CN106148442B - Method for producing gamma-polyglutamic acid by adding leucine in fermentation process - Google Patents
Method for producing gamma-polyglutamic acid by adding leucine in fermentation process Download PDFInfo
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- CN106148442B CN106148442B CN201610825876.9A CN201610825876A CN106148442B CN 106148442 B CN106148442 B CN 106148442B CN 201610825876 A CN201610825876 A CN 201610825876A CN 106148442 B CN106148442 B CN 106148442B
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Abstract
The invention provides a method for producing gamma-polyglutamic acid by adding leucine in a fermentation process, which comprises the following steps: 1) activation of CGMCC3336 strain; 2) preparing a seed solution: 3) fermenting in a fermentation tank, and adding leucine with different concentrations in the fermentation process. The invention can obviously improve the yield of the gamma-polyglutamic acid, and can simultaneously control the proportion of L/D glutamic acid in the gamma-polyglutamic acid, thereby obtaining the gamma-polyglutamic acid with different purposes.
Description
Technical Field
The invention belongs to the technical field of biological agents, and relates to a production method of gamma-polyglutamic acid, in particular to a method for producing gamma-polyglutamic acid by adding leucine in a fermentation process.
Background
Gamma-polyglutamic acid is a multifunctional biodegradable high molecular material formed by combining D-glutamic acid and L-glutamic acid through amido bond between alpha-amino and gamma-carboxyl.
The gamma-polyglutamic acid has excellent water solubility, super-strong adsorbability and biodegradability, the degradation product is pollution-free glutamic acid, the gamma-polyglutamic acid is an excellent environment-friendly high polymer material, can be used as a water-retaining agent, a heavy metal ion adsorbent, a flocculating agent, a slow-release agent, a drug carrier and the like, and has high commercial value and social value in industries such as cosmetics, environmental protection, food, medicine, agriculture, desert control and the like.
At present, the research on the properties of polyglutamic acid mainly comprises the research on the properties of the polyglutamic acid, the improvement and gene research on producing bacteria, the research on a fermentation process and an extraction and purification process, and the research on the production and properties of derivatives. In recent years, due to the enhancement of people's environmental awareness and the requirement of national sustainable development strategy, it is an industrial trend to develop environmentally friendly materials and develop products for improving environmental problems, which also promotes the process of industrial research and exploration of polyglutamic acid.
In research, the gamma-polyglutamic acid with different configuration compositions has different applications, for example, the gamma-polyglutamic acid with higher content of L-glutamic acid is widely applied in the cosmetic industry due to the compatibility of the gamma-polyglutamic acid with the skin, and the gamma-polyglutamic acid with higher content of D-glutamic acid is widely applied in other fields due to the slower degradation of the gamma-polyglutamic acid. In addition, the pollution-free and environment-friendly high polymer materials are increasingly demanded in the current market, and the application range is also increasingly wide. Therefore, in the present stage, it is becoming more and more important to improve the yield of polyglutamic acid and to control the configuration.
Disclosure of Invention
The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. To this end, an object of the present invention is to provide a method for producing gamma-polyglutamic acid by adding leucine during fermentation, which increases precursors during fermentation metabolism by adding leucine to a fermentation broth at a concentration such that the yield of gamma-polyglutamic acid is increased, and controls the configuration of gamma-polyglutamic acid by the addition amount and the addition time.
In one aspect of the present invention, the present invention provides a method for producing gamma-polyglutamic acid by adding leucine during fermentation, comprising the steps of:
1) activation of CGMCC3336 strain: culturing on a solid nutrient-rich medium slant at 37 deg.C for 16 hr to obtain mature slant seed, wherein the solid culture medium comprises: 10g/L of peptone, 5g/L of yeast powder, 10g/L of NaCl, 20g/L of agar and pH 7.2;
2) preparing a seed solution:
transferring a ring of the seeds into a triangular flask filled with a liquid seed culture medium, and culturing at 37 ℃ and 220rpm for 16 hours to logarithmic phase; wherein, the seed liquid culture medium comprises the following components in percentage by weight (g/L): 30 parts of glucose; yeast extract 7; tryptone 10; dipotassium phosphate 0.5; magnesium sulfate 0.5, pH 7.2; 50ml of culture medium is subpackaged in a 500ml triangular flask;
3) fermentation in a fermentation tank:
the components of the fermentation tank culture medium are as follows (g/L): 80 portions of glucose, 80 portions of sodium glutamate, 18 portions of ammonium nitrate, 10 portions of sodium chloride, 0.5 portion of magnesium sulfate, 1.0 portion of calcium chloride, 0.01 portion of ferrous sulfate, 0.44 portion of arginine, 0.26 portion of histidine, 0.5 portion of threonine, 0.4 portion of methionine, 0.5 portion of glutamine, 0.001 portion of pyridoxine and pH 7.2;
fermentation conditions are as follows: the liquid loading amount of a 5L fermentation tank is 3L, the inoculation amount of the seed liquid is 10%, the fermentation temperature is 37 ℃, leucine is added in the fermentation, the ventilation rate is 1.0vvm, the rotation speed is 400rpm, and the initial pH is 7.2.
In addition, the novel dosage form for children according to the above embodiment of the present invention may have the following additional technical features:
in some embodiments of the invention, leucine is added to the fermentation in step (3) at a concentration of 0.2 ~ 1.0.0 g/L.
In some embodiments of the invention, in the fermentation in step (3), the leucine is added for a period of 20 ~ 48 hours of fermentation.
The invention adopts Bacillus licheniformis (Bacillus licheniformis) to produce gamma-polyglutamic acid. The strain source is as follows: the strain used in the invention is a patent applied by Tianjin North ocean Baichuan biotechnology Limited company with the name of a method for producing gamma-polyglutamic acid by an inert carrier solid state fermentation method, and the application number is as follows: 200910228297. the strain is Bacillus licheniformis (Bacillus licheniformis), which is preserved in China general microbiological culture Collection center (CGMCC), and has a strain number of CGMCC 3336.
The invention adopts a high performance liquid chromatograph to measure the concentration of D-glutamic acid and the concentration of L-glutamic acid, and the working conditions of the high performance liquid chromatograph are as follows:
the instrument comprises the following steps: an Agilent1200 high performance liquid chromatograph, which is provided with an ultraviolet visible detector and a workstation;
a chromatographic column: a xylonite CR (+)/(CR-) chiral column;
mobile phase: aqueous perchloric acid solution at pH 2.0;
flow rate: 0.4 mL/min;
column temperature: 25 ℃;
detection wavelength: UV200nm
Sample preparation: and (3) after the fermentation liquor is redissolved by secondary alcohol precipitation, hydrolyzing the fermentation liquor with 6mol/L hydrochloric acid with the same volume at 110 ℃, diluting a sample by a proper multiple by using a mobile phase before hydrolysis, diluting the sample by a proper multiple by using the mobile phase after hydrolysis, filtering the sample by using a 0.22 mu m filter membrane, and analyzing the filtrate by using liquid chromatography.
The invention has the advantages and positive effects that:
the invention can obviously improve the yield of the gamma-polyglutamic acid, and can simultaneously control the proportion of L/D glutamic acid in the gamma-polyglutamic acid, thereby obtaining the gamma-polyglutamic acid with different purposes.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following detailed description, and it should be understood that the described embodiments are a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In one aspect of the present invention, the present invention provides a method for producing gamma-polyglutamic acid by adding leucine during fermentation. The method for producing gamma-polyglutamic acid by adding leucine during fermentation according to an embodiment of the present invention will be described in detail below.
Example (b):
the invention provides a method for producing gamma-polyglutamic acid by adding leucine in a fermentation process, which comprises the following steps:
1) activation of CGMCC3336 strain: culturing on a solid nutrient-rich medium slant at 37 deg.C for 16 hr to obtain mature slant seed, wherein the solid culture medium comprises: 10g/L of peptone, 5g/L of yeast powder, 10g/L of NaCl, 20g/L of agar and pH 7.2;
2) preparing a seed solution:
transferring a ring of the seeds into a triangular flask filled with a liquid seed culture medium, and culturing at 37 ℃ and 220rpm for 16 hours to logarithmic phase; wherein, the seed liquid culture medium comprises the following components in percentage by weight (g/L): 30 parts of glucose; yeast extract 7; tryptone 10; dipotassium phosphate 0.5; magnesium sulfate 0.5, pH 7.2; 50ml of culture medium is subpackaged in a 500ml triangular flask;
3) fermentation in a fermentation tank:
the components of the fermentation tank culture medium are as follows (g/L): 80 portions of glucose, 80 portions of sodium glutamate, 18 portions of ammonium nitrate, 10 portions of sodium chloride, 0.5 portion of magnesium sulfate, 1.0 portion of calcium chloride, 0.01 portion of ferrous sulfate, 0.44 portion of arginine, 0.26 portion of histidine, 0.5 portion of threonine, 0.4 portion of methionine, 0.5 portion of glutamine, 0.001 portion of pyridoxine and pH 7.2;
fermentation conditions are as follows: the liquid loading amount of a 5L fermentation tank is 3L, the inoculation amount of the seed liquid is 10%, the fermentation temperature is 37 ℃, leucine is added in the fermentation, the ventilation rate is 1.0vvm, the rotation speed is 400rpm, and the initial pH is 7.2.
The invention adopts Bacillus licheniformis (Bacillus licheniformis) to produce gamma-polyglutamic acid. The strain source is as follows: the strain used in the invention is a patent applied by Tianjin North ocean Baichuan biotechnology Limited company with the name of a method for producing gamma-polyglutamic acid by an inert carrier solid state fermentation method, and the application number is as follows: 200910228297. the strain is Bacillus licheniformis (Bacillus licheniformis), which is preserved in China general microbiological culture Collection center (CGMCC), and has a strain number of CGMCC 3336.
The invention adopts a high performance liquid chromatograph to measure the concentration of D-glutamic acid and the concentration of L-glutamic acid, and the working conditions of the high performance liquid chromatograph are as follows:
the instrument comprises the following steps: an Agilent1200 high performance liquid chromatograph, which is provided with an ultraviolet visible detector and a workstation;
a chromatographic column: a xylonite CR (+)/(CR-) chiral column;
mobile phase: aqueous perchloric acid solution at pH 2.0;
flow rate: 0.4 mL/min;
column temperature: 25 ℃;
detection wavelength: UV200nm
Sample preparation: and (3) after the fermentation liquor is redissolved by secondary alcohol precipitation, hydrolyzing the fermentation liquor with 6mol/L hydrochloric acid with the same volume at 110 ℃, diluting a sample by a proper multiple by using a mobile phase before hydrolysis, diluting the sample by a proper multiple by using the mobile phase after hydrolysis, filtering the sample by using a 0.22 mu m filter membrane, and analyzing the filtrate by using liquid chromatography.
Control test: the difference from the examples is that leucine is not added during the fermentation process; after fermentation, the yield of the gamma-polyglutamic acid is detected to be 19g/L, and the content ratio of the L-glutamic acid to the D-glutamic acid in the gamma-polyglutamic acid is 1: 1.
example 1
After 24 hours of fermentation, 0.5g/L leucine was added, and the results of the experiment: the yield of the gamma-polyglutamic acid is improved to 22g/L from 19g/L of a control group, and the ratio of L/D configuration is 1.5: 1.
example 2
After 24 hours of fermentation, 1g/L leucine was added, and the results of the experiment: the yield of the gamma-polyglutamic acid is improved to 25g/L from 19g/L of a control group, and the ratio of L/D configuration is 1.8: 1.2.
example 3
Fermentation is carried out for 48 hours, 0.5g/L leucine is added, and the experimental result is as follows: addition of leucine at fermentation hours results: the yield of the gamma-polyglutamic acid is improved to 25g/L from 19g/L of a control group, and the ratio of L/D configuration is 1: 1.8.
example 4
Fermentation is carried out for 48 hours, 1g/L leucine is added, and the experimental result is as follows: the yield of the gamma-polyglutamic acid is improved to 28g/L from 19g/L of a control group, and the ratio of L/D configuration is 1: 2.
finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (2)
1. A method for producing gamma-polyglutamic acid by adding leucine in a fermentation process is characterized by comprising the following steps:
1) activation of CGMCC3336 strain: culturing on a solid nutrient-rich medium slant at 37 deg.C for 16 hr to obtain mature slant seed, wherein the solid culture medium comprises: 10g/L of peptone, 5g/L of yeast powder, 10g/L of NaCl, 20g/L of agar and pH 7.2;
2) preparing a seed solution:
transferring a ring of the seeds into a triangular flask filled with a seed liquid culture medium, and culturing at 37 ℃ and 220rpm for 16 hours to logarithmic phase; wherein the seed liquid culture medium comprises the following components in percentage by weight (g/L): 30 parts of glucose; yeast extract 7; tryptone 10; dipotassium phosphate 0.5; magnesium sulfate 0.5, pH 7.2; 50ml of culture medium is subpackaged in a 500ml triangular flask;
3) fermentation in a fermentation tank:
the components of the fermentation tank culture medium are as follows (g/L): 80 portions of glucose, 80 portions of sodium glutamate, 18 portions of ammonium nitrate, 10 portions of sodium chloride, 0.5 portion of magnesium sulfate, 1.0 portion of calcium chloride, 0.01 portion of ferrous sulfate, 0.44 portion of arginine, 0.26 portion of histidine, 0.5 portion of threonine, 0.4 portion of methionine, 0.5 portion of glutamine, 0.001 portion of pyridoxine and pH 7.2;
fermentation conditions are as follows: the liquid filling amount of a 5L fermentation tank is 3L, the inoculation amount of the seed liquid is 10%, the fermentation temperature is 37 ℃, leucine with the final concentration of 0.2-1.0 g/L is added in 20-48 hours of fermentation, the ventilation amount is 1.0vvm, the rotation speed is 400rpm, and the initial pH is 7.2.
2. The method for producing gamma-polyglutamic acid by adding leucine in a fermentation process according to claim 1, characterized in that, further comprising determining the concentration of D-glutamic acid and the concentration of L-glutamic acid by using high performance liquid chromatography, the working conditions of the high performance liquid chromatography and the preparation of samples:
the instrument comprises the following steps: an Agilent1200 high performance liquid chromatograph, which is provided with an ultraviolet visible detector and a workstation;
a chromatographic column: a xylonite CR (+)/(CR-) chiral column;
mobile phase: aqueous perchloric acid solution at pH 2.0;
flow rate: 0.4 mL/min;
column temperature: 25 ℃;
detection wavelength: UV200 nm;
sample preparation: and (3) after the fermentation liquor is redissolved by secondary alcohol precipitation, hydrolyzing the fermentation liquor with 6mol/L hydrochloric acid with the same volume at 110 ℃, diluting a sample by a proper multiple by using a mobile phase before hydrolysis, diluting the sample by a proper multiple by using the mobile phase after hydrolysis, filtering the sample by using a 0.22 mu m filter membrane, and analyzing the filtrate by using liquid chromatography.
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| CN103525879A (en) * | 2013-10-22 | 2014-01-22 | 天津北洋百川生物技术有限公司 | Method for producing gama-polyglutamic acid by adding hydrogen peroxide in fermentation process |
| CN103613753A (en) * | 2013-11-14 | 2014-03-05 | 天津北洋百川生物技术有限公司 | Method for separating and purifying polyglutamic acid by using additive-free organic solvent |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103525879A (en) * | 2013-10-22 | 2014-01-22 | 天津北洋百川生物技术有限公司 | Method for producing gama-polyglutamic acid by adding hydrogen peroxide in fermentation process |
| CN103613753A (en) * | 2013-11-14 | 2014-03-05 | 天津北洋百川生物技术有限公司 | Method for separating and purifying polyglutamic acid by using additive-free organic solvent |
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