阅读说明：本技术 一种发酵生产α型谷氨酸的方法 (Method for producing α -type glutamic acid by fermentation ) 是由 程士清 尤学波 赵杰 王小平 于 2019-12-16 设计创作，主要内容包括：本发明属于氨基酸生产技术领域,公开了一种优化的谷氨酸发酵培养基,其包括发酵培养基A和发酵培养基B；所述发酵培养基A首先添加,然后间隔12h以上添加发酵培养基B。本发明发酵培养基采用两部分组成,发酵培养基A侧重于菌株增殖的提升,发酵培养基B侧重于谷氨酸的合成和分泌；两种发酵相互配合,提高了谷氨酸产量。(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.)

1. A method for producing α -type glutamic acid by fermentation is characterized by comprising the following steps of inoculating a glutamic acid-producing brevibacterium flavum seed solution into a fermentation tank filled with a fermentation medium A for fermentation culture for 12-24h, adding a fermentation medium B, continuing the fermentation culture for 24-36h, and collecting fermentation liquor.

2. Method according to claim 1, characterized in that it comprises the following steps:

inoculating 8-10% of inoculum size of Brevibacterium flavum for producing glutamic acid into a 100L fermentation tank filled with 60L of fermentation medium A for fermentation culture for 24h, then adding 10L of fermentation medium B, continuing the fermentation culture for 24h, collecting fermentation liquid for producing α type glutamic acid, controlling the fermentation temperature at 30-36 ℃, the ventilation ratio at 1: 0.7-0.9, the stirring speed at 200-.

3. The method according to claim 1 or 2, wherein the fermentation medium a is prepared by: 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.

4. The method according to claim 1 or 2, wherein fermentation medium B is prepared by: 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.

5. The method according to claim 3, wherein the fermentation medium A is prepared by: taking the following raw materials: glucose 50-100g/L, yeast extract 10-30g/L, K₂HPO₄1-5g/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.

6. The method of claim 5, wherein fermentation medium A is prepared by:

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.

7. The method of claim 4, wherein fermentation medium B is prepared by:

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.

8. The method of claim 7, wherein 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.

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 is an acidic amino acid, contains two carboxyl groups in a molecule, has a chemical name of α -aminoglutaric acid, is discovered in 1856 by Lixon, is colorless crystal, has delicate flavor, is slightly soluble in water, is soluble in hydrochloric acid solution, has an isoelectric point of 3.22, is greatly contained in cereal protein, and is also more contained in animal brain, the glutamic acid plays an important role in protein metabolism in organisms, and participates in a plurality of important chemical reactions in animals, plants and microorganisms, sodium glutamate is commonly called monosodium glutamate, is an important flavor enhancer and has an enhancing effect on flavor, the sodium glutamate is widely used in food flavoring agents, can be used independently and can be used together with other amino acids and the like in food, has a flavor enhancing effect, the concentration of the sodium glutamate in the food is 0.2-0.5%, the daily intake of 0-120 micrograms/kg (calculated by the glutamic acid) is allowed for each person, and the general dosage of 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

Based on the existing fermentation medium, the applicant continuously improves the characteristics of microbial fermentation to improve the fermentation efficiency, and accordingly, proposes a method for producing α -type glutamic acid by fermentation.

The invention is realized by the following technical scheme:

a method for producing α -type glutamic acid by fermentation is characterized in that the method comprises the following steps of inoculating a strain seed solution for producing glutamic acid into a fermentation tank filled with a fermentation culture medium A for fermentation culture for 12-24h, adding a fermentation culture medium B, continuing the fermentation culture for 24-36h, and collecting fermentation liquor.

Further, the method comprises the steps of:

inoculating 8-10% of inoculum size of Brevibacterium flavum for producing glutamic acid into a 100L fermentation tank filled with 60L of fermentation medium A for fermentation culture for 24h, then adding 10L of fermentation medium B, continuing the fermentation culture for 24h, collecting fermentation liquid for producing α type glutamic acid, controlling the fermentation temperature at 30-36 ℃, the ventilation ratio at 1: 0.7-0.9, the stirring speed at 200-.

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 the following raw materials: glucose 50-100g/L, yeast extract 10-30g/L, K₂HPO₄1-5g/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, 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.

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 components of the cell wall of phosphatidylethanolamine, so that the proliferation rate of the strain is increased, the later strain proliferation is slow, acid production is mainly performed, and 2-hydroxyethylamine can also be used as a cationic surfactant, so that the cell wall is loosened, the cell permeability is improved, and glutamic acid is promoted to be released to the outside of the cell;

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 period of fermentation, the proliferation speed of the strain is slowed down, acid production is taken as the main part, amino on chitosan is combined with teichoic acid or lipopolysaccharide with negative charges in the bacterial cell wall, and metal cations are chelated, so that the permeability of the cell wall is changed, and the glutamic acid is promoted to be secreted out of cells.

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.