A corynebacterium glutamicum mutant, a microbial inoculant thereof and application thereof in L-arginine fermentation

Abstract

The application discloses a corynebacterium glutamicum mutant strain, a bacterial agent thereof and application of the bacterial agent in L-arginine fermentation; the mutant strain has the characteristics of high L-arginine yield and low mixed acid, and the application further includes the bacterial agent containing the mutant strain and application of the bacterial agent in L-arginine fermentation. 562 In the fermentation process, the method for controlling the CO2 concentration in tail gas in stages is adopted according to the cell concentration OD The fermentation method has high acid yield, less mixed acid, the arginine yield reaches 12.5%, the sugar acid conversion rate is 52.3%, and the total amount of mixed acid is 0.4%; compared with the fermentation control by calculating the OUR, CER and RQ values, the method for controlling only the CO2 concentration is more rapid and practical, can more accurately judge the strain state, is an efficient and practical L-arginine fermentation process, and has remarkable industrial application value.

Description

Technical Field

[0001] This invention relates to the field of fermentation engineering technology, and in particular to a mutant strain of Corynebacterium glutamicum, its inoculum, and its application in L-arginine fermentation. Background Technology

[0002] L-arginine, also known as 2-amino-5-guanidinovalerate, is a semi-essential amino acid in the human body. It can improve the nitrogen metabolism balance in the body and can be used as an antidote for ammonia poisoning-induced hepatic coma. Therefore, arginine is very important for maintaining liver health. Furthermore, medical research has shown that arginine also plays a significant role in enhancing human immunity and delaying aging.

[0003] The main methods for producing L-arginine include chemical catalysis, protein hydrolysis, and microbial fermentation, with microbial fermentation being the primary method. Corynebacterium glutamicum, a Gram-positive bacterium, is the main microorganism used in L-arginine production and meets food safety standards, making it suitable for industrial fermentation production of glutamic acid.

[0004] The following is a partial list of patent information related to arginine fermentation and tail gas analysis technologies. It can be seen that the main hot topics and processes in the field of amino acid fermentation in the prior art are as follows:

[0005] A process for producing arginine by fermentation using fed-batch culture medium, CN 112430633 A: This patent discloses a process for fermenting arginine using fed-batch culture medium, in which arginine is produced by feeding ammonium sulfate aqueous solution and glucose solution during the fermentation process;

[0006] A strain of *Escherichia coli* and its application in the fermentation production of L-arginine, CN 116355814 A: This patent discloses an *E. coli* strain cultured in a 30L fermenter for 48 hours, producing 136.65 g / L of L-arginine, with a conversion rate of 48.58%. The culture medium contains 1 g / L glucose, 1 g / L yeast extract, 1 g / L ammonium sulfate, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 0.01 g / L manganese sulfate, and 0.01 g / L zinc sulfate.

[0007] The process for improving L-arginine fermentation acid production, CN 10540626 A: This patent discloses a yellow short bacillus that has obtained high acid production capacity through mutagenesis. Trend control and strict feeding are adopted during the fermentation process, and the final acid production can reach 3-4%.

[0008] A method for producing S-adenosylmethionine (S-methionine) using a biochemical method based on a tail gas analysis system, CN 115927516 A: This patent discloses a method for producing S-methionine using a biochemical method based on a tail gas analysis system. This production method is based on a fermentation tail gas analysis system, which regulates cell metabolism at the cellular level through the physiological metabolic parameter RQ to promote S-methionine production.

[0009] Currently, domestic fermentation methods for amino acids such as arginine generally employ dissolved oxygen control. While this method can achieve the desired process objectives to some extent, the accuracy of dissolved oxygen electrodes is affected by various conditions in the fermentation broth. Furthermore, using parameters such as OUR, CER, and RQ values ​​for fermentation control involves numerous calculation formulas that can lead to larger errors and are not suitable for ordinary fermentation workshop operations. Therefore, this patent develops a control method that can replace dissolved oxygen control, utilizing the correlation between bacterial concentration and CO2 concentration in fermentation tail gas. This method solves the aforementioned control difficulties, increases arginine yield and sugar-acid conversion rate, and reduces the yield of other acids. Summary of the Invention

[0010] This invention addresses the shortcomings of existing technologies by disclosing a mutant strain of *Corynebacterium glutamicum*, its inoculum, and its application in L-arginine fermentation. Through mutagenesis, this invention successfully obtained a high-yielding strain. Furthermore, during process development, by gradient control of the CO2 concentration in the fermentation tail gas, a process with high acid production, high sugar-acid conversion rate, and low miscellaneous acid content was achieved, solving the problem of low acid production efficiency in existing technologies.

[0011] First, the Corynebacterium glutamicum mutant strain described in this application is Corynebacterium glutamicum IBBZ-01.

[0012] (Corynebacterium glutamicum), accession number CCTCC NO:M 2022765; deposited at the China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China; deposit date: May 30, 2022.

[0013] Furthermore, the Corynebacterium glutamicum IBBZ-01 is a high-yielding L-arginine producing strain with low paraacidity, selected from Corynebacterium glutamicum (CICC No. 20160) through induced mutagenesis and directional culture.

[0014] The present invention provides a bacterial agent containing Corynebacterium glutamicum IBBZ-01, comprising: a colony of Corynebacterium glutamicum IBBZ-01, and / or its culture, and / or its extract.

[0015] On the other hand, this application also discloses the application of Corynebacterium glutamicum IBBZ-01 in L-arginine fermentation;

[0016] For the technical solution described above, in a further preferred embodiment, the L-arginine fermentation method in the application includes determining the fermentation concentration (OD) based on the bacterial strain concentration. 562 The process involves controlling the CO2 concentration in the fermentation tail gas at different stages. This significantly increases the yield of arginine.

[0017] A further preferred embodiment of the above-described technical solution is the fermentation method, which includes the following steps:

[0018] (1) During fermentation, when the cell concentration reaches OD 562 When the concentration is 10-20, the CO2 concentration should be controlled between 0.5-1.5%.

[0019] (2) OD concentration of the strain 562 When the concentration is 20-30, the CO2 concentration should be controlled between 2.0-3.5%.

[0020] (3) During fermentation, if the residual sugar drops to 0.5-1.5%, feed sugar solution is added to maintain the residual sugar content at 0.5-1.5%.

[0021] (4) The bacterial cell concentration OD 562 When the concentration is 30-40%, the CO2 concentration should be controlled between 4.0-5.0%.

[0022] (5) OD concentration of the fermentation broth to be tested 562 When the concentration is greater than 40, the CO2 concentration should be controlled between 5.5% and 6.5%.

[0023] In a further preferred embodiment of the above-described technical solution, the supplementary sugar solution is a 50-80 wt% glucose solution.

[0024] For the technical solution described above, a further preferred embodiment is that, in the initial stage of the fermentation process of step (1), the fermentation conditions are controlled as follows: aeration rate of 90-120 LPM, temperature of 29-33℃, and pH of 7-7.3. Even more preferred is that the fermentation conditions are: aeration rate of 100 LPM, temperature of 31℃, and pH of 7.0.

[0025] For the technical solution described above, in a further preferred embodiment, the CO2 concentration range in step (1) is 0.8-1.2%, the CO2 concentration range in step (2) is 2.5-3.0%, the CO2 concentration range in step (4) is 4.3-4.7%, and the CO2 concentration range in step (5) is 5.8-6.2%.

[0026] For the technical solution described above, in a further preferred embodiment, in the steps described, the control change of CO2 concentration can be based on the OD value of the bacterial cell concentration. 562 Dissolved oxygen levels can be regulated. While monitoring dissolved oxygen is a possible control method, it is not essential. In fact, carbon dioxide concentration can be controlled by adjusting the fermentation turbine. This control method relies entirely on the CO2 concentration in the fermentation exhaust gas. Typically, the turbine speed is set between 200 and 450 rpm. This allows for CO2 concentration regulation based on cell concentration, while also enabling flexible management of the fermentation process through turbine speed adjustments, with dissolved oxygen monitoring serving as a optional but not necessary control measure.

[0027] Regarding the technical solution described above, further, the culture medium used in step (1) during the process of gradually increasing the inoculation volume of the L-arginine-producing bacterium IBBZ-01 includes:

[0028] Primary seed culture medium: glucose 3-5 wt%, corn steep liquor powder 1.0-1.2 wt%, molasses 1.0-1.5 wt%, ammonium sulfate 0.3-0.5 wt%, KH2PO4 0.1-0.13 wt%, MgSO4·7H2O 0.05-0.07 wt%, calcium carbonate 0.5-1.0 wt%, pH 7.0-7.2;

[0029] Secondary seed culture medium: glucose 3-5 wt%, corn steep liquor powder 1.0-1.2 wt%, molasses 1.0-1.5 wt%, ammonium sulfate 0.3-0.5 wt%, KH2PO4 0.1-0.13 wt%, MgSO4·7H2O 0.05-0.07 wt%, pH 6.8-7.2;

[0030] Initial fermentation medium: glucose 6-8 wt%, corn steep liquor powder 1.0-1.3 wt%, molasses 2.0-2.4 wt%, yeast powder 0.01-0.05 wt%, ammonium sulfate 0.3-0.5 wt%, KH2PO4 0.1-0.13 wt%, MgSO4·7H2O 0.05-0.07 wt%.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. This invention, through screening and optimization, obtained a high-yielding L-arginine strain with high acid production and low levels of other acids. The test results in Example 1 showed an arginine yield of 12.5%, a sugar-acid conversion rate of 52.3%, and a total amount of other acids such as citrulline and ornithine of 0.4%. Comparative Example 6 shows that the *Corynebacterium glutamicum* IBBZ-01 strain of this invention, compared to its original strain *Corynebacterium glutamicum* CICC No. 20160, exhibits a 3-fold increase in arginine production and a 3.37-fold increase in sugar-acid conversion rate. It also shows a significant reduction in other acid production, with the amount of other acids in *Corynebacterium glutamicum* IBBZ-01 reduced to 26% of that in its original strain, indicating a significant improvement in fermentation performance.

[0033] 2. During the microbial synthesis of arginine, the CO2 concentration in the fermentation tail gas directly reflects the growth, reproduction, and metabolic state of the strain, especially the strength of the strain's aerobic and anaerobic metabolic processes. By combining the control of fermentation tail gas with the metabolic process, the difficulty of fermentation control can be significantly reduced, and the arginine acid production can reach about 12%, with a sugar-acid conversion rate of over 50%.

[0034] 3. This invention is based on the bacterial cell concentration OD 562 By closely linking the CO2 concentration with the fermentation tail gas, and then controlling the CO2 concentration in stages, this method can better regulate the metabolic activity of the bacteria based on the different characteristics of each stage of production, significantly improving the acid production and sugar-acid conversion rate of arginine and reducing the amount of miscellaneous acids.

[0035] 4. This invention replaces the conventional fermentation method of dissolved oxygen control by controlling the CO2 concentration in the fermentation tail gas. Compared with dissolved oxygen control, it has the advantages of higher accuracy and greater practicality in controlling CO2 concentration.

[0036] 5. Currently, the detection of fermentation exhaust gas is gradually being applied in fermentation process development. Generally, fermentation control is carried out by calculating OUR, CER, and RQ values, and the main direction of fermentation metabolic flow is determined. In this invention, only the method of controlling CO2 concentration is used. This method is faster and more practical than fermentation control by calculating OUR, CER, and RQ values. This is mainly because when CO2 in fermentation exhaust gas changes, the calculated OUR, CER, and RQ values ​​may not change or change very little, thus leading to an incorrect judgment of the strain's state at that time. Detailed Implementation

[0037] The present invention will be further described below with reference to the embodiments, but it should be understood that the scope of protection of the present invention is not limited to the embodiments.

[0038] In this invention, unless otherwise explicitly stated, percentages and contents are all by mass. Unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available.

[0039] The L-arginine-producing strain IBBZ-01 with low paraacidity was deposited on May 30, 2022, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M2022765.

[0040] Characteristics of Corynebacterium glutamicum:

[0041] Starting with L-arginine-producing Corynebacterium glutamicum (CICC No. 20160), a high-yield L-arginine-producing strain, Corynebacterium glutamicum IBBZ-01 (CCTCC NO: M 2022765), was obtained through mutagenesis screening; genetic markers: sulfaguanidine (SG) resistance, arginine oxime resistance, D-Arg resistance, and L-homogeneous arginine (L-HA) resistance;

[0042] The culture medium composition is as follows:

[0043] Complete culture medium (CM): glucose 1.0 wt%, beef extract 1 wt%, peptone 0.5 wt%, yeast extract 0.5 wt%, sodium chloride 0.5 wt%, urea 0.5 wt%, pH 7.0–7.2;

[0044] Basic culture medium (MM): glucose 3wt%, ammonium sulfate 0.5wt%, KH2PO4 0.1wt%, MgSO4·7H2O 0.05wt%, pH 7.0–7.2;

[0045] Supplemental medium (SM): Add amino acid structure analogs to the basal medium (MM), pH 7.0-7.2; amino acid structure analogs include sulfaguanidine, arginine, and D-arginine;

[0046] Seed culture medium: glucose 3wt%, corn steep liquor powder 1.0wt%, molasses 1.0wt%, ammonium sulfate 0.3wt%, KH2PO4 0.1wt%, MgSO4·7H2O 0.05wt%, calcium carbonate 0.5wt%, pH 7.0–7.2;

[0047] Shake flask fermentation medium: glucose 10wt%, corn steep liquor powder 1.1wt%, ammonium sulfate 1wt%, KH2PO4 0.1wt%, MgSO4·7H2O 0.05wt%, calcium carbonate 1wt%;

[0048] The breeding process is as follows:

[0049] (1) Pre-mutation treatment:

[0050] Take one loopful of activated bacterial culture and inoculate it into an Erlenmeyer flask containing 25 mL of liquid complete culture medium. Incubate the flask at 30°C and 85 rpm for 12 hours. Then, take another 1 mL of culture medium and inoculate it into another Erlenmeyer flask containing 25 mL of liquid complete culture medium. Incubate the flask at 30°C and 90 rpm for 4 hours to bring the cells into the logarithmic growth phase.

[0051] (2) Preparation of bacterial suspension:

[0052] Transfer 10 mL of culture medium to a sterile centrifuge tube, centrifuge at 5000 rpm for 10 min, discard the supernatant, resuspend the cells in physiological saline, pour into an Erlenmeyer flask, shake for 10 min, and adjust the cell concentration to 5*10⁻⁶ cells / mL. 8 per mL.

[0053] (3) Ultraviolet mutagenesis:

[0054] The prepared bacterial suspension was mutagenized under ultraviolet light for a certain period of time, and samples were taken, diluted, plated, and cultured to calculate the lethality rate. Wild-type and concentrated defective types were eliminated by starvation culture and penicillin method, defective types were detected by sound-based culture method, and fermentation acid production capacity was verified.

[0055] (4) Diethyl sulfate mutagenesis:

[0056] Add 0.5 wt% diethyl sulfate (DES) to the prepared bacterial suspension, shake at 30°C for 20 min, stop the treatment, spread on screening medium SM plates, and incubate statically at 31°C for 4–6 days. Select single colonies of mutant strains growing on SM and transfer them to MM plates supplemented with sulfaguanidine, high arginine, and D-arginine, respectively. After static incubation at 31°C for 24 hours, transfer to shake flasks for rescreening.

[0057] (5) ARTP mutagenesis:

[0058] A suitable amount of bacterial suspension was evenly spread onto a sterile metal slide and placed in the mutagenesis chamber of an ARTP biological mutagenesis breeding machine with helium as the working gas, a power supply of 100W, a working gas flow rate of 10L / min, and a treatment distance of 2mm. Mutagenesis treatments were performed for 0, 20, 30, 60, and 120 seconds, respectively. After treatment was stopped, the suspension was spread onto screening medium SM plates and incubated statically at 31°C for 4–6 days. Single colonies of mutant strains growing on SM were selected and transferred to MM plates supplemented with sulfaguanidine, high arginine, and D-arginine, respectively. After static incubation at 31°C for 24 hours, the samples were then subjected to shake flask re-screening.

[0059] (6) Shake-flask re-sieving:

[0060] One loop of the mutant strain was inoculated into a shake-flask fermentation medium and cultured at 31°C and 110 rpm for 72 hours. The L-arginine accumulated in the fermentation broth of the mutant strain was then detected by high performance liquid chromatography. Among all mutant strains, the strain with the highest L-leucine production was IBBZ-01.

[0061] (7) Shake flask culture process:

[0062] Preparation of shake-flask seed culture: One loop of Corynebacterium glutamicum mutant strain IBBZ-01 was inoculated into seed culture medium and cultured at 30℃ and 90 rpm for 16 h. At this time, the bacterial concentration OD 562 Approximately 0.5–0.6 * 20, for later use;

[0063] The seed culture was inoculated into the shake flask fermentation medium at 8% (V / V) and cultured at 30℃ and 120 rpm for 72 h. The L-arginine production was detected by high performance liquid chromatography. The original strain CICC No.20160 produced 4.5 g / L, while the mutant strain IBBZ-01 produced 34.2 g / L. It can be seen that the mutant strain has a stronger acid production capacity and a better L-arginine production capacity than the original strain.

[0064] Example 1

[0065] 1. The seed culture medium consists of the following:

[0066] Primary seed culture medium:

[0067] 3 wt% glucose, 1.0 wt% corn steep liquor powder, 1.0 wt% molasses, 0.3 wt% ammonium sulfate, 0.1 wt% KH2PO4, 0.05 wt% MgSO4·7H2O, 1.0 wt% calcium carbonate; dispensed in 500 ml / 3000 ml containers; sterilized at 121℃ for 20 min.

[0068] Secondary seed tank culture medium:

[0069] 3 wt% glucose, 1.0 wt% corn steep liquor powder, 1.0 wt% molasses, 0.3 wt% ammonium sulfate, 0.1 wt% KH2PO4, 0.05 wt% MgSO4·7H2O, pH 7.0–7.2, sterilization conditions 121℃ / 20 min;

[0070] 2. The fermentation medium is as follows:

[0071] The initial culture medium for standard fermenters consisted of: glucose 7 wt%, corn steep liquor powder 1.0 wt%, molasses 2.0 wt%, yeast powder 0.01%, ammonium sulfate 0.3 wt%, KH₂PO₄ 0.1 wt%, and MgSO₄·7H₂O 0.05 wt%.

[0072] Feeding solution: 60wt% glucose;

[0073] 3. Fermentation Tank Process

[0074] Step 1:

[0075] One-loop Corynebacterium glutamicum IBBZ-01 was inoculated into the primary seed culture medium in a shake flask and cultured at 30℃ and 90 rpm for 16 h with shaking to obtain 500 mL of seed culture. The maturity indicator of the primary seed culture was OD. 562 It is 0.5 to 0.6 * 20.

[0076] Step 2:

[0077] The shake-flask seed culture was inoculated into a 50L secondary seed tank culture medium at a ratio of 0.3% (V / V). After 16 hours of incubation at 30℃, pH 6.8, and dissolved oxygen not less than 20%, the maturity indicator of the secondary seed culture was determined by OD. 562 The concentration was 0.5–0.6 × 20; a secondary seed solution was obtained.

[0078] Step 3:

[0079] 1) Inoculate 10% of the secondary seed culture into a 500L fermenter, with an initial fermentation volume of 60%. Fermentation temperature: 31℃, pH: 7.0, rotation speed: 200 RPM, aeration rate: 100 LPM, tank pressure: 0.05 MPa. During the process, CO2 concentration in the fermentation exhaust gas is the primary control standard; dissolved oxygen is no longer monitored or controlled. OD after inoculation... 562 No adjustments are needed for values ​​1 through 10;

[0080] 2) At the bacterial cell concentration OD 562 When the growth reaches between 10 and 20, the CO2 concentration is controlled at 0.9% by adjusting the rotation speed;

[0081] 3) OD concentration of the strain 562 When the speed is 20-30, the CO2 concentration is controlled at 2.8% by adjusting the rotation speed;

[0082] 4) When the residual sugar drops to 0.8 wt%, start adding feed sugar solution, and control the residual sugar at around 0.8 wt%.

[0083] 5) OD concentration of the strain 562 When the speed is 30-40, the CO2 concentration can be controlled at 4.5% by adjusting the rotation speed;

[0084] 6) OD concentration of the bacterial cells to be detected 562 When the concentration exceeds 40, the CO2 concentration is controlled at 6.0% by adjusting the rotation speed.

[0085] 7) After the sugar solution is replenished and the residual sugar is exhausted, the feed is transferred to the next tank. The yield of arginine is 12.5 wt%, the sugar-acid conversion rate is 52.3%, and the total amount of miscellaneous acids such as citrulline and ornithine is 0.4 wt%.

[0086] As shown in Example 1, the core of this process is to carry out arginine fermentation by controlling the CO2 concentration of fermentation tail gas in stages according to the growth trend of the production agent. This method abandons the original method of controlling dissolved oxygen. According to this control method, the acid production and conversion rate of arginine are significantly improved, and the impurities are significantly reduced.

[0087] In the fermentation process described above, acid production refers to the actual concentration of arginine in the fermentation broth. The formula for calculating the sugar-acid conversion rate is as follows:

[0088] Sugar-acid conversion rate = (Actual arginine concentration × Actual fermentation volume) / (Actual total glucose consumed × 100%)

[0089] Meanwhile, heteroacids refer to the sum of the concentrations of citrulline and ornithine in the fermentation broth. These two parameters are used together to evaluate the efficiency of the fermentation process and the composition of the products.

[0090] Example 2 (Different CO2 control concentration ranges)

[0091] Based on Example 1: In step 3, the CO2 concentration in the fermentation exhaust gas was controlled within different ranges; the methods and results are shown in Table 1.

[0092] Table 1. Control range of different CO2 concentration gradients

[0093]

[0094]

[0095] As shown in the table, according to the bacterial cell concentration OD 562 The most crucial step in gradient control of CO2 concentration is: OD 562 In the 10–20 stage, CO2 concentration needs to be controlled within 0.5–1.5%; OD 562 During the 20-30 stage, CO2 concentration needs to be controlled between 2.0% and 3.5%; OD 562 In the 30-40 stage, CO2 concentration needs to be controlled between 4.0% and 5.0%, OD 562 At concentrations above 40, CO2 concentration needs to be controlled between 5.5% and 6.5%. Too low or too high CO2 concentrations will lead to reduced acid production and increased mixed acids.

[0096] Acid production: It can be seen that the closer the CO2 concentration is to Example 1, the higher the acid production, and the greater the deviation, the lower the acid production.

[0097] Miscellaneous acids: are also closely related to the stage control of CO2 concentration; the further away from Example 1, the higher the level of miscellaneous acids.

[0098] As shown in groups 1-3, this group of data was analyzed for bacterial cell concentration OD. 562 The optimization experiment, which controlled the CO2 concentration at 10-20°C between 0.5-1.5%, showed that within this range, as the CO2 concentration increased, the acid production and sugar-acid ratio both showed a trend of first increasing and then decreasing, while the miscellaneous acid first decreased and then increased. The closer the CO2 concentration was to 0.9% in Example 1, the higher the acid production and the better the results.

[0099] As shown in data sets 4-6, this set of data was analyzed for bacterial cell concentration OD. 562 In the optimization experiment where the CO2 concentration was controlled at 2.0-3.5% at 20-30°C, similarly, as the CO2 concentration increased, the acid production and sugar-acid ratio showed a trend of first increasing and then decreasing, while the miscellaneous acid ratio first decreased and then increased. The closer the CO2 concentration was to 2.8% in Example 1, the higher the acid production and sugar-acid conversion rate, and the lower the miscellaneous acid ratio.

[0100] As shown in groups 7-9, this group of data was analyzed for bacterial cell concentration OD. 562 In the optimization experiment where the CO2 concentration was controlled at 4.0-5.0% at 30-40°C, as the CO2 concentration increased, the acid production and sugar-acid ratio both showed an initial increase followed by a decrease, while the miscellaneous acid ratio first decreased and then increased. The closer the CO2 concentration was to 4.5% in Example 1, the higher the acid production and sugar-acid conversion rate, and the lower the miscellaneous acid ratio.

[0101] As shown in groups 10-12, this group of data was analyzed for bacterial cell concentration OD. 562 In the optimization experiment where the CO2 concentration was controlled at 5.5-6.5% when the CO2 concentration was above 40, the acid production and sugar-acid ratio both showed an initial increase followed by a decrease as the CO2 concentration increased, while the miscellaneous acid ratio both decreased first and then increased. The closer the CO2 concentration was to 6.0% in Example 1, the higher the acid production and sugar-acid conversion rate, and the lower the miscellaneous acid ratio.

[0102] Conclusion: Experiments have shown that controlling CO2 concentration by gradient can improve and promote acid production and sugar-acid conversion rates, while inhibiting the synthesis of other acids.

[0103] Example 3 (Different ventilation rates)

[0104] Based on Example 1, the ventilation rate in step 3 was controlled under different conditions, and the results are shown in Table 2:

[0105] Table 2. Different ventilation rates

[0106] 1 90 10.8 47.0 0.6 2 95 11.8 51.3 0.5 3 105 11.5 50.0 0.4 4 115 10.5 45.7 0.8 5 120 10.2 44.3 0.9

[0107] As shown in data 1-5, an experiment was conducted to optimize the ventilation rate. The results showed that within the range of 90-120 LPM, the acid production and sugar-acid conversion rate first increased and then decreased as the ventilation rate increased. The effect was best at 95 LPM. Meanwhile, the mixed acid production first decreased and then increased, with the lowest mixed acid production at 105 LPM.

[0108] The amount of aeration determines the amount of oxygen entering the fermenter, thus affecting the oxygen transfer coefficient, leading to changes in the respiratory metabolism of the strain, and also affecting the ability of the rotation speed to control the CO2 concentration in the fermentation exhaust gas.

[0109] Assuming that the rate of CO2 production (CER) during the acid-producing phase of the bacteria is stable, increasing the aeration rate will dilute the CO2 concentration in the fermentation exhaust gas, thus narrowing the CO2 gradient control range. Consequently, it becomes impossible to distinguish the CO2 gradient control at different stages, leading to experimental failure. Conversely, decreasing the aeration rate will cause the CO2 concentration to suddenly increase in the early stage of fermentation, or make the rotation speed too sensitive to control the CO2 concentration, with a slight increase in rotation speed causing a sharp rise in CO2 concentration, which will adversely affect the stability of the process.

[0110] In summary, controlling CO2 concentration requires strict control of the ventilation rate to ensure that the CO2 concentration remains stable and controllable throughout the process.

[0111] Example 4 (Different fermentation temperatures and pH levels)

[0112] Based on Example 1: the fermentation temperature and pH conditions in step 3 were controlled at different levels, and the results are shown in Table 3 below:

[0113] Table 3. Different temperatures and pH values

[0114]

[0115]

[0116] Acid production rate: The acid production rate of the four groups was around 11%, which was lower than that of Example 1, proving that temperature and pH have a certain influence on acid production.

[0117] Sugar-acid conversion rate: Has an impact, with a slight decrease.

[0118] Mixed acids: Unaffected.

[0119] Based on the above data analysis, we can conclude that changes in temperature and pH have a certain impact on acid production and sugar-acid conversion rates, but a smaller impact on miscellaneous acids.

[0120] Example 5 (Control of different residual sugar concentrations)

[0121] Based on Example 1: The concentration of residual sugar in step 3 was controlled under different conditions, and the results are shown in Table 4 below:

[0122] Table 4. Different residual sugar concentrations

[0123] 1 0.5 11.4 49.57 0.4 2 1.0 11.9 51.74 0.5 3 1.5 10.9 47.39 0.5

[0124] Acid production: Lowering or increasing the residual sugar control concentration has a certain impact on acid production. Based on the results of the three groups, compared with the 0.8% concentration control in Example 1, the residual sugar deviation will cause a certain fluctuation in acid production.

[0125] Sugar-acid conversion rate: Consistent with the acid production trend, both sugar-acid conversion rates decreased to varying degrees.

[0126] Mixed acids: Unaffected.

[0127] Based on the above data analysis, it can be concluded that the variation in the residual sugar control range of 0.5–1.5 wt% has a certain impact on acid production and sugar-acid conversion rate, but has a smaller impact on miscellaneous acids.

[0128] Comparative Example 1 (Comparative Example 1)

[0129] Based on Example 1, step 3 was carried out using the same control method, with dissolved oxygen controlled during fermentation and the dissolved oxygen level maintained between 5% and 25%. The results are shown in Table 5 below:

[0130] Table 5. Control by different dissolved oxygen levels

[0131] 1 5 5.6 24.35 3.5 2 15 8.5 36.96 1.9 3 25 7.2 31.30 2.5

[0132] Based on the current results of the three groups, the highest acid production was 8.5 wt% under the 15% dissolved oxygen control method, while the other two conditions were 5.6 wt% and 7.2 wt%, respectively. At the same time, the sugar-acid conversion rate was also low, with a maximum of about 37%. Neither of them could reach the level of Example 1, and the amount of miscellaneous acid was much higher than 0.4 wt%.

[0133] Based on the above data analysis, it can be concluded that fermentation using dissolved oxygen control methods cannot achieve the results of fermentation using gradient control of CO2 concentration in the fermentation tail gas as described in Example 1. Gradient control of CO2 in arginine fermentation has the advantages of significantly improving acid production and sugar-acid conversion rates while reducing miscellaneous acids.

[0134] Comparative Example 2 (Comparative Example 2)

[0135] Based on Example 2, the CO2 control range of the fermentation tail gas in step 3 was modified, and the results are shown in Table 6:

[0136] Table 6. Different CO2 control ranges

[0137]

[0138] Based on the current results from the eight groups, gradient control of CO2 concentration in fermentation exhaust gas needs to strictly follow the concentration OD. 562 Adjustments are needed; controlling the CO2 concentration too high or too low at each stage will reduce acid production and the sugar-acid ratio, and may even increase the production of miscellaneous acids.

[0139] The experimental data from sets 1 and 2 show that at the bacterial cell concentration OD... 562 If the CO2 concentration is controlled between 0.5% and 1.5% between 10 and 20, the acid production and sugar-acid conversion rate will decrease, but the increase in miscellaneous acids will not be significant.

[0140] Experimental data from sets 3-8 show that its concentration OD 562 At each stage, it is necessary to control the process within the range expressed in Example 2. Exceeding this range will result in reduced acid production and sugar-acid conversion, and will also lead to an increase in heteroacids.

[0141] Therefore, Comparative Example 2 proves that the CO2 concentration gradient control of fermentation tail gas needs to be carried out according to Example 2.

[0142] Comparative Example 3 (Comparative Example 3)

[0143] Based on Example 3, the ventilation range in step 3 was further reduced or increased, and the results are shown in Table 7 below:

[0144] Table 7. Different ventilation rates

[0145] 1 70 8.5 36.96 1.2 2 80 9.5 41.30 0.8 3 130 9.6 41.74 1.0 4 140 9.4 40.87 1.5

[0146] Based on the results of the four experiments, the increase or decrease in ventilation volume has a significant impact on acid production, sugar-acid conversion rate, and miscellaneous acids.

[0147] In terms of acid production and sugar-acid conversion rate, outside the range specified in Implementation 3, the lower the ventilation rate, the more likely it is to lead to a decrease in acid production and sugar-acid conversion rate and an increase in miscellaneous acids. Similarly, the higher the ventilation rate, the more likely it is to lead to a decrease in acid production and sugar-acid conversion rate and an increase in miscellaneous acids.

[0148] Comparative Example 3 demonstrates that the ventilation rate needs to be controlled within the range of 90–120 LPM.

[0149] Comparative Example 4 (Comparative Example 4)

[0150] Based on Example 4, the pH and temperature control ranges in step 3 were further reduced or increased, and the results are shown in Table 8 below:

[0151] Table 8. Different temperatures and pH values

[0152] 1 28 7.1 8.9 38.70 0.4 2 34 7.1 5.1 22.17 0.5 3 31 6.9 8.5 36.96 0.5 4 31 7.4 9.1 39.57 0.4

[0153] Based on the above four sets of data, fermentation temperature and pH both affect fermentation acid production and sugar-acid ratio, but have no effect on miscellaneous acids;

[0154] Based on data from sets 1 and 2, when the fermentation temperature is lower or higher than the range of Example 4, the acid production and sugar-acid conversion rate begin to decrease, especially when the fermentation temperature is increased to 34°C, the acid production decreases significantly.

[0155] In the experimental data of groups 3 and 4, the acid production of the producing bacteria was adversely affected at pH 6.9 and 7.4, but the production of other acids did not increase as a result.

[0156] In summary, the fermentation temperature and pH need to be controlled at 29–33℃ and 7.0–7.3, respectively.

[0157] Comparative Example 5 (Comparative Example 5)

[0158] Based on Example 5, the residual sugar control range in step 3 was further reduced or increased, and the results are shown in Table 9 below:

[0159] Table 9. Different residual sugar concentrations

[0160] 1 0.2 8.2 35.65 0.4 2 0.4 9.5 41.30 0.4 3 1.6 9.3 40.43 0.8 4 2 8.1 35.22 0.9

[0161] Based on the above four sets of data, compared with Example 5, the acid production rate and sugar-acid conversion rate are both at a low level. When the residual sugar is below 0.5 wt%, the acid production and sugar-acid ratio decrease, but the miscellaneous acid does not increase. When the residual sugar is above 1.5 wt%, the acid production and sugar-acid ratio decrease, and the miscellaneous acid begins to increase slightly. It is speculated that the main reason is that there is more residual sugar, which leads to changes in the sugar metabolism process, and thus leads to a slight increase in miscellaneous acid.

[0162] In summary, residual sugar should be controlled within the range of 0.5–1.5 wt% for optimal results.

[0163] Comparative Example 6

[0164] Based on Example 1, the fermentation strain was changed to *Corynebacterium glutamicum* CICC No. 20160. The results showed that arginine yield decreased to 3.5 wt%, sugar-acid conversion rate decreased to 15.5%, and miscellaneous acid content increased to 1.50 wt%. Through comparative experiments, the following conclusions can be drawn:

[0165] The effect of strain selection on arginine fermentation: In this experiment, it was found that when Corynebacterium glutamicum CICCNo.20160 was used as the fermentation strain, the yield of arginine and the sugar-acid conversion rate were significantly lower than those when Corynebacterium glutamicum IBBZ-01 was used. This may be because there are metabolic differences between different strains, resulting in different efficiencies in arginine production.

[0166] Relationship between heteroacid production and bacterial strain: In this experiment, it was also observed that the heteroacid content was higher when using Corynebacterium glutamicum CICCNo.20160 than when using Corynebacterium glutamicum IBBZ-01. This may be because different strains produce different amounts of byproducts during metabolism, thus affecting the production of heteroacids.

[0167] In summary, based on the experimental data from Comparative Example 6, we can conclude that selecting a suitable bacterial strain is crucial for improving arginine fermentation efficiency. *Corynebacterium glutamicum* IBBZ-01 exhibits superior performance compared to *Corynebacterium glutamicum* CICC No. 20160 in terms of arginine yield, sugar-acid conversion rate, and reduction of miscellaneous acid formation. Therefore, in practical applications, *Corynebacterium glutamicum* IBBZ-01 should be the preferred choice for arginine fermentation production.

[0168] The above description, in conjunction with specific preferred embodiments of the present invention, further illustrates the invention and should not be construed as limiting the scope of the invention to these descriptions. Any modifications or alterations made by those skilled in the art without departing from the technical scope of the invention will be considered to be covered within the scope of the claims of the present invention.

Claims

1. A mutant strain of Corynebacterium glutamicum, characterized in that, The mutant strain was Corynebacterium glutamicum ( Corynebacterium glutamicum IBBZ-01, with accession number CCTCC NO:M 2022765.

2. A bacterial agent containing the mutant strain of claim 1, comprising: Corynebacterium glutamicum ( Corynebacterium glutamicum The bacterial flora of IBBZ-01, and / or its cultures, and / or its extracts.

3. The application of the mutant strain described in claim 1 in L-arginine fermentation.

4. The application according to claim 3, characterized in that: The L-arginine fermentation method includes adjusting the bacterial concentration (OD) based on the microbial concentration. 562 The steps for controlling the CO2 concentration in the fermentation tail gas in different stages.

5. The application according to claim 4, characterized in that: The method includes the following steps: (1) During fermentation, when the cell concentration reaches OD 562 When the concentration is 10-20, the CO2 concentration should be controlled between 0.5-1.5%. (2) OD concentration of the strain 562 When the concentration is 20-30, the CO2 concentration should be controlled between 2.0-3.5%. (3) During fermentation, if the residual sugar drops to 0.5-1.5%, feed sugar solution is added to maintain the residual sugar at 0.5-1.5%; (4) OD of bacterial cell concentration 562 When the concentration is 30-40, the CO2 concentration should be controlled between 4.0-5.0%. (5) OD of the fermentation broth to be tested 562 When the concentration is greater than 40, the CO2 concentration should be controlled between 5.5% and 6.5%.

6. The application according to claim 5, characterized in that: The supplemental sugar solution is a 50-80 wt% glucose solution.

7. The application according to claim 5, characterized in that: The CO2 concentration range in step (1) is 0.8~1.2%, the CO2 concentration range in step (2) is 2.5~3.0%, the CO2 concentration range in step (4) is 4.3~4.7%, and the CO2 concentration range in step (5) is 5.8~6.2%.

8. The application according to claim 5, characterized in that: In the initial stage of the fermentation process of step (1), the fermentation conditions are controlled as follows: aeration rate of 90~120 LPM, temperature of 29~33℃, and pH of 7.0~7.

3.

9. The application according to claim 5, characterized in that: The initial fermentation culture medium in step (1) is: 6-8 wt% glucose, 1.0-1.3 wt% corn steep liquor powder, 2.0-2.4 wt% molasses, 0.01-0.05 wt% yeast powder, 0.3-0.5 wt% ammonium sulfate, 0.1-0.13 wt% KH2PO4, and 0.05-0.07 wt% MgSO4•7H2O.

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