CN110878325B - Optimized glutamic acid fermentation medium - Google Patents
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Abstract
The invention belongs to the technical field of amino acid production, and discloses an optimized glutamic acid fermentation medium which comprises a fermentation medium A and a fermentation medium B; the fermentation medium A is added firstly, and then the fermentation medium B is added at an interval of more than 12 h. The fermentation medium of the invention consists of two parts, wherein the fermentation medium A emphasizes on the improvement of the proliferation of strains, and the fermentation medium B emphasizes on the synthesis and the secretion of glutamic acid; the two fermentations are mutually matched, and the yield of the glutamic acid is improved.
Description
Technical Field
The invention belongs to the technical field of amino acid production, and particularly relates to an optimized glutamic acid fermentation medium.
Background
Glutamic acid, an acidic amino acid. The molecule contains two carboxyl groups, and the chemical name of the molecule is alpha-aminoglutaric acid. Glutamic acid was found in ryxon 1856 as a colorless crystal, umami-tasting, slightly soluble in water, and soluble in hydrochloric acid solution with an isoelectric point of 3.22. It is abundant in cereal protein, and is also abundant in animal brain. Glutamate plays an important role in protein metabolism in organisms, and is involved in many important chemical reactions in animals, plants, and microorganisms. Sodium glutamate, commonly known as monosodium glutamate, is an important flavoring agent and has an enhancing effect on flavor. Sodium glutamate is widely used as a food flavoring agent, and can be used alone or in combination with other amino acids. It can be used in food for flavoring. The concentration of the food is 0.2% -0.5%, and the daily intake of each person is 0-120 microgram/kg (calculated as glutamic acid). The amount is generally 0.2-1.5 g/kg in food processing.
The glutamic acid is mainly prepared by microbial fermentation, and the proliferation rate of thalli is improved and the yield of amino acid can be improved by optimizing a fermentation culture medium. In the prior art, a great deal of research is carried out on the optimization of a glutamic acid fermentation medium, for example, in document 1, "the fermentation medium of corynebacterium glutamicum CNl021 is optimized based on a PB test and a response surface analysis method, and in 2014, the fermentation medium composition is optimized by a steepest-grade test and a response surface analysis method to obtain an optimal fermentation medium group of corynebacterium glutamicum, and the acid yield of the fermentation medium is increased by 22.75% compared with that of a fermentation medium of an unoptimized medium. Document 2 "influence of mixed rare earth nitrates on glutamic acid-producing bacteria, amino acid journal" found that addition of a certain amount of rare earth elements to a fermentation medium can increase the yield of glutamic acid. The prior patent technology of the applicant, namely 'a preparation method of a glutamic acid fermentation culture medium', is improved on the basis of a conventional culture medium, and a yeast extract nitrogen source is replaced by adding a mycoprotein extract, so that the cost is saved, the amino acid fermentation yield can be improved, and two purposes are achieved.
Disclosure of Invention
On the basis of the existing fermentation medium, the applicant continuously improves the characteristics of microbial fermentation to improve the fermentation efficiency, and accordingly, provides an optimized glutamic acid fermentation medium.
The invention is realized by the following technical scheme:
an optimized glutamic acid fermentation medium comprises a fermentation medium A and a fermentation medium B; the fermentation medium A is added firstly, and then the fermentation medium B is added at an interval of more than 12 h.
Further, the preparation method of the fermentation medium A comprises the following steps: taking the following raw materials: glucose, Yeast extract, K2HPO4，MgSO4·7H2O, 2-hydroxyethylamine, CeCl3，MnSO4·H2O，FeSO4·7H2O，VB1Biotin; stirring the raw materials uniformly, adjusting the pH value, and sterilizing to obtain the fermentation medium A.
Further, the preparation method of the fermentation medium B comprises the following steps: taking the following raw materials: succinic acid, urea, chitosan; and (3) uniformly stirring all the raw materials, adjusting the pH value, and sterilizing to obtain a fermentation medium B.
Preferably, the preparation method of the fermentation medium A comprises the following steps: taking each raw material: glucose 50-100g/L, yeast extract 10-30g/L, K2HPO4 1-5g/L，MgSO4·7H2O20-200 mg/L, 2-hydroxyethylamine 10-50mg/L, CeCl31-20mg/L，MnSO4·H2O 1-10mg/L，FeSO4·7H2O 1-10mg/L，VB15-50mg/L, biotin 1-10 mug/L; stirring the raw materials uniformly, adjusting pH to 6-7, sterilizing at 121 deg.C, and naturally cooling to obtain fermentation culture medium A.
More preferably, the preparation method of the fermentation medium A comprises the following steps:
taking the following raw materials: 80g/L glucose, 20g/L yeast extract, K2HPO4 2g/L，MgSO4·7H2O50 mg/L, 2-hydroxyethylamine 40mg/L, CeCl310mg/L，MnSO4·H2O 3mg/L，FeSO4·7H2O 3mg/L，VB110mg/L, biotin 7 mu g/L; stirring the raw materials uniformly, adjusting pH to 6.5, sterilizing at 121 deg.C for 15min, and naturally cooling to obtain fermentation culture medium A.
Preferably, the preparation method of the fermentation medium B comprises the following steps:
taking the following raw materials: 1-10g/L of succinic acid, 1-5g/L of urea and 20-100mg/L of chitosan; stirring the raw materials uniformly, adjusting the pH value, and sterilizing to obtain a fermentation medium B;
more preferably, the preparation method of the fermentation medium B comprises the following steps:
taking the following raw materials: 5g/L of succinic acid, 2g/L of urea and 80mg/L of chitosan; stirring the raw materials uniformly, adjusting pH to 6.5, sterilizing at 121 deg.C for 15min, and naturally cooling to obtain fermentation medium B.
Compared with the prior art, the invention has the advantages that the following aspects are mainly included but not limited:
the fermentation medium of the invention consists of two parts, wherein the fermentation medium A emphasizes on the improvement of the proliferation of strains, and the fermentation medium B emphasizes on the synthesis and the secretion of glutamic acid;
during early cell proliferation, 2-hydroxyethylamine can promote synthesis of phosphatidylethanolamine cell wall components, so that the proliferation rate of strains is increased, later-stage strain proliferation is slowed down, acid production is mainly performed, and 2-hydroxyethylamine can also be used as a cationic surfactant, so that cell walls are loosened, cell permeability is improved, and glutamic acid is promoted to be released into fermentation liquor;
CeCl3the rare earth salt can promote the proliferation of strains, improve the activity of the related synthetase of the glutamic acid and improve the yield of the glutamic acid; however, the excessive concentration can cause the strains to proliferate and die, and the yield of the glutamic acid is correspondingly reduced;
in the middle and later stages of fermentation, the proliferation speed of the strain is slowed down, mainly acid production, amino on chitosan is combined with teichoic acid or lipopolysaccharide with negative charges in the bacterial cell wall, and cations such as Mg2+, Ca2+ and the like are chelated, so that the permeability of the cell wall is changed, and the secretion of glutamic acid to the outside of cells is promoted.
Succinic acid is added into a fermentation medium, so that the tricarboxylic acid cycle has a certain promotion effect, and the glyoxylate cycle pathway is inhibited, so that the intermediate metabolite flows to the tricarboxylic acid cycle pathway more, and the increase of the glutamic acid yield is promoted.
Drawings
FIG. 1: CeCl3Influence of rare earth salts on the concentration of the bacteria;
FIG. 2: CeCl3Influence of rare earth salts on glutamic acid content;
FIG. 3: influence of 2-hydroxyethylamine on the concentration of the bacteria;
FIG. 4: the effect of 2-hydroxyethylamine on glutamic acid content;
FIG. 5: influence of 2-hydroxyethylamine on the conversion of sugar acids.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present application, the technical solutions in the present application will be clearly and completely described below with reference to specific embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all 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.
Example 1
An optimized glutamic acid fermentation medium comprises a fermentation medium A and a fermentation medium B; the fermentation medium A was added first and then the fermentation medium B was added at 24h intervals.
The preparation method of the fermentation medium A comprises the following steps: taking the following raw materials: 80g/L glucose, 20g/L yeast extract, K2HPO42g/L，MgSO4·7H2O50 mg/L, 2-hydroxyethylamine 40mg/L, CeCl310mg/L，MnSO4·H2O 3mg/L，FeSO4·7H2O 3mg/L，VB110mg/L, biotin 7 mu g/L; stirring the raw materials uniformly, adjusting pH to 6.5, sterilizing at 121 deg.C for 15min, and naturally cooling to obtain fermentation culture medium A;
the preparation method of the fermentation medium B comprises the following steps: taking the following raw materials: 5g/L of succinic acid, 2g/L of urea and 80mg/L of chitosan; stirring the raw materials uniformly, adjusting pH to 6.5, sterilizing at 121 deg.C for 15min, and naturally cooling to obtain fermentation medium B;
adopting a conventional fermentation process: inoculating Brevibacterium flavum GDK-9 with 8% of inoculum size to obtain seed solution (OD)600nm13.5) inoculating into a 100L fermentation tank filled with 60L fermentation medium A for fermentation culture for 24h, then adding 10L fermentation medium B, continuing fermentation culture for 24h, and collecting fermentation liquor; in the whole fermentation culture process, the fermentation temperature is controlled to be 35 ℃, the ventilation ratio is 1: 0.7, the stirring speed is 300r/min, the dissolved oxygen is maintained at 20 percent, glucose with the fed-batch mass percentage concentration of 20 percent is fed-batch to maintain the residual sugar to be not less than 1.0 percent, and the fed-batch defoaming agent is fed-batch for defoaming.
Example 2
An optimized glutamic acid fermentation medium comprises a fermentation medium A and a fermentation medium B; the fermentation medium A was added first and then the fermentation medium B was added at 24h intervals.
The preparation method of the fermentation medium A comprises the following steps: taking the following raw materials: 100g/L glucose, 25g/L yeast extract, K2HPO4 1g/L，MgSO4·7H2O70 mg/L, 2-hydroxyethylamine 20mg/L, CeCl35mg/L，MnSO4·H2O 2mg/L，FeSO4·7H2O 2mg/L，VB1 5mg/L, biotin 5 mu g/L; stirring the raw materials uniformly, adjusting pH to 6.5, sterilizing at 121 deg.C for 15min, and naturally cooling to obtain fermentation culture medium A;
the preparation method of the fermentation medium B comprises the following steps: taking the following raw materials: 7g/L of succinic acid, 2g/L of urea and 50mg/L of chitosan; stirring the raw materials uniformly, adjusting pH to 6.5, sterilizing at 121 deg.C for 15min, and naturally cooling to obtain fermentation medium B;
adopting a conventional fermentation process: inoculating Brevibacterium flavum GDK-9 with 8% of inoculum size to obtain seed solution (OD)600nm13.5) inoculating into a 100L fermentation tank filled with 60L fermentation medium A for fermentation culture for 24h, then adding 10L fermentation medium B, continuing fermentation culture for 24h, and collecting fermentation liquor; in the whole fermentation culture process, the fermentation temperature is controlled to be 35 ℃, the ventilation ratio is 1: 0.7, the stirring speed is 300r/min, the dissolved oxygen is maintained at 20 percent, glucose with the fed-batch mass percentage concentration of 20 percent is fed-batch to maintain the residual sugar to be not less than 1.0 percent, and the fed-batch defoaming agent is fed-batch for defoaming.
Example 3
Mono, CeCl3Influence of rare earth salts on thallus concentration, glutamic acid content and saccharic acid conversion rate.
The fermentation medium is as follows: 80g/L glucose, 20g/L yeast extract, K2HPO4 2g/L，MgSO4·7H2O 50mg/L，CeCl30-40mg/L，MnSO4·H2O 3mg/L，FeSO4·7H2O 3mg/L，VB110mg/L, biotin 7 mu g/L;
the fermentation process was the same as in example 1, with 70L of fermentation medium in a 100L fermentor.
Setting up CeCl3Is added at a concentration of 0, 2.5,5,10,20,40mg/L, as shown in FIGS. 1-2, with CeCl3The addition amount is increased, the thallus concentration and the glutamic acid content are both improved, when the addition amount is 10mg/L, the thallus concentration and the glutamic acid content reach peak values, then a descending trend appears, but the saccharic acid conversion rate is not obviously changed in the whole process (not shown in the attached drawing); description of CeCl3The rare earth salt can promote the proliferation of the bacterial strain, improve the activity of the relative synthetase of the glutamic acid,the yield of the glutamic acid is improved, but the excessive concentration can cause the strain to proliferate and die, and the yield of the glutamic acid is correspondingly reduced.
Second, the determination of CeCl by the above experiment3The effect of 2-hydroxyethylamine on the cell concentration, glutamic acid content and conversion rate of saccharic acid was investigated on the basis of the addition amount of 10 mg/L. Setting the concentration of 2-hydroxyethylamine to be 2.5,5,10,20,40, 80 and 160mg/L, as shown in fig. 3-4, increasing the thallus concentration with the increase of the addition amount of 2-hydroxyethylamine, correspondingly increasing the glutamic acid content and the saccharic acid conversion rate, when the addition amount is 40mg/L, the thallus concentration and the glutamic acid content reach peak values, continuously increasing the concentration of 2-hydroxyethylamine, generating obvious bacteriostasis effect and obviously reducing the density of the strain; the reason is that the 2-hydroxyethylamine with low concentration can promote the synthesis of phosphatidylethanolamine cell wall components, so that the proliferation rate of the strain is improved, but the bacterial inhibition phenomenon can be caused due to the excessive concentration, the proliferation of the strain is slowed down in the later period, the acid production is taken as a main part, and the 2-hydroxyethylamine can also be used as a cationic surfactant, so that the cell wall is loosened, the cell permeability is improved, the release of glutamic acid to fermentation liquor is promoted, and the yield of glutamic acid and the conversion rate of saccharic acid are improved.
Thirdly, the determination of CeCl by the above experiment3The influence of the fermentation medium B on the cell concentration, glutamic acid content and sugar-acid conversion rate was investigated on the basis of 10mg/L and 40mg/L of 2-hydroxyethylamine.
The control group 1 was fermented with fermentation medium A without fermentation medium B, and a 100L fermenter contained 70L fermentation medium A; the fermentation process is referred to example 1.
Control group 2: the fermentation medium B was the same as in example 1 except that succinic acid was not added.
Control group 3: the fermentation medium B was the same as in example 1 except that chitosan was not added.
Control group 4: 5g/L succinic acid was added to the fermentation medium A, and the remainder was the same as in control 1.
The experimental group is example 1.
Specific results are shown in table 1.
TABLE 1
| Group of | Bacterial concentration OD600nm | Glutamic acid output g/L | Conversion rate of sugar and acid% |
| Control group 1 | 53.9 | 137.4 | 59.1 |
| Control group 2 | 54.2 | 141.2 | 61.8 |
| Control group 3 | 54.5 | 145.8 | 62.6 |
| Control group 4 | 53.8 | 137.9 | 59.2 |
| Experimental group | 55.3 | 150.7 | 64.1 |
And (4) conclusion: the control group 1 adopts a single fermentation medium for fermentation, the yield of glutamic acid and the conversion rate of saccharic acid are obviously lower than those of the experimental group, and the concentration difference of thalli of each group is not large; the control group 4 is added with succinic acid on the basis of the control group 1, so that the concentration of thalli is not influenced, and the yield of glutamic acid and the conversion rate of saccharic acid are not obviously different, probably because the thalli proliferation is taken as the main part in the early stage of fermentation, the acid production is less, and the succinic acid has no obvious stimulation effect on the thalli proliferation; the experimental group and the control group 3 adopt succinic acid added in the middle of fermentation, at the moment, the proliferation of thalli is slow, acid production is mainly performed, succinic acid has positive promotion effect on tricarboxylic acid cycle and has inhibition effect on glyoxylic acid cycle, so that the yield of glutamic acid is increased; gradient tests show that the addition amount of succinic acid is too large (larger than 10 g/L), the yield of glutamic acid cannot be further improved, comprehensive cost is considered, and the addition amount of succinic acid lower than 10g/L is more suitable. The chitosan is added in the middle and later fermentation stages of the control group 2 and the experimental group, so that the permeability of cell walls can be changed, and the secretion of glutamic acid to the outside of cells is promoted, thereby improving the yield of the glutamic acid and the conversion rate of saccharic acid; however, the excessive addition amount (more than 100 mg/L) of the chitosan can cause the occurrence of bacteriostasis, thereby causing the death of the strain.
Although the invention has been described in detail with respect to the general description and the specific embodiments, it will be apparent to those skilled in the art that modifications and improvements can be made to the invention or the method can be practiced without the specific embodiments. Accordingly, it is intended that all such modifications, improvements and extensions that do not depart from the spirit of the invention, be considered within the scope of the invention as claimed.
Claims (3)
1. An optimized glutamic acid fermentation medium comprises a fermentation medium A and a fermentation medium B; the fermentation medium A is added firstly, and then the fermentation medium B is added at intervals of more than 12 h;
the preparation method of the fermentation medium A comprises the following steps: taking the following raw materials: grape50-100g/L glucose, 10-30g/L yeast extract, K2HPO4 1-5g/L，MgSO4·7H2O20-200 mg/L, 2-hydroxyethylamine 10-50mg/L, CeCl31-20mg/L，MnSO4·H2O 1-10mg/L，FeSO4·7H2O 1-10mg/L，VB1 5-50mg/L, biotin 1-10 mug/L; stirring the raw materials uniformly, adjusting the pH to 6-7, sterilizing at 121 ℃, and naturally cooling to obtain a fermentation medium A;
the preparation method of the fermentation medium B comprises the following steps:
taking the following raw materials: 1-10g/L of succinic acid, 1-5g/L of urea and 20-100mg/L of chitosan; and (3) uniformly stirring all the raw materials, adjusting the pH value, and sterilizing to obtain a fermentation medium B.
2. The glutamic acid fermentation medium according to claim 1, wherein the fermentation medium A is prepared by:
taking the following raw materials: 80g/L glucose, 20g/L yeast extract, K2HPO4 2g/L，MgSO4·7H2O50 mg/L, 2-hydroxyethylamine 40mg/L, CeCl310mg/L，MnSO4·H2O 3mg/L，FeSO4·7H2O 3mg/L，VB1 10mg/L, biotin 7 mu g/L; stirring the raw materials uniformly, adjusting pH to 6.5, sterilizing at 121 deg.C for 15min, and naturally cooling to obtain fermentation culture medium A.
3. The glutamic acid fermentation medium according to claim 1, wherein the fermentation medium B is prepared by:
taking the following raw materials: 5g/L of succinic acid, 2g/L of urea and 80mg/L of chitosan; stirring the raw materials uniformly, adjusting pH to 6.5, sterilizing at 121 deg.C for 15min, and naturally cooling to obtain fermentation medium B.
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| WO1999061645A1 (en) * | 1998-05-28 | 1999-12-02 | Fujisawa Pharmaceutical Co., Ltd. | Novel compound, wf00144 |
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| WO2009062119A3 (en) * | 2007-11-07 | 2009-09-24 | Sustainable Green Technologies, Inc. | Microorganisms and methods for increased hydrogen production |
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Effective date of registration: 20210427 Address after: 162650 the Inner Mongolia Autonomous Region Hulun Buir Lingdong Industrial Development Zone (Zhalantun) a street Applicant after: HULUN BUIR NORTHEAST FUFENG BIOLOGICAL TECHNOLOGY Co.,Ltd. Address before: 021000 Hulunbuir northeast Fufeng Biotechnology Co., Ltd., Kaichuang street, Lingdong Industrial Development Zone, Hulunbuir City, Inner Mongolia Autonomous Region Applicant before: Zhao Lankun |
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