Method for increasing glutamic acid production by feeding a nutrient solution

Abstract

The application belongs to the field of biotechnology, and discloses a method for improving glutamic acid yield by feeding a nutrient solution, which comprises the following steps: feeding a nutrient solution containing glucose in a fermentation process. The application reduces the inhibition of cell growth and activity caused by high temperature in the middle and late stages of glutamic acid fermentation.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for increasing glutamic acid production through fed-batch nutrient solution. Background Technology

[0002] Optimizing amino acid fermentation conditions is a complex process involving extensive experimental research; it is time-consuming, labor-intensive, and costly. Most glutamic acid production utilizes temperature-sensitive bacteria, requiring optimization of key influencing factors (carbon source, nitrogen source, phosphorus salts, etc.) during fermentation to determine optimal acid-producing conditions. This addresses issues such as slow cell growth, weakened activity in the later stages of fermentation, and long fermentation cycles encountered with temperature-sensitive *Corynebacterium glutamicum*, ultimately aiming to improve the cell viability and acid-producing capacity of glutamic acid fermentation bacteria. In actual fermentation processes, the applicant has found that a large amount of heat is generated inside the fermenter during the later stages of fermentation, causing temperatures to rise, sometimes exceeding 40°C. High temperatures inhibit cell proliferation and activity. Overcoming this inhibition and increasing the fermentation rate and single-tank fermentation capacity is a key technical problem that needs to be solved. CN202210790067.4, the applicant's previous patent, improved the heat resistance of glutamic acid fermentation bacteria by optimizing the fermentation medium composition; however, above 38.5°C, the bacterial proliferation and acid-producing performance significantly decreased. Therefore, further research on heat resistance is necessary. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a method for increasing glutamic acid production by feeding nutrient solution, which further reduces the inhibition of cell growth and activity caused by high temperature in the middle and late stages of glutamic acid fermentation.

[0004] The present invention is achieved through the following technical solution.

[0005] The method for increasing glutamic acid production by feeding nutrient solution includes the following steps: feeding nutrient solution containing glucose during fermentation.

[0006] Specifically,

[0007] The method includes the following steps: transferring the Corynebacterium glutamicum seed culture into a fermenter containing fermentation medium for fermentation culture at a temperature of 32°C; and waiting for the fermentation broth to oxidize to an OD value of 100%. 600 When the temperature reaches 30°C, the fermentation temperature is increased to above 38°C until the fermentation ends. The residual sugar content in the fermentation broth is maintained at 1% by adding a nutrient solution containing glucose until the fermentation ends. The total fermentation time is 36 hours.

[0008] Furthermore,

[0009] The glucose-containing nutrient solution comprises glucose, sodium malate, and sodium alginate, with water as the solvent. Preferably, the glucose-containing nutrient solution is composed of the following components: 800 g / L glucose, 10 g / L sodium malate, 6 g / L sodium alginate, with water as the solvent.

[0010] Preferably, the nutrient solution containing glucose is composed of the following components: 800 g / L glucose, 12 g / L sodium malate, 7 g / L sodium alginate, and water as the solvent.

[0011] Preferably, the nutrient solution containing glucose is composed of the following components: 700 g / L glucose, 11 g / L sodium malate, 5 g / L sodium alginate, and water as the solvent.

[0012] Preferably, the nutrient solution containing glucose is composed of the following components: 700 g / L glucose, 10 g / L sodium malate, 7 g / L sodium alginate, and water as the solvent.

[0013] Preferably, the fermentation medium comprises: glucose 80 g / L, lactose 5 g / L, MnSO4·H2O 3 mg / L, FeSO4·7H2O 3 mg / L, MgSO4·7H2O 2 g / L, Na2HPO4·12H2O 4 g / L, KCl 2 g / L, VB1 10 mg / L, biotin 7 μg / L, biogenic nitrogen 2 g / L, methionine 0.6 g / L, Tween-80 0.2 g / L, and water as the solvent. Attached Figure Description

[0014] Figure 1 The effect of different nutrient solutions on the concentration of fermentation cells under three high-temperature conditions;

[0015] Figure 2 The effect of different nutrient solutions on the glutamic acid content in fermentation broth under three high-temperature conditions. Detailed Implementation

[0016] To enable those skilled in the art to better understand the technical solutions of this application, the technical solutions of this application will be clearly and completely described below in conjunction with specific embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0017] Example 1

[0018] A nutrient solution for the production of glutamic acid by glutamic acid fermentation bacteria is composed of the following components: 800 g / L glucose, 10 g / L sodium malate, 6 g / L sodium alginate, and water as the solvent.

[0019] Example 2

[0020] A nutrient solution for the production of glutamic acid by glutamic acid fermentation bacteria is composed of the following components: 800 g / L glucose, 12 g / L sodium malate, 7 g / L sodium alginate, and water as the solvent.

[0021] Example 3

[0022] A nutrient solution for the production of glutamic acid by glutamic acid fermentation bacteria is composed of the following components: 700 g / L glucose, 11 g / L sodium malate, 5 g / L sodium alginate, and water as the solvent.

[0023] Example 4

[0024] A nutrient solution for the production of glutamic acid by glutamic acid fermentation bacteria is composed of the following components: 700 g / L glucose, 10 g / L sodium malate, 7 g / L sodium alginate, and water as the solvent.

[0025] Comparative Example 1

[0026] A nutrient solution for the production of glutamic acid by glutamic acid fermentation bacteria is composed of the following components: 800 g / L glucose and water as the solvent.

[0027] Comparative Example 2

[0028] A nutrient solution for the production of glutamic acid by glutamic acid fermentation bacteria is composed of the following components: 800 g / L glucose, 10 g / L sodium malate, and water as the solvent.

[0029] Comparative Example 3

[0030] A nutrient solution for the production of glutamic acid by glutamic acid fermentation bacteria is composed of the following components: 800 g / L glucose, 6 g / L sodium alginate, and water as the solvent.

[0031] Example 5

[0032] The acid production process of glutamic acid fermentation bacteria includes the following steps:

[0033] Cell activation: Cryopreserved Corynebacterium glutamicum GDK-9 was inoculated onto slant culture for activation, and passaged twice.

[0034] The slant culture medium consisted of 5 g / L peptone, 10 g / L beef extract, 4 g / L yeast powder, 25 g / L corn steep liquor powder, 1 g / L KH2PO4, 0.2 g / L MgSO4, 1 g / L NaCl, 25 g / L agar powder, 0.2 g / L methionine, and pH 6.8.

[0035] Seed culture: The activated strains were washed with sterile water and then inoculated into the prepared seed culture medium for seed culture.

[0036] The seed culture medium is as follows: glucose 40 g / L, corn steep liquor powder 10 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.8 g / L, MnSO4 5 mg / L, FeSO4 5 mg / L, threonine 1 g / L, V H 10 mg / L.

[0037] The seed culture conditions are as follows: temperature maintained at around 32℃, dissolved oxygen controlled at 20%, and pH controlled at around 7.0 using ammonia water.

[0038] Fermentation culture: The seed culture of Corynebacterium glutamicum GDK-9 (OD) was used to ferment the culture. 600 =20) Inoculate 10% of the culture medium into a fermenter and carry out fermentation culture at a temperature of 32℃.

[0039] High-temperature incubation: OD of fermentation broth 600 When the temperature reaches 30°C, the fermentation temperature is increased to 38.5°C until the fermentation ends. Throughout the fermentation process, the ventilation ratio is controlled at 1:0.8, the stirring speed is 200 rpm, the dissolved oxygen is maintained at 20%, a nutrient solution containing glucose is added to maintain the residual sugar at 1%, an antifoaming agent is added to defoam, and ammonia water is added to adjust the pH of the fermentation broth to 7.2 until the fermentation ends. The total fermentation time is 36 hours.

[0040] The fermentation medium consisted of: glucose 80 g / L, lactose 5 g / L, MnSO4·H2O 3 mg / L, FeSO4·7H2O 3 mg / L, MgSO4·7H2O 2 g / L, Na2HPO4·12H2O 4 g / L, KCl 2 g / L, VB1 10 mg / L, biotin 7 μg / L, biogenic nitrogen 2 g / L, methionine 0.6 g / L, and Tween-80 0.2 g / L.

[0041] Example 6

[0042] The acid production process of glutamic acid fermentation bacteria includes the following steps:

[0043] Cell activation: Cryopreserved Corynebacterium glutamicum GDK-9 was inoculated onto slant culture for activation, and passaged twice.

[0044] The slant culture medium consisted of 5 g / L peptone, 10 g / L beef extract, 4 g / L yeast powder, 25 g / L corn steep liquor powder, 1 g / L KH2PO4, 0.2 g / L MgSO4, 1 g / L NaCl, 25 g / L agar powder, 0.2 g / L methionine, and pH 6.8.

[0045] Seed culture: The activated strains were washed with sterile water and then inoculated into the prepared seed culture medium for seed culture.

[0046] The seed culture medium is as follows: glucose 40 g / L, corn steep liquor powder 10 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.8 g / L, MnSO4 5 mg / L, FeSO4 5 mg / L, threonine 1 g / L, V H 10 mg / L.

[0047] The seed culture conditions are as follows: temperature maintained at around 32℃, dissolved oxygen controlled at 20%, and pH controlled at around 7.0 using ammonia water.

[0048] Fermentation culture: The seed culture of Corynebacterium glutamicum GDK-9 (OD) was used to ferment the culture. 600 =20) Inoculate 10% of the culture medium into a fermenter and carry out fermentation culture at a temperature of 32℃.

[0049] High-temperature incubation: OD of fermentation broth 600 When the temperature reaches 30°C, the fermentation temperature is increased to 39.5°C until the fermentation ends. Throughout the fermentation process, the ventilation ratio is controlled at 1:0.8, the stirring speed is 200 rpm, the dissolved oxygen is maintained at 20%, a nutrient solution containing glucose is added to maintain the residual sugar at 1%, an antifoaming agent is added to defoam, and ammonia water is added to adjust the pH of the fermentation broth to 7.2 until the fermentation ends. The total fermentation time is 36 hours.

[0050] The fermentation medium consisted of: glucose 80 g / L, lactose 5 g / L, MnSO4·H2O 3 mg / L, FeSO4·7H2O 3 mg / L, MgSO4·7H2O 2 g / L, Na2HPO4·12H2O 4 g / L, KCl 2 g / L, VB1 10 mg / L, biotin 7 μg / L, biogenic nitrogen 2 g / L, methionine 0.6 g / L, and Tween-80 0.2 g / L.

[0051] Example 7

[0052] The acid production process of glutamic acid fermentation bacteria includes the following steps:

[0053] Cell activation: Cryopreserved Corynebacterium glutamicum GDK-9 was inoculated onto slant culture for activation, and passaged twice.

[0054] The slant culture medium consisted of 5 g / L peptone, 10 g / L beef extract, 4 g / L yeast powder, 25 g / L corn steep liquor powder, 1 g / L KH2PO4, 0.2 g / L MgSO4, 1 g / L NaCl, 25 g / L agar powder, 0.2 g / L methionine, and pH 6.8.

[0055] Seed culture: The activated strains were washed with sterile water and then inoculated into the prepared seed culture medium for seed culture.

[0056] The seed culture medium is as follows: glucose 40 g / L, corn steep liquor powder 10 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.8 g / L, MnSO4 5 mg / L, FeSO4 5 mg / L, threonine 1 g / L, V H 10 mg / L.

[0057] The seed culture conditions are as follows: temperature maintained at around 32℃, dissolved oxygen controlled at 20%, and pH controlled at around 7.0 using ammonia water.

[0058] Fermentation culture: The seed culture of Corynebacterium glutamicum GDK-9 (OD) was used to ferment the culture. 600 =20) Inoculate 10% of the culture medium into a fermenter and carry out fermentation culture at a temperature of 32℃.

[0059] High-temperature incubation: OD of fermentation broth 600 When the temperature reaches 30°C, the fermentation temperature is increased to 40.5°C until the fermentation ends. Throughout the fermentation process, the ventilation ratio is controlled at 1:0.8, the stirring speed is 200 rpm, the dissolved oxygen is maintained at 20%, a nutrient solution containing glucose is added to maintain the residual sugar at 1%, an antifoaming agent is added to defoam, and ammonia water is added to adjust the pH of the fermentation broth to 7.2 until the fermentation ends. The total fermentation time is 36 hours.

[0060] The fermentation medium consisted of: glucose 80 g / L, lactose 5 g / L, MnSO4·H2O 3 mg / L, FeSO4·7H2O 3 mg / L, MgSO4·7H2O 2 g / L, Na2HPO4·12H2O 4 g / L, KCl 2 g / L, VB1 10 mg / L, biotin 7 μg / L, biogenic nitrogen 2 g / L, methionine 0.6 g / L, and Tween-80 0.2 g / L.

[0061] Example 8

[0062] The effects of the nutrient solutions of Examples 1-4 and Comparative Examples 1-3 on cell concentration and fermentation acid production were verified under the temperature conditions of Example 5 (38.5℃), Example 6 (39.5℃), and Example 7 (40.5℃).

[0063] like Figure 1-2 As shown, with the increase of temperature, the cell concentration and glutamic acid production of Examples 1-4 and Comparative Examples 1-3 all decreased, and the trends were relatively consistent. The difference was that the decrease in Examples 1-4 was smaller, while the decrease in Comparative Example 1 was the most obvious, followed by Comparative Examples 2-3.

[0064] Specifically, in Example 5 (38.5℃), sodium malate and sodium alginate were added to the nutrient solutions of Examples 1-4, and the differences in cell concentration and fermentation acid production were small. Comparative Example 1 only added glucose, and its cell concentration and fermentation acid production were significantly lower than those of Examples 1-4. Compared with Example 1, the cell concentration and fermentation acid production of Comparative Example 1 decreased by 2% and 10%, respectively. This indicates that at 38.5℃, the nutrient solution components had a significantly smaller impact on the concentration of the bacterial strain than on the fermentation acid production. This may be because at this temperature, the effect on the growth and proliferation of the bacterial strain is small, while the effect on the acid production activity of the strain is large. Comparative Examples 2-3 added sodium malate and sodium alginate to the nutrient solution, respectively, and both the cell concentration and acid production were significantly increased compared to Comparative Example 1.

[0065] As the fermentation temperature increased, the cell concentration and acid production of Comparative Example 1 decreased more significantly under the temperature conditions of Example 6 (39.5℃) and Example 7 (40.5℃). At 40.5℃, compared to Example 1, the cell concentration and acid production of Comparative Example 1 decreased by 7.5% and 19.7%, respectively. This indicates that higher temperatures significantly inhibited the growth and proliferation of the strains, and may even have led to the death of some strains. The combined effect of decreased growth and vitality resulted in a more pronounced decline in acid production.

[0066] Specifically, under the temperature conditions of Example 7 (40.5℃), sodium malate 10g / L was added to the nutrient solution of Comparative Example 2, and sodium alginate 6g / L was added to Comparative Example 3. The fermentation yields of Comparative Example 1 increased by 16g / L and 11g / L, respectively, compared with Comparative Example 1. In contrast, the group of Example 1, which added both sodium malate 10g / L and sodium alginate 6g / L, increased by 35g / L compared with Comparative Example 1, which exceeded the sum of Comparative Examples 2-3. This indicates that under the high-temperature fermentation conditions of 40.5℃, the synergistic effect of sodium malate and sodium alginate is better.

[0067] Analysis of the above reasons reveals that increasing the content of saturated fatty acids in the bacterial cell membrane and enhancing the expression level of related glycoproteins in the cell wall are important factors for bacterial production under various stress conditions. Sodium malate may have increased the content of saturated fatty acids in the cell membrane of Corynebacterium glutamicum GDK-9, thereby improving its heat resistance; while sodium alginate may have promoted the expression of capsule-associated glycoproteins in Corynebacterium glutamicum GDK-9, thereby improving its high-temperature resistance.

[0068] The above examples are merely preferred embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A method for increasing glutamic acid production by fed-batch nutrient solution, characterized in that, The method includes the following steps: transferring the Corynebacterium glutamicum GDK-9 seed culture into a fermenter containing fermentation medium for fermentation culture at a temperature of 32°C; and waiting for the fermentation broth to oxidize to an OD value of 100%. 600 When the temperature reaches 30°C, increase the fermentation temperature to 38.5°C until fermentation ends; maintain the residual sugar content in the fermentation broth at 1% by adding a nutrient solution containing glucose until fermentation ends; the total fermentation time is 36 hours. The glucose-containing nutrient solution is composed of the following components: 800 g / L glucose, 10 g / L sodium malate, 6 g / L sodium alginate, and water as the solvent. The fermentation medium consisted of: glucose 80 g / L, lactose 5 g / L, MnSO4·H2O 3 mg / L, FeSO4·7H2O 3 mg / L, MgSO4·7H2O 2 g / L, Na2HPO4·12H2O 4 g / L, KCl 2 g / L, VB1 10 mg / L, biotin 7 μg / L, biogenic nitrogen 2 g / L, methionine 0.6 g / L, and Tween-80 0.2 g / L.

Images