Method for producing glutamic acid through fermentation, culture medium and application of culture medium

Abstract

The invention relates to the technical field of microbial fermentation culture, and particularly discloses a method for producing glutamic acid through fermentation, a culture medium and application of the culture medium. According to the method for producing glutamic acid through fermentation, catechol is added into a seed culture medium and a fermentation culture medium, during fermentation production, first fed-batch liquid starts to be fed after bottom sugar is completely consumed, and second fed-batch liquid starts to be fed when the concentration of produced acid in fermentation liquid reaches 140-165 g / L; the first fed-batch liquid is a glucose solution containing propyl gallate, the concentration of glucose in the first fed-batch liquid is 68-72%, the concentration of propyl gallate is 10-50 mg / 100 g, and the concentration of residual sugar in the fermentation liquid is controlled to be smaller than 5 g / L through fed-batch of the first fed-batch liquid; the fed-batch liquid II is a sorbitol solution, and the concentration of sorbitol in the fermentation liquid is controlled to be 0.5-4g / L through fed-batch of the fed-batch liquid II. The method can improve the fermentation conversion rate and is suitable for industrial production.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation and culture, and in particular to a method for producing glutamic acid by fermentation, a culture medium and application thereof. Background Art

[0002] Glutamic acid, also known as α-aminoglutaric acid, contains one amino group and two carboxyl groups in its molecule and is an acidic amino acid. Glutamic acid is the precursor of synthetic monosodium glutamate (monosodium glutamate) and has a large market demand. Glutamic acid is widely used in food, medicine, cosmetics and agriculture. It can be used as a flavoring agent and nutrient in food; it can also be used as a medicine to treat hepatic coma, etc.

[0003] Glutamic acid is currently mainly produced by fermentation. Its biosynthesis pathway includes the following: (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 through the pyruvate dehydrogenase system, and one molecule of pyruvate is converted into oxaloacetate through the carbon dioxide fixation pathway; (3) Oxaloacetate and acetyl-CoA are synthesized into citric acid under the catalysis of aldolase; (4) Citric acid enters the TCA cycle and further generates α-ketoglutarate; (5) α-ketoglutarate is converted into glutamate through the glutamate dehydrogenase and NH4 + Glutamate is generated in the presence of glucose; (6) Glutamate is finally transported to the extracellular space through the mechanosensitive channels on the cell membrane. Theoretically, without considering microbial growth and respiratory consumption, 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 bacteria that produce glutamic acid by fermentation, suboptimal strains of biotin and thermosensitive strains. Since thermosensitive strains are not limited by the amount of biotin, they are easy to control fermentation, and their fermentation index is higher than that of suboptimal strains of biotin, so currently the industrial production of glutamic acid mainly uses thermosensitive strains.

[0005] Although the level of glutamate fermentation has made significant progress after decades of development, there is still much room for improvement compared with the theoretical conversion rate. The main bottlenecks that limit the level of glutamate fermentation are: (1) Since glutamate fermentation is aerobic fermentation, as the fermentation time increases, oxidative metabolism is enhanced, oxygen free radicals increase, and free radicals attack macromolecules such as DNA, proteins and lipids in cells, causing damage to cells, resulting in a rapid decrease in the activity of the strains in the late fermentation period or even death; (2) As the concentration of glutamate fermentation increases, the osmotic pressure of the fermentation medium continues to increase, the cells lose water, and the activity of the intracellular glutamate synthase system decreases rapidly, resulting in a rapid decrease in the final glutamate synthesis rate.

[0006] At present, the industry mainly improves and optimizes the glutamate fermentation formula and process control, by reducing the concentration of harmful substances in the fermentation medium or by adding osmotic pressure compatible solutes to the fermentation medium to achieve the purpose of prolonging the activity of the bacteria. Chinese patent CN111621530A discloses a semi-continuous fermentation method for producing glutamate, by intermittently releasing part of the fermentation liquid when the acid production concentration reaches 14%-17% in the middle of the fermentation, and adding fresh culture medium to dilute the concentration of glutamate and harmful substances in the fermentation liquid, thereby slowing down the decline in bacterial activity and improving fermentation efficiency. There is also a solution (CN107227324A) that uses a fermentation tank and membrane coupling technology to filter and dialyze during the fermentation process to separate glutamate and harmful byproducts in the fermentation liquid in time, thereby prolonging the glutamate fermentation acid production cycle and improving bacterial utilization and sugar-acid conversion rate.

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

[0008] However, although the use of semi-continuous fermentation process or membrane coupling technology can reduce the concentration of glutamate and harmful substances in the culture medium, the membrane coupling technology requires the addition of membrane equipment, which will increase equipment investment and operational difficulty on the one hand, and membrane separation during the fermentation process will increase the risk of contamination on the other hand. The semi-continuous fermentation process has a low concentration of glutamate in the fermentation liquid separated during the fermentation process, which will increase the cost of extraction and concentration. Adding betaine hydrochloride to the culture medium will cause low pH and high chloride ion concentration, which will cause serious corrosion to the 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 purposes of the present invention is to provide a new method for improving the conversion rate of glutamic acid fermentation production.

[0011] In order to achieve this object, the technical solution of the present invention is as follows:

[0012] A method for producing glutamic acid by fermentation, wherein catechol is added to a seed culture medium and a fermentation culture medium, and during fermentation production, a first feeding liquid is started after the substrate sugar is completely consumed, and a second feeding liquid is started when the acid production concentration in the fermentation liquid reaches 140-165 g / L;

[0013] The feed liquid 1 is a glucose solution containing propyl gallate, the concentration of glucose in the feed liquid 1 is 68-72%, the concentration of propyl gallate is 10-50 mg / 100 g, and the residual sugar concentration of the fermentation liquid is controlled to be less than 5 g / L by the feed liquid 1.

[0014] The second feeding liquid is a sorbitol solution, and the sorbitol concentration in the fermentation liquid is controlled to be 0.5-4 g / L by feeding the second feeding liquid.

[0015] Antioxidants are a class of substances that can help capture and neutralize free radicals and prevent the adverse effects of oxygen. Their mechanism of action can be to act directly on free radicals or to indirectly consume substances that easily generate free radicals. Adding antioxidants to the culture medium can help improve bacterial growth. Commonly used antioxidants include phenolic antioxidants, amine antioxidants, and sulfur-containing compound antioxidants. Different microorganisms and fermentation raw materials are different, so the most suitable antioxidants are also different. Even for the same antioxidant, different addition concentrations and addition times will have unpredictable effects on bacterial growth and fermentation indicators.

[0016] The present invention has found that propyl gallate is a phenolic antioxidant with relatively stable properties, but it reacts with metal ions such as copper and iron to form a color reaction, turning purple or dark green, is unstable to light, decomposes, and has poor high temperature resistance. Propyl gallate will react with metal ions when added directly to the culture medium, and will decompose after high temperature sterilization, resulting in poor application effect in the fermentation industry.

[0017] To this end, the present invention first adds propyl gallate after the fermentation starts by adding it together with the flowing sugar. On the one hand, the sterilization temperature of the flowing sugar is low, which can reduce the high-temperature decomposition of propyl gallate. On the other hand, the flowing propyl gallate can prevent the damage of oxygen free radicals to the bacteria during the fermentation process, delay the aging of the bacteria, and prolong the duration of high acid production of the bacteria. In coordination with this, the present invention also particularly adds catechol to the seeds and the fermentation medium. It is found that catechol can chelate metal ions such as iron and copper in the culture medium system of the present invention, improve the absorption and utilization effect of the bacteria on them, promote the growth of the bacteria, and can also reduce the reaction of propyl gallate with metal ions, thereby improving the effect of propyl gallate.

[0018] In addition, the present invention also feeds sorbitol in the middle and late stages of fermentation. It is found that sorbitol, in combination with the basal culture medium and the feed liquid in the fermentation system of the present invention, can protect cells from environmental pressure, 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 feed liquid 1 of the present invention is 110-115° C., and the time is 18-22 minutes.

[0020] The sterilization temperature of the second flow-addition liquid is 121-123°C, and the time is 18-22 minutes.

[0021] The fermentation time of the present invention is 32-36h.

[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 feed liquid 1 is 30-50 mg / 100 g, a better conversion rate improvement effect can be obtained.

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

[0025] Preferably, the fermentation medium further comprises: 50 g / L glucose, 30 g / L corn steep liquor, 15 g / L corn steep liquor hydrolyzate, 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 the present invention is 121-125°C, the sterilization time is 20-25 minutes, the temperature is quickly lowered to 32°C after the sterilization, 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 comprises: 28-32 g / L glucose, 4.8-5.2 g / L yeast powder, 0.48-0.52 g / L dipotassium hydrogen phosphate, 0.9-1.1 g / L magnesium sulfate, 14-16 mg / L ferrous sulfate, 14-16 mg / L manganese sulfate, 190-210 μg / L biotin, and 290-310 μg / L thiamine;

[0028] Preferably, the seed culture medium further comprises: 30 g / L glucose, 5 g / L yeast powder, 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 the present invention, the OD562nm of the seed solution is 10-15.

[0030] In the present invention, the fermentation bacteria are first activated and cultured, and then the culture medium is switched to a seed medium with catechol added thereto for shaking culture to obtain a seed solution.

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

[0032] The present invention also provides a culture medium for producing glutamic acid by fermentation, which comprises a basic fermentation medium, a first feeding liquid and a second feeding liquid, wherein the fermentation medium, the first feeding liquid and the second feeding liquid are respectively as described above.

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

[0034] The beneficial effects of the present invention are at least:

[0035] The method of the present invention provides a new and effective way to improve the fermentation production of glutamic acid through the specific combination of the basic culture medium and the feeding culture medium. The method is simple to operate, has no corrosion to the equipment, saves equipment investment and energy consumption, and is conducive to industrial production. DETAILED DESCRIPTION

[0036] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

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

[0038] The thermosensitive Corynebacterium glutamicum used in the specific implementation mode of the present invention is Corynebacterium glutamicum MHZ-0112-8, which is disclosed in Chinese patent CN112322594A, with a deposit number of CGMCC No.11941, and was deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee on December 25, 2015 (referred to as CGMCC, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101).

[0039] Example 1

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

[0041] (1) Seed preparation: First, spread the thermosensitive Corynebacterium glutamicum on LB plate culture medium, place it in a 30°C incubator for activation and culture overnight, then scrape the colonies on the plate and inoculate them into a sterilized shake flask seed culture medium, place it in a 32°C shaker at 220 rpm for shaking culture, take samples every 2 hours to detect OD562nm, and terminate the culture when OD562nm reaches 10.

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

[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 then pour into the fermenter for sterilization. The sterilization temperature is 121°C and the sterilization time is 20 min. After sterilization, quickly cool to 32°C, add ammonia water to adjust the pH of the fermentation medium to 7.0 and wait for inoculation. Glucose is sterilized by splitting and poured into the fermenter before inoculation.

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

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

[0046] Feed liquid 2: 10% sorbitol solution, sterilized at 121°C for 20 min and set aside.

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

[0048] Example 2

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

[0050] (1) Seed preparation: First, spread the thermosensitive Corynebacterium glutamicum on LB plate culture medium, place it in a 30°C incubator for activation and culture overnight, then scrape the colonies on the plate and inoculate them into a sterilized shake flask seed culture medium, place it in a 32°C shaker at 220 rpm for shaking culture, take samples every 2 hours to detect OD562nm, and terminate the culture when OD562nm reaches 15.

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

[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 then pour into the fermenter for sterilization. The sterilization temperature is 122°C and the sterilization time is 25 min. After sterilization, quickly cool to 32°C, add ammonia water to adjust the pH of the fermentation medium to 7.2 and wait for inoculation. Glucose is sterilized by splitting and poured into the fermenter before inoculation.

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

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

[0055] Feed liquid 2: 10% sorbitol solution, sterilized at 122°C for 20 min and set aside.

[0056] (3) Glutamic acid fermentation method: The cultured seed liquid is inoculated into a 10L fermentation tank at an inoculation ratio of 12% (v / v), the initial culture temperature is 32°C, and when the fermentation OD562nm reaches 35, the temperature is raised to 37°C, and the temperature is maintained until the end of fermentation. The pH is controlled at 6.8-7.2 by automatically adding ammonia water; the air volume, rotation speed and tank pressure are adjusted to control DO, and the DO control range is 20-40%. After the bottom sugar is completely consumed, the first flow addition is started to control the residual sugar concentration of the fermentation liquid to less than 5g / L. After 20h of fermentation (the acid production concentration reaches 165g / L), the second flow addition is started to control the sorbitol concentration in the fermentation liquid to 1g / L. The fermentation is terminated after 32h of fermentation. The OD value, residual sugar and glutamic acid content of the fermentation liquid are sampled every 2h to detect the OD value, residual sugar and glutamic acid content of the fermentation liquid. At the end of fermentation, the final acid production is 188g / L, and the conversion rate is 66.8%.

[0057] Example 3

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

[0059] (1) Seed preparation: First, spread the thermosensitive Corynebacterium glutamicum on LB plate culture medium, place it in a 30°C incubator for activation and culture overnight, then scrape the colonies on the plate and inoculate them into a sterilized shake flask seed culture medium, place it in a 32°C shaker at 220 rpm for shaking culture, take samples every 2 hours to detect OD562nm, and terminate the culture when OD562nm reaches 10.

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

[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 then pour into the fermenter for sterilization. The sterilization temperature is 125°C and the sterilization time is 20 min. After sterilization, quickly cool to 32°C, add ammonia water to adjust the pH of the fermentation medium to 7.1 and wait for inoculation. Glucose is sterilized by splitting and poured into the fermenter before inoculation.

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

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

[0064] Feed liquid 2: 10% sorbitol solution, sterilized at 123°C for 20 min and set aside.

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

[0066] Example 4

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

[0068] (1) Seed preparation: First, spread the thermosensitive Corynebacterium glutamicum on LB plate culture medium, place it in a 30°C incubator for activation and culture overnight, then scrape the colonies on the plate and inoculate them into a sterilized shake flask seed culture medium, place it in a 32°C shaker at 220 rpm for shaking culture, take samples every 2 hours to detect OD562nm, and terminate the culture when OD562nm reaches 10.

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

[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 then pour into the fermenter for sterilization. The sterilization temperature is 123°C and the sterilization time is 20 min. After sterilization, quickly cool to 32°C, add ammonia water to adjust the pH of the fermentation medium to 7.0 and wait for inoculation. Glucose is sterilized by splitting and poured into the fermenter before inoculation.

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

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

[0073] Feed liquid 2: 10% sorbitol solution, sterilized at 123°C for 20 min and set aside.

[0074] (3) Glutamic acid fermentation method: the cultured seed liquid is inoculated into a 10L fermentation tank according to an inoculation ratio of 12% (v / v), the initial culture temperature is 32°C, and when the fermentation OD562nm reaches 40, the temperature is raised to 38°C, and the temperature is maintained until the end of fermentation, and the pH is controlled at 6.8-7.2 by automatically adding ammonia water; the air volume, rotation speed and tank pressure are adjusted to control DO, and the DO control range is 20-40%. After the bottom sugar is completely consumed, the first flow addition is started to control the residual sugar concentration of the fermentation liquid to less than 5g / L. After 16h of fermentation (the acid production concentration reaches 140g / L), the second flow addition is started to control the sorbitol concentration in the fermentation liquid to 1g / L. The fermentation is terminated after 34h of fermentation. The OD value, residual sugar and glutamic acid content of the fermentation liquid are sampled every 2h to detect the OD value, residual sugar and glutamic acid content of the fermentation liquid. At the end of fermentation, the final acid production is 193g / L, and the conversion rate is 67.9%.

[0075] Comparative Example 1

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

[0077] (1) Seed preparation: First, spread the thermosensitive Corynebacterium glutamicum on LB plate culture medium, place it in a 30°C incubator for activation and culture overnight, then scrape the colonies on the plate and inoculate them into a sterilized shake flask seed culture medium, place it in a 32°C shaker at 220 rpm for shaking culture, take samples every 2 hours to detect OD562nm, and terminate the culture when OD562nm reaches 10.

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

[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 then pour into the fermenter for sterilization. The sterilization temperature is 121°C and the sterilization time is 20 min. After sterilization, quickly cool to 32°C, add ammonia water to adjust the pH of the fermentation medium to 7.0 and wait for inoculation. Glucose is sterilized by splitting and poured into the fermenter before inoculation.

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

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

[0082] (3) Glutamic acid fermentation method: the cultured seed liquid is added to the fermentation tank at an inoculation ratio of 12%, the initial culture temperature is 32°C, and when the fermentation OD562nm reaches 40, the temperature is raised to 38°C and maintained until the end of fermentation. The pH is controlled at 6.8-7.2 by automatically adding ammonia water; the air volume, rotation speed and tank pressure are adjusted to control DO, and the DO control range is 20-40%. After the base sugar is completely consumed, a glucose solution with a mass concentration of 70% is started to be added to control the residual sugar concentration of the fermentation liquid to be less than 5g / L. The fermentation is terminated after 34h of fermentation. The OD value, residual sugar and glutamic acid content of the fermentation liquid are sampled every 2h to detect the OD value, residual sugar and glutamic acid content of the fermentation liquid. At the end of fermentation, the final acid production is 176g / L, and the conversion rate is 64.1%.

[0083] Comparative Example 2

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

[0085] (1) Seed preparation: First, spread the thermosensitive Corynebacterium glutamicum on LB plate culture medium, place it in a 30°C incubator for activation and culture overnight, then scrape the colonies on the plate and inoculate them into a sterilized shake flask seed culture medium, place it in a 32°C shaker at 220 rpm for shaking culture, take samples every 2 hours to detect OD562nm, and terminate the culture when OD562nm reaches 10.

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

[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 then pour into the fermenter for sterilization. The sterilization temperature is 121°C and the sterilization time is 25 min. After sterilization, quickly cool to 32°C, add ammonia water to adjust the pH of the fermentation medium to 7.0 and wait for inoculation. Glucose is sterilized by splitting and poured into the fermenter before inoculation.

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

[0089] Feed liquid: 70% glucose solution containing propyl gallate, the composition is 70g glucose and 30mg propyl gallate per 100g solution, sterilized at 115℃ for 20min for use.

[0090] (3) Glutamic acid fermentation method: the cultured seed liquid is added to the fermentation tank at an inoculation ratio of 12%, the initial culture temperature is 32°C, and when the fermentation OD562nm reaches 40, the temperature is raised to 38°C, and the temperature is maintained until the end of fermentation. The pH is controlled at 6.8-7.2 by automatically adding ammonia water; the air volume, rotation speed and tank pressure are adjusted to control DO, and the DO control range is 20-40%. After the bottom sugar is completely consumed, the flow addition is started to control the residual sugar concentration of the fermentation liquid to less than 5g / L. The fermentation is terminated after 34h of fermentation culture. The OD value, residual sugar and glutamic acid content of the fermentation liquid are sampled every 2h to detect the OD value, residual sugar and glutamic acid content of the fermentation liquid. At the end of fermentation, the final acid production is 180g / L, and the conversion rate is 65.0%.

[0091] Comparative Example 3

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

[0093] (1) Seed preparation: First, spread the thermosensitive Corynebacterium glutamicum on LB plate culture medium, place it in a 30°C incubator for activation and culture overnight, then scrape the colonies on the plate and inoculate them into a sterilized shake flask seed culture medium, place it in a 32°C shaker at 220 rpm for shaking culture, take samples every 2 hours to detect OD562nm, and terminate the culture when OD562nm reaches 10.

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

[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 then pour into the fermenter for sterilization. The sterilization temperature is 125°C and the sterilization time is 25 min. After sterilization, quickly cool to 32°C, add ammonia water to adjust the pH of the fermentation medium to 7.0 and wait for inoculation. Glucose is sterilized by splitting and poured into the fermenter before inoculation.

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

[0097] 70% glucose solution, composed of 70g of glucose per 100g of solution, sterilized at 110℃ for 20min before use.

[0098] (3) Glutamic acid fermentation method: the cultured seed liquid is added to the fermentation tank at an inoculation ratio of 12%, the initial culture temperature is 32°C, and when the fermentation OD562nm reaches 40, the temperature is raised to 38°C, and the temperature is maintained until the end of fermentation. The pH is controlled at 6.8-7.2 by automatically adding ammonia water; the air volume, rotation speed and tank pressure are adjusted to control DO, and the DO control range is 20-40%. After the bottom sugar is completely consumed, a glucose solution with a mass concentration of 70% is started to be added to control the residual sugar concentration of the fermentation liquid to be less than 5g / L. The fermentation is terminated after 34h of fermentation culture. The OD value, residual sugar and glutamic acid content of the fermentation liquid are sampled every 2h to detect the OD value, residual sugar and glutamic acid content of the fermentation liquid. At the end of fermentation, the final acid production is 178g / L, and the conversion rate is 64.5%.

[0099] Comparative Example 4

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

[0101] (1) Seed preparation: First, spread the thermosensitive Corynebacterium glutamicum on LB plate culture medium, place it in a 30°C incubator for activation and culture overnight, then scrape the colonies on the plate and inoculate them into a sterilized shake flask seed culture medium, place it in a 32°C shaker at 220 rpm for shaking culture, take samples every 2 hours to detect OD562nm, and terminate the culture when OD562nm reaches 10.

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

[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 then pour into the fermenter for sterilization. The sterilization temperature is 121°C and the sterilization time is 20 min. After sterilization, quickly cool to 32°C, add ammonia water to adjust the pH of the fermentation medium to 7.0 and wait for inoculation. Glucose is sterilized by splitting and poured into the fermenter before inoculation.

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

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

[0106] Feed liquid 2: 10% sorbitol solution, sterilized at 122°C for 20 min and set aside.

[0107] (3) Glutamic acid fermentation method: the cultured seed liquid is added to the fermentation tank at an inoculation ratio of 12%, the initial culture temperature is 32°C, and when the fermentation OD562nm reaches 40, the temperature is raised to 38°C, and the temperature is maintained until the end of fermentation. The pH is controlled at 6.8-7.2 by automatically adding ammonia water; the air volume, rotation speed and tank pressure are adjusted to control DO, and the DO control range is 20-40%. After the bottom sugar is completely consumed, the first flow addition is started to control the residual sugar concentration of the fermentation liquid to less than 5g / L. After 18h of fermentation (the acid production concentration reaches 150g / L), the second flow addition is started to control the sorbitol concentration in the fermentation liquid to 1g / L. The fermentation is terminated after 34h of fermentation. The OD value, residual sugar and glutamic acid content of the fermentation liquid are sampled every 2h to detect the OD value, residual sugar and glutamic acid content of the fermentation liquid. At the end of fermentation, the final acid production is 182g / L, and the conversion rate is 65.3%.

[0108] Comparative experiment 1

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

[0110] Table 1 Fermentation indexes at different catechol concentrations

[0111]

[0112] According to the results of comparative examples 1-4 and comparative experiment 5, after adding catechol and propyl gallate to the fermentation medium at the same time, the fermentation index is significantly higher than that of the single addition of one of them and the experimental group without addition. The reason for the analysis may be that the addition of catechol can chelate metal ions and promote bacterial growth on the one hand, and can reduce the reaction of propyl gallate and metal ions on the other hand; secondly, propyl gallate is added together with the flow sugar, which can better play an antioxidant effect, because with the increase of sugar consumption, oxidative metabolism is enhanced, oxygen free radicals are increased, and cell damage is enhanced, and at the same time, the concentration of propyl gallate is also increased, which can better eliminate free radicals and maintain bacterial metabolic activity. The fermentation index of the scheme of the present invention is significantly improved, and the acid production can be increased by 15g / L compared with the group without addition, and the conversion rate can be increased by 3.7%.

[0113] Comparative experiment 2

[0114] 10% sorbitol was added to control the sorbitol concentration in the fermentation broth to 0-4 g / L. The other conditions were the same as those in Example 1. The fermentation indicators under different conditions are shown in Table 2.

[0115] Table 2 Fermentation indexes 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 show that by adding a sorbitol solution in the fermentation system of the present invention and controlling the sorbitol concentration in the fermentation broth, the fermentation index can be significantly improved. As the fermentation time increases, the acid production concentration increases, the osmotic pressure increases, and the bacterial activity decreases rapidly. By adding sorbitol in the middle and late fermentation, the cells can be protected from environmental pressure, the cell survival ability is enhanced, and the metabolic load is reduced, the activity of pyruvate dehydrogenase, isocitrate dehydrogenase and cytochrome C oxidase is maintained, and the acid production level in the late fermentation is improved. Compared with the group without adding sorbitol, the acid production can be increased by 16g / L, and the conversion rate can be 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 Example 1, except that protocatechuic acid is used to replace catechol in seeds and fermentation medium. The final acid production after fermentation is 183 g / L, and the conversion rate is 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 Example 1, except that methyl gallate is used to replace propyl gallate in the feed liquid 1. The final acid production at the end of fermentation is 185 g / L, and the conversion rate is 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 Example 1, except that glycerol is used to replace sorbitol in the second feed liquid. The final acid production at the end of fermentation is 186 g / L, and the conversion rate is 66.1%.

[0124] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all 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 catechol to the seed culture medium and the fermentation culture medium, and during fermentation production, start feeding the first feeding liquid after the substrate sugar is completely consumed, and start feeding the second feeding liquid when the acid production concentration in the fermentation liquid reaches 140-165g / L; The feed liquid 1 is a glucose solution containing propyl gallate, the concentration of glucose in the feed liquid 1 is 68-72%, the concentration of propyl gallate is 10-50 mg / 100 g, and the residual sugar concentration of the fermentation liquid is controlled to be less than 5 g / L by the feed liquid 1. The second feeding liquid is a sorbitol solution, and the sorbitol concentration in the fermentation liquid is controlled to be 0.5-4 g / L by feeding the second feeding liquid.

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: 45-55 g / L glucose, 28-32 g / L corn steep liquor, 14-16 g / L corn steep liquor hydrolyzate, 3.8-4.2 g / L dipotassium hydrogen phosphate, 1.4-1.6 g / L magnesium sulfate, 9-11 mg / L ferrous sulfate, 9-11 mg / L manganese sulfate, 480-520 μg / L biotin, and 580-620 μg / L thiamine.

4. The method according to claim 3, characterized in that The fermentation medium also includes: 50 g / L glucose, 30 g / L corn steep liquor, 15 g / L corn steep liquor hydrolyzate, 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.

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

6. The method according to claim 5, characterized in that The seed culture medium also includes: 30 g / L glucose, 5 g / L yeast powder, 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.

7. The method according to any one of claims 1 to 6, characterized in that: The OD562nm of the seed solution is 10-15.

8. The method according to any one of claims 1 to 7, characterized in that: The fermentation bacteria is a thermosensitive Corynebacterium glutamicum, and the fermentation control conditions are: pH value 6.8-7.2, DO 20-40%, initial culture temperature 32°C, temperature change OD562nm 35-50, and temperature after temperature change 37-39°C.

9. A culture medium for producing glutamic acid by fermentation, characterized in that: The method comprises a basic fermentation medium, a first feeding liquid and a second feeding liquid, wherein the fermentation medium, the first feeding liquid and the second feeding liquid are respectively as described in any one of claims 1 to 4.

10. Use of the method according to any one of claims 1 to 8 or the culture medium according to claim 9 in improving the conversion rate of glutamate produced by fermentation.

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