Method for reducing potassium ion content of culture medium

Abstract

The invention belongs to the technical field of amino acid production, and discloses a method for reducing the content of potassium ions in a culture medium, which comprises the following steps: dividing a fermentation culture medium for producing glutamic acid by microorganisms into a fermentation culture medium A and a fermentation culture medium B, and maintaining the content of potassium ions in the fermentation culture medium A to be 50% or less of that in the fermentation culture medium B. By optimizing the addition amount of potassium ions in the non-fermentation stage, the glutamic acid production rate is increased, and the fermentation period is shortened.

Description

Technical Field

[0001] The invention belongs to the technical field of amino acid production and relates to a method for reducing the potassium ion content of a culture medium. Background Art

[0002] The process of glutamate fermentation is actually a process of comprehensive action of Corynebacterium glutamicum under multiple factors, which is affected by ventilation, temperature, pH, nutrients, fermentation environment and other factors. Traditional fermentation control strategies often adopt extensive control of ventilation, temperature, pH and other conditions, and the control method is single and constant. With the development of glutamate fermentation technology, this control strategy can no longer meet the needs of the market and has a significant gap with the international fermentation level. Through in-depth research on the microbial metabolic fermentation process, analyzing the effects of bacterial growth metabolic requirements, nutritional changes, product accumulation, etc. on bacterial acid production at different stages, and continuously optimizing the formula on the basis of the existing fermentation medium, the comprehensive technology of glutamate fermentation is gradually improved, the control of domestic glutamate production costs is enhanced, and international competitiveness is improved, which has very important practical significance for revitalizing the domestic glutamate fermentation industry.

[0003] Potassium ion is an important component in the fermentation medium. It does not participate in the composition of cell structural substances, but it is an activator of many enzymes. Various metal ions may have a greater impact on the fermentation of glutamate by Corynebacterium glutamicum. The applicant's previous patent application "CN2024107209948, a method for improving glutamate fermentation using sodium ions" studied the effect of sodium ions on the fermentation of glutamate by Brevibacterium flavum. In the early stage of fermentation, high concentrations of sodium ions were added to increase the osmotic pressure to improve the synthesis of glutamate. In the middle and late stages of fermentation, sodium chloride in the fermentation medium was removed to reduce the osmotic pressure of the fermentation system, so that the strain cells quickly secreted glutamate, which reduced the intracellular osmotic pressure, thereby protecting the cells. On the basis of the above research, the present invention continues to explore the effect of potassium ions on the fermentation of glutamate by Brevibacterium flavum. Summary of the invention

[0004] In order to further optimize the efficiency of glutamic acid production by Brevibacterium flavum fermentation, the present invention will increase the glutamic acid production rate as much as possible and shorten the fermentation cycle without affecting the quality of the fermentation liquid by comparing the experimental results of different potassium ion addition amounts in the non-fermentation stage.

[0005] The present invention is achieved through the following technical solutions.

[0006] A method for reducing potassium ion content in a culture medium comprises: dividing a fermentation medium for producing glutamic acid by microorganisms into a fermentation medium A and a fermentation medium B, and maintaining the potassium ion content in the fermentation medium A to be less than 50% of that in the fermentation medium B.

[0007] Preferably, the microorganism is Brevibacterium flavum GDK-9.

[0008] Preferably, the components of the fermentation medium A are: glucose 50 g / L, corn steep liquor 60 g / L, K2HPO4 2 g / L, MgSO4·7H2O 1 g / L, MnSO4·H2O 3 mg / L, FeSO4·7H2O 3 mg / L, V B1 10mg / L, biotin 7μg / L, sodium chloride 50 / L.

[0009] Preferably, the components of the fermentation medium B are: glucose 50 g / L, corn steep liquor 60 g / L, K2HPO4 6 g / L, MgSO4·7H2O 1 g / L, MnSO4·H2O 3 mg / L, FeSO4·7H2O 3 mg / L, V B1 10mg / L, biotin 7μg / L, sodium chloride 50 / L.

[0010] As another specific embodiment of the present invention, it relates to a method for improving glutamate fermentation efficiency by adjusting potassium ions, which comprises the following steps:

[0011] The seed liquid of Brevibacterium flavum GDK-9 was inoculated with 10% of the inoculation amount into a 1000L fully automatic fermenter filled with 700L of fermentation medium A for fermentation culture, the fermentation temperature was 38°C, the ventilation ratio was 1:0.7, the stirring speed was 500r / min, the dissolved oxygen was maintained at 20%, the fermenter was coupled with a ceramic membrane, and when the fermentation reached 20h, the fermentation liquid A in the fermenter was separated by the ceramic membrane to obtain filtrate A and concentrated bacteria, the filtrate A was discharged, the concentrated bacteria were returned to the fermenter, and the fermentation tank medium B was added to the fermenter at the same time to make the volume of the fermentation liquid the same as that before the ceramic membrane separation, the fermentation time was continued for more than 16h, the fermentation was completed, the fermentation liquid B was obtained, the ceramic membrane was separated, and the filtrate B was collected; during the whole fermentation process, the pH value of the fermentation liquid was controlled to 7.0 by adding a foaming agent, and 25% ammonia water was added at the same time, and the reducing sugar content was controlled to be not less than 1% by adding a glucose solution; the filtrate A and the filtrate B were combined to obtain a solution rich in glutamic acid.

[0012] Preferably, the fermentation medium A is: glucose 50 g / L, corn steep liquor 60 g / L, K2HPO4 2 g / L, MgSO4·7H2O 1 g / L, MnSO4·H2O 3 mg / L, FeSO4·7H2O 3 mg / L, V B1 10mg / L, biotin 7μg / L, sodium chloride 50 / L.

[0013] Preferably, the fermentation medium B is: glucose 50 g / L, corn steep liquor 60 g / L, K2HPO4 4-8 g / L, MgSO4·7H2O 1 g / L, MnSO4·H2O 3 mg / L, FeSO4·7H2O 3 mg / L, V B1 10mg / L, biotin 7μg / L.

[0014] Preferably, the fermentation medium B is: glucose 50 g / L, corn steep liquor 60 g / L, K2HPO4 6 g / L, MgSO4·7H2O 1 g / L, MnSO4·H2O 3 mg / L, FeSO4·7H2O 3 mg / L, V B1 10mg / L, biotin 7μg / L.

[0015] Preferably, the fermentation is continued for 18 hours.

[0016] Preferably, the molecular weight cut-off of the ceramic membrane is 20,000 Da.

[0017] Compared with the prior art, the beneficial effects achieved by the present invention mainly include but are not limited to the following aspects:

[0018] During the fermentation of glutamic acid by Brevibacterium flavum, the early stage is mainly dominated by strain proliferation, and the later stage is dominated by fermentation. The present invention interprets the influence of potassium ions on fermentation according to the fermentation characteristics at different stages. Fermentation medium A is used for the early stage of fermentation, during which the strain proliferation is the main one. K2HPO4 has a great influence on the glutamic acid production and bacterial concentration. In the group without adding K2HPO4, the glutamic acid production and bacterial concentration have a significant decline. When it increases to 2g / L, the glutamic acid production and bacterial concentration of K2HPO4 maintain a rapid growth. Continuing to increase the concentration of K2HPO4 has little effect on the glutamic acid production and bacterial concentration.

[0019] In the late fermentation period, without adding K2HPO4, the fermentation period was significantly extended, and the glutamate production was low. Taking the fermentation period of 40h as an example, the glutamate production was greatly reduced compared with the conventional 2g / L group, and K2HPO4 had a great influence on glutamate synthesis. When K2HPO4 in fermentation medium B increased to 4g / L, it was doubled compared with the fermentation medium. At this time, the glutamate production was improved to a certain extent, and the fermentation period was also shortened. It was the best time to terminate the fermentation at 38h. The addition of K2HPO4 was continued to increase to 6g / L, and the glutamate production continued to increase, and the fermentation period was maintained at 38h. It can be seen that the potassium content required for the large-scale production of glutamate fermentation products is much higher than the amount required for rapid growth of the bacteria. Compared with the acid-producing medium (fermentation medium B), it is completely feasible to reduce the addition of potassium ions in the proliferation medium (fermentation medium A). DETAILED DESCRIPTION

[0020] In order to enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in this application will be clearly and completely described below in conjunction with the specific embodiments of this application. Obviously, the described embodiments are only part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of this invention.

[0021] Example 1

[0022] A method for adjusting potassium ions to improve glutamate fermentation efficiency comprises the following steps:

[0023] Brevibacterium flavum GDK-9 seed solution (OD 600nm 8) inoculating 10% of the inoculum into a 1000L fully automatic fermenter containing 700L of fermentation medium A for fermentation and culturing, the fermentation temperature is 38°C, the ventilation ratio is 1:0.7, the stirring speed is 500r / min, the dissolved oxygen is maintained at 20%, the fermenter is coupled with a ceramic membrane (molecular weight cutoff is 20000Da), and when the fermentation reaches 20h, the fermentation liquid A in the fermenter is separated by the ceramic membrane to obtain a filtrate A and concentrated bacterial bodies, the filtrate A is discharged, the concentrated bacterial bodies are returned to the fermenter, and the fermentation tank medium B is added to the fermenter to make the volume of the fermentation liquid equal to that before the ceramic membrane separation, and the fermentation is continued for 20h to complete the fermentation to obtain a fermentation liquid B, which is separated by the ceramic membrane and the filtrate B is collected; during the whole fermentation process, the pH value of the fermentation liquid is controlled to 7.0 by adding a foaming agent to defoam, and 25% ammonia water is added to control the reducing sugar content to be not less than 1% by adding a glucose solution; the filtrate A and the filtrate B are combined to obtain a solution rich in glutamic acid.

[0024] The fermentation medium A is: glucose 50g / L, corn steep liquor 60g / L, K2HPO4 0-4g / L, MgSO4·7H2O 1g / L, MnSO4·H2O 3mg / L, FeSO4·7H2O 3mg / L, V B1 10mg / L, biotin 7μg / L, sodium chloride 50 / L, sterilized at 115℃ for 15min.

[0025] The fermentation medium B is: glucose 50g / L, corn steep liquor 60g / L, K2HPO4 2g / L, MgSO4·7H2O 1g / L, MnSO4·H2O 3mg / L, FeSO4·7H2O 3mg / L, V B1 10mg / L, biotin 7μg / L, sterilized at 115℃ for 15min.

[0026] Example 2

[0027] Based on Example 1, the effects of different concentrations of K2HPO4 in fermentation medium A on the concentrations of glutamate and bacteria in fermentation broth A were studied.

[0028] The concentration of K2HPO4 in fermentation medium A was set to 0, 1, 2, 3, 4, in g / L, with a total of five concentration gradients. The effects of different K2HPO4 concentrations in fermentation medium A on the glutamate and bacterial concentrations in fermentation broth A were studied, as shown in Table 1.

[0029] Table 1

[0030]

[0031] As shown in Table 1 above, K2HPO4 in fermentation medium A has a great influence on glutamate production and bacterial concentration. In the group without K2HPO4, glutamate production and bacterial concentration have declined significantly. When it is increased to 2g / L, glutamate production and bacterial concentration of K2HPO4 maintain rapid growth. Continuing to increase the concentration of K2HPO4 has little effect on glutamate production and bacterial concentration. In the process of microbial glutamate fermentation, the early stage is mainly the stage of rapid bacterial proliferation. Fermentation medium A is a proliferation medium. Since the demand for K2HPO4 is not too large during bacterial proliferation, it is more appropriate to maintain it at 2g / L.

[0032] Example 3

[0033] A method for improving glutamate fermentation efficiency by adjusting potassium ions, comprising the following steps: 600nm 8) inoculating 10% of the inoculum into a 1000L fully automatic fermenter filled with 700L of fermentation medium A for fermentation and culturing, the fermentation temperature is 38°C, the ventilation ratio is 1:0.7, the stirring speed is 500r / min, the dissolved oxygen is maintained at 20%, the fermenter is coupled with a ceramic membrane (molecular weight cutoff is 20000Da), and when the fermentation reaches 20h, the fermentation liquid A in the fermenter is separated by the ceramic membrane to obtain a filtrate A and concentrated bacterial bodies, the filtrate A is discharged, the concentrated bacterial bodies are returned to the fermenter, and the fermentation tank medium B is added to the fermenter to make the volume of the fermentation liquid equal to that before the ceramic membrane separation, and the fermentation is continued for 16-22h to complete the fermentation to obtain a fermentation liquid B, which is separated by the ceramic membrane and the filtrate B is collected; during the whole fermentation process, the pH value of the fermentation liquid is controlled to 7.0 by adding a foaming agent to defoam, and 25% ammonia water is added to control the reducing sugar content to be not less than 1% by adding a glucose solution; the filtrate A and the filtrate B are combined to obtain a solution rich in glutamic acid.

[0034] The fermentation medium A is composed of 50 g / L glucose, 60 g / L corn steep liquor, 2 g / L K2HPO4, 1 g / L MgSO4·7H2O, 3 mg / L MnSO4·H2O, 3 mg / L FeSO4·7H2O, V B1 10mg / L, biotin 7μg / L, sodium chloride 50 / L, sterilized at 115℃ for 15min.

[0035] The fermentation medium B is: glucose 50g / L, corn steep liquor 60g / L, K2HPO4 0-8g / L, MgSO4·7H2O1g / L, MnSO4·H2O 3mg / L, FeSO4·7H2O 3mg / L, V B1 10mg / L, biotin 7μg / L, sterilized at 115℃ for 15min.

[0036] Example 4

[0037] Based on Example 3, the effects of different concentrations of K2HPO4 in fermentation medium B on glutamate concentration and fermentation cycle were studied.

[0038] The concentration gradient of K2HPO4 was set to 0, 2, 4, 6, 8, in g / L; the change of glutamate production under different total fermentation times was verified, in g / L, as shown in Table 2.

[0039] Table 2

[0040]

[0041]

[0042] As shown in Table 2 above, without adding K2HPO4, the fermentation cycle is significantly extended, and the glutamic acid output is low. Taking the fermentation cycle as 40h as an example, the output of glutamic acid is reduced by 15.6% compared with the conventional 2g / L group. It can be seen that K2HPO4 has a greater impact on glutamic acid synthesis. When K2HPO4 in fermentation medium B is increased to 4g / L, the output of glutamic acid is improved to a certain extent, and the fermentation cycle is also shortened. It is the best time to terminate the fermentation at 38h. When the fermentation is 38h, the output of glutamic acid is increased by 6.4% compared with the 2g / L group. Continue to increase the addition of K2HPO4 to 6g / L, the output of glutamic acid continues to increase, and the fermentation cycle is maintained at 38h. It can be seen that the potassium content required for the large-scale generation of glutamic acid fermentation products is much higher than the amount required when the bacterial cell grows rapidly. Compared with the acid-producing medium (fermentation medium B), it is feasible to reduce the addition of potassium ions in the proliferation medium (fermentation medium A).

[0043] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for reducing the potassium ion content of a culture medium, the method comprising: The fermentation medium for producing glutamic acid by microorganisms is divided into a fermentation medium A and a fermentation medium B, and the potassium ion content in the fermentation medium A is maintained at less than 50% of that in the fermentation medium B.

2. The method according to claim 1, characterized in that The microorganism is Brevibacterium flavum GDK-9.

3. The method according to claim 1, characterized in that: The components of the fermentation medium A are: glucose 50 g / L, corn steep liquor 60 g / L, K2HPO4 2 g / L, MgSO4·7H2O 1 g / L, MnSO4·H2O 3 mg / L, FeSO4·7H2O 3 mg / L, V B1 10mg / L, biotin 7μg / L, sodium chloride 50 / L.

4. The method according to claim 1, characterized in that The components of the fermentation medium B are: glucose 50 g / L, corn steep liquor 60 g / L, K2HPO4 6 g / L, MgSO4·7H2O 1 g / L, MnSO4·H2O 3 mg / L, FeSO4·7H2O 3 mg / L, V B1 10mg / L, biotin 7μg / L, sodium chloride 50 / L.

5. A method for improving glutamate fermentation efficiency by adjusting potassium ions, comprising the following steps: inoculating a 10% inoculum of Brevibacterium flavum GDK-9 seed solution into a 1000L fully automatic fermentation tank filled with 700L fermentation medium A for fermentation culture, the fermentation temperature is 38°C, the ventilation ratio is 1:0.7, the stirring speed is 500r / min, the dissolved oxygen is maintained at 20%, the fermentation tank is coupled with a ceramic membrane, and when the fermentation reaches 20h, the fermentation liquid A in the fermentation tank is separated through the ceramic membrane to obtain a filtrate A and concentrated bacteria, and the fermentation temperature is 38°C, the ventilation ratio is 1:0.7, the stirring speed is 500r / min, and the dissolved oxygen is maintained at 20%. The filtrate A is discharged, and the concentrated bacteria are returned to the fermentation tank. At the same time, the fermentation tank culture medium B is added to the fermentation tank to make the volume of the fermentation liquid equal to that before ceramic membrane separation. The fermentation time is continued for more than 16 hours, and the fermentation is completed to obtain the fermentation liquid B. The filtrate B is separated by the ceramic membrane and collected. During the whole fermentation process, the pH value of the fermentation liquid is controlled to 7.0 by adding 25% ammonia water through the flow addition for defoaming, and the reducing sugar content is controlled to be not less than 1% by adding glucose solution through the flow addition. The filtrate A and the filtrate B are combined to obtain a solution rich in glutamic acid.

6. The method according to claim 5, characterized in that The fermentation medium A is: glucose 50g / L, corn steep liquor 60g / L, K2HPO4 2g / L, MgSO4·7H2O 1g / L, MnSO4·H2O 3mg / L, FeSO4·7H2O 3mg / L, V B1 10mg / L, biotin 7μg / L, sodium chloride 50 / L.

7. The method according to claim 5, characterized in that The fermentation medium B is: glucose 50g / L, corn steep liquor 60g / L, K2HPO4 4-8g / L, MgSO4·7H2O 1g / L, MnSO4·H2O 3mg / L, FeSO4·7H2O 3mg / L, V B1 10mg / L, biotin 7μg / L.

8. The method according to claim 5, characterized in that The fermentation medium B is: glucose 50g / L, corn steep liquor 60g / L, K2HPO4 6g / L, MgSO4·7H2O 1g / L, MnSO4·H2O 3mg / L, FeSO4·7H2O 3mg / L, V B1 10mg / L, biotin 7μg / L.

9. The method according to claim 5, characterized in that The fermentation time is 18 hours.

10. The method according to claim 5, characterized in that The molecular weight cut-off of the ceramic membrane is 20000Da.

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