Method for producing L-glutamic acid

Abstract

The invention belongs to the technical field of biology, and discloses a method for producing L-glutamic acid, which comprises the following steps: fermenting by using a fermentation culture medium containing oligopeptide PSGPR, then obtaining an L-glutamic acid wet crystal and a mother liquor through an evaporation and concentration process, separating and extracting the mother liquor, and recovering again to obtain the L-glutamic acid wet crystal. The method can improve the glutamic acid fermentation efficiency, optimize the separation and purification process and improve the glutamic acid yield.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for producing L-glutamic acid. Background Technology

[0002] Glutamic acid, an important industrial raw material, is mainly produced through microbial fermentation. Its yield and efficiency are influenced by various factors, including strain performance, culture conditions, and downstream processing techniques. With the development of biotechnology, traditional processes are constantly being replaced by new technologies to achieve efficient, energy-saving, and environmentally friendly industrial production. Existing technologies offer numerous optimization schemes for fermentation conditions and culture media, some of which are listed below.

[0003] CN110885866A designed fermentation medium A (containing glucose, yeast extract, biotin, and rare earth elements) and medium B (containing succinic acid, urea, and chitosan), achieving a two-step fermentation process through phased addition: first, medium A is added to promote cell growth for 24 hours, followed by the addition of medium B for continuous acid production for 24 hours, resulting in a 9.2% improvement over traditional processes. The rare earth element Ce³⁺ may enhance metabolic flux by activating key enzyme activity, while chitosan can regulate cell membrane permeability and promote glutamate efflux. By dividing the medium into dedicated formulas for the growth and acid production stages, premature nutrient inhibition or depletion is avoided.

[0004] After 30 hours of fermentation, CN110904168A was fed with a 15-25 g / L dipotassium hydrogen phosphate solution, which could inhibit phosphofructokinase activity by regulating intracellular ATP levels, thus preventing excessive carbon metabolism flux towards the EMP pathway and further increasing the sugar-acid conversion rate by 3%-5%.

[0005] CN101319238A discloses the application of apigenin stearate in glutamic acid fermentation. Adding 0.05-0.2‰ of the total volume of apigenin stearate to the fermentation broth during the 10th to 40th hour of fermentation using Corynebacterium glutamicum can increase the yield of glutamic acid with almost no effect on the survival and growth of the strain. Its effect is better than adding Tween-60, and the dosage is smaller.

[0006] "Optimization of Culture Medium for Solid-State Fermentation of Pork Bone Extract to Produce Glutamic Acid by Compound Microorganisms, Food Industry Science and Technology 2016" In solid-state fermentation by compound microorganisms, using pork bone extract (600 g / kg) and wheat bran (100 g / kg) as the substrate, and adding ammonium sulfate (15 g / kg) and triglyceride monostearate (3 g / kg), the glutamic acid yield can be increased to 167.8 g / kg (dry weight) through the synergistic metabolism of Aspergillus oryzae and Monascus purpureus, and the total amino acid yield reaches 728.6 g / kg, providing a new approach for the industrialization of solid-state fermentation.

[0007] Based on the aforementioned technical issues, the applicant proposed a process to improve the fermentation rate of glutamic acid (CN2025118460760). This process uses a fermentation medium containing specific oligopeptides, thereby improving fermentation efficiency. To further improve the yield and productivity of glutamic acid, further optimization of the separation and extraction process is needed.

[0008] Document CN118272465A discloses a production process for high-quality glutamic acid, which includes the following steps: step 1) glutamic acid fermentation, step 2) centrifugation, step 3) ultrafiltration, step 4) decolorization, step 5) isoelectric crystallization, step 6) drying, and step 7) sieving. The product obtained by the above production process is granular, uniform in size, and has a purity of over 95%, but the recovery and extraction rate is relatively low.

[0009] Document CN110437088A discloses a method for recovering glutamic acid from an isoelectric mother liquor of glutamic acid, comprising the following steps: treating the isoelectric mother liquor of glutamic acid through an ultrafiltration membrane, then subjecting the ultrafiltration membrane filtrate to electrodialysis, and evaporating, concentrating, cooling, crystallizing, and separating the electrodialysis retentate to obtain glutamic acid crystals, thereby achieving the purpose of recovering glutamic acid from the isoelectric mother liquor; the recovery rate of glutamic acid from the isoelectric mother liquor can reach more than 50%, and the total recovery rate of glutamic acid can reach more than 90%. Summary of the Invention

[0010] In order to further improve the production efficiency of glutamic acid, the present invention provides a method for producing L-glutamic acid, which can improve the fermentation efficiency of glutamic acid, optimize the separation and purification process, and increase the yield of glutamic acid.

[0011] The present invention is achieved through the following scheme.

[0012] A method for producing L-glutamic acid includes the following steps: Fermentation was carried out using a fermentation medium containing the oligopeptide PSGPR. Then, L-glutamic acid wet crystals and mother liquor were obtained by evaporation and concentration. The mother liquor was separated and extracted, and L-glutamic acid wet crystals were recovered again.

[0013] Specifically, the method includes the following steps: Step 1) Fermentation of the strain: The seed liquid of the engineered bacteria that produces L-glutamic acid is inoculated into a fermenter containing fermentation medium for fermentation culture. The fermentation culture is carried out for 40 hours, and the fermentation broth is collected. The fermentation medium contains the oligopeptide PSGPR. Step 2) Separation and extraction: The fermentation broth is centrifuged using a high-speed disc separator to collect the supernatant liquid and wet bacterial cells. The wet bacterial cells are sent to the by-product workshop for drying, and the dried product is bacterial protein. The supernatant liquid is evaporated and concentrated to obtain a concentrated solution. The concentrated solution is then introduced into a cooling crystallization tank, where the pH is adjusted to 3.2-3.3, stirred, and cooled to 10°C at a rate of 2°C / h. After standing for 6 hours, sedimentation separation is performed to obtain wet glutamic acid crystals and mother liquor. The dried solution yields L-glutamic acid. The mother liquor was filtered through an inorganic ceramic membrane, and the filtrate was collected and processed in an electrodialysis system. The filtrate was then evaporated and concentrated to obtain a concentrated solution. The concentrated solution was then introduced into a cooling crystallization tank, where the pH was adjusted to 3.2-3.3. The solution was stirred and cooled to 8°C at a rate of 2°C / h. Finally, the solution was allowed to stand for 10 hours for sedimentation and separation to obtain wet crystals of glutamic acid. The crystals were then dried to obtain L-glutamic acid.

[0014] Preferably, the fermentation medium comprises: 85 g / L glucose, 45 g / L corn steep liquor, 3 g / L potassium dihydrogen phosphate, 3 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 0.2 g / L ferrous sulfate heptahydrate, 0.05 g / L manganese sulfate monohydrate, 5 mg / L vitamin B1, 20 μg / L biotin, and 0.5 g / L oligopeptide.

[0015] Preferably, the high-speed disc drive operates at a speed of 5000 rpm for 3 minutes.

[0016] Preferably, the electrodialysis treatment system uses alternating cation-anion membranes, with each pair of membranes operating at a voltage of 0.5V, a flow rate of 10cm / s, and a temperature of 30℃.

[0017] Preferably, the inorganic ceramic membrane has a molecular weight cutoff of 10,000 Da.

[0018] Preferably, the upper liquid enters a double-effect evaporation system to evaporate and concentrate it five times to obtain a concentrated liquid.

[0019] Preferably, the electrodialysis retentate is evaporated and concentrated 10 times in a double-effect evaporation system to obtain a concentrated solution.

[0020] Preferably, during the fermentation process of the strain, the fermentation temperature is controlled at 35°C. The ventilation volume is 0.9. The vvm tank pressure is 0.05 MPa, the stirring speed is 200 r / min, the residual sugar is maintained at no less than 1.0% by adding a 60% glucose solution, the pH is controlled at 6.5 by adding ammonia, and defoaming agent is added to defoam.

[0021] Compared with the prior art, the beneficial effects achieved by the present invention mainly include, but are not limited to, the following aspects: In glutamate fermentation, bacterial strains directly influence metabolic flux distribution, product synthesis rates, and resource utilization efficiency, becoming a core factor limiting fermentation efficiency. Strain proliferation mainly occurs in the early and middle stages of fermentation, with acid production being the primary focus in the later stages. This invention experimentally verifies the effect of sea cucumber oligopeptides on strain performance and optimizes the truncated oligopeptides. Although conventional short peptides can be used as nitrogen sources for strain fermentation, experimental results show that the oligopeptides of this invention do not act as nitrogen sources, but rather function as signaling molecules. When the extracellular oligopeptide concentration reaches a certain level, it binds to cell surface receptors, activating the phosphokinase system, allowing the signal to enter the cell, ultimately regulating the expression of corresponding glutamate synthesis-related regulators, thereby promoting glutamate synthesis.

[0022] This invention employs a two-stage separation and extraction process, which enables the yield of L-glutamic acid to reach 96.4%, significantly higher than the 75%-90% yield in the traditional isoelectric exchange process. Furthermore, it reduces the production of wastewater and the consumption of raw materials such as sulfuric acid and liquid ammonia, thereby lowering production costs. Detailed Implementation

[0023] To enable those skilled in the art to better understand the technical solutions of this application, the technical solutions of this application will be clearly and completely described below in conjunction with specific embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0024] Example 1 The optimization scheme for L-glutamic acid fermentation process includes the following steps: Seed culture of *Bacillus flavus* GDK-9 was obtained using conventional methods, and the OD value of the seed culture was controlled within the specified range. 600nm The inoculum was 18, and then 6% was inoculated into a fermenter containing fermentation medium for fermentation culture. Fermentation was carried out for 40 hours, and the fermentation broth was collected. Throughout the fermentation process, the fermentation temperature was controlled at 35℃. The ventilation volume is 0.9. The fermentation tank pressure is 0.05 MPa, the stirring speed is 200 r / min, the residual sugar is maintained at no less than 1.0% by adding a 60% glucose solution, the pH is controlled at 6.5 by adding ammonia, and defoaming is achieved by adding an antifoaming agent. The components of the fermentation tank culture medium are: glucose 85 g / L, corn steep liquor 45 g / L, potassium dihydrogen phosphate 3 g / L, dipotassium hydrogen phosphate 3 g / L, magnesium sulfate heptahydrate 0.5 g / L, ferrous sulfate heptahydrate 0.2 g / L, manganese sulfate monohydrate 0.05 g / L, vitamin B1 5 mg / L, and biotin 20 μg / L.

[0025] 1. The oligopeptides of this invention were synthesized by Jier Biochemical Co., Ltd. using a solid-phase synthesis method. The performance and effects of the oligopeptides of this invention were verified using the above-described fermentation process. Oligopeptides were added to the fermentation medium at concentrations of 0.25, 0.5, 1, and 2 g / L, with each batch repeated three times, and the average value was taken. In the glutamic acid fermentation process, the bacterial density reached its peak at approximately 15-20 hours. In the early stage of fermentation, bacterial proliferation was dominant; in the middle and later stages, the bacterial density maintained this peak and decreased to some extent in the later stages. Therefore, the bacterial density in the fermentation broth was measured at 16 hours of fermentation to determine the effect of the oligopeptides on bacterial growth. OD600 was used as an indicator of bacterial growth density in the culture medium. See Tables 1-4 for details.

[0026] Table 1 (0.25 g / L)

[0027] Table 2 (0.5 g / L)

[0028] Table 3 (1g / L)

[0029] Table 4 (2g / L)

[0030] As shown in Tables 1-4, PSGPR showed the best growth-promoting effect on the strain, followed by GPSGPR, while the other oligopeptides had little or no effect. PSGPR oligopeptides were more beneficial to strain proliferation within the concentration range of 0.5-2 g / L in the culture medium, but the differences within this range were not significant; therefore, a concentration of 0.5 g / L was chosen for subsequent experiments. Comparison of the effects of different oligopeptides revealed that in some oligopeptide addition groups, there was no significant improvement in strain growth. This indicates that the role of oligopeptides in the culture medium is not as a nitrogen source, but rather as signal transduction factors.

[0031] 2. The fermentation time was 40 h. The content of glutamic acid in the fermentation broth of the experimental group with oligopeptide addition of 0.5 g / L was detected to verify the effect of oligopeptide on the fermentation efficiency of glutamic acid. See Table 5 for details.

[0032] Table 5

[0033] As shown in Table 5, PSGPR showed the best positive regulatory effect on glutamate fermentation yield, increasing it by 24.5% compared to the control group (without addition). GPSGPR was the second best, increasing it by 17.2% compared to the control group (without addition). Comparing the effects of the above oligopeptides on strain density, PSGPR increased the strain density by 9.6% compared to the control group (without addition), and GPSGPR increased the strain density by 7.0% compared to the control group (without addition). In conclusion, the oligopeptides of this invention have a stronger effect on glutamate fermentation efficiency than on strain density. This indicates that the oligopeptides of this invention can not only improve strain proliferation but also potentially act as extracellular signal regulators, binding to cells and promoting glutamate expression by regulating intracellular glutamate synthesis and secretion-related signaling pathways.

[0034] Example 2

[0035] The optimized process for L-glutamic acid separation and purification includes the following steps: 100L of glutamic acid fermentation broth with a glutamic acid concentration of 140.9g / L was centrifuged using a high-speed disc centrifuge at 5000rpm for 3 minutes. The supernatant and wet bacterial cells were collected. The wet bacterial cells were sent to the by-product workshop for drying, yielding bacterial protein. The supernatant was then concentrated 5 times using a double-effect evaporation system. The concentrated liquid was then transferred to a cooling crystallization tank, where the pH was adjusted to 3.2. The mixture was stirred at 100rpm and cooled to 10℃ at a rate of 2℃ / h. After standing for 6 hours, sedimentation separation was performed to obtain wet glutamic acid crystals and mother liquor. After drying, 10.93kg of L-glutamic acid was obtained. The mother liquor was filtered through an inorganic ceramic membrane with a molecular weight cutoff of 10,000 Da. The filtrate was collected and fed into an electrodialysis system (using alternating cation-anion membranes, 10 pairs of membranes, each pair operating at 0.5V, current density of 15mA / cm², flow rate of 10cm / s, and temperature of 30℃). The electrodialysis retentate was collected and then concentrated 10 times in a double-effect evaporation system. The concentrate was then fed into a cooling crystallizer, where the pH was adjusted to 3.2, stirred at 100 rpm, and cooled to 8℃ at a rate of 2℃ / h. Finally, the mixture was allowed to stand for 10 hours for sedimentation and separation to obtain wet glutamic acid crystals. After drying, 2.65 kg of L-glutamic acid was obtained.

[0036] Calculations show that the yield of L-glutamic acid can reach 96.4%, which is significantly higher than the 75%-90% yield in the traditional isoelectric exchange process. It can also reduce the production of wastewater and the consumption of raw materials such as sulfuric acid and liquid ammonia, thereby reducing production costs.

[0037] The above examples are merely preferred embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A method for producing L-glutamic acid, comprising the following steps: Fermentation was carried out using a fermentation medium containing the oligopeptide PSGPR. Then, L-glutamic acid wet crystals and mother liquor were obtained by evaporation and concentration. The mother liquor was separated and extracted, and L-glutamic acid wet crystals were recovered again.

2. The method according to claim 1, characterized in that, The method includes the following steps: Step 1) Fermentation of the strain: The seed liquid of the engineered bacteria that produces L-glutamic acid is inoculated into a fermenter containing fermentation medium for fermentation culture for 40 hours, and the fermentation broth is collected; the fermentation medium contains the oligopeptide PSGPR. Step 2) Separation and extraction: The fermentation broth is centrifuged using a high-speed disc separator to collect the supernatant liquid and wet bacterial cells. The wet bacterial cells are sent to the by-product workshop for drying, and the dried product is bacterial protein. The supernatant liquid is evaporated and concentrated to obtain a concentrated solution. The concentrated solution is then introduced into a cooling crystallization tank, where the pH is adjusted to 3.2-3.3, stirred, and cooled to 10°C at a rate of 2°C / h. After standing for 6 hours, sedimentation separation is performed to obtain wet glutamic acid crystals and mother liquor. The dried solution yields L-glutamic acid. The mother liquor was filtered through an inorganic ceramic membrane, and the filtrate was collected and processed in an electrodialysis system. The filtrate was then evaporated and concentrated to obtain a concentrated solution. The concentrated solution was then introduced into a cooling crystallization tank, where the pH was adjusted to 3.2-3.

3. The solution was stirred and cooled to 8°C at a rate of 2°C / h. Finally, the solution was allowed to stand for 10 hours for sedimentation and separation to obtain wet crystals of glutamic acid. The crystals were then dried to obtain L-glutamic acid.

3. The method according to claim 2, characterized in that, The components of the fermentation medium are: Glucose 85g / L, corn steep liquor 45g / L, potassium dihydrogen phosphate 3g / L, dipotassium hydrogen phosphate 3g / L, magnesium sulfate heptahydrate 0.5g / L, ferrous sulfate heptahydrate 0.2g / L, manganese sulfate monohydrate 0.05g / L, vitamin B1 5mg / L, biotin 20μg / L, oligopeptide 0.5g / L.

4. The method according to claim 2, characterized in that, The high-speed disc drive operates at 5000 rpm for 3 minutes.

5. The method according to claim 2, characterized in that, The electrodialysis treatment system uses alternating cation-anion membranes, with each pair of membranes operating at a voltage of 0.5V, a current density of 15mA / cm², a flow rate of 10cm / s, and a temperature of 30℃.

6. The method according to claim 2, characterized in that, The inorganic ceramic membrane has a molecular weight cutoff of 10,000 Da.

7. The method according to claim 2, characterized in that, The upper liquid enters a double-effect evaporation system to evaporate and concentrate it five times to obtain a concentrated liquid.

8. The method according to claim 2, characterized in that, The electrodialysis retentate is then evaporated and concentrated 10 times in a double-effect evaporation system to obtain a concentrated solution.

9. The method according to claim 2, characterized in that, During the fermentation process of the strain, the fermentation temperature was controlled at 35℃. The ventilation volume is 0.

9. The vvm tank pressure is 0.05 MPa, the stirring speed is 200 r / min, the residual sugar is maintained at no less than 1.0% by adding a 60% glucose solution, the pH is controlled at 6.5 by adding ammonia, and defoaming agent is added to defoam.

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