A fermentation method of l-lysine

Abstract

The application discloses a kind of L-lysine fermentation methods, belong to L-lysine fermentation technical field, the fermentation method is, secondary glutamic acid corynebacterium seed liquid is inoculated to the fermenter containing 1L fermentation medium according to 9.8-10.2% inoculation amount, the temperature in the fermenter is kept at 34-36 ℃, tank pressure is kept at 0.07-0.08MPa, dissolved oxygen is kept at 20-25%, pH is kept at 7-7.2 by adding ammonia water, when residual sugar is reduced to 2g / L, residual sugar is controlled to 5-8g / L by adding glucose aqueous solution, when fermentation is to 8h, betaine makeup liquid is added, when fermentation is to 10h, medium makeup liquid is added.The fermentation method of the application can improve the L-lysine production rate of corynebacterium glutamicum, L-lysine yield and sugar acid conversion rate.

Description

Technical Field

[0001] This invention relates to a fermentation method for L-lysine, belonging to the field of L-lysine fermentation technology. Background Technology

[0002] L-Lysine, one of the eight essential amino acids for humans and animals, has been widely used in the medical, food, and animal husbandry fields. In the medical field, L-lysine can be used to treat viral herpes and enhance the body's resistance to viruses. Complex salts of L-lysine and glutamic acid can also protect brain tissue and reduce bone density loss. In the food field, L-lysine is used as a nutritional supplement, widely applied in grain products, and also as a flavoring agent. Animal husbandry is currently the most important application area for L-lysine, where it is used as a feed additive to help improve the disease resistance and growth rate of poultry and livestock, with huge demand.

[0003] There are three main methods for producing L-lysine: chemical synthesis, protein hydrolysis, and microbial fermentation. Because L-lysine produced through chemical synthesis and protein hydrolysis is difficult to meet veterinary standards and suffers from high production costs and severe environmental pollution, microbial fermentation is currently the primary method for L-lysine production. Among microbial fermentation methods, *Corynebacterium glutamicum* and *Escherichia coli* are commonly used microorganisms for L-lysine production due to their rapid chassis cell growth rate and clear genetic background. *Corynebacterium glutamicum* is considered a relatively safer production strain, hence the extensive research on it.

[0004] Corn steep liquor, a byproduct of corn starch production, contains abundant sulfites, peptides, polysaccharides, proteins, and various amino acids, such as arginine, glutamic acid, histidine, phenylalanine, and alanine. It can be used as water-soluble plant protein, compound fertilizer additive, fiber feed, and a fermentation nitrogen source. In the production of amino acids via microbial fermentation, corn steep liquor, as a cheap and efficient fermentation nitrogen source, can supply organic nitrogen and growth factors, reducing the production cost of amino acids.

[0005] However, existing technologies for producing L-lysine via microbial fermentation, using Corynebacterium glutamicum as the production strain and corn steep liquor as the fermentation nitrogen source, have the following problems: Application and Process Optimization of Small Molecule Peptides from Corn in Lysine Fermentation. Wang Jinduo; Li Lanxiao; Xu Qingyang. China Condiments. 2023. This paper discusses the use of nutrient-rich but complex agricultural byproducts, such as corn steep liquor, in fermentation processes. However, corn steep liquor contains excessive amounts of impurities and proteins. During fermentation, Corynebacterium glutamicum needs to compensate by breaking down proteins to obtain the nitrogen source necessary for growth. L-Lysine fermentation is a typical growth-coupled process. Therefore, when using corn steep liquor as the nitrogen source, the proteins it contains are not easily utilized by Corynebacterium glutamicum. Furthermore, after inoculation, Corynebacterium glutamicum does not easily adapt to the fermentation medium, resulting in a slow lag phase, slow logarithmic growth rate, and slow L-lysine production rate.

[0006] Research on the Feed-on Fermentation Process of L-Lysine. Wang Jinduo; Li Lanxiao; Xu Qingyang. China Condiments. 2022. In the fermentation process, the current general method is fed-batch fermentation, which involves adding the substrate culture medium at once and feeding glucose and other carbon sources during the process to maintain the fermentation of Corynebacterium glutamicum. However, as the fermentation of Corynebacterium glutamicum progresses, the number of Corynebacterium glutamicum keeps doubling, and the concentration of nutrients used to support the growth of Corynebacterium glutamicum gradually decreases. This often leads to a decrease in the activity of Corynebacterium glutamicum in the middle and late stages of fermentation, which in turn leads to a short cell stationary period, a slow L-lysine production rate, and low L-lysine yield and sugar-acid conversion rate.

[0007] Research on Fermentation Process Control and Optimization of L-Lysine. Wang Jinduo. Master's Thesis, Tianjin University of Science and Technology. Published in 2023. It was found that during the fermentation process, the osmotic pressure generated by high concentrations of nutrients and high concentrations of sulfate in the middle and late stages of fermentation can damage Corynebacterium glutamicum, which in turn leads to a slow production rate of L-lysine and low yield and sugar-acid conversion rate of L-lysine.

[0008] The existing technology addresses the above problems using the following solutions: Application and Process Optimization of Corn Small Molecule Peptides in Lysine Fermentation. Wang Jinduo; Li Lanxiao; Xu Qingyang. China Condiments. 2023. This paper discloses the use of corn small molecule peptides to replace corn steep liquor powder as the nitrogen source for fermentation. Compared with corn steep liquor powder, corn small molecule peptides contain a higher amino acid content. Since single-molecule amino acids are more easily utilized by Corynebacterium glutamicum, they adapt to the fermentation medium more easily after inoculation, have a shorter lag phase, and a faster logarithmic growth rate, thereby increasing the production rate of L-lysine. However, a comparison of pretreatment methods for corn gluten meal and the decolorization process of its enzymatic hydrolysate. Zhao Mouming; Ma Mei; Su Guowan; Zheng Lin; Liu Yang. Modern Food Science and Technology. 2019. This paper discloses the process of obtaining corn peptides (i.e., corn small molecule peptides) after enzymatic hydrolysis of corn gluten meal. However, the close connection between protein and starch in corn gluten meal causes many protein cleavage sites to be coated, affecting the enzymatic hydrolysis efficiency of corn gluten meal. Pretreatment is required, but pretreatment will cause protein loss. The application of enzymatic hydrolysis of corn steep liquor in glutamic acid fermentation process. Chen Xiaoni; Li Xuepeng; Fu Guanghao; Zhou Jing; Xu Na. Fermentation Technology Communications. 2021 published the use of phytase and acidic protease to enzymatically hydrolyze corn steep liquor, which can improve the utilization of corn steep liquor and solve the problem that corn steep liquor is not easily utilized by Corynebacterium glutamicum.

[0009] Research on Feed-on Fermentation Technology of L-Lysine with Complete Nutrients. Wang Jinduo; Li Lanxiao; Xu Qingyang. China Condiments. 2022. This paper discloses that feeding-on complete nutrient medium during fermentation can effectively increase the biomass and logarithmic growth rate of *Corynebacterium glutamicum*, and also prolong the stationary phase and high-speed acid production phase of *Corynebacterium glutamicum*, solving the problem of insufficient cell activity caused by nutrient deficiency in the later stage of fermentation, thereby improving the L-lysine production rate, L-lysine yield, and sugar-acid conversion rate. It also discloses that a 50% substrate concentration feed-on fermentation medium yields the best results after 10 hours of fermentation.

[0010] Research on Fermentation Process Control and Optimization of L-Lysine. Wang Jinduo. Master's Thesis, Tianjin University of Science and Technology. 2023. It was published that adding betaine during the fermentation process can improve the resistance of Corynebacterium glutamicum to osmotic pressure, and the L-lysine yield and sugar-acid conversion rate are highest when the amount of betaine added is 8 g / L.

[0011] However, the existing technical solution, which uses microbial fermentation with Corynebacterium glutamicum as the production strain, enzymatically hydrolyzed corn steep liquor as the fermentation nitrogen source, and adds a 50% total nutrient medium to the fermentation medium after 10 hours of fermentation, along with the addition of betaine at a concentration of 8 g / L, still has the following problems: The Effect of Cottonseed Protein Hydrolysate on L-Lysine Fermentation. Zhai Yanling; Chang Liqun; Xu Qingyang. Fermentation Technology Communications. 2015. The study published that cottonseed protein hydrolysate significantly promotes cell growth, and the rate of sugar consumption by the cells increases with the concentration of the hydrolysate. Because cottonseed protein hydrolysate is rich in nutrients, microorganisms utilize it rapidly. However, if too much hydrolysate is added, the cell growth rate accelerates, sugar consumption increases, leading to rapid depletion of nutrients in the culture medium and a shortened cell stationary phase, which is detrimental to later fermentation stages. Similar problems exist in the use of enzymatically hydrolyzed corn steep liquor. Because enzymatically hydrolyzed corn steep liquor contains a large amount of amino acids, it causes Corynebacterium glutamicum to grow too quickly, also resulting in a shortened cell stationary phase, which is detrimental to later fermentation stages, thus reducing L-lysine yield and sugar-acid conversion rate.

[0012] Research on L-Lysine Fermentation Process Control and Optimization. Wang Jinduo. Master's Thesis, Tianjin University of Science and Technology, 2023. The thesis published in this paper highlighted the problem of insufficient dissolved oxygen in the later stages of fermentation caused by increased cell viability due to total nutrient feeding. The paper also disclosed that limited dissolved oxygen supply in the fermenter restricts acid production by the cells; excessive biomass and high viscosity of the fermentation broth limit the oxygen uptake capacity of the cells.

[0013] The study, *Impact of osmotic stress on volume regulation, cytoplasmic solute composition and lysine production in *Corynebacterium glutamicum* MH20-22B. H.R. Consch; R. Kramer; S. Morbach. *Journal of Biotechnology*, 2003, disclosed the use of *Corynebacterium glutamicum* as the production strain. Under low osmotic stress, betaine did not increase L-lysine production; its promoting effect on L-lysine production was only observed under high osmotic stress. Although betaine shortened the time required to reach the stationary phase and allowed cells to begin synthesizing L-lysine earlier, producing a beneficial effect on the timescale of lysine production, the final L-lysine concentration was not increased.

[0014] Chinese patent CN119120608B, "A Method for Increasing L-Lysine Fermentation Yield," discloses the use of an osmotic pressure protectant composed of tetrahydropyrimidine and betaine to maintain the activity of the bacteria in the middle and late stages, thereby increasing the yield of L-lysine per fermentation tank and the sugar-acid conversion rate. However, the patent also discloses the effect of exogenous arginine on the growth and fermentation characteristics of Corynebacterium glutamicum under high glucose stress. A 2017 issue of *Microbiology Bulletin* by Lü Hongfang, Wang Hao, Xu Ning, Ju Jiansong, and Liu Jun states that tetrahydropyrimidine can increase the biomass of Corynebacterium glutamicum. Therefore, in this patent, in the later stages of fermentation, due to the high biomass of Corynebacterium glutamicum, there is a problem of excessive biomass, leading to high viscosity of the fermentation broth, which limits the oxygen uptake capacity of the bacteria, thus reducing the yield of L-lysine and the sugar-acid conversion rate. Summary of the Invention

[0015] To address the shortcomings of existing technologies, this invention provides a fermentation method for L-lysine that can improve the L-lysine production rate, L-lysine yield, and sugar-acid conversion rate of Corynebacterium glutamicum.

[0016] To solve the above technical problems, the technical solution adopted by the present invention is as follows: A fermentation method for L-lysine includes: enzymatic hydrolysis of corn steep liquor, preparation of mung bean protein, preparation of corn steep liquor dry powder, preparation of activation culture medium, preparation of seed culture medium, preparation of fermentation culture medium, preparation of culture medium replenishment solution, preparation of betaine replenishment solution, activation, primary seed culture, secondary seed culture, and fermentation. The enzymatic hydrolysis of corn steep liquor is carried out by mixing corn steep liquor and distilled water, stirring at 50-52℃ for 30-60 min, adjusting the pH to 3.7-3.9 with hydrochloric acid aqueous solution, adding phytase and acidic protease, enzymatic hydrolysis for 15.5-16.5 h, inactivating the enzyme, filtering, and spray drying the filtrate to obtain enzymatic hydrolyzed corn steep liquor powder. Preferably, in the enzymatically hydrolyzed corn steep liquor, the ratio of corn steep liquor, distilled water, phytase, and acidic protease is 2000g:3-3.2L:3-3.2g:6-6.5g; Preferably, in the enzymatic hydrolysis of corn steep liquor, the enzyme inactivation temperature is 90-92℃ and the time is 20-30 min; Preferably, in the enzymatically hydrolyzed corn steep liquor, the phytase activity is ≥50,000 U / g; Preferably, in the enzymatically hydrolyzed corn steep liquor, the enzyme activity of the acidic protease is ≥100,000 U / g; Preferably, in the enzymatically hydrolyzed corn steep liquor, the molar concentration of the hydrochloric acid aqueous solution is 0.2 mol / L; The preparation of mung bean protein involves using mung bean protein powder as raw material and preparing mung bean protein using an alkali dissolution and acid precipitation method. Preferably, the preparation of mung bean protein involves mixing mung bean protein powder with a first portion of distilled water, stirring at room temperature for 30-60 minutes, adjusting the pH to 9-9.2 using sodium hydroxide aqueous solution, continuing to stir for 30-60 minutes, centrifuging for the first time, collecting the supernatant, adjusting the pH to 4.4-4.6 using hydrochloric acid aqueous solution, stirring at room temperature for 30-60 minutes, allowing to stand at room temperature for 6-7 hours, centrifuging for the second time, collecting the precipitate, mixing it with a second portion of distilled water, adjusting the pH to 6.9-7.1 using sodium hydroxide aqueous solution, stirring at room temperature for 30-60 minutes, centrifuging for the third time, collecting the supernatant, and freeze-drying to obtain mung bean protein. More preferably, in the preparation of mung bean protein, the ratio of mung bean protein powder, the first part of distilled water, and the second part of distilled water is 100g:9-10L:0.8-1L; More preferably, in the preparation of mung bean protein, the molar concentration of the sodium hydroxide aqueous solution is 2 mol / L. More preferably, in the preparation of mung bean protein, the molar concentration of the hydrochloric acid aqueous solution is 6 mol / L; More preferably, in the preparation of mung bean protein, the centrifugation speed of the first centrifugation is 10000-11000 rpm and the time is 20-25 min; More preferably, in the preparation of mung bean protein, the second centrifugation speed is 6000-6500 rpm and the time is 20-25 min; More preferably, in the preparation of mung bean protein, the centrifugation speed of the third centrifugation is 4000-4500 rpm and the time is 10-12 min; More preferably, in the preparation of mung bean protein, the freeze-drying cold trap temperature is -50°C to -45°C, the pressure is 15-20 Pa, and the time is 45-48 h; To prepare corn steep liquor powder, corn steep liquor, mung bean protein, and distilled water are mixed and stirred at room temperature for 30-60 minutes. The pH is adjusted to 12-12.2 using sodium hydroxide aqueous solution, and stirring is continued for 1-1.5 hours. The pH is then adjusted to 6.9-7.1 using hydrochloric acid aqueous solution, and stirring is continued for 30-60 minutes. Trehalose is added, and stirring is continued for 1-1.5 hours. The mixture is then freeze-dried to obtain corn steep liquor powder. Preferably, in the preparation of corn steep liquor powder, the ratio of corn steep liquor, mung bean protein, distilled water, and trehalose is 2000g:500-550g:2-2.2L:40-42g; Preferably, in the preparation of corn steep liquor powder, the molar concentration of the sodium hydroxide aqueous solution is 2 mol / L. Preferably, in the preparation of corn steep liquor powder, the molar concentration of the hydrochloric acid aqueous solution is 0.2 mol / L; Preferably, in the preparation of corn steep liquor powder, the temperature of the freeze-drying cold trap is -50°C to -45°C, the pressure is 15-20 Pa, and the time is 24-26 h. The activation medium is prepared according to the following components: tryptone 11.8-12.2 g / L, yeast extract 6.8-7.2 g / L, NaCl 4.8-5.2 g / L, sucrose 19-21 g / L, agar 14.5-15.5 g / L, with distilled water as the solvent and a pH of 7.4-7.6. The seed culture medium is prepared according to the following components: Glucose 39-41 g / L, enzymatically hydrolyzed corn steep liquor powder 14-16 g / L, NaCl 4.8-5.2 g / L, KH2PO4 2.9-3.1 g / L, MgSO4 1.1-1.3 g / L, (NH4)2SO4 4.8-5.2 g / L, MnSO4 0.009-0.011 g / L, FeSO4 0.009-0.011 g / L, VB1 9-11 mg / L, VB3 19-21 mg / L, biotin 9-11 mg / L, solvent is distilled water, pH is 7.4-7.6; The fermentation medium is prepared according to the following components: Glucose 39-41 g / L, enzymatically hydrolyzed corn steep liquor powder 39-41 g / L, KH2PO4 4.8-5.2 g / L, (NH4)2SO4 3.8-4.2 g / L, MgSO4 0.38-0.42 g / L, KCl 0.58-0.62 g / L, MnSO4 0.0058-0.0062 g / L, FeSO4 0.0058-0.0062 g / L, VB1 9-11 mg / L, VB3 19-21 mg / L, biotin 9-11 mg / L, solvent is distilled water, pH is 7-7.2; The prepared culture medium replenishment solution is prepared according to the following components: Glucose 19-21 g / L, corn steep liquor powder 19-21 g / L, KH2PO4 2.4-2.6 g / L, (NH4)2SO4 1.9-2.1 g / L, MgSO4 0.18-0.22 g / L, KCl 0.28-0.32 g / L, MnSO4 0.0028-0.0032 g / L, FeSO4 0.0028-0.0032 g / L, VB1 4.8-5.2 mg / L, VB3 9-11 mg / L, biotin 4.8-5.2 mg / L, solvent is distilled water, pH is 7-7.2; The culture medium replenishment solution is prepared and used immediately. To prepare the betaine replenishment solution, n-dodecane, mung bean protein, and distilled water are mixed and stirred at room temperature for 1-1.5 hours. Then, betaine is added, and stirring is continued for 30-60 minutes to obtain the betaine replenishment solution. Preferably, in the preparation of the betaine replenishment solution, the ratio of n-dodecane, mung bean protein, distilled water, and betaine is 50g:90-100g:0.9-1.1L:28-30g; The activation process involves streaking Corynebacterium glutamicum stored in cryovials at -80°C onto an activation medium, culturing it at 31.5-32.5°C for 24-25 hours, and transferring the streaking process three times to obtain activated Corynebacterium glutamicum. The Corynebacterium glutamicum mentioned is a strain owned by the applicant and has been disclosed in Chinese Patent Publication No. CN103992964B, with the Latin name being... Corynebacterium glutamicum The depositary institution is the China General Microbiological Culture Collection Center (CGMCC), the accession number is CGMCC No. 8184, and the deposit date is September 13, 2013; The primary seed culture involves picking a single colony from activated Corynebacterium glutamicum and inoculating it into 480-520 mL of seed culture medium, then incubating it on a shaker at a temperature of 34-36℃ and a shaker speed of 180-220 rpm until OD (October Expiratory Scale) is reached. 562nm The value was 12, and a primary Corynebacterium glutamicum seed culture was obtained; The secondary seed culture involves inoculating the primary Corynebacterium glutamicum seed culture at a rate of 1.9-2.1% (volume fraction) into a 3L fermenter containing 1L of fermentation medium. The temperature inside the fermenter is maintained at 34-36℃, and the pressure is maintained at 0.07-0.08MPa. Dissolved oxygen is maintained at 20-25% by adjusting the aeration rate and rotation speed, and the pH is maintained at 7-7.2 by adding ammonia. Fermentation continues until the OD reaches [value missing]. 562nm The value was 15, and secondary Corynebacterium glutamicum seed culture was obtained; The fermentation process involves inoculating a secondary Corynebacterium glutamicum seed culture at a rate of 15-16% into a fermenter containing 1L of fermentation medium. The temperature inside the fermenter is maintained at 34-36℃, and the pressure is maintained at 0.07-0.08MPa. Dissolved oxygen is maintained at 20-25% by adjusting the aeration rate and rotation speed, and the pH is maintained at 7-7.2 by adding ammonia. When the residual sugar decreases to 2g / L, a glucose solution is added to control the residual sugar to 5-8g / L. After 8 hours of fermentation, 0.18-0.2L of betaine supplement is added. After 10 hours of fermentation, a culture medium supplement is added. After 30 hours of fermentation, the L-lysine concentration is monitored every hour. Fermentation is terminated when the L-lysine concentration no longer increases, yielding the fermentation broth. Preferably, the fermentation tank used in the fermentation process has a capacity of 3L; Preferably, in the fermentation process, the mass concentration of the ammonia water is 25-28%. Preferably, in the fermentation, the concentration of the glucose aqueous solution is 700-800 g / L; Preferably, during the fermentation, the flow rate of the culture medium replenishment solution is 9-10 mL / h.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: The fermentation method for L-lysine of the present invention can improve the L-lysine production rate, L-lysine yield and sugar-acid conversion rate of Corynebacterium glutamicum. Specifically, when the total fermentation time is 40h, the mass concentration of L-lysine in the fermentation broth is 242.3-244.3g / L and the sugar-acid conversion rate is 72.0-72.6%. Detailed Implementation

[0018] To provide a clearer understanding of the technical features, objectives, and effects of the present invention, specific embodiments of the present invention are now described.

[0019] In Examples 1-3 and Comparative Examples 1-3, the corn steep liquor used came from the same batch, meaning that the indicators of the corn steep liquor used were the same. Specifically, the indicators were: pH value of corn steep liquor was 3.78, dry matter content was 45.80%, total nitrogen content was 3.58%, free amino acid content was 10.27%, crude protein content was 17.51%, phosphorus (P2O5) content was 3.85%, and potassium (K2O) content was 2.21%.

[0020] In Examples 1-3 and Comparative Examples 1-3, the room temperature was specifically 22±2℃.

[0021] In Examples 1-3 and Comparative Examples 1-3, during fermentation, the 3L fermenter used was an automatic fermenter with automatic temperature, pH and dissolved oxygen control functions. When the volume of the fermentation liquid in the fermenter reached 75% of the fermenter's volume, it could automatically discharge the material, and the discharge volume was 5% of the volume of the fermentation liquid in the fermenter.

[0022] In Examples 1-3 and Comparative Examples 1-3, the alkaline dissolution and acid precipitation method was used when preparing mung bean protein.

[0023] In Examples 1-3 and Comparative Examples 1-3, the residual sugar content was detected using Fehling's reagent titration.

[0024] In Examples 1-3 and Comparative Examples 1-3, the L-lysine content was detected using the ninhydrin colorimetric method.

[0025] In Examples 1-3 and Comparative Examples 1-3, the formulas for calculating the sugar-acid conversion rate are as follows: Sugar-acid conversion rate = (mass concentration of L-lysine in fermentation broth × volume of fermentation broth + mass concentration of L-lysine in feed × volume of feed) / glucose consumption × 100%.

[0026] Example 1 A fermentation method for L-lysine includes the following steps: 1. Enzymatic hydrolysis of corn steep liquor: Add 2000g of corn steep liquor and 3L of distilled water to a reaction vessel. Maintain the temperature inside the reaction vessel at 50℃ and the stirring speed at 200rpm for 30min. Adjust the pH to 3.7 using hydrochloric acid aqueous solution. Add 3g of phytase and 6g of acidic protease. Enzymatic hydrolysis is carried out for 15.5h. Maintain the temperature inside the reaction vessel at 90℃ and inactivate the enzyme for 20min. Filter the solution and spray dry the filtrate to obtain enzymatically hydrolyzed corn steep liquor powder. The phytase activity is 50,000 U / g; The enzyme activity of the acidic protease is 100,000 U / g; The molar concentration of the hydrochloric acid aqueous solution is 0.2 mol / L; 2. Preparation of mung bean protein: Add 100g of mung bean protein powder and 9L of distilled water to a reaction vessel. Maintain the stirring speed in the reaction vessel at 200rpm and stir for 30min at room temperature. Adjust the pH to 9 using sodium hydroxide solution, and continue stirring for 30min. Transfer to a centrifuge and centrifuge at 10000rpm for 20min. Collect the supernatant and transfer it to a reaction vessel. Maintain the stirring speed in the reaction vessel at 100rpm and adjust the pH to 4.4 using hydrochloric acid solution. Stir for 30min at room temperature and allow to stand at room temperature. After 6 hours, transfer the mixture to a centrifuge and centrifuge at 6000 rpm for 20 minutes. Collect the precipitate and transfer it to a reaction vessel. Add 0.8 L of distilled water and adjust the pH to 6.9 using sodium hydroxide solution. Stir at room temperature for 30 minutes, then transfer the mixture to a centrifuge and centrifuge at 4000 rpm for 10 minutes. Collect the supernatant and transfer it to a vacuum freeze dryer. Maintain the cold trap temperature in the vacuum freeze dryer at -50℃ and the pressure at 15 Pa. Freeze dry for 45 hours to obtain mung bean protein. The molar concentration of the sodium hydroxide aqueous solution was 2 mol / L. The molar concentration of the hydrochloric acid aqueous solution is 6 mol / L; 3. Preparation of corn steep liquor powder: Add 2000g corn steep liquor, 500g mung bean protein, and 2L distilled water to a reaction vessel. Maintain the stirring speed in the reaction vessel at 200rpm and stir at room temperature for 30min. Adjust the pH to 12 using sodium hydroxide aqueous solution and continue stirring for 1h. Adjust the pH to 6.9 using hydrochloric acid aqueous solution and continue stirring for 30min. Add 40g trehalose and continue stirring for 1h. Transfer to a vacuum freeze dryer and maintain the cold trap temperature in the vacuum freeze dryer at -50℃ and the pressure at 15Pa. Freeze dry for 24h to obtain corn steep liquor powder. The molar concentration of the sodium hydroxide aqueous solution was 2 mol / L. The molar concentration of the hydrochloric acid aqueous solution is 0.2 mol / L; 4. Prepare the activation medium: Prepare the activation medium according to the following components: Tryptone 12 g / L, yeast extract 7 g / L, NaCl 5 g / L, sucrose 20 g / L, agar 15 g / L, solvent: distilled water, pH 7.5; 5. Prepare seed culture medium: Prepare seed culture medium according to the following components: Glucose 40 g / L, enzymatically hydrolyzed corn steep liquor powder 15 g / L, NaCl 5 g / L, KH2PO4 3 g / L, MgSO4 1.2 g / L, (NH4)2SO4 5 g / L, MnSO4 0.01 g / L, FeSO4 0.01 g / L, VB1 10 mg / L, VB3 20 mg / L, biotin 10 mg / L, solvent: distilled water, pH 7.5; 6. Prepare the fermentation medium: Prepare the fermentation medium according to the following components: Glucose 40 g / L, enzymatically hydrolyzed corn steep liquor powder 40 g / L, KH2PO4 5 g / L, (NH4)2SO4 4 g / L, MgSO4 0.4 g / L, KCl 0.6 g / L, MnSO4 0.006 g / L, FeSO4 0.006 g / L, VB1 10 mg / L, VB3 20 mg / L, biotin 10 mg / L, solvent: distilled water, pH 7; 7. Preparation of culture medium replenishment solution: Prepare the culture medium replenishment solution according to the following components: Glucose 20 g / L, corn steep liquor powder 20 g / L, KH2PO4 2.5 g / L, (NH4)2SO4 2 g / L, MgSO4 0.2 g / L, KCl 0.3 g / L, MnSO4 0.003 g / L, FeSO4 0.003 g / L, VB1 5 mg / L, VB3 10 mg / L, biotin 5 mg / L, solvent: distilled water, pH 7; The culture medium replenishment solution is prepared and used immediately. 8. Preparation of betaine replenishment solution: Add 50g n-dodecane, 90g mung bean protein and 0.9L distilled water to the reaction vessel, keep the stirring speed in the reaction vessel at 200rpm, stir at room temperature for 1h, add 28g betaine, and continue stirring for 30min to obtain betaine replenishment solution; 9. Activation: The Corynebacterium glutamicum stored in cryovials at -80℃ was streaked onto activation medium and cultured at 32℃ for 24 hours. The streaking was repeated 3 times to obtain activated Corynebacterium glutamicum. The Corynebacterium glutamicum mentioned is a strain owned by the applicant and has been disclosed in Chinese Patent Publication No. CN103992964B, with the Latin name being... Corynebacterium glutamicum The depositary institution is the China General Microbiological Culture Collection Center (CGMCC), the accession number is CGMCC No. 8184, and the deposit date is September 13, 2013; 10. Primary seed culture: A single colony from the activated Corynebacterium glutamicum was inoculated into 500 mL of seed culture medium and cultured on a shaker at 35°C and 200 rpm until OD500. 562nm The value was 12, and a primary Corynebacterium glutamicum seed culture was obtained; 11. Secondary seed culture: Inoculate the primary Corynebacterium glutamicum seed culture at a rate of 2% (volume fraction) into a 3L fermenter containing 1L of fermentation medium. Maintain the temperature in the fermenter at 35℃ and the pressure at 0.08MPa. Adjust the aeration rate and rotation speed to maintain dissolved oxygen at 20-25%, and maintain the pH at 7 by adding ammonia. Fermentation continues until the OD reaches [the desired concentration]. 562nm The value was 15, and secondary Corynebacterium glutamicum seed culture was obtained; 12. Fermentation: The secondary Corynebacterium glutamicum seed culture was inoculated into a 3L fermenter containing 1L of fermentation medium at a 10% (volume fraction) inoculation rate. The temperature inside the fermenter was maintained at 35℃ and the pressure was maintained at 0.08MPa. The dissolved oxygen was maintained at 20-25% by adjusting the aeration rate and rotation speed, and the pH was maintained at 7 by adding ammonia. When the residual sugar decreased to 2g / L, glucose aqueous solution was added to control the residual sugar to 5-8g / L. After 8 hours of fermentation, 0.18L of betaine supplement solution was added. After 10 hours of fermentation, culture medium supplement solution was added and the flow rate was controlled at 10mL / h. After 30 hours of fermentation, the L-lysine concentration was detected every 1 hour. When the L-lysine concentration no longer increased, the fermentation was terminated, and the fermentation broth was obtained. The total fermentation time and glucose consumption were statistically analyzed, and the total acid production and sugar-acid conversion rate were calculated. The mass concentration of the ammonia solution is 25%. The concentration of the glucose aqueous solution is 750 g / L.

[0027] Example 2 A fermentation method for L-lysine includes the following steps: 1. Enzymatic hydrolysis of corn steep liquor: Add 2000g of corn steep liquor and 3.1L of distilled water to a reaction vessel. Maintain the temperature inside the reaction vessel at 50℃ and the stirring speed at 200rpm for 40min. Adjust the pH to 3.8 using hydrochloric acid aqueous solution. Add 3.1g of phytase and 6.2g of acidic protease. Enzymatic hydrolysis for 16h. Maintain the temperature inside the reaction vessel at 90℃ and inactivate the enzyme for 25min. Filter and spray dry the filtrate to obtain enzymatically hydrolyzed corn steep liquor powder. The phytase activity is 50,000 U / g; The enzyme activity of the acidic protease is 100,000 U / g; The molar concentration of the hydrochloric acid aqueous solution is 0.2 mol / L; 2. Preparation of mung bean protein: Add 100g of mung bean protein powder and 9.5L of distilled water to a reaction vessel. Maintain the stirring speed in the reaction vessel at 250rpm and stir for 40min at room temperature. Adjust the pH to 9 using sodium hydroxide solution, and continue stirring for 40min. Transfer to a centrifuge and centrifuge at 10000rpm for 20min. Collect the supernatant and transfer it to a reaction vessel. Maintain the stirring speed in the reaction vessel at 150rpm and adjust the pH to 4.5 using hydrochloric acid solution. Stir for 40min at room temperature. After standing for 6.5 hours, the mixture was transferred to a centrifuge and centrifuged at 6000 rpm for 20 minutes. The precipitate was collected and transferred to a reaction vessel. 0.9 L of distilled water was added, and the pH was adjusted to 7 using sodium hydroxide solution. The mixture was stirred at room temperature for 40 minutes, then transferred to a centrifuge and centrifuged at 4000 rpm for 15 minutes. The supernatant was collected and transferred to a vacuum freeze dryer. The cold trap temperature in the vacuum freeze dryer was maintained at -50℃ and the pressure was maintained at 20 Pa. The mixture was freeze-dried for 46 hours to obtain mung bean protein. The molar concentration of the sodium hydroxide aqueous solution was 2 mol / L. The molar concentration of the hydrochloric acid aqueous solution is 6 mol / L; 3. Preparation of corn steep liquor powder: Add 2000g corn steep liquor, 520g mung bean protein, and 2.1L distilled water to a reaction vessel. Maintain the stirring speed in the reaction vessel at 200rpm and stir at room temperature for 40min. Adjust the pH to 12.1 using sodium hydroxide aqueous solution and continue stirring for 1.5h. Adjust the pH to 7 using hydrochloric acid aqueous solution and continue stirring for 40min. Add 41g trehalose and continue stirring for 1.5h. Transfer to a vacuum freeze dryer. Maintain the cold trap temperature in the vacuum freeze dryer at -50℃ and the pressure at 20Pa. Freeze dry for 25h to obtain corn steep liquor powder. The molar concentration of the sodium hydroxide aqueous solution was 2 mol / L. The molar concentration of the hydrochloric acid aqueous solution is 0.2 mol / L; 4. Prepare the activation medium: Prepare the activation medium according to the following components: Tryptone 12 g / L, yeast extract 7 g / L, NaCl 5 g / L, sucrose 20 g / L, agar 15 g / L, solvent: distilled water, pH 7.5; 5. Prepare seed culture medium: Prepare seed culture medium according to the following components: Glucose 40 g / L, enzymatically hydrolyzed corn steep liquor powder 15 g / L, NaCl 5 g / L, KH2PO4 3 g / L, MgSO4 1.2 g / L, (NH4)2SO4 5 g / L, MnSO4 0.01 g / L, FeSO4 0.01 g / L, VB1 10 mg / L, VB3 20 mg / L, biotin 10 mg / L, solvent: distilled water, pH 7.5; 6. Prepare the fermentation medium: Prepare the fermentation medium according to the following components: Glucose 40 g / L, enzymatically hydrolyzed corn steep liquor powder 60 g / L, KH2PO4 5 g / L, (NH4)2SO4 4 g / L, MgSO4 0.4 g / L, KCl 0.6 g / L, MnSO4 0.006 g / L, FeSO4 0.006 g / L, VB1 10 mg / L, VB3 20 mg / L, biotin 10 mg / L, solvent: distilled water, pH 7; 7. Preparation of culture medium replenishment solution: Prepare the culture medium replenishment solution according to the following components: Glucose 500 g / L, corn steep liquor powder 30 g / L, KH2PO4 2.5 g / L, (NH4)2SO4 2 g / L, MgSO4 0.2 g / L, KCl 0.3 g / L, MnSO4 0.003 g / L, FeSO4 0.003 g / L, VB1 5 mg / L, VB3 10 mg / L, biotin 5 mg / L, solvent: distilled water, pH 7; The culture medium replenishment solution is prepared and used immediately. 8. Preparation of betaine replenishment solution: Add 50g n-dodecane, 95g mung bean protein and 1L distilled water to the reaction vessel, keep the stirring speed in the reaction vessel at 250rpm, stir at room temperature for 1.5h, add 29g betaine, and continue stirring for 40min to obtain betaine replenishment solution; 9. Activation: The Corynebacterium glutamicum stored in cryovials at -80℃ was streaked onto activation medium and cultured at 32℃ for 24 hours. The streaking was repeated 3 times to obtain activated Corynebacterium glutamicum. The Corynebacterium glutamicum mentioned is a strain owned by the applicant and has been disclosed in Chinese Patent Publication No. CN103992964B, with the Latin name being... Corynebacterium glutamicum The depositary institution is the China General Microbiological Culture Collection Center (CGMCC), the accession number is CGMCC No. 8184, and the deposit date is September 13, 2013; 10. Primary seed culture: A single colony from the activated Corynebacterium glutamicum was inoculated into 500 mL of seed culture medium and cultured on a shaker at 35°C and 200 rpm until OD500. 562nm The value was 12, and a primary Corynebacterium glutamicum seed culture was obtained; 11. Secondary seed culture: Inoculate the primary Corynebacterium glutamicum seed culture at a rate of 2% (volume fraction) into a 3L fermenter containing 1L of fermentation medium. Maintain the temperature in the fermenter at 35℃ and the pressure at 0.08MPa. Adjust the aeration rate and rotation speed to maintain dissolved oxygen at 20-25%, and maintain the pH at 7 by adding ammonia. Fermentation continues until the OD reaches [the desired concentration]. 562nm The value was 15, and secondary Corynebacterium glutamicum seed culture was obtained; 12. Fermentation: The secondary Corynebacterium glutamicum seed culture was inoculated into a 3L fermenter containing 1L of fermentation medium at a 10% (volume fraction) inoculation rate. The temperature inside the fermenter was maintained at 35℃ and the pressure was maintained at 0.08MPa. The dissolved oxygen was maintained at 20-25% by adjusting the aeration rate and rotation speed, and the pH was maintained at 7 by adding ammonia. When the residual sugar decreased to 2g / L, glucose aqueous solution was added to control the residual sugar to 5-8g / L. After 8 hours of fermentation, 0.18L of betaine supplement solution was added. After 10 hours of fermentation, culture medium supplement solution was added and the flow rate was controlled at 10mL / h. After 30 hours of fermentation, the L-lysine concentration was detected every 1 hour. When the L-lysine concentration no longer increased, the fermentation was terminated, and the fermentation broth was obtained. The total fermentation time and glucose consumption were statistically analyzed, and the total acid production and sugar-acid conversion rate were calculated. The mass concentration of the ammonia solution is 25%. The concentration of the glucose aqueous solution is 750 g / L.

[0028] Example 3 A fermentation method for L-lysine includes the following steps: 1. Enzymatic hydrolysis of corn steep liquor: Add 2000g of corn steep liquor and 3.2L of distilled water to a reaction vessel. Maintain the temperature inside the reaction vessel at 52℃ and the stirring speed at 250rpm for 60min. Adjust the pH to 3.9 using hydrochloric acid aqueous solution. Add 3.2g of phytase and 6.5g of acidic protease. Enzymatic hydrolysis is carried out for 16.5h. Maintain the temperature inside the reaction vessel at 92℃ and inactivate the enzyme for 30min. Filter the solution and spray dry the filtrate to obtain enzymatically hydrolyzed corn steep liquor powder. The phytase activity is 50,000 U / g; The enzyme activity of the acidic protease is 100,000 U / g; The molar concentration of the hydrochloric acid aqueous solution is 0.2 mol / L; 2. Preparation of mung bean protein: Add 100g of mung bean protein powder and 10L of distilled water to a reaction vessel. Maintain the stirring speed in the reaction vessel at 300rpm and stir at room temperature for 60min. Adjust the pH to 9.2 using sodium hydroxide solution, and continue stirring for 60min. Transfer to a centrifuge and centrifuge at 11000rpm for 25min. Collect the supernatant and transfer it to a reaction vessel. Maintain the stirring speed in the reaction vessel at 200rpm and adjust the pH to 4.6 using hydrochloric acid solution. Stir at room temperature for 60min. After standing at room temperature for 7 hours, the mixture was transferred to a centrifuge and centrifuged at 6000 rpm for 25 minutes. The precipitate was collected and transferred to a reaction vessel. 1 L of distilled water was added, and the pH was adjusted to 7.1 using sodium hydroxide solution. The mixture was stirred at room temperature for 60 minutes, then transferred to a centrifuge and centrifuged at 4000 rpm for 12 minutes. The supernatant was collected and transferred to a vacuum freeze dryer. The cold trap temperature in the vacuum freeze dryer was maintained at -50℃ and the pressure was maintained at 20 Pa. The mixture was freeze-dried for 48 hours to obtain mung bean protein. The molar concentration of the sodium hydroxide aqueous solution was 2 mol / L. The molar concentration of the hydrochloric acid aqueous solution is 6 mol / L; 3. Preparation of corn steep liquor powder: Add 2000g corn steep liquor, 550g mung bean protein, and 2.2L distilled water to a reaction vessel. Maintain the stirring speed in the reaction vessel at 300rpm and stir at room temperature for 60min. Adjust the pH to 12.2 using sodium hydroxide aqueous solution and continue stirring for 1.5h. Adjust the pH to 7.1 using hydrochloric acid aqueous solution and continue stirring for 60min. Add 42g trehalose and continue stirring for 1.5h. Transfer to a vacuum freeze dryer. Maintain the cold trap temperature in the vacuum freeze dryer at -50℃ and the pressure at 20Pa. Freeze dry for 26h to obtain corn steep liquor powder. The molar concentration of the sodium hydroxide aqueous solution was 2 mol / L. The molar concentration of the hydrochloric acid aqueous solution is 0.2 mol / L; 4. Prepare the activation medium: Prepare the activation medium according to the following components: Tryptone 12 g / L, yeast extract 7 g / L, NaCl 5 g / L, sucrose 20 g / L, agar 15 g / L, solvent: distilled water, pH 7.5; 5. Prepare seed culture medium: Prepare seed culture medium according to the following components: Glucose 40 g / L, enzymatically hydrolyzed corn steep liquor powder 15 g / L, NaCl 5 g / L, KH2PO4 3 g / L, MgSO4 1.2 g / L, (NH4)2SO4 5 g / L, MnSO4 0.01 g / L, FeSO4 0.01 g / L, VB1 10 mg / L, VB3 20 mg / L, biotin 10 mg / L, solvent: distilled water, pH 7.5; 6. Prepare the fermentation medium: Prepare the fermentation medium according to the following components: Glucose 40 g / L, enzymatically hydrolyzed corn steep liquor powder 60 g / L, KH2PO4 5 g / L, (NH4)2SO4 4 g / L, MgSO4 0.4 g / L, KCl 0.6 g / L, MnSO4 0.006 g / L, FeSO4 0.006 g / L, VB1 10 mg / L, VB3 20 mg / L, biotin 10 mg / L, solvent: distilled water, pH 7; 7. Preparation of culture medium replenishment solution: Prepare the culture medium replenishment solution according to the following components: Glucose 500 g / L, corn steep liquor powder 30 g / L, KH2PO4 2.5 g / L, (NH4)2SO4 2 g / L, MgSO4 0.2 g / L, KCl 0.3 g / L, MnSO4 0.003 g / L, FeSO4 0.003 g / L, VB1 5 mg / L, VB3 10 mg / L, biotin 5 mg / L, solvent: distilled water, pH 7; The culture medium replenishment solution is prepared and used immediately. 8. Preparation of betaine replenishment solution: Add 50g n-dodecane, 100g mung bean protein and 1.1L distilled water to the reaction vessel, keep the stirring speed in the reaction vessel at 300rpm, stir at room temperature for 1.5h, add 30g betaine, and continue stirring for 60min to obtain betaine replenishment solution; 9. Activation: The Corynebacterium glutamicum stored in cryovials at -80℃ was streaked onto activation medium and cultured at 32℃ for 24 hours. The streaking was repeated 3 times to obtain activated Corynebacterium glutamicum. The Corynebacterium glutamicum mentioned is a strain owned by the applicant and has been disclosed in Chinese Patent Publication No. CN103992964B, with the Latin name being... Corynebacterium glutamicumThe depositary institution is the China General Microbiological Culture Collection Center (CGMCC), the accession number is CGMCC No. 8184, and the deposit date is September 13, 2013; 10. Primary seed culture: A single colony from the activated Corynebacterium glutamicum was inoculated into 500 mL of seed culture medium and cultured on a shaker at 35°C and 200 rpm until OD500. 562nm The value was 12, and a primary Corynebacterium glutamicum seed culture was obtained; 11. Secondary seed culture: Inoculate the primary Corynebacterium glutamicum seed culture at a rate of 2% (volume fraction) into a 3L fermenter containing 1L of fermentation medium. Maintain the temperature in the fermenter at 35℃ and the pressure at 0.08MPa. Adjust the aeration rate and rotation speed to maintain dissolved oxygen at 20-25%, and maintain the pH at 7 by adding ammonia. Fermentation continues until the OD reaches [the desired concentration]. 562nm The value was 15, and secondary Corynebacterium glutamicum seed culture was obtained; 12. Fermentation: The secondary Corynebacterium glutamicum seed culture was inoculated into a 3L fermenter containing 1L of fermentation medium at a 10% (volume fraction) inoculation rate. The temperature inside the fermenter was maintained at 35℃ and the pressure was maintained at 0.08MPa. The dissolved oxygen was maintained at 20-25% by adjusting the aeration rate and rotation speed, and the pH was maintained at 7 by adding ammonia. When the residual sugar decreased to 2g / L, glucose aqueous solution was added to control the residual sugar to 5-8g / L. After 8 hours of fermentation, 0.18L of betaine supplement solution was added. After 10 hours of fermentation, culture medium supplement solution was added and the flow rate was controlled at 10mL / h. After 30 hours of fermentation, the L-lysine concentration was detected every 1 hour. When the L-lysine concentration no longer increased, the fermentation was terminated, and the fermentation broth was obtained. The total fermentation time and glucose consumption were statistically analyzed, and the total acid production and sugar-acid conversion rate were calculated. The mass concentration of the ammonia solution is 25%. The concentration of the glucose aqueous solution is 750 g / L.

[0029] Comparative Example 1 The fermentation method for L-lysine is basically the same as in Example 1, except that the addition of mung bean protein in step 3 (preparing corn steep liquor powder) is omitted. In other words, step 3 (preparing corn steep liquor powder) is replaced with: Add 2000g of corn steep liquor and 2L of distilled water to a reaction vessel. Maintain the stirring speed in the reaction vessel at 200rpm and stir at room temperature for 30min. Add 40g of trehalose and continue stirring for 1h. Transfer the mixture to a vacuum freeze dryer. Maintain the cold trap temperature in the vacuum freeze dryer at -50℃ and the pressure at 15Pa. Freeze dry for 24h to obtain corn steep liquor powder.

[0030] Comparative Example 2 The fermentation method for L-lysine is basically the same as in Example 1, except that the order of adding trehalose in step 3 (preparing corn steep liquor powder) is placed after freeze-drying, i.e., step 3 (preparing corn steep liquor powder) is changed to: Add 2000g corn steep liquor, 500g mung bean protein, and 2L distilled water to a reaction vessel. Maintain the stirring speed in the reaction vessel at 200rpm and stir at room temperature for 30min. Adjust the pH to 12 using sodium hydroxide aqueous solution and continue stirring for 1h. Adjust the pH to 6.9 using hydrochloric acid aqueous solution and continue stirring for 30min. Transfer the mixture to a vacuum freeze dryer and maintain the cold trap temperature in the vacuum freeze dryer at -50℃ and the pressure at 15Pa. Freeze dry for 24h. Transfer the mixture to a mixing vessel, add 40g trehalose, and maintain the stirring speed in the mixing vessel at 300rpm and stir at room temperature for 30min to obtain corn steep liquor powder. The molar concentration of the sodium hydroxide aqueous solution was 2 mol / L. The molar concentration of the hydrochloric acid aqueous solution is 0.2 mol / L.

[0031] Comparative Example 3 The fermentation method for L-lysine is basically the same as in Example 1, except that the addition of mung bean protein in step 8 (preparing the betaine replenishment solution) is omitted. That is, step 8 (preparing the betaine replenishment solution) is replaced with: Add 50g of n-dodecane and 0.9L of distilled water to the reaction vessel, maintain the stirring speed in the reaction vessel at 200rpm, stir at room temperature for 1h, add 28g of betaine, and continue stirring for 30min to obtain betaine replenishment solution; In step 12 of the fermentation process, when adding 0.18L of betaine supplement solution, an additional 0.18L of mung bean protein aqueous dispersion with a concentration of 100g / L was added.

[0032] Results and Analysis The results of total fermentation time, glucose consumption, L-lysine concentration in fermentation broth at the end of fermentation, total acid production, and sugar-acid conversion rate in Examples 1-3 and Comparative Examples 1-3 are shown in Table 1: Table 1

[0033] The results above show that the mass concentration of L-lysine, total acid production, and sugar-acid conversion rate of Example 1 are higher than those of Comparative Examples 1-3.

[0034] Further analysis revealed the following reasons: Compared with Comparative Example 1, Example 1 added mung bean protein in the step of preparing corn steep liquor powder, as disclosed in the paper "Construction and Properties of Rice Protein-Mung Bean Protein Co-assembled Complex" by Jiang Weiwei, Wang Xufeng, Xu Qiancheng, Wu Taoying, and He Xiaohong, published in the Chinese Journal of Food Science and Technology in 2025. Mung bean protein can co-assemble with rice protein through hydrophobic interactions. In this invention, the hydrophobic groups in mung bean protein can co-assemble with the hydrophobic groups of impurities in corn steep liquor through hydrophobic interactions, thereby avoiding the influence of impurities on fermentation. Through the combination of enzymatically hydrolyzed corn steep liquor powder and mung bean protein in the fermentation medium, the stepwise absorption capacity of Corynebacterium glutamicum on protein can be controlled, thereby controlling the cell growth rate. Compared to Comparative Example 2, Example 1 added trehalose in the corn steep liquor powder preparation step. For example, the use of betaine to enhance lysine fermentation and its metabolic flux analysis (Hong Ming; Li Yan; Zhang Sulong; Li Xuesong; Feng Bin. Fermentation Technology Communications. 2019, published). Although betaine can effectively improve the osmotic pressure tolerance of *E. coli*, it inhibits the osmotic pressure tolerance mechanism of the *E. coli* cells themselves. Activating the cells' own osmotic pressure tolerance mechanism can improve fermentation indicators. Studies have shown that *Corynebacterium glutamicum* cells can also resist high osmotic pressure environments through a compatible solute accumulation mechanism, that is, by synthesizing small molecules such as sugars, alcohols, amino acids and their derivatives that are uncharged and highly water-soluble to resist high osmotic pressure environments. For example, trehalose's protective effect on proteins and its application in the food industry. Wang Yu; Yun Xueyan; Zhang Xiaoyan; Wang Lili; Liang Xiaohong. Food Science and Technology. Published in 2015. Trehalose forms hydrogen bonds with proteins during freeze-drying, thus fixing the trehalose. During fermentation, the controlled release of trehalose can be achieved as proteins are consumed, thereby avoiding inhibition of the osmotic tolerance mechanism of Corynebacterium glutamicum. Compared with Comparative Example 3, Example 1 added mung bean protein in the preparation of betaine replenishment solution, as disclosed in the paper "Construction and Properties of Rice Protein-Mung Bean Protein Co-assembled Complex" by Jiang Weiwei, Wang Xufeng, Xu Qiancheng, Wu Taoying, and He Xiaohong, published in the Chinese Journal of Food Science and Technology, 2025. Mung bean protein can improve the hydrophilicity of rice protein; as disclosed in the paper "Research on L-Lysine Fermentation Process Control and Optimization" by Wang Jinduo, published in the Tianjin University of Science and Technology, 2023. n-Dodecane can act as an oxygen carrier to improve the oxygen uptake capacity of bacteria. In the preparation of betaine replenishment solution, mung bean protein can improve the dispersibility of n-dodecane, thus ensuring the effect of n-dodecane as an oxygen carrier in improving the oxygen uptake capacity of bacteria.

Claims

1. A fermentation method for L-lysine, characterized in that, include: Enzymatic hydrolysis of corn steep liquor to prepare mung bean protein, preparation of corn steep liquor dry powder, preparation of activation culture medium, preparation of seed culture medium, preparation of fermentation culture medium, preparation of culture medium replenishment solution, preparation of betaine replenishment solution, activation, primary seed culture, secondary seed culture, fermentation; The enzymatic hydrolysis of corn steep liquor is carried out by mixing corn steep liquor and distilled water, stirring at 50°C, adjusting the pH to 3.7, adding phytase and acidic protease, hydrolyzing for 15.5 hours, inactivating the enzymes, filtering, and spray drying the filtrate to obtain enzymatic hydrolyzed corn steep liquor powder. To prepare corn steep liquor powder, corn steep liquor, mung bean protein, and distilled water are mixed, stirred at room temperature, the pH is adjusted to 12, stirred, the pH is adjusted to 6.9, stirred, trehalose is added, stirred, and then freeze-dried to obtain corn steep liquor powder. In the preparation of corn steep liquor powder, the ratio of corn steep liquor, mung bean protein, distilled water, and trehalose is 2000g:500g:2L:40g. The activation culture medium is prepared according to the following components: 12 g / L tryptone, 7 g / L yeast extract, 5 g / L NaCl, 20 g / L sucrose, 15 g / L agar, with distilled water as the solvent and a pH of 7.

5. The seed culture medium is prepared according to the following components: Glucose 40 g / L, enzymatically hydrolyzed corn steep liquor powder 15 g / L, NaCl 5 g / L, KH2PO4 3 g / L, MgSO4 1.2 g / L, (NH4)2SO4 5 g / L, MnSO4 0.01 g / L, FeSO4 0.01 g / L, VB1 10 mg / L, VB3 20 mg / L, biotin 10 mg / L, solvent: distilled water, pH 7.5; The fermentation medium is prepared according to the following components: Glucose 40 g / L, enzymatically hydrolyzed corn steep liquor powder 40 g / L, KH2PO4 5 g / L, (NH4)2SO4 4 g / L, MgSO4 0.4 g / L, KCl 0.6 g / L, MnSO4 0.006 g / L, FeSO4 0.006 g / L, VB1 10 mg / L, VB3 20 mg / L, biotin 10 mg / L, solvent: distilled water, pH 7; The prepared culture medium replenishment solution is prepared according to the following components: Glucose 20 g / L, corn steep liquor powder 20 g / L, KH2PO4 2.5 g / L, (NH4)2SO4 2 g / L, MgSO4 0.2 g / L, KCl 0.3 g / L, MnSO4 0.003 g / L, FeSO4 0.003 g / L, VB1 5 mg / L, VB3 10 mg / L, biotin 5 mg / L, solvent: distilled water, pH 7; To prepare the betaine replenishment solution, n-dodecane, mung bean protein, and distilled water are mixed and stirred at room temperature. Then, betaine is added and stirred to obtain the betaine replenishment solution. In the preparation of the betaine replenishment solution, the ratio of n-dodecane, mung bean protein, distilled water, and betaine is 50g:90g:0.9L:28g. The activation process involves streaking Corynebacterium glutamicum stored in cryovials at -80°C onto an activation medium, culturing it at 32°C for 24 hours, and transferring it three times to obtain activated Corynebacterium glutamicum. The accession number of the Corynebacterium glutamicum is CGMCC No. 8184; The primary seed culture involves picking a single colony from activated Corynebacterium glutamicum and inoculating it into 500 mL of seed culture medium, then incubating it on a shaker at 35°C and 200 rpm until OD500 is reached. 562nm The value was 12, and a primary Corynebacterium glutamicum seed culture was obtained; The secondary seed culture involves inoculating the primary Corynebacterium glutamicum seed culture at a 2% inoculation rate into a 3L fermenter containing 1L of fermentation medium. The temperature inside the fermenter is maintained at 35℃, and the pressure is maintained at 0.08MPa. Dissolved oxygen is maintained at 20-25% by adjusting the aeration rate and rotation speed, and the pH is maintained at 7 by adding ammonia. Fermentation proceeds until the OD reaches [the desired concentration]. 562nm The value was 15, and secondary Corynebacterium glutamicum seed culture was obtained; The fermentation process involved inoculating a secondary Corynebacterium glutamicum seed culture at a rate of 10% into a fermenter containing 1L of fermentation medium. The temperature inside the fermenter was maintained at 35°C, the pressure at 0.08MPa, and the dissolved oxygen at 20-25%. The pH was maintained at 7 by adding ammonia. When the residual sugar decreased to 2g / L, a glucose solution was added to control the residual sugar to 5-8g / L. After 8 hours of fermentation, 0.18L of betaine supplement was added. After 10 hours of fermentation, a culture medium supplement was added. The fermentation process uses a 3L fermentation tank. The mass concentration of the ammonia solution is 25%. During 30 hours of fermentation, the concentration of L-lysine was measured every hour. Fermentation was terminated when the concentration of L-lysine stopped increasing, and the fermentation broth was obtained. The concentration of the glucose aqueous solution is 750 g / L; The culture medium replenishment solution was added at a flow rate of 10 mL / h.

2. The fermentation method for L-lysine according to claim 1, characterized in that, In the enzymatically hydrolyzed corn steep liquor, the ratio of corn steep liquor, distilled water, phytase, and acidic protease is 2000g:3L:3g:6g. The enzyme inactivation temperature was 90℃, and the time was 20 minutes. The phytase activity is 50,000 U / g; The enzyme activity of the acidic protease is 100,000 U / g.

3. The fermentation method for L-lysine according to claim 1, characterized in that, The preparation of mung bean protein involves using mung bean protein powder as raw material and preparing mung bean protein using an alkali dissolution and acid precipitation method.

4. The fermentation method for L-lysine according to claim 3, characterized in that, To prepare mung bean protein, mung bean protein powder and a first portion of distilled water are mixed, stirred at room temperature, and the pH is adjusted to 9-9.

2. After stirring and centrifugation, the supernatant is collected, the pH is adjusted to 4.4, stirred at room temperature, allowed to stand at room temperature, centrifuged, the precipitate is collected, mixed with a second portion of distilled water, the pH is adjusted to 6.9, stirred at room temperature, centrifuged, the supernatant is collected, and freeze-dried to obtain mung bean protein. In the preparation of mung bean protein, the ratio of mung bean protein powder, the first part of distilled water, and the second part of distilled water is 100g:9L:0.8L. The freeze-drying cold trap temperature is -50℃, the pressure is 15Pa, and the time is 45h.

5. The fermentation method for L-lysine according to claim 1, characterized in that, In the preparation of corn steep liquor powder, the freeze-drying cold trap temperature is -50℃, the pressure is 15Pa, and the time is 24h.

6. The fermentation method for L-lysine according to claim 1, characterized in that, The culture medium replenishment solution is prepared and used immediately.

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