CN114606282A - Method for reducing 4' -oxygen-isovaleryl tylosin component by changing dissolved oxygen in tylosin fermentation process - Google Patents

Method for reducing 4' -oxygen-isovaleryl tylosin component by changing dissolved oxygen in tylosin fermentation process Download PDF

Info

Publication number
CN114606282A
CN114606282A CN202210092868.3A CN202210092868A CN114606282A CN 114606282 A CN114606282 A CN 114606282A CN 202210092868 A CN202210092868 A CN 202210092868A CN 114606282 A CN114606282 A CN 114606282A
Authority
CN
China
Prior art keywords
tylosin
fermentation
dissolved oxygen
isovaleryl
component
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202210092868.3A
Other languages
Chinese (zh)
Other versions
CN114606282B (en
Inventor
时鹏飞
黄科学
高岭
杨耀刚
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Qilu Pharmaceutical Inner Mongolia Co ltd
Original Assignee
Qilu Pharmaceutical Inner Mongolia Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Qilu Pharmaceutical Inner Mongolia Co ltd filed Critical Qilu Pharmaceutical Inner Mongolia Co ltd
Priority to CN202210092868.3A priority Critical patent/CN114606282B/en
Publication of CN114606282A publication Critical patent/CN114606282A/en
Application granted granted Critical
Publication of CN114606282B publication Critical patent/CN114606282B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
    • C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin

Landscapes

  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

The invention relates to a method for reducing 4' -oxygen-isovaleryl tylosin component by changing dissolved oxygen in the fermentation process of tylosin, wherein streptomyces thermotolerans is used as a production strain to carry out fermentation production of tylosin in a fermentation tank, the pH value is regulated at the initial stage of fermentation, then tylosin dry powder and L-leucine are supplemented, and glucose solution is supplemented until the fermentation is finished; and controlling the dissolved oxygen of different fermentation times in the whole fermentation process, wherein the dissolved oxygen is more than 50% in 0-35 h, the dissolved oxygen is 20-30% in 35-70 h, and the dissolved oxygen is more than 70% in the period from 70h to tank placement. The invention changes the air distribution form in the tylosin fermentation process, controls the dissolved oxygen amount in different fermentation time periods, solves the problems of hypha aging autolysis and large foam caused by the pressure increase of a single stirrer of the tylosin, effectively reduces the content of the 4' -O-isovaleryl tylosin component in the fermentation liquor, further improves the content of the tylosin in the fermentation liquor, and realizes that the proportion of the tylosin component in the fermentation liquor is higher than 84%.

Description

Method for reducing 4' -oxygen-isovaleryl tylosin component by changing dissolved oxygen in tylosin fermentation process
Technical Field
The invention relates to a method for reducing 4' -oxygen-isovaleryl tylosin component by changing dissolved oxygen in a tylosin fermentation process, belonging to the technical field of fermentation.
Background
Tylosin, also known as Acetylisovaleryltylosin (AIV), 3-O-acetyl-4' -O-isovaleryltartrate, has a structure shown in formula I, and is a special macrolide antibiotic for livestock. As a brand new macrolide antibiotic, the tavermectin overcomes the defects of other macrolide medicines, has the advantages of high efficiency, low toxicity, low residue, no generation of drug resistance among the macrolide medicines and the like, and is popular in the market.
Figure RE-GDA0003596143590000011
Many tylosin derivatives have been produced in the prior art mainly by the addition of tylosin and different acyl donors, including acetylisovaleryltylosin, i.e. tylosin, as a third generation upgrade of tylosin. By selective acylation modification of tylosin at the 3' -OH and 4' -OH positions, it has been elucidated that acetylisovaleryltylosin genes are synthesized including two genes acyA and acyB which are responsible for the acylation function on the basis of tylosin, wherein acyB comprises Bl and B2, wherein gene acyA is mainly responsible for the acetylation function, gene acyB is mainly responsible for the isovalerylation function, gene B1 is responsible for the isovalerylation of tylosin at the 4' -OH, and gene B2 is a positive regulator which activates B1. The specific biosynthesis pathway is as follows:
Figure RE-GDA0003596143590000012
fermentation biosynthetic pathway of AIV
The tylosin fermentation broth is complex in composition, and contains 3-O-acetyl-4 ' -O-isovaleryl tylosin (T2i5) as a main component, but also contains intermediate products such as 3-O-acetyl tylosin (T20), 4' -O-isovaleryl tylosin (T0i5), 3-O-acetyl-4 ' -O-isovaleryl ralosin (R2i5), and the like, and the acetyl isovaleryl tylosin related substances are shown in the following table.
Figure RE-GDA0003596143590000021
The current main fermentation method of the tulathromycin is as follows: after inoculation of a fermentation tank, after the pH naturally rises to 7.50, acid is continuously supplemented, the offline is controlled to be between 7.45 and 7.55, the pH of fermentation liquor is naturally metabolized and reduced to be below 6.9 when the fermentation time is 35 to 45 hours, then substrate tylosin dry powder and L-leucine are fed in continuously, and glucose solution is supplemented to ensure that the pH is between 6.95 and 7.25 until the fermentation is finished. In the prior art, the combination of the stirring paddles of the fermentation tank is generally three-layer stirring, wherein a parabolic stirrer is adopted at the bottom layer, a straight blade disc stirrer is adopted at the middle layer, and an axial flow type stirring paddle is adopted at the upper layer. Although the combination has higher energy consumption and gas-liquid dispersion effect, the combination is not beneficial to the liquid level control of fermentation, and larger foam is easily generated after the surface layer of the liquid level is stirred and contacted with the upper layer, thereby further influencing the volume of material supplement and tank discharge. Meanwhile, the existing fermentation process only analyzes liquid phase results of T20 and T2i5 in the metabolic process of the tylosin and further controls the feeding rate of the next step, but for the tylosin, hypha is easy to break and generate foam due to mechanical stress such as stirring and the like, so that secondary metabolism is influenced, the volume is controlled to be below 85% of the volume (close to the upper layer of stirring), and the fermentation liquid surface and the upper layer of stirring in the later period are contacted to aggravate the rolling of fermentation liquid, so that the foam is rapidly increased, the fermentation state is uncontrollable, and the content of 4' -oxygen-isovaleryl tylosin is overhigh.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for reducing the 4' -O-isovaleryl tylosin component by changing the dissolved oxygen in the fermentation process of tylosin.
The technical scheme of the invention is as follows:
a method for changing dissolved oxygen in a tylosin fermentation process to reduce 4' -oxygen-isovaleryl tylosin component comprises the steps of carrying out tylosin fermentation production in a fermentation tank by taking streptomyces thermotolerans as a production strain, regulating and controlling the pH of fermentation liquor to 7.00-7.50 at the initial stage of fermentation, then feeding supplemented substrate tylosin dry powder and L-leucine, and then supplementing a glucose solution to ensure that the pH of the fermentation liquor is between 6.95-7.25 until the fermentation is finished;
controlling the dissolved oxygen of the fermentation liquor in different fermentation times in the whole fermentation process, controlling the dissolved oxygen of the fermentation liquor to be more than 50% within 0-35 h, controlling the dissolved oxygen of the fermentation liquor to be 20-30% within 35-70 h, and controlling the dissolved oxygen of the fermentation liquor to be more than 70% during 70h to tank discharge.
According to the optimization of the invention, the fermentation tank comprises a fermentation tank body, wherein a stirring device is arranged in the fermentation tank body, and an annular air distributor is arranged below the stirring device; the annular air distributor comprises an annular pipe and an air inlet pipe, the annular pipe is communicated with the air inlet pipe, and the annular pipe is uniformly provided with 4 large air outlet holes and 30 small air outlet holes; the intake pipe stretches out outside the fermentation cylinder jar body, and fermentation cylinder jar body bottom is provided with the bin outlet.
Further preferably, the stirring device is fixed on a motor base at the top end of the fermentation tank body, a motor is arranged on the motor base, a speed reducer is connected to the side face of the motor, and a sealing ring, a stirring shaft penetrating through a top cover of the fermentation tank, a coupler, a straight blade disc stirrer, a middle bearing and a parabolic stirrer are sequentially connected below the speed reducer.
Further preferably, the distance between the parabolic stirrer and the annular air distributor is 5-20 cm, and the width of the parabolic stirrer is the same as the diameter of the annular air distributor.
Further preferably, the annular tube has a wall thickness phi 125 x 4mm and is fixed to the fermentation tank by a flange.
Further preferably, the size of the large air outlet holes is phi 22mm x 4, and the size of the small air outlet holes is phi 18mm x 30.
Further preferably, the sum of the areas of the large air outlet and the small air outlet is less than or equal to 80% of the sectional area of the annular pipe.
According to the invention, the material culture medium adopted in the fermentation production is preferably preparedThe ratio is as follows: 10-20 g/L of starch, 1-10 g/L of yeast extract, 0.1-0.8 g/L of fish extract and K2HPO4 0.01～0.5g/L。
According to the invention, preferably, the initial fermentation stage is to adjust the pH by using a mixture of glycerol and citric acid;
further preferably, the mass ratio of glycerol to citric acid is 1: 1.
According to the invention, the mass ratio of the tylosin dry powder to the L-leucine is (5-6): 1, wherein the dosage of the tylosin dry powder is 800-1000 mu g/mL.
Has the advantages that:
1. the invention changes the air distribution form in the tylosin fermentation process, effectively controls the dissolved oxygen in different fermentation time periods, solves the problems of hypha aging autolysis and large foam caused by the pressure increase of a single stirrer of the tylosin, effectively reduces the component proportion of 4' -O-isovaleryl tylosin in the fermentation liquor, further improves the component proportion of the tylosin in the fermentation liquor, and realizes that the component proportion of the tylosin in the fermentation liquor is more than 84 percent.
2. The invention changes the traditional fermentation process that the straight pipe is introduced with air from the bottom of the fermentation tank, the axial flow type stirring paddle on the upper layer in the fermentation tank is removed, the straight pipe air distributor is changed into the annular air distributor, the injection pressure is improved after the air is divided, and the diameter of the air bubbles is far smaller than that of the straight pipe injection type air outlet after the air passes through the air distributor provided with the large air outlet and the small air outlet. Meanwhile, the air outlet is downwards sprayed, and after the air outlet is in stirring contact with the lower layer, the air outlet can be smashed by the stirring paddle and forms an 'air pocket' on the back surface of the paddle blade, and after the air pocket is smashed, an enriched small air bubble area can be formed, so that the contact area of the gas and the liquid is further increased, and the dissolved oxygen content in the fermentation liquor in the later period is improved.
3. The invention removes the upper layer stirring, realizes that the power consumption of a single batch of fermentation tanks is reduced by 5-10%, and effectively reduces the production cost.
Drawings
FIG. 1 is a schematic view of a fermenter according to the present invention.
FIG. 2 is a schematic view of the configuration of the annular air distributor of the present invention.
FIG. 3 is a graph showing the effect of different stirring frequencies on the 4 "-O-isovaleryl tylosin (T0i5) composition of tylosin.
FIG. 4 shows the change in cell density under different dissolved oxygen conditions.
FIG. 5 shows the change of 4 "-O-isovaleryl tylosin (T0i5) under different dissolved oxygen conditions.
FIG. 6 shows the changes of 3-O-acetyl-4' -O-isovaleryl tylosin (T2i5) under the same dissolved oxygen conditions.
FIG. 7 is a comparison of potency under different dissolved oxygen conditions.
In the figure: 1. fermentation cylinder jar body, 2, motor cabinet, 3, motor, 4, reduction gear, 5, sealing washer, 6, fermentation cylinder top cap, 7, (mixing) shaft, 8, shaft coupling, 9, straight leaf disc agitator, 10, intermediate bearing, 11, parabola type agitator, 12, intake pipe, 13, annular air distributor, 14, discharge gate, 15, the ring pipe, 16, big venthole, 17, little venthole.
Detailed Description
The technical solution of the present invention is further described below with reference to the following examples and drawings, but the scope of the present invention is not limited thereto. Reagents and medicines involved in the examples are all common commercial products unless otherwise specified; the experimental procedures referred to in the examples are those conventional in the art unless otherwise specified.
The equipment used in the examples is conventional and existing equipment.
The raw materials used in the examples were starch from jin he ltd, indomongol county, glucose from bio-technology ltd, monyof, inner mongol, fish extract from bio-engineering ltd, conlino, jenan, and tylosin dry powder from zilu pharmaceuticals (inner mongol).
As shown in figures 1-2, the fermentation tank used in the fermentation process of the invention comprises a fermentation tank body 1, a stirring device is arranged in the fermentation tank body 1, the stirring device comprises a motor base 2 fixed at the top end of the fermentation tank body 1, a motor 3 is arranged on the motor base 2, and a speed reducer 4 is connected to the side surface of the motor 3.
And a sealing ring 5, a stirring shaft 7 penetrating through a fermentation tank top cover 6, a coupler 8, a straight blade disc stirrer 9, an intermediate bearing 10 and a parabolic stirrer 11 are sequentially connected below the speed reducer 4. An annular air distributor 13 is arranged at a position 10cm below the parabolic stirrer 11; the annular air distributor 13 comprises an annular pipe 15 and an air inlet pipe 12, the annular pipe 15 is communicated with the air inlet pipe 12, and 4 large air outlet holes 16 and 30 small air outlet holes 17 are uniformly formed in the annular pipe 15; the air inlet pipe 12 extends out of the fermentation tank body 1, and a discharge hole 14 is formed in the bottom of the fermentation tank body 1.
The wall thickness of the annular air distributor 13 is phi 125 x 4mm, and the annular air distributor is fixed on the fermentation tank through a flange. The size of the large air outlet 16 is phi 22mm, and the size of the small air outlet 17 is phi 18 mm. The sum of the areas of the large air outlet holes 16 and the small air outlet holes 17 is less than or equal to 80 percent of the sectional area of the annular pipe 15.
Example 1
A method for changing dissolved oxygen in a tylosin fermentation process to reduce 4' -oxygen-isovaleryl tylosin component comprises the steps of carrying out tylosin fermentation production in a fermentation tank by taking streptomyces thermotolerans as a production strain, adjusting and controlling the pH to 7.0 by adopting a mixture of glycerol and citric acid at the initial stage of fermentation, feeding supplemented substrates of tylosin dry powder and L-leucine, causing metabolism change due to substrate supplementation, starting to increase the fermentation pH, and then supplementing a glucose solution to enable the pH of a fermentation liquid to be 7.2 until the fermentation is completed;
and (3) combining the growth condition of hypha in the whole fermentation process, adjusting the stirring frequency, and controlling the dissolved oxygen metabolism of different fermentation times step by step, wherein the stirring frequency is controlled to be 0-30 Hz within 0-35 h, the dissolved oxygen content of the fermentation liquor is controlled to be 55%, the stirring frequency is controlled to be 50 Hz within 35-70 h to maintain the dissolved oxygen content of the fermentation liquor to be 30%, and the dissolved oxygen content of the fermentation liquor with the stirring frequency of 40-50 Hz is controlled to be 75% during 70h to tank release.
The material culture medium adopted in the fermentation production comprises the following components in percentage by weight: 15g/L of starch, 5g/L of yeast extract, 0.5g/L of fish extract and K2HPO40.3 g/L. The mass ratio of the glycerol to the citric acid is 1: 1.
The mass ratio of the tylosin dry powder to the L-leucine is 5:1, wherein the dosage of the tylosin dry powder is 900 mu g/mL.
A fermentation liquid after fermentation is sampled and measured, the titer is 24265 mu g/mL, the component proportion of the tylosin (T2i5) is 84.17 percent, and the component proportion of the 4' -O-isovaleryl tylosin (T0i5) is 4 percent.
Example 2
A method for changing dissolved oxygen in a tylosin fermentation process to reduce 4' -oxygen-isovaleryl tylosin component comprises the steps of carrying out tylosin fermentation production in a fermentation tank by taking streptomyces thermotolerans as a production strain, adjusting and controlling the pH to 7.0 by adopting a mixture of glycerol and citric acid at the initial stage of fermentation, feeding supplemented substrates, namely tylosin dry powder and L-leucine, starting to increase the pH, and supplementing a glucose solution to enable the pH of a fermentation liquor to be 7.1 until the fermentation is finished;
and (3) combining the growth condition of hypha in the whole fermentation process, adjusting the stirring frequency, and controlling the dissolved oxygen metabolism of different fermentation times step by step, wherein the stirring frequency is controlled to be 0-30 Hz within 0-35 h, the dissolved oxygen content of the fermentation liquor is controlled to be 60%, the stirring frequency is controlled to be 50 Hz within 35-70 h to maintain the dissolved oxygen content of the fermentation liquor to be 20%, and the dissolved oxygen content of the fermentation liquor with the stirring frequency of 40-50 Hz is controlled to be 80% during 70h to tank release.
The material culture medium adopted in the fermentation production comprises the following components in proportion: 15g/L of starch, 5g/L of yeast extract, 0.5g/L of fish extract and K2HPO40.3 g/L. The mass ratio of the glycerol to the citric acid is 1: 1.
The mass ratio of the tylosin dry powder to the L-leucine is 6:1, wherein the dosage of the tylosin dry powder is 800 mu g/mL.
Example 3
A method for changing dissolved oxygen in a tylosin fermentation process to reduce 4' -oxygen-isovaleryl tylosin component comprises the steps of carrying out tylosin fermentation production in a fermentation tank by taking streptomyces thermotolerans as a production strain, adjusting and controlling the pH to 7.2 by adopting a mixture of glycerol and citric acid at the initial stage of fermentation, feeding supplemented substrate tylosin dry powder and L-leucine, and then supplementing a glucose solution to enable the pH of a fermentation broth to be 7.0 until the fermentation is finished;
and (3) combining the growth condition of hypha in the whole fermentation process, adjusting the stirring frequency, and controlling the dissolved oxygen metabolism of different fermentation times step by step, wherein the stirring frequency is controlled to be 0-30 Hz within 0-35 h, the dissolved oxygen content of the fermentation liquor is controlled to be 65%, the stirring frequency is controlled to be 50 Hz within 35-70 h to maintain the dissolved oxygen content of the fermentation liquor to be 25%, and the dissolved oxygen content of the fermentation liquor with the stirring frequency of 40-50 Hz is controlled to be 85% during 70h to tank release.
The material culture medium adopted in the fermentation production comprises the following components in proportion: 15g/L of starch, 5g/L of yeast extract, 0.5g/L of fish extract and K2HPO40.3 g/L. The mass ratio of the glycerol to the citric acid is 1: 1.
The mass ratio of the tylosin dry powder to the L-leucine is 6:1, wherein the dosage of the tylosin dry powder is 1000 mu g/mL.
Comparative example 1
A method for reducing 4' -O-isovaleryl tylosin component by changing dissolved oxygen in the fermentation process of tylosin is the same as that in example 1, and is different from the method in that an upper axial flow type stirring paddle in the prior art is reserved.
A fermentation liquid after fermentation is sampled and measured, the titer is 20116 mu g/mL, the component proportion of the tavermectin (T2i5) is 81.98 percent, and the component proportion of the 4' -O-isovaleryl tylosin (T0i5) is 6.59 percent.
Comparative example 2
A method for reducing 4' -O-isovaleryl tylosin component by changing dissolved oxygen in a tylosin fermentation process is the same as that in example 1, and is characterized in that stirring frequency is adjusted so that the dissolved oxygen in the period from 0-35 h and 70h in the fermentation process to tank placing is 40-60%.
The fermentation broth after the fermentation was sampled and measured to have a titer of 22018. mu.g/mL, a component proportion of the tylosin (T2i5) of 83.52%, and a component proportion of the 4' -O-isovaleryl tylosin (T0i5) of 4.42%.
Comparative example 3
A method for reducing 4' -O-isovaleryl tylosin component by changing dissolved oxygen in a tylosin fermentation process is the same as that in example 1, and is characterized in that stirring frequency is adjusted so that the dissolved oxygen in the whole fermentation process is 20-40%.
Sampling to determine the titer of the fermentation broth after the fermentation is finished to 10708 mu g/mL, the component proportion of the tylosin (T2i5) is 72.25 percent, and the component proportion of the 4' -O-isovaleryl tylosin (T0i5) is 8.24 percent
Comparative example 4
A method for reducing 4' -O-isovaleryl tylosin component by changing dissolved oxygen in the fermentation process of tylosin, which is the same as that in example 1, keeps an upper axial flow type stirring paddle in the prior art, and adjusts the stirring frequency to ensure that the dissolved oxygen in the whole fermentation process is 0-20%.
Sampling and measuring the fermentation broth after fermentation, wherein the titer is 9164 mu g/mL, the component proportion of the tylosin (T2i5) is 32.36 percent, and the component proportion of the 4' -O-isovaleryl tylosin (T0i5) is 15.11 percent
Comparative example 5
The existing three-layer stirring fermentation tank is adopted to produce the taiwan bacterin by fermentation.
The specific method comprises the following steps: streptomyces thermotolerans is taken as a production strain, aerated and stirred in a liquid fermentation medium containing starch, yeast powder, fish extract, yeast extract and inorganic salt for culture, when the pH value of a fermentation broth is reduced to 7.00-7.20, substrate tylosin dry powder and L-leucine are added, and in the middle and later stages (100 plus 160h) of the fermentation process, the dissolved oxygen change in the fermentation process is adjusted by adjusting the stirring batch to 40-50 Hz, three batches are determined to find that the instability problem of T0i5 is directly related to the reduction of the rotating speed in process control and the insufficient dissolved oxygen, and T0i5 is negatively related to the dissolved oxygen.
Sampling and measuring a fermentation liquid obtained by completing the fermentation of 01 batches, wherein the titer is 21098 mu g/mL, the component proportion of the tylosin (T2i5) is 82.68 percent, and the component proportion of the 4' -O-isovaleryl tylosin (T0i5) is 5.62 percent
The results of the test in example 1 and comparative examples 1 to 5 are shown in Table 1 and FIGS. 3 to 7 below:
TABLE 1
Figure RE-GDA0003596143590000071
As can be seen from table 1 and fig. 3, when tavermectin is produced by fermentation using the existing three-layer stirred fermentor, in the same fermentation period, the control effect of air flow on dissolved oxygen is low, and fermentation control may be difficult due to foam, the control effect of stirring speed on dissolved oxygen is the highest, insufficient dissolved oxygen may be caused by reducing frequency, and through three-batch experiments, it is found that the instability problem of T0i5 is directly related to the reduction of speed in process control, and insufficient dissolved oxygen has a negative correlation with dissolved oxygen, T0i 5. Therefore, the change of dissolved oxygen in the fermentation process can be effectively controlled to reduce the prenylation route to below 4.5% by controlling the rotating speed. Compared with the comparative example 5, in the embodiment 1 of the invention, the circular-pipe type air distributor is adopted, the air flow and the stirring speed are adjusted at different fermentation time, so that the dissolved oxygen level is controlled, the upper-layer stirring blades are removed, and the component proportion of the 4' -oxygen-isovaleryl tylosin is effectively reduced.
As can be seen from FIGS. 4 to 7, in example 1 of the present invention, compared with comparative examples 2 to 4, the component of 4 '-O-isovaleryl tylosin (T0i5) can be significantly reduced, and the component of 3-O-acetyl-4' -O-isovaleryl tylosin (T2i5) and the fermentation titer can be improved without changing the feeding manner by the dissolved oxygen amount in different time periods during the fermentation process. In comparative examples 2-4, due to low dissolved oxygen control in the process, the growth of Taiwan hyphae in the early stage is poor, the whole thallus concentration is low, and the obvious negative correlation between 4' -oxygen-isovaleryl tylosin and the dissolved oxygen control can be seen.
Compared with the comparative example 1, only the upper layer stirring is changed, but the components of 4 '-oxygen-isovaleryl tylosin in a tank are higher than those in the example 1, the titer and the components of 3-O-acetyl-4' -O-isovaleryl tylosin are reduced, and the reason is that hyphae in the middle and later stages of fermentation are seriously broken due to the shearing force of the upper layer stirring, the hyphae are vacuolated, and the addition of a substrate is influenced along with uncontrollable aggravation of foam. In comparative example 2, although the decrease control of dissolved oxygen has a small influence on the 3-O-acetyl-4 ″ -O-isovaleryl tylosin component, the 4 ″ -O-isovaleryl tylosin component is increased, increasing the difficulty of the extraction process.
Therefore, the invention effectively slows down the later period hypha fracture autolysis, reduces the component content of the fermented 4' -O-isovaleryl tylosin by modifying and adjusting the fermentation tank and controls the dissolved oxygen amount in the fermentation process by stages through the stirring frequency, improves the conversion rate, improves the total valence of tank placing by 5-10 percent, and improves the AIV component of the fermentation liquor by more than or equal to 84 percent, and simultaneously removes the upper stirring leaves, thereby realizing the reduction of the power consumption of a single batch of fermentation tank by 5-10 percent and realizing the increase of economic benefits.

Claims (10)

1. A method for changing dissolved oxygen in a tylosin fermentation process to reduce 4' -oxygen-isovaleryl tylosin component is characterized in that streptomyces thermotolerans is taken as a production strain to carry out fermentation production of tylosin in a fermentation tank, the pH of fermentation liquor is regulated to 7.00-7.50 at the initial stage of fermentation, then substrate tylosin dry powder and L-leucine are fed in a flowing manner, and glucose solution is fed in to ensure that the pH of the fermentation liquor is between 6.95-7.25 until the fermentation is finished;
controlling the dissolved oxygen of the fermentation liquor in different fermentation times in the whole fermentation process, controlling the dissolved oxygen of the fermentation liquor to be more than 50% within 0-35 h, controlling the dissolved oxygen of the fermentation liquor to be 20-30% within 35-70 h, and controlling the dissolved oxygen of the fermentation liquor to be more than 70% during 70h to tank discharge.
2. The method of changing dissolved oxygen in a tylosin fermentation process to reduce the 4 "-O-isovaleryl tylosin component of claim 1, wherein the fermentor comprises a fermentor tank with a stirring device disposed therein and an annular air distributor disposed below the stirring device; the annular air distributor comprises an annular pipe and an air inlet pipe, the annular pipe is communicated with the air inlet pipe, and the annular pipe is uniformly provided with 4 large air outlet holes and 30 small air outlet holes; the intake pipe stretches out outside the fermentation cylinder jar body, and fermentation cylinder jar body bottom is provided with the bin outlet.
3. The method for reducing the 4' -O-isovaleryl tylosin component by changing the dissolved oxygen in the tylosin fermentation process according to claim 2, wherein the stirring device is fixed on a motor base at the top end of the fermentation tank body, a motor is arranged on the motor base, a speed reducer is connected to the side surface of the motor, and a sealing ring, a stirring shaft penetrating through a top cover of the fermentation tank, a coupler, a straight-blade disc stirrer, a middle bearing and a parabolic stirrer are sequentially connected below the speed reducer.
4. The method for changing dissolved oxygen in a tylosin fermentation process to reduce 4' -O-isovaleryl tylosin component according to claim 2, wherein the distance between the parabolic stirrer and the annular air distributor is 5-20 cm, and the width of the parabolic stirrer and the diameter of the annular air distributor are the same.
5. The method of changing dissolved oxygen in a tylosin fermentation process to reduce the 4 "-O-isovaleryl tylosin component of claim 2, wherein the annular tube wall thickness is phi 125 x 4mm and is flanged to the fermentor.
6. The method of changing dissolved oxygen in a tylosin fermentation process to reduce the 4 "-O-isovaleryl tylosin component of claim 2, wherein the large exit orifice size is 22mm 4 and the small exit orifice size is 18mm 30.
7. The method of changing dissolved oxygen in a tylosin fermentation process to reduce the 4 "-O-isovaleryl tylosin component of claim 2, wherein the sum of the areas of the large and small gas exit holes is less than or equal to 80% of the cross-sectional area of the annular tube.
8. The method for reducing the content of 4' -O-isovaleryl tylosin by changing the dissolved oxygen in the fermentation process of tylosin according to claim 1, wherein the ratio of material culture media adopted in the fermentation production is as follows: 10-20 g/L of starch, 1-10 g/L of yeast extract, 0.1-0.8 g/L of fish extract and K2HPO4 0.01～0.5g/L。
9. The method of changing dissolved oxygen in a tylosin fermentation process to lower the 4 "-O-isovaleryl tylosin component of claim 1 wherein the initial fermentation stage is a pH adjustment with a mixture of glycerol and citric acid; the mass ratio of the glycerol to the citric acid is 1: 1.
10. The method for changing dissolved oxygen in the tylosin fermentation process to reduce the components of 4' -O-isovaleryl tylosin according to claim 1, wherein the mass ratio of tylosin dry powder to L-leucine is (5-6): 1, and the tylosin dry powder is added according to the dosage of 800-1000 μ g/mL.
CN202210092868.3A 2022-01-26 2022-01-26 Method for reducing 4' -oxygen-isovaleryl tylosin component by changing dissolved oxygen in fermentation process of tylosin Active CN114606282B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210092868.3A CN114606282B (en) 2022-01-26 2022-01-26 Method for reducing 4' -oxygen-isovaleryl tylosin component by changing dissolved oxygen in fermentation process of tylosin

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210092868.3A CN114606282B (en) 2022-01-26 2022-01-26 Method for reducing 4' -oxygen-isovaleryl tylosin component by changing dissolved oxygen in fermentation process of tylosin

Publications (2)

Publication Number Publication Date
CN114606282A true CN114606282A (en) 2022-06-10
CN114606282B CN114606282B (en) 2023-08-25

Family

ID=81859261

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210092868.3A Active CN114606282B (en) 2022-01-26 2022-01-26 Method for reducing 4' -oxygen-isovaleryl tylosin component by changing dissolved oxygen in fermentation process of tylosin

Country Status (1)

Country Link
CN (1) CN114606282B (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103224872A (en) * 2013-04-27 2013-07-31 齐鲁制药（内蒙古）有限公司 Fermentation tank for improving tylosin production level
CN103276031A (en) * 2013-06-28 2013-09-04 宁夏泰瑞制药股份有限公司 Culture medium for producing acetylisovaleryltylosin by fermenting streptomyces thermotolerans and fermentation method
CN112126667A (en) * 2020-09-30 2020-12-25 宁夏泰益欣生物科技有限公司 Fermentation method for improving titer of tulathromycin

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103224872A (en) * 2013-04-27 2013-07-31 齐鲁制药（内蒙古）有限公司 Fermentation tank for improving tylosin production level
CN103276031A (en) * 2013-06-28 2013-09-04 宁夏泰瑞制药股份有限公司 Culture medium for producing acetylisovaleryltylosin by fermenting streptomyces thermotolerans and fermentation method
CN112126667A (en) * 2020-09-30 2020-12-25 宁夏泰益欣生物科技有限公司 Fermentation method for improving titer of tulathromycin

Also Published As

Publication number Publication date
CN114606282B (en) 2023-08-25

Similar Documents

Publication Publication Date Title
Jolicoeur et al. Development of a helical‐ribbon impeller bioreactor for high‐density plant cell suspension culture
Drapeau et al. Growth kinetics of Dioscorea deltoidea and Catharanthus roseus in batch culture
Chao et al. Bacterial cellulose production under oxygen-enriched air at different fructose concentrations in a 50-liter, internal-loop airlift reactor
König et al. Strategies for penicillin fermentation in tower–loop reactors
US20080233631A1 (en) Ventilating/Stirring Culture Tank
CN112608831B (en) Industrial fermentation production process for improving yield of biosurfactant
CN104894183B (en) A kind of method that ansamitocin P-3 is prepared with precious orange synnema actinomycetes
CN219297482U (en) Biological medicine fermentation cylinder
CN102408991B (en) Bioreactor system for large scale culture of medicinal plants
CN102321537A (en) Bubbling bioreactor used for plant tissue culture
CN114606282A (en) Method for reducing 4' -oxygen-isovaleryl tylosin component by changing dissolved oxygen in tylosin fermentation process
CN117229881B (en) Bioreactor for filamentous fungi and application thereof
CN112126667B (en) Fermentation method for improving titer of teicoplanin
Sousa et al. Development of a bioprocess for the production of actinomycin-D
CN108396045B (en) High-yield fermentation production method of doramectin
CN111500510A (en) Constant-volume fermentation process for improving production efficiency of valine
CN108315230B (en) Reactor and method for preparing yeast culture by using reactor
CN114015604B (en) Fermentation medium and method for producing spinosad by fermentation
CN209669179U (en) The equipment of filamentous fungi bacterium ball size in a kind of mechanical control fermentation process
CN203741308U (en) Liquid table vinegar fermentation tank
Sakurai et al. Effect of operational conditions on the rate of citric acid production by rotating disk contactor using Aspergillus niger
Wang et al. Productivity enhancement in L-lysine fermentation using oxygen-enhanced bioreactor and oxygen vector
CN217838917U (en) Preparation facilities based on acetic acid is single cell protein of raw materials production
CN112301071A (en) Method for producing adenine by fermentation method
EP1613759B1 (en) Fermentation processes with low concentrations of carbon- and nitrogen-containing nutrients

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant