Material-supplementing method for increasing tylosin fermentation units

Abstract

The present invention relates to a material-supplementing method for increasing tylosin fermentation units. The method uses Streptomyces fradiae as a fermentation strain, and comprises inoculating a seed liquid into a fermentation tank filled with a liquid fermentation culture medium and using a material-supplementing fermentation process to produce tylosin: at fermentation hour 12, adding a disodium malonate solution to react for 2 hours, then adding a VB12 solution, and during fermentation hours 30-130, adding a methylcobalamin solution at intervals of 12 hours. Using the material-supplementing process effectively increases tylosin fermentation units by 2-4% or more, and the method is suitable for industrial production.

Description

A feeding method for improving tylosin fermentation unit

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application number 202410006152.6, filed with the State Intellectual Property Office of China on January 3, 2024, entitled “A method for improving the feeding of tylosin fermentation units”, the entire contents of which are incorporated herein by reference. Technical Field

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

[0004] Tylosin is a 16-membered macrolide antibiotic produced by Streptomyces freundii. It can be used as a therapeutic drug, selectively inhibiting protein synthesis and effectively preventing and treating respiratory and digestive tract infections in livestock and poultry. It can also be used as a feed additive to promote animal growth and development. Recent applications have demonstrated that it leaves very little residue in animals and is a certified animal-specific antibiotic by the EU CEP. Tilmicosin and acetylisovaleryl tylosin are downstream products of tylosin. These semi-synthetic macrolide antibiotics for animals are highly value-added and have broad prospects in both the export and domestic markets.

[0005] Existing technologies typically use Streptomyces freundii as a fermentation strain and improve its fermentation unit by optimizing the culture medium formula. However, this fermentation method has the following problems: the energy consumption of the tylosin bacterial synthesis metabolism process increases, the bacteria are prone to aging and autolysis in the later stages of fermentation, the synthesis rate of tylosin slows down, and the lack of stamina in the culture medium leads to a decline in production capacity. Currently, there is little research on the tylosin fermentation feeding process. Industrial production of tylosin generally adopts sugar and oil supplementation during the fermentation process to delay bacterial aging and autolysis. However, excessive sugar and oil supplementation in the middle and late stages of fermentation can lead to sugar blockage and mycelial regreening. Excessive dilution leads to a decrease in the culture medium matrix concentration, affecting the absorption of nitrogen sources and other substances, and has little effect on improving the tylosin fermentation unit. Therefore, optimizing the tylosin feeding process and effectively improving the tylosin fermentation unit are urgent issues that manufacturers need to address.

[0006] Summary of the Invention

[0007] Aiming at the problems in the prior art of low utilization of tylosin biosynthesis and low fermentation units caused by bacterial autolysis and insufficient culture medium stamina, the present invention provides a feeding method for improving tylosin fermentation units.

[0008] Streptomyces fradiae was used as the fermentation strain, the seed liquid was inoculated into a fermentation tank filled with liquid fermentation medium, and a fed-batch fermentation process was adopted to add sodium malonate solution, VB12 solution and methylcobalamin solution respectively to produce tylosin. This can effectively improve the fermentation titer of tylosin and is suitable for industrial production.

[0009] The technical solution adopted to achieve the above-mentioned purpose of the invention is: a method for improving the feed-batch fermentation unit of tylosin, using Streptomyces fradiae as the fermentation strain, inoculating 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 168-170 hours, wherein the fermentation process is fed according to AC flow:

[0010] A. Ferment for 12 hours and add sodium malonate solution;

[0011] B. Add sodium malonate solution and react for 2 hours, then add VB12 solution;

[0012] C. Ferment for 30-130 hours, adding methylcobalamin solution every 12 hours.

[0013] The methylcobalamin solution is added at intervals of 12 hours, that is, the methylcobalamin solution is added once after 30-60 hours of fermentation, and the methylcobalamin solution is added twice after 60-130 hours of fermentation.

[0014] Wherein, the concentration of the added sodium malonate solution is 0.1-0.5%.

[0015] Wherein, the additional VB 12 The concentration of the solution is 0.1 to 0.3%.

[0016] Wherein, the concentration of the added methylcobalamin solution is 0.05-0.25%.

[0017] The volume of the added sodium malonate solution is 3.0-3.2% of the volume of the initial fermentation liquid.

[0018] The volume of the added VB12 solution is 2.4-2.8% of the volume of the initial fermentation liquid.

[0019] Wherein, the volume of the added methylcobalamin solution is 4.7-5.2% of the volume of the initial fermentation broth.

[0020] The fermentation medium is composed of: 500g corn flour, 500g corn gluten powder, 220g cottonseed protein powder, 110g calcium carbonate, 25g betaine hydrochloride, 150g yeast powder, 65g corn steep liquor, 35g potassium chloride, and 600g soybean oil.

[0021] The fermentation culture conditions are as follows: culture temperature 31°C, pH 6.3-6.9, stirring speed 250 rpm / min, air flow 40-50 m 3 / h.

[0022] The technical solution of the present invention has at least the following beneficial technical effects:

[0023] 1) During the biosynthesis of tylosin, small molecules such as propionic acid participate in its synthesis. Therefore, the addition of sodium malonate during the fermentation process can provide a precursor for the synthesis of tylosin, while the addition of VB12 can promote the decomposition of sodium malonate, providing a rich precursor for tylosin to promote its synthesis. Methylcobalamin is a coenzyme factor that can assist VB12 in promoting its decomposition activity. Therefore, the present invention adds VB12, which promotes the decomposition of the precursor sodium malonate, and the cofactor methylcobalamin during the optimal feeding cycle of tylosin fermentation to promote tylosin biosynthesis, thereby increasing the fermentation unit by 2 to 4%.

[0024] 2) The present invention overcomes the problems of bacterial autolysis and insufficient culture medium stamina in the late fermentation stage by feeding in the early and middle stages of fermentation, accelerates the tylosin production rate, and stabilizes the fermentation cycle. DETAILED DESCRIPTION

[0025] The Streptomyces fradiae described in the present invention can be any of the existing tylosin-producing Streptomyces fradiae, preferably Streptomyces fradiae, with a strain collection number of CGMCC No. 6717.

[0026] The reagents and raw materials used in the present invention are all commercially available. Unless otherwise specified, the experimental materials and reagents used in the following examples can be obtained from commercial sources.

[0027] The fermentation culture of the embodiments of the present invention and the comparative examples was carried out with a fermentation volume of 28 m3 (m / v); the titer detection method (HPLC method) was as follows: chromatographic column: C18 column (250 mm×4.6 mm, 5 μm); mobile phase: 0.85 mol / L sodium perchlorate solution-acetonitrile solution 6:4; pH: 2.5; flow rate: 1 mL / min; column temperature: 35°C; injection volume: 10 μL; detection wavelength: 290 nm.

[0028] Example 1

[0029] After the first-stage and second-stage seeds of Streptomyces freundii mature, they are transferred to a 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 12Methylcobalamin solution was added to the fermenter between 30 and 130 hours of operation, with additional additions every 12 hours. Between 30 and 60 hours, 680 mL of 0.05% methylcobalamin solution was added, and between 60 and 130 hours, 780 mL of 0.05% methylcobalamin solution was added. Supplementation was stopped after 130 hours of operation. The titer at the end of fermentation was 17,950 μg / mL, and the fermentation cycle was 171 hours.

[0030] Example 2

[0031] After the first and second seed cultures of Streptomyces freundii mature, they are transferred to a fermentation tank for fermentation. After the tylosin fermentation tank has been running for 12 hours, 875 mL of 0.20% sodium malonate solution is added; after 2 hours of reaction, 750 mL of 0.15% VB 12 Methylcobalamin solution was added to the fermenter between 30 and 130 hours of operation, with additional additions every 12 hours. Between 30 and 60 hours, 660 mL of 0.10% methylcobalamin solution was added, and between 60 and 130 hours, 750 mL of 0.10% methylcobalamin solution was added. Supplementation was stopped after 130 hours of operation. The titer at the end of fermentation was 17,975 μg / mL, and the fermentation cycle was 170 hours.

[0032] Example 3:

[0033] After the first and second seed cultures of Streptomyces freundii mature, they are transferred to a fermentation tank for fermentation. After the tylosin fermentation tank has been running for 12 hours, 900 mL of 0.25% sodium malonate solution is added; after 2 hours of reaction, 780 mL of 0.20% VB 12 Methylcobalamin solution was added to the fermenter between 30 and 130 hours of operation, with additional additions every 12 hours. Between 30 and 60 hours, 680 mL of 0.15% methylcobalamin solution was added, and between 60 and 130 hours, 780 mL of 0.15% methylcobalamin solution was added. Supplementation was stopped after 130 hours of operation. The titer at the end of fermentation was 18215 μg / mL, and the fermentation cycle was 169 hours.

[0034] Example 4:

[0035] After the first and second seed cultures of Streptomyces freundii mature, they are transferred to a fermentation tank for fermentation. After the tylosin fermentation tank has been running for 12 hours, 850 mL of 0.30% sodium malonate solution is added; after 2 hours of reaction, 680 mL of 0.25% VB 12Methylcobalamin solution was added to the fermenter between 30 and 130 hours of operation, with additional additions every 12 hours. Between 30 and 60 hours, 640 mL of 0.20% methylcobalamin solution was added, and between 60 and 130 hours, 680 mL of 0.20% methylcobalamin solution was added. Supplementation was stopped after 130 hours of operation. The titer at the end of fermentation was 18025 μg / mL, and the fermentation cycle was 170 hours.

[0036] Example 5:

[0037] After the first and second seed cultures of Streptomyces freundii mature, they are transferred to a fermentation tank for fermentation. After the tylosin fermentation tank has been running for 12 hours, 850 mL of 0.50% sodium malonate solution is added; after 2 hours of reaction, 680 mL of 0.30% VB 12 Methylcobalamin solution was added to the fermenter between 30 and 130 hours of operation, with additional additions every 12 hours. Between 30 and 60 hours, 640 mL of 0.25% methylcobalamin solution was added, and between 60 and 130 hours, 680 mL of 0.25% methylcobalamin solution was added. Supplementation was stopped after 130 hours of operation. The titer at the end of fermentation was 18025 μg / mL, and the fermentation cycle was 170 hours.

[0038] Comparative Example 1:

[0039] During the tylosin fermentation process, no feed was added, and the other culture conditions were the same as in Example 1. The titer was 16485 μg / mL at the end of the fermentation, and the fermentation period was 178 h.

[0040] Comparative Example 2:

[0041] During the tylosin fermentation process, 900 mL of 0.25% sodium malonate solution was added to the fermenter after 12 h of operation. The remaining culture conditions were the same as those in Example 1. The titer was 16515 μg / mL at the end of the fermentation, and the fermentation period was 177 h.

[0042] Comparative Example 3:

[0043] During the fermentation of tylosin, 900 mL of 0.25% sodium malonate solution was added to the fermenter after 12 hours of operation, and 780 mL of 0.20% VB was added after 2 hours of reaction. 12 The other culture conditions were the same as those in Example 1. The titer was 16587 μg / mL at the end of fermentation, and the fermentation period was 175 h.

[0044] Comparative Example 4:

[0045] During the tylosin fermentation process, 680 mL of 0.15% methylcobalamin solution was added to the fermenter after 30 h of operation. The other culture conditions were the same as those in Example 1. The titer was 16610 μg / mL at the end of the fermentation, and the fermentation period was 176 h.

[0046] Comparative Example 5:

[0047] During the fermentation of tylosin, 900 mL of 0.25% sodium malonate solution was added to the fermenter after 12 h of operation, and 780 mL of 0.20% VB was added after 2 h of reaction. 12 The solution was fermented for 30 hours, and 680 mL of 0.15% methylcobalamin solution was added. The other culture conditions were the same as those in Example 1. The titer was 16795 μg / mL at the end of the fermentation, and the fermentation period was 174 hours.

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 A-C fed-batch addition: A: Ferment for 12 h and add sodium malonate solution. B: React for 2 h after adding sodium malonate solution, and then 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, wherein 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 to 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, characterized in that 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 protein powder, 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: the culture temperature is 31°C, the pH value is 6.3 - 6.9, the stirring speed is 250 rpm / min, and the air flow rate is 40 - 50 m 3 / h.