A method for producing glutamic acid by fermentation, a culture medium and application thereof

Abstract

The present application relates to the technical field of microbial fermentation culture, and specifically discloses a method for fermentatively producing glutamic acid, a culture medium and application thereof. The method for fermentatively producing glutamic acid adds catechol to seed culture medium and fermentation culture medium, starts to add flow-adding liquid one after complete consumption of the base sugar, and starts to add flow-adding liquid two when the acid concentration in the fermentation broth reaches 140-165 g / L; the flow-adding liquid one is a glucose solution containing propyl gallate, the concentration of glucose in the flow-adding liquid one is 68-72%, and the concentration of propyl gallate is 10-50 mg / 100 g; the residual sugar concentration in the fermentation broth is controlled to be less than 5 g / L by adding the flow-adding liquid one; and the flow-adding liquid two is a sorbitol solution, and the sorbitol concentration in the fermentation broth is controlled to be 0.5-4 g / L by adding the flow-adding liquid two. The method can improve the fermentation conversion rate and is suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation culture technology, and more specifically, to a method for fermenting and producing glutamic acid, a culture medium, and its applications. Background Technology

[0002] Glutamic acid, also known as α-aminoglutarate, is an acidic amino acid containing one amino group and two carboxyl groups. It is a precursor to monosodium glutamate (MSG) and is in high demand in the market. Glutamic acid has wide applications in food, medicine, cosmetics, and agriculture. It can be used as a flavoring agent and nutritional supplement in food, and also as a medicine to treat hepatic coma, among other conditions.

[0003] Glutamic acid is currently mainly produced by fermentation. Its biosynthetic pathway includes: (1) glucose is converted into pyruvate through the glycolysis pathway (EMP) and the pentose phosphate pathway (HMP); (2) one molecule of pyruvate is converted into acetyl-CoA by pyruvate dehydrogenase system, and one molecule of pyruvate is converted into oxaloacetate through carbon dioxide fixation pathway; (3) oxaloacetate and acetyl-CoA are converted into citrate under the catalysis of aldolase; (4) citrate enters the TCA cycle to further generate α-ketoglutarate; (5) α-ketoglutarate is converted into oxaloacetate by glutamate dehydrogenase and NH4+. + Glutamic acid is generated in the presence of glucose; (6) Glutamic acid is eventually transported to the extracellular space through mechanosensitive channels on the cell membrane. Theoretically, without considering microbial growth and respiration, one molecule of glucose will eventually produce one molecule of glutamate, and the theoretical sugar-acid conversion rate is 81.7%.

[0004] There are two types of microbial strains used in the fermentation process to produce glutamic acid: biotin-dependent strains and thermosensitive strains. Because thermosensitive strains are not limited by the amount of biotin used, are easier to control during fermentation, and have higher fermentation performance than biotin-dependent strains, industrial production of glutamic acid currently mainly uses thermosensitive strains.

[0005] Although the level of glutamic acid fermentation has made significant progress over the past few decades, there is still considerable room for improvement compared to the theoretical conversion rate. The main bottlenecks currently limiting the level of glutamic acid fermentation are: (1) Since glutamic acid fermentation is an aerobic fermentation, as the fermentation time increases, oxidative metabolism intensifies, oxygen free radicals increase, and free radicals attack macromolecules such as DNA, proteins, and lipids in cells, causing damage to cells and leading to a rapid decline in the activity of the strain or even death in the later stage of fermentation; (2) As the concentration of glutamic acid fermentation increases, the osmotic pressure of the fermentation medium continuously increases, cells lose water, and the activity of intracellular glutamic acid synthase system rapidly decreases, resulting in a rapid decrease in the final glutamic acid synthesis rate.

[0006] Currently, industrial improvements and optimizations mainly focus on the fermentation formulation and process control of glutamic acid. This is achieved by reducing the concentration of harmful substances in the fermentation medium or by adding osmotically compatible solutes to prolong cell activity. Chinese patent CN111621530A discloses a semi-continuous fermentation method for producing glutamic acid. In this method, when the acid concentration reaches 14%-17% during the middle stage of fermentation, a portion of the fermentation broth is intermittently released while fresh culture medium is added to dilute the concentration of glutamic acid and harmful substances in the fermentation broth, thereby slowing down the decline in cell activity and improving fermentation efficiency. Another approach (CN107227324A) utilizes fermenters and membrane coupling technology to perform filtration and dialysis during fermentation, promptly separating glutamic acid and harmful byproducts from the fermentation broth, thus prolonging the glutamic acid fermentation acid production cycle and improving cell utilization and sugar-acid conversion rate.

[0007] Chinese patent CN103243131B discloses a method for preparing L-glutamic acid by fermentation. By adding a certain concentration of betaine hydrochloride as an osmotic pressure protectant to the culture medium during the initial and temperature-changing stages, the cells can absorb betaine to maintain normal osmotic pressure balance when the external osmotic pressure increases, thereby preventing water outflow and salt intrusion and ensuring normal cell function.

[0008] While semi-continuous fermentation or membrane coupling technology can reduce the concentration of glutamate and harmful substances in the culture medium, membrane coupling technology requires additional membrane equipment, increasing both equipment investment and operational complexity. Furthermore, membrane separation during fermentation increases the risk of contamination. Semi-continuous fermentation, on the other hand, produces fermentation broth with low glutamate concentrations, increasing extraction and concentration costs. Adding betaine hydrochloride to the culture medium results in a low pH and high chloride ion concentration, which can severely corrode fermentation equipment.

[0009] Therefore, it is necessary to conduct further research on how to better balance the improvement of fermentation conversion rate and the needs of industrial production. Summary of the Invention

[0010] One of the objectives of this invention is to provide a new method that can improve the conversion rate of glutamic acid fermentation production.

[0011] To achieve this objective, the technical solution of the present invention is as follows:

[0012] A method for producing glutamic acid by fermentation involves adding catechol to a seed culture medium and a fermentation culture medium. During fermentation, feed solution one is started after the base sugar is completely consumed, and feed solution two is started when the acid concentration in the fermentation broth reaches 140-165 g / L.

[0013] The first feed solution is a glucose solution containing propyl gallate. The concentration of glucose in the first feed solution is 68-72%, and the concentration of propyl gallate is 10-50 mg / 100g. By feeding the first feed solution, the residual sugar concentration in the fermentation broth is controlled to be less than 5g / L.

[0014] The second feed solution is a sorbitol solution. By feeding the second feed solution, the concentration of sorbitol in the fermentation broth is controlled to be 0.5-4 g / L.

[0015] Antioxidants are substances that help capture and neutralize free radicals, preventing the adverse effects of oxygen. Their mechanism of action can be either direct action on free radicals or indirect consumption of substances that easily generate free radicals. Adding antioxidants to the culture medium can promote cell growth. Commonly used antioxidants include phenolic antioxidants, amine antioxidants, and sulfur-containing compound antioxidants. Different microorganisms and different raw materials used in fermentation require different optimal antioxidants. Even with the same antioxidant, different concentrations and timing of addition can have unpredictable effects on cell growth and fermentation parameters.

[0016] This invention reveals that propyl gallate is a phenolic antioxidant with relatively stable properties. However, it reacts with metal ions such as copper and iron, turning purple or dark green. It is unstable to light, decomposes, and has poor heat resistance. Adding propyl gallate directly to the culture medium will cause it to react with metal ions, and it will decompose after high-temperature sterilization, resulting in poor performance in the fermentation industry.

[0017] Therefore, this invention first adds propyl gallate after fermentation begins, co-adding it with fed-batch sugar. On one hand, the low sterilization temperature of fed-batch sugar reduces the high-temperature decomposition of propyl gallate; on the other hand, the addition of propyl gallate prevents damage to the bacterial cells from oxygen free radicals during fermentation, delaying cell aging and extending the duration of high acid production. In conjunction with this, this invention also specifically adds catechol to the seed culture and fermentation medium. It has been found that catechol can chelate metal ions such as iron and copper in the culture medium system of this invention, improving the absorption and utilization of these ions by the bacteria, promoting cell growth, and also reducing the reaction between propyl gallate and metal ions, thus enhancing the effect of propyl gallate.

[0018] In addition, the present invention also added sorbitol in the middle and late stages of fermentation. It was found that sorbitol, in combination with the basal culture medium and the fed-batch liquid in the fermentation system of the present invention, can protect cells from environmental stress, enhance cell viability, reduce metabolic load, maintain the activity of pyruvate dehydrogenase, isocitrate dehydrogenase and cytochrome C oxidase, and improve the fermentation effect.

[0019] The sterilization temperature of the first fed-batch liquid in this invention is 110-115℃, and the time is 18-22 min.

[0020] The sterilization temperature of the second feed solution is 121-123℃, and the time is 18-22 min.

[0021] The fermentation time of this invention is 32-36 hours.

[0022] In the method of the present invention, the concentration of catechol in the seed culture medium is 0.8-1.2 mg / L; the concentration of catechol in the fermentation culture medium is 2-10 mg / L.

[0023] When the concentration of catechol in the fermentation medium is 4-10 mg / L and the concentration of propyl gallate in the first fed broth is 30-50 mg / 100g, a better conversion rate can be obtained.

[0024] In the method of the present invention, the fermentation culture medium further includes: glucose 45-55 g / L, corn steep liquor 28-32 g / L, corn steep liquor hydrolysate 14-16 g / L, dipotassium hydrogen phosphate 3.8-4.2 g / L, magnesium sulfate 1.4-1.6 g / L, ferrous sulfate 9-11 mg / L, manganese sulfate 9-11 mg / L, biotin 480-520 μg / L, and thiamine 580-620 μg / L.

[0025] Preferably, the fermentation medium further includes: 50 g / L glucose, 30 g / L corn steep liquor, 15 g / L corn steep liquor hydrolysate, 4 g / L dipotassium hydrogen phosphate, 1.5 g / L magnesium sulfate, 10 mg / L ferrous sulfate, 10 mg / L manganese sulfate, 500 μg / L biotin, and 600 μg / L thiamine.

[0026] The sterilization temperature of the fermentation medium of this invention is 121-125℃, the sterilization time is 20-25min, and after sterilization, the temperature is rapidly reduced to 32℃, and ammonia water is added to adjust the pH of the fermentation medium to 7.0-7.2.

[0027] In the method of the present invention, the seed culture medium further includes: glucose 28-32 g / L, yeast powder 4.8-5.2 g / L, dipotassium hydrogen phosphate 0.48-0.52 g / L, magnesium sulfate 0.9-1.1 g / L, ferrous sulfate 14-16 mg / L, manganese sulfate 14-16 mg / L, biotin 190-210 μg / L, and thiamine 290-310 μg / L;

[0028] Preferably, the seed culture medium further includes: 30 g / L glucose, 5 g / L yeast extract, 0.5 g / L dipotassium hydrogen phosphate, 1 g / L magnesium sulfate, 15 mg / L ferrous sulfate, 15 mg / L manganese sulfate, 200 μg / L biotin, and 300 μg / L thiamine.

[0029] In the method of this invention, the OD562nm of the seed liquid is 10-15.

[0030] In this invention, the fermentation bacteria are first activated and cultured, and then transferred to a seed culture medium containing catechins and shaken to obtain a seed liquid.

[0031] In the method of this invention, the fermentation bacteria is thermosensitive Corynebacterium glutamicum, and the fermentation control conditions are: pH 6.8-7.2, DO 20-40%, initial culture temperature 32℃, temperature-dependent OD562nm 35-50, and temperature after temperature-dependent fermentation 37-39℃.

[0032] The present invention also provides a culture medium for fermenting and producing glutamic acid, which includes a basic fermentation medium, a first feed solution and a second feed solution, wherein the fermentation medium, the first feed solution and the second feed solution are respectively as described above.

[0033] The present invention further provides an application of the above-mentioned method or culture medium in improving the conversion rate of glutamic acid produced by fermentation.

[0034] The beneficial effects of this invention are at least as follows:

[0035] The method of this invention provides a new and effective approach to improving the fermentation production of glutamic acid through a specific combination of basal and fed-batch culture media. It is simple to operate, non-corrosive to equipment, saves on equipment investment and energy consumption, and is suitable for industrial production. Detailed Implementation

[0036] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available or prepared according to conventional methods in the art.

[0038] The thermosensitive Corynebacterium glutamicum used in the specific embodiments of this invention is Corynebacterium glutamicum MHZ-0112-8, disclosed in Chinese Patent CN112322594A, with accession number CGMCC No.11941, and deposited on December 25, 2015 at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postcode 100101).

[0039] Example 1

[0040] This embodiment provides a fermentation method for high glutamic acid production, comprising the following steps:

[0041] (1) Seed preparation: First, spread the thermosensitive Corynebacterium glutamicum on LB agar plates and incubate overnight at 30°C. Then, scrape the colonies from the plates and inoculate them into sterilized shake flask seed culture medium. Incubate at 32°C on a shaker at 220 rpm. Take samples every 2 hours to detect OD562nm. End the culture when OD562nm reaches 10.

[0042] The seed culture medium consisted of: 30 g / L glucose, 5 g / L yeast extract, 0.5 g / L dipotassium hydrogen phosphate, 1 g / L magnesium sulfate, 15 mg / L ferrous sulfate, 15 mg / L manganese sulfate, 200 μg / L biotin, 300 μg / L thiamine, and 1 mg / L catechol.

[0043] (2) Fermentation preparation: Weigh each component of the fermentation medium according to the initial volume of the fermenter, add tap water to dissolve, and pour into the fermenter for sterilization. The sterilization temperature is 121℃ and the sterilization time is 20min. After sterilization, quickly cool down to 32℃, add ammonia water to adjust the pH of the fermentation medium to 7.0, and wait for inoculation. Glucose is sterilized separately and poured into the fermenter before inoculation.

[0044] The fermentation medium consisted of: 50 g / L glucose, 30 g / L corn steep liquor, 15 g / L corn steep liquor hydrolysate, 4 g / L dipotassium hydrogen phosphate, 1.5 g / L magnesium sulfate, 10 mg / L ferrous sulfate, 10 mg / L manganese sulfate, 500 μg / L biotin, 600 μg / L thiamine, and 4 mg / L catechol.

[0045] Feed solution 1: 70% glucose solution containing propyl gallate, with a composition of 70g glucose and 20mg propyl gallate per 100g of solution, sterilized at 110℃ for 20min for later use.

[0046] Flow-through solution 2: 10% sorbitol solution, sterilized at 121℃ for 20 min for later use.

[0047] (3) Glutamic acid fermentation method: The cultured seed liquid was inoculated into a 10L fermenter at an inoculation ratio of 12% (v / v). The initial culture temperature was 32℃. When the fermentation OD562nm reached 40, the temperature was raised to 38℃ and maintained until the end of fermentation. The pH was controlled at 6.8-7.2 by automatically adding ammonia. The air volume, rotation speed and tank pressure were adjusted to control DO, with the DO control range of 20-40%. After the bottom sugar was completely consumed, the first feed solution was started, and the residual sugar concentration of the fermentation liquid was controlled to be less than 5g / L. After 18h of fermentation (when the acid concentration reached 152g / L), the second feed solution was started, and the sorbitol concentration in the fermentation liquid was controlled to be 1g / L. Fermentation was stopped after 34h. The OD value, residual sugar and glutamic acid content of the fermentation liquid were sampled every 2h. The final acid production was 191g / L, and the conversion rate was 67.4%.

[0048] Example 2

[0049] This embodiment provides a fermentation method for high glutamic acid production, comprising the following steps:

[0050] (1) Seed preparation: First, spread the thermosensitive Corynebacterium glutamicum on LB agar plates and incubate overnight at 30°C. Then, scrape the colonies from the plates and inoculate them into sterilized shake flask seed culture medium. Incubate at 32°C on a shaker at 220 rpm. Take samples every 2 hours to detect OD562nm. End the culture when OD562nm reaches 15.

[0051] The seed culture medium consisted of: 30 g / L glucose, 5 g / L yeast extract, 0.5 g / L dipotassium hydrogen phosphate, 1 g / L magnesium sulfate, 15 mg / L ferrous sulfate, 15 mg / L manganese sulfate, 200 μg / L biotin, 300 μg / L thiamine, and 1 mg / L catechol.

[0052] (2) Fermentation preparation: Weigh each component of the fermentation medium according to the initial volume of the fermenter, add tap water to dissolve, and pour into the fermenter for sterilization. The sterilization temperature is 122℃ and the sterilization time is 25min. After sterilization, quickly cool down to 32℃, add ammonia water to adjust the pH of the fermentation medium to 7.2, and wait for inoculation. Glucose is sterilized separately and poured into the fermenter before inoculation.

[0053] The fermentation medium consisted of: 50 g / L glucose, 30 g / L corn steep liquor, 15 g / L corn steep liquor hydrolysate, 4 g / L dipotassium hydrogen phosphate, 1.5 g / L magnesium sulfate, 10 mg / L ferrous sulfate, 10 mg / L manganese sulfate, 500 μg / L biotin, 600 μg / L thiamine, and 4 mg / L catechol.

[0054] Feed solution 1: 70% glucose solution containing propyl gallate, with a composition of 70g glucose and 20mg propyl gallate per 100g of solution, sterilized at 112℃ for 20min for later use.

[0055] Flow-through solution 2: 10% sorbitol solution, sterilized at 122℃ for 20 min for later use.

[0056] (3) Glutamic acid fermentation method: The cultured seed liquid was inoculated into a 10L fermenter at an inoculation ratio of 12% (v / v). The initial culture temperature was 32℃. When the fermentation OD562nm reached 35, the temperature was raised to 37℃ and maintained until the end of fermentation. The pH was controlled at 6.8-7.2 by automatically adding ammonia water. The DO was controlled by adjusting the air volume, rotation speed and tank pressure. The DO control range was 20-40%. After the bottom sugar was completely consumed, the first feed solution was started. The residual sugar concentration of the fermentation liquid was controlled to be less than 5g / L. After 20h of fermentation (the acid concentration reached 165g / L), the second feed solution was started. The sorbitol concentration in the fermentation liquid was controlled to be 1g / L. Fermentation was stopped after 32h. The OD value, residual sugar and glutamic acid content of the fermentation liquid were sampled every 2h. The final acid production was 188g / L, and the conversion rate was 66.8%.

[0057] Example 3

[0058] This embodiment provides a fermentation method for high glutamic acid production, comprising the following steps:

[0059] (1) Seed preparation: First, spread the thermosensitive Corynebacterium glutamicum on LB agar plates and incubate overnight at 30°C. Then, scrape the colonies from the plates and inoculate them into sterilized shake flask seed culture medium. Incubate at 32°C on a shaker at 220 rpm. Take samples every 2 hours to detect OD562nm. End the culture when OD562nm reaches 10.

[0060] The seed culture medium consisted of: 30 g / L glucose, 5 g / L yeast extract, 0.5 g / L dipotassium hydrogen phosphate, 1 g / L magnesium sulfate, 15 mg / L ferrous sulfate, 15 mg / L manganese sulfate, 200 μg / L biotin, 300 μg / L thiamine, and 1 mg / L catechol.

[0061] (2) Preparation of fermentation medium: Weigh each component of the fermentation medium according to the initial volume of the fermenter, add tap water to dissolve, and pour into the fermenter for sterilization. The sterilization temperature is 125℃ and the sterilization time is 20min. After sterilization, quickly cool down to 32℃, add ammonia water to adjust the pH of the fermentation medium to 7.1 and wait for inoculation. Glucose is sterilized separately and poured into the fermenter before inoculation.

[0062] The fermentation medium consisted of: 50 g / L glucose, 30 g / L corn steep liquor, 15 g / L corn steep liquor hydrolysate, 4 g / L dipotassium hydrogen phosphate, 1.5 g / L magnesium sulfate, 10 mg / L ferrous sulfate, 10 mg / L manganese sulfate, 500 μg / L biotin, 600 μg / L thiamine, and 4 mg / L catechol.

[0063] Feed solution 1: 70% glucose solution containing propyl gallate, with a composition of 70g glucose and 40mg propyl gallate per 100g of solution, sterilized at 115℃ for 20min for later use.

[0064] Flow-through solution 2: 10% sorbitol solution, sterilized at 123℃ for 20 min for later use.

[0065] (3) Glutamic acid fermentation method: The cultured seed liquid was inoculated into a 10L fermenter at an inoculation ratio of 12% (v / v). The initial culture temperature was 32℃. When the fermentation OD562nm reached 50, the temperature was raised to 39℃ and maintained until the end of fermentation. The pH was controlled at 6.8-7.2 by automatically adding ammonia. The air volume, rotation speed and tank pressure were adjusted to control DO, with the DO control range of 20-40%. After the bottom sugar was completely consumed, the first feed solution was started, and the residual sugar concentration of the fermentation liquid was controlled to be less than 5g / L. After 16h of fermentation (when the acid concentration reached 142g / L), the second feed solution was started, and the sorbitol concentration in the fermentation liquid was controlled to be 1g / L. Fermentation was stopped after 36h. The OD value, residual sugar and glutamic acid content of the fermentation liquid were sampled every 2h. The final acid production was 195g / L, and the conversion rate was 68.2%.

[0066] Example 4

[0067] This embodiment provides a fermentation method for high glutamic acid production, comprising the following steps:

[0068] (1) Seed preparation: First, spread the thermosensitive Corynebacterium glutamicum on LB agar plates and incubate overnight at 30°C. Then, scrape the colonies from the plates and inoculate them into sterilized shake flask seed culture medium. Incubate at 32°C on a shaker at 220 rpm. Take samples every 2 hours to detect OD562nm. End the culture when OD562nm reaches 10.

[0069] The seed culture medium consisted of: 30 g / L glucose, 5 g / L yeast extract, 0.5 g / L dipotassium hydrogen phosphate, 1 g / L magnesium sulfate, 15 mg / L ferrous sulfate, 15 mg / L manganese sulfate, 200 μg / L biotin, 300 μg / L thiamine, and 1 mg / L catechol.

[0070] (2) Preparation of fermentation medium: Weigh each component of the fermentation medium according to the initial volume of the fermenter, add tap water to dissolve, and pour into the fermenter for sterilization. The sterilization temperature is 123℃ and the sterilization time is 20min. After sterilization, quickly cool down to 32℃, add ammonia water to adjust the pH of the fermentation medium to 7.0, and wait for inoculation. Glucose is sterilized separately and poured into the fermenter before inoculation.

[0071] The fermentation medium consisted of: 50 g / L glucose, 30 g / L corn steep liquor, 15 g / L corn steep liquor hydrolysate, 4 g / L dipotassium hydrogen phosphate, 1.5 g / L magnesium sulfate, 10 mg / L ferrous sulfate, 10 mg / L manganese sulfate, 500 μg / L biotin, 600 μg / L thiamine, and 4 mg / L catechol.

[0072] Feed solution 1: 70% glucose solution containing propyl gallate, with a composition of 70g glucose and 30mg propyl gallate per 100g of solution, sterilized at 113℃ for 20min for later use.

[0073] Flow-through solution 2: 10% sorbitol solution, sterilized at 123℃ for 20 min for later use.

[0074] (3) Glutamic acid fermentation method: The cultured seed liquid was inoculated into a 10L fermenter at an inoculation ratio of 12% (v / v). The initial culture temperature was 32℃. When the fermentation OD562nm reached 40, the temperature was raised to 38℃ and maintained until the end of fermentation. The pH was controlled at 6.8-7.2 by automatically adding ammonia water. The DO was controlled by adjusting the air volume, rotation speed and tank pressure. The DO control range was 20-40%. After the bottom sugar was completely consumed, the first feed solution was started. The residual sugar concentration of the fermentation liquid was controlled to be less than 5g / L. After 16h of fermentation (the acid concentration reached 140g / L), the second feed solution was started. The sorbitol concentration in the fermentation liquid was controlled to be 1g / L. Fermentation was stopped after 34h. The OD value, residual sugar and glutamic acid content of the fermentation liquid were sampled every 2h. The final acid production was 193g / L, and the conversion rate was 67.9%.

[0075] Comparative Example 1

[0076] This comparative example provides a fermentation method for high glutamic acid production, comprising the following steps:

[0077] (1) Seed preparation: First, spread the thermosensitive Corynebacterium glutamicum on LB agar plates and incubate overnight at 30°C. Then, scrape the colonies from the plates and inoculate them into sterilized shake flask seed culture medium. Incubate at 32°C on a shaker at 220 rpm. Take samples every 2 hours to detect OD562nm. End the culture when OD562nm reaches 10.

[0078] The seed culture medium consisted of: 30 g / L glucose, 5 g / L yeast extract, 0.5 g / L dipotassium hydrogen phosphate, 1 g / L magnesium sulfate, 15 mg / L ferrous sulfate, 15 mg / L manganese sulfate, 200 μg / L biotin, and 300 μg / L thiamine.

[0079] (2) Preparation of fermentation medium: Weigh each component of the fermentation medium according to the initial volume of the fermenter, add tap water to dissolve, and pour into the fermenter for sterilization. The sterilization temperature is 121℃ and the sterilization time is 20min. After sterilization, quickly cool down to 32℃, add ammonia water to adjust the pH of the fermentation medium to 7.0, and wait for inoculation. Glucose is sterilized separately and poured into the fermenter before inoculation.

[0080] The fermentation medium consisted of: 50 g / L glucose, 30 g / L corn steep liquor, 15 g / L corn steep liquor hydrolysate, 4 g / L dipotassium hydrogen phosphate, 1.5 g / L magnesium sulfate, 10 mg / L ferrous sulfate, 10 mg / L manganese sulfate, 500 μg / L biotin, and 600 μg / L thiamine.

[0081] The 70% glucose solution is composed of 70g of glucose per 100g of solution, and is sterilized at 110℃ for 20min before use.

[0082] (3) Glutamic acid fermentation method: The cultured seed liquid was inoculated into the fermenter at an inoculation ratio of 12%. The initial culture temperature was 32℃. When the fermentation OD562nm reached 40, the temperature was raised to 38℃ and maintained until the end of fermentation. The pH was controlled at 6.8-7.2 by automatically adding ammonia water. The DO was controlled by adjusting the air volume, rotation speed and tank pressure. The DO control range was 20-40%. After the bottom sugar was completely consumed, a 70% glucose solution was added to control the residual sugar concentration of the fermentation broth to be less than 5g / L. Fermentation was stopped after 34h. The OD value, residual sugar and glutamic acid content of the fermentation broth were sampled every 2h. The final acid production was 176g / L, and the conversion rate was 64.1%.

[0083] Comparative Example 2

[0084] This comparative example provides a fermentation method for high glutamic acid production, comprising the following steps:

[0085] (1) Seed preparation: First, spread the thermosensitive Corynebacterium glutamicum on LB agar plates and incubate overnight at 30°C. Then, scrape the colonies from the plates and inoculate them into sterilized shake flask seed culture medium. Incubate at 32°C on a shaker at 220 rpm. Take samples every 2 hours to detect OD562nm. End the culture when OD562nm reaches 10.

[0086] The seed culture medium consisted of: 30 g / L glucose, 5 g / L yeast extract, 0.5 g / L dipotassium hydrogen phosphate, 1 g / L magnesium sulfate, 15 mg / L ferrous sulfate, 15 mg / L manganese sulfate, 200 μg / L biotin, and 300 μg / L thiamine.

[0087] (2) Preparation of fermentation medium: Weigh each component of the fermentation medium according to the initial volume of the fermenter, add tap water to dissolve, and pour into the fermenter for sterilization. The sterilization temperature is 121℃ and the sterilization time is 25min. After sterilization, quickly cool down to 32℃, add ammonia water to adjust the pH of the fermentation medium to 7.0 and wait for inoculation. Glucose is sterilized separately and poured into the fermenter before inoculation.

[0088] The fermentation medium consisted of: 50 g / L glucose, 30 g / L corn steep liquor, 15 g / L corn steep liquor hydrolysate, 4 g / L dipotassium hydrogen phosphate, 1.5 g / L magnesium sulfate, 10 mg / L ferrous sulfate, 10 mg / L manganese sulfate, 500 μg / L biotin, and 600 μg / L thiamine.

[0089] Flow-through solution: 70% glucose solution containing propyl gallate, with a composition of 70g glucose and 30mg propyl gallate per 100g of solution, sterilized at 115℃ for 20min for later use.

[0090] (3) Glutamic acid fermentation method: The cultured seed liquid was inoculated into the fermenter at an inoculation ratio of 12%. The initial culture temperature was 32℃. When the fermentation OD562nm reached 40, the temperature was raised to 38℃ and maintained until the end of fermentation. The pH was controlled at 6.8-7.2 by automatically adding ammonia water. The DO was controlled by adjusting the air volume, rotation speed and tank pressure. The DO control range was 20-40%. After the bottom sugar was completely consumed, the feed liquid was started. The residual sugar concentration of the fermentation liquid was controlled to be less than 5g / L. Fermentation was stopped after 34h. The OD value, residual sugar and glutamic acid content of the fermentation liquid were sampled every 2h. The final acid production was 180g / L, and the conversion rate was 65.0%.

[0091] Comparative Example 3

[0092] This comparative example provides a fermentation method for high glutamic acid production, comprising the following steps:

[0093] (1) Seed preparation: First, spread the thermosensitive Corynebacterium glutamicum on LB agar plates and incubate overnight at 30°C. Then, scrape the colonies from the plates and inoculate them into sterilized shake flask seed culture medium. Incubate at 32°C on a shaker at 220 rpm. Take samples every 2 hours to detect OD562nm. End the culture when OD562nm reaches 10.

[0094] The seed culture medium consisted of: 30 g / L glucose, 5 g / L yeast extract, 0.5 g / L dipotassium hydrogen phosphate, 1 g / L magnesium sulfate, 15 mg / L ferrous sulfate, 15 mg / L manganese sulfate, 200 μg / L biotin, 300 μg / L thiamine, and 1 mg / L catechol.

[0095] (2) Preparation of fermentation medium: Weigh each component of the fermentation medium according to the initial volume of the fermenter, add tap water to dissolve, and pour into the fermenter for sterilization. The sterilization temperature is 125℃ and the sterilization time is 25min. After sterilization, quickly cool down to 32℃, add ammonia water to adjust the pH of the fermentation medium to 7.0, and wait for inoculation. Glucose is sterilized separately and poured into the fermenter before inoculation.

[0096] The fermentation medium consisted of: 50 g / L glucose, 30 g / L corn steep liquor, 15 g / L corn steep liquor hydrolysate, 4 g / L dipotassium hydrogen phosphate, 1.5 g / L magnesium sulfate, 10 mg / L ferrous sulfate, 10 mg / L manganese sulfate, 500 μg / L biotin, 600 μg / L thiamine, and 4 mg / L catechol.

[0097] A 70% glucose solution, with a composition of 70g glucose per 100g of solution, is sterilized at 110℃ for 20min before use.

[0098] (3) Glutamic acid fermentation method: The cultured seed liquid was inoculated into the fermenter at an inoculation ratio of 12%. The initial culture temperature was 32℃. When the fermentation OD562nm reached 40, the temperature was raised to 38℃ and maintained until the end of fermentation. The pH was controlled at 6.8-7.2 by automatically adding ammonia water. The DO was controlled by adjusting the air volume, rotation speed and tank pressure. The DO control range was 20-40%. After the bottom sugar was completely consumed, a 70% glucose solution was added. The residual sugar concentration of the fermentation broth was controlled to be less than 5g / L. Fermentation was stopped after 34h. The OD value, residual sugar and glutamic acid content of the fermentation broth were sampled every 2h. The final acid production was 178g / L, and the conversion rate was 64.5%.

[0099] Comparative Example 4

[0100] This comparative example provides a fermentation method for high glutamic acid production, comprising the following steps:

[0101] (1) Seed preparation: First, spread the thermosensitive Corynebacterium glutamicum on LB agar plates and incubate overnight at 30°C. Then, scrape the colonies from the plates and inoculate them into sterilized shake flask seed culture medium. Incubate at 32°C on a shaker at 220 rpm. Take samples every 2 hours to detect OD562nm. End the culture when OD562nm reaches 10.

[0102] The seed culture medium consisted of: 30 g / L glucose, 5 g / L yeast extract, 0.5 g / L dipotassium hydrogen phosphate, 1 g / L magnesium sulfate, 15 mg / L ferrous sulfate, 15 mg / L manganese sulfate, 200 μg / L biotin, and 300 μg / L thiamine.

[0103] (2) Preparation of fermentation medium: Weigh each component of the fermentation medium according to the initial volume of the fermenter, add tap water to dissolve, and pour into the fermenter for sterilization. The sterilization temperature is 121℃ and the sterilization time is 20min. After sterilization, quickly cool down to 32℃, add ammonia water to adjust the pH of the fermentation medium to 7.0, and wait for inoculation. Glucose is sterilized separately and poured into the fermenter before inoculation.

[0104] The fermentation medium consisted of: 50 g / L glucose, 30 g / L corn steep liquor, 15 g / L corn steep liquor hydrolysate, 4 g / L dipotassium hydrogen phosphate, 1.5 g / L magnesium sulfate, 10 mg / L ferrous sulfate, 10 mg / L manganese sulfate, 500 μg / L biotin, and 600 μg / L thiamine.

[0105] Feed solution 1: 70% glucose solution, with a composition of 70g glucose per 100g of solution, sterilized at 115℃ for 20min for later use.

[0106] Flow-through solution 2: 10% sorbitol solution, sterilized at 122℃ for 20 min for later use.

[0107] (3) Glutamic acid fermentation method: The cultured seed liquid was inoculated into the fermenter at an inoculation ratio of 12%. The initial culture temperature was 32℃. When the fermentation OD562nm reached 40, the temperature was raised to 38℃ and maintained until the end of fermentation. The pH was controlled at 6.8-7.2 by automatically adding ammonia water. The air volume, rotation speed and tank pressure were adjusted to control DO. The DO control range was 20-40%. After the bottom sugar was completely consumed, the first feed solution was started. The residual sugar concentration of the fermentation liquid was controlled to be less than 5g / L. After 18h of fermentation (when the acid concentration reached 150g / L), the second feed solution was started. The sorbitol concentration in the fermentation liquid was controlled to be 1g / L. Fermentation was stopped after 34h. The OD value, residual sugar and glutamic acid content of the fermentation liquid were sampled every 2h. The final acid production was 182g / L, and the conversion rate was 65.3%.

[0108] Comparative Experiment 1

[0109] The concentration of catechol in the fermentation medium was adjusted to 0-10 mg / L, and the amount of propyl gallate added was 0-50 mg per 100 g of 70% glucose solution. When the concentration of catechol in the fermentation medium was 0, no catechol was added to the seed culture medium. When the concentration of catechol in the fermentation medium was not 0, the concentration of catechol in the seed culture medium was the same as described in Example 1. All other conditions were the same as in Example 1, and the fermentation parameters under different conditions are shown in Table 1.

[0110] Table 1 Fermentation indices at different catechin concentrations

[0111]

[0112] The results of Comparative Examples 1-4 and Comparative Experiment 5 show that the fermentation indicators were significantly higher when both catechol and propyl gallate were added simultaneously to the fermentation medium compared to the groups with only one of them added or without any addition. This is likely because the addition of catechol can chelate metal ions, promoting cell growth, and also reduce the reaction between propyl gallate and metal ions. Secondly, adding propyl gallate along with the added sugar allows for better antioxidant activity. As sugar consumption increases, oxidative metabolism intensifies, leading to increased oxygen free radicals and greater cell damage. Simultaneously, the increased concentration of propyl gallate helps eliminate free radicals and maintain cell metabolic activity. The fermentation indicators using this invention are significantly improved, with acid production increasing by 15 g / L and conversion rate increasing by 3.7% compared to the group without addition.

[0113] Comparative Experiment 2

[0114] Add 10% sorbitol to control the concentration of sorbitol in the fermentation broth to 0-4 g / L. Other conditions are the same as in Example 1. Fermentation indicators under different conditions are shown in Table 2.

[0115] Table 2 Fermentation indices at different sorbitol concentrations

[0116] serial number Sorbitol concentration (g / L) Peak OD Acid production (g / L) Conversion rate (%) 1 0 100 186 65.9％ 2 0.5 102 188 66.2％ 3 (Example 1) 1 102 191 67.4％ 4 2 103 200 69.8％ 5 4 102 202 70.2％

[0117] The results showed that by adding sorbitol solution to the fermentation system of this invention and controlling the concentration of sorbitol in the fermentation broth, fermentation indicators could be significantly improved. With prolonged fermentation time, acid production concentration increased, osmotic pressure increased, and cell activity rapidly decreased. Adding sorbitol in the middle and late stages of fermentation protected cells from environmental stress, enhanced cell viability, reduced metabolic load, maintained the activities of pyruvate dehydrogenase, isocitrate dehydrogenase, and cytochrome C oxidase, and increased acid production in the later stages of fermentation. Compared with the group without sorbitol addition, acid production was increased by 16 g / L, and the conversion rate was increased by 4.3%.

[0118] Comparative Example 5

[0119] This comparative example provides a method for producing glutamic acid by fermentation, which is basically the same as that in Example 1, except that protocatechuic acid is used to replace catechins in the seeds and fermentation medium. The final acid production after fermentation is 183 g / L, with a conversion rate of 65.4%.

[0120] Comparative Example 6

[0121] This comparative example provides a method for producing glutamic acid by fermentation, which is basically the same as that in Example 1, except that methyl gallate is used to replace propyl gallate in the feed liquid. The final acid production after fermentation is 185 g / L, with a conversion rate of 66.0%.

[0122] Comparative Example 7

[0123] This comparative example provides a method for producing glutamic acid by fermentation, which is basically the same as that in Example 1, except that glycerol is used to replace sorbitol in the feed liquid 2. The final acid production after fermentation is 186 g / L, with a conversion rate of 66.1%.

[0124] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for producing glutamic acid by fermentation, characterized in that, Add catechins to the seed culture medium and fermentation culture medium. During fermentation production, start adding feed solution one after the base sugar is completely consumed, and start adding feed solution two when the acid concentration in the fermentation broth reaches 140-165 g / L. The first feed solution is a glucose solution containing propyl gallate. The concentration of glucose in the first feed solution is 68-72%, and the concentration of propyl gallate is 10-50 mg / 100g. By feeding the first feed solution, the residual sugar concentration in the fermentation broth is controlled to be less than 5g / L. The second feed solution is a sorbitol solution. By feeding the second feed solution, the concentration of sorbitol in the fermentation broth is controlled to be 0.5-4 g / L.

2. The method according to claim 1, characterized in that, The concentration of catechol in the seed culture medium is 0.8-1.2 mg / L; the concentration of catechol in the fermentation culture medium is 2-10 mg / L.

3. The method according to claim 2, characterized in that, The fermentation medium also includes: glucose 45-55 g / L, corn steep liquor 28-32 g / L, corn steep liquor hydrolysate 14-16 g / L, dipotassium hydrogen phosphate 3.8-4.2 g / L, magnesium sulfate 1.4-1.6 g / L, ferrous sulfate 9-11 mg / L, manganese sulfate 9-11 mg / L, biotin 480-520 μg / L, and thiamine 580-620 μg / L.

4. The method according to claim 2, characterized in that, The fermentation medium also includes: glucose 50 g / L, corn steep liquor 30 g / L, corn steep liquor hydrolysate 15 g / L, dipotassium hydrogen phosphate 4 g / L, magnesium sulfate 1.5 g / L, ferrous sulfate 10 mg / L, manganese sulfate 10 mg / L, biotin 500 μg / L, and thiamine 600 μg / L.

5. The method according to any one of claims 2-4, characterized in that, The seed culture medium also includes: glucose 28-32 g / L, yeast extract 4.8-5.2 g / L, dipotassium hydrogen phosphate 0.48-0.52 g / L, magnesium sulfate 0.9-1.1 g / L, ferrous sulfate 14-16 mg / L, manganese sulfate 14-16 mg / L, biotin 190-210 μg / L, and thiamine 290-310 μg / L.

6. The method according to any one of claims 2-4, characterized in that, The seed culture medium also includes: glucose 30 g / L, yeast powder 5 g / L, dipotassium hydrogen phosphate 0.5 g / L, magnesium sulfate 1 g / L, ferrous sulfate 15 mg / L, manganese sulfate 15 mg / L, biotin 200 μg / L, and thiamine 300 μg / L.

7. The method according to any one of claims 1-4, characterized in that, The OD562nm of the seed solution is 10-15.

8. The method according to claim 5, characterized in that, The OD562nm of the seed solution is 10-15.

9. The method according to claim 6, characterized in that, The OD562nm of the seed solution is 10-15.

10. The method according to any one of claims 1-4 and 8-9, characterized in that, The fermentation bacteria is thermosensitive Corynebacterium glutamicum. The fermentation control conditions are: pH 6.8-7.2, DO 20-40%, initial culture temperature 32℃, OD562nm 35-50 during temperature variation, and temperature 37-39℃ after temperature variation.

11. The method according to claim 5, characterized in that, The fermentation bacteria is thermosensitive Corynebacterium glutamicum. The fermentation control conditions are: pH 6.8-7.2, DO 20-40%, initial culture temperature 32℃, OD562nm 35-50 during temperature variation, and temperature 37-39℃ after temperature variation.

12. The method according to claim 6, characterized in that, The fermentation bacteria is thermosensitive Corynebacterium glutamicum. The fermentation control conditions are: pH 6.8-7.2, DO 20-40%, initial culture temperature 32℃, OD562nm 35-50 during temperature variation, and temperature 37-39℃ after temperature variation.

13. The method according to claim 7, characterized in that, The fermentation bacteria is thermosensitive Corynebacterium glutamicum. The fermentation control conditions are: pH 6.8-7.2, DO 20-40%, initial culture temperature 32℃, OD562nm 35-50 during temperature variation, and temperature 37-39℃ after temperature variation.

14. A culture medium for fermenting and producing glutamic acid, characterized in that, It includes a basic fermentation medium, a first feed solution, and a second feed solution, wherein the fermentation medium, the first feed solution, and the second feed solution are as described in any one of claims 1-4.

15. The application of the method according to any one of claims 1-13 or the culture medium according to claim 14 in improving the conversion rate of glutamic acid produced by fermentation.

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