Method for increasing fermentation unit of acetylisovaleryltylosin tartrate

Abstract

The invention provides a method for improving the fermentation unit of acetylisovaleryltylosin tartrate, which comprises the following steps: adding an oxygen carrier and a surfactant in the fermentation process of acetylisovaleryltylosin tartrate, and fermenting in a fermentation medium by utilizing thermotolerant streptomyces to produce acetylisovaleryltylosin tartrate. The dissolved oxygen level of the fermentation liquor in different fermentation periods is effectively regulated and controlled, the oxygen dissolving capacity of the thermostable streptomyces is improved, synthesis of the acetylisovaleryltylosin tartrate is promoted, and the fermentation unit of the acetylisovaleryltylosin tartrate can be remarkably increased.

Description

Technical Field

[0001] The present invention relates to a method for improving the fermentation unit of tylvalosin, belonging to the technical field of antibiotic fermentation. Background Art

[0002] Tylvalosin (acetyl-isovaleryl tylosin, AIV) is an important derivative of tylosin. It was obtained by Japanese scholar Okamoto in 1979 through acetylation at the 3-position of the tylosin A structure and isovaleryl acylation at the 4''-position by Streptomyces thermotolirans ATCC 11416. Like tylosin, tylvalosin belongs to the 16-membered macrolide antibiotics, and its antibacterial spectrum is similar to that of tylosin. It has good therapeutic effects on swine enzootic pneumonia, porcine reproductive and respiratory syndrome virus, proliferative enteritis, and chicken mycoplasma infection. In addition, tylvalosin can also enhance the chemotaxis, phagocytosis ability of phagocytes, increase the number of cells and activate cells, and improve the non-specific immune ability of the body. It is a new type of antibiotic specifically for livestock and poultry. Its efficacy against mycoplasma is 4-5 times that of tylosin, and it is an ideal drug for treating mycoplasma diseases in livestock and poultry currently known. Tylvalosin has no cross-resistance with other antibiotics, has less residue after administration, lower toxicity, and can be used for a long time, and has broad market prospects.

[0003] The closest prior art (a method for reducing the component of 4''-oxo-isovaleryl tylosin by changing the dissolved oxygen in the tylvalosin fermentation process, CN 114606282A) regulates the pH of the fermentation broth in the initial stage of fermentation, and then adds the substrate tylosin dry powder and L-leucine by feeding, removes the axial flow stirring paddle on the upper layer of the fermentation tank, and uses a fermentation tank with a stirring device to control the dissolved oxygen in the fermentation to reduce the component of 4''-oxo-isovaleryl tylosin. However, in this prior art, through a mechanical stirring type fermentation tank, during the fermentation process, the faster the stirring speed, the higher the dissolved oxygen. However, the shear force generated by too high a stirring speed will cause damage to the bacteria, and the mycelia are easily broken, forming short and short branches, which is not conducive to fermentation. Therefore, to solve the problems such as obvious autolysis of tylvalosin bacteria, large foam in the fermentation broth, and low fermentation unit, it is necessary to regulate the dissolved oxygen level in different fermentation cycles of the fermentation broth, improve the dissolved oxygen capacity of Streptomyces thermotolirans, so as to achieve the improvement of the fermentation unit. Summary of the Invention

[0004] In view of the problems in the prior art, such as the damage to bacterial cells caused by the shear force generated by the stirring speed, the easy breakage of hyphae, and the low fermentation unit, the present invention provides a method for improving the fermentation unit of tylvalosin, belonging to the technical field of antibiotic fermentation. Using Streptomyces thermotolerans as the production strain, during the fermentation process, an oxygen carrier and a surfactant are added in stages to regulate the dissolved oxygen level in different fermentation cycles of the fermentation broth, improve the dissolved oxygen capacity of Streptomyces thermotolerans, promote the synthesis of tylvalosin, and enhance the fermentation unit of tylvalosin.

[0005] To achieve the above-mentioned invention purpose, the present invention provides a method for improving the fermentation unit of tylvalosin, which uses tylvalosin-producing bacteria to ferment and produce tylvalosin in a fermentation medium, and an oxygen carrier and a surfactant are added to the fermentation medium.

[0006] Among them, 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.

[0007] Among them, the added oxygen carrier is n-hexadecane.

[0008] Among them, the added oxygen carrier concentration is 1.5-2.5%.

[0009] Among them, the addition amount of the oxygen carrier is 0.1-0.3% of the volume of the fermentation broth.

[0010] Among them, the addition time of the surfactant is 30-60 h of fermentation culture.

[0011] Among them, the added surfactant is span-20.

[0012] Among them, the added surfactant concentration is 0.1-0.3%.

[0013] Among them, the addition amount of the surfactant is 0.2-0.3% of the volume of the fermentation broth.

[0014] Among them, the dissolved oxygen amount during 0-30 h of fermentation culture is more than 45%, the dissolved oxygen amount during 30-60 h of fermentation culture is 25-40%, and the dissolved oxygen amount during 60-100 h of fermentation culture is more than 65%.

[0015] The technical solution of the present invention has at least the following beneficial technical effects:

[0016] By adding an oxygen carrier and a surfactant to the fermentation broth of Streptomyces thermotolerans in stages to regulate the dissolved oxygen amount in different fermentation cycles, the present invention reduces the oxygen transfer resistance between the gas-liquid two phases, thereby improving the dissolved oxygen capacity of Streptomyces thermotolerans, promoting the synthesis of tylvalosin, and enhancing the fermentation unit of tylvalosin. Specific embodiments

[0017] In the following embodiments of the present invention, those skilled in the art know that the strain cultured in the tylosin fermentation medium described in the present invention is generally Streptomyces thermotolirans, preferably Streptomyces thermotolirans ATCC 25500.

[0018] Detection method of titer (HPLC method): C18 (250mm×4.6mm, 5μm); mobile phase: acetonitrile (0.15mol / L), ammonium acetate solution, acetic acid = 45:45:10; flow rate: 1.0mL / min; column temperature: 30°C; injection volume: 10μL; wavelength of ultraviolet detector: 280nm.

[0019] Example 1

[0020] After the Streptomyces thermotolirans is matured by seed culture, it is transferred to a fermenter for fermentation culture. Taking 20m 3 As the fermentation volume unit, when the fermenter runs to 0h, 20ml of 1.5% n-hexadecane is added to the fermentation broth, and the dissolved oxygen content of the fermentation broth is detected to be 58%. At 30h, 40ml of 0.1% span-20 is added, and the dissolved oxygen content of the fermentation broth is detected to be 30%. At 60h, 18ml of 1.5% n-hexadecane is added, and the dissolved oxygen content of the fermentation broth is detected to be 78%. At the end of fermentation, the tylosin fermentation unit is detected to be 39274μg / mL.

[0021] Example 2

[0022] After the Streptomyces thermotolirans is matured by seed culture, it is transferred to a fermenter for fermentation culture. Taking 20m 3 As the fermentation volume unit, when the fermenter runs to 15h, 40ml of 2.0% n-hexadecane is added to the fermentation broth, and the dissolved oxygen content of the fermentation broth is detected to be 62%. At 45h, 50ml of 0.2% span-20 is added, and the dissolved oxygen content of the fermentation broth is detected to be 36%. At 80h, 36ml of 2.0% n-hexadecane is added, and the dissolved oxygen content of the fermentation broth is detected to be 83%. At the end of fermentation, the tylosin fermentation unit is detected to be 39685μg / mL.

[0023] Example 3:

[0024] After the Streptomyces thermotolirans is matured by seed culture, it is transferred to a fermenter for fermentation culture. Taking 20m 3 As the fermentation volume unit, when the fermenter runs to 30h, 60ml of 2.5% n-hexadecane is added to the fermentation broth, and the dissolved oxygen content of the fermentation broth is detected to be 68%. At 60h, 60ml of 0.3% span-20 is added, and the dissolved oxygen content of the fermentation broth is detected to be 39%. At 100h, 54ml of 2.5% n-hexadecane is added, and the dissolved oxygen content of the fermentation broth is detected to be 85%. At the end of fermentation, the tylosin fermentation unit is detected to be 39210μg / mL.

[0025] Comparative Example 1:

[0026] During the fermentation process of tylosin, no substances were added, and the remaining culture conditions were the same as those in Example 1. When the fermentation ended, the fermentation unit of tylosin was detected to be 37410 μg / mL.

[0027] Comparative Example 2:

[0028] After the seed culture of Streptomyces thermotolerans was mature, it was transferred to a fermenter for fermentation culture. Taking 20 m 3 as the fermentation volume unit, when the fermenter ran for 45 h, 40 ml of 2.0% n-hexadecane was added to the fermentation broth, and the dissolved oxygen content of the fermentation broth was detected to be 60%. When it ran for 80 h, 36 ml of 2.0% n-hexadecane was added, and the dissolved oxygen content of the fermentation broth was detected to be 82%. When the fermentation ended, the fermentation unit of tylosin was detected to be 38225 μg / mL.

[0029] Comparative Example 3:

[0030] After the seed culture of Streptomyces thermotolerans was mature, it was transferred to a fermenter for fermentation culture. Taking 20 m 3 as the fermentation volume unit, when the fermenter ran for 45 h, 50 ml of 0.2% span-20 was added, and the dissolved oxygen content of the fermentation broth was detected to be 70%. When the fermentation ended, 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, wherein 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, wherein 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, characterized in that, 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, wherein 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. The method for increasing the fermentation unit of tylvalosin according to claim 1, characterized in that, The dissolved oxygen content during 0 - 30 h of fermentation culture is above 45%, the dissolved oxygen content during 30 - 60 h of fermentation culture is 25 - 40%, and the dissolved oxygen content during 60 - 100 h of fermentation culture is above 65%.