Accelerator for industrial production of GA1 through fermentation method and fermentation method for co-production of GA1 and GA3
By adding urea and citric acid as GA1 accelerator to the fermentation method of GA3, the content and purification efficiency of GA1 are improved, the problem that GA1 cannot be produced in industrial form is solved, and efficient purification and separation of GA1 is achieved.
Patent Information
- Application Number
- CN202411399047.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-09
- Publication Date
- 2024-12-24
AI Technical Summary
The prior art cannot produce the highly active gibberellic acid compound GA1 through fermentation method, and GA1 has a low content in fermentation production of GA3 and cannot be purified by separation, resulting in its inability to be effectively utilized.
When GA3 is produced by fermentation, urea and citric acid are added as GA1 accelerator, and GA1 is purified and separated by membrane filtration and concentration, and the GA1 content in the fermentation broth reaches more than 1000 ppm.
The high content purification and separation of GA1 is achieved, which solves the problem that GA1 cannot be produced in industrialized, and improves the production efficiency and product utilization rate of fermentation.
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Abstract
Description
Technical Field
[0001] The present invention relates to the production of gibberellic acid, and in particular to an accelerator for industrial production of GA1 by fermentation and a fermentation method for co-producing GA1 and GA3. Background Art
[0002] Gibberellic acid is a type of tetracyclic diterpene carboxylic acid compound that can be produced by green plants, fungi and bacteria. Together with auxin, abscisic acid, cytokinin and ethylene, it is known as the five major plant hormones. Since the first discovery of gibberellic acid in 1935, 136 GAs with different structures have been identified. They are named GA1, GA2, GA136 in the order of discovery, collectively known as gibberellic acids (GAs). Among them, the biologically active gibberellic acids mainly include GA1, GA2, GA3, GA4, GA7, etc.
[0003] In 1935, gibberellic acid was first isolated from the fermentation broth of Gibberella fujikura. Subsequently, gibberellic acid was also found in a variety of fungi and bacteria. For example, Khan et al. isolated a bamboo basal rot pathogen Arthrinium phaeos permum from the root of Carex kobomugi and found that it could produce a variety of active gibberellic acids, but the yield was very low, all below 10 ng / mL. Kang et al. isolated a strain of Acinetobacter calcoaceticus from the soil and found that it had the ability to produce gibberellic acid and dissolve phosphate, but the yield was lower, among which the yields of biologically active GA1, GA3 and GA4 were only 0.45, 6.2 and 2.8 ng / 100 mL, respectively. Therefore, other strains cannot meet the requirements of industrial production of gibberellic acid, while the yield of GA3 produced by Gibberella fujikura can reach 1000-1200 mg / L, far exceeding other strains that can produce gibberellic acid. Therefore, Gibberella fujikura has broad application prospects.
[0004] There are three main methods for producing gibberellic acid, namely plant extraction, chemical synthesis and microbial fermentation. Although most plants can synthesize gibberellic acid, the extraction rate is very low, far from meeting production needs; and the chemical synthesis method has the disadvantages of high cost, low efficiency and high pollution. Therefore, the large-scale industrial production of gibberellic acid is mainly carried out by microbial fermentation. At present, the most widely used gibberellic acid is GA3, and the main industrial production strain is Fujikura Gibberella. The domestic industrial production of GA3 is 1200-2000 mg / L, while foreign countries use liquid deep fermentation, and the GA3 production can reach 3900 mg / L.
[0005] GA1 is a highly active gibberellic acid compound with significant plant growth regulating effects. Currently, there is no fermentation method for producing GA1. In the fermentation production of GA3 (such as CN108285915A, CN113817794A, CN113957115A and CN113755343A), the GA1 content is lower than 300 ppm. GA1 cannot be purified and separated by separation methods such as membrane filtration and concentration of GA3. GA1 can only be treated and discharged as an impurity together with the waste liquid. GA1 cannot be industrially produced by fermentation.
[0006] Therefore, how to industrially produce GA1 through fermentation has become an urgent problem that the industry needs to solve.
[0007] The above background technology is for facilitating the understanding of the present invention and is not a known technology disclosed to the general public before the application of the present invention. Summary of the invention
[0008] In view of the above-mentioned defects, the present invention provides a GA1 promoter. When GA3 is produced by fermentation, the GA1 promoter is added to co-produce GA1. The GA1 content in the fermentation broth reaches more than 1000 ppm. GA1 can be purified and separated by separation methods such as membrane filtration and concentration.
[0009] The technical solution is: a promoter for industrial production of GA1 by fermentation, the promoter comprising:
[0010] Urea 1-99 wt%; and
[0011] Citric acid 1-99wt%.
[0012] Furthermore, the urea is 20-80wt%, and the citric acid is 20-80wt%.
[0013] Furthermore, the urea is 25 wt % and the citric acid is 75 wt %.
[0014] The present invention also provides a fermentation method for co-producing GA1 and GA3.
[0015] The technical solution is: a fermentation method for co-producing GA1 and GA3, the method comprising the following steps:
[0016] S1, take seed solution and fermentation culture medium.
[0017] S2, adding seed solution and fermentation culture medium into the fermentation tank.
[0018] S3, fermentation reaction;
[0019] The fermentation culture medium is composed of a fermentation base culture medium and the above-mentioned promoter for industrial production of GA1 by fermentation, and the amount of the promoter added is 0.5-2 g / L based on the volume of the fermentation culture medium.
[0020] Furthermore, the amount of the seed solution added is 5-15wt% of the amount of the fermentation culture solution.
[0021] Compared with the prior art, the present invention has the following beneficial effects:
[0022] The present invention adds a GA1 promoter to the existing fermentation culture medium (i.e., the fermentation basic culture medium) during the fermentation process, so that GA1 can be co-produced when producing GA3 by fermentation. The GA1 content in the fermentation liquid is close to 1000 ppm or even exceeds 1000 ppm, so that GA1 can be subsequently purified and separated by separation methods such as membrane filtration and concentration. DETAILED DESCRIPTION
[0023] The present invention will be further described below.
[0024] Example 1 Preparation of Seed Liquid before Fermentation
[0025] The seed liquid before fermentation was prepared according to the process 1.1-1.3 in Example 1 of CN108285915A, wherein the GA3 strain, process and culture medium were exactly the same. The GA3 strain was Gibberella fujikura, and the deposit number was CGMCC No. 23265 (General Microbiological Center of China Microbiological Culture Collection Administration Committee).
[0026] The seed liquid test data is shown in Table 1.
[0027] Table 1 Seed liquid test data before fermentation
[0028] Wet weight PH Mycelium length Mycelium thickness Is the cytoplasm uniform? Whether there are cavitation bubbles Fermentation broth viscosity 17-25％ 4.9-5.5 Longer Thinner More uniform Small amount ++++
[0029] The seed solution prepared in Example 1 was used to prepare the fermentation solution of the following Example 2-Example.
[0030] In Examples 2 to 8 and Comparative Examples 1 to 2, the concentration and dosage of the seed solution are equal.
[0031] In Example 2 to Example 8 and Comparative Example 1 to Comparative Example 2, the volume of the fermentation tank was 50 L, and the volume of the fermentation broth was 35 L.
[0032] In Example 2-Example and Comparative Example 1-Comparative Example, the fermentation basal culture medium is as shown in Table 2 below.
[0033] Table 2 Fermentation basal culture medium and addition amount
[0034]
[0035] Example 2
[0036] A gibberellic acid fermentation method comprises the following steps:
[0037] S1, take the seed solution and fermentation culture medium of Example 1 (the fermentation culture medium consists of the fermentation basic culture medium in Table 2 and GA1 promoter), the mass of the seed solution is 10% of the mass of the fermentation culture medium.
[0038] S2, adding seed solution and fermentation culture medium into the fermentation tank.
[0039] S3, fermentation reaction.
[0040] In the fermentation reaction of S3, the carbon source was supplemented by feeding and the pH was controlled by adding ammonia water. The fermentation reaction time was 198h.
[0041] In S3, the air flow is 2200Nm 3 / h, the pressure was controlled at 0.035MPa, the temperature was controlled at 29±0.2℃, the pH was 5.0-5.2, and the feeding method was the same as that in Example 1 of CN108285915A.
[0042] The GA1 promoter in this embodiment is shown in Table 3.
[0043] After the fermentation was completed, samples were taken for testing. The results are shown in Table 4.
[0044] Example 3
[0045] A gibberellic acid fermentation method comprises the following steps:
[0046] S1, take the seed solution and fermentation culture medium of Example 1 (the fermentation culture medium consists of the fermentation basic culture medium in Table 2 and GA1 promoter), the mass of the seed solution is 10% of the mass of the fermentation culture medium.
[0047] S2, adding seed solution and fermentation culture medium into the fermentation tank.
[0048] S3, fermentation reaction.
[0049] In the fermentation reaction of S3, the carbon source was supplemented by feeding and the pH was controlled by adding ammonia water. The fermentation reaction time was 198h.
[0050] In S3, the air flow is 2200Nm 3 / h, the pressure was controlled at 0.035MPa, the temperature was controlled at 29±0.2℃, the pH was 5.0-5.2, and the feeding method was the same as that in Example 1 of CN108285915A.
[0051] The GA1 promoter in this embodiment is shown in Table 3.
[0052] After the fermentation was completed, samples were taken for testing. The results are shown in Table 4.
[0053] Example 4
[0054] A gibberellic acid fermentation method comprises the following steps:
[0055] S1, take the seed solution and fermentation culture medium of Example 1 (the fermentation culture medium consists of the fermentation basic culture medium in Table 2 and GA1 promoter), the mass of the seed solution is 10% of the mass of the fermentation culture medium.
[0056] S2, adding seed solution and fermentation culture medium into the fermentation tank.
[0057] S3, fermentation reaction.
[0058] In the fermentation reaction of S3, the carbon source was supplemented by feeding and the pH was controlled by adding ammonia water. The fermentation reaction time was 198h.
[0059] In S3, the air flow is 2200Nm 3 / h, the pressure was controlled at 0.035MPa, the temperature was controlled at 29±0.2℃, the pH was 5.0-5.2, and the feeding method was the same as that in Example 1 of CN108285915A.
[0060] The GA1 promoter in this embodiment is shown in Table 3.
[0061] After the fermentation was completed, samples were taken for testing. The results are shown in Table 4.
[0062] Example 5
[0063] A gibberellic acid fermentation method comprises the following steps:
[0064] S1, take the seed solution and fermentation culture medium of Example 1 (the fermentation culture medium consists of the fermentation basic culture medium in Table 2 and GA1 promoter), the mass of the seed solution is 10% of the mass of the fermentation culture medium.
[0065] S2, adding seed solution and fermentation culture medium into the fermentation tank.
[0066] S3, fermentation reaction.
[0067] In the fermentation reaction of S3, the carbon source was supplemented by feeding and the pH was controlled by adding ammonia water. The fermentation reaction time was 198h.
[0068] In S3, the air flow is 2200Nm 3 / h, the pressure was controlled at 0.035MPa, the temperature was controlled at 29±0.2℃, the pH was 5.0-5.2, and the feeding method was the same as that in Example 1 of CN108285915A.
[0069] The GA1 promoter in this embodiment is shown in Table 3.
[0070] After the fermentation was completed, samples were taken for testing. The results are shown in Table 4.
[0071] Example 6
[0072] A gibberellic acid fermentation method comprises the following steps:
[0073] S1, take the seed solution and fermentation culture medium of Example 1 (the fermentation culture medium consists of the fermentation basic culture medium in Table 2 and GA1 promoter), the mass of the seed solution is 10% of the mass of the fermentation culture medium.
[0074] S2, adding seed solution and fermentation culture medium into the fermentation tank.
[0075] S3, fermentation reaction.
[0076] In the fermentation reaction of S3, the carbon source was supplemented by feeding and the pH was controlled by adding ammonia water. The fermentation reaction time was 198h.
[0077] In S3, the air flow is 2200Nm 3 / h, the pressure was controlled at 0.035MPa, the temperature was controlled at 29±0.2℃, the pH was 5.0-5.2, and the feeding method was the same as that in Example 1 of CN108285915A.
[0078] The GA1 promoter in this embodiment is shown in Table 3.
[0079] After the fermentation was completed, samples were taken for testing. The results are shown in Table 4.
[0080] Example 7
[0081] A gibberellic acid fermentation method comprises the following steps:
[0082] S1, take the seed solution and fermentation culture medium of Example 1 (the fermentation culture medium consists of the fermentation basic culture medium in Table 2 and GA1 promoter), the mass of the seed solution is 10% of the mass of the fermentation culture medium.
[0083] S2, adding seed solution and fermentation culture medium into the fermentation tank.
[0084] S3, fermentation reaction.
[0085] In the fermentation reaction of S3, the carbon source was supplemented by feeding and the pH was controlled by adding ammonia water. The fermentation reaction time was 198h.
[0086] In S3, the air flow is 2200Nm 3 / h, the pressure was controlled at 0.035MPa, the temperature was controlled at 29±0.2℃, the pH was 5.0-5.2, and the feeding method was the same as that in Example 1 of CN108285915A.
[0087] The GA1 promoter in this embodiment is shown in Table 3.
[0088] After the fermentation was completed, samples were taken for testing. The results are shown in Table 4.
[0089] Comparative Example 1
[0090] A gibberellic acid fermentation method comprises the following steps:
[0091] S1, take the seed solution and fermentation broth of Example 1 (the fermentation basal broth in Table 2 is the fermentation broth), the mass of the seed solution is 10% of the mass of the fermentation broth.
[0092] S2, adding seed solution and fermentation culture medium into the fermentation tank.
[0093] S3, fermentation reaction.
[0094] In the fermentation reaction of S3, the carbon source was supplemented by feeding and the pH was controlled by adding ammonia water. The fermentation reaction time was 198h.
[0095] In S3, the air flow is 2200Nm 3 / h, the pressure was controlled at 0.035MPa, the temperature was controlled at 29±0.2℃, the pH was 5.0-5.2, and the feeding method was the same as that in Example 1 of CN108285915A.
[0096] After the fermentation was completed, samples were taken for testing. The results are shown in Table 4.
[0097] Example 8
[0098] A gibberellic acid fermentation method comprises the following steps:
[0099] S1, take the seed solution and fermentation culture medium of Example 1 (the fermentation culture medium consists of the fermentation basic culture medium in Table 5 and GA1 promoter), the mass of the seed solution is 10% of the mass of the fermentation culture medium.
[0100] S2, adding seed solution and fermentation culture medium into the fermentation tank.
[0101] S3, fermentation reaction.
[0102] In the fermentation reaction of S3, the carbon source was supplemented by feeding and the pH was controlled by adding ammonia water. The fermentation reaction time was 198h.
[0103] In S3, the air flow is 2200Nm 3 / h, the pressure was controlled at 0.035MPa, the temperature was controlled at 29±0.2℃, the pH was 5.0-5.2, and the feeding method was the same as that in Example 1 of CN108285915A.
[0104] The GA1 promoter in this embodiment is shown in Table 3.
[0105] After the fermentation was completed, samples were taken for testing. The results are shown in Table 4.
[0106] Comparative Example 2
[0107] A gibberellic acid fermentation method comprises the following steps:
[0108] S1, take the seed solution and fermentation broth of Example 1 (the fermentation basal broth in Table 5 is the fermentation broth), the mass of the seed solution is 10% of the mass of the fermentation broth.
[0109] S2, adding seed solution and fermentation culture medium into the fermentation tank.
[0110] S3, fermentation reaction.
[0111] In the fermentation reaction of S3, the carbon source was supplemented by feeding and the pH was controlled by adding ammonia water. The fermentation reaction time was 198h.
[0112] In S3, the air flow is 2200Nm 3 / h, the pressure was controlled at 0.035MPa, the temperature was controlled at 29±0.2℃, the pH was 5.0-5.2, and the feeding method was the same as that in Example 1 of CN108285915A.
[0113] After the fermentation was completed, samples were taken for testing. The results are shown in Table 4.
[0114] Table 3 GA1 accelerator and addition amount
[0115]
[0116]
[0117] Table 4 Fermentation broth test data
[0118]
[0119] Table 5 Fermentation basal culture medium and addition amount of Example 8
[0120]
[0121] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A promoter for industrial production of GA1 by fermentation, characterized in that: The accelerator includes: Urea 1-99 wt%; and Citric acid 1-99wt%.
2. The promoter for industrial production of GA1 by fermentation according to claim 1, characterized in that: The urea is 20-80 wt % and the citric acid is 20-80 wt %.
3. The promoter for industrial production of GA1 by fermentation according to claim 2, characterized in that: The urea is 25wt% and the citric acid is 75wt%.
4. A fermentation method for co-producing GA1 and GA3, characterized in that: The method comprises the following steps: S1, take seed solution and fermentation culture medium. S2, adding seed solution and fermentation culture medium into the fermentation tank. S3, fermentation reaction; The fermentation broth is composed of a fermentation basal broth and a promoter for industrial production of GA1 by fermentation as claimed in any one of claims 1 to 3, and the amount of the promoter added is 0.5-2 g / L based on the volume of the fermentation broth.
5. The fermentation method for co-producing GA1 and GA3 according to claim 4, characterized in that: The amount of seed liquid added is 5-15wt% of the fermentation culture liquid.
6. The fermentation method for co-producing GA1 and GA3 according to claim 4, characterized in that: The fermentation basal culture solution is: