Pantoea agglomerans with high glutamic acid yield, method and application
By optimizing the fermentation process and using the clump-forming pantothenic acid PA-GLU-10, the problems of high liquid ammonia consumption and difficulty in increasing yield in existing technologies have been solved, achieving efficient glutamic acid production in a low pH environment and reducing production costs.
Patent Information
- Application Number
- CN202511064838.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-31
- Publication Date
- 2025-11-21
AI Technical Summary
Existing glutamic acid fermentation production suffers from problems such as high liquid ammonia consumption, difficulty in increasing yield, and high costs. Furthermore, pH adjustment during fermentation increases production costs and affects cell growth and glutamic acid production.
A high-glutamic acid-producing pantoea strain, PA-GLU-10, was used to reduce the amount of liquid ammonia used and improve fermentation efficiency and yield by optimizing pH control and feeding methods during the fermentation process.
It enables efficient production of glutamic acid in a low pH environment, reduces the amount of liquid ammonia used, increases fermentation rate and yield, and lowers production costs, making it suitable for continuous fermentation and industrial production.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial technology, and in particular to a high-glutamic acid-producing pantothenic bacterium, its method, and its application. Background Technology
[0002] Glutamic acid is a traditional, widely fermented product with applications in food, medicine, industry, and agriculture. Its monosodium glutamate (MSG), its salt, is an important flavoring agent. Superior high-yield industrial strains of glutamate are key to its fermentation production.
[0003] In the industrial fermentation production of glutamic acid, Corynebacterium glutamicum is commonly used as the production strain. Its optimal growth pH is between 7 and 8. As fermentation progresses and glutamic acid accumulates, the pH of the fermentation system continuously decreases. To make the environment more suitable for glutamic acid fermentation, liquid ammonia is typically added to adjust the pH of the fermentation broth, increasing production costs. Furthermore, during the initial stage of glutamic acid production, the bacterial cells proliferate rapidly while glutamic acid accumulates in small amounts. However, in the later stages, as glutamic acid is produced in large quantities, the continuously increasing glutamic acid concentration inhibits bacterial growth and affects glutamic acid yield. Therefore, selecting superior, high-yield industrial production strains is one of the important ways to solve the common problems in industrial glutamic acid production, such as high liquid ammonia consumption, difficulty in further increasing yield, and high costs. Summary of the Invention
[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a high-glutamic acid-producing pantothenic bacterium, its method, and its application.
[0005] The technical solution adopted by this invention to solve its technical problem is:
[0006] A high-glutamic acid-producing Pantoea agglomerans strain, named PA-GLU-10, classified as Pantoea agglomerans, with accession number GDMCC 65358, accession date October 28, 2024, deposited at Guangdong Provincial Center for Microbial Culture Collection, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0007] Furthermore, the yield of glutamic acid during the 36-hour shake-flask fermentation of the Pantothenic agglomerates was 134 g / L, and the decolorization time of methylene blue during the 96-hour shake-flask fermentation of the Pantothenic agglomerates was 20-25 min.
[0008] Application of a type of pantothenic bacteria as described above in glutamic acid production.
[0009] An application of the pantothenic bacteria as described in claim 1 or 2 in the continuous fermentation production of glutamic acid.
[0010] The method for producing glutamic acid using the above-described method of pantothenic agglomerate fermentation includes the following steps:
[0011] (1) Seed culture: Inoculate the clustered pantothecin into the seed culture medium and culture at 35℃ and 220 r / min for 16 h to obtain the seed culture solution;
[0012] (2) Shake-flask fermentation: The seed culture was transferred to the fermentation medium at a rate of 20%, with an initial pH of 6. The culture was carried out at 35°C and a rotation speed of 220 r / min. The pH was adjusted to 5.8-6 every 2 hours using ammonia. After 12 hours of fermentation, when the glutamic acid content in the fermentation reached more than 50 g / L, the addition of ammonia was reduced, and the fermentation pH was controlled to 4.5-4.7. 0.1 g of glutamic acid crystals were added to the fermentation system. After that, the pH was adjusted to 4.5-4.7 every 2 hours using ammonia. At the same time, the glucose and glutamic acid content in the fermentation broth was sampled and tested. After the glucose was depleted, high-glucose fermentation replenishment medium was used to replenish the broth so that the glucose in the fermentation system was maintained below 10 g / L. The fermentation was completed after 36 hours.
[0013] Furthermore, the seed culture medium is formulated as follows: molasses 80 g / L, magnesium sulfate 0.25 g / L, ammonium sulfate 3 g / L, potassium dihydrogen phosphate 2.5 g / L, yeast extract 2 g / L, pH = 6;
[0014] The fermentation medium is formulated as follows: glucose 75g / L, ammonium chloride 15g / L, magnesium sulfate 0.25g / L, corn steep liquor 4ml / L, ferric sulfate 20mg / L, manganese sulfate 20mg / L, sodium dihydrogen phosphate 5g / L, potassium chloride 5g / L, and phosphate to adjust the pH to 7.
[0015] The formula for the high-sugar fermentation replenishment medium is as follows: glucose 700g / L, ammonium chloride 4g / L, magnesium sulfate 0.25g / L, corn steep liquor 5ml / L, ferric sulfate 20mg / L, manganese sulfate 20mg / L, sodium dihydrogen phosphate 5g / L, potassium chloride 5g / L, and phosphate to adjust the pH to 6.
[0016] The solvent for all the above culture media is water.
[0017] The method for producing glutamic acid using the above-described method of pantothenic agglomerate fermentation includes the following steps:
[0018] (1) Seed culture: Inoculate the clustered pantothecin into the seed culture medium and culture at 35℃ and 220 r / min for 16 h to obtain the seed culture solution;
[0019] (2) Shake-flask fermentation: The seed culture was transferred to the fermentation medium at a rate of 20%, with an initial pH of 6. The culture was carried out at 35°C and a rotation speed of 220 r / min. The pH was adjusted to 5.8-6 every 2 hours using ammonia. After 12 hours of fermentation, when the glutamic acid content in the fermentation reached more than 50 g / L, the addition of ammonia was reduced, and the fermentation pH was controlled to 4.5-4.7. 0.1 g of glutamic acid crystals were added to the fermentation system. After that, the pH was adjusted to 4.5-4.7 every 2 hours using ammonia. At the same time, the glucose and glutamic acid content in the fermentation broth was sampled and tested. After the glucose was depleted, high-glucose fermentation replenishment medium was used to replenish the broth so that the glucose in the fermentation system was maintained below 10 g / L. The fermentation was completed after 96 hours.
[0020] Furthermore, the seed culture medium is formulated as follows: molasses 80 g / L, magnesium sulfate 0.25 g / L, ammonium sulfate 3 g / L, potassium dihydrogen phosphate 2.5 g / L, yeast extract 2 g / L, pH = 6;
[0021] The fermentation medium is formulated as follows: glucose 75g / L, ammonium chloride 15g / L, magnesium sulfate 0.25g / L, corn steep liquor 4ml / L, ferric sulfate 20mg / L, manganese sulfate 20mg / L, sodium dihydrogen phosphate 5g / L, potassium chloride 5g / L, and phosphate to adjust the pH to 7.
[0022] The formula for the high-sugar fermentation replenishment medium is as follows: glucose 700g / L, ammonium chloride 4g / L, magnesium sulfate 0.25g / L, corn steep liquor 5ml / L, ferric sulfate 20mg / L, manganese sulfate 20mg / L, sodium dihydrogen phosphate 5g / L, potassium chloride 5g / L, and phosphate to adjust the pH to 6.
[0023] The solvent for all the above culture media is water.
[0024] The advantages and positive effects of this invention are as follows:
[0025] 1. This invention provides a strain of Pantoea agglomerans, which can be used for the fermentation production of glutamic acid. It has the advantages of being tolerant to low pH during fermentation, producing glutamic acid quickly, and being suitable for continuous culture. It overcomes the problems of poor acid tolerance and high cost of existing fermentation strains, and is expected to reduce the production cost of glutamic acid.
[0026] 2. Compared with Corynebacterium glutamicum, the Pantotheca agglomeratum strain of this invention can tolerate a lower fermentation pH (6.0-4.0), thereby reducing the amount of liquid ammonia used during fermentation; it has a faster fermentation rate and can produce glutamic acid more efficiently (Pantobacterium agglomeratum yields 124-134 g / L in a 24-hour small-scale fermentation, while Corynebacterium glutamicum yields 90-100 g / L in a 24-hour small-scale fermentation); it can maintain good strain activity even during continuous fermentation, making it suitable for continuous fermentation of glutamic acid and extending the fermentation cycle (Table 3), which helps to reduce production costs and is suitable for industrial production.
[0027] 3. The glutamic acid producing strain PA-GLU-10 of the present invention exhibits excellent glutamic acid production capacity under acidic conditions and maintains good cell viability during a long fermentation period. Therefore, the present invention provides an excellent industrial strain for high-yield glutamic acid production that is acid-resistant, high-yielding, and low-cost. Detailed Implementation
[0028] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.
[0029] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.
[0030] A high-glutamic acid-producing Pantoea agglomerans strain, named PA-GLU-10, classified as Pantoea agglomerans, with accession number GDMCC 65358, accession date October 28, 2024, deposited at Guangdong Provincial Center for Microbial Culture Collection, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0031] Preferably, the yield of glutamic acid in the Pantotheca agglomerata shake flask fermentation after 36 hours is 134 g / L, and the decolorization time of methylene blue in the Pantotheca agglomerata shake flask fermentation after 96 hours is 20-25 minutes.
[0032] Application of a type of pantothenic bacteria as described above in glutamic acid production.
[0033] An application of the pantothenic bacteria as described in claim 1 or 2 in the continuous fermentation production of glutamic acid.
[0034] The method for producing glutamic acid using the above-described method of pantothenic agglomerate fermentation includes the following steps:
[0035] (1) Seed culture: Inoculate the clustered pantothecin into the seed culture medium and culture at 35℃ and 220 r / min for 16 h to obtain the seed culture solution;
[0036] (2) Shake-flask fermentation: The seed culture was transferred to the fermentation medium at a rate of 20%, with an initial pH of 6. The culture was carried out at 35°C and a rotation speed of 220 r / min. The pH was adjusted to 5.8-6 every 2 hours using ammonia. After 12 hours of fermentation, when the glutamic acid content in the fermentation reached more than 50 g / L, the addition of ammonia was reduced, and the fermentation pH was controlled to 4.5-4.7. 0.1 g of glutamic acid crystals were added to the fermentation system. After that, the pH was adjusted to 4.5-4.7 every 2 hours using ammonia. At the same time, the glucose and glutamic acid content in the fermentation broth was sampled and tested. After the glucose was depleted, high-glucose fermentation replenishment medium was used to replenish the broth so that the glucose in the fermentation system was maintained below 10 g / L. The fermentation was completed after 36 hours.
[0037] Preferably, the seed culture medium is formulated as follows: molasses 80 g / L, magnesium sulfate 0.25 g / L, ammonium sulfate 3 g / L, potassium dihydrogen phosphate 2.5 g / L, yeast extract 2 g / L, pH = 6;
[0038] The fermentation medium is formulated as follows: glucose 75g / L, ammonium chloride 15g / L, magnesium sulfate 0.25g / L, corn steep liquor 4ml / L, ferric sulfate 20mg / L, manganese sulfate 20mg / L, sodium dihydrogen phosphate 5g / L, potassium chloride 5g / L, and phosphate to adjust the pH to 7.
[0039] The formula for the high-sugar fermentation replenishment medium is as follows: glucose 700g / L, ammonium chloride 4g / L, magnesium sulfate 0.25g / L, corn steep liquor 5ml / L, ferric sulfate 20mg / L, manganese sulfate 20mg / L, sodium dihydrogen phosphate 5g / L, potassium chloride 5g / L, and phosphate to adjust the pH to 6.
[0040] The solvent for all the above culture media is water.
[0041] The method for producing glutamic acid using the above-described method of pantothenic agglomerate fermentation includes the following steps:
[0042] (1) Seed culture: Inoculate the clustered pantothecin into the seed culture medium and culture at 35℃ and 220 r / min for 16 h to obtain the seed culture solution;
[0043] (2) Shake-flask fermentation: The seed culture was transferred to the fermentation medium at a rate of 20%, with an initial pH of 6. The culture was carried out at 35°C and a rotation speed of 220 r / min. The pH was adjusted to 5.8-6 every 2 hours using ammonia. After 12 hours of fermentation, when the glutamic acid content in the fermentation reached more than 50 g / L, the addition of ammonia was reduced, and the fermentation pH was controlled to 4.5-4.7. 0.1 g of glutamic acid crystals were added to the fermentation system. After that, the pH was adjusted to 4.5-4.7 every 2 hours using ammonia. At the same time, the glucose and glutamic acid content in the fermentation broth was sampled and tested. After the glucose was depleted, high-glucose fermentation replenishment medium was used to replenish the broth so that the glucose in the fermentation system was maintained below 10 g / L. The fermentation was completed after 96 hours.
[0044] Preferably, the seed culture medium is formulated as follows: molasses 80 g / L, magnesium sulfate 0.25 g / L, ammonium sulfate 3 g / L, potassium dihydrogen phosphate 2.5 g / L, yeast extract 2 g / L, pH = 6;
[0045] The fermentation medium is formulated as follows: glucose 75g / L, ammonium chloride 15g / L, magnesium sulfate 0.25g / L, corn steep liquor 4ml / L, ferric sulfate 20mg / L, manganese sulfate 20mg / L, sodium dihydrogen phosphate 5g / L, potassium chloride 5g / L, and phosphate to adjust the pH to 7.
[0046] The formula for the high-sugar fermentation replenishment medium is as follows: glucose 700g / L, ammonium chloride 4g / L, magnesium sulfate 0.25g / L, corn steep liquor 5ml / L, ferric sulfate 20mg / L, manganese sulfate 20mg / L, sodium dihydrogen phosphate 5g / L, potassium chloride 5g / L, and phosphate to adjust the pH to 6.
[0047] The solvent for all the above culture media is water.
[0048] Specifically, the relevant preparation and testing methods are as follows:
[0049] The culture medium used in this invention is as follows:
[0050] (1) Acid-resistant bacteria enrichment plate: 5 g / L peptone, 2.5 g / L yeast powder, 5 g / L sodium chloride, 38.5 g / L BHI (brain and heart extract broth), 20 g / L glucose, 20 g / L agar, and pH adjusted to 4.5 with hydrochloric acid.
[0051] (2) High glutamic acid screening medium: peptone 5g / L, yeast powder 2.5g / L, sodium chloride 5g / L, BHI (brain and heart extract broth) 38.5g / L, glucose 20g / L, L-glutamic acid 30g / L, pH 4.5.
[0052] (3) Activation culture medium: peptone 5g / L, yeast powder 2.5g / L, sodium chloride 5g / L, BHI (brain and heart extract broth) 38.5g / L, glucose 20g / L, pH 6.
[0053] (4) Glutamine liquid culture medium: peptone 5g / L, yeast powder 2.5g / L, sodium chloride 5g / L, BHI (brain and heart extract broth) 38.5g / L, glucose 20g / L, glutamine 5g / L, pH 4.5.
[0054] (5) Succinic acid as the sole carbon source solid culture medium: peptone 5g / L, yeast powder 2.5g / L, sodium chloride 5g / L, BHI (brain and heart extract broth) 38.5g / L, succinic acid 30g / L, agar 20g / L, pH 4.5.
[0055] (6) Seed culture medium: molasses 80g / L, magnesium sulfate 0.25g / L, ammonium sulfate 3g / L, potassium dihydrogen phosphate 2.5g / L, yeast extract 2g / L, pH=6.
[0056] (7) Fermentation medium: glucose 75g / L, ammonium chloride 15g / L, magnesium sulfate 0.25g / L, corn steep liquor 4ml / L, ferric sulfate 20mg / L, manganese sulfate 20mg / L, sodium dihydrogen phosphate 5g / L, potassium chloride 5g / L, and phosphate to adjust pH to 7.
[0057] (8) High sugar fermentation replenishment medium: glucose 700g / L, ammonium chloride 4g / L, magnesium sulfate 0.25g / L, corn steep liquor 5ml / L, ferric sulfate 20mg / L, manganese sulfate 20mg / L, sodium dihydrogen phosphate 5g / L, potassium chloride 5g / L, and phosphate to adjust pH 6.
[0058] (9) PBS buffer: sodium chloride 8.0 g / L, potassium dihydrogen phosphate 0.2 g / L, disodium hydrogen phosphate dodecahydrate 2.9 g / L, potassium chloride 0.2 g / L, pH adjusted to 7.4, solvent is water.
[0059] The solvent for all the above culture media is water.
[0060] Example 1
[0061] Samples were taken from the rice used in the brewing process of Shaoxing rice wine (November 2019, Shaoxing City, Zhejiang Province, COFCO Shaoxing Wine Co., Ltd., the raw material for Shaoxing rice wine). After serial dilution with physiological saline, the samples were spread onto acid-resistant bacteria enrichment plates and incubated at 32°C until approximately 100–200 single colonies grew. The best-growing colonies were selected and inoculated into 96-well deep-well plates containing high-glutamic acid screening medium, and incubated at 32°C for 48 hours at a rotation speed of 800 rpm. Vigorous-growing strains were selected and inoculated into seed culture medium. The seed culture was transferred to the fermentation medium at a 10% inoculum volume and incubated at 32°C and 220 rpm for 48 hours. The glutamic acid content of the fermentation broth in each well was detected using an SBA-40C biosensor analyzer. Glutamic acid was detectable in more than 10 strains, with the highest glutamic acid yield being 9.3 g / L.
[0062] The strains selected in the final screening had round, raised colonies with smooth, regular edges, a slightly yellow color, and were Gram-negative.
[0063]
[0064] The above-mentioned strains were clumped together as Pantothenia glutinosa PA-96 and cultured in activation medium at 32°C and 220 rpm until the logarithmic phase. Nitrosoguanidine (NTG) was added to the bacterial suspension at a final concentration of 0.4–0.6 mg / mL and incubated at 32°C for 15–20 min. After treatment, the suspension was centrifuged at 5000 rpm for 3 min, the supernatant was removed, and the suspension was washed three times with activation medium. The suspension was then inoculated into pH 4.5 high-glutamic acid selection medium and cultured at 32°C and 220 rpm for 48 h as one generation. Three generations were continuously cultured to obtain a first-screen bacterial suspension.
[0065] The bacterial suspension was diluted with 0.9% physiological saline to prepare a bacterial suspension with OD=0.1. It was then subjected to UV mutagenesis (5 mL of bacterial suspension was evenly spread on a 6 cm sterile petri dish and irradiated with a 30 W UV lamp at a distance of 20 cm for 45-60 s). All the mutagenic bacteria were inoculated into glutamine liquid medium at pH 4.5 and cultured at 32℃ and 220 rpm for 48 h as one generation. After three generations, the second bacterial suspension was obtained.
[0066] The second-screened bacterial suspension was diluted with 0.9% physiological saline to prepare a bacterial suspension with OD=0.1. ARTP mutagenesis was then performed (10 μL bacterial suspension, helium as the working gas for the plasma, power 120 W, gas flow rate 10 slpm, treatment time 25 s and 30 s). The mutagenized bacteria were plated on a solid medium containing succinic acid as the sole carbon source at pH 4.5 and incubated at 35°C for 24-48 hours until a series of single colonies grew on the plates.
[0067] Single colonies grown on the above plates were inoculated into 96-well deep-well plates containing 0.3 mL of seed culture medium and incubated at 35°C for 24 h at a rotation speed of 800 rpm. The seed culture was then transferred at a 10% inoculum to 96-well deep-well plates containing 0.8 mL of fermentation medium per well and incubated at 35°C and 800 rpm for 24 h. Finally, urea at a final concentration of 3% (w / w) was added to each well, and fermentation continued for another 48 h to complete the fermentation. The SBA-40C biosensor analyzer detected the glutamic acid content in the fermentation broth of each well and selected the top 10 strains with the highest glutamic acid production. The glutamic acid production was as follows: 1# 20 g / L, 2# 19 g / L, 3# 21 g / L, 4# 16 g / L, 5# 23 g / L, 6# 26 g / L, 7# 20 g / L, 8# 25 g / L, 9# 21 g / L, and 10# 24 g / L (Table 1).
[0068] Table 196 Deep-well Plate Fermentation Production of Glutamic Acid
[0069] strain number Fermented glutamic acid production (g / L) 1# 20 2# 19 3# 21 4# 16 5# 23 6# 26 7# 20 8# 25 9# 21 10# 24
[0070] Example 2
[0071] Shake flask fermentation screening: (1) Seed culture: Use a 250mL baffled Erlenmeyer flask (if equipped with a baffle), fill it with 20mL of seed culture medium, and inoculate the 10 strains with the highest glutamic acid production, i.e., strains 1# to 10#. Culture at 35℃ and 220r / min for 16h. (2) Shake flask fermentation: Use a 500mL concave Erlenmeyer flask, fill it with 75mL of fermentation culture medium, add 20% transfer volume to the fermentation culture medium, initial pH 6, culture at 35℃ and 220r / min. Adjust the pH to 5.8-6 with ammonia every 2 hours. When the fermentation reaches about 12h, and the glutamic acid content in the fermentation reaches more than 50g / L, reduce the addition of ammonia, control the fermentation pH to 4.5-4.7, and add 0.1g of glutamic acid crystals to the fermentation system. Then, the pH was adjusted to 4.5-4.7 with ammonia every 2 hours, and samples were taken for testing. The samples were diluted 200 times with PBS buffer at pH 7.4, and the glucose and glutamate contents were detected using an SBA-40C biosensor analyzer. After the glucose was depleted, high-glucose fermentation replenishment medium was added to maintain the glucose in the fermentation system below 10 g / L. Fermentation ended after 36 hours.
[0072] When the shake-flask fermentation had been underway for about 24 hours, it was found that strains #2 and #4 produced excessive foam, which splashed and clogged the sealing film. This high foaming characteristic is detrimental to industrial production, therefore, shake-flask fermentation of strains #2 and #4 was discontinued. The glutamic acid yields of the remaining eight strains were as follows: #1 130 g / L, #3 124 g / L, #5 134 g / L, #6 136 g / L, #7 128 g / L, #8 132 g / L, #9 128 g / L, and #10 134 g / L (Table 2).
[0073] Table 2 Production of Glutamic Acid by Shake Flask Fermentation
[0074] strain number Fermented glutamic acid production (g / L) 1# 130 2# —— 3# 124 4# —— 5# 134 6# 136 7# 128 8# 132 9# 128 10# 134
[0075] Example 3
[0076] The above eight strains were subjected to extended-cycle shake-flask fermentation experiments (the process was the same as in Example 2, except that the fermentation time was 96 hours). Fermentation samples were taken every 12 hours for methylene blue staining. 50 μL of 0.1% methylene blue staining solution was added to 1 mL of fermentation sample. The methylene blue fading time of the samples after 24 hours was about 5 minutes. Samples with a fading time of more than 30 minutes were considered to have significantly reduced strain activity.
[0077] The results are shown in Table 3. As can be seen from Table 3, the activity of strain #3 decreased at 48h, strain #7 decreased at 60h, strains #5, #6 and #9 decreased at 72h, strain #8 decreased at 84h, and strain #1 showed a severe decrease in activity at 96h of fermentation. Only strain #10 had a decolorization time of about 20-25min (Table 3). Based on comprehensive evaluation, strain #10 exhibited high glutamic acid production and the best continuous culture activity, making it a suitable strain for continuous fermentation production of glutamic acid. It was named PA-GLU-10 and deposited with the accession number GDMCC 65358. Identification confirmed it as *Pantoea agglomerans*, with the name PA-GLU-10, classification name *Pantoea agglomerans*, accession number GDMCC 65358, deposit date October 28, 2024, and depositary institution: Guangdong Provincial Microbial Culture Collection Center, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0078] Table 3. Strain Viability Detection
[0079]
[0080] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.
Claims
1. A highly glutamic acid-producing pantothenic aerosol strain, characterized by: The name of the Pantoea agglomerans species is PA-GLU-10, its classification name is Pantoea agglomerans, its accession number is GDMCC 65358, its accession date is October 28, 2024, and its depository is Guangdong Provincial Center for Microbial Culture Collection, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
2. The pantothecin clump according to claim 1, characterized in that: The yield of glutamic acid from the Pantotheca agglomerata shake flask fermentation for 36 hours was 134 g / L, and the methylene blue fading time was 20-25 min during the Pantotheca agglomerata shake flask fermentation for 96 hours.
3. The application of the pantothenic bacteria as described in claim 1 or 2 in the production of glutamic acid.
4. An application of the pantothenic bacteria as described in claim 1 or 2 in the continuous fermentation production of glutamic acid.
5. The method for producing glutamic acid by fermentation of pantothenic acid as described in claim 1 or 2, characterized in that: Includes the following steps: (1) Seed culture: Inoculate the clustered pantothecin into the seed culture medium and culture at 35℃ and 220 r / min for 16 h to obtain the seed culture solution; (2) Shake-flask fermentation: The seed culture was transferred to the fermentation medium at a rate of 20%, with an initial pH of 6. The culture was carried out at 35°C and a rotation speed of 220 r / min. The pH was adjusted to 5.8-6 every 2 hours using ammonia. After 12 hours of fermentation, when the glutamic acid content in the fermentation reached more than 50 g / L, the addition of ammonia was reduced, and the fermentation pH was controlled to 4.5-4.
7. 0.1 g of glutamic acid crystals were added to the fermentation system. After that, the pH was adjusted to 4.5-4.7 every 2 hours using ammonia. At the same time, the glucose and glutamic acid content in the fermentation broth was sampled and tested. After the glucose was depleted, high-glucose fermentation replenishment medium was used to replenish the broth so that the glucose in the fermentation system was maintained below 10 g / L. The fermentation was completed after 36 hours.
6. The method according to claim 5, characterized in that: The seed culture medium formula is as follows: molasses 80 g / L, magnesium sulfate 0.25 g / L, ammonium sulfate 3 g / L, potassium dihydrogen phosphate 2.5 g / L, yeast extract 2 g / L, pH=6; The fermentation medium is formulated as follows: glucose 75g / L, ammonium chloride 15g / L, magnesium sulfate 0.25g / L, corn steep liquor 4ml / L, ferric sulfate 20mg / L, manganese sulfate 20mg / L, sodium dihydrogen phosphate 5g / L, potassium chloride 5g / L, and phosphate to adjust the pH to 7. The formula for the high-sugar fermentation replenishment medium is as follows: glucose 700g / L, ammonium chloride 4g / L, magnesium sulfate 0.25g / L, corn steep liquor 5ml / L, ferric sulfate 20mg / L, manganese sulfate 20mg / L, sodium dihydrogen phosphate 5g / L, potassium chloride 5g / L, and phosphate to adjust the pH to 6. The solvent for all the above culture media is water.
7. The method for producing glutamic acid by fermentation of pantothenic acid as described in claim 1 or 2, characterized in that: Includes the following steps: (1) Seed culture: Inoculate the clustered pantothecin into the seed culture medium and culture at 35℃ and 220 r / min for 16 h to obtain the seed culture solution; (2) Shake-flask fermentation: The seed culture was transferred to the fermentation medium at a rate of 20%, with an initial pH of 6. The culture was carried out at 35°C and a rotation speed of 220 r / min. The pH was adjusted to 5.8-6 every 2 hours using ammonia. After 12 hours of fermentation, when the glutamic acid content in the fermentation reached more than 50 g / L, the addition of ammonia was reduced, and the fermentation pH was controlled to 4.5-4.
7. 0.1 g of glutamic acid crystals were added to the fermentation system. After that, the pH was adjusted to 4.5-4.7 every 2 hours using ammonia. At the same time, the glucose and glutamic acid content in the fermentation broth were sampled and tested. After the glucose was depleted, high-glucose fermentation replenishment medium was used to replenish the broth so that the glucose in the fermentation system was maintained below 10 g / L. The fermentation was completed after 96 hours.
8. The method according to claim 7, characterized in that: The seed culture medium formula is as follows: molasses 80 g / L, magnesium sulfate 0.25 g / L, ammonium sulfate 3 g / L, potassium dihydrogen phosphate 2.5 g / L, yeast extract 2 g / L, pH=6; The fermentation medium is formulated as follows: glucose 75g / L, ammonium chloride 15g / L, magnesium sulfate 0.25g / L, corn steep liquor 4ml / L, ferric sulfate 20mg / L, manganese sulfate 20mg / L, sodium dihydrogen phosphate 5g / L, potassium chloride 5g / L, and phosphate to adjust the pH to 7. The formula for the high-sugar fermentation replenishment medium is as follows: glucose 700g / L, ammonium chloride 4g / L, magnesium sulfate 0.25g / L, corn steep liquor 5ml / L, ferric sulfate 20mg / L, manganese sulfate 20mg / L, sodium dihydrogen phosphate 5g / L, potassium chloride 5g / L, and phosphate to adjust the pH to 6. The solvent for all the above culture media is water.