Lysine total nutrient fed-batch fermentation method
By using a mixed feed-through method with a complete nutrient solution, the problem of solution homogenization in lysine fermentation was solved, improving fermentation efficiency and yield, and achieving more efficient lysine production.
Patent Information
- Application Number
- CN202411162199.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-23
- Publication Date
- 2026-03-03
AI Technical Summary
In the current lysine fermentation process, the fed-batch solution cannot be mixed quickly, which affects the fermentation efficiency and quality. Furthermore, the consumption of nutrients in the later stages is insufficient and cannot meet the fermentation requirements of the strain.
The total nutrient solution, including ammonium sulfate solution, glucose solution and second-generation culture medium, is mixed and fed into the fermenter via a solenoid valve to control the flow rate. This ensures rapid and uniform mixing, with the stirring speed and temperature controlled within the optimal range. The reaction time is 8-12 hours.
It increased the yield of lysine, improved fermentation efficiency and quality, and increased the yield by 0.64%-0.79%.
Abstract
Description
Technical Field
[0001] This invention belongs to the field of bioengineering technology and relates to a fed-batch fermentation method for lysine. Background Technology
[0002] L-Lysine (Lysine for short) is a major biotechnology product, primarily used as a food and feed additive, and widely applied in the food, livestock, and aquaculture industries. Currently, it is obtained by preparing a culture medium from organic matter such as soybeans and corn, adding other chemical agents, and then inoculating it with microbial strains for fermentation. The main problems are: to maintain the viability of the microbial strains, various solutions need to be added in a continuous flow; however, due to the slow stirring during fermentation, the added preparations cannot be quickly and evenly mixed, thus affecting efficiency and quality.
[0003] During fermentation, it is necessary to strictly control nitrogen source, carbon source, pH, conversion rate, and fresh air. Currently, the added solutions such as glucose and ammonia cannot be quickly mixed with other materials in the tank, which is insufficient to achieve the optimal fermentation environment. Furthermore, as nutrients are consumed, they cannot meet the needs of the microbial culture during the later stages of fermentation.
[0004] Application No.: 2022112797781, provides a method for fermenting to produce L-lysine, the method comprising the following steps: (1) pretreating and hydrolyzing lignocellulose raw materials to obtain hydrolysate; (2) fermenting L-lysine using a fermentation medium containing the hydrolysate, wherein during the fermentation process, the glucose content in the fermentation system is maintained above 1 g / L by feeding. This invention utilizes lignocellulose raw materials for hydrolysis and fermentation to produce lysine, and simultaneously achieves a fed-batch fermentation process for lysine by adding concentrated hydrolysate of the lignocellulose raw materials, thereby increasing lysine yield and possessing broad industrial application prospects.
[0005] The above-mentioned scheme, which involves adding concentrated hydrolysate of glucose and lignocellulose raw materials, still cannot overcome the aforementioned defects. Summary of the Invention
[0006] To address the aforementioned problems and overcome the shortcomings of existing technologies, this invention discloses a fed-batch fermentation method for lysine, which prepares lysine by reacting for a certain period of time.
[0007] This invention is achieved through the following technical solution: A method for fed-batch fermentation of lysine using total nutrition, the method comprising the following steps: adding a certain amount of culture medium to a fermenter, inoculating with a molten liquid, and carrying out fermentation; when the NH concentration in the fermentation broth is less than 2 g / L, or the glucose concentration is less than 5 g / L, feeding the total nutrient solution at a flow rate of 60 mL / (L·h). The total nutrient solution includes ammonium sulfate solution, glucose solution and second-generation culture medium; the concentration of ammonium sulfate solution is 350 g / L; the concentration of glucose solution is 500 g / L.
[0008] Preferably, The culture medium comprises: 3.5% soybean meal hydrolysate, 1.5% corn steep liquor, 1% peptone, 1% yeast extract (FM905), 1.5% glucose, 1.5% K2HPO4•3H2O, 1.5% MgSO4, with the remainder being water, by mass percentage.
[0009] Preferably, The second-generation culture medium comprises: 5% soybean meal hydrolysate, 3.5% corn steep liquor, 2% peptone, 3% yeast extract (FM905), 3% K2HPO4•3H2O, 1.5% MgSO4, and the remainder is water, by mass percentage.
[0010] Preferably, In the total nutrient solution, the mass ratio of ammonium sulfate solution, glucose solution and second-generation culture medium is 1-5:1-5:5-30.
[0011] Preferably, The ammonium sulfate solution, glucose solution, and second-generation culture medium are placed in separate compartments and connected to the main feed pipeline via solenoid valves.
[0012] The main feed pipe supplies the solution via a spray method at the top of the tank, and solenoid valves are used to open and close each solution and control the flow rate.
[0013] Preferably, The reaction system solution has a pH of 6.0-8.0, a temperature of 35-45℃, a stirring speed of 120-300 rpm, and a reaction time of 8-12 h. Detailed Implementation
[0014] To enable those skilled in the art to better understand the technical solutions in this application, the technical solutions will be fully described below in conjunction with specific embodiments of this application. Example
[0015] A method for fed-batch fermentation of lysine, comprising the following steps: Add 100L of culture medium to the fermenter, inoculate the inoculum, and begin fermentation; When the NH concentration in the fermentation broth is less than 2 g / L or the glucose concentration is less than 5 g / L, the total nutrient solution is fed in at a flow rate of 6 L / h; when the NH concentration is greater than 10 g / L or the glucose concentration is greater than 20 g / L, the feeding is stopped. The culture medium comprises: 3.5% soybean meal hydrolysate, 1.5% corn steep liquor, 1% peptone, 1% yeast extract (FM905), 1.5% glucose, 1.5% K2HPO4•3H2O, 1.5% MgSO4, with the remainder being water, by mass percentage; The total nutrient solution comprises ammonium sulfate solution, glucose solution, and second-generation culture medium; the concentration of ammonium sulfate solution is 350 g / L; the concentration of glucose solution is 500 g / L. The second-generation culture medium comprises: 5% soybean meal hydrolysate, 3.5% corn steep liquor, 2% peptone, 3% yeast extract (FM905), 3% K2HPO4•3H2O, 1.5% MgSO4, and the remainder is water, by mass percentage.
[0016] In the total nutrient solution, the mass ratio of ammonium sulfate solution, glucose solution and second-generation culture medium is 2:5:20; The reaction system solution has a pH of 7.0, a temperature of 45°C, a stirring speed of 180 rpm, and a reaction time of 8 hours.
[0017] After fermentation, the fermentation broth was centrifuged and filtered. The supernatant was then filtered again, decolorized, and subjected to ion extraction to obtain lysine. Example
[0018] A method for fed-batch fermentation of lysine, comprising the following steps: Add 100L of culture medium to the fermenter, inoculate the inoculum, and begin fermentation; When the NH concentration in the fermentation broth is less than 2 g / L or the glucose concentration is less than 5 g / L, the total nutrient solution is fed in at a flow rate of 6 L / h; when the NH concentration is greater than 10 g / L or the glucose concentration is greater than 20 g / L, the feeding is stopped. The culture medium comprises: 3.5% soybean meal hydrolysate, 1.5% corn steep liquor, 1% peptone, 1% yeast extract (FM905), 1.5% glucose, 1.5% K2HPO4•3H2O, 1.5% MgSO4, with the remainder being water, by mass percentage; The total nutrient solution comprises ammonium sulfate solution, glucose solution, and second-generation culture medium; the concentration of ammonium sulfate solution is 350 g / L; the concentration of glucose solution is 500 g / L. The second-generation culture medium comprises: 5% soybean meal hydrolysate, 3.5% corn steep liquor, 2% peptone, 3% yeast extract (FM905), 3% K2HPO4•3H2O, 1.5% MgSO4, and the remainder is water, by mass percentage.
[0019] The mass ratio of the total nutrient solution is controlled by a solenoid valve. The mass ratio of ammonium sulfate solution, glucose solution and second-generation culture medium is 1:5:5 in the early stage of fermentation, 2:3:20 in the middle stage of fermentation and 3:2:30 in the late stage of fermentation.
[0020] The reaction system solution has a pH of 7.0, a temperature of 45°C, a stirring speed of 240 rpm, and a reaction time of 8 hours.
[0021] After fermentation, the fermentation broth was centrifuged and filtered. The supernatant was then filtered again, decolorized, and subjected to ion extraction to obtain lysine. Example
[0022] In the experimental comparison, the experimental group was Example 1, and the control group was a quantitatively fed high-concentration glucose solution and ammonium sulfate solution.
[0023] In the control group, 10L of fermentation broth was used, 400ml of feed liquid was consumed, and 9.6L of supernatant was obtained. After extraction and purification, 1806g of lysine was obtained, with a yield of 18.06%.
[0024] In the experimental group, 10L of fermentation broth was used, and 1600ml of total nutrient fed-batch solution (including 1200ml of second-generation culture medium) was consumed to produce 10.8L of supernatant. After extraction and purification, 2094g of lysine was obtained, with a yield of 18.7%.
[0025] In conclusion, the lysine fed-batch fermentation method proposed in this invention, which involves feeding the lysine mixture with a complete nutrient medium, results in a 0.64% increase in yield.
[0026] When using Example 2, the experimental group consumed 1600 ml of total nutrient fed-batch solution (including 1000 ml of second-generation culture medium) to prepare 10.6 L of supernatant. After extraction and purification, 2074 g of lysine was obtained, with a yield of 18.85%.
[0027] In conclusion, compared with Example 1, Example 2 controlled the ratio of total nutrients in the later stage, reduced sugar and increased the addition of second-generation culture medium, making the fermentation more thorough and complete, and increasing the yield by 0.79% compared with the prior art.
[0028] While the specific embodiments of the present invention have been described above in conjunction with examples, they are not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that any modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection of the present invention.
Claims
1. A method for fed-batch fermentation of lysine, characterized in that, The method includes the following steps: adding a certain amount of culture medium to a fermenter, inoculating with the inoculum, and fermenting; when the NH concentration in the fermentation broth is less than 2 g / L, or the glucose concentration is less than 5 g / L, adding the total nutrient solution at a flow rate of 60 mL / (L·h); The total nutrient solution includes ammonium sulfate solution, glucose solution and second-generation culture medium; the concentration of ammonium sulfate solution is 350 g / L; the concentration of glucose solution is 500 g / L.
2. The method according to claim 1, characterized in that, The culture medium comprises: 3.5% soybean meal hydrolysate, 1.5% corn steep liquor, 1% peptone, 1% yeast extract (FM905), 1.5% glucose, 1.5% K2HPO4•3H2O, 1.5% MgSO4, with the remainder being water, by mass percentage.
3. The method according to claim 1, characterized in that, The second-generation culture medium comprises: 5% soybean meal hydrolysate, 3.5% corn steep liquor, 2% peptone, 3% yeast extract (FM905), 3% K2HPO4•3H2O, 1.5% MgSO4, and the balance being water, by mass percentage.
4. The method according to claim 1, characterized in that, In the total nutrient solution, the mass ratio of ammonium sulfate solution, glucose solution and second-generation culture medium is 1-5:1-5:5-30.
5. The method according to claim 1 or 4, characterized in that, The ammonium sulfate solution, glucose solution, and second-generation culture medium are placed in separate compartments and connected to the main feed pipeline via solenoid valves.
6. The method according to claim 1, characterized in that, The reaction system solution has a pH of 6.0-8.0, a temperature of 35-45℃, a stirring speed of 120 rpm, and a reaction time of 8-12 h.