Material supplementing method for improving fermentation unit of tylosin

Abstract

The invention relates to a feeding method for improving the fermentation unit of tylosin, which comprises the following steps: inoculating a seed solution into a fermentation tank filled with a liquid fermentation culture medium by taking streptomyces fradiae as a fermentation strain, producing tylosin by adopting a fed-batch fermentation process, fermenting for 12 hours, adding a sodium malonate solution, reacting for 2 hours, adding a VB12 solution, fermenting for 30-130 hours, and then adding a VB12 solution to react for 2 hours. And supplementing the mecobalamin solution every 12 hours. By adopting the material supplementing process, the fermentation unit of tylosin can be effectively increased by 2-4% or above, and the material supplementing process is suitable for industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of antibiotic fermentation, and in particular relates to a feeding method for improving a tylosin fermentation unit. Background Art

[0002] Tylosin is a 16-membered macrolide antibiotic produced by Streptomyces freundii. It can be used as a therapeutic drug to selectively inhibit protein synthesis, effectively prevent and treat respiratory and digestive tract infections in livestock and poultry, and can also be used as a feed additive to promote animal growth and development. After years of application, it has been proven that it has very little residue in animals and is an animal-specific antibiotic certified by the EU CEP. Tilmicosin and acetylisovaleryl tylosin are downstream products of tylosin. They are semi-synthetic macrolide animal-specific antibiotics from tylosin, with high added value and broad prospects in both the export and domestic markets.

[0003] The existing technology usually uses Streptomyces freundii as the fermentation strain, and improves its fermentation unit by optimizing the culture medium formula. However, the problem with this fermentation method is that the energy consumption of the tylosin bacterial body synthesis metabolism process increases, the bacterial body is prone to aging and autolysis in the later stage of fermentation, the rate of synthesizing tylosin slows down, and the lack of stamina of the culture medium leads to a decline in production capacity. At present, there is little research on the tylosin fermentation feeding process. The industrial production of tylosin generally adopts sugar and oil supplementation during the fermentation process to delay the aging and autolysis of the bacterial body, but excessive sugar and oil supplementation in the middle and late stages of fermentation will lead to sugar resistance, mycelium turning green, etc.; too thin will lead to a decrease in the concentration of the culture medium matrix, affecting the absorption of nitrogen sources and other substances, and the effect on improving the tylosin fermentation unit is not obvious. Therefore, optimizing the tylosin feeding process and effectively improving the tylosin fermentation unit are problems that production enterprises need to solve urgently. Summary of the invention

[0004] Aiming at the problems in the prior art that the biosynthesis utilization rate of tylosin is low and the fermentation unit is low due to the autolysis of bacterial bodies and the insufficient stamina of the culture medium, the present invention provides a feeding method for improving the fermentation unit of tylosin. Streptomyces fradiae is used as the fermentation strain, seed liquid is connected to a fermentation tank filled with a liquid fermentation medium, and a sodium malonate solution, a VB12 solution and a methylcobalamin solution are respectively added by a fed-batch fermentation process to produce tylosin, which can effectively improve the fermentation titer of tylosin and is suitable for industrial production.

[0005] The technical solution adopted to achieve the above-mentioned purpose of the invention is: a method for improving the feeding of tylosin fermentation units, using Streptomyces fradiae as the fermentation strain, accessing the seed liquid into a fermentation tank filled with liquid fermentation medium, and producing tylosin by a fed-batch fermentation process, the fermentation culture time is 168-170h, wherein the fermentation process is fed by AC flow:

[0006] A. Ferment for 12 h and add sodium malonate solution.

[0007] B. React for 2 h after adding sodium malonate solution and add VB12 solution.

[0008] C. Ferment for 30 - 130 h and add methylcobalamin solution every 12 h.

[0009] Among them, the methylcobalamin solution added every 12 h means adding methylcobalamin solution once during fermentation for 30 - 60 h and adding methylcobalamin solution a second time during fermentation for 60 - 130 h.

[0010] Among them, the concentration of the added sodium malonate solution is 0.1 - 0.5%.

[0011] Among them, the concentration of the added VB 12 solution is 0.1 - 0.3%.

[0012] Among them, the concentration of the added methylcobalamin solution is 0.05 - 0.25%.

[0013] Among them, the volume of the added sodium malonate solution is 3.0 - 3.2% of the volume of the initial fermentation broth.

[0014] Among them, the volume of the added VB12 solution is 2.4 - 2.8% of the volume of the initial fermentation broth.

[0015] Among them, the volume of the added methylcobalamin solution is 4.7 - 5.2% of the volume of the initial fermentation broth.

[0016] Among them, the fermentation medium composition is: 500 g of corn flour, 500 g of corn gluten meal, 220 g of cottonseed protein powder, 110 g of calcium carbonate, 25 g of betaine hydrochloride, 150 g of yeast powder, 65 g of corn steep liquor, 35 g of potassium chloride, 600 g of soybean oil.

[0017] Among them, the fermentation culture conditions are: culture temperature 31°C, pH value 6.3 - 6.9, stirring speed 250 rpm / min, air flow rate 40 - 50 m 3 / h.

[0018] The technical solution of the present invention has at least the following beneficial technical effects:

[0019] 1) In the biosynthesis process of tylosin, propionic acid and other small molecules participate in the synthesis, so the addition of sodium malonate in the fermentation process can provide a precursor for the synthesis of tylosin, and the addition of VB12 can promote the decomposition of sodium malonate, providing tylosin with abundant precursors to promote its synthesis. Methylcobalamin is a coenzyme factor that can assist VB12 in promoting the decomposition activity, so the present invention adds VB12 and cofactor methylcobalamin that promote the decomposition of the precursor sodium malonate in the optimal feeding cycle of tylosin fermentation, promotes the biosynthesis of tylosin, and thus increases the fermentation unit by 2-4%.

[0020] 2) The present invention overcomes the problems of bacterial autolysis and insufficient stamina of the culture medium in the late fermentation period by feeding in the early and middle stages of fermentation, accelerates the tylosin production rate, and stabilizes the fermentation cycle. DETAILED DESCRIPTION

[0021] The Streptomyces fradiae described in the present invention can be any existing tylosin-producing Streptomyces fradiae, preferably Streptomyces fradiae, with a strain collection number of CGMCC No.6717.

[0022] The reagents and raw materials used in the present invention are all commercially available. Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0023] The fermentation culture of the embodiments of the present invention and the comparative examples is carried out with a fermentation volume of 28m3 as the unit (m / v); the detection method of the potency (HPLC method) is as follows: chromatographic column: C18 column (250mm×4.6mm, 5μm); mobile phase: 0.85mol / L sodium perchlorate solution-acetonitrile solution 6:4; pH value: 2.5; flow rate: 1mL / min; column temperature: 35°C; injection volume: 10μL; detection wavelength: 290nm.

[0024] Example 1

[0025] After the first-level and second-level seeds of Streptomyces freundii mature, they are transferred to the fermentation tank for fermentation. After the tylosin fermentation tank has been running for 12 hours, 900 mL of 0.10% sodium malonate solution is added; after 2 hours of reaction, 780 mL of 0.10% VB 12 Solution; add methylcobalamin solution when the fermentation tank runs for 30-130 hours, and add once every 12 hours, of which 680mL 0.05% methylcobalamin solution is added at 30-60 hours, and 780mL 0.05% methylcobalamin solution is added at 60-130 hours. Stop adding when the fermentation tank runs for 130 hours. The titer detected at the end of fermentation is 17950μg / mL, and the fermentation cycle is 171h.

[0026] Example 2

[0027] After Streptomyces fradiae is matured through primary and secondary seed cultures, it is transferred to a fermenter for fermentation. After the tylosin fermenter runs for 12 h, 875 mL of 0.20% sodium malonate solution is added; after reacting for 2 h, 750 mL of 0.15% VB 12 solution is added; methylcobalamin solution is added when the fermenter runs for 30 - 130 h, and it is replenished every 12 h. Among them, 660 mL of 0.10% methylcobalamin solution is added for 30 - 60 h, 750 mL of 0.10% methylcobalamin solution is added for 60 - 130 h, and the addition stops when the fermenter runs for 130 h. The titer is detected to be 17975 μg / mL at the end of fermentation, and the fermentation cycle is 170 h.

[0028] Example 3:

[0029] After Streptomyces fradiae is matured through primary and secondary seed cultures, it is transferred to a fermenter for fermentation. After the tylosin fermenter runs for 12 h, 900 mL of 0.25% sodium malonate solution is added; after reacting for 2 h, 780 mL of 0.20% VB 12 solution is added; methylcobalamin solution is added when the fermenter runs for 30 - 130 h, and it is replenished every 12 h. Among them, 680 mL of 0.15% methylcobalamin solution is added for 30 - 60 h, 780 mL of 0.15% methylcobalamin solution is added for 60 - 130 h, and the addition stops when the fermenter runs for 130 h. The titer is detected to be 18215 μg / mL at the end of fermentation, and the fermentation cycle is 169 h.

[0030] Example 4:

[0031] After Streptomyces fradiae is matured through primary and secondary seed cultures, it is transferred to a fermenter for fermentation. After the tylosin fermenter runs for 12 h, 850 mL of 0.30% sodium malonate solution is added; after reacting for 2 h, 680 mL of 0.25% VB 12 solution is added; methylcobalamin solution is added when the fermenter runs for 30 - 130 h, and it is replenished every 12 h. Among them, 640 mL of 0.20% methylcobalamin solution is added for 30 - 60 h, 680 mL of 0.20% methylcobalamin solution is added for 60 - 130 h, and the addition stops when the fermenter runs for 130 h. The titer is detected to be 18025 μg / mL at the end of fermentation, and the fermentation cycle is 170 h.

[0032] Example 5:

[0033] After Streptomyces fradiae is matured through primary and secondary seed cultures, it is transferred to a fermenter for fermentation. After the tylosin fermenter runs for 12 h, 850 mL of 0.50% sodium malonate solution is added; after reacting for 2 h, 680 mL of 0.30% VB 12Solution; During the operation of the fermenter for 30 - 130 h, add methylcobalamin solution, and replenish it every 12 h. Among them, add 640 mL of 0.25% methylcobalamin solution from 30 - 60 h, and add 680 mL of 0.25% methylcobalamin solution from 60 - 130 h. Stop replenishing when the fermenter runs to 130 h. The titer was detected to be 18025 μg / mL at the end of fermentation, and the fermentation cycle was 170 h.

[0034] Comparative Example 1:

[0035] During the tylosin fermentation process, no supplementary feeding was added, and the remaining culture conditions were the same as those in Example 1. The titer was detected to be 16485 μg / mL at the end of fermentation, and the fermentation cycle was 178 h.

[0036] Comparative Example 2:

[0037] During the tylosin fermentation process, add 900 mL of 0.25% sodium malonate solution when the fermenter runs to 12 h, and the remaining culture conditions are the same as those in Example 1. The titer was detected to be 16515 μg / mL at the end of fermentation, and the fermentation cycle was 177 h.

[0038] Comparative Example 3:

[0039] During the tylosin fermentation process, add 900 mL of 0.25% sodium malonate solution when the fermenter runs to 12 h, and add 780 mL of 0.20% VB 12 solution after reacting for 2 h. The remaining culture conditions are the same as those in Example 1. The titer was detected to be 16587 μg / mL at the end of fermentation, and the fermentation cycle was 175 h.

[0040] Comparative Example 4:

[0041] During the tylosin fermentation process, add 680 mL of 0.15% methylcobalamin solution when the fermenter runs to 30 h, and the remaining culture conditions are the same as those in Example 1. The titer was detected to be 16610 μg / mL at the end of fermentation, and the fermentation cycle was 176 h.

[0042] Comparative Example 5:

[0043] During the tylosin fermentation process, add 900 mL of 0.25% sodium malonate solution when the fermenter runs to 12 h, and add 780 mL of 0.20% VB 12 solution, and add 680 mL of 0.15% methylcobalamin solution when fermenting to 30 h. The remaining culture conditions are the same as those in Example 1. The titer was detected to be 16795 μg / mL at the end of fermentation, and the fermentation cycle was 174 h.

Claims

1. A feeding method for increasing the fermentation unit of tylosin, using Streptomyces fradiae as the fermentation strain, inoculating the seed liquid into a fermenter containing a liquid fermentation medium, and adopting a fed-batch fermentation process to produce tylosin, with the fermentation culture time being 169 - 171 h, characterized in that Among them, the fermentation process is carried out by feeding in a A-C mode: A: Ferment for 12 h, and add sodium malonate solution; B: React for 2 h after adding sodium malonate solution, and add VB12 solution; C: Ferment for 30 - 130 h, and add methylcobalamin solution every 12 h.

2. The feeding method for increasing the tylosin fermentation unit according to claim 1, characterized in that The addition of methylcobalamin solution every 12 h means adding methylcobalamin solution once during fermentation for 30 - 60 h and adding methylcobalamin solution a second time during fermentation for 60 - 130 h.

3. The feeding method for increasing the tylosin fermentation unit according to claim 1, characterized in that The concentration of the added sodium malonate solution is 0.1 - 0.5%.

4. The feeding method for increasing the tylosin fermentation unit according to claim 1, characterized in that The added VB 12 solution has a concentration of 0.1-0.3%.

5. The feeding method for increasing the tylosin fermentation unit according to claim 1, characterized in that The concentration of the added methylcobalamin solution is 0.05 - 0.25%.

6. The feeding method for increasing the tylosin fermentation unit according to claim 1, characterized in that The volume of the added sodium malonate solution is 3.0 - 3.2% of the volume of the initial fermentation broth.

7. The feeding method for increasing the tylosin fermentation unit according to claim 1, wherein The volume of the added VB12 solution is 2.4 - 2.8% of the volume of the initial fermentation broth.

8. The feeding method for increasing the tylosin fermentation unit according to claim 1, characterized in that The volume of the added methylcobalamin solution is 4.7 - 5.2% of the volume of the initial fermentation broth.

9. The feeding method for increasing the tylosin fermentation unit according to any one of claims 1-8, characterized in that The composition of the fermentation medium is as follows: 500 g of corn flour, 500 g of corn gluten meal, 220 g of cottonseed protein powder, 110 g of calcium carbonate, 25 g of betaine hydrochloride, 150 g of yeast powder, 65 g of corn steep liquor, 35 g of potassium chloride, and 600 g of soybean oil.

10. The feeding method for increasing the tylosin fermentation unit according to claim 9, characterized in that The fermentation culture conditions are as follows: culture temperature is 31°C, pH value is 6.3 - 6.9, stirring speed is 250 rpm / min, and air flow rate is 40 - 50 m 3 / h.