Special culture medium for glutamic acid fermentation in low-pH environment as well as preparation method and application of special culture medium

Abstract

The invention discloses a special culture medium for glutamic acid fermentation in a low-pH environment as well as 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 salt with specific composition, trace elements and a glycine-citric acid buffer system. By systematically reconstructing a carbon source, a nitrogen source, inorganic salt, trace elements and a 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 and nitrogen source utilization obstacle, key enzyme activity inhibition and intracellular metabolic flow imbalance caused by pH reduction are remarkably relieved; therefore, high-efficiency fermentation under the low-pH condition is realized.

Description

Technical Field

[0001] This invention relates to the field of bio-fermentation and microbial culture technology, specifically to a special culture medium for glutamic acid fermentation under low pH conditions, its preparation method, and its application. Background Technology

[0002] Corynebacterium glutamicum is currently the main microbial species used in the industrial fermentation production of glutamic acid. Its fermentation process is typically carried out under neutral or weakly acidic conditions to achieve high acid production efficiency and conversion rate. However, while fermentation under low pH conditions can reduce the risk of contamination, decrease the amount of neutralizing agent used in subsequent extraction processes, and potentially simplify wastewater treatment, the acidic environment significantly inhibits the normal growth and metabolism of the microorganisms, leading to obstructed glutamic acid synthesis pathways, decreased production intensity, and increased raw material consumption.

[0003] In traditional glutamic acid fermentation processes, the culture medium formulation is primarily optimized for a typical pH range (usually pH 6.8–7.2). When the pH of the fermentation system decreases, the efficiency of cell absorption and utilization of carbon sources, nitrogen sources, and inorganic salts changes significantly. The original culture medium components often fail to meet the physiological and metabolic needs of the cells in an acidic environment. Specifically, this manifests as reduced carbon source conversion, insufficient nitrogen source utilization, inhibition of key enzyme activity, and intracellular metabolic imbalance, ultimately leading to a decrease in glutamic acid yield while significantly increasing raw material consumption per unit of product.

[0004] Currently, there is a lack of systematic, dedicated culture media for glutamate fermentation under low pH conditions. Existing technologies mostly focus on obtaining acid-tolerant strains through mutagenesis and selection, or alleviating acid stress through fed-batch strategies and pH segmentation control, but fail to fundamentally address the mismatch between culture medium components and the metabolic needs of the microorganisms under low pH conditions. In particular, the selection and ratio of carbon and nitrogen sources, as well as the synergistic effect of inorganic salt ions, have a crucial impact on maintaining cell membrane stability, energy supply, and precursor accumulation in acidic environments, but relevant targeted research remains insufficient.

[0005] Chinese patent application CN110878325A discloses an optimized glutamic acid fermentation medium, comprising fermentation medium A and fermentation medium B. Fermentation medium A is added first, followed by fermentation medium B at intervals of at least 12 hours. This application increases yield by adding different functional media in stages, focusing on cell proliferation in the early stage of fermentation and glutamic acid synthesis in the later stage. However, this scheme does not consider changes in the solubility of nutrients, ion balance, and transmembrane transport capacity of cells under low pH fermentation conditions. The composition of the culture medium is still designed based on conventional pH conditions, which may cause precipitation of some nutrients or a decrease in bioavailability under acidic conditions, limiting its applicability in low pH fermentation.

[0006] Chinese patent application CN115404248A discloses a method for glutamic acid fermentation without the addition of inorganic phosphorus. This method involves adding protease and phytase to corn steep liquor for dual enzymatic hydrolysis, converting phytic acid into inorganic phosphorus that can be directly utilized by microorganisms, thus replacing the need for exogenous phosphate. This method achieves phosphorus self-sufficiency under conventional pH conditions, reducing costs. However, in low pH environments, phytase activity may be inhibited, leading to incomplete phytic acid hydrolysis and consequently affecting the effective release of phosphorus.

[0007] Therefore, developing a specialized culture medium that can adapt to low-pH fermentation environments, effectively relieve metabolic inhibition, and significantly improve raw material utilization efficiency is of great practical significance for promoting the glutamic acid fermentation industry towards a more energy-efficient, emission-reducing, and high-efficiency direction. This invention aims to support stable and efficient fermentation production of Corynebacterium glutamicum under low pH conditions by systematically reconstructing the culture medium composition and optimizing key nutrients. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide a special culture medium for glutamic acid fermentation under low pH conditions, as well as its preparation method and application. The aim is to adapt the carbon source, nitrogen source and inorganic salt system to the metabolic needs of Corynebacterium glutamicum under acidic stress conditions by directionally reconstructing the system, thereby relieving the problems of nutrient absorption obstacles, key enzyme activity inhibition and intracellular metabolic flow disorder caused by pH reduction, and thus significantly improving the glutamic acid yield and reducing the raw material consumption while maintaining normal cell growth.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] A special culture medium for glutamic acid fermentation under low pH conditions, comprising the following components:

[0011] The carbon source is composed of glucose and sorbitol in a mass ratio of 70-90:10-30.

[0012] The nitrogen source is composed of liquid ammonia and corn steep liquor hydrolysate in a mass ratio of 40-60:40-60, wherein the corn steep liquor hydrolysate is obtained by hydrolyzing corn steep liquor with acidic protease and phytase.

[0013] Inorganic salts, including potassium dihydrogen phosphate 1.5~3.0 g / L, magnesium sulfate 0.4~0.8 g / L, potassium chloride 0.8~1.5 g / L, and sodium citrate 0.5~1.2 g / L;

[0014] Trace elements, including ferrous sulfate 0.005~0.015 g / L, zinc sulfate 0.002~0.006 g / L, manganese chloride 0.001~0.004 g / L, and sodium molybdate 0.0005~0.0015 g / L;

[0015] The buffer regulator is a glycine-citric acid complex buffer with a total concentration of 8-15 g / L, wherein the molar ratio of glycine to citric acid is 1-2:1.

[0016] Furthermore, the preparation method of the corn steep liquor hydrolysate is as follows: the corn steep liquor is hydrolyzed using a combination of acidic protease and phytase, under the following conditions: pH 4.5~5.5, temperature 45~55℃, and hydrolysis time 2~4 hours.

[0017] Furthermore, when preparing corn steep liquor hydrolysate, the initial solids content of the corn steep liquor is 10%~15%, the amount of acidic protease added is 0.8~1.5 U per gram of dry matter, the amount of phytase added is 20~40 U per gram of dry matter, and the pH is adjusted and maintained at the set value using dilute hydrochloric acid or ammonia during the hydrolysis reaction.

[0018] The present invention also provides a method for preparing the special culture medium as described above, comprising the following steps:

[0019] S1: Dissolve glucose and sorbitol in water in a certain proportion, add corn steep liquor hydrolysate, mix and sterilize to obtain a basic carbon-nitrogen solution;

[0020] S2: Preparation of inorganic salt mother liquor: Weigh out potassium dihydrogen phosphate, magnesium sulfate, potassium chloride and sodium citrate respectively, dissolve them in deionized water, make up to the required concentration, and filter to remove bacteria;

[0021] S3: Preparation of trace element stock solution: Weigh ferrous sulfate, zinc sulfate, manganese chloride and sodium molybdate respectively, dissolve them in deionized water, make up to volume and then filter to remove bacteria;

[0022] S4: Prepare a glycine-citric acid buffer solution, adjust the pH to 5.2, and filter to sterilize;

[0023] S5: Cool the sterilized basic carbon-nitrogen solution, then add the inorganic salt mother liquor, buffer regulator solution and trace element mother liquor in sequence, and mix thoroughly.

[0024] Furthermore, the trace elements were dissolved in deionized water containing 0.1% dilute hydrochloric acid, and ferrous sulfate was pre-complexed with sodium citrate.

[0025] On the other hand, the present invention also provides the application of the above-mentioned special culture medium in the fermentation of Corynebacterium glutamicum to produce glutamic acid, wherein the pH is controlled at 4.8~5.5 and the fermentation temperature is 32~34℃ during the fermentation process, and liquid ammonia is used as a nitrogen source and pH adjuster.

[0026] Furthermore, no external pH adjustment is performed during the fermentation process. When the system pH is below 4.8, liquid ammonia is added to raise the pH back to above 5.0.

[0027] Furthermore, the fermentation cycle is 36-48 hours.

[0028] Furthermore, the *Corynebacterium glutamicum* is a conventional production strain that has not undergone acid-resistant mutagenesis, and no exogenous growth factors or vitamins are added during the fermentation process.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) By systematically reconstructing carbon source, nitrogen source, inorganic salt, trace elements and buffer system, the present invention provides a culture medium that can effectively meet the metabolic needs of Corynebacterium glutamicum under acidic stress of pH 4.8~5.5, significantly alleviate the problems of carbon and nitrogen source utilization obstacles, key enzyme activity inhibition and intracellular metabolic imbalance caused by pH reduction, thereby achieving efficient fermentation under low pH conditions.

[0031] (2) In this invention, the carbon source is compounded with sorbitol, which can enhance the stability of cell membrane in acidic environment and reduce the inhibition of glucose metabolism pathway by acid stress. The nitrogen source adopts a compound form of liquid ammonia and double enzyme hydrolysis of corn steep liquor, which provides both fast-acting and slow-acting nitrogen sources. The enzymatic hydrolysis process, which still maintains high efficiency under acidic conditions, ensures the effective release and utilization of key nutrients such as phosphorus and small peptides.

[0032] (3) The types, concentrations and addition methods of inorganic salts and trace elements in this invention are designed to maintain high solubility and bioavailability under low pH conditions, avoiding phosphate precipitation and metal ion hydrolysis; the pre-complexation of iron ions with sodium citrate further enhances their stability in acidic systems, ensuring a continuous supply of key cofactors.

[0033] (4) The present invention uses a glycine-citric acid composite buffer system and is linked with liquid ammonia flow, which not only stabilizes the pH of the fermentation system, but also achieves precise replenishment of nitrogen source without the need for external neutralizing agent, thus reducing the complexity of operation and the consumption of chemical raw materials.

[0034] (5) The special culture medium provided by the present invention supports a glutamic acid yield of not less than 120 g / L, a sugar-acid conversion rate of not less than 86%, a liquid ammonia consumption of less than 120 kg / t, and a glucose consumption of less than 1400 kg / t under low pH conditions. Its comprehensive performance is better than that of conventional neutral and unoptimized low pH fermentation processes.

[0035] (6) The culture medium provided by the present invention is suitable for conventional Corynebacterium glutamicum production strains, does not require acid-resistant mutagenic strains, and can directly enter the extraction process without neutralization after fermentation, simplifying downstream treatment, reducing wastewater treatment load, and has good industrial applicability and economic benefits.

[0036] (7) By reducing the consumption of liquid ammonia and glucose, eliminating the neutralization step, and reducing the discharge of organic wastewater, this invention helps to achieve energy conservation and emission reduction in the glutamic acid fermentation process, which meets the requirements of green manufacturing and sustainable development. Detailed Implementation

[0037] The present invention will be further described below with reference to the embodiments. The embodiments of the present invention include, but are not limited to, the following embodiments.

[0038] Existing glutamate fermentation processes typically operate within a pH range of 6.8–7.2, requiring large amounts of alkali to neutralize metabolic acid production, leading to byproduct accumulation, complex downstream processing, and high raw material consumption. If the fermentation pH is directly lowered to below 5.5, conventional culture media cannot meet the nutrient absorption and metabolic needs of the bacteria under acidic stress, resulting in decreased glucose uptake rates, suppressed activity of key dehydrogenases, and insufficient supply of TCA cycle intermediates, ultimately leading to a significant reduction in glutamate synthesis efficiency. To address these issues, this invention provides a specialized culture medium for glutamate fermentation under low pH conditions, along with its preparation method and applications. By systematically reconstructing the carbon source, nitrogen source, inorganic salts, trace elements, and buffer system, *Corynebacterium glutamicum* maintains a high metabolic flux under pH conditions of 4.8–5.5, achieving high yield and low raw material consumption.

[0039] The culture medium provided by this invention comprises the following components: carbon source, nitrogen source, inorganic salts, trace elements, and buffer regulator;

[0040] The carbon source is composed of glucose and sorbitol in a mass ratio of 70-90:10-30. According to calculations, the mass percentage of sorbitol in the carbon source is 10%-25%. This amount of addition can ensure that sorbitol can effectively stabilize the cell membrane structure without inhibiting the main glucose metabolism pathway due to excessive addition.

[0041] The nitrogen source is composed of liquid ammonia and corn steep liquor hydrolysate in a mass ratio of 40-60:40-60. The corn steep liquor hydrolysate is obtained by hydrolyzing corn steep liquor with acidic protease and phytase for 2-4 hours, with a hydrolysis pH of 4.5-5.5 and a temperature of 45-55℃.

[0042] Inorganic salts include 1.5–3.0 g / L potassium dihydrogen phosphate, 0.4–0.8 g / L magnesium sulfate, 0.8–1.5 g / L potassium chloride, and 0.5–1.2 g / L sodium citrate;

[0043] Trace elements include 0.005~0.015 g / L ferrous sulfate, 0.002~0.006 g / L zinc sulfate, 0.001~0.004 g / L manganese chloride, and 0.0005~0.0015 g / L sodium molybdate;

[0044] The buffer regulator is a glycine-citric acid complex buffer with a total concentration of 8-15 g / L, wherein the molar ratio of glycine to citric acid is 1-2:1.

[0045] In some embodiments, the preparation method of corn steep liquor hydrolysate specifically involves: the initial solids content of the corn steep liquor is 10%~15%, followed by the addition of acidic protease and phytase for hydrolysis; wherein the amount of acidic protease added is 0.8~1.5 U / g dry matter, and the amount of phytase added is 20~40 U / g dry matter. The hydrolysis reaction is carried out in a jacketed stainless steel reactor, with circulating water circulating through the jacket for temperature control. During the reaction, the pH is adjusted and maintained at the set value using 1 mol / L hydrochloric acid or ammonia.

[0046] In some implementations, the initial pH of the culture medium is adjusted to 5.0–5.5, and no external pH adjustment is performed during fermentation; the system pH is maintained at no lower than 4.8 solely by liquid ammonia feeding. Liquid ammonia serves as both a nitrogen source and a pH adjuster, achieving a dual-function integration.

[0047] The present invention also provides a method for preparing the above-mentioned special culture medium, comprising the following steps:

[0048] S1: Prepare the basic carbon-nitrogen solution by dissolving glucose and sorbitol in deionized water in a specific ratio, heating to 50-60°C and stirring until completely dissolved, cooling to room temperature, and then adding corn steep liquor hydrolysate treated with dual enzymes and mixing thoroughly. The total concentration of glucose and sorbitol in this step is 140-160 g / L, and the amount of corn steep liquor hydrolysate added is 8-12 g / L (on a dry matter basis). The basic carbon-nitrogen solution is sterilized by moist heat sterilization at 121°C for 30 minutes, followed by rapid cooling to prevent caramelization of the sugars.

[0049] S2: Prepare inorganic salt stock solutions by weighing potassium dihydrogen phosphate, magnesium sulfate, potassium chloride, and sodium citrate separately, dissolving them in deionized water, and bringing the volume to the required concentration. Filter to remove bacteria. These inorganic salt stock solutions should be prepared by dissolving them separately in individual bottles and mixing them according to the specified ratio before use to avoid precipitation caused by the coexistence of phosphate ions with calcium and magnesium ions. Each stock solution should be 10 times the working concentration and stored at 4℃ for later use.

[0050] S3: Prepare the trace element stock solution. Weigh out ferrous sulfate, zinc sulfate, manganese chloride, and sodium molybdate, and dissolve them in deionized water containing 0.1% dilute hydrochloric acid to prevent the hydrolysis and precipitation of metal ions. After bringing the solution to volume, filter to remove bacteria. Before preparing the trace element stock solution, pre-form a complex between ferrous sulfate and sodium citrate to enhance the stability of iron ions at low pH. Specifically, dissolve sodium citrate in 0.1% dilute hydrochloric acid, then add ferrous sulfate and stir for 30 minutes to ensure complete complexation. Finally, add the remaining trace elements.

[0051] S4: Prepare a buffer solution by dissolving glycine and citric acid in deionized water at a molar ratio, adjusting the pH to 5.2, and then filtering to remove bacteria.

[0052] S5: After sterilizing the basic carbon-nitrogen solution obtained in step S1, cool it to 35~40℃. Under aseptic conditions, first add the inorganic salt mother liquor, then add the buffer regulator solution, and finally add the trace element mother liquor to prevent metal ions from combining with organic acids too early to form insoluble salts. After the solutions are mixed evenly, the special culture medium is obtained.

[0053] S6: Introduce sterile air or nitrogen into the dedicated culture medium to adjust dissolved oxygen to 20%–30% saturation. Inoculate with *Corynebacterium glutamicum* seed culture, control the fermentation temperature at 32–34°C, and maintain a stirring speed of 300–500 rpm for low-pH fermentation. Liquid ammonia is used as the sole nitrogen source during fermentation. The feed rate is controlled via online pH feedback. When the pH drops below 4.8, liquid ammonia feeding is initiated, with the feed rate controlled at 0.5–2.0 L / h·m³. 3 The fermentation broth was stopped once the pH rose to 5.0. The liquid ammonia storage tank is equipped with a mass flow meter and a pneumatic regulating valve to achieve precise feed.

[0054] When using the above-mentioned special culture medium to produce glutamic acid by low-pH fermentation of Corynebacterium glutamicum, the pH is 4.8~5.5, the fermentation cycle is 36~48 hours, the glutamic acid yield at the end of fermentation is not less than 120 g / L, the sugar-acid conversion rate is not less than 86%, the liquid ammonia consumption is less than 120 kg / t, and the glucose consumption is less than 1400 kg / t glutamic acid.

[0055] The production strain used in this invention is the conventional production strain *Corynebacterium glutamicum*. This strain has not undergone acid-resistant mutagenesis, and no exogenous growth factors or vitamins are added during fermentation; all nutrients are provided by the culture medium components. After fermentation, the fermentation broth is centrifuged to remove the bacterial cells, and the supernatant directly enters the isoelectric point crystallization process without neutralization. The crystallization mother liquor is nanofiltration to recover residual sugars and then reused for preparing new batches of culture medium.

[0056] The above-mentioned special culture medium is suitable for 50 L~200 m 3 The fermenters are of a certain scale, with a liquid filling coefficient of 0.7 to 0.85. The following specific embodiments and comparative examples are provided only to explain this application and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments or comparative examples, conventional operations in the art are used; reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0057] Example 1

[0058] Preparation of corn steep liquor hydrolysate: Take 100 kg of corn steep liquor with a solid content of 12%, add deionized water to adjust the total volume to 1 m³. 3 Adjust the pH to 5.0 with 1 mol / L hydrochloric acid, heat to 50℃, add acidic protease (1.0 U per gram of dry matter) and phytase (30 U per gram of dry matter), keep warm and stir for 3 hours, boil for 10 minutes to inactivate the enzymes after the reaction is complete, cool to room temperature to obtain corn steep liquor hydrolysate.

[0059] Preparation of special culture medium:

[0060] S1: Weigh 112 kg of glucose and 48 kg of sorbitol (mass ratio 70:30), add 800 L of deionized water, heat to 55°C and stir to dissolve, cool to 25°C, then add 10 kg (dry matter basis) of the above corn steep liquor hydrolysate, and bring the volume to 1 mL. 3 Sterilize at 121℃ for 30 minutes, then cool to 38℃;

[0061] S2: Prepare 10 times the inorganic salt stock solution respectively: potassium dihydrogen phosphate 25 g / L, magnesium sulfate 6 g / L, potassium chloride 12 g / L, sodium citrate 9 g / L, and mix 100 mL of each.

[0062] S3: Prepare a 1000-fold trace element stock solution: 0.01 g / L ferrous sulfate, 0.004 g / L zinc sulfate, 0.0025 g / L manganese chloride, and 0.001 g / L sodium molybdate. The solvent is deionized water containing 0.1% HCl. Take 1 L.

[0063] S4: Prepare 100 L of a mixed solution of glycine 70 g / L and citric acid 45 g / L, adjust the pH to 5.2, filter and sterilize to obtain a buffer conditioning solution;

[0064] S5: Aseptically add 1 L of inorganic salt stock solution, 100 L of buffer solution, and 1 L of trace element stock solution to the sterilized basic carbon-nitrogen solution in sequence, and mix well.

[0065] S6: Inoculation with Corynebacterium glutamicum seed culture (OD) 600=15 (inoculum 10%), sterile air circulation, dissolved oxygen controlled at 25%, temperature 33℃, stirring at 400 rpm, liquid ammonia added to maintain pH ≥ 4.8. Fermentation ended after 42 hours, and samples were taken for analysis.

[0066] Example 2

[0067] The preparation of corn steep liquor hydrolysate is the same as in Example 1.

[0068] Preparation of special culture medium:

[0069] S1: Glucose to sorbitol mass ratio 80:20, total carbon source 150 g / L, corn steep liquor hydrolysate addition amount 9 g / L (dry basis).

[0070] S2: Potassium dihydrogen phosphate 2.2 g / L, magnesium sulfate 0.6 g / L, potassium chloride 1.1 g / L, sodium citrate 0.9 g / L;

[0071] S3: Trace element concentration is the same as in Example 1;

[0072] S4: Glycine 65 g / L, Citric acid 48 g / L, Total buffer 11.3 g / L;

[0073] The remaining steps are the same as in Example 1. Fermentation is completed after 40 hours.

[0074] Example 3

[0075] Preparation of corn steep liquor hydrolysate: pH 4.8, temperature 52℃, acidic protease 1.2 U / g, phytase 35 U / g, reaction time 2.5 hours.

[0076] Preparation of special culture medium:

[0077] S1: Glucose to sorbitol mass ratio 75:25, total carbon source 155 g / L, corn steep liquor hydrolysate 11 g / L (dry basis).

[0078] S2: Potassium dihydrogen phosphate 2.6 g / L, magnesium sulfate 0.55 g / L, potassium chloride 1.25 g / L, sodium citrate 0.85 g / L;

[0079] S3: Ferrous sulfate 0.012 g / L, zinc sulfate 0.005 g / L, manganese chloride 0.003 g / L, sodium molybdate 0.0012 g / L;

[0080] S4: Glycine 75 g / L, Citric acid 42 g / L;

[0081] The rest is the same as in Example 1. Fermentation ends after 44 hours.

[0082] Comparative Example 1

[0083] In this comparative example, a standard culture medium with a pH of 7.0 was used. The specific components of the culture medium are as follows:

[0084] Carbon source: 150 g / L glucose;

[0085] Nitrogen source: 40 g / L urea + 10 g / L corn steep liquor (unhydrolyzed);

[0086] Inorganic salts: KH₂PO₄ 2.0 g / L, MgSO₄ 0.6 g / L, KCl 1.0 g / L;

[0087] No buffer;

[0088] The pH was adjusted to 7.0 using NaOH during the fermentation process.

[0089] Comparative Example 2

[0090] In this comparative example, the culture medium was low pH but without sorbitol, and the carbon source was glucose 160 g / L; the rest was the same as in Example 1. During fermentation, the pH naturally dropped below 4.5, and no liquid ammonia was added.

[0091] Comparative Example 3

[0092] In this comparative example, corn steep liquor was added to the culture medium, but the corn steep liquor was not subjected to double-enzyme hydrolysis. The nitrogen source was a mixture of liquid ammonia and unhydrolyzed corn steep liquor in a 50:50 ratio; the rest was the same as in Example 1.

[0093] The above-described examples and comparative examples were run in parallel for three batches in a 50 L fermenter (containing 35 L of liquid), and the average value was taken. The measured indicators are shown in the table below:

[0094]

[0095] The results showed that the embodiments of this application achieved higher glutamic acid yield and conversion rate than Comparative Example 1 under low pH conditions, while significantly reducing the consumption of liquid ammonia and glucose. Comparative Example 2 suffered from low carbon source utilization efficiency due to the lack of sorbitol; Comparative Example 3 had insufficient nitrogen and phosphorus release due to the lack of hydrolysis of corn steep liquor, which limited the metabolic activity of the microorganisms.

[0096] The above embodiments are merely one of the preferred embodiments of the present invention and should not be used to limit the scope of protection of the present invention. Any modifications or refinements made to the main design concept and spirit of the present invention that are not of substantial significance, but solve the same technical problem as the present invention, should be included within the scope of protection of the present invention.

Claims

1. A special culture medium for glutamic acid fermentation under low pH conditions, characterized in that, It includes the following components: The carbon source is composed of glucose and sorbitol in a mass ratio of 70-90:10-30. The nitrogen source is composed of liquid ammonia and corn steep liquor hydrolysate in a mass ratio of 40-60:40-60, wherein the corn steep liquor hydrolysate is obtained by hydrolyzing corn steep liquor with acidic protease and phytase. Inorganic salts, including potassium dihydrogen phosphate 1.5~3.0 g / L, magnesium sulfate 0.4~0.8 g / L, potassium chloride 0.8~1.5 g / L, and sodium citrate 0.5~1.2 g / L; Trace elements, including ferrous sulfate 0.005~0.015 g / L, zinc sulfate 0.002~0.006 g / L, manganese chloride 0.001~0.004 g / L, and sodium molybdate 0.0005~0.0015 g / L; The buffer regulator is a glycine-citric acid complex buffer with a total concentration of 8-15 g / L, wherein the molar ratio of glycine to citric acid is 1-2:

1.

2. The special culture medium for glutamic acid fermentation under low pH environment according to claim 1, characterized in that, The method for preparing the corn steep liquor hydrolysate is as follows: the corn steep liquor is hydrolyzed using a combination of acidic protease and phytase under the following conditions: pH 4.5-5.5, temperature 45-55℃, and hydrolysis time 2-4 hours.

3. The special culture medium for glutamic acid fermentation under low pH environment according to claim 2, characterized in that, When preparing corn steep liquor hydrolysate, the initial solids content of the corn steep liquor is 10%~15%, the amount of acidic protease added is 0.8~1.5 U per gram of dry matter, the amount of phytase added is 20~40 U per gram of dry matter, and the pH is adjusted to the set value by dilute hydrochloric acid or ammonia during the hydrolysis reaction.

4. A method for preparing a special culture medium as described in any one of claims 1 to 3, characterized in that, Includes the following steps: S1: Dissolve glucose and sorbitol in water in a certain proportion, add corn steep liquor hydrolysate, mix and sterilize to obtain a basic carbon-nitrogen solution; S2: Preparation of inorganic salt mother liquor: Weigh out potassium dihydrogen phosphate, magnesium sulfate, potassium chloride and sodium citrate respectively, dissolve them in deionized water, make up to the required concentration, and filter to remove bacteria; S3: Preparation of trace element stock solution: Weigh ferrous sulfate, zinc sulfate, manganese chloride and sodium molybdate respectively, dissolve them in deionized water, make up to volume and then filter to remove bacteria; S4: Prepare a glycine-citric acid buffer solution, adjust the pH to 5.2, and filter to sterilize; S5: Cool the sterilized basic carbon-nitrogen solution, then add the inorganic salt mother liquor, buffer regulator solution and trace element mother liquor in sequence, and mix thoroughly.

5. The preparation method according to claim 4, characterized in that, In step S3, when preparing the trace element stock solution, deionized water containing 0.1% dilute hydrochloric acid is used to dissolve each trace element, and ferrous sulfate is pre-complexed with sodium citrate.

6. The application of a special culture medium as described in any one of claims 1 to 3 in the fermentation of *Corynebacterium glutamicum* to produce glutamic acid, characterized in that, During fermentation, the pH was controlled at 4.8-5.5, the fermentation temperature was 32-34℃, and liquid ammonia was used as the nitrogen source and pH adjuster.

7. The application according to claim 6, characterized in that, No external pH adjustment is performed during fermentation. When the system pH is below 4.8, liquid ammonia is added to raise the pH to above 5.

0.

8. The application according to claim 6, characterized in that, The fermentation cycle is 36-48 hours.

9. The application according to claim 6, characterized in that, The *Corynebacterium glutamicum* strain is a conventional production strain that has not undergone acid-resistant mutagenesis, and no exogenous growth factors or vitamins are added during the fermentation process.

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