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CN-122012641-A - Special culture medium for glutamic acid fermentation in low pH environment and preparation method and application thereof

CN122012641ACN 122012641 ACN122012641 ACN 122012641ACN-122012641-A

Abstract

The invention discloses a special culture medium for glutamic acid fermentation in a low pH environment, and a preparation method and application thereof, and relates to the technical field of biological fermentation and microbial culture. The culture medium comprises a carbon source compounded by glucose and sorbitol, a nitrogen source compounded by liquid ammonia and double-enzyme hydrolyzed corn steep liquor, inorganic salts and microelements with specific compositions, and a glycine-citric acid buffer system. According to the invention, through systematically reconstructing the carbon source, the nitrogen source, the inorganic salt, the trace elements and the buffer system, the provided culture medium can effectively meet the metabolic demand of corynebacterium glutamicum under the acidic stress of pH 4.8-5.5, and the problems of carbon-nitrogen source utilization disorder, key enzyme activity inhibition and intracellular metabolic flow unbalance caused by pH reduction are remarkably relieved, so that the efficient fermentation under the low pH condition is realized.

Inventors

  • ZHAO CHUNXIAO
  • YOU XUEBO
  • KAN JINGYU
  • CHENG YUNHUA
  • SHI ZHIGANG
  • ZHANG YUHONG
  • GAO LEI

Assignees

  • 内蒙古阜丰生物科技有限公司

Dates

Publication Date
20260512
Application Date
20251216

Claims (9)

  1. 1. A special culture medium for glutamic acid fermentation in a low pH environment is characterized by comprising the following components: the carbon source is formed by compounding glucose and sorbitol according to the mass ratio of 70-90:10-30; the nitrogen source is formed by compounding liquid ammonia and corn steep liquor in a mass ratio of 40-60:40-60, wherein the corn steep liquor is obtained by jointly hydrolyzing corn steep liquor by acid protease and phytase; inorganic salts, including 1.5-3.0 g/L of monopotassium phosphate, 0.4-0.8 g/L of magnesium sulfate, 0.8-1.5 g/L of potassium chloride and 0.5-1.2 g/L of sodium citrate; microelements including 0.005-0.015 g/L of ferrous sulfate, 0.002-0.006 g/L of zinc sulfate, 0.001-0.004 g/L of manganese chloride and 0.0005-0.0015 g/L of sodium molybdate; The buffer regulator is glycine-citric acid composite buffer solution, the total concentration is 8-15 g/L, and the molar ratio of glycine to citric acid is 1-2:1.
  2. 2. The special culture medium for glutamic acid fermentation under the low-pH environment according to claim 1, wherein the preparation method of the corn steep liquor hydrolysate is characterized in that corn steep liquor is hydrolyzed by combining acid protease and phytase, the hydrolysis condition is that the pH is 4.5-5.5, the temperature is 45-55 ℃, and the hydrolysis time is 2-4 hours.
  3. 3. The special culture medium for glutamic acid fermentation under low pH environment according to claim 2, wherein when the corn steep liquor is prepared, the initial solid content of the corn steep liquor is 10% -15%, the addition amount of acid protease is 0.8-1.5U per gram of dry matter, the addition amount of phytase is 20-40U per gram of dry matter, and the pH is regulated to be at a set value by dilute hydrochloric acid or ammonia water in the hydrolysis reaction process.
  4. 4. A method for preparing the special culture medium according to any one of claims 1 to 3, comprising the steps of: s1, glucose and sorbitol are dissolved in water according to a proportion, corn steep liquor hydrolysate is added, and the mixture is mixed and sterilized to obtain basic carbon-nitrogen solution; S2, preparing inorganic salt mother liquor, namely respectively weighing monopotassium phosphate, magnesium sulfate, potassium chloride and sodium citrate, dissolving the monopotassium phosphate, the magnesium sulfate, the potassium chloride and the sodium citrate by deionized water, fixing the volume to the required concentration, filtering and sterilizing; s3, preparing microelement mother liquor, namely respectively weighing ferrous sulfate, zinc sulfate, manganese chloride and sodium molybdate, dissolving with deionized water, and filtering and sterilizing after constant volume; S4, preparing glycine-citric acid buffer regulator solution, regulating the pH value to 5.2, and filtering and sterilizing; and S5, cooling the sterilized basic carbon-nitrogen solution, sequentially adding the inorganic salt mother solution, the buffer regulator solution and the trace element mother solution, and uniformly mixing.
  5. 5. The method according to claim 4, wherein the trace element mother liquor is prepared in step S3, each trace element is dissolved by deionized water containing 0.1% of diluted hydrochloric acid, and ferrous sulfate is pre-complexed with sodium citrate.
  6. 6. The use of a special culture medium according to any one of claims 1-3 for the fermentative production of glutamic acid by corynebacterium glutamicum, characterized in that the pH is controlled to be 4.8-5.5 during the fermentation, the fermentation temperature is 32-34 ℃, and liquid ammonia is used as nitrogen source and pH regulator.
  7. 7. The use according to claim 6, wherein no external pH adjustment is performed during fermentation, and liquid ammonia is fed when the pH of the system is below 4.8, so that the pH is raised to above 5.0.
  8. 8. The use according to claim 6, wherein the fermentation period is 36-48 hours.
  9. 9. The use according to claim 6, wherein the corynebacterium glutamicum is a conventional strain of production which has not been subjected to acid-resistant mutagenesis, and no exogenous growth factors or vitamins are added during fermentation.

Description

Special culture medium for glutamic acid fermentation in low pH environment and preparation method and application thereof Technical Field The invention relates to the technical field of biological fermentation and microbial culture, in particular to a special culture medium for glutamic acid fermentation in a low pH environment, and a preparation method and application thereof. Background Corynebacterium glutamicum (Corynebacterium glutamicum) is the main strain for industrial fermentation production of glutamic acid at present, and the fermentation process is usually carried out under neutral or weak acidic conditions so as to achieve higher acid production efficiency and conversion rate. However, fermentation under low pH conditions can reduce the risk of contamination, reduce the amount of neutralizing agent used in the subsequent extraction process, and is expected to simplify the wastewater treatment process, but the acidic environment can significantly inhibit the normal growth and metabolism of the thalli, resulting in a hindered glutamate synthesis pathway, reduced production strength and increased raw material consumption. In the traditional glutamic acid fermentation process, the formula design of the culture medium is mainly optimized for the conventional pH range (usually pH 6.8-7.2). When the pH of the fermentation system is reduced, the absorption and utilization efficiency of the carbon source, the nitrogen source and the inorganic salt by the thalli can be obviously changed, and the original culture medium components often cannot meet the physiological and metabolic demands of the thalli in an acidic environment. The method is characterized in that the conversion rate of the carbon source is reduced, the nitrogen source is not fully utilized, the activity of key enzyme is inhibited, the intracellular metabolic flow is unbalanced, the yield of glutamic acid is finally reduced, and the raw material consumption of unit product is obviously increased. Currently, for glutamic acid fermentation in a low pH environment, a systematic special culture medium is not available in the industry. The prior art focuses on obtaining acid-resistant strains through strain mutation breeding, or relieving acid stress through process adjustment such as feed supplement strategy, pH sectional control and the like, but fails to fundamentally solve the problem that the components of a culture medium are not matched with the metabolic demands of thalli under low pH. Particularly, the selection of carbon and nitrogen source types, proportion collocation and the synergistic effect of inorganic salt ions have key effects on maintaining the stability of cell membranes, energy supply and precursor accumulation in an acidic environment, but related targeted researches are still lacking. The invention patent application CN110878325A discloses an optimized glutamic acid fermentation medium, which comprises a fermentation medium A and a fermentation medium B, wherein the fermentation medium A is added firstly, and then the fermentation medium B is added at intervals of more than 12 hours. According to the application, culture mediums with different functions are added in stages, so that the strain is emphasized in the early stage of fermentation, and the glutamic acid synthesis is emphasized in the later stage, and the yield is improved. However, this approach does not take into account the solubility of the nutrient components, the ion balance and the change in the transmembrane transport capacity of the cells in the low pH fermentation environment, and the composition of the culture medium used is still designed based on conventional pH conditions, which may cause precipitation of part of the nutrient elements or reduced bioavailability under acidic conditions, limiting its applicability in low pH fermentation. The invention patent application CN115404248A discloses a glutamic acid fermentation method without adding inorganic phosphorus, which is characterized in that protease and phytase are added into corn steep liquor for double enzymolysis, and phytic acid is hydrolyzed into inorganic phosphorus which can be directly utilized by microorganisms, so that the addition of exogenous phosphate is replaced. The method realizes the self-sufficiency of the phosphorus source under the conventional pH condition, and reduces the cost. However, in low pH environments, phytase activity may be inhibited, resulting in incomplete hydrolysis of the phytic acid, which in turn affects the efficient release of phosphorus. Therefore, the development of the special culture medium which can adapt to the low pH fermentation environment, effectively relieve the metabolic inhibition and remarkably improve the utilization efficiency of the raw materials has important practical significance for promoting the glutamic acid fermentation industry to develop towards more energy conservation, emission reduction and high efficiency. The invention aims to