosin according to claim 1, wherein The addition time of the surfactant is 30 - 60 h of fermentation culture.

7. The method for increasing the fermentation unit of tylvalosin according to claim 1, characterized in that, The added surfactant is span-20.

8. The method for increasing the fermentation unit of tylvalosin according to claim 1, characterized in that, The added concentration of the surfactant is 0.1 - 0.3%.

9. The method for increasing the fermentation unit of tylvalosin according to claim 1, characterized in that, The addition amount of the surfactant is 0.2 - 0.3% of the volume of the fermentation broth.

10. A method for increasing the fermentation unit of tylvalosin according to claim 1, characterized in that the dissolved oxygen content is above 45% during 0 - 30 h of fermentation culture, the dissolved oxygen content is 25 - 40% during 30 - 60 h of fermentation culture, and the dissolved oxygen content is above 65% during 60 - 100 h of fermentation culture.