CN112852896A - Fermentation production method of L-arginine - Google Patents
Fermentation production method of L-arginine Download PDFInfo
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- CN112852896A CN112852896A CN202110253935.0A CN202110253935A CN112852896A CN 112852896 A CN112852896 A CN 112852896A CN 202110253935 A CN202110253935 A CN 202110253935A CN 112852896 A CN112852896 A CN 112852896A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/10—Citrulline; Arginine; Ornithine
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention provides a fermentation production method of L-arginine. The method comprises the following steps: inoculating the seed liquid of the L-arginine producing strain into a sterilized fermentation culture medium for fermentation, wherein the concentration of glucose in the fermentation culture medium is 5-6 wt%, and feeding glucose and ammonium sulfate in the fermentation process to produce the L-arginine. According to the invention, glucose and ammonium sulfate are fed in a continuous flow manner in the fermentation process, and other process steps and conditions are reasonably matched, so that the conversion rate is effectively improved, and the L-arginine content at the fermentation end point is increased.
Description
Technical Field
The invention belongs to the technical field of fermentation engineering, and particularly relates to a fermentation production method of L-arginine.
Background
L-arginine is a semi-essential amino acid in the human and animal body, not only a constituent of body proteins, but also a synthetic precursor of various bioactive substances. L-arginine participates in the urea cycle and is an important intermediate metabolite of the urea cycle of organisms; l-arginine also affects the cardiovascular system, nervous system and immune system through the nitric oxide pathway; l-arginine also has important roles in regulating reproductive functions and gene expression. In starvation, trauma or emergency situations, L-arginine is an essential amino acid. With the continuous and intensive research and understanding of the biological functions of L-arginine, L-arginine has increasingly wide application in the pharmaceutical and food industries, and particularly shows wide application prospects in the aspects of nutrition regulation and endocrine regulation.
The production method of L-arginine includes hydrolysis method and fermentation method. Among them, the hydrolysis method is time-consuming to operate, low in yield, poor in process stability and serious in environmental pollution, and is not suitable for large-scale production. The fermentation method overcomes the defects of complex process, large pollution and the like of the protein hydrolysis extraction method, and has wide development prospect. The process of producing L-arginine by fermentation is a very complex comprehensive process of chemical change and physiological change. There are usually two ways to improve the fermentation level, one is to select good strains, and the other is to select a proper fermentation culture process. The former is a strain breeding technology established on the research of metabolism control fermentation, and the latter is a fermentation control technology established on the basis of biochemical reaction engineering. Only a close combination of the two ultimately achieves a high level of fermentative production.
For a long time, people pay more attention to the breeding of high-yield strains, and research on fermentation process control is less. At present, the fermentation method for producing the L-arginine generally adopts a mode of supplementing raw materials in batches, and the conversion rate and the yield of the L-arginine are lower. Therefore, the research on improving the fermentation conversion rate and the yield of the L-arginine is of great significance.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a fermentation production method of L-arginine. The method can effectively improve the conversion rate and the L-arginine content at the fermentation end point.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method for the fermentative production of L-arginine, comprising the steps of:
inoculating the seed solution of the L-arginine producing strain into a sterilized fermentation medium, wherein the concentration of glucose in the fermentation medium is 5-6 wt% (for example, 5 wt%, 5.2 wt%, 5.3 wt%, 5.5 wt%, 5.6 wt%, 5.8 wt%, 6 wt%, etc.), and feeding glucose and ammonium sulfate during the fermentation process to produce L-arginine.
Compared with a fermentation process for supplementing raw materials in batches, the method disclosed by the invention has the advantages that the glucose and the ammonium sulfate are continuously fed in the fermentation process, and other process steps are combined, so that the conversion rate is effectively improved, and the content of the L-arginine at the fermentation end point is improved.
In the present invention, the concentration of glucose in the fermentation medium is preferably 5 to 6% by weight. If the glucose concentration is too low, nutrient substances are insufficient, so that the proliferation of bacteria at the initial fermentation stage is not facilitated, and the conversion rate and the yield of the L-arginine are reduced; if the glucose concentration is too high, the osmotic pressure of the fermentation medium is too high, which is also unfavorable for the proliferation of bacteria, and the conversion rate and yield of L-arginine are reduced.
In some embodiments of the present invention, the method for preparing the seed liquid comprises the following steps:
inoculating L-arginine producing strain into sterilized seed culture medium, wherein the inoculation amount is 8% -12% of the total volume of the strain and the seed culture medium, culturing is carried out for 20-24h (for example, 20h, 21h, 22h, 23h or 24 h) under the conditions that the temperature is 32 + -0.5 ℃ (for example, 31.5 ℃, 31.6 ℃, 31.8 ℃, 32 ℃, 32.2 ℃, 32.3 ℃ or 32.5 ℃, and the like), the pressure is 0.05 + -0.01 Mpa (for example, 0.04Mpa, 0.042Mpa, 0.045Mpa, 0.048Mpa, 0.05Mpa, 0.052Mpa, 0.055, 0.058Mpa or 0.06Mpa, and the like), and the aeration ratio is 1:0.8-1:1.2 (for example, 1:0.8, 1:0.9, 1:1, 1:1.1 or 1:1.2, and the like), and the seed solution is obtained.
Wherein the aeration ratio is the volume ratio of the feed liquid to the sterile air introduced per minute.
Preferably, the seed culture medium comprises the following components: 25-35g/L of glucose, 15-25g/L of ammonium sulfate, 15-25g/L of corn steep liquor, 0.4-0.6g/L of magnesium sulfate heptahydrate, 1.2-1.8g/L of potassium dihydrogen phosphate and 1.2-1.8g/L of urea, and the solvent is water.
Preferably, the seed solution has an OD at 562nm of 5 to 6 (e.g., may be 5, 5.2, 5.3, 5.5, 5.6, 5.8, or 6, etc.) and a pH of 7.0 to 7.2 (e.g., may be 7.0, 7.02, 7.05, 7.08, 7.1, 7.12, 7.15, 7.18, or 7.2, etc.).
In some embodiments of the invention, the seed solution is inoculated in an amount of 8-12% of the total volume of the seed solution and the fermentation medium; for example, it may be 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, or the like.
In some embodiments of the invention, the L-arginine producing strain is Corynebacterium Glutamicum (CGMCC), which was deposited in 1972 in China general microbiological culture Collection center (CGMCC) with a collection number of CGMCC
1.728. The strain is a model strain, and can be directly purchased from the common microorganism center of the China Committee for culture Collection of microorganisms.
In some embodiments of the invention, the fermentation medium is prepared by: and (3) sterilizing the fermentation substrate at high temperature, cooling, and adding glucose until the final concentration of the glucose is 5-6 wt% to obtain the fermentation medium.
The fermentation medium can cause partial carbon source loss during high-temperature sterilization, and the method helps to ensure that the concentration of glucose in the fermentation medium is more accurate by performing high-temperature sterilization on the fermentation substrate and then adding glucose sterilized by other methods to the preset concentration.
Preferably, the fermentation substrate comprises the following components: 25-35g/L of corn steep liquor, 1.2-1.8g/L of magnesium sulfate heptahydrate, 1.2-1.8g/L of potassium dihydrogen phosphate, 0.8-1.2g/L of dipotassium hydrogen phosphate, 0.8-1.2mg/L of biotin and 0.8-1.2mg/L of thiamine, and the solvent is water.
In some embodiments of the present invention, the fermentation process is performed at 32 ± 0.5 ℃ (e.g., 31.5 ℃, 31.6 ℃, 31.8 ℃, 32 ℃, 32.2 ℃, 32.3 ℃ or 32.5 ℃) and at 0.1 ± 0.02Mpa (e.g., 0.08Mpa, 0.085Mpa, 0.09Mpa, 0.095Mpa, 0.1Mpa, 0.105Mpa, 0.11Mpa, 0.115Mpa or 0.12 Mpa).
In some embodiments of the invention, the initial aeration ratio of the fermentation is 1:0.8 to 1:1 (e.g., can be 1:0.8, 1:0.85, 1:0.9, 1:0.95, or 1:1, etc.) and the stirring speed is 50 to 60 rpm; for example, 50rpm, 52rpm, 53rpm, 55rpm, 56rpm, 58rpm, 60rpm, or the like may be used.
In some embodiments of the invention, ammonia water is fed in the whole fermentation process to maintain the pH of the fermentation liquor at 7.0-7.2; for example, it may be 7.0, 7.02, 7.05, 7.08, 7.1, 7.12, 7.15, 7.18, 7.2, or the like.
In some embodiments of the invention, foam removal is performed by feeding foam remover during the fermentation process.
In some embodiments of the present invention, during the fermentation process, when the dissolved oxygen in the fermentation broth is increased back to 80% or more of the initial dissolved oxygen and the residual reducing sugar content is reduced to 0.5 wt% or less, the feeding of glucose and ammonium sulfate is started, and the aeration ratio and the stirring speed are adjusted to maintain the dissolved oxygen in the fermentation broth at 30-35% (for example, 30%, 31%, 32%, 33%, 34%, 35%, etc.) of the initial dissolved oxygen.
Wherein, glucose and ammonium sulfate can be fed separately in the form of solution, and exemplarily, the concentration of the glucose solution can be 50-55 wt%, and the concentration of the ammonium sulfate solution can be 40-45 wt%.
Preferably, in the fermentation process, the content of residual reducing sugar in the fermentation liquor is maintained to be 1-1.2 wt% by feeding glucose; for example, it may be 1 wt%, 1.02 wt%, 1.05 wt%, 1.08 wt%, 1.1 wt%, 1.12 wt%, 1.15 wt%, 1.18 wt%, 1.2 wt%, or the like.
Preferably, in the fermentation process, ammonium sulfate is fed back to maintain the ammonium nitrogen content in the fermentation liquor at 0.05-0.15 wt%; for example, it may be 0.05 wt%, 0.06 wt%, 0.07 wt%, 0.08 wt%, 0.09 wt%, 0.1 wt%, 0.11 wt%, 0.12 wt%, 0.13 wt%, 0.14 wt%, 0.15 wt%, or the like.
In the present invention, the ammonium nitrogen refers to nitrogen element in ammonium ions, wherein the source of the ammonium ions includes ammonium sulfate and ammonia water for adjusting pH.
In some embodiments of the present invention, when the average accumulated amount of L-arginine in the fermentation broth is less than 1.5g/L per hour, the feeding of glucose and ammonium sulfate is stopped, and the fermentation is terminated after the content of residual reducing sugar in the fermentation broth is reduced to less than 0.5 wt%.
In some embodiments of the invention, the method comprises the steps of:
(1) feeding a sterilized seed medium into an empty seed tank, wherein the seed medium comprises the following components: 25-35g/L of glucose, 15-25g/L of ammonium sulfate, 15-25g/L of corn steep liquor, 0.4-0.6g/L of magnesium sulfate heptahydrate, 1.2-1.8g/L of potassium dihydrogen phosphate and 1.2-1.8g/L of urea, wherein the solvent is water;
inoculating L-arginine producing bacteria with an inoculation amount of 8-12% of the total volume of the L-arginine producing bacteria and the seed culture medium, culturing at 32 + -0.5 deg.C under 0.05 + -0.01 Mpa and at a ventilation ratio of 1:0.8-1:1.2 for 20-24h, wherein OD at 562nm is 5-6, and pH is 7.0-7.2 to obtain a seed solution of the L-arginine producing bacteria;
(2) sterilizing the fermentation substrate at high temperature, cooling, adding into an empty fermentation tank, and adding glucose until the final concentration of glucose is 5-6 wt% to obtain a fermentation culture medium; the fermentation substrate comprises the following components: 25-35g/L of corn steep liquor, 1.2-1.8g/L of magnesium sulfate heptahydrate, 1.2-1.8g/L of monopotassium phosphate, 0.8-1.2g/L of dipotassium phosphate, 0.8-1.2mg/L of biotin and 0.8-1.2mg/L of thiamine, and the solvent is water;
inoculating the seed solution obtained in the step (1) for fermentation, wherein the inoculation amount is 8-12% of the total volume of the seed solution and a fermentation culture medium, the temperature is controlled to be 32 +/-0.5 ℃, the pressure is 0.1 +/-0.02 Mpa, the initial aeration ratio is 1:0.8-1:1, and the initial stirring rotation speed is 50-60 rpm;
in the fermentation process, ammonia water is fed in the whole process to maintain the pH of the fermentation liquor at 7.0-7.2, and foam is removed by feeding foam killer; when the dissolved oxygen in the fermentation liquor rises to more than 80 percent of the initial dissolved oxygen and the content of the residual reducing sugar is reduced to less than 0.5 percent by weight, glucose and ammonium sulfate are fed in a flowing mode, so that the content of the residual reducing sugar in the fermentation liquor is maintained to be 1-1.2 percent by weight, the content of free nitrogen elements is maintained to be 0.05-0.15 percent by weight, and meanwhile, the aeration ratio and the stirring speed are adjusted, so that the dissolved oxygen in the fermentation liquor is maintained to be 30-35 percent of the initial dissolved oxygen;
when the average accumulation amount of the L-arginine in the fermentation liquid is less than 1.5g/L per hour, stopping feeding the glucose and the ammonium sulfate, and ending the fermentation after the content of the residual reducing sugar in the fermentation liquid is reduced to be less than 0.5 wt%.
Compared with the prior art, the invention has the following beneficial effects:
compared with a fermentation process for supplementing raw materials in batches, the method disclosed by the invention has the advantages that glucose and ammonium sulfate are continuously fed in a flowing manner in the process of producing the L-arginine by fermentation, and other process steps and conditions are reasonably matched, so that the fermentation period is less than 65 hours, the conversion rate is more than 27%, the L-arginine content at the fermentation end point is more than 59g/L, and the conversion rate and the L-arginine content at the fermentation end point are effectively improved.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the specific embodiments are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Example 1
The embodiment provides a fermentation production method of L-arginine, which comprises the following steps:
(1) feeding a sterilized seed medium into an empty seed tank, wherein the seed medium comprises the following components: 30g/L glucose, 20g/L ammonium sulfate, 20g/L corn steep liquor, 0.5g/L magnesium sulfate heptahydrate, 1.5g/L potassium dihydrogen phosphate and 1.5g/L urea, wherein the solvent is water;
inoculating corynebacterium glutamicum (with the preservation number of CGMCC 1.728), wherein the inoculation amount is 8% of the total volume of a strain and a seed culture medium, culturing is carried out at the temperature of 32 +/-0.5 ℃, the pressure of 0.05 +/-0.01 MPa and the ventilation ratio of 1:0.8, sampling is carried out once every 4 hours after 12 hours of inoculation (at the moment, a high-power microscope is mainly used for observing whether a seed tank is infected with bacteria or not, a medium-power microscope is used for observing the growth condition of bacteria), after 20 hours of culture, the microscopic examination is carried out, the bacteria are free of foreign bacteria and peculiar smell, the bacteria are robust and full, the OD value at 562nm is 5-6, and the pH is 7.0-7.2, so that the qualified seed solution is obtained;
(2) sterilizing the fermentation substrate at high temperature, cooling, adding into an empty 50L fermentation tank, and adding glucose until the final concentration of glucose is 5.6 wt% to obtain a fermentation culture medium; the fermentation substrate comprises the following components: 30g/L of corn steep liquor, 1.5g/L of magnesium sulfate heptahydrate, 1.5g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 1mg/L of biotin and 1mg/L of thiamine, wherein the solvent is water;
inoculating the seed solution obtained in the step (1) for fermentation, wherein the inoculation amount is 10% of the total volume of the seed solution and a fermentation culture medium, the initial constant volume is 20L, ammonia water is added to adjust the initial pH value to be 7.08, the temperature is controlled to be 32 +/-0.5 ℃, the pressure is 0.1 +/-0.02 Mpa, the initial aeration ratio is 1:0.8, the initial stirring rotating speed is 50rpm, and the initial dissolved oxygen amount is calibrated to be 100%;
in the fermentation process, ammonia water is fed in the whole process to maintain the pH of the fermentation liquor at 7.0-7.2, and foam is removed by feeding foam killer; when the dissolved oxygen in the fermentation liquor rises to more than 80 percent of the initial dissolved oxygen and the content of the residual reducing sugar is reduced to less than 0.5 percent by weight, a 50 percent by weight glucose solution and a 40 percent by weight ammonium sulfate solution are fed in a flowing mode, so that the content of the residual reducing sugar in the fermentation liquor is maintained at 1 to 1.2 percent by weight, the content of ammonium nitrogen is maintained at 0.08 to 0.12 percent by weight, and the aeration ratio and the stirring speed are adjusted at the same time, so that the dissolved oxygen in the fermentation liquor is maintained at 30 to 35 percent of the initial dissolved oxygen;
sampling every two hours after the fermentation time reaches 50 hours, detecting the content of L-arginine in the fermentation liquid, stopping adding glucose and ammonium sulfate in a flowing manner when the increase amplitude of the content of L-arginine in every two hours is less than 3g/L, and ending the fermentation after the content of residual reducing sugar in the fermentation liquid is reduced to be less than 0.5 wt%.
Example 2
The embodiment provides a fermentation production method of L-arginine, which comprises the following steps:
(1) feeding a sterilized seed medium into an empty seed tank, wherein the seed medium comprises the following components: 30g/L glucose, 20g/L ammonium sulfate, 20g/L corn steep liquor, 0.5g/L magnesium sulfate heptahydrate, 1.5g/L potassium dihydrogen phosphate and 1.5g/L urea, wherein the solvent is water;
inoculating corynebacterium glutamicum (with the preservation number of CGMCC 1.728), wherein the inoculation amount is 8% of the total volume of strains and a seed culture medium, culturing is carried out at the temperature of 32 +/-0.5 ℃, the pressure of 0.05 +/-0.01 MPa and the ventilation ratio of 1:1, sampling and microscopic examination are carried out every 4 hours after 12 hours of inoculation (at the moment, a high-power microscope is mainly used for observing whether a seed tank is infected with bacteria or not, a medium-power microscope is used for observing the growth condition of bacteria), after 24 hours of culture, the microscopic examination is free of foreign bacteria and peculiar smell, the bacteria are robust and full, the OD value at 562nm is 5-6, and the pH is 7.0-7.2, so that the qualified seed solution is obtained;
(2) sterilizing the fermentation substrate at high temperature, cooling, adding into an empty 50L fermentation tank, and adding glucose until the final concentration of glucose is 5.0 wt% to obtain a fermentation culture medium; the fermentation substrate comprises the following components: 30g/L of corn steep liquor, 1.5g/L of magnesium sulfate heptahydrate, 1.5g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 1mg/L of biotin and 1mg/L of thiamine, wherein the solvent is water;
inoculating the seed solution obtained in the step (1) for fermentation, wherein the inoculation amount is 10% of the total volume of the seed solution and a fermentation culture medium, the initial constant volume is 20L, ammonia water is added to adjust the initial pH value to be 7.12, the temperature is controlled to be 32 +/-0.5 ℃, the pressure is 0.1 +/-0.02 Mpa, the initial aeration ratio is 1:1, the initial stirring rotation speed is 60rpm, and the initial dissolved oxygen amount is calibrated to be 100%;
in the fermentation process, ammonia water is fed in the whole process to maintain the pH of the fermentation liquor at 7.0-7.2, and foam is removed by feeding foam killer; when the dissolved oxygen in the fermentation liquor rises to more than 80 percent of the initial dissolved oxygen and the content of the residual reducing sugar is reduced to less than 0.5 percent by weight, a 50 percent by weight glucose solution and a 40 percent by weight ammonium sulfate solution are fed in a flowing mode, so that the content of the residual reducing sugar in the fermentation liquor is maintained at 1 to 1.2 percent by weight, the content of ammonium nitrogen is maintained at 0.08 to 0.12 percent by weight, and the aeration ratio and the stirring speed are adjusted at the same time, so that the dissolved oxygen in the fermentation liquor is maintained at 30 to 35 percent of the initial dissolved oxygen;
sampling every two hours after the fermentation time reaches 50 hours, detecting the content of L-arginine in the fermentation liquid, stopping adding glucose and ammonium sulfate in a flowing manner when the increase amplitude of the content of L-arginine in every two hours is less than 3g/L, and ending the fermentation after the content of residual reducing sugar in the fermentation liquid is reduced to be less than 0.5 wt%.
Example 3
The embodiment provides a fermentation production method of L-arginine, which comprises the following steps:
(1) feeding a sterilized seed medium into an empty seed tank, wherein the seed medium comprises the following components: 30g/L glucose, 20g/L ammonium sulfate, 20g/L corn steep liquor, 0.5g/L magnesium sulfate heptahydrate, 1.5g/L potassium dihydrogen phosphate and 1.5g/L urea, wherein the solvent is water;
inoculating corynebacterium glutamicum (with the preservation number of CGMCC 1.728), wherein the inoculation amount is 8% of the total volume of a strain and a seed culture medium, culturing is carried out at the temperature of 32 +/-0.5 ℃, the pressure of 0.05 +/-0.01 MPa and the ventilation ratio of 1:0.9, sampling is carried out once every 4 hours after 12 hours of inoculation (at the moment, a high-power microscope is mainly used for observing whether a seed tank is infected with bacteria or not, a medium-power microscope is used for observing the growth condition of bacteria), after 22 hours of culture, the microscopic examination is carried out, the bacteria are free of foreign bacteria and peculiar smell, the bacteria are robust and full, the OD value at 562nm is 5-6, and the pH is 7.0-7.2, so that the qualified seed solution is obtained;
(2) sterilizing the fermentation substrate at high temperature, cooling, adding into an empty 50L fermentation tank, and adding glucose until the final concentration of glucose is 5.3 wt% to obtain a fermentation culture medium; the fermentation substrate comprises the following components: 30g/L of corn steep liquor, 1.5g/L of magnesium sulfate heptahydrate, 1.5g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 1mg/L of biotin and 1mg/L of thiamine, wherein the solvent is water;
inoculating the seed solution obtained in the step (1) for fermentation, wherein the inoculation amount is 10% of the total volume of the seed solution and a fermentation culture medium, the initial constant volume is 20L, ammonia water is added to adjust the initial pH value to be 7.03, the temperature is controlled to be 32 +/-0.5 ℃, the pressure is 0.1 +/-0.02 Mpa, the initial aeration ratio is 1:0.9, the initial stirring rotating speed is 55rpm, and the initial dissolved oxygen amount is calibrated to be 100%;
in the fermentation process, ammonia water is fed in the whole process to maintain the pH of the fermentation liquor at 7.0-7.2, and foam is removed by feeding foam killer; when the dissolved oxygen in the fermentation liquor rises to more than 80 percent of the initial dissolved oxygen and the content of the residual reducing sugar is reduced to less than 0.5 percent by weight, a 50 percent by weight glucose solution and a 40 percent by weight ammonium sulfate solution are fed in a flowing mode, so that the content of the residual reducing sugar in the fermentation liquor is maintained at 1 to 1.2 percent by weight, the content of ammonium nitrogen is maintained at 0.05 to 0.08 percent by weight, and the aeration ratio and the stirring speed are adjusted at the same time, so that the dissolved oxygen in the fermentation liquor is maintained at 30 to 35 percent of the initial dissolved oxygen;
sampling every two hours after the fermentation time reaches 50 hours, detecting the content of L-arginine in the fermentation liquid, stopping adding glucose and ammonium sulfate in a flowing manner when the increase amplitude of the content of L-arginine in every two hours is less than 3g/L, and ending the fermentation after the content of residual reducing sugar in the fermentation liquid is reduced to be less than 0.5 wt%.
Example 4
The embodiment provides a fermentation production method of L-arginine, which comprises the following steps:
(1) feeding a sterilized seed medium into an empty seed tank, wherein the seed medium comprises the following components: 30g/L glucose, 20g/L ammonium sulfate, 20g/L corn steep liquor, 0.5g/L magnesium sulfate heptahydrate, 1.5g/L potassium dihydrogen phosphate and 1.5g/L urea, wherein the solvent is water;
inoculating corynebacterium glutamicum (with the preservation number of CGMCC 1.728), wherein the inoculation amount is 8% of the total volume of strains and a seed culture medium, culturing is carried out at the temperature of 32 +/-0.5 ℃, the pressure of 0.05 +/-0.01 MPa and the ventilation ratio of 1:1, sampling and microscopic examination are carried out every 4 hours after 12 hours of inoculation (at the moment, a high-power microscope is mainly used for observing whether a seed tank is infected with bacteria or not, a medium-power microscope is used for observing the growth condition of bacteria), after 24 hours of culture, the microscopic examination is free of foreign bacteria and peculiar smell, the bacteria are robust and full, the OD value at 562nm is 5-6, and the pH is 7.0-7.2, so that the qualified seed solution is obtained;
(2) sterilizing the fermentation substrate at high temperature, cooling, putting into an empty 50L fermentation tank, and adding glucose until the final concentration of glucose is 6 wt% to obtain a fermentation medium; the fermentation substrate comprises the following components: 30g/L of corn steep liquor, 1.5g/L of magnesium sulfate heptahydrate, 1.5g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 1mg/L of biotin and 1mg/L of thiamine, wherein the solvent is water;
inoculating the seed solution obtained in the step (1) for fermentation, wherein the inoculation amount is 10% of the total volume of the seed solution and a fermentation culture medium, the initial constant volume is 20L, ammonia water is added to adjust the initial pH value to be 7.2, the temperature is controlled to be 32 +/-0.5 ℃, the pressure is 0.1 +/-0.02 Mpa, the initial aeration ratio is 1:1, the initial stirring rotation speed is 50rpm, and the initial dissolved oxygen amount is calibrated to be 100%;
in the fermentation process, ammonia water is fed in the whole process to maintain the pH of the fermentation liquor at 7.0-7.2, and foam is removed by feeding foam killer; when the dissolved oxygen in the fermentation liquor rises to more than 80 percent of the initial dissolved oxygen and the content of the residual reducing sugar is reduced to less than 0.5 percent by weight, a 50 percent by weight glucose solution and a 40 percent by weight ammonium sulfate solution are fed in a flowing mode, so that the content of the residual reducing sugar in the fermentation liquor is maintained at 1 to 1.2 percent by weight, the content of ammonium nitrogen is maintained at 0.12 to 0.15 percent by weight, and the aeration ratio and the stirring speed are adjusted at the same time, so that the dissolved oxygen in the fermentation liquor is maintained at 30 to 35 percent of the initial dissolved oxygen;
sampling every two hours after the fermentation time reaches 50 hours, detecting the content of L-arginine in the fermentation liquid, stopping adding glucose and ammonium sulfate in a flowing manner when the increase amplitude of the content of L-arginine in every two hours is less than 3g/L, and ending the fermentation after the content of residual reducing sugar in the fermentation liquid is reduced to be less than 0.5 wt%.
Example 5
The embodiment provides a fermentation production method of L-arginine, which comprises the following steps:
(1) feeding a sterilized seed medium into an empty seed tank, wherein the seed medium comprises the following components: 30g/L glucose, 20g/L ammonium sulfate, 20g/L corn steep liquor, 0.5g/L magnesium sulfate heptahydrate, 1.5g/L potassium dihydrogen phosphate and 1.5g/L urea, wherein the solvent is water;
inoculating corynebacterium glutamicum (with the preservation number of CGMCC 1.728), wherein the inoculation amount is 8% of the total volume of strains and a seed culture medium, culturing is carried out at the temperature of 32 +/-0.5 ℃, the pressure of 0.05 +/-0.01 MPa and the ventilation ratio of 1:1, sampling and microscopic examination are carried out every 4 hours after 12 hours of inoculation (at the moment, a high-power microscope is mainly used for observing whether a seed tank is infected with bacteria or not, a medium-power microscope is used for observing the growth condition of bacteria), after 24 hours of culture, the microscopic examination is free of foreign bacteria and peculiar smell, the bacteria are robust and full, the OD value at 562nm is 5-6, and the pH is 7.0-7.2, so that the qualified seed solution is obtained;
(2) sterilizing the fermentation substrate at high temperature, cooling, adding into an empty 50L fermentation tank, and adding glucose until the final concentration of glucose is 5.9 wt% to obtain a fermentation culture medium; the fermentation substrate comprises the following components: 30g/L of corn steep liquor, 1.5g/L of magnesium sulfate heptahydrate, 1.5g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 1mg/L of biotin and 1mg/L of thiamine, wherein the solvent is water;
inoculating the seed solution obtained in the step (1) for fermentation, wherein the inoculation amount is 10% of the total volume of the seed solution and a fermentation culture medium, the initial constant volume is 20L, ammonia water is added to adjust the initial pH value to be 7.2, the temperature is controlled to be 32 +/-0.5 ℃, the pressure is 0.1 +/-0.02 Mpa, the initial aeration ratio is 1:1, the initial stirring rotation speed is 50rpm, and the initial dissolved oxygen amount is calibrated to be 100%;
in the fermentation process, ammonia water is fed in the whole process to maintain the pH of the fermentation liquor at 7.0-7.2, and foam is removed by feeding foam killer; when the dissolved oxygen in the fermentation liquor rises to more than 80 percent of the initial dissolved oxygen and the content of the residual reducing sugar is reduced to less than 0.5 percent by weight, a 50 percent by weight glucose solution and a 40 percent by weight ammonium sulfate solution are fed in a flowing mode, so that the content of the residual reducing sugar in the fermentation liquor is maintained to be 0.5 to 0.7 percent by weight, the content of ammonium nitrogen is maintained to be 0.02 to 0.04 percent by weight, and the aeration ratio and the stirring speed are adjusted at the same time, so that the dissolved oxygen in the fermentation liquor is maintained to be 30 to 35 percent of the initial dissolved oxygen;
sampling every two hours after the fermentation time reaches 50 hours, detecting the content of L-arginine in the fermentation liquid, stopping adding glucose and ammonium sulfate in a flowing manner when the increase amplitude of the content of L-arginine in every two hours is less than 3g/L, and ending the fermentation after the content of residual reducing sugar in the fermentation liquid is reduced to be less than 0.5 wt%.
Example 6
The embodiment provides a fermentation production method of L-arginine, which comprises the following steps:
(1) feeding a sterilized seed medium into an empty seed tank, wherein the seed medium comprises the following components: 30g/L glucose, 20g/L ammonium sulfate, 20g/L corn steep liquor, 0.5g/L magnesium sulfate heptahydrate, 1.5g/L potassium dihydrogen phosphate and 1.5g/L urea, wherein the solvent is water;
inoculating corynebacterium glutamicum (with the preservation number of CGMCC 1.728), wherein the inoculation amount is 8% of the total volume of strains and a seed culture medium, culturing is carried out at the temperature of 32 +/-0.5 ℃, the pressure of 0.05 +/-0.01 MPa and the ventilation ratio of 1:1, sampling and microscopic examination are carried out every 4 hours after 12 hours of inoculation (at the moment, a high-power microscope is mainly used for observing whether a seed tank is infected with bacteria or not, a medium-power microscope is used for observing the growth condition of bacteria), after 24 hours of culture, the microscopic examination is free of foreign bacteria and peculiar smell, the bacteria are robust and full, the OD value at 562nm is 5-6, and the pH is 7.0-7.2, so that the qualified seed solution is obtained;
(2) sterilizing the fermentation substrate at high temperature, cooling, putting into an empty 50L fermentation tank, and adding glucose until the final concentration of glucose is 6 wt% to obtain a fermentation medium; the fermentation substrate comprises the following components: 30g/L of corn steep liquor, 1.5g/L of magnesium sulfate heptahydrate, 1.5g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 1mg/L of biotin and 1mg/L of thiamine, wherein the solvent is water;
inoculating the seed solution obtained in the step (1) for fermentation, wherein the inoculation amount is 10% of the total volume of the seed solution and a fermentation culture medium, the initial constant volume is 20L, ammonia water is added to adjust the initial pH value to be 7.2, the temperature is controlled to be 32 +/-0.5 ℃, the pressure is 0.1 +/-0.02 Mpa, the initial aeration ratio is 1:1, the initial stirring rotation speed is 50rpm, and the initial dissolved oxygen amount is calibrated to be 100%;
in the fermentation process, ammonia water is fed in the whole process to maintain the pH of the fermentation liquor at 7.0-7.2, and foam is removed by feeding foam killer; when the dissolved oxygen in the fermentation liquor rises to more than 80 percent of the initial dissolved oxygen and the content of the residual reducing sugar is reduced to less than 0.5 percent by weight, a 50 percent by weight glucose solution and a 40 percent by weight ammonium sulfate solution are fed in a flowing mode, so that the content of the residual reducing sugar in the fermentation liquor is maintained at 1.3 to 1.5 percent by weight, the content of ammonium nitrogen is maintained at 0.18 to 0.20 percent by weight, and the aeration ratio and the stirring speed are adjusted at the same time, so that the dissolved oxygen in the fermentation liquor is maintained at 30 to 35 percent of the initial dissolved oxygen;
sampling every two hours after the fermentation time reaches 50 hours, detecting the content of L-arginine in the fermentation liquid, stopping adding glucose and ammonium sulfate in a flowing manner when the increase amplitude of the content of L-arginine in every two hours is less than 3g/L, and ending the fermentation after the content of residual reducing sugar in the fermentation liquid is reduced to be less than 0.5 wt%.
Comparative example 1
Provided is a method for the fermentative production of L-arginine, which is different from example 1 in that in step (2), aqueous ammonia is added to adjust the initial pH to 7.2, and then 2L of a 40 wt% ammonium sulfate solution is added; when the dissolved oxygen in the fermentation liquor is increased back to more than 80 percent of the initial dissolved oxygen and the content of the residual reducing sugar is reduced to less than 0.5 percent by weight, supplementing 3L of 50 percent by weight glucose solution and 1.5L of 40 percent by weight ammonium sulfate solution at one time; when the dissolved oxygen amount rises to 80% again, 3L of 50 wt% glucose solution and 1.5L of 40 wt% ammonium sulfate solution are supplemented once again;
sampling every two hours after the fermentation time reaches 50 hours, detecting the content of L-arginine in the fermentation liquid, stopping adding glucose and ammonium sulfate in a flowing manner when the increase amplitude of the content of L-arginine in every two hours is less than 3g/L, and ending the fermentation after the content of residual reducing sugar in the fermentation liquid is reduced to be less than 0.5 wt%.
Comparative example 2
Provided is a fermentation production method of L-arginine, which is different from example 2 in that ammonia water is added in step (2) to adjust the initial pH value to 7.2, and then 2L of 40 wt% ammonium sulfate solution is added; when the dissolved oxygen in the fermentation liquor is increased back to more than 80 percent of the initial dissolved oxygen and the content of the residual reducing sugar is reduced to less than 0.5 percent by weight, supplementing 3L of 50 percent by weight glucose solution and 1.5L of 40 percent by weight ammonium sulfate solution at one time; when the dissolved oxygen amount rises to 80% again, 3L of 50 wt% glucose solution and 1.5L of 40 wt% ammonium sulfate solution are supplemented once again;
sampling every two hours after the fermentation time reaches 50 hours, detecting the content of L-arginine in the fermentation liquid, stopping adding glucose and ammonium sulfate in a flowing manner when the increase amplitude of the content of L-arginine in every two hours is less than 3g/L, and ending the fermentation after the content of residual reducing sugar in the fermentation liquid is reduced to be less than 0.5 wt%.
The fermentation cycles (i.e. the fermentation times at the end of the fermentation) of the above examples and comparative examples were recorded; and (3) measuring the absorbance of the fermentation broth at the position of 525nm by using a spectrophotometer when the fermentation is finished, measuring the content of the L-arginine in the fermentation broth when the fermentation is finished by combining an absorbance-L-arginine concentration standard curve, and calculating the conversion rate, wherein the conversion rate is the mass of the L-arginine in the fermentation broth/the total glucose consumption mass.
The results of the above tests are shown in table 1 below:
TABLE 1
As can be seen from Table 1, the fermentation period of the fermentation production method of L-arginine provided by the embodiment of the invention is below 65h, the L-arginine content at the end of fermentation is above 48g/L, and the conversion rate is above 22%; by controlling the content of residual reducing sugar and ammonium nitrogen in the fermentation liquor to be within the range, the method is favorable for further improving the yield and the conversion rate of the L-arginine, so that the content of the L-arginine at the fermentation end point reaches more than 59g/L, and the conversion rate reaches more than 27%.
Compared with the example 1, the content of residual reducing sugar and the content of free nitrogen element controlled in the fermentation process of the example 5 are lower, most of carbon source and nitrogen source are consumed by the propagation of thalli, and only a small part of carbon source and nitrogen source is converted into thalli metabolite L-arginine, so that the content of L-arginine at the fermentation end point and the conversion rate are obviously reduced; example 6 the controlled content of residual reducing sugar and free nitrogen element in the fermentation process is higher, because the thallus growth has certain dependence on water, the content of residual reducing sugar and free nitrogen element is higher, the growth of thallus cells begins to decrease due to cell dehydration, one or more enzymes influencing product synthesis (which is called carbon catabolite repression) can be repressed, the fermentation period is increased, and the L-arginine content and the conversion rate at the fermentation end point are obviously reduced.
Compared with the examples 1 and 2, the comparative examples 1 and 2 adopt the mode of supplementing glucose and ammonium sulfate in batches, and although the fermentation period is reduced, the L-arginine content and the conversion rate at the fermentation end point are reduced obviously.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A method for producing L-arginine by fermentation, which comprises the following steps:
inoculating the seed liquid of the L-arginine producing strain into a sterilized fermentation culture medium for fermentation, wherein the concentration of glucose in the fermentation culture medium is 5-6 wt%, and feeding glucose and ammonium sulfate in the fermentation process to produce the L-arginine.
2. The method of claim 1, wherein the method of preparing the seed liquid comprises the steps of:
inoculating the L-arginine producing strain into a sterilized seed culture medium, wherein the inoculation amount is 8-12% of the total volume of the strain and the seed culture medium, and culturing for 20-24h under the conditions that the temperature is 32 +/-0.5 ℃, the pressure is 0.05 +/-0.01 Mpa and the aeration ratio is 1:0.8-1:1.2 to obtain a seed solution;
preferably, the seed culture medium comprises the following components: 25-35g/L of glucose, 15-25g/L of ammonium sulfate, 15-25g/L of corn steep liquor, 0.4-0.6g/L of magnesium sulfate heptahydrate, 1.2-1.8g/L of potassium dihydrogen phosphate and 1.2-1.8g/L of urea, wherein the solvent is water;
preferably, the OD value of the seed liquid at 562nm is 5-6, and the pH value is 7.0-7.2.
3. The method of claim 2, wherein the seed solution is inoculated in an amount of 8-12% of the total volume of the seed solution and the fermentation medium.
4. The method according to any one of claims 1 to 3, wherein the L-arginine producing bacterium is Corynebacterium glutamicum (Corynebacterium glutamicum), which was deposited in 1972 at CGMCC (CGMCC), with a collection number of CGMCC 1.728.
5. The method according to any one of claims 1 to 4, wherein the fermentation medium is prepared by: sterilizing the fermentation substrate at high temperature, cooling, and adding glucose until the final concentration of glucose is 5-6 wt% to obtain the fermentation medium;
preferably, the fermentation substrate comprises the following components: 25-35g/L of corn steep liquor, 1.2-1.8g/L of magnesium sulfate heptahydrate, 1.2-1.8g/L of potassium dihydrogen phosphate, 0.8-1.2g/L of dipotassium hydrogen phosphate, 0.8-1.2mg/L of biotin and 0.8-1.2mg/L of thiamine, and the solvent is water.
6. The method according to any one of claims 1 to 5, wherein the temperature is controlled to be 32 ± 0.5 ℃ and the pressure is controlled to be 0.1 ± 0.02MPa during the fermentation;
and/or the initial aeration ratio of the fermentation is 1:0.8-1:1, and the stirring speed is 50-60 rpm.
7. The method according to any one of claims 1 to 6, wherein ammonia is fed in during the fermentation process to maintain the pH of the fermentation broth at 7.0 to 7.2;
and/or adding foam killer during the fermentation process for defoaming.
8. The method according to any one of claims 1 to 7, wherein during the fermentation, when the dissolved oxygen in the fermentation broth rises back to more than 80% of the initial dissolved oxygen and the residual reducing sugar content falls to less than 0.5 wt%, feeding of glucose and ammonium sulfate is started, and the aeration ratio and the stirring speed are adjusted to maintain the dissolved oxygen in the fermentation broth at 30-35% of the initial dissolved oxygen;
preferably, in the fermentation process, the content of residual reducing sugar in the fermentation liquor is maintained to be 1-1.2 wt% by feeding glucose;
preferably, during the fermentation process, ammonium nitrogen content in the fermentation liquor is maintained at 0.05-0.15 wt% by feeding ammonium sulfate.
9. The method according to any one of claims 1 to 8, wherein when the average accumulated amount of L-arginine in the fermentation broth is less than 1.5g/L per hour, the feeding of glucose and ammonium sulfate is stopped, and the fermentation is terminated after the content of residual reducing sugars in the fermentation broth has decreased to less than 0.5 wt%.
10. A method according to any of claims 1-9, characterized in that the method comprises the steps of:
(1) feeding a sterilized seed medium into an empty seed tank, wherein the seed medium comprises the following components: 25-35g/L of glucose, 15-25g/L of ammonium sulfate, 15-25g/L of corn steep liquor, 0.4-0.6g/L of magnesium sulfate heptahydrate, 1.2-1.8g/L of potassium dihydrogen phosphate and 1.2-1.8g/L of urea, wherein the solvent is water;
inoculating L-arginine producing bacteria with an inoculation amount of 8-12% of the total volume of the L-arginine producing bacteria and the seed culture medium, culturing at 32 + -0.5 deg.C under 0.05 + -0.01 Mpa and at a ventilation ratio of 1:0.8-1:1.2 for 20-24h, wherein OD at 562nm is 5-6, and pH is 7.0-7.2 to obtain a seed solution of the L-arginine producing bacteria;
(2) sterilizing the fermentation substrate at high temperature, cooling, adding into an empty fermentation tank, and adding glucose until the final concentration of glucose is 5-6 wt% to obtain a fermentation culture medium; the fermentation substrate comprises the following components: 25-35g/L of corn steep liquor, 1.2-1.8g/L of magnesium sulfate heptahydrate, 1.2-1.8g/L of monopotassium phosphate, 0.8-1.2g/L of dipotassium phosphate, 0.8-1.2mg/L of biotin and 0.8-1.2mg/L of thiamine, and the solvent is water;
inoculating the seed solution obtained in the step (1) for fermentation, wherein the inoculation amount is 8-12% of the total volume of the seed solution and a fermentation culture medium, the temperature is controlled to be 32 +/-0.5 ℃, the pressure is 0.1 +/-0.02 Mpa, the initial aeration ratio is 1:0.8-1:1, and the initial stirring rotation speed is 50-60 rpm;
in the fermentation process, ammonia water is fed in the whole process to maintain the pH of the fermentation liquor at 7.0-7.2, and foam is removed by feeding foam killer; when the dissolved oxygen in the fermentation liquor rises to more than 80 percent of the initial dissolved oxygen and the content of the residual reducing sugar is reduced to less than 0.5 percent by weight, glucose and ammonium sulfate are fed in a flowing mode, so that the content of the residual reducing sugar in the fermentation liquor is maintained to be 1-1.2 percent by weight, the content of free nitrogen elements is maintained to be 0.05-0.15 percent by weight, and meanwhile, the aeration ratio and the stirring speed are adjusted, so that the dissolved oxygen in the fermentation liquor is maintained to be 30-35 percent of the initial dissolved oxygen;
when the average accumulation amount of the L-arginine in the fermentation liquid is less than 1.5g/L per hour, stopping feeding the glucose and the ammonium sulfate, and ending the fermentation after the content of the residual reducing sugar in the fermentation liquid is reduced to be less than 0.5 wt%.
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