CN117903983A

CN117903983A - Corynebacterium glutamicum mutant strain, microbial inoculum thereof and application of corynebacterium glutamicum mutant strain in L-arginine fermentation

Info

Publication number: CN117903983A
Application number: CN202410043166.5A
Authority: CN
Inventors: 刘军; 范超; 洪皓; 陈剑彬; 吴文忠
Original assignee: Heilongjiang Nuoshu Biotechnology Co ltd; Innobio Corp ltd
Current assignee: Heilongjiang Nuoshu Biotechnology Co ltd; Innobio Corp ltd
Priority date: 2024-01-11
Filing date: 2024-01-11
Publication date: 2024-04-19

Abstract

The invention discloses a corynebacterium glutamicum mutant strain, a microbial inoculum thereof and application thereof in L-arginine fermentation; the mutant strain has the characteristics of high L-arginine yield and low mixed acid, and the invention also comprises a bacterial agent containing the mutant strain and application thereof in L-arginine fermentation. In the fermentation process, according to the thallus concentration OD ₅₆₂, a method for controlling the concentration of CO ₂ in fermentation tail gas in stages is adopted, so that the arginine yield and the sugar acid conversion rate are obviously improved. The fermentation method has high acid yield, less mixed acid, 12.5% of arginine yield, 52.3% of sugar acid conversion rate and 0.4% of mixed acid total amount; compared with the method for calculating OUR, CER and RQ values to perform fermentation control, the method for controlling the concentration of CO ₂ is quicker and more practical, can more accurately judge the strain state, is an efficient and practical L-arginine fermentation process, and has remarkable industrial application value.

Description

Corynebacterium glutamicum mutant strain, microbial inoculum thereof and application of corynebacterium glutamicum mutant strain in L-arginine fermentation

Technical Field

The invention relates to the technical field of fermentation engineering, in particular to a corynebacterium glutamicum mutant strain, a microbial inoculum thereof and application of the corynebacterium glutamicum mutant strain in L-arginine fermentation.

Background

The L-arginine system is named as 2-amino-5-guanidyl valeric acid, is a semi-essential amino acid in human body, can improve nitrogen metabolism balance in human body, and can be used as an antidote for treating ammonia poisoning hepatic coma, so that arginine is very important for maintaining liver health, and medical research proves that arginine has obvious effects in enhancing human body immunity and delaying aging.

The production methods of L-arginine mainly include chemical catalysis, proteolysis and microbial fermentation, wherein microbial fermentation is the main method for producing L-arginine. Corynebacterium glutamicum is a gram-positive bacterium as a main fungus for producing L-arginine, accords with food production safety, and can be used for producing glutamic acid by industrial fermentation.

The following are some patents listing the related art of arginine fermentation and tail gas analysis, and it can be seen that the main hot spots and processes in the amino acid fermentation field in the prior art are as follows:

A process for producing arginine by fermenting fed-batch culture solution comprises CN 112430633A: the patent discloses a process for fermenting arginine by using a fed-batch culture solution, wherein arginine is produced by feeding an ammonium sulfate aqueous solution and a glucose solution in the fermentation process;

Escherichia coli and application thereof in fermentation production of L-arginine, CN 116355814A: the patent discloses an escherichia coli which is cultured in the mode, a 30L fermentation tank is adopted for culturing, fermentation culture is carried out for 48 hours, the yield of L-arginine is 136.65g/L, and the conversion rate is 48.58%. Culture medium: comprises 1g/L glucose, 1g/L yeast powder, 1g/L ammonium sulfate, 1g/L monopotassium phosphate, 0.5g/L magnesium sulfate, 0.01g/L manganese sulfate and 0.01g/L zinc sulfate;

The technology for improving the fermentation and acid production of L-arginine is disclosed as CN 10540626A: the patent discloses that the Brevibacterium flavum obtains high acid production capacity through mutagenesis, trend control is adopted in the fermentation process, and the final acid production capacity of strict feed supplement reaches 3-4%;

A biological method for producing adenosylmethionine based on an exhaust gas analysis system comprises the steps of CN 115927516A: the patent discloses a method for producing adenosylmethionine based on a tail gas analysis system biological method. The production method is a method for promoting the yield of S-adenosylmethionine by regulating and controlling cell metabolism at the cellular level through a physiological metabolism parameter RQ based on a fermentation tail gas analysis system.

At present, the domestic fermentation method of amino acids such as arginine generally adopts a mode of controlling dissolved oxygen and the like, although the method can achieve the process aim to a certain extent, as the dissolved oxygen electrode is influenced by various conditions in fermentation liquor, the dissolved oxygen electrode is interfered in detection accuracy, and when parameters such as OUR, CER, RQ values and the like are adopted for fermentation control, the error becomes larger due to excessive calculation formulas, and the method is not suitable for common fermentation workshop operation, so the patent develops a control method which can replace the dissolved oxygen control process, utilizes the correlation of bacterial liquid concentration and fermentation tail gas CO ₂ concentration, solves the control difficulty, increases the arginine yield and the sugar acid conversion rate, and reduces the impurity acid yield.

Disclosure of Invention

Aiming at the defects in the prior art, the invention discloses a corynebacterium glutamicum mutant strain, a microbial inoculum thereof and application thereof in L-arginine fermentation. The invention obtains a high-yield strain through mutagenesis, and in the process of technological development, the concentration of CO ₂ in the fermentation tail gas is controlled in a gradient manner, so that a process with high acid yield, high sugar acid conversion rate and low mixed acid is obtained, and the problem of low acid production efficiency in the prior art is solved.

Firstly, the mutant strain of the corynebacterium glutamicum is corynebacterium glutamicum IBBZ-01

(Corynebacterium glutamicum) the preservation number is CCTCC NO: M2022765; the preservation unit is China center for type culture Collection, and the address is Wu Changou eight paths of Wuhan City in Hubei province, and Wuhan university; the zip code 430072; the preservation date is 2022, 05 and 30.

Furthermore, the corynebacterium glutamicum IBBZ-01 (Corynebacterium glutamicum) is a production strain with high yield of L-arginine and less side acid, which is screened out by taking corynebacterium glutamicum (CICC No. 20160) as an initial strain and performing induced mutation and directional culture.

The invention provides a microbial inoculum containing the corynebacterium glutamicum IBBZ-01 (Corynebacterium glutamicum), which comprises the following components: the flora of Corynebacterium glutamicum IBBZ-01 (Corynebacterium glutamicum), and/or cultures thereof, and/or extracts thereof.

In another aspect, the application also discloses an application of the corynebacterium glutamicum IBBZ-01 in L-arginine fermentation;

With respect to the technical scheme, it is further preferred that in the application, the method for fermenting L-arginine comprises the step of controlling the concentration of CO ₂ in the fermentation tail gas in stages according to the condition of the strain concentration OD ₅₆₂. Thereby greatly improving the yield of arginine;

With respect to the technical solution described above, it is further preferred that the fermentation method comprises the steps of:

(1) In the fermentation process, when the concentration OD ₅₆₂ of the thallus is 10-20, the concentration of CO ₂ is controlled to be 0.5-1.5%;

(2) When the strain concentration OD ₅₆₂ is 20-30, controlling the concentration of CO ₂ to be 2.0-3.5%;

(3) In the fermentation process, if the residual sugar is reduced to 0.5-1.5%, feeding a feed supplement sugar solution, wherein the residual sugar is controlled to be 0.5-1.5%;

(4) When the concentration OD ₅₆₂ of the thalli is 30-40, controlling the concentration of CO ₂ to be 4.0-5.0%;

(5) When the concentration OD ₅₆₂ of the fermentation liquid to be detected is larger than 40, the concentration of CO ₂ is controlled to be 5.5-6.5%.

For the technical scheme, the feeding sugar solution is preferably 50-80 wt% of glucose solution.

For the above technical solution, it is further preferable that the fermentation conditions are controlled in the initial stage of the fermentation process in the step (1): ventilation is 90-120 LPM, temperature is 29-33 ℃, pH is 7-7.3. Still more preferably, the fermentation conditions are aeration rate of 100LPM, temperature of 31℃and pH 7.0;

For the above technical solution, it is further preferred that the concentration range of the CO ₂ in the step (1) is 0.8-1.2%, the concentration range of the CO ₂ in the step (2) is 2.5-3.0%, the concentration range of the CO ₂ in the step (4) is 4.3-4.7%, and the concentration range of the CO ₂ in the step (5) is 5.8-6.2%.

For the above technical solution, it is further preferable that in the step, the control change of the concentration of CO ₂ can be controlled according to the OD ₅₆₂ value of the concentration of the bacterial cells. In addition, although detecting the dissolved oxygen value is one possible control means, it is not necessary. In practice, we can achieve control of carbon dioxide concentration by adjusting the rotational speed. This control is entirely dependent on the concentration of CO ₂ in the fermentation tail gas. Typically, the rotational speed is set to 200 to 450rpm. In this way, the concentration of CO ₂ can be regulated by means of the concentration of the thallus, the fermentation process can be flexibly managed by the rotation speed regulation, and the monitoring of the dissolved oxygen value is used as an optional control means.

For the technical scheme, further, the culture medium in the process of expanding the inoculation volume step by step of the L-arginine producing strain IBBZ-01 in the step (1) comprises:

Primary seed medium: 3 to 5 weight percent of glucose, 1.0 to 1.2 weight percent of corn steep liquor dry powder, 1.0 to 1.5 weight percent of molasses, 0.3 to 0.5 weight percent of ammonium sulfate, 0.05 to 0.07 weight percent of KH ₂PO₄ 0.1～0.13wt％,MgSO₄·7H₂ O, 0.5 to 1.0 weight percent of calcium carbonate and 7.0 to 7.2 weight percent of pH 7;

Secondary seed medium: 3 to 5 weight percent of glucose, 1.0 to 1.2 weight percent of corn steep liquor dry powder, 1.0 to 1.5 weight percent of molasses, 0.3 to 0.5 weight percent of ammonium sulfate, 0.05 to 0.07 weight percent of KH ₂PO₄ 0.1～0.13wt％,MgSO₄·7H₂ O and pH of 6.8 to 7.2;

Fermentation initial medium: 6 to 8 weight percent of glucose, 1.0 to 1.3 weight percent of corn steep liquor dry powder, 2.0 to 2.4 weight percent of molasses, 0.01 to 0.05 weight percent of yeast powder, 0.3 to 0.5 weight percent of ammonium sulfate and 0.05 to 0.07 weight percent of KH ₂PO₄ 0.1～0.13wt％,MgSO₄·7H₂ O.

Compared with the prior art, the invention has the following beneficial effects:

1. According to the invention, a strain with high L-arginine yield is obtained through screening and optimization, the acid yield is high, the detection result in the example 1 with less mixed acid reaches 12.5% of arginine yield, the sugar-acid conversion rate is 52.3%, and the total amount of mixed acid such as citrulline, ornithine and the like is 0.4%; as is clear from comparative example 6, corynebacterium glutamicum IBBZ-01 of the present invention had a 3-fold increase in arginine production compared to its starting strain Corynebacterium glutamicum CICC No.20160 and a 3.37-fold increase in sugar acid conversion compared to its starting strain. The production of the hetero acid is obviously reduced, and the hetero acid amount of the corynebacterium glutamicum IBBZ-01 is reduced to 26% of that of the original strain, which shows that the fermentation performance is obviously improved;

2. In the process of synthesizing arginine by microorganisms, the concentration of CO ₂ in fermentation tail gas directly reflects the growth, propagation and metabolism states of the strain, especially reflects the strength of aerobic and anaerobic metabolic processes of the strain, can obviously reduce the difficulty of fermentation control by combining the control mode of fermentation tail gas and metabolic processes, and can ensure that the acid production of arginine reaches about 12% at most and the sugar acid conversion rate reaches more than 50%;

3. According to the method, the concentration OD ₅₆₂ of the thalli is closely related to the concentration of CO ₂ in the fermentation tail gas, and then the concentration of CO ₂ is controlled in stages, so that the method can better regulate the metabolic activity of the thalli according to different characteristics of each stage of the production bacteria, obviously improve the acid production and sugar acid conversion rate of arginine and reduce the mixed acid;

4. the invention replaces the conventional fermentation mode of dissolved oxygen control by controlling the concentration of the fermentation tail gas CO ₂, and has the characteristics of higher accuracy, higher practicability and the like by controlling the concentration of CO ₂ compared with the dissolved oxygen control;

5. At present, the detection of fermentation tail gas is gradually started to be applied to fermentation process development, the OUR, CER and RQ values are generally calculated to perform fermentation control, and the main direction of fermentation metabolic flow is judged, and in the method, only the way of controlling the concentration of CO ₂ is adopted.

Detailed Description

The present invention will be further described with reference to examples, but it should be understood that the scope of the present invention is not limited by the examples.

In the present invention, percentages and percentages are by mass unless explicitly stated otherwise. Unless otherwise specified, all experimental procedures used are conventional and all materials, reagents, etc. used are commercially available.

L-arginine and less by-product strain IBBZ-01 deposited in China center for type culture Collection, at a site of 2022, month 05, 30: china, university of Wuhan, and preservation number is CCTCC NO: M2022765.

Characteristics of Corynebacterium glutamicum:

The corynebacterium glutamicum (Corynebacterium glutamicum) IBBZ-01 (CCTCC NO: M2022765) which is a high-yield L-arginine production strain is obtained by taking the corynebacterium glutamicum (CICC No. 20160) of L-arginine as an initial strain and screening the initial strain through mutagenesis; genetic markers: sulfaguanidine (SG) resistance, arginine hydroxamic acid resistance, D-Arg resistance, L-homoarginine (L-HA) resistance;

the composition of the culture medium is as follows:

Complete Medium (CM): 1.0wt% of glucose, 1wt% of beef extract, 0.5wt% of peptone, 0.5wt% of yeast extract, 0.5wt% of sodium chloride, 0.5wt% of urea and pH 7.0-7.2;

minimal Medium (MM): glucose 3wt%, ammonium sulfate 0.5wt%, KH ₂PO₄ 0.1wt％,MgSO₄·7H₂ O0.05 wt%, pH 7.0-7.2;

Supplemented Medium (SM): adding a structural analogue of an amino acid to a Minimal Medium (MM), pH 7.0-7.2; structural analogues of amino acids include sulfaguanidine, homoarginine, D-arginine;

Seed culture medium: 3wt% of glucose, 1.0wt% of corn steep liquor dry powder, 1.0wt% of molasses, 0.3wt% of ammonium sulfate, 0.05wt% of KH ₂PO₄ 0.1wt％,MgSO₄·7H₂ O, 0.5wt% of calcium carbonate and pH 7.0-7.2;

Shake flask fermentation medium: 10wt% of glucose, 1.1wt% of corn steep liquor dry powder, 1wt% of ammonium sulfate, 0.05wt% of KH ₂PO₄0.1wt％,MgSO₄·7H₂ O and 1wt% of calcium carbonate;

the breeding process is as follows:

(1) Pretreatment of mutagenesis:

a ring of activated strain is inoculated into a conical flask filled with 25mL of liquid complete medium, and placed in a reciprocating shaker at 30 ℃ and 85rpm for shaking culture for 12h. Then 1mL of culture solution is taken and is connected into another conical flask containing 25mL of liquid complete culture medium, and the conical flask is placed in a reciprocating shaking table at 30 ℃ and 90rpm to oscillate for 4 hours, so that cells are in logarithmic growth phase.

(2) Preparation of bacterial suspension:

10mL of culture solution is taken in a sterile centrifuge tube, centrifuged at 5000rpm for 10min, the supernatant is discarded, cells are resuspended by adopting physiological saline and then poured into a conical flask, the shaking is carried out for 10min, and the cell concentration is adjusted to 5X 10 ⁸ cells/mL.

(3) Ultraviolet mutagenesis:

And (3) taking the prepared bacterial suspension, carrying out mutagenesis on the bacterial suspension for a certain time under ultraviolet light, sampling, diluting, coating and culturing the bacterial suspension on a flat plate, and calculating the mortality rate. And (3) eliminating the wild type and concentrated defect by adopting starvation culture and penicillin method, detecting the defect by using an audio-visual culture method, and verifying the acid production capacity of fermentation.

(4) Diethyl sulfate mutagenesis:

Adding 0.5wt% diethyl sulfate (DES) into the prepared bacterial suspension, oscillating at 30 ℃ for 20min, stopping treatment, coating on a screening culture medium SM flat plate, and standing at 31 ℃ for 4-6 days. The single colony of the mutant strain growing on SM is selected and transferred to MM plates added with sulfaguanidine, homoarginine and D-arginine respectively, and the mutant strain is subjected to static culture at 31 ℃ for 24 hours and then enters a shaking bottle for rescreening.

(5) Art mutagenesis:

A proper amount of bacterial suspension is absorbed and uniformly coated on a sterile metal slide, and the sterile metal slide is placed in a mutagenesis room of an ARTP biological mutagenesis breeding machine with helium as working gas, power of a power supply of 100W, working gas flow of 10L/min and treatment distance of 2mm, and mutagenesis treatment is carried out for 0, 20, 30, 60 and 120 seconds respectively; after stopping the treatment, the cells are spread on a screening medium SM plate and are subjected to static culture at 31 ℃ for 4 to 6 days. Selecting single colony of mutant strain growing on SM, transferring to MM plate added with sulfaguanidine, homoarginine and D-arginine respectively, standing at 31deg.C for 24 hr, and shaking for re-screening;

(6) Shaking and re-screening:

Inoculating the mutant strain to shake flask fermentation medium, shake culturing at 31deg.C and 110rpm for 72 hr, and detecting accumulated L-arginine in the mutant strain fermentation broth by high performance liquid phase method, wherein the strain producing highest L-leucine in all mutants is IBBZ-01.

(7) The shake flask culture process comprises the following steps:

Preparing shake flask seed liquid: inoculating a corynebacterium polyglutamic acid mutant strain IBBZ-01 into a seed culture medium, and culturing for 16 hours at 30 ℃ and 90rpm, wherein the bacterial concentration OD ₅₆₂ is about 0.5-0.6 x 20 for later use;

shake flask seeds were inoculated into a shake flask fermentation medium at 8% (V/V), cultured at 30℃for 72 hours at 120rpm, and the L-arginine production was examined by high performance liquid chromatography, with the yield of original strain CICC No.20160 being 4.5g/L, and the yield of mutant strain IBBZ-01 being 34.2g/L, and the mutant strain was found to have a stronger acid-producing ability and more excellent L-arginine-producing ability than the original strain.

Example 1

1. The seed medium consisted of:

primary seed medium:

3wt% of glucose, 1.0wt% of corn steep liquor dry powder, 1.0wt% of molasses, 0.3wt% of ammonium sulfate, 0.05wt% of KH ₂PO₄0.1wt％,MgSO₄·7H₂ O and 1.0wt% of calcium carbonate; split charging 500 ml/3000 ml; sterilizing at 121 ℃/20min;

secondary seed pot medium:

3wt% of glucose, 1.0wt% of corn steep liquor dry powder, 1.0wt% of molasses, 0.3wt% of ammonium sulfate, 0.05wt% of KH ₂PO₄0.1wt％,MgSO₄·7H₂ O, pH 7.0-7.2 and sterilization conditions of 121 ℃/20min;

2. The fermentation medium was as follows:

Standard fermenter fermentation initial medium: 7wt% of glucose, 1.0wt% of corn steep liquor dry powder, 2.0wt% of molasses, 0.01 wt% of yeast powder, 0.3wt% of ammonium sulfate and 0.05wt% of KH ₂PO₄ 0.1wt％,MgSO₄·7H₂ O

Feed supplement sugar solution: 60wt% glucose;

3. Fermentation tank process

Step 1:

Inoculating corynebacterium glutamicum IBBZ-01 into a primary seed culture medium of a shake flask, and performing shake culture at 30 ℃ and 90rpm for 16 hours to obtain 500mL of shake flask seed liquid, wherein the primary seed liquid is a maturation mark: OD ₅₆₂ is 0.5-0.6 x 20.

Step 2:

Inoculating the shake flask seed liquid into a 50L secondary seed tank culture medium at a ratio of 0.3% (V/V), wherein the pH is 6.8 at 30 ℃, the dissolved oxygen is not less than 20%, and culturing for 16h, wherein the secondary seed liquid is mature as a sign: OD ₅₆₂ is 0.5-0.6x20; obtaining a secondary seed liquid;

step 3:

1) 10% of secondary seed liquid is inoculated into a 500L fermentation tank, the initial fermentation liquid loading amount is 60%, the fermentation temperature is 31 ℃, the pH value is 7.0, the rotating speed is 200RPM, the ventilation amount is 100LPM, the tank pressure is 0.05Mpa, the concentration of fermentation tail gas CO ₂ is used as a main control standard in the process, dissolved oxygen is not monitored and controlled any more, and the OD ₅₆₂ does not need to be adjusted in the process of 1-10 after inoculation;

2) When the concentration OD ₅₆₂ of the thalli grows to between 10 and 20, the concentration of CO ₂ is controlled to be 0.9 percent by adjusting the rotating speed;

3) When the strain concentration OD ₅₆₂ is 20-30, controlling the concentration of CO ₂ to 2.8% through the rotating speed;

4) When the residual sugar is reduced to 0.8wt%, feeding of the feed supplement sugar solution is started, and the residual sugar is controlled to be about 0.8 wt%.

5) When the strain concentration OD ₅₆₂ is 30-40, the concentration of CO ₂ is controlled to be 4.5% by adjusting the rotating speed;

6) After the concentration OD ₅₆₂ of the detected thalli is more than 40, controlling the concentration of CO ₂ to be 6.0% by adjusting the rotating speed;

7) The sugar liquor is fed completely and the residual sugar is exhausted, and the tank is put down, the arginine yield is 12.5wt%, the sugar acid conversion rate is 52.3%, and the total amount of the mixed acid such as citrulline, ornithine and the like is 0.4wt%.

As shown in example 1, the core of the process is to perform arginine fermentation in a mode of controlling the concentration of CO ₂ in fermentation tail gas according to the growth trend of the production microbial inoculum, and the mode is abandoned according to the original dissolved oxygen control mode; according to the control mode, the acid production and conversion rate of arginine are obviously improved, and the mixed acid is obviously reduced;

During the fermentation process, the acid production refers to the actual concentration of arginine in the fermentation broth. The sugar acid conversion was calculated as follows:

Sugar acid conversion = (actual arginine concentration x actual fermentation volume)/(actual glucose consumption total x 100%)

Meanwhile, the hetero acid refers to the sum of the concentration of citrulline and ornithine in the fermentation broth. These two parameters are used together to evaluate the efficiency and product composition of the fermentation process.

Example 2 (different CO ₂ control concentration ranges)

Based on example 1: in the step 3, the concentration of CO ₂ in the fermentation tail gas is respectively controlled in different ranges; the method and results are shown in Table 1:

TABLE 1 gradient control ranges for different concentrations of CO ₂

As shown in the table results, the key steps are the most in gradient control of CO ₂ concentration according to the cell concentration OD ₅₆₂: OD ₅₆₂ is 10-20, and the concentration of CO ₂ is controlled to be 0.5-1.5%; OD ₅₆₂ is in the stage of 20-30, and the concentration of CO ₂ needs to be controlled at 2.0-3.5%; OD ₅₆₂ is controlled at 30-40 stage, CO ₂ concentration is controlled to be 4.0-5.0%, OD ₅₆₂ is controlled to be more than 40, CO ₂ concentration is controlled to be 5.5-6.5%, and too low or too high CO ₂ concentration can lead to reduced acid production and increased hetero acid.

Acid production: it can be seen that the control of the CO ₂ concentration stage is close to example 1, the higher the acid production, the lower the acid production, the larger the deviation.

Hybrid acid: also closely related to the CO ₂ concentration stage control, the more deviate from example 1, the higher its hetero-acid;

As shown in the data of the group 1-3, the data of the group are subjected to an optimization experiment that the concentration of CO ₂ is controlled to be 0.5-1.5% when the concentration of the bacterial cells OD ₅₆₂ -20, and the result shows that the acid production and the sugar acid ratio of the CO ₂ are in a trend of increasing firstly and then decreasing as the concentration of the CO ₂ is increased, the mixed acid is decreased firstly and then increased, and the concentration of the CO ₂ is higher as the concentration of the CO ₂ is close to 0.9% in the embodiment 1, so that the result is better.

As shown in the data of the group 4-6, the optimized experiment that the concentration of CO ₂ is controlled to be 2.0-3.5% when the concentration of the bacteria OD ₅₆₂ -30 is carried out in the data of the group, and similarly, as the concentration of CO ₂ is increased, the acid production and the sugar acid ratio of the bacteria are in a trend of increasing firstly and then decreasing, the mixed acid is decreased firstly and then increased, and the concentration of CO ₂ is higher as the concentration is closer to 2.8% in the embodiment 1, the acid production and the sugar acid conversion rate is higher, and the mixed acid is lower.

As shown in the data of the group 7-9, the optimized experiment that the concentration of CO ₂ is controlled to be 4.0-5.0% when the concentration of the bacteria OD ₅₆₂ -40 is carried out in the data of the group, as the concentration of CO ₂ is increased, the acid production ratio and the sugar acid ratio of the bacteria are increased firstly and then reduced, the mixed acid is reduced firstly and then increased, and the closer the concentration of CO ₂ is to 4.5% in the embodiment 1, the acid production and the sugar acid conversion rate are higher, and the mixed acid is lower.

As shown in the data of the group 10-12, the optimized experiment that the concentration of CO ₂ is controlled to be 5.5-6.5% when the concentration of the bacteria is over OD ₅₆₂ and the concentration of the acid and the sugar acid are increased and then reduced along with the increase of the concentration of CO ₂, the mixed acid is reduced and then increased, and the concentration of CO ₂ is closer to 6.0% in the embodiment 1, the acid and the sugar acid are converted more and the mixed acid is reduced.

Conclusion: experiments prove that the concentration of CO ₂ is controlled by gradient, so that the acid production and the sugar acid conversion rate can be improved and promoted, and the synthesis of the mixed acid can be inhibited.

Example 3 (different ventilation)

Based on example 1, the ventilation in step 3 was controlled to be performed under different conditions, and the results are shown in table 2:

TABLE 2 different ventilation

Sequence number	Ventilation LPM	Acid production wt%	Sugar acid ratio%	Hetero acid wt%
					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

As shown in data 1-5, ventilation optimization experiments were performed, and the results showed that: in the range of 90-120 LPM, the acid production and sugar acid conversion rate show a trend of increasing and then decreasing with increasing ventilation, wherein 95LPM has the best effect, and the mixed acid shows a trend of decreasing and then increasing, and the mixed acid is the lowest at 105 LPM.

The amount of ventilation determines how much oxygen it enters the fermenter, thereby affecting the oxygen transfer coefficient, resulting in changes in bacterial strain respiratory metabolism, and affecting the ability of the rotational speed to control the concentration of fermentation tail gas CO ₂.

On the premise that the rate of CO ₂ generated by thalli in the acid production period, namely CER, is stable, increasing ventilation volume can generate the effect of diluting the concentration of CO ₂ in fermentation tail gas, so that the gradient control range of CO ₂ is narrowed, and the gradient control of CO ₂ in each stage cannot be distinguished, thereby causing experimental failure; decreasing ventilation can lead to a sudden increase in the concentration of CO ₂ during the early stages of fermentation, or too sensitive when the CO ₂ concentration is controlled at a slightly elevated speed, resulting in a steep increase in the concentration of CO ₂, which can adversely affect process stability.

In summary, in controlling the concentration of CO ₂, the range of ventilation needs to be strictly controlled, so that the concentration of CO ₂ in the whole process is in a stable and controllable state.

Example 4 (different fermentation temperatures and pH)

Based on example 1: the fermentation temperature and pH in step 3 were controlled to different conditions, and the results are shown in Table 3 below:

TABLE 3 different temperatures and pH

Acid yield: the 4 groups of acid production are respectively about 11%, and compared with the example 1, the acid production is reduced to some extent, and the temperature and the pH are proved to have a certain influence on the acid production.

Sugar acid conversion: has an effect and slightly drops.

Hybrid acid: is not affected.

Combining the above data analysis, it can be concluded that: the change of temperature and pH has a certain influence on the acid production and the conversion rate of the sugar acid, but has a small influence on the mixed acid;

example 5 (control of different residual sugar concentrations)

Based on example 1: the fermentation residual sugar concentration in the step 3 is controlled under different conditions, and the results are shown in the following table 4:

TABLE 4 different residual sugar concentrations

Sequence number	Residual sugar wt%	Acid production wt%	Sugar acid ratio%	Hetero acid wt%
					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

Acid production: reducing or increasing the residual sugar control concentration had some effect on acid production, and from the 3-group results, the residual sugar deviation resulted in some fluctuation in acid production compared to the 0.8% concentration control in example 1.

Sugar acid conversion: consistent with the acid production trend, the sugar acid conversion rate is reduced to different degrees.

Hybrid acid: is not affected.

Combining the above data analysis, it can be concluded that: the control range of residual sugar ranging from 0.5 to 1.5wt% has a certain effect on both acid production and sugar acid conversion, but less on the hetero acid.

Comparative example 1 (comparative example 1)

Based on example 1, the control method of step 3 was adopted to perform fermentation by using a dissolved oxygen control method, and the dissolved oxygen was controlled to be 5-25%, and the results are shown in the following table 5:

TABLE 5 control with different dissolved oxygen

Sequence number	Dissolved oxygen%	Acid production wt%	Sugar acid ratio%	Hetero acid wt%
					1	5	5.6	24.35	3.5
2	15	8.5	36.96	1.9
					3	25	7.2	31.30	2.5

As shown in the current 3 groups of results, the maximum acid production rate of a 15% dissolved oxygen control mode is 8.5wt%, the other two conditions are 5.6wt% and 7.2wt%, the sugar-acid conversion rate is lower, the maximum sugar-acid conversion rate is about 37%, the maximum acid production rate cannot reach the level of the example 1, and the mixed acid is far higher than 0.4wt%.

Combining the above data analysis, it can be concluded that: the fermentation by the dissolved oxygen control method cannot reach the fermentation result of the gradient control of the concentration of the fermentation tail gas CO ₂ in the embodiment 1, and the adoption of the gradient control of the concentration of the CO ₂ for arginine fermentation has the advantages of obviously improving the acid production and the sugar acid conversion rate and reducing the mixed acid.

Comparative example 2 (comparative example 2)

Based on example 2, the control range of the fermentation tail gas CO ₂ in step 3 was modified, and the results are shown in Table 6:

TABLE 6 different CO ₂ control ranges

As seen from the current 8 groups of results, the concentration of CO ₂ in the gradient control fermentation tail gas needs to be strictly regulated according to the concentration OD ₅₆₂, and too high or too low concentration of CO ₂ in each stage can reduce the ratio of acid production to sugar acid, and even increase the content of mixed acid.

1. The 2 groups of experimental data show that the acid production and sugar acid conversion rate can be reduced, but the increase of the mixed acid is not obvious if the concentration of CO ₂ is controlled to be outside 0.5-1.5% in the range of the cell concentration OD ₅₆₂ -20.

The 3-8 sets of experimental data indicate that the concentration OD ₅₆₂ at each stage needs to be controlled within the range expressed in example 2, beyond which it results in reduced acid production and sugar acid conversion, and in increased hetero acids.

Comparative example 2 thus demonstrates that fermentation tail gas CO ₂ concentration gradient control needs to be performed as in example 2.

Comparative example 3 (comparative example 3)

Based on example 3, the ventilation range in step 3 was further reduced or increased, resulting in the following table 7:

TABLE 7 different ventilation

Sequence number	Ventilation LPM	Acid production wt%	Sugar acid ratio%	Hetero acid wt%
					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

According to the current 4 groups of experimental results, with the increase or decrease of ventilation, the method has obvious influence on acid production and sugar acid conversion rate and mixed acid;

from the point of view of acid production and sugar acid conversion, outside the range specified in example 3, lower aeration levels will result in lower acid production and sugar acid conversion, increased hetero acids, and similarly higher aeration levels will also result in lower acid production and sugar acid conversion, increased hetero acids;

comparative example 3 demonstrates that ventilation is required to be controlled in the range of 90 to 120 LPM.

Comparative example 4 (comparative example 4)

Based on example 4, the pH and temperature control ranges in step 3 were further reduced or increased, resulting in the following table 8:

TABLE 8 different temperatures and pH

Sequence number	Temperature (DEG C)	pH	Acid production wt%	Sugar acid ratio%	Hetero acid wt%
						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

From the data of the 4 groups, the fermentation temperature and the pH can influence the fermentation acid production and the sugar acid ratio, but the mixed acid has no influence;

1. As seen from the data set 2, the fermentation temperature was lower or higher than the range of example 4, and the acid and sugar acid conversion began to decrease, especially as the fermentation temperature increased to 34 ℃ and the acid production was greatly decreased;

3. the acid production of the producing bacteria is adversely affected at pH6.9 and 7.4, but the acid is not increased as a result of the 4 sets of experimental data;

in summary, the fermentation temperature and pH are controlled to be 29-33 ℃ and 7.0-7.3 respectively.

Comparative example 5 (comparative example 5)

Based on example 5, the residual sugar control range in step 3 was further reduced or increased, resulting in the following table 9:

TABLE 9 different residual sugar concentrations

Sequence number	Residual sugar%	Acid production wt%	Sugar acid ratio%	Hetero acid wt%
					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

From the above 4 sets of data, both acid production and sugar acid conversion were at lower levels than in example 5, with lower acid and sugar acid ratios but no increase in the hetero acids when the residual sugar was below 0.5 wt%; when the residual sugar is higher than 1.5wt%, the acid production and sugar acid ratio is reduced, and the mixed acid starts to rise slightly, presumably mainly because of more residual sugar, the sugar metabolic process is changed, and the mixed acid is increased slightly;

in summary, the residual sugar is required to be controlled within the range of 0.5 to 1.5wt%, which is a suitable condition.

Comparative example 6

Based on example 1, the fermentation strain was replaced with Corynebacterium glutamicum CICC No.20160. The results showed that arginine production was reduced to 3.5wt%, sugar acid conversion was reduced to 15.5 wt% and the hetero acid content was increased to 1.50wt%. By comparison experiments, the following conclusions can be drawn:

Influence of strain selection on arginine fermentation process: in this experiment, it was found that when Corynebacterium glutamicum CICCNo.20160 was used as the fermentation strain, the arginine yield and the sugar acid conversion rate were significantly lower than those of Corynebacterium glutamicum IBBZ-01, probably because of the metabolic differences between the different strains, resulting in different efficiencies in arginine production.

Relationship between heteroacid formation and strain: in this experiment, it was also observed that when Corynebacterium glutamicum CICCNo.20160 was used, the content of hetero acids was higher than when Corynebacterium glutamicum IBBZ-01 was used, probably because the amount of by-products produced during metabolism was different from strain to strain, thus affecting the formation of hetero acids.

From the above, by the experimental data of comparative example 6, it can be concluded that: the selection of a proper strain is of great significance for improving the arginine fermentation efficiency. Corynebacterium glutamicum IBBZ-01 shows better performance in terms of arginine yield, sugar acid conversion and reduction of the formation of hetero acids than Corynebacterium glutamicum CICC No. 20160. Therefore, in practical operation, corynebacterium glutamicum IBBZ-01 should be preferred for the fermentative production of arginine.

The foregoing is a further description of the invention in connection with specific preferred embodiments thereof, and is not intended to limit the practice of the invention to such description. It is intended that all such variations and modifications as would be included within the scope of the invention are within the scope of the following claims.

Claims

1. A corynebacterium glutamicum mutant is characterized in that the mutant is corynebacterium glutamicum IBBZ-01 (Corynebacterium glutamicum) and has a preservation number of CCTCC NO: M2022765.

2. A microbial agent comprising the mutant strain of claim 1, comprising: corynebacterium glutamicum IBBZ-01

The flora of (Corynebacterium glutamicum), and/or a culture thereof, and/or an extract thereof.

3. Use of the mutant strain of claim 1 in fermentation of L-arginine.

4. A use according to claim 3, characterized in that: comprising fermentation of L-arginine using Corynebacterium glutamicum IBBZ-01 (Corynebacterium glutamicum).

5. The use according to claim 4, characterized in that: the L-arginine fermentation method comprises the step of controlling the concentration of CO ₂ in fermentation tail gas in stages according to the condition of the strain concentration OD ₅₆₂.

6. The use according to claim 5, characterized in that: the method comprises the following steps:

7. The use according to claim 6, characterized in that: the feed supplement sugar solution is glucose solution with the weight percent of 50-80.

8. The use according to claim 6, characterized in that: the concentration range of CO ₂ in the step (1) is 0.8-1.2%, the concentration range of CO ₂ in the step (2) is 2.5-3.0%, the concentration range of CO ₂ in the step (4) is 4.3-4.7%, and the concentration range of CO ₂ in the step (5) is 5.8-6.2%.

9. The use according to claim 6, characterized in that: the fermentation condition is controlled at the initial stage of the fermentation process in the step (1): ventilation is 90-120 LPM, temperature is 29-33 ℃, pH is 7.0-7.3.

10. The use according to claim 6, characterized in that: the fermentation initial medium in the step (1) is as follows: 6 to 8 weight percent of glucose, 1.0 to 1.3 weight percent of corn steep liquor dry powder, 2.0 to 2.4 weight percent of molasses, 0.01 to 0.05 weight percent of yeast powder, 0.3 to 0.5 weight percent of ammonium sulfate and 0.05 to 0.07 weight percent of KH ₂PO₄0.1～0.13wt％,MgSO₄·7H₂ O.