Ultra-high molecular weight gamma-polyglutamic acid synthesizing strain and application thereof

Abstract

The application belongs to the field of microorganisms, and particularly relates to a strain of ultrahigh molecular weight gamma-polyglutamic acid synthesis strain and application thereof. The specific technical scheme is as follows: a strain of Bacillus subtilis FB-3 is preserved in the China General Microbiological Culture Collection Center on April 29, 2022, and the preservation number is CGMCC NO. 24826. The Bacillus subtilis is inoculated into a gamma-PGA fermentation medium, and is subjected to early fermentation under the conditions of a glucose concentration of 5-20 g / L and a ventilation amount of 0.2-3 vvm for 6-18 hours. After the early fermentation is completed, the oxygen supply level in the fermentation system is adjusted in real time, and qO2 is maintained constant at 15-40 mmol / (g.h). The application provides a new strain of Bacillus subtilis, which can produce gamma-PGA with a molecular weight of up to 25000 KDa, far exceeding the existing level of the same kind of microorganism. The method provided by the application is simple to operate, can be automatically monitored and calculated, has low requirements on operators, is suitable for large-scale fermentation production of ultrahigh molecular weight gamma-PGA, and has industrial popularization and application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microorganisms, and particularly relates to a strain of super-high molecular weight gamma-polyglutamic acid synthesis and application thereof. BACKGROUND

[0002] Gamma-polyglutamic acid (Poly-γ-glutamic acid, γ-PGA) is a linear polymer composed of glutamic acid monomers through γ-amide bonds. The molecular weight of γ-PGA is distributed in 10-10000 KDa, and γ-PGA with different molecular weights has different application fields. The super-high molecular weight (>10000 KDa) γ-PGA is mainly used for the synthesis of new functional resins and gels, and has important application value in the cosmetics and pharmaceutical industries. The molecular weight of γ-PGA obtained by microbial fermentation mainly depends on the genetic characteristics of the production strain. The molecular weight of γ-PGA synthesized by most strains is about 1000 KDa, and no microorganism capable of producing γ-PGA with a molecular weight of 20000 KDa or more has been found.

[0003] When preparing γ-PGA by microbial fermentation, the yield of γ-PGA is also limited in addition to the limitation of molecular weight. Since γ-PGA is a macromolecular polymer, the viscosity of the fermentation broth will increase sharply with the accumulation of γ-PGA, which will cause the diameter of the gas bubbles to increase, the gas-liquid exchange area to decrease, and the bubble residence time to be shortened, resulting in dissolved oxygen limitation. At the same time, the higher the molecular weight of γ-PGA, the greater the viscosity of the fermentation broth, and the more serious the dissolved oxygen limitation compared with ordinary γ-PGA fermentation, which results in very limited production efficiency of super-high molecular weight γ-PGA.

[0004] In summary, if a new strain capable of producing super-high molecular weight γ-PGA can be provided, and the problem of limited yield can be solved, it will have excellent industrial application prospects. SUMMARY

[0005] The purpose of the present application is to provide a strain of super-high molecular weight gamma-polyglutamic acid synthesis and application thereof.

[0006] To achieve the above-mentioned purpose of the application, the technical solution adopted by the present application is as follows: a strain of Bacillus subtilis FB-3, which was preserved in the China General Microbiological Culture Collection Center on April 29, 2022, and the preservation number is CGMCC NO. 24826.

[0007] Preferably, the 16S rDNA sequence thereof is shown in SEQ ID NO. 1.

[0008] Correspondingly, the application of the Bacillus subtilis in the preparation of γ-PGA.

[0009] Preferably, the glucose concentration in the fermentation broth is controlled to be 5-20 g / L, the aeration rate is controlled to be 0.2-3 vvm, and the fermentation is performed for 6-18 hours to complete the early-stage fermentation.

[0010] Preferably, after the early-stage fermentation is completed, the specific oxygen consumption rate in the middle-late stage of fermentation is controlled to be 15-40 mmol / (g·h).

[0011] Preferably, the application comprises the following steps:

[0012] (1) inoculating the Bacillus subtilis into a γ-PGA fermentation medium, and performing fermentation for 6-18 hours under the condition of a glucose concentration of 5-20 g / L and an aeration rate of 0.2-3 vvm to complete the early-stage fermentation;

[0013] (2) after the early-stage fermentation is completed, the oxygen supply level in the fermentation system is adjusted in real time to maintain the qO2 constant at 15-40 mmol / (g·h).

[0014] Preferably, the composition of the γ-PGA fermentation medium comprises: glucose 5-70 g / L, sodium glutamate 10-50 g / L, citric acid 5-10 g / L, (NH4)2SO4 5-10 g / L, K2HPO4 0.5-1 g / L, FeCl3·6H2O 0.02 g / L, MnSO4·H2O 0.1 g / L, MgSO4·7H2O 0.5 g / L, CaCl2 0.2 g / L, and the pH is 6.5-7.5.

[0015] Correspondingly, a fermentation method of γ-PGA comprises the following steps:

[0016] (1) inoculating the microorganism into a γ-PGA fermentation medium, and performing fermentation for 6-18 hours under the condition of a glucose concentration of 5-20 g / L and an aeration rate of 0.2-3 vvm to complete the early-stage fermentation;

[0017] (2) after the early-stage fermentation is completed, the oxygen supply level in the fermentation system is adjusted in real time to maintain the qO2 constant at 15-40 mmol / (g·h).

[0018] Preferably, the composition of the γ-PGA fermentation medium comprises: glucose 5-70 g / L, sodium glutamate 10-50 g / L, citric acid 5-10 g / L, (NH4)2SO4 5-10 g / L, K2HPO4 0.5-1 g / L, FeCl3·6H2O 0.02 g / L, MnSO4·H2O 0.1 g / L, MgSO4·7H2O 0.5 g / L, CaCl2 0.2 g / L, and the pH is 6.5-7.5.

[0019] Preferably, the method for adjusting the oxygen supply level in the fermentation system in real time is as follows: by on-line detection of the specific oxygen consumption rate of unit cell, according to the size of the specific oxygen consumption rate, the oxygen supply level of the system is adjusted in real time;

[0020] The method for on-line detection of the specific oxygen consumption rate comprises:

[0021] ① The oxygen consumption rate (OUR) is determined on-line, and the value of the OUR is calculated by formula (1):

[0022]

[0023] Wherein, ΔDO / Δt is the accumulation rate of dissolved oxygen in the fermentation broth, Q is the aeration rate, and V is the volume of the fermentation broth; and are the oxygen concentrations in the inlet gas and outlet gas of the fermenter respectively;

[0024] ② The cell density is detected on-line;

[0025] ③ The specific oxygen consumption rate of unit cell is calculated by formula (2):

[0026]

[0027] Wherein, OUR and X are the oxygen consumption rate and cell density detected on-line in steps ① and ②.

[0028] The present application has the following beneficial effects: the present application provides a new Bacillus subtilis strain, which can produce γ-PGA with a maximum molecular weight of 25000 KDa, far exceeding the existing level of the same kind of microorganism. Meanwhile, in view of the problem that the oxygen supply is limited in the middle and later stages of the fermentation of the microorganism for preparing ultra-high molecular weight γ-PGA, resulting in limited production of ultra-high molecular weight γ-PGA, the present application provides a method for real-time detection and effective characterization of the supply and utilization of oxygen in the fermentation process of γ-PGA, and based on this, the oxygen supply level is adjusted in real time, so that the microorganism can be provided with appropriate oxygen supply in a more timely, scientific and accurate manner, thereby exerting the maximum synthesis capacity of ultra-high molecular weight γ-PGA.

[0029] The method provided by the present application is simple to operate, can be automatically monitored and calculated, has low requirements for the operator, is suitable for large-scale fermentation production of ultra-high molecular weight γ-PGA, and has industrial popularization and application value. DETAILED DESCRIPTION

[0030] The present application provides a Bacillus subtilis FB-3, the 16S rDNA sequence of which is shown as SEQ ID NO. 1. The microorganism was preserved in the China General Microbiological Culture Collection Center on April 29, 2022, and the preservation number is CGMCC NO. 24826.

[0031] The maximum molecular weight of the gamma-PGA produced by the Bacillus subtilis is 2.5 x 10 4 KDa, which is much higher than the molecular weight of all existing publicly disclosed microorganisms producing gamma-PGA.

[0032] In order to better utilize the gamma-PGA produced by the Bacillus subtilis and improve the fermentation efficiency, the present application further provides a fermentation method capable of real-time oxygen supply regulation, which specifically comprises the following steps:

[0033] 1. The Bacillus subtilis FB-3 is inoculated into a culture medium for activation (cultured at 35℃ for 18-24 hours) to obtain a seed culture by scale-up culture. The activation culture medium is LB slant medium, and the components include: 10 g / L of proteose peptone, 5 g / L of yeast powder, 10 g / L of NaCl, 20 g / L of agar, and pH is 7.0-7.4. The scale-up culture medium is LB liquid medium, which is obtained by removing agar from the LB slant medium. The scale-up culture conditions are as follows: the activated strain is inoculated into the scale-up culture medium in a flask, cultured at 35℃ for 18-24 hours to obtain a first-stage seed; the first-stage seed is inoculated into a new scale-up culture medium in a 10-500 L seed tank at an inoculation amount of 1-10% (v / v), and cultured at 35℃, 150-200 rpm, and a ventilation amount of 1.0-2.0 vvm for 24-48 hours. Unless otherwise specified, the ventilation is sterile clean air.

[0034] 2. The seed culture obtained in step 1 is inoculated into a gamma-PGA fermentation medium. In the early stage of fermentation, a low glucose concentration is maintained, and the fermentation is carried out under low oxygen supply (ventilation amount) for 6-18 hours to control the growth of the bacterial cells and avoid the bacterial cell density being too high to exceed the maximum oxygen supply limit of the fermentation tank.

[0035] The components of the γ-PGA fermentation medium include: glucose 5-70 g / L (the specific concentration is adjusted according to the period of fermentation), sodium glutamate 10-50 g / L, citric acid 5-10 g / L, (NH4)2SO4 5-10 g / L, K2HPO40.5-1 g / L, FeCl3·6H2O 0.02 g / L, MnSO4·H2O 0.1 g / L, MgSO4·7H2O 0.5 g / L, CaCl20.2 g / L, and the pH is 6.5-7.5, wherein the glucose is sterilized separately. The γ-PGA fermentation conditions are as follows: the volume of the fermentation tank is 10 L-5 m 3 , the sample loading amount is 50%-75%, the inoculation amount is 3%-10%, the fermentation temperature is 35°C, the stirring speed is 50-600 rpm, and the aeration amount is 0.2-3 vvm (the specific aeration amount is adjusted according to the period of fermentation).

[0036] During the early stage of the γ-PGA fermentation, the glucose concentration is maintained at a low level, preferably 5-20 g / L, and the maintenance method is as follows: sample is taken every 2 hours, the glucose concentration in the fermentation broth is determined using a biosensor, and the glucose concentration is maintained substantially constant by metering feeding. During the early stage of the γ-PGA fermentation, the oxygen supply level is maintained at a low level, and one embodiment is as follows: the stirring speed is controlled at 50-200 rpm, and the air amount is controlled at 0.2-1 vvm.

[0037] After the synthesis of the ultra-high molecular weight γ-PGA starts (the content of the γ-PGA in the fermentation broth is determined using high-performance gel chromatography (GPC) to determine whether the synthesis of the γ-PGA starts), the fermentation enters the later stage, the glucose concentration in the medium is increased to 20-70 g / L by feeding, and the specific oxygen consumption rate (qO2) of the unit cell is detected online, the oxygen supply level of the γ-PGA fermentation is adjusted in real time according to the size of qO2, and qO2 is maintained constant at 15-40 mmol / (g·h). The oxygen supply amount of the unit cell is ensured, and efficient fermentation of the γ-PGA is realized. The method for real-time adjustment of the oxygen supply level of the γ-PGA fermentation is as follows: according to the actual situation, one or more parameters such as the stirring speed of the fermentation tank, the aeration rate, the tank pressure, and the aeration oxygen partial pressure are changed to change the size of the oxygen supply, so that qO2 is maintained in the set range.

[0038] The method for online detection of the specific aerobic rate includes:

[0039] ① Online determination of oxygen consumption rate (OUR). Fermentation tail gas analyzer using mass spectrometry, paramagnetic, infrared, chemical electrode principle, online detection of oxygen content in the gas inlet and exhaust of fermenter; using fermenter online weighing device, online detection of the volume of fermentation broth; using gas mass flow meter, online detection of the ventilation rate of fermenter; using dissolved oxygen electrode, online determination of the accumulation rate of dissolved oxygen in fermentation broth; the OUR value is calculated by formula (1):

[0040]

[0041] Wherein ΔDO / Δt is the accumulation rate of dissolved oxygen in the fermentation broth (mmol / (L·h)); Q is the ventilation rate (L / min), V is the volume of fermentation broth (L); and The oxygen concentration in the gas inlet and exhaust of fermenter (mmol / L) respectively.

[0042] ② Online detection of cell density. Using online cell detection electrode, the total cell concentration in fermentation broth is measured by turbidity principle, and the cell density is converted into cell dry weight content g / L through turbidity-dry weight standard curve.

[0043] ③ Specific oxygen consumption rate of unit cell (qO2) is calculated by formula (2):

[0044]

[0045] Wherein OUR and X are the oxygen consumption rate and cell density detected online in steps ① and ②.

[0046] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present application, not all the embodiments. If not specifically mentioned, the technical means used in the embodiments are the conventional means familiar to those skilled in the art.

[0047] Example 1: Screening and identification of microorganisms

[0048] 1. Microbial source and screening. Take farmland soil samples from Shuangliu District, Sichuan Province, accurately weigh 10 g of the sample into 100 mL of sterile water, stir for 30 minutes, and then take 10 mL of the liquid for pretreatment in a 80℃ water bath for 15 minutes. Gradient dilution of the sample was performed using sterile water, and 0.1 mL of each gradient dilution was spread on a separation plate medium and incubated in an incubator at 35℃ for 20 hours. The more viscous and convex single colonies on the plate were picked and further purified, numbered, and inoculated into a slant medium, which was temporarily stored in a 4℃ refrigerator. The separation plate medium and the storage slant medium were both LB-sodium glutamate medium, with the following components: 10 g / L proteose peptone, 5 g / L yeast powder, 10 g / L NaCl, 10 g / L sodium glutamate, and 20 g / L agar, with a pH of 7.0-7.4.

[0049] Each strain preliminarily screened was inoculated into LB liquid medium (10 g / L proteose peptone, 5 g / L yeast powder, 10 g / L NaCl, pH 7.0-7.4) and cultured at 35℃ and 200 rpm for 18 hours to obtain seed culture. Each seed culture was inoculated at 10% (v / v) into a triangular flask containing γ-PGA fermentation medium, which was cultured at 35℃ and 240 rpm for 48 hours. The components of the γ-PGA fermentation medium were: 40 g / L glucose, 5 g / L citric acid, 25 g / L sodium glutamate, 10 g / L ammonium sulfate, 2 g / L K2HPO4, 0.02 g / L FeCl3·6H2O, 1 g / L MgSO4·7H2O, 0.05 g / L MnSO4·H2O, 0.5 g / L MgSO4·7H2O, and 0.2 g / L CaCl2, with a pH of 7.0-7.2.

[0050] The yield and molecular weight of γ-PGA in each fermentation broth were detected, and strain FB-3 with high yield and molecular weight was selected. The yield of γ-PGA in the shake flask fermentation of strain FB-3 was 25 g / L after 36 hours and 30 g / L after 48 hours, and the highest molecular weight of the produced γ-PGA reached 2.5×10 4 KDa, and the weight average molecular weight also reached 1.45×10 4 KDa. 16S rDNA sequencing molecular biology and routine physiological and biochemical identification showed that the microorganism was Bacillus subtilis, and its 16S rDNA sequence is shown as SEQ ID NO. 1. The microorganism was preserved in the China General Microbiological Culture Collection Center on April 29, 2022, with the preservation number of CGMCC NO. 24826.

[0051] 2. Physiological and biochemical characteristics. After identification and testing, the physiological and biochemical characteristics of strain FB-3 were as shown in Table 1. In Table 1, "+" represents a positive reaction, and "-" represents a negative reaction.

[0052] Table 1 Comparison of physiological and biochemical characteristics of strain FB-3

[0053] Physiological and biochemical tests Results Physiological and biochemical tests Results Contact enzyme + Lecithinase test + D-xylose acid production + Propionate utilization - Methyl red test + V-P test + Glucose gas production - D-glucose acid production + Citrate utilization + D-mannitol acid production + Nitrate reduction + Casein hydrolysis + Starch hydrolysis + Indole test - Gelatin liquefaction + Anaerobic growth -

[0054] Example 2: Effect of fermentation in a 100L fermenter to produce γ-PGA

[0055] 1. Activation of the strain and seed expansion culture. The Bacillus licheniformis LJG-1 preserved at -80°C was thawed, and a loopful was streaked on an LB agar slant and incubated at 35°C for 18 hours for activation. The activated strain was transferred to a new LB liquid medium, and incubated at 35°C, 200 rpm for 18 hours to obtain a first-stage seed solution. The first-stage seed solution was transferred to a 10L seed fermenter containing 5L of sterile LB medium, and inoculated at 10% (v / v), and incubated at 35°C, 200 rpm, with aeration at 1vvm for 20 hours to obtain a second-stage seed culture.

[0056] 2. Fermentation to produce γ-PGA. The second-stage seed culture was transferred to a 100L fermenter containing 50L of sterile γ-PGA medium (20g / L of glucose as the initial concentration, and other components were the same as in the γ-PGA fermentation medium in Example 1) through a sterile pipeline, and the fermentation temperature was 35°C. In the early stage of fermentation, the oxygen supply was maintained at a low level, with aeration at 1vvm and an initial stirring speed of 200 rpm, while the glucose concentration was controlled at 20g / L to control the cell density in the fermentation broth and avoid exceeding the oxygen supply limit of the fermenter. After 6 hours of fermentation (after the start of γ-PGA synthesis), the glucose concentration was increased to 30g / L. The specific oxygen consumption rate (qO2) of strain FB-3 in the γ-PGA fermentation was determined in real time by means of an online cell detection device, a tail gas analyzer, and an air mass flow meter, and based on this, the oxygen supply level was adjusted by gradually increasing the stirring speed to maintain the qO2 of strain FB-3 at about 20mmol / (g·h), until the maximum stirring speed of 600 rpm was reached.

[0057] Meanwhile, a control group (fixed oxygen supply) was set up, in which the conditions were the same as above, the glucose concentration was maintained at 20g / L, but the oxygen supply level was not adjusted, and the stirring speed was kept constant at 600 rpm.

[0058] The fermentation was completed after 48 hours. During the fermentation, samples were taken every 6 hours to determine the γ-PGA concentration, cell content, and record the changes in the stirring speed and the specific oxygen consumption rate (qO2) of strain FB-3.

[0059] The results show that in the control group, the synthesis of γ-PGA stagnates after 18 hours, and the final yield is only 12.44 g / L. In the experimental group (real-time oxygen supply group), the stirring speed is gradually increased as the fermentation progresses, and the oxygen supply is gradually increased. The synthesis of γ-PGA continues, and the final yield reaches 24.11 g / L, which is increased by 93.81% compared with the control group. It proves that the real-time oxygen supply regulation method can effectively solve the problem of dissolved oxygen limitation in the later stage of γ-PGA fermentation, and can significantly improve the yield of γ-PGA.

[0060] Example Three: Effect of Different Specific Oxygen Consumption Rates on γ-PGA Fermentation

[0061] To further optimize the real-time oxygen supply regulation method, γ-PGA fermentation under different specific oxygen consumption rates (qO2) was set up in a 100L fermenter. The strain activation, seed expansion culture, and real-time oxygen supply regulation fermentation parameters were exactly the same as in Example Two. Only after the start of γ-PGA synthesis (after 6 hours of fermentation), the oxygen supply level was regulated by changing the stirring speed, so that the qO2 of strain FB-3 was maintained at 10 mmol / (g·h), 15 mmol / (g·h), 20 mmol / (g·h), 25 mmol / (g·h), and 30 mmol / (g·h), respectively. After 48 hours of fermentation, the final cell mass and γ-PGA yield of each group were measured, and the results are shown in Table 2.

[0062] Table 2 Effect of Different Specific Oxygen Consumption Rates on Fermentation

[0063] [Table 2] q02 maintenance levels (mmol / (g-h)) Final cell mass (g / L) γ-PGA production (g / L) 10 1.60 10.51 15 1.91 18.45 20 2.22 24.79 25 2.27 33.60 30 3.49 17.11

[0064] Example Four: Effect of Fermentation in a 5 Ton Fermenter to Produce γ-PGA

[0065] A 5 ton (5m 3 ) fermenter was used to scale up the fermentation process of strain FB-3. The fermenter is 3.8 meters high and 1.2 meters in diameter. The stirring paddle is a three-stage paddle, with straight six-blade turbine paddles at the top and bottom, and a four-blade propeller in the middle. The stirring paddle is 0.5 meters in diameter. The inner side of the tank wall is equipped with a coil for circulating condensate water. At the same time, it is equipped with online cell detection electrodes, liquid level electrodes, and tail gas detection devices for real-time detection of specific oxygen consumption rate (qO2).

[0066] The strain activation and seed expansion culture refer to Example 2, except that the secondary seed is cultured in a 500L fermenter, the inoculation amount is 1% (v / v), the temperature is 35°C, the stirring speed is 200rpm, the aeration amount is 1vvm, and the culture time is 12 hours. After the medium in the 5-ton fermenter is sterilized and cooled, the strain is transferred to the 5-ton fermenter under sterile conditions through a pipeline, the sample loading amount is 70% (v / v), the temperature is 35°C, the initial stirring speed is 80rpm, and the initial aeration is 0.4vvm. The glucose concentration is maintained at about 20g / L. After 10 hours, the γ-PGA starts to be synthesized, the glucose concentration is increased to 40g / L, the stirring, aeration amount and tank pressure are adjusted, and the specific oxygen consumption rate is maintained at about 25mmol / (g·h). The fermentation lasts for 60 hours, and then the cell amount and γ-PGA content are detected. Because the fermentation scale is expanded in this example, the lag phase is prolonged, and the γ-PGA synthesis time is postponed.

[0067] The results show that the final cell amount is 3.12g / L, and the final γ-PGA yield is 29.07g / L at the end of 60 hours of fermentation, and the industrial scale-up production of γ-PGA fermentation is successfully achieved.

[0068] Example Five: Optimization of γ-PGA production in a 5-ton fermenter

[0069] In order to further optimize the real-time oxygen supply regulation of the γ-PGA fermentation process in a 5-ton fermenter, the oxygen supply level in the fermentation is regulated by adding oxygen-rich air. The strain activation, seed expansion culture and γ-PGA fermentation conditions in the 5-ton fermenter in this example are the same as those in Example Four, except that the sodium glutamate addition amount in the γ-PGA fermentation medium is increased to 35g / L, and after the γ-PGA starts to be synthesized (after 10 hours of fermentation), pure oxygen is prepared using an oxygen generator and mixed with air, and the oxygen supply level is regulated by changing the oxygen partial pressure in the aeration, rather than by adjusting the stirring speed to control the oxygen supply level, so as to maintain the specific oxygen consumption rate at about 25mmol / (g·h). After 60 hours of fermentation, the final cell amount and γ-PGA yield in the fermentation broth are determined.

[0070] The results show that the final cell amount is 4.18g / L, and the final γ-PGA yield is 40.34g / L.

[0071] The above examples only describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications, variations, modifications and replacements of the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A method for preparing γ-PGA using Bacillus subtilis, characterized in that: The Bacillus subtilis described was Bacillus subtilis FB-3, which was deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 29, 2022, with accession number CGMCC NO.24826. The Bacillus subtilis was inoculated into a γ-PGA fermentation medium. The γ-PGA fermentation medium consisted of: glucose 5–70 g / L, monosodium glutamate 10–50 g / L, citric acid 5–10 g / L, (NH4)2SO4 5–10 g / L, K2HPO4 0.5–1 g / L, FeCl3·6H2O 0.02 g / L, MnSO4·H2O 0.1 g / L, MgSO4·7H2O 0.5 g / L, and CaCl2 0.2 g / L, with a pH of 6.5–7.

5. The method includes the following steps: (1) The Bacillus subtilis was inoculated into γ-PGA fermentation medium and fermented for 6 to 18 hours under the conditions of 5 to 20 g / L glucose concentration and 0.2 to 3 vvm aeration rate to complete the initial fermentation; (2) After the initial fermentation is completed, the concentration of glucose in the culture medium is increased by feeding to maintain it at 20-70 g / L; the oxygen supply level in the fermentation system is adjusted in real time to maintain the specific oxygen consumption rate at 15-40 mmol / (g·h).

2. The method according to claim 1, characterized in that: The method for adjusting the oxygen supply level in the fermentation system in real time is as follows: by detecting the specific oxygen consumption rate per unit cell online, the oxygen supply level of the system is adjusted in real time according to the magnitude of the specific oxygen consumption rate. Online methods for detecting specific aerobic rates include: ① The oxygen consumption rate, OUR, is measured online, and the value of OUR is calculated using formula (1): Where ΔDO / Δt is the accumulation rate of dissolved oxygen in the fermentation broth; Q is the aeration rate; and V is the volume of the fermentation broth. and These represent the oxygen concentrations in the inlet and outlet air of the fermenter, respectively. ② Online detection of bacterial cell density; ③ The specific oxygen consumption rate per unit cell is calculated using formula (2): Where OUR and X are the oxygen consumption rate and cell density detected online in steps ① and ②, respectively.

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