Fermentation culture process for improving titer of tylosin

Abstract

The invention discloses a fermentation culture process for improving the titer of tylosin, which comprises the following steps: S1, fermentation preparation: adding a proper amount of drinking water into a barrel filled with materials, uniformly stirring and mixing, pouring the material liquid into a fermentation tank, and then adding a proper amount of drinking water; heating the tank to 60.0-80.0 DEG C, gelatinizing for 20 minutes, unscrewing an inoculation cap, adding water through an inoculation opening to fix the volume, tightening the inoculation cap, sampling, measuring the pH value, adjusting the pH value, then adding a small amount of calcium carbonate dissolved by water and soybean oil, and starting self-control sterilization; after sterilization is completed, the temperature of the tank is reduced to 30.0 + / -2.0 DEG C before inoculation is prepared, inoculation is carried out according to a plan, and a seed solution is transferred into a fermentation tank. According to the method, the gelatinization temperature, the sterilization parameters and the inoculation conditions are accurately regulated and controlled, so that a proper initial environment is provided for thallus growth; the tank temperature, the air flow and the stirring rotating speed are regulated and controlled in sections, and the metabolic requirements of thalli in different growth stages are met.

Description

Technical Field

[0001] This invention relates to the field of bio-fermentation technology, and in particular to a fermentation culture process for improving the potency of tylosin. Background Technology

[0002] Tylosin, also known as tylosin, is a macrolide antibiotic obtained in the United States in the 1950s from the culture medium of *Streptomyces fradiae*. Tylosin is a white, plate-like crystal, slightly soluble in water and alkaline. In aqueous solution at 25°C and pH 5.5-7.5, tylosin has a shelf life of up to 3 months. As a highly effective, residue-free antibacterial and growth-promoting agent specifically for poultry and livestock, tylosin is particularly effective against Gram-positive bacteria and mycoplasma infections, and also significantly promotes the growth of poultry and livestock. It is a key veterinary drug product in the national development plan.

[0003] Dissolved oxygen is an important factor affecting metabolism during fermentation. Many factors influence dissolved oxygen, including aeration rate, stirring speed, and temperature.

[0004] Currently, a constant temperature control method of 30℃ is commonly used in the fermentation production of tylosin. However, antibiotic fermentation generally cannot rely on a single optimal culture temperature throughout the entire fermentation cycle. The optimal growth temperature for tylosin is 32℃, while the optimal temperature for tylosin synthesis is around 28℃. Therefore, using a constant temperature control method will affect the fermentation potency.

[0005] Stirring speed affects the fermentation potency of tylosin. If the stirring speed is too fast in the early stages, it will damage its structure; if the stirring speed is too slow in the later stages, it will inhibit cell growth and thus affect the fermentation yield. Therefore, adjusting the stirring speed is essential.

[0006] Aeration rate regulation affects the fermentation potency of tylosin. Excessive airflow generates excessive shear force, damaging the cell structure and adversely affecting the cell, thus impacting fermentation potency. Therefore, regulating the aeration rate is crucial. Summary of the Invention

[0007] The purpose of this invention is to solve the above-mentioned technical problems by proposing a fermentation culture process to improve the potency of tylosin.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A fermentation culture process for improving the potency of tylosin includes the following steps: S1, Fermentation preparation: Add an appropriate amount of drinking water to the bucket containing the material, stir and mix well, then pour the liquid into the fermentation tank, and add an appropriate amount of drinking water; raise the tank temperature to 60.0-80.0℃ and gelatinize for 20 minutes, unscrew the inoculation cap and add water through the inoculation port to make up the volume, tighten the inoculation cap, take a sample to measure the pH and adjust the pH, then add a small amount of water-dissolved calcium carbonate and soybean oil, and start the automatic sterilization; after sterilization is completed, lower the tank temperature to 30.0±2.0℃ before inoculation, inoculate according to the plan and transfer the seed liquid into the fermentation tank; S2, segmented cultivation: control the tank pressure at 0.04-0.05MPa, adjust the tank temperature, air flow and stirring speed according to the fermentation stage, and the cultivation time is 160-180h; S3, Fermentation Termination: Take samples every 6-8 hours for testing. Stop fermentation when tylosin A component >85%, tylosin C component <5%, cell concentration 30-50%, fermentation units above 13000u / mL, and pH value 6.0-7.0.

[0009] Preferably, the temperature of the tank in S2 is controlled in segments as follows: 32±1℃ for 0-40h, 28±2℃ for 40-120h, 30±2℃ from 120h until component conversion, and 37±2℃ from the start of component conversion to the end of fermentation.

[0010] Preferably, the segmented control method of airflow in S2 is: 30-32m for 0-10h. 3 / h, after 10 hours the tank will be 34-36m. 3 / h.

[0011] Preferably, the segmented control method of the stirring speed in S2 is as follows: 20-25HZ for 0-5h, 30-40HZ for 5-20h, and 45-55HZ for releasing the mixture after 20h.

[0012] Preferably, the conditions for automatic sterilization in S1 are as follows: based on the characteristics of the material in the fermenter, the sterilization temperature is maintained at 115-121℃, the sterilization pressure at 0.10-0.15MPa, and the sterilization time at 20-30 minutes by the automatic control system.

[0013] Preferably, the inoculation amount of the seed liquid in S1 is 5-10% of the volume of the culture medium in the fermenter, and the mycelial concentration of the seed liquid before inoculation is 25-35%.

[0014] Preferably, the pH adjuster used in step S1 is hydrochloric acid or sodium hydroxide, and the pH value is controlled at 6.5-7.0 after adjustment.

[0015] Preferably, the amount of calcium carbonate added in S1 is 0.5-1.0% of the total mass of the materials, and the amount of soybean oil added is 1.0-2.0% of the total mass of the materials.

[0016] Preferably, the criterion for determining the component transformation in S2 is: when sampling and testing reveal that the proportion of tylosin A component begins to rise rapidly and the proportion of tylosin C component begins to fall rapidly, the component transformation is determined to have begun.

[0017] Compared with the prior art, the beneficial effects of this invention are as follows: Significantly improved fermentation potency: By precisely controlling the gelatinization temperature, sterilization parameters and inoculation conditions, a suitable initial environment for cell growth was provided; the tank temperature, air flow and stirring speed were controlled in stages to match the metabolic needs of different growth stages of the cells, ultimately enabling the tylosin fermentation unit to reach 13200-13400u / mL, which is more than 10% higher than the existing process.

[0018] Product quality optimization: By controlling the temperature during the component conversion period and using precise termination criteria, the conversion of component C to component A is effectively promoted, so that after fermentation, the proportion of component A in tylosin reaches 87-90% and the proportion of component C is less than 5%, significantly improving product purity and safety.

[0019] Enhanced production stability: This invention quantifies and defines the parameters of each stage of fermentation, reducing human error. Through repeated experiments, the batch-to-batch variation rate of the process is less than 3%, and production stability is greatly improved.

[0020] Improved production efficiency: The optimized process controls the total cultivation time to 160-180 hours, which is 10-20 hours shorter than some existing processes. At the same time, it reduces the consumption of auxiliary materials and lowers production costs, resulting in significant economic benefits. Detailed Implementation

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0022] This invention discloses a fermentation culture process for improving the potency of tylosin, specifically including the following steps: 1. Fermentation preparation (1) Material mixing and transfer: Add an appropriate amount of drinking water to the bucket containing the fermentation material, and stir for 15-20 minutes with a mechanical stirring device until the material is completely mixed and a uniform liquid is formed; pour the liquid into the fermentation tank through the conveying pipe, and add drinking water to the tank according to the volume of the fermentation tank so that the initial volume of the liquid reaches 60-70% of the rated volume of the fermentation tank.

[0023] (2) Gelatinization treatment: Start the heating system of the fermenter and raise the temperature of the tank to 60.0-80.0℃ at a rate of 2-3℃ / min. Then, keep the tank at this temperature for 20 minutes to gelatinize. During the gelatinization process, maintain the stirring speed at 15-20HZ to ensure that the liquid is heated evenly.

[0024] (3) Volume adjustment and pH adjustment: Turn off the heating system, unscrew the inoculation cap of the fermenter, add drinking water through the inoculation port to make up the volume so that the liquid volume reaches 80-85% of the rated volume of the fermenter; after tightening the inoculation cap, take 50-100mL of liquid through the sampling port, use a pH meter to detect its pH value, and use hydrochloric acid or sodium hydroxide solution with a concentration of 1-2mol / L to adjust the pH value to 6.5-7.0.

[0025] (4) Addition and sterilization of auxiliary materials: Weigh calcium carbonate at 0.5-1.0% of the total mass of the material, dissolve it in 5-10 times the mass of drinking water, and inject it into the fermentation tank together with soybean oil at 1.0-2.0% of the total mass of the material through the auxiliary material addition port; start the automatic sterilization system, and based on the viscosity, composition and other characteristics of the material in the tank, automatically adjust the heating power and pressure through the automatic control system to maintain the sterilization temperature at 115-121℃, the sterilization pressure at 0.10-0.15MPa, and the sterilization time at 20-30 minutes; after sterilization, turn on the cooling system to reduce the tank temperature to 30.0±2.0℃.

[0026] (5) Inoculation operation: Select the seed liquid of Streptomyces freundii in the logarithmic growth phase, test its mycelial concentration, and ensure that the mycelial concentration is 25-35%; transfer the seed liquid into the fermenter through the inoculation port at an inoculation amount of 5-10% of the culture medium volume in the fermenter. Maintain a slight positive pressure in the tank during the inoculation process to prevent contamination by other microorganisms.

[0027] 2. Segmented cultivation After inoculation, activate the fermenter's automatic control system to maintain a stable pressure of 0.04-0.05 MPa. Adjust the tank temperature, air flow rate, and stirring speed according to the fermentation stage. The total cultivation time is 160-180 hours. Specific control parameters are as follows: (1) Temperature control: 0-40h is the vigorous growth period of the mycelium, and the temperature is controlled at 32±1℃ to promote rapid mycelial proliferation; 40-120h is the initial stage of product synthesis, and the temperature is lowered to 28±2℃ to inhibit the generation of miscellaneous metabolites; 120h-before component conversion is the product accumulation period, and the temperature is adjusted to 30±2℃ to accelerate the synthesis of tylosin precursor substances; the period from the start of component conversion to the end of fermentation is the component optimization period, and the temperature is raised to 37±2℃ to promote the conversion of component C to component A. The criteria for judging component conversion are: sampling and testing every 6-8h, and when the proportion of tylosin component A begins to rise rapidly and the proportion of component C begins to fall rapidly, it is judged that component conversion has started.

[0028] (2) Airflow regulation: During the mycelial adaptation period of 0-10h, the airflow should be controlled at 30-32m³ / h. 3 / h, maintain the dissolved oxygen concentration in the tank at 20-30%; 10h - after the tank is opened, it is the period of mycelial growth and product synthesis, and the air flow rate is increased to 34-36m 3 / h, to keep the dissolved oxygen concentration stable at 30-40%, meeting the metabolic needs of the bacteria.

[0029] (3) Stirring speed control: 0-5h is the seed germination period, and a low stirring speed of 20-25HZ is used to avoid damage to the mycelium; 5-20h is the rapid growth period of mycelium, and the speed is increased to 30-40HZ to enhance the mixing effect of the liquid; 20h-discharge is the product synthesis and conversion period, and the speed is maintained at 45-55HZ to ensure uniform distribution of oxygen and nutrients.

[0030] 3. Fermentation terminated During fermentation, the fermentation liquid was sampled every 6-8 hours through the sampling port. The proportions of tylosin A and C components were determined using high-performance liquid chromatography (HPLC), the cell concentration was determined using a hemocytometer, and the fermentation units were determined using a microbial assay. The pH value of the fermentation liquid was also measured. When the test results met the following criteria: tylosin A component >85%, tylosin C component <5%, cell concentration 30-50%, fermentation units above 13000 u / mL, and pH value 6.0-7.0, the heating, stirring, and aeration systems of the fermenter were shut off, fermentation was stopped, and a high-potency tylosin fermentation broth was obtained.

[0031] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited by the embodiments.

[0032] Example 1 A fermentation culture process for improving the potency of tylosin includes the following steps: Fermentation preparation: Add 200L of drinking water to a bucket containing 100kg of fermentation material, stir for 20 minutes until well mixed, pour the liquid into a 500L fermentation tank, and add drinking water to 300L; heat to 70℃ for gelatinization for 20 minutes at 18HZ; unscrew the inoculation cap and add water to make up to 420L, take a sample to measure the pH, which is 6.2, and adjust to 6.8 with 1mol / L sodium hydroxide solution; add 0.8kg of calcium carbonate (dissolved in 8L of water) and 1.5kg of soybean oil, start automatic sterilization, and maintain sterilization at 121℃ and 0.15MPa for 25 minutes; cool to 30℃, select a seed liquid with a mycelial concentration of 30%, and transfer it into the fermentation tank at an inoculation rate of 5% (21L).

[0033] Segmented culture: Maintain tank pressure at 0.045 MPa; tank temperature controlled at 32℃ for 0-40h, 28℃ for 40-120h, 30℃ from 120h until component conversion, and 37℃ from the start to the end of component conversion; air flow rate 31 m³ / h for 0-10h.3 / h, 10h - release tank 35m 3 / h; stirring speed 0-5h 22HZ, 5-20h 35HZ, 20h-release 50HZ; incubation 170h.

[0034] Fermentation termination: Samples were taken every 6 hours for testing. Fermentation was stopped when component A reached 88%, component C reached 4%, cell concentration reached 40%, fermentation units reached 13300 u / mL, and pH reached 6.5.

[0035] Example 2 A fermentation culture process for improving the potency of tylosin includes the following steps: Fermentation preparation: Add 300L of drinking water to a bucket containing 150kg of fermentation material, stir for 18 minutes until well mixed, pour the liquid into an 800L fermentation tank, and add drinking water to 480L; heat to 65℃ for gelatinization for 20 minutes at 15HZ; unscrew the inoculation cap and add water to make up to 680L, take a sample to measure the pH, which is 6.0, and adjust to 7.0 with 2mol / L sodium hydroxide solution; add 1.2kg of calcium carbonate (dissolved in 12L of water) and 2.5kg of soybean oil, start automatic sterilization, and maintain sterilization at 118℃ and 0.12MPa for 30 minutes; cool to 31℃, select seed liquid with a mycelial concentration of 35%, and transfer it into the fermentation tank at an inoculation rate of 8% (54.4L).

[0036] Segmented culture: Maintain tank pressure at 0.04 MPa; tank temperature controlled at 33℃ for 0-40h, 29℃ for 40-120h, 31℃ from 120h until component conversion, and 38℃ from the start to the end of component conversion; air flow rate 30 m³ / h for 0-10h. 3 / h, 10h - Release tank 34m 3 / h; stirring speed 0-5h 20HZ, 5-20h 30HZ, 20h-release 45HZ; incubation 165h.

[0037] Fermentation termination: Samples were taken every 8 hours for testing. Fermentation was stopped when component A reached 87%, component C reached 3.5%, cell concentration reached 35%, fermentation units reached 13200 u / mL, and pH reached 6.3.

[0038] Example 3 A fermentation culture process for improving the potency of tylosin includes the following steps: Fermentation preparation: Add 160L of drinking water to a bucket containing 80kg of fermentation material, stir for 15 minutes until well mixed, pour the liquid into a 400L fermentation tank, and add drinking water to 240L; heat to 75℃ for gelatinization for 20 minutes at 20HZ; unscrew the inoculation cap and add water to make up to 340L, take a sample to measure the pH, which is 6.9, and adjust to 6.5 with 1mol / L hydrochloric acid solution; add 0.4kg of calcium carbonate (dissolved in 4L of water) and 1.6kg of soybean oil, start automatic sterilization, and maintain sterilization at 115℃ and 0.10MPa for 20 minutes; cool to 29℃, select seed liquid with a mycelial concentration of 25%, and transfer it into the fermentation tank at an inoculation rate of 10% (34L).

[0039] Segmented culture: Maintain tank pressure at 0.05 MPa; tank temperature controlled at 31℃ for 0-40h, 27℃ for 40-120h, 29℃ from 120h until component conversion, and 36℃ from the start to the end of component conversion; air flow rate at 32 m³ / h for 0-10h. 3 / h, 10h - Release tank 36m 3 / h; stirring speed 0-5h 25HZ, 5-20h 40HZ, 20h-release 55HZ; incubation 175h.

[0040] Fermentation termination: Samples were taken every 7 hours for testing. Fermentation was stopped when component A reached 90%, component C reached 4.5%, cell concentration reached 45%, fermentation units reached 13400 u / mL, and pH reached 6.8.

[0041] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A fermentation culture process for improving the potency of tylosin, characterized in that, Includes the following steps: S1, Fermentation preparation: Add an appropriate amount of drinking water to the bucket containing the material, stir and mix well, then pour the liquid into the fermentation tank, and add an appropriate amount of drinking water; raise the tank temperature to 60.0-80.0℃ and gelatinize for 20 minutes, unscrew the inoculation cap and add water through the inoculation port to make up the volume, tighten the inoculation cap, take a sample to measure the pH and adjust the pH, then add a small amount of water-dissolved calcium carbonate and soybean oil, and start the automatic sterilization; after sterilization is completed, lower the tank temperature to 30.0±2.0℃ before inoculation, inoculate according to the plan and transfer the seed liquid into the fermentation tank; S2, segmented cultivation: control the tank pressure at 0.04-0.05MPa, adjust the tank temperature, air flow and stirring speed according to the fermentation stage, and the cultivation time is 160-180h; S3, Fermentation Termination: Take samples every 6-8 hours for testing. Stop fermentation when tylosin A component >85%, tylosin C component <5%, cell concentration 30-50%, fermentation units above 13000u / mL, and pH value 6.0-7.

0.

2. The fermentation culture process for improving the potency of tylosin according to claim 1, characterized in that, The temperature control method of the tank in S2 is as follows: 32±1℃ for 0-40h, 28±2℃ for 40-120h, 30±2℃ from 120h to before component conversion, and 37±2℃ from the start of component conversion to the end of fermentation.

3. The fermentation culture process for improving the potency of tylosin according to claim 1, characterized in that, The segmented control method of airflow in S2 is as follows: 30-32m for 0-10h. 3 / h, after 10 hours the tank will be 34-36m. 3 / h.

4. The fermentation culture process for improving the potency of tylosin according to claim 1, characterized in that, The segmented control method of the stirring speed in S2 is as follows: 20-25HZ for 0-5h, 30-40HZ for 5-20h, and 45-55HZ for 20h after discharge.

5. The fermentation culture process for improving the potency of tylosin according to claim 1, characterized in that, The conditions for automatic sterilization in S1 are as follows: based on the characteristics of the material inside the fermenter, the sterilization temperature is maintained at 115-121℃, the sterilization pressure at 0.10-0.15MPa, and the sterilization time at 20-30 minutes by the automatic control system.

6. The fermentation culture process for improving the potency of tylosin according to claim 1, characterized in that, The inoculation amount of the seed liquid in S1 is 5-10% of the volume of the culture medium in the fermenter, and the mycelial concentration of the seed liquid before inoculation is 25-35%.

7. The fermentation culture process for improving the potency of tylosin according to claim 1, characterized in that, The pH adjuster used in S1 is hydrochloric acid or sodium hydroxide, and the pH value is controlled between 6.5 and 7.0 after adjustment.

8. The fermentation culture process for improving the potency of tylosin according to claim 1, characterized in that, The amount of calcium carbonate added in S1 is 0.5-1.0% of the total mass of the material, and the amount of soybean oil added is 1.0-2.0% of the total mass of the material.

9. The fermentation culture process for improving the potency of tylosin according to claim 1, characterized in that, The criteria for determining the component transformation in S2 are as follows: when sampling and testing reveal that the proportion of tylosin A component begins to rise rapidly and the proportion of tylosin C component begins to fall rapidly, the component transformation is determined to have begun.