Method for producing L-citrulline through fermentation

Abstract

The invention provides a method for producing L-citrulline through fermentation, and belongs to the technical field of fermentation. When the L-citrulline is produced by fermentation, an arginine solution is used as a pH regulator, the arginine solution is fed to replace ammonia water to regulate the pH value, and meanwhile, a glucose solution is fed to carry out fermentation culture. According to the method, fermentation and conversion are synchronously carried out, ammonia water and sulfuric acid can be omitted, ammonium ions are utilized to the maximum extent, the production period can be shortened, the production cost is remarkably reduced, and the environment is protected.

Description

Technical Field

[0001] The invention belongs to the technical field of fermentation, and particularly relates to a method for producing L-citrulline through fermentation. Background Art

[0002] As a functional amino acid, L-citrulline has the functions of inhibiting oxidation and arteriosclerosis, scavenging free radicals, dilating blood vessels, enhancing male sexual function, improving human pancreatic metabolic function, regulating cholesterol and blood pressure levels, etc. It is often added to functional beverages and foods as a nutritional supplement.

[0003] In the modern food industry, L-citrulline is generally produced by fermentation. This method uses Escherichia coli that produces arginine deiminase as the fermentation strain, glucose as the carbon source, supplemented by nitrogen sources such as yeast extract, peptone, ammonium chloride, and inorganic salts such as magnesium sulfate and potassium dihydrogen phosphate. Ammonia is used to regulate the pH of the process, and the residual sugar level is controlled during the fermentation process. Aerobic fermentation is carried out, and the bacteria secrete arginine deiminase, which uses arginine as a substrate and catalyzes the conversion of arginine deiminase into L-citrulline in one step. Patent publication number CN116004740A discloses a method for producing L-citrulline, and its arginine-to-citrulline conversion rate can reach 100%. However, the fermentation process requires the maintenance of a pH of 7.0 by ammonia or liquid ammonia, and a feed medium containing yeast extract and peptone is required to supplement the nitrogen source. After 50-55 hours of fermentation, the enzyme conversion needs to be continued for 10-12 hours. The ammonium radicals released during the conversion process are wasted, and the addition of nitrogen sources increases production costs. For another example, CN118931987A patent discloses a method for producing L-citrulline using L-arginine fermentation waste liquid as raw material. The fermentation and conversion end conversion rate exceeds 98%, but it is still necessary to add an alkaline substance to control the stability of the fermentation pH. After the fermentation obtains the enzyme liquid, it needs to undergo 12 to 16 hours of conversion, the production efficiency is low, and the ammonium ions released during the conversion process are wasted. It can be seen that the previous fermentation method for producing L-citrulline generally has the problems of reagent waste, low production efficiency and ammonium ion waste. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a method for producing L-citrulline by fermentation, wherein the pH value is adjusted by feeding an L-arginine solution instead of ammonia water, thereby eliminating the consumption of subsequent acid reagents, and utilizing the ammonium ions released when arginine deiminase is converted into L-arginine. This is used as an inorganic nitrogen source for fermentation, thereby achieving waste recycling, shortening the fermentation cycle, and improving production efficiency.

[0005] The present invention provides a method for producing L-citrulline by fermentation, comprising the following steps: inoculating a fermentation bacterium that produces arginine deiminase into a fermentation medium for fermentation and culturing; During the fermentation, arginine solution and glucose solution were added simultaneously until the fermentation broth OD 600nm No more increase; The fermentation is terminated when the mass concentration of glucose in the fermentation system is ≤0.05%; The amount of the arginine solution added maintains the pH value of the fermentation system at neutral.

[0006] Preferably, the fermentation medium comprises the following components: yeast extract 4-6 g / L, peptone 10-12 g / L, potassium dihydrogen phosphate 6-8 g / L, disodium hydrogen phosphate dodecahydrate 18-20 g / L, ammonium sulfate 4-6 g / L, glucose 18-20 g / L, kanamycin 30-40 mg / L and IPTG 0.04-0.05 g / L.

[0007] Preferably, the flow acceleration of the glucose solution is such that the glucose concentration in the fermentation system is maintained within the range of 1-6 g / L.

[0008] Preferably, the fermentation temperature is 36-37°C; the pH value of the fermentation system is 6.9-7.1; The fermentation pressure is 0.10-0.12 MPa; the ventilation ratio is 0.5-0.6 vvm; the rotation speed is 300-400 r / min; and the fermentation cycle is 28-32 h.

[0009] Preferably, the OD of the fermentation system 600nm When it reaches 20, add IPTG; The fermentation temperature was lowered to 29-30° C. before IPTG addition.

[0010] Preferably, the fermentation bacteria producing arginine deiminase is Escherichia coli with a deposit number of CCTCC NO: M 2025599 ( Escherichia coli ) strain DXMC015.

[0011] Preferably, the seed liquid of the fermentation bacteria producing arginine deiminase is obtained by subjecting the fermentation bacteria producing arginine deiminase to shake flask culture, primary seed culture and secondary seed culture.

[0012] Preferably, the shake flask culture medium comprises the following components: yeast extract 4-6 g / L, tryptone 9-11 g / L, sodium chloride 9-11 g / L and kanamycin 40-50 mg / L; The rotation speed of the shake flask culture is 180-200 r / min, the temperature of the shake flask culture is 36-37° C., the pH value of the shake flask culture system is 6.9-7.1, and the cycle of the shake flask culture is 8-12 hours.

[0013] Preferably, the culture medium for primary seed culture comprises the following components: yeast extract 4-6 g / L, tryptone 9-11 g / L, sodium chloride 9-11 g / L and kanamycin 40-50 mg / L; The temperature of the primary culture is 36~37°C, the pressure of the primary culture is 0.08~0.10MPa, the ventilation ratio of the primary culture is 0.5~0.6vvm, the stirring speed of the primary culture is 300~400r / min, and the cycle of the primary culture is 6~8h.

[0014] Preferably, the culture medium for the secondary seed culture comprises the following components: yeast extract 4-6 g / L, peptone 10-12 g / L, potassium dihydrogen phosphate 2-4 g / L, dipotassium hydrogen phosphate 11-13 g / L, magnesium sulfate heptahydrate 1-3 g / L, glucose 20-22 g / L, and kanamycin 30-40 mg / L; The temperature of the secondary seed culture is 36~37°C, the system pH value of the secondary seed culture is 6.9~7.1, the pressure of the secondary seed culture is 0.08~0.10MPa, the ventilation ratio of the secondary seed culture is 0.5~0.6vvm, the stirring speed of the secondary seed culture is 300~400r / min, and the cycle of the secondary seed culture is 6~8h.

[0015] The present invention provides a method for producing L-citrulline by fermentation, comprising the following steps: inoculating a fermentation bacterium capable of producing arginine deiminase into a fermentation medium for fermentation and culturing; during the fermentation and culturing, simultaneously adding an arginine solution and a glucose solution to the fermentation liquid OD 600nmNo longer increases; when the mass concentration of glucose in the fermentation system is ≤0.05%, the fermentation is terminated; the amount of the arginine solution added is such that the pH value of the fermentation system is maintained at a neutral value. The present invention aims to solve the problem that the fermentation bacteria producing arginine deiminase metabolize glucose to reduce the pH value of the fermentation system during the aerobic fermentation process, and ammonia water is needed to adjust the pH value in order to produce arginine deiminase. At the same time, arginine, as an alkaline substrate, needs to be supplemented with sulfuric acid to adjust the system to neutral or acidic in order to ensure the catalytic efficiency of arginine deiminase, and a large amount of sulfuric acid and ammonia water is wasted in the process. The present invention changes the way and timing of adding raw materials. Arginine solution is added at the beginning of fermentation culture, thereby replacing the role of ammonia water in adjusting the pH to neutrality, omitting the addition of ammonia water, and omitting the step of adding sulfuric acid to adjust the pH in the step-by-step fermentation conversion. The addition of sulfuric acid is omitted, saving the investment cost of ammonia water and sulfuric acid. At the same time, by adding arginine at the beginning of fermentation, the conversion and fermentation are carried out simultaneously, not only ensuring a 100% conversion rate of arginine, but also greatly shortening the fermentation cycle and improving production efficiency. In addition, in the fermentation method of the prior art, fermentation and conversion distribution are carried out and there is also the problem of ammonium ion removal causing waste. The technical solution of the present invention will be carried out synchronously with conversion and fermentation, and the arginine deiminase catalytic conversion L-citrulline process will remove ammonium ions as the growth of inorganic nitrogen source supply fermentation bacteria, thereby omitting the removal step of ammonium ions and making waste conversion utilization, while meeting the concept of sustainable development and green environmental protection. The results of the embodiments show that the technical solution of the present invention is used to ferment and produce L-citrulline. According to the calculation of 10,000 tons per year of an L-citrulline workshop, 3000~4000 tons of ammonia and 1500~1800 tons of sulfuric acid can be saved, reducing costs by 2.5~4.5 million yuan / year. At the same time, when the acid production level and conversion rate level are not reduced, the time required for independent conversion is saved. Relative to the prior art, each batch of production can be shortened by 4~6h. According to the calculation of 10,000 tons per year of an L-citrulline workshop, 1000~1500 tons of output can be increased, and the output value is increased by 4500~70 million yuan.

[0016] Biological deposit information Escherichia coli ( Escherichia coli ) strain DXMC015, deposited with the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on March 27, 2025. The deposit number is CCTCC NO: M 2025599. DETAILED DESCRIPTION

[0017] The present invention provides a method for producing L-citrulline by fermentation, comprising the following steps: inoculating a fermentation bacterium that produces arginine deiminase into a fermentation medium for fermentation and culturing; During the fermentation, arginine solution and glucose solution were added simultaneously until the fermentation broth OD 600nm No more increase; The fermentation is terminated when the mass concentration of glucose in the fermentation system is ≤0.05%; The amount of the arginine solution added is such as to maintain the pH value of the fermentation system at neutral.

[0018] The invention inoculates fermentation bacteria producing arginine deiminase into a fermentation medium for fermentation culture.

[0019] In the present invention, the type of the fermentation bacteria producing arginine deiminase is not particularly limited, and any fermentation bacteria producing arginine deiminase known in the art can be used, such as the bacteria for producing L-citrulline disclosed in Patent Publication No. CN11893198A. In the embodiment of the present invention, the fermentation bacteria producing arginine deiminase is Escherichia coli ( Escherichia coli ) strain DXMC015.

[0020] In the present invention, the seed solution of the arginine deiminase-producing fermentation bacteria is preferably obtained by subjecting the fermentation bacteria to shake flask culture, primary seed culture, and secondary seed culture. During the shake flask culture, the inoculum size of the arginine deiminase-producing fermentation bacteria is 1 to 3 rings per shake flask, and can be 2 rings per shake flask. The shake flask culture medium preferably includes the following components: 4 to 6 g / L yeast extract, 9 to 11 g / L tryptone, 9 to 11 g / L sodium chloride, and 40 to 50 mg / L kanamycin; alternatively, it can include 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, and 45 mg / L kanamycin. The shake flask culture speed is preferably 180 to 200 r / min, and can be 190 r / min. The shake flask culture temperature is preferably 36 to 37°C, and can be 36.5°C. The pH value of the shake flask culture system is preferably 6.9-7.1, and may be 7.0. The shake flask culture cycle is preferably 8-12 hours, and may be 9-11 hours, or even 10 hours. The culture medium for the primary seed culture preferably includes the following components: 4-6 g / L yeast extract, 9-11 g / L tryptone, 9-11 g / L sodium chloride, and 40-50 mg / L kanamycin; alternatively, it may include 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, and 45 mg / L kanamycin. The temperature of the primary seed culture is preferably 36-37°C, and may be 36.5°C. The pressure of the primary seed culture is preferably 0.08-0.10 MPa, and may be 0.09 MPa. The ventilation ratio of the primary seed culture is preferably 0.5-0.6 vvm, and may be 0.55 vvm. The stirring speed of the primary seed culture is preferably 300-400 r / min, and may be 350 r / min. The primary seed culture cycle is preferably 6-8 hours, and can be 7 hours. The secondary seed culture medium preferably includes the following components: 4-6 g / L yeast extract, 10-12 g / L peptone, 2-4 g / L potassium dihydrogen phosphate, 11-13 g / L dipotassium hydrogen phosphate, 1-3 g / L magnesium sulfate heptahydrate, 20-22 g / L glucose, and 30-40 mg / L kanamycin; alternatively, it can include 5 g / L yeast extract, 11 g / L peptone, 3 g / L potassium dihydrogen phosphate, 12 g / L dipotassium hydrogen phosphate, 2 g / L magnesium sulfate heptahydrate, 21 g / L glucose, and 35 mg / L kanamycin. The secondary seed culture temperature is preferably 36-37°C, and can be 36.5°C. The secondary seed culture system pH is preferably 6.9-7.1, and can be 7.0. The secondary seed culture pressure is preferably 0.08-0.10 MPa, and can be 0.09 MPa. The ventilation ratio of the secondary seed culture is preferably 0.5-0.6 vvm, and may be 0.55 vvm. The stirring speed of the secondary seed culture is preferably 300-400 r / min, and may be 350 r / min.The cycle of the secondary seed culture is preferably 6 to 8 hours, and can be 7 hours.

[0021] In the present invention, the inoculum size of the arginine deiminase-producing fermentation bacteria in the fermentation medium is 1% to 3%, and may be 2%. The fermentation medium preferably comprises the following components: 4 to 6 g / L yeast extract, 10 to 12 g / L peptone, 6 to 8 g / L potassium dihydrogen phosphate, 18 to 20 g / L disodium hydrogen phosphate dodecahydrate, 4 to 6 g / L ammonium sulfate, 18 to 20 g / L glucose, 30 to 40 mg / L kanamycin, and 0.04 to 0.05 g / L IPTG; alternatively, the inoculum size may comprise 5 g / L yeast extract, 11 g / L peptone, 7 g / L potassium dihydrogen phosphate, 19 g / L disodium hydrogen phosphate dodecahydrate, 5 g / L ammonium sulfate, 19 g / L glucose, 35 mg / L kanamycin, and 0.045 g / L IPTG. The fermentation culture temperature is preferably 36 to 37°C, and may be 36.5°C. The pH value of the fermentation system is preferably 6.9-7.1, and may be 7.0. The pressure of the fermentation culture is 0.10-0.12 MPa, and may be 0.11 MPa. The ventilation ratio of the fermentation culture is 0.5-0.6 vvm, and may be 0.55 vvm. The rotation speed of the fermentation culture is 300-400 r / min, and may be 350 r / min. The fermentation culture cycle is 28-32 hours, and may be 29-31 hours, and may also be 30 hours.

[0022] In the present invention, the flow acceleration of the glucose solution is preferably such that the glucose concentration of the fermentation system is maintained within the range of 1 to 6 g / L, and may be 2 to 5 g / L, or may be 3 to 4 g / L. The concentration of the glucose solution is preferably 60% to 70%, or may be 65%. The glucose solution provides a carbon source for the growth of the fermentation bacteria producing arginine deiminase.

[0023] In the present invention, the concentration of the arginine solution is preferably 35% to 40%, and may be 38%. The flow acceleration of the arginine solution is not specifically limited, so as to achieve a neutral pH value of the fermentation system, for example, in the range of 6.9 to 7.1.

[0024] In the present invention, the OD of the fermentation system 600nm When the temperature reaches 20°C, IPTG is added. Before adding IPTG, the fermentation temperature is lowered to 29-30°C. The purpose of adding IPTG is to induce the expression of arginine deiminase and promote secretion.

[0025] In the present invention, the method for producing L-citrulline by fermentation uses an arginine solution as a pH regulator, and the pH is adjusted by feeding the arginine solution instead of ammonia water. At the same time, arginine deiminase converts arginine into L-citrulline while separating ammonium ions. The ammonium ions that should be treated as waste in a subsequent extraction process can be used as an inorganic nitrogen source for fermentation, thereby saving ammonia water and effectively utilizing the waste ammonium ions. At the same time, fermentation and conversion are carried out simultaneously, which can shorten the production cycle, significantly reduce production costs, and protect the environment.

[0026] The following is a detailed description of the method for producing L-citrulline by fermentation provided by the present invention in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1 A method for producing L-citrulline by fermentation, comprising the following steps: Step 1: Cultivation of E. coli seeds In a sterile environment, two loops of single colonies were picked from a plate or slant of the Escherichia coli strain with a deposit number of CCTCC NO: M 2025599, and transferred to a shake flask containing a seed culture medium. The culture was shaken at a pH of 7, 37°C, and 200 rpm for 8 hours to obtain shake flask seeds. The seed culture medium formula was: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 9 g / L, and kanamycin 50 mg / L.

[0028] The shake flask seeds were inoculated into a first-level seed tank filled with culture medium to obtain first-level cultured seeds. The first-level seed tank culture medium formula was: 5g / L yeast extract powder, 10g / L tryptone, 9g / L sodium chloride, and 50mg / L kanamycin. The culture conditions for the first-level seed tank were: temperature 37°C, tank pressure 0.08MPa, ventilation ratio 0.5vvm, stirring 400r / min, and culture period 6h. The end condition for culture in the first-level seed tank was: absorbance was measured at a wavelength of 600nm using a spectrophotometer, and the seed solution was cultured when the OD value reached 3. The primary culture seeds were inoculated into the secondary seed tank via an inoculation pipe at an inoculum volume of 3% of the secondary seed tank culture medium volume to obtain E. coli seed solution. The secondary seed tank culture medium composition was: 5g / L yeast extract powder, 10g / L peptone, 3g / L potassium dihydrogen phosphate, 12g / L dipotassium hydrogen phosphate, 2g / L magnesium sulfate heptahydrate, 20g / L glucose, and 40mg / L kanamycin. The culture conditions in the secondary seed tank were: temperature 37°C, pH 7.1, tank pressure 0.1MPa, ventilation ratio 0.5vvm, agitation 400r / min, and culture period 8h.

[0029] Step 2: L-citrulline fermentation E. coli seed culture was transferred to a 7 m³ fermenter containing 3 m³ of fermentation medium for cultivation. The fermentation medium composition consisted of 5 g / L yeast extract, 10 g / L peptone, 6 g / L potassium dihydrogen phosphate, 18 g / L sodium hydrogen phosphate dodecahydrate, 5 g / L ammonium sulfate, 20 g / L glucose, 30 mg / L kanamycin, and 0.048 g / L IPTG. Transplantation conditions were as follows: absorbance was measured at 600 nm using a spectrophotometer, and the seed culture was transferred when the OD value reached 3. The transfer volume for the secondary seed culture was 2% of the fermenter medium volume. Fermentation conditions were as follows: initial temperature 37°C, pH 7.0, tank pressure 0.1 MPa, ventilation ratio 0.5 vvm, and agitation 300 rpm.

[0030] During the fermentation process, 35% arginine solution was added to maintain the pH value at 7.0, and 65% glucose solution was added to control the glucose concentration at 2~5g / L. 600nm When the OD value was 20.25, the temperature was lowered to 30°C, and IPTG solution was pumped into the fermentation tank to a final concentration of 0.048 g / L. The culture was continued for 30 hours. When the OD value began to decrease, the addition of glucose solution and arginine solution was stopped. The culture period was 32 hours. When the glucose concentration of the fermentation liquid reached 0.022%, the fermentation was terminated, and L-citrulline was obtained.

[0031] After the fermentation is completed, the content of L-citrulline and L-arginine obtained is determined, with specific reference to the following existing technologies ([1] Li Wei, Tao Qianyi, Li Wenlian. Fermentation production of L-glutamine and L-arginine [M]. Beijing: Chemical Industry Press, 2021: 191-192. [2] Ma Yue, Su Lingqia, Wu Dan, et al. Optimization of process conditions for preparing L-citrulline by recombinant arginine deiminase [J]. Biotechnology Bulletin, 2015, 31(8): 180-18.) The L-citrulline concentration in the fermentation broth was 110.20 g / L, the L-arginine concentration was 0, and the L-citrulline conversion rate was 100%. No ammonia or sulfuric acid was consumed during the fermentation process.

[0032] Example 2 A method for producing L-citrulline through fermentation, comprising the following steps: Step 1: Cultivation of E. coli seeds In a sterile environment, a single colony is picked from a plate or slant of the Escherichia coli strain with a deposit number of CCTCC No: M 2025599, and a loopful of the colony is transferred to a shake flask containing a seed culture medium to obtain a shake flask seed; the seed culture medium formula is: yeast extract powder 4 g / L, tryptone 9 g / L, sodium chloride 9 g / L, and kanamycin 45 mg / L; the shake flask culture conditions are: temperature 37°C, rotation speed 180 r / min, pH 7, and culture period 10 hours.

[0033] The shake flask culture seeds were transplanted to the first-level seed tank and cultured until OD 600nm When the value reaches 3, the first-level culture seed is obtained. The medium formula for the first-level seed tank is: yeast extract powder 6g / L, tryptone 10g / L, sodium chloride 10g / L, and kanamycin 45mg / L. The culture conditions for the first-level seed tank are: temperature 37°C, tank pressure 0.08MPa, ventilation ratio 0.6vvm, stirring 350r / min, and culture period 7h.

[0034] The primary culture seeds were transferred to the secondary seed tank via a transfer pipe to obtain E. coli seed solution. The transfer volume of the primary culture seeds was 2.5% of the volume of the secondary seed tank culture medium. The secondary seed tank culture medium formula was: 4g / L yeast extract powder, 12g / L peptone, 2.5g / L potassium dihydrogen phosphate, 11g / L dipotassium hydrogen phosphate, 2.5g / L magnesium sulfate heptahydrate, 22g / L glucose, and 30mg / L kanamycin. The culture conditions in the secondary seed tank were: temperature 37°C, pH 7.0, tank pressure 0.09MPa, ventilation ratio 0.55vvm, agitation 350r / min, and a culture period of 6h.

[0035] Step 2: L-citrulline fermentation The E. coli seed liquid was transferred to a 7m³ fermentation tank containing 3m³ fermentation medium for cultivation. The fermentation medium formula included: 6g / L yeast extract powder, 10g / L peptone, 7g / L potassium dihydrogen phosphate, 19g / L sodium hydrogen phosphate dodecahydrate, 4g / L ammonium sulfate, 19g / L glucose, 32mg / L kanamycin, and 0.045g / L IPTG. The transplanting conditions were as follows: absorbance was measured at a wavelength of 600nm using a spectrophotometer, and the seed liquid was transplanted when the OD value reached 3. The transplant volume of the secondary seed culture was 2.5% of the fermentation tank medium volume. The fermentation culture conditions were: initial temperature 37°C, pH 6.9, tank pressure 0.12 MPa, ventilation ratio 0.55vvm, and agitation 350r / min. During the fermentation process, a 38% arginine solution was fed to maintain the pH at 6.9. A 60% glucose solution was also fed to maintain the glucose concentration between 3 and 5 g / L. After 8 hours of fermentation, when the fermentation broth reached an OD value of 21.1, the temperature was lowered to 29°C and IPTG was pumped into the fermentor to a final concentration of 0.045 g / L. Cultivation continued for 28 hours. When the OD value decreased, the addition of glucose and arginine solution was stopped. After 30 hours of culture, the glucose concentration in the fermentation broth reached 0.035%, and fermentation was completed to obtain L-citrulline.

[0036] After fermentation, the fermentation broth had an L-citrulline concentration of 109.80 g / L, an L-arginine concentration of 0, and an L-citrulline conversion rate of 100%. The fermentation process did not involve the use of ammonia and sulfuric acid.

[0037] Example 3 A method for producing L-citrulline through fermentation, comprising the following steps: Step 1: Cultivation of E. coli seeds In a sterile environment, three loops of single colonies were picked from a plate or slant of the Escherichia coli strain with a deposit number of CCTCC No: M 2025599 and transferred to a shake flask containing seed culture medium to obtain shake flask seeds; the seed culture medium formula was: yeast extract powder 6 g / L, tryptone 11 g / L, sodium chloride 11 g / L, and kanamycin 42 mg / L; the shake flask culture conditions were: temperature 36.5°C, rotation speed 190 r / min, and culture period 8 hours.

[0038] The shake flask culture seeds were transplanted to a first-stage seed tank for cultivation to obtain first-stage culture seeds. The first-stage seed tank culture medium composition was: yeast extract powder 6g / L, tryptone 10g / L, sodium chloride 10g / L, and kanamycin 42mg / L. The first-stage seed tank culture conditions were: temperature 36.5°C, tank pressure 0.09MPa, ventilation ratio 0.45vvm, agitation 300r / min, and incubation period 8h. The first-stage seed tank transplant conditions were: absorbance was measured at a wavelength of 600nm using a spectrophotometer, and the seed solution was transplanted when the OD value reached 3.

[0039] The primary culture seeds were transferred to the secondary seed tank via a transfer pipe to obtain E. coli seed solution. The transfer volume of the primary culture seeds was 2.5% of the volume of the secondary seed tank culture medium. The secondary seed tank culture medium composition was: yeast extract powder 4g / L, peptone 11g / L, potassium dihydrogen phosphate 2.5g / L, potassium dihydrogen phosphate 11g / L, magnesium sulfate heptahydrate 2.5g / L, glucose 22g / L, and kanamycin 30mg / L. The culture conditions in the secondary seed tank were: temperature 37°C, pH 6.9, tank pressure 0.09MPa, ventilation ratio 0.55vvm, agitation 350r / min, and culture period 7h.

[0040] Step 2: L-citrulline fermentation The E. coli seed liquid was transferred to a 7m³ fermentation tank containing 3m³ fermentation medium for cultivation. The fermentation medium formula was: 6g / L yeast extract powder, 12g / L peptone, 8g / L potassium dihydrogen phosphate, 20g / L sodium hydrogen phosphate dodecahydrate, 4.5g / L ammonium sulfate, 20g / L glucose, 35mg / L kanamycin, and 0.04g / L IPTG. The transplanting conditions were: absorbance was measured at a wavelength of 600nm using a spectrophotometer, and the seed liquid was transplanted when the OD value reached 3. The transplant volume for the secondary seed culture was 1.5% of the fermentation tank medium volume. The fermentation conditions were: initial temperature 37°C, pH 7.0, tank pressure 0.11MPa, ventilation ratio 0.6vvm, and agitation 400r / min. During the fermentation process, a 36% arginine solution was fed to maintain the pH at 7.0. A 70% glucose solution was also fed to maintain the glucose concentration between 1 and 3 g / L. After 9 hours of fermentation, the fermentation broth reached an OD value of 20.6. The fermentation broth was cooled to 30°C, and IPTG was pumped into the fermentor to a final concentration of 0.040 g / L. Cultivation continued for 31 hours. When the OD value began to decrease, the addition of glucose and arginine solutions was stopped. After 32 hours of culture, the glucose concentration in the fermentation broth reached 0.046%, completing the fermentation and yielding L-citrulline.

[0041] After fermentation, the fermentation broth had an L-citrulline concentration of 111.20 g / L, an L-arginine concentration of 0, and an L-citrulline conversion rate of 100%. No ammonia or sulfuric acid was consumed during the fermentation process.

[0042] Comparative Example 1 On the basis of Example 1, 20% ammonia water was used to maintain the fermentation pH at 7.0 during the fermentation process. After the fermentation was completed, an arginine solution with the same amount as in Example 1 was prepared, and the pH was adjusted to 7.0 using concentrated sulfuric acid. The solution was added to the fermentation broth, and the fermentation conditions were maintained unchanged. The conversion was carried out for 5 h, and the L-arginine concentration was 0, and the conversion was stopped.

[0043] After 32 hours of fermentation, the fermentation broth had an L-citrulline concentration of 107.80 g / L, an L-arginine concentration of 0, and an L-citrulline conversion rate of 100%. The process consumed 148 L of ammonia and 55 L of sulfuric acid.

[0044] Comparative Example 2 On the basis of Example 2, 20% ammonia water was used to maintain the fermentation pH during the fermentation process. After the fermentation was completed, an arginine solution with the same amount as in Example 2 was prepared, and the pH was adjusted to 6.9 using concentrated sulfuric acid. The solution was added to the fermentation broth, and the fermentation conditions were maintained unchanged. The conversion was carried out for 4.5 h, and the L-arginine concentration was 0, and the conversion was stopped.

[0045] After 30 hours of fermentation, the fermentation broth had an L-citrulline concentration of 106.20 g / L, an L-arginine concentration of 0, and an L-citrulline conversion rate of 100%. The fermentation process consumed 127 L of ammonia and 51 L of concentrated sulfuric acid.

[0046] Comparative Example 3 On the basis of Example 3, 20% ammonia water was used to maintain the fermentation pH during the fermentation process. After the fermentation was completed, an arginine solution with the same amount as in Example 2 was prepared, and the pH was adjusted to 6.9 using concentrated sulfuric acid. The solution was added to the fermentation broth, and the fermentation conditions were maintained unchanged. The conversion was carried out for 5.5 h, and the L-arginine concentration was 0, and the conversion was stopped.

[0047] After 32 hours of fermentation, the fermentation broth had an L-citrulline concentration of 108.60 g / L, an L-arginine concentration of 0, and an L-citrulline conversion rate of 100%. The fermentation process consumed 135 L of ammonia and 57 L of concentrated sulfuric acid.

[0048] The fermentation results of the three embodiments and comparative examples 1 to 3 show that: 1. The present invention adjusts the pH by feeding arginine solution instead of ammonia water, which can save ammonia water and sulfuric acid without reducing the acid production level and conversion rate. Based on the annual output of 10,000 tons of L-citrulline in a workshop, 3,000 to 4,000 tons of ammonia water and 1,500 to 1,800 tons of sulfuric acid can be saved, reducing costs by 2.5 to 4.5 million yuan per year.

[0049] 2. The present invention adjusts the pH by feeding arginine solution instead of ammonia water, saving the time required for separate conversion without reducing the acid production level and conversion rate. Compared with the existing technology, each batch production can be shortened by 4 to 6 hours. Based on the annual output of 10,000 tons of L-citrulline in a workshop, the output can be increased by 1,000 to 1,500 tons, and the output value can be increased by 45 to 70 million yuan.

[0050] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for producing L-citrulline by fermentation, characterized in that: The following steps are involved: inoculating a fermentation bacterium that produces arginine deiminase into a fermentation medium for fermentation and culturing; During the fermentation, arginine solution and glucose solution were added simultaneously until the fermentation broth OD 600nm No more increase; The fermentation is terminated when the mass concentration of glucose in the fermentation system is ≤0.05%; The amount of the arginine solution added is such as to maintain the pH value of the fermentation system at neutral.

2. The method for producing L-citrulline by fermentation according to claim 1, wherein The fermentation medium comprises the following components: 4-6 g / L yeast extract, 10-12 g / L peptone, 6-8 g / L potassium dihydrogen phosphate, 18-20 g / L disodium hydrogen phosphate dodecahydrate, 4-6 g / L ammonium sulfate, 18-20 g / L glucose, 30-40 mg / L kanamycin, and 0.04-0.05 g / L IPTG.

3. The method for producing L-citrulline by fermentation according to claim 1, wherein The flow acceleration of the glucose solution maintains the glucose concentration of the fermentation system within the range of 1-6 g / L.

4. The method for producing L-citrulline by fermentation according to claim 1, wherein The fermentation culture temperature is 36-37°C; the pH value of the fermentation system is 6.9-7.1; The fermentation pressure is 0.10-0.12 MPa; the ventilation ratio is 0.5-0.6 vvm; the rotation speed is 300-400 r / min; and the fermentation cycle is 28-32 h.

5. The method for producing L-citrulline by fermentation according to claim 1, wherein The OD of the fermentation system 600nm When it reaches 20, add IPTG; The fermentation temperature was lowered to 29-30° C. before IPTG addition.

6. The method for producing L-citrulline by fermentation according to any one of claims 1 to 5, wherein: The fermentation bacteria producing arginine deiminase is Escherichia coli with a preservation number of CCTCC NO: M 2025599 ( Escherichia coli ) strain DXMC015.

7. The method for producing L-citrulline by fermentation according to claim 6, wherein: The seed liquid of the fermentation bacteria producing arginine deiminase is obtained by subjecting the fermentation bacteria producing arginine deiminase to shaking flask culture, primary seed culture and secondary seed culture.

8. The method for producing L-citrulline by fermentation according to claim 7, wherein: The shake flask culture medium comprises the following components: yeast extract 4-6 g / L, tryptone 9-11 g / L, sodium chloride 9-11 g / L and kanamycin 40-50 mg / L; The rotation speed of the shake flask culture is 180-200 r / min, the temperature of the shake flask culture is 36-37° C., the pH value of the shake flask culture system is 6.9-7.1, and the cycle of the shake flask culture is 8-12 hours.

9. The method for producing L-citrulline by fermentation according to claim 7, wherein: The culture medium for primary seed culture comprises the following components: 4-6 g / L yeast extract, 9-11 g / L tryptone, 9-11 g / L sodium chloride, and 40-50 mg / L kanamycin; The temperature of the primary culture is 36~37°C, the pressure of the primary culture is 0.08~0.10MPa, the ventilation ratio of the primary culture is 0.5~0.6vvm, the stirring speed of the primary culture is 300~400r / min, and the cycle of the primary culture is 6~8h.

10. The method for producing L-citrulline by fermentation according to claim 7, wherein: The medium for secondary seed culture comprises the following components: 4-6 g / L yeast extract, 10-12 g / L peptone, 2-4 g / L potassium dihydrogen phosphate, 11-13 g / L dipotassium hydrogen phosphate, 1-3 g / L magnesium sulfate heptahydrate, 20-22 g / L glucose, and 30-40 mg / L kanamycin. The temperature of the secondary seed culture is 36~37°C, the system pH value of the secondary seed culture is 6.9~7.1, the pressure of the secondary seed culture is 0.08~0.10MPa, the ventilation ratio of the secondary seed culture is 0.5~0.6vvm, the stirring speed of the secondary seed culture is 300~400r / min, and the cycle of the secondary seed culture is 6~8h.