A semi-continuous fermentation method for improving the production of L-isoleucine

Abstract

The application belongs to the technical field of microorganisms, and provides a semi-continuous fermentation method for improving L-isoleucine yield, including the following steps: (1) seed culture; (2) fermentation culture; (3) flow addition; and L-isoleucine fermentation liquor is obtained. The semi-continuous fermentation is used, and the L-isoleucine fermentation acid production efficiency is improved by flow adding nutrients such as sugar beet molasses.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a semi-continuous fermentation method for increasing L-isoleucine production. Background Technology

[0002] A byproduct of the sugar-making industry, beet molasses contains vitamins, minerals, microbial proteins, nucleic acids, biotin, surfactants, and growth factors (bioactive substances), and can be used as a raw material in the microbial fermentation industry.

[0003] Isoleucine is one of the eight essential amino acids for the human body and also one of the three branched-chain amino acids, playing a particularly important role in human metabolism. L-Isoleucine is mainly used in the large-scale production of various amino acid infusions, liver-protecting syrups, and liver-strengthening oral solutions, showing significant therapeutic effects on various liver diseases. In recent years, L-Isoleucine has been widely used in biomedicine, tissue engineering, photochemistry, electrochemistry, food industry, and cosmetics. Abroad, it is also widely used as a lactation promoter in dairy cows, as a feed additive, and added to beverages to produce functional drinks. In industrial production, L-Isoleucine is mainly produced using fermentation.

[0004] Semi-continuous fermentation, also known as fed-batch fermentation or continuous fermentation, refers to a fermentation technique in which a certain nutrient or complete culture medium is added to the fermentation system in a specific way during the batch fermentation process of microorganisms. In early industrial production, the feeding methods were very simple, limited to intermittent and constant-rate fed-batch methods, and fermentation control was mainly based on experience. Compared with traditional batch fermentation, semi-continuous fermentation can solve problems such as substrate inhibition, glucose effect, and metabolic downregulation, resulting in higher acid production and conversion rates, lower probability of contamination, and optimized control of the fermentation process.

[0005] Chinese patent "CN2025117345252, A preparation process for increasing L-isoleucine yield" identified a strain that, after multiple mutagenesis screenings, can withstand high osmotic pressure and has a wider range of optimal proliferation and acid production temperatures. This strain has the accession number CGMCC No. 32505. Unexpectedly, it was found that this strain can synthesize isoleucine using inorganic nitrogen sources, which is superior to fermentation using organic nitrogen sources, thus significantly reducing production costs. Summary of the Invention

[0006] To further improve the acid production and sugar-acid conversion rate of L-isoleucine fermentation, and to solve problems such as substrate inhibition, metabolic inhibition, and susceptibility to bacterial contamination during fermentation, this invention provides a semi-continuous fermentation method to increase L-isoleucine production.

[0007] This invention is achieved through the following technical solution: A semi-continuous fermentation method for increasing L-isoleucine yield, comprising the following steps: The seed culture of Corynebacterium glutamicum producing L-isoleucine was inoculated into a fermentation medium and fermented for more than 40 hours. The fermentation medium contained aminosulfonic acid and sodium bisulfite.

[0008] Preferably, the carbon source of the fermentation medium is a mixture of glucose and beet molasses.

[0009] Preferably, the amount of aminosulfonic acid and sodium bisulfite added to the fermentation medium is 0.01 g / L.

[0010] Preferably, the total amount of carbon source added to the fermentation medium is 90 g / L.

[0011] More preferably, the fermentation medium is formulated as follows: 40 g / L glucose, 50 g / L beet molasses, 20 g / L nitrogen source, 2 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, 0.01 g / L sulfamic acid, and 0.01 g / L sodium bisulfite. The nitrogen source is corn steep liquor and ammonium sulfate, and the mass ratio of corn steep liquor to ammonium sulfate is 1:3.

[0012] Preferably, the inoculation amount of the Corynebacterium glutamicum seed solution is 5-15%.

[0013] Preferably, the fermentation culture time is 48 hours.

[0014] Specifically, the Corynebacterium glutamicum is Corynebacterium glutamicum YY2410A-1, with accession number CGMCCNo.32505.

[0015] Furthermore, during the fermentation process, when the residual sugar content in the fermenter is 0.5%, 2.3% glucose solution and 0.6% beet molasses are added; when the residual sugar content is 0.2%, 3% glucose solution and 0.8% beet molasses are added.

[0016] Preferably, during the feeding process, the flow rate of 2.3% glucose solution and 0.6% beet molasses is 4 ml / Lh per L of fermentation broth; and the flow rate of 2.3% glucose solution and 0.6% beet molasses is 5 ml / Lh per L of fermentation broth.

[0017] The technical solution of the present invention has the following outstanding advantages and uniqueness: This invention addresses the problem of low acid production efficiency in existing L-isoleucine fermentation. By using semi-continuous fermentation and adding nutrients such as beet molasses, the acid production and sugar-acid conversion rate of L-isoleucine fermentation are improved, solving problems such as easy contamination during fermentation, strong foaming of the fermentation broth, substrate inhibition, and metabolic regression. By replacing corn steep liquor with beet molasses during the addition process, the probability of contamination during fermentation is reduced, and the problem of insufficient nutrients in the later stages is solved through a nutrient addition strategy.

[0018] This invention replaces part of the glucose in the fermentation medium with beet molasses, reducing fermentation costs and increasing enterprise profits. Sulfamic acid is added to the fermentation medium at an appropriate concentration, acting as an antioxidant and stabilizer, suitable for high-density fermentation systems. Sodium bisulfite can remove reactive oxygen species from the fermentation broth, protecting the cell membranes and enzyme proteins of the strain from oxidative damage, maintaining metabolic pathway stability, and may also affect the carbon flux of sugar metabolism pathways, promoting the accumulation of isoleucine precursors. The addition of sulfamic acid and sodium bisulfite increases the yield of L-isoleucine by more than 10% compared to when no addition is made. Detailed Implementation

[0019] This invention describes in detail the specific implementation methods of the technical solution. It should be noted that the embodiments shown are only some representative implementation methods and do not exhaust all possibilities. Based on the core technical solution disclosed in this patent, any equivalent implementation method derived by those skilled in the art through conventional technical means falls within the protection scope claimed by this patent.

[0020] The strain used in this invention is *Corynebacterium glutamicum* YY2410A-1, accession number CGMCC No. 32505, deposited on November 6, 2024, at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. It should be noted that the entire technical content of Chinese patent "CN2025117345252, A preparation process for increasing L-isoleucine yield" is incorporated herein by reference. Example

[0021] A semi-continuous fermentation method for increasing L-isoleucine yield, comprising the following steps: Prepare the seed culture medium: glucose 10 g / L, corn steep liquor 5 g / L, yeast extract 3 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 0.5 g / L, and adjust the pH of the seed culture medium to 7.0. Then, use activated Corynebacterium glutamicum YY2410A-1, inoculate the strain into a seed tank containing the seed culture medium for large-scale cultivation. Maintain the cultivation temperature at 32℃ and the tank pressure at 0.1 MPa until OD reaches [value missing]. 600 Once the level reaches 2, the seed liquid is obtained.

[0022] The expanded seed culture was inoculated into the initial fermentation medium in a 100-ton fermenter at an inoculum size of 8%. The fermentation conditions were: pH 7.1; initial aeration 1500m³. 3 The fermentation conditions were as follows: fermentation temperature: 34℃; fermentation time: 48h; fermentation culture medium: glucose 40g / L, beet molasses 50g / L, nitrogen source 20g / L, potassium dihydrogen phosphate 2g / L, magnesium sulfate heptahydrate 1g / L, sulfamic acid 0.01g / L, sodium bisulfite 0.01g / L. The nitrogen source was corn steep liquor and ammonium sulfate, with a mass ratio of corn steep liquor to ammonium sulfate of 1:3.

[0023] When the residual sugar content in the fermenter is 0.5%, begin adding 2.3% glucose solution and 0.6% beet molasses, with a flow rate of 4 ml / L / h for both solutions per liter of fermentation broth. When the residual sugar content is below 0.2%, add 3% glucose solution and 0.8% beet molasses, with a flow rate of 5 ml / L / h for both solutions per liter of fermentation broth. Maintain an aeration rate of 2500 m³ / h during the addition process. 3 / h, tank pressure 0.05MPa. Example

[0024] A semi-continuous fermentation method for increasing L-isoleucine yield, comprising the following steps: Prepare the seed culture medium: glucose 10 g / L, corn steep liquor 5 g / L, yeast extract 3 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 0.5 g / L, and adjust the pH of the seed culture medium to 7.0. Then, use activated Corynebacterium glutamicum YY2410A-1, inoculate the strain into a seed tank containing the seed culture medium for large-scale cultivation. Maintain the cultivation temperature at 32℃ and the tank pressure at 0.1 MPa until OD reaches [value missing]. 600 When the concentration reaches around 2, the seed liquid is obtained.

[0025] The expanded seed culture was inoculated into the initial fermentation medium in a 100-ton fermenter at an inoculum size of 8%. The fermentation conditions were: pH 7.1; initial aeration 1500m³. 3 The fermentation conditions were: 1 / h, 0.05 MPa, 34℃, 48h total fermentation time, and 20g / L glucose + beet molasses, 2g / L nitrogen source, 2g / L potassium dihydrogen phosphate, and 1g / L magnesium sulfate heptahydrate. The nitrogen source was corn steep liquor and ammonium sulfate, with a mass ratio of 1:3.

[0026] When the residual sugar content in the fermenter is 0.5%, begin adding 2.3% glucose solution and 0.6% beet molasses, with a flow rate of 4 ml / L / h for both solutions per liter of fermentation broth. When the residual sugar content is below 0.2%, add 3% glucose solution and 0.8% beet molasses, with a flow rate of 5 ml / L / h for both solutions per liter of fermentation broth. Maintain an aeration rate of 2500 m³ / h during the addition process. 3 / h, tank pressure 0.05MPa.

[0027] The optimal amounts of glucose and beet molasses added to the fermentation medium were verified, with a total addition of 90 g / L. Three replicates were set up for each group, and the average values ​​were calculated. See Table 1 for details.

[0028] Table 1 glucose 80 70 60 50 40 30 20 Beet Molasses 10 20 30 40 50 60 70 L-Isoleucine 43.7 44.3 44.1 44.8 44.9 43.8 40.4 As shown in Table 1, both glucose and beet molasses can be used as carbon sources for fermentation. However, if the ratio of glucose to beet molasses is too low, the yield of L-isoleucine will be reduced. When the ratio of the two is similar, the fermentation yield of L-isoleucine can be increased slightly. Considering that beet molasses is cheaper, it is more appropriate to choose 40g / L glucose + 50g / L beet molasses. Example

[0029] A semi-continuous fermentation method for increasing L-isoleucine yield, comprising the following steps: Prepare the seed culture medium: glucose 10 g / L, corn steep liquor 5 g / L, yeast extract 3 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 0.5 g / L, and adjust the pH of the seed culture medium to 7.0. Then, use activated Corynebacterium glutamicum YY2410A-1, inoculate the strain into a seed tank containing the seed culture medium for large-scale cultivation. Maintain the cultivation temperature at 32℃ and the tank pressure at 0.1 MPa until OD reaches [value missing]. 600 Once the level reaches 2, the seed liquid is obtained.

[0030] The expanded seed culture was inoculated into the initial fermentation medium in a 100-ton fermenter at an inoculum size of 8%. The fermentation conditions were: pH 7.1; initial aeration 1500m³. 3 The fermentation conditions were as follows: fermentation temperature: 34℃; fermentation time: 48h; fermentation medium: glucose 90g / L, nitrogen source 20g / L, potassium dihydrogen phosphate 2g / L, magnesium sulfate heptahydrate 1g / L, aminosulfonic acid 0.01g / L, sodium bisulfite 0.01g / L. The nitrogen source was corn steep liquor and ammonium sulfate, with a mass ratio of corn steep liquor to ammonium sulfate of 1:3.

[0031] When the residual sugar content in the fermenter is 0.5%, begin adding 2.3% glucose solution at a flow rate of 8 ml / L / h per liter of fermentation broth. When the residual sugar content is below 0.2%, add 3% glucose solution at a flow rate of 10 ml / L / h per liter of fermentation broth. Maintain an aeration rate of 2500 m³ / h during the addition process. 3 / h, tank pressure 0.05MPa. Example

[0032] A semi-continuous fermentation method for increasing L-isoleucine yield, comprising the following steps: Prepare the seed culture medium: glucose 10 g / L, corn steep liquor 5 g / L, yeast extract 3 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 0.5 g / L, and adjust the pH of the seed culture medium to 7.0. Then, use activated Corynebacterium glutamicum YY2410A-1, inoculate the strain into a seed tank containing the seed culture medium for large-scale cultivation. Maintain the cultivation temperature at 32℃ and the tank pressure at 0.1 MPa until OD reaches [value missing]. 600 Once the level reaches 2, the seed liquid is obtained.

[0033] The expanded seed culture was inoculated into the initial fermentation medium in a 100-ton fermenter at an inoculum size of 8%. The fermentation conditions were: pH 7.1; initial aeration 1500m³. 3 The fermentation conditions were as follows: fermentation temperature was 34℃, fermentation time was 48h, fermentation culture medium was 90g / L glucose, 20g / L nitrogen source, 2g / L potassium dihydrogen phosphate, and 1g / L magnesium sulfate heptahydrate. The nitrogen source was corn steep liquor and ammonium sulfate, and the mass ratio of corn steep liquor to ammonium sulfate was 1:3.

[0034] When the residual sugar content in the fermenter is 0.5%, begin adding 2.3% glucose solution at a flow rate of 8 ml / L / h per liter of fermentation broth. When the residual sugar content is below 0.2%, add 3% glucose solution at a flow rate of 10 ml / L / h per liter of fermentation broth. Maintain an aeration rate of 2500 m³ / h during the addition process. 3 / h, tank pressure 0.05MPa. Example

[0035] Comparative Examples 1-4 were used to verify the effects of beet molasses, sulfamic acid, and sodium bisulfite on the L-isoleucine yield fermented by Corynebacterium glutamicum YY2410A-1. Three time points were set at 24, 36, and 48 (h). Each group of experiments was repeated in triplicate, and the average value was calculated.

[0036] Table 2 Example 1 26.5 41.8 51.3 Example 2 23.9 39.7 44.9 Example 3 24.1 40.6 49.2 Example 4 23.7 38.4 43.1 As shown in Table 2, compared with Example 4, beet molasses (Example 2) had a limited effect on increasing the yield of L-isoleucine fermented by Corynebacterium glutamicum YY2410A-1. However, beet molasses is cheaper than glucose, which can significantly reduce fermentation costs and increase the added value of large-scale production. Sulfamic acid, added at an appropriate concentration, can act as an antioxidant and stabilizer, suitable for high-density fermentation systems. Sodium bisulfite can remove reactive oxygen species in the fermentation broth, protect the cell membrane and enzyme proteins of the strain from oxidative damage, maintain the stability of metabolic pathways, and may also affect the carbon flux of sugar metabolism pathways, promoting the accumulation of isoleucine precursors. The addition of sulfamic acid and sodium bisulfite increased the yield of L-isoleucine by 11.8% compared to Example 4. Example 1 reduced costs by replacing some glucose with beet molasses and increased fermentation yield by adding sulfamic acid and sodium bisulfite. After optimization of the fermentation process, the fermentation method of the present invention is suitable for industrial-scale production.

[0037] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the disclosed technical content without departing from the scope of the technical solution of the present invention, resulting in equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall fall within the scope of the technical solution of the present invention.

Claims

1. A semi-continuous fermentation method for increasing L-isoleucine yield, comprising the following steps: The seed culture of Corynebacterium glutamicum producing L-isoleucine was inoculated into a fermentation medium and fermented for more than 40 hours. The fermentation medium contained aminosulfonic acid and sodium bisulfite.

2. The semi-continuous fermentation method according to claim 1, characterized in that, The carbon source of the fermentation medium is a mixture of glucose and beet molasses.

3. The semi-continuous fermentation method according to claim 1, characterized in that, The amount of aminosulfonic acid and sodium bisulfite added to the fermentation medium is 0.01 g / L.

4. The semi-continuous fermentation method according to claim 2, characterized in that, The total amount of carbon source added to the fermentation medium is 90 g / L.

5. The semi-continuous fermentation method according to any one of claims 1-4, characterized in that, The fermentation medium is formulated as follows: glucose 40 g / L, beet molasses 50 g / L, nitrogen source 20 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 1 g / L, aminosulfonic acid 0.01 g / L, sodium bisulfite 0.01 g / L. The nitrogen source is corn steep liquor and ammonium sulfate, and the mass ratio of corn steep liquor to ammonium sulfate is 1:

3.

6. The semi-continuous fermentation method according to any one of claims 1-4, characterized in that, The inoculation amount of the Corynebacterium glutamicum seed solution is 5-15%.

7. The semi-continuous fermentation method according to any one of claims 1-4, characterized in that, The fermentation culture time was 48 hours.

8. The semi-continuous fermentation method according to any one of claims 1-4, characterized in that, The Corynebacterium glutamicum is Corynebacterium glutamicum YY2410A-1, with accession number CGMCC No.32505.

9. The semi-continuous fermentation method according to any one of claims 1-4, characterized in that, During the fermentation process, when the residual sugar content in the fermenter is 0.5%, 2.3% glucose solution and 0.6% beet molasses are added; when the residual sugar content is 0.2%, 3% glucose solution and 0.8% beet molasses are added.

10. The semi-continuous fermentation method according to claim 9, characterized in that, During the feeding process, the flow rate of 2.3% glucose solution and 0.6% beet molasses was 4 ml / Lh per L of fermentation broth; the flow rate of 2.3% glucose solution and 0.6% beet molasses was 5 ml / Lh per L of fermentation broth.