CN110551783A

CN110551783A - Fermentation method for producing gentamicin by using continuous flow feeding material with oxygen consumption rate as key control parameter

Info

Publication number: CN110551783A
Application number: CN201910901442.6A
Authority: CN
Inventors: 李敏超; 张嗣良; 庄英萍; 储炬; 杭海峰
Original assignee: Qingdao Institute Of Innovation East China University Of Technology; East China University of Science and Technology
Current assignee: Qingdao Institute Of Innovation East China University Of Technology; East China University of Science and Technology
Priority date: 2019-09-23
Filing date: 2019-09-23
Publication date: 2019-12-10
Anticipated expiration: 2039-09-23
Also published as: CN110551783B

Abstract

The invention relates to a fermentation method for producing gentamicin by continuous fed-batch with oxygen consumption rate as a key control parameter, which comprises a primary seed preparation process, a secondary seed preparation process and a fermentation production process, wherein in the fermentation production process, the purpose of regulating and controlling the oxygen consumption rate is realized by accurately controlling the sugar supplement rate or the nitrogen supplement rate in the fermentation process, when the OUR in the early stage of the fermentation process is reduced from a peak to a target OUR, the feeding is started, and the OUR is maintained within the range of 15-30 in the feeding process, so that the metabolic activity of thalli can keep the capability of continuously synthesizing the gentamicin. Compared with the prior art, the method realizes continuous feeding after the OUR in the process is reduced to different OUR from the peak after initial culture and utilization by the continuous feeding, maintains the metabolic activity of thalli, continuously supplements nutrition after entering a secondary metabolic resistance generation stage in the middle and later stages, has stable product synthesis, continuously increases the titer, and has very good continuous resistance generation maintenance. After adopting automatic control, continuous feeding is adopted to maintain OUR, which is convenient for industrial production scale control.

Description

Fermentation method for producing gentamicin by using continuous flow feeding material with oxygen consumption rate as key control parameter

Technical Field

The invention belongs to the technical field of fermentation, and particularly relates to a fermentation method for producing gentamicin by continuous flow feeding with oxygen consumption rate as a key control parameter.

Background

Gentamicin is a broad-spectrum aminoglycoside antibiotic with independent intellectual property rights and widely applied to agriculture, medicine and feed industries in China; at present, almost half of the global aminoglycoside antibiotics market is occupied, and the annual output value of domestic gentamicin bulk drug market is close to the output value scale of 10 hundred million. Therefore, the method has important social and practical significance for improving the fermentation level and the product quality of gentamicin in China by researching the process technology.

The oxygen demand of the thalli in different fermentation stages is different, and is particularly obvious in the antibiotic fermentation process; the supply of oxygen during fermentation not only directly affects the growth of the microorganisms but also has a major impact on the activity of the enzyme system, metabolic pathways and product yields and their composition during fermentation. Therefore, the research on the influence and control of the oxygen supply on the fermentation has important significance on improving the production efficiency, improving the product quality and the like. In the general Oxygen-consuming fermentation process, higher Oxygen supply is controlled to avoid the Oxygen limitation, the Oxygen supply level is represented by Dissolved Oxygen (DO), and the DO can be effectively controlled by controlling the stirring speed and the air flow. Dissolved oxygen levels are affected by both oxygen supply rate (OTR) and oxygen consumption rate (OUR). Wherein OTR reflects the oxygen supply capacity of the equipment, and the main influencing factors mainly relate to the rheological property and the operating parameters of the fermentation liquid, such as flow, stirring speed, the number of stirring layers, stirring paddle type and the like. While the OUR is obviously different from DO and OTR, as a typical physiological parameter, the OUR reflects the (millimolar) number of oxygen consumed by a unit volume (mass) of fermentation liquor in unit time, and generally the OUR can represent the energy metabolism state of thalli or cells in the fermentation process and can be used as a key parameter for controlling the fermentation process. Currently, some patents report that OUR is adopted as a key parameter for fermentation process control to control the fermentation process of fermentation products and realize high yield (patent number: CN 103642870A, CN 105154490A, CN 108060144A, CN 101560535A, CN 102703540A), but reports that OUR for controlling the fermentation process by continuous feeding realizes optimized control of the gentamicin fermentation process, improves the gentamicin fermentation yield and improves the components of the gentamicin fermentation process are not reported. At present, the actual fermentation unit of gentamicin in China is basically maintained at the fermentation level of 1300-1600 u/ml; the production method basically adopts the fermentation process that the whole materials are supplemented intermittently in a fixed period, the viscosity is generally controlled by adopting a water supplementing mode under the condition of high viscosity after the materials are supplemented, and the fermentation process is relatively simple to control.

Disclosure of Invention

the invention aims to overcome the defects of the prior art and provide a fermentation method for producing gentamicin by continuous fed-batch feeding with oxygen consumption rate (or oxygen uptake rate, OUR) as a key control parameter. The method can stably improve the fermentation production level and improve various components of gentamicin.

The purpose of the invention can be realized by the following technical scheme:

A fermentation method for producing gentamicin by continuous flow feeding with oxygen consumption rate as key control parameter is a method for producing gentamicin by pure culture and three-stage fermentation of Micromonospora echinospora as an initial strain, and comprises the specific processes of first-stage seed preparation, second-stage seed preparation and fermentation production,

In the fermentation production process, the purpose of regulating and controlling the oxygen consumption rate is realized by accurately controlling the sugar supplement rate or the nitrogen supplement rate in the fermentation process, the feeding is started when the OUR is reduced from a high peak to the target OUR in the early stage of the fermentation process, and the OUR is maintained within the range of 15-30 in the feeding process, so that the metabolic activity of thalli can keep the capacity of continuously synthesizing the gentamicin.

In the fermentation production process, in order to prevent the oxygen concentration in the fermentation process from being lower than the critical oxygen concentration, stirring linkage is adopted, and the dissolved oxygen is controlled to be not lower than 40%.

In the fermentation production process, the fermentation liquor volume control strategy is to discharge 5kg when the fermentation liquor reaches 35kg, then continue to feed after the volume reaches 35 kg.

The rate of oxygen consumption during fermentation decreasing from the pre-fermentation peak to the target OUR is the OUR level at which feed control is prepared.

In one embodiment of the invention, OUR can be controlled in stages, generally from high to low, based on metabolic activity in a fermentative production process.

In one embodiment of the present invention, a preferred case of OUR control is: OUR is maintained at 30-28 for 14-48 h, 28-25 for 48-72 h, and 20-25 for 72-96 h.

In one embodiment of the present invention, the process mass spectrometer BP400 from THERMAL FISHER was used in the fermentation production process to perform off-gas detection and calculate the physiological parameters of the fermentation process OUR based on the "star of fermentation" software package.

The software package of the 'star of fermentation' is composed of a star of fermentation, a fermentation network message and auxiliary software, has the main functions of online acquisition of fermentation process data, online calculation, offline data input, event record input and image input, is mature known operating software, and can be obtained by the national research center of biochemical engineering technology (Shanghai), wherein relevant information of the software can refer to http:// www.doc88.com/p-9943458130154.html and http:// www.nc-bio.com/products/retrieval-projects/352. html.

In one embodiment of the present invention, the OUR feed is controlled in the fermentative production process to be fed with a feed nitrogen medium (g/L) comprising: silkworm chrysalis meal 22, corn steep liquor 30, high-temperature soybean meal 120(100 meshes), citric acid 20, ammonium sulfate 0.76, potassium nitrate 3.8, cobalt chloride 0.004, natural enemy 0.04 and organic silicone oil 0.04; the pH was adjusted to 9.0 using sodium hydroxide before sterilization.

in one embodiment of the invention, the feed sugar composition fed to the OUR is controlled to glucose (500 g/L concentration) in the fermentative production process.

In one embodiment of the present invention, the primary seed medium composition (g/L): 1.4 parts of glucose, 15 parts of corn starch, 20 parts of corn flour, 2.6 parts of Angel peptone, 20 parts of low-temperature soybean flour, 2.5 parts of ammonium sulfate, 0.054 part of potassium nitrate, 6.2 parts of calcium carbonate, 0.03 part of natural sodium nitrite and 0.05 part of silicone oil, wherein the pH value is 7.2;

Secondary seed medium (g/L): corn starch 23, corn flour 30, Angel peptone 13, bean cake powder 13, ammonium sulfate 5, magnesium sulfate 7.1, potassium nitrate 0.5, calcium carbonate 7, cobalt oxide 0.002, sodium chloride 0.05 and silicone oil 0.03, and pH 7.8;

Fermentation basal medium (g/L): 50 parts of corn starch, 20 parts of corn flour, 25 parts of medium-temperature soybean flour, 23 parts of Angel peptone, 5 parts of ammonium sulfate, 7.1 parts of magnesium sulfate, 0.5 part of potassium nitrate, 7 parts of calcium carbonate, 0.002 part of cobalt oxide, 0.05 part of sodium alginate and 0.03 part of organic silicone oil, wherein the pH value is 9.0.

In one embodiment of the present invention, the primary seed culture conditions are: proportioning the components of a first-grade seed culture medium in a 15L seeding tank, sterilizing the first-grade seed culture medium, keeping the pressure of about 0.05MPa by using sterile air, inoculating a cultured shake flask seed solution into the first-grade seeding tank for culture, wherein the culture process comprises the following steps: the liquid loading amount is 10L/25; the inoculation amount is 60 ml; the culture temperature is 35.5 +/-0.5 ℃; the aeration ratio is 1: 1; stirring and rotating speed: 0-30h 200rpm, 30-48h 400rpm, pot pressure: 0.03 to 0.05 MPa; and (3) culture period: 48 h;

The secondary seed culture conditions are as follows: proportioning the components of a secondary seed culture medium in a 30L seeding tank, sterilizing and cooling the secondary seed culture medium, keeping the pressure at about 0.05MPa by using sterile air, then transferring 5L of the primary seed culture medium into the secondary seeding tank for culture, and filling the liquid in the secondary seeding tank by 5L/20L. The culture temperature is 35.5 +/-0.5 ℃; the aeration ratio is 1: 1; stirring speed: 300rpm for 0-10h, 400rpm for 10-20h, 450rpm for 20-30h, tank pressure: 0.03 to 0.05 MPa; and (3) culture period: and (5) 48 h.

The 50L fermentation tank culture conditions were: the materials are fed in a 50L seeding tank according to the proportion of a fermentation basic culture medium (the material is 20 kg). Sterilizing the culture medium, cooling, transferring 10kg of the secondary seed solution into a fermentation tank, and culturing. The whole culture temperature is 35.5 +/-0.5 ℃, and the aeration ratio is 1: 1; adjusting the stirring speed according to dissolved oxygen content not less than 30%, and adjusting the tank pressure: 0.03 to 0.05 MPa; and (3) culture period: and (6) 96 h.

The invention realizes the adjustment of the metabolic activity of the thalli by controlling the material supplementing rate, and achieves the purpose of controlling the OUR.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. Compared with the conventional multiple-time feeding mode, the method has the advantages that continuous feeding is started after the OUR in the process is reduced from the peak after initial culture and utilization through the continuous feeding, the metabolic activity of thalli is maintained, nutrition is continuously supplemented after the secondary metabolic resistance generation stage is started in the middle and later stages, the product synthesis is stable, the titer is continuously increased, and the continuous production resistance is well maintained. After adopting automatic control, continuous feeding is adopted to maintain OUR, which is convenient for industrial production scale control.

2. The invention can realize the adjustment of component proportion by controlling different OUR during continuous material feeding, and is beneficial to the control of product quality. Particularly, according to the actual industrialized production requirement, OUR in different stages can be controlled to realize different component proportions, the content of C1a is obviously improved under the condition of lower OUR, and part of C1a can be extracted from gentamicin products, for example, C1 can reach more than 25% when proper OUR is maintained at about 22-23. The method has good operability, and the product quality can meet the requirements of pharmacopeia on gentamicin components or improve the component proportion according to the actual component requirements of industrial production.

Detailed Description

The present invention will be described in detail with reference to specific examples.

Example 1

The strain used in this example was Micromonospora echinospora, a mature commercial strain.

The whole fermentation process route is as follows: shake flask seed → primary seed culture → secondary seed culture → fermentation culture.

The culture medium comprises the following components:

Primary seed medium (g/L): 1.4 parts of glucose, 15 parts of corn starch, 20 parts of corn flour, 2.6 parts of Angel peptone, 20 parts of low-temperature soybean flour, 2.5 parts of ammonium sulfate, 0.054 parts of potassium nitrate, 6.2 parts of calcium carbonate, 0.03 parts of natural enemy, and 0.05 part of silicone oil with the pH value of 7.2.

Secondary seed medium (g/L): corn starch 23, corn flour 30, Angel peptone 13, bean cake powder 13, ammonium sulfate 5, magnesium sulfate 7.1, potassium nitrate 0.5, calcium carbonate 7, cobalt oxide 0.002, sodium chloride 0.05 and silicone oil 0.03, pH 7.8

Fermentation basal medium (g/L): 50 parts of corn starch, 20 parts of corn flour, 25 parts of medium-temperature soybean flour, 23 parts of Angel peptone, 5 parts of ammonium sulfate, 7.1 parts of magnesium sulfate, 0.5 part of potassium nitrate, 7 parts of calcium carbonate, 0.002 part of cobalt oxide, 0.05 part of sodium alginate and 0.03 part of organic silicone oil, wherein the pH value is 9.0

Feed nitrogen medium (g/L) composition: silkworm chrysalis meal 22, corn steep liquor 30, high-temperature bean flour 120(100 meshes), citric acid 20, ammonium sulfate 0.76, potassium nitrate 3.8, cobalt chloride 0.004, natural enemy 0.04 and organic silicone oil 0.04; the pH was adjusted to 9.0 using sodium hydroxide prior to sterilization.

The sugar supplement composition is as follows: the glucose concentration was 500 g/L.

The culture conditions in the fermentation process are as follows:

First-order seed culture: the mixture ratio is carried out in a 15L seeding tank according to the components of the first-class seed culture medium. Sterilizing a first-stage seed culture medium, keeping the pressure at about 0.05MPa by using sterile air, and then inoculating a cultured shake flask seed solution into a first-stage seed tank for culture, wherein the liquid loading amount is 10L/25 in the culture process; the inoculation amount is 60 ml; the culture temperature is 35.5 +/-0.5 ℃; the aeration ratio is 1: 1; stirring speed: 0-30h 200rpm, 30-48h 400rpm, pot pressure: 0.03 to 0.05 MPa; and (3) culture period: and (5) 48 h.

Secondary seed culture: proportioning according to the components of the secondary seed culture medium in a 30L seeding tank. Sterilizing the secondary seed culture medium, cooling, maintaining the pressure at 0.05MPa with sterile air, culturing the primary seed to obtain 5L, transferring into a secondary seed tank, and culturing to obtain 5L/20L liquid. The culture temperature is 35.5 +/-0.5 ℃; the aeration ratio is 1: 1; stirring speed: 300rpm for 0-10h, 400rpm for 10-20h, 450rpm for 20-30h, tank pressure: 0.03 to 0.05 MPa; and (3) culture period: and (5) 48 h.

50L fermentation tank culture: the materials are fed in a 50L seeding tank according to the proportion of a fermentation basic culture medium (the material is 20 kg). Sterilizing the culture medium, cooling, transferring 10kg of the secondary seed solution into a fermentation tank, and culturing. The whole culture temperature is 35.5 +/-0.5 ℃, and the aeration ratio is 1: 1; adjusting the stirring speed according to dissolved oxygen content not less than 30%, and adjusting the tank pressure: 0.03 to 0.05 MPa; and (3) culture period: and (6) 96 h.

And (3) a feeding control strategy: according to the OUR reduction to 32mmol/kg/h before OUR fermentation, sugar and nitrogen supplement are started, and the OUR is maintained within the range of 28-30 during the fermentation process. The volume is controlled to be 30-35kg according to the weighing of the fermentation tank, and 5kg is discharged when the volume reaches about 35 kg.

After the fermentation process is finished, the relevant indexes of the fermentation liquor are measured and analyzed, and the fermentation efficiency of the result is 2884 ug/ml.

Example 2

the primary seed culture and the secondary seed culture were the same as in example 1, and the primary seed culture medium, the secondary seed culture medium and the fermentation medium were the same as in example 1, except for the OUR of the continuous feed control. And (3) a feeding control strategy: according to the OUR reduction to about 22mmol/kg/h in the earlier stage of OUR fermentation, sugar and nitrogen are supplemented, and the OUR is maintained within the range of 18-20 in the fermentation process. The volume is controlled to be 30-35kg according to the weighing of the fermentation tank, and 5kg of materials is discharged when the volume reaches about 35 kg.

Example 3

The primary seed culture and the secondary seed culture were the same as in example 1, and the primary seed culture medium, the secondary seed culture medium and the fermentation medium were the same as in example 1, except for the OUR of the continuous feed control. And (3) a feeding control strategy: according to the OUR reduction to about 28mmol/kg/h in the earlier stage of OUR fermentation, sugar and nitrogen are supplemented, and the OUR is maintained within the range of 22-25 in the fermentation process. The volume is controlled to be 30-35kg according to the weighing of the fermentation tank, and 5kg of materials is discharged when the volume reaches about 35 kg.

Example 4

The primary seed culture and the secondary seed culture were the same as in example 1, and the primary seed culture medium, the secondary seed culture medium and the fermentation medium were the same as in example 1, except for the OUR of the continuous feed control. And (3) a feeding control strategy: according to the OUR in the earlier stage of OUR fermentation, when the OUR is reduced to about 32mmol/kg/h, sugar and nitrogen are supplemented, the OUR is maintained at 30-28 within 14-48 hOUR, 28-25 within 48-72 h, and the OUR is maintained at 20-25 within 72-tank discharge. The volume is controlled to be 30-35kg according to the weighing of the fermentation tank, and 5kg is discharged when about 35kg is reached.

The results of the 4 examples are shown in the table, and the requirements (Chinese pharmacopoeia C1: 25-50%, C1a 15-40%, C2+ C2a 20-50%, English pharmacopoeia C1: 25-50%, C1a 10-35%, C2+ C2a 25-55%) on the ratio of the components of gentamycin specified by Chinese pharmacopoeia and English pharmacopoeia are compared, so that the final product concentration of high OUR in the fermentation process in example 1 can be maintained to be 2884ug/ml, the belt release amount of 65% is reached, the single-tank capacity is maximum, and the C1 in the only defect component ratio can not reach the standard. In example 2, the lower OUR in the maintenance process obviously shows that the C1a component is greatly improved, and the extraction part C1a can meet the market demand through the process control. Maintaining an intermediate OUR level in example 3 can achieve higher fermentation units in the fermentation process, with more suitable component ratios. In example 4, a phased OUR control strategy is adopted, which can give consideration to the effects of high yield and component improvement in adjusting the fermentation unit and component ratio, and is an optimal control scheme.

TABLE 1 results of four examples

the implementation cases in the present specification are described in a progressive manner, each implementation case is mainly described as different from other embodiments, and the same and similar parts in the implementation cases are referred to each other.

The invention is not limited to four examples, and the improvement of components or the improvement of fermentation titer by the similar process strategy of the invention is within the protection scope of the invention.

The embodiments described above are described to facilitate an understanding and appreciation of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make modifications and alterations without departing from the scope of the present invention.

Claims

1. A fermentation method for producing gentamicin by continuous flow feeding with oxygen consumption rate as key control parameter is characterized by comprising a first-stage seed preparation process, a second-stage seed preparation process and a fermentation production process,

2. The fermentation method for producing gentamicin by continuous fed-batch feeding with oxygen consumption rate as a key control parameter according to claim 1, wherein in the fermentation production process, stirring linkage is adopted, and dissolved oxygen is controlled to be not less than 40%.

3. The fermentation method for producing gentamicin by continuous fed-batch feeding with oxygen consumption rate as key control parameter as claimed in claim 1, wherein in the fermentation production process, the fermentation broth volume control strategy is to discharge 5kg when the fermentation broth reaches 35kg, then continue feeding after the volume reaches 35 kg.

4. The fermentation process of claim 1, wherein the decrease in oxygen consumption rate from the pre-fermentation peak to the target OUR during fermentation is the OUR level for which feed control is prepared.

5. the fermentation method for producing gentamicin by continuous fed-batch feeding with oxygen consumption rate as key control parameter as claimed in claim 1, wherein OUR is controlled in stages according to metabolic activity in the fermentation production process to decrease from high to low.

6. A fermentation process for the production of gentamicin by continuous fed batch with oxygen consumption rate as the key control parameter as claimed in claim 5 wherein one way of OUR control is: OUR is maintained at 30-28 for 14-48 h, 28-25 for 48-72 h, and 20-25 for 72-96 h.

7. The fermentation method for producing gentamicin by continuous fed-batch feeding with oxygen consumption rate as key control parameter as claimed in claim 1, wherein in the fermentation production process, a process mass spectrometer BP400 of THERMAL FISHER company is used, and the detection of tail gas and the calculation of OUR physiological parameters of the fermentation process are realized based on a software package of fermentation Star.

8. The fermentation method for producing gentamicin by continuous fed-batch feeding with oxygen consumption rate as key control parameter as claimed in claim 1, wherein in the fermentation production process, the feeding nitrogen culture medium (g/L) for controlling OUR feeding is: silkworm chrysalis meal 22, corn steep liquor 30, high-temperature bean flour 120, citric acid 20, ammonium sulfate 0.76, potassium nitrate 3.8, cobalt chloride 0.004, natural enemy 0.04 and organic silicone oil 0.04; the pH was adjusted to 9.0 using sodium hydroxide prior to sterilization.

9. The fermentation process for the production of gentamicin by continuous fed batch with oxygen consumption rate as key control parameter as claimed in claim 1, wherein the feed sugar composition of the controlled OUR feed is glucose.

10. The fermentation method for producing gentamicin by continuous fed-batch feeding with oxygen consumption rate as key control parameter as claimed in claim 1, wherein the primary seed culture medium composition (g/L): 1.4 parts of glucose, 15 parts of corn starch, 20 parts of corn flour, 2.6 parts of Angel peptone, 20 parts of low-temperature soybean flour, 2.5 parts of ammonium sulfate, 0.054 parts of potassium nitrate, 6.2 parts of calcium carbonate, 0.03 part of natural enemy, and 0.05 part of silicone oil, wherein the pH value is 7.2;