A fermentation process for gibberellin GA4

Abstract

The present invention discloses a fermentation process of gibberellin GA4, which involves a strain producing gibberellin GA4, and the strain is taxonomically named: Fusarium fujikuroi NRF01, which was deposited at the China Center for Type Culture Collection on May 19, 2022, with the deposit number CCTCC NO. M2022683). During the fermentation process, the fermentation conditions are controlled in stages. The total sugar concentration in the fermentation broth is controlled at 0.5 - 1.0% by feeding and supplementing sugar, and the amino nitrogen concentration in the fermentation broth is controlled at 84 - 126 mg / L by feeding and supplementing nitrogen source. After fermentation for 5 - 7 days, the fermentation level of gibberellin GA4 is 2186 mg / L. The present invention provides a fermentation process of gibberellin GA4 and its application research. This strain can produce a high yield of gibberellin GA4 within a relatively short fermentation cycle. Through the step-by-step cultivation of this strain, the fermentation conditions are controlled in stages during the fermentation process. The total sugar concentration in the fermentation broth is controlled by feeding and supplementing sugar, and the amino nitrogen concentration in the fermentation broth is controlled by feeding and supplementing nitrogen source.

Description

Technical Field

[0001] The present invention relates to the technical field of bioengineering, and specifically relates to a fermentation process of gibberellin GA4. Background Art

[0002] The present invention relates to gibberellins GA1, GA4, GA7, and the structural formula of the main product GA4 is as follows:

[0003]

[0004] Gibberellin GA4

[0005] Gibberellins (GAs) are a class of plant hormones belonging to diterpenoid compounds, which are widely present in higher plants, fungi, and bacteria. There are many types of them. Currently, a total of 136 kinds of gibberellins have been discovered, collectively called gibberellin classes (GAs). Among them, the main ones with biological activity are GA1, GA3, GA4, and GA7. GA4 has relatively mild activity and has a variety of physiological functions. For example, it stimulates the elongation of plant cells, making the plants taller and the leaves larger; breaks the dormancy of seeds, tubers, and roots, and promotes their germination; stimulates fruit growth, increases the fruiting rate or forms seedless fruits; promotes early flower bud differentiation in some plants that require low temperature to pass through the growth stage; enables some plants to bolt and flower under short-day conditions; can induce the formation of α-amylase and accelerate the hydrolysis of storage substances in endosperm cells; increases the fruit setting rate, promotes fruit growth, and delays fruit senescence. GA4 is receiving increasing attention due to its unique activity and advantages. Since the 1990s in China, represented by China Agricultural University, in-depth series of studies on gibberellin GA4 have been carried out. Currently, gibberellin GA4 is mainly obtained through liquid deep fermentation. However, there are two prominent problems in the production process. One is that the fermentation level of gibberellin GA4 is low; the other is that there are many by-products, mainly GA1 and GA7. GA4 is converted into GA7 under the action of dehydrogenase, and GA7 and GA4 are converted into GA3 and GA1 under the action of 13-hydroxylase, resulting in a high cost of separation and purification of GA4, which seriously hinders its large-scale commercial application. There are few research reports on GA4 at home and abroad. Zhejiang Qianjiang Biochemical Co., Ltd. obtained a strain of Gibberella fujikuroi (CN201510630480.4) through mutagenesis screening and breeding. After culturing in a shake flask for 9 days, the yield of gibberellin GA4 reached 1447 mg / L, and the content of gibberellin GA7 was less than 3%, with a long fermentation cycle. Jin Zhimin et al. from Zhejiang University of Technology (CN200810061778.8) disclosed a method for separating gibberellin GA4 from the gibberellin GA4+7 mixture. Yang Wenge et al. from Nanjing Tech University (CN201210464476.1) disclosed a method for separating and purifying GA4 and / or GA7, that is, obtaining a single GA4 or GA7 from the GA4+7 mixture. Yang Wenge et al. from Nanjing Tech University (CN201710214169.0) disclosed a process for fermenting Gibberella fujikuroi to produce gibberellin GA1 and GA4. After fermenting in a shake flask for 9 days, the fermentation level of gibberellin GA1 reached 2950 mg / L, and the fermentation level of gibberellin GA4 reached 1441 mg / L. There is also a problem of a long fermentation cycle, and a large amount of by-product gibberellin GA1 is produced, increasing the cost of separation and purification.Geetanjali et al. obtained a morphological mutant strain Gibberella fujikuroi Mor-189 through ultraviolet mutagenesis. In a medium with wheat gluten as the sole nitrogen source, fed-batch culture was carried out for 7 days, and the yield of gibberellin GA4 reached 600 mg / L (Geetanjali Lale, Ramchandra Gadre. Enhanced production of gibberellin A4 (GA4) by a mutant of Gibberella fujikuroi in wheat gluten medium. Journal of Industrial Microbiology & Biotechnology. 2010, 37: 297-306).

[0006] Considering the overall production performance, these strains or processes are not suitable for the large-scale industrial production of gibberellin GA4. Summary of the Invention

[0007] The purpose of the present invention is to provide a fermentation process for gibberellin GA4. By controlling the fermentation conditions in two stages, adding carbon source during the fermentation process to control the total sugar concentration, and adding nitrogen source to control the amino nitrogen concentration, the yield of gibberellin GA4 is increased.

[0008] To achieve the above purpose, the present invention provides the following technical solution: A fermentation process for gibberellin GA4, which involves a strain producing gibberellin GA4, and the strain is classified and named as: Fusarium fujikuroi NRF01, which was deposited at the China Center for Type Culture Collection on May 19, 2022. The deposit address is: Wuhan, China, and the deposit number is CCTCC NO. M2022683). During the fermentation process, the fermentation conditions are controlled in stages. The total sugar concentration in the fermentation broth is controlled at 0.5-1.0% by feeding and adding sugar, and the amino nitrogen concentration in the fermentation broth is controlled at 84-126 mg / L by feeding and adding nitrogen source. After fermentation for 5-7 days, the fermentation level of gibberellin GA4 is 2186 mg / L.

[0009] Further, it includes the following steps:

[0010] (1) Use an inoculation knife to scrape a mycelium block of Fusarium fujikuroi NRF01 about 1 cm long and 1 cm wide on the inclined plane, and inoculate it into a shake flask containing a seed medium. The composition of the seed medium is as follows: glucose 2.0 - 5.0%, soybean cake powder 1.0 - 3.0%, peanut cake powder 0.8 - 1.5%, dextrin 2.0 - 4.0%, KH2PO4 0.08 - 0.16%, (NH4)2SO4 0.01 - 0.03%, MgSO4·7H2O 0.05 - 0.15%. Incubate at a temperature of 28 - 30°C and a shaker speed of 300 - 400 rpm for 24 - 48 hours to obtain 150 mL of activated seed liquid of Fusarium fujikuroi NRF01.

[0011] (2) Inoculate 150 mL of the activated seed liquid into a 50 L fermenter (30 L / 50 L) containing the seed medium. The composition of the seed medium is as follows: glucose 2.0 - 4.0%, soybean cake powder 1.5 - 2.5%, peanut cake powder 1.0 - 2.0%, dextrin 2.0 - 4.0%, KH 2 PO 4 0.10 - 0.20%, (NH 4 )2SO 4 0.02 - 0.04%, MgSO 4 .7H 2 O 0.05 - 0.15%. Incubate at a temperature of 28 - 30°C, a stirring speed of 250 - 350 rpm, and an aeration rate of 1.5 - 3.0 m3 / h for 18 - 36 hours to obtain the seed liquid.

[0012] (3) Inoculate 8 - 15% (V / V) of the seed liquid into a 100 L fermenter (70 L / 100 L) containing the fermentation medium. The composition of the fermentation medium is as follows: starch liquefied liquid 4.0% - 12.0%, initial nitrogen source 2.5 - 4.0%, soybean oil 0.08 - 0.16%, MgSO4·7H2O 0.08 - 0.16%, (NH4)2SO4 0.02 - 0.05%, KH2PO4 0.15 - 0.40%, ZnSO4 0.005 - 0.01%, MnSO4 0.005 - 0.01%.

[0013] Furthermore, in the early stage of fermentation, the culture temperature is 28 - 30°C, the aeration rate is 7 - 8 m3 / h, the stirring speed is 350 - 450 rpm, and the culture time is 24 - 48 hours; in the late stage of fermentation, the culture temperature is 31 - 33°C, the aeration rate is 5 - 6 m3 / h, the stirring speed is 200 - 350 rpm, and the culture time is 120 - 144 hours.

[0014] Preferably, the initial nitrogen source in the fermentation medium is one or a combination of soybean cake powder, peanut cake powder, corn protein powder, rice protein powder, defatted cottonseed protein, (NH 4 )2SO 4 , NaNO 3 one or a combination of several kinds.

[0015] Preferably, the sugar supplemented during the fermentation process is one or a combination of maltose, maltose, lactose, sucrose, dextrin, and starch liquefied liquid.

[0016] Preferably, the nitrogen source supplemented during the fermentation process can be one or a combination of peanut powder, soybean powder, ammonium sulfate, ammonia water, and ammonium acetate.

[0017] Compared with the prior art, the present invention provides a fermentation process for gibberellin GA4, which has the following beneficial effects: The present invention provides a fermentation process for gibberellin GA4 and its application research. This strain can produce a high yield of gibberellin GA4 within a short fermentation period. Through the step-by-step cultivation of this strain, the fermentation conditions are controlled in stages during the fermentation process. Sugar is added in a fed-batch manner to control the total sugar concentration in the fermentation broth, and nitrogen source is added in a fed-batch manner to control the amino nitrogen concentration in the fermentation broth. The fermentation period is 5 - 7 days, and the highest fermentation level can reach 2186 mg / L, which has good application prospects and plays a positive role in promoting the industrial production of gibberellin GA4. Specific Embodiments

[0018] Next, the technical solutions in the embodiments of the present invention will be described clearly and completely. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0019] Example 1:

[0020] Seed activation: Use an inoculation knife to scrape a mycelium block of Fusarium fujikuroi NRF01 about 1 cm long and wide on the slant, and inoculate it into a shake flask containing a seed medium. The composition of the seed medium is as follows: glucose 2.0%, soybean cake powder 1.5%, peanut cake powder 1.5%, dextrin 2.0%, KH 2 PO 4 0.12%, (NH 4 )2SO 4 0.015%, MgSO 4 .7H 2 O 0.12%, temperature 29°C, shaker speed 300 rpm, cultivate for 36 hours to obtain 150 mL of activated seed liquid of Fusarium fujikuroi NRF01;

[0021] Seed culture: 150 mL of the activated seed liquid was inoculated into a 50 L fermenter containing the seed medium (30 L / 50 L). The composition of the seed medium was as follows: glucose 3.0%, soybean cake powder 2.0%, peanut cake powder 1.5%, dextrin 3.0%, KH2PO4 0.16%, (NH 4 )2SO 4 0.04%, MgSO 4 .7H 2 O 0.15%, temperature 29 °C, stirring speed 300 rpm, aeration rate 2.5 m3 / h, cultured for 24 hours to obtain the seed liquid;

[0022] Fermentation culture:

[0023] The composition of the fermentation medium was as follows: starch liquefied liquid 5.0%, peanut powder 1.5%, soybean powder 1.0%, soybean oil 0.12%, MgSO 4 .7H 2 O 0.15%, (NH 4 )2SO 4 0.025%, KH 2 PO 4 0.20%, ZnSO 4 0.005%, MnSO 4 0.0075%;

[0024] Seeding: 10% (7 L) of the seed liquid was inoculated into a 100 L fermenter containing the fermentation medium (70 L / 100 L);

[0025] Fermentation parameter control: In the early stage of fermentation, the culture temperature was 29 °C, the aeration rate was 7 m 3 / h, the stirring speed was 350 rpm, and the culture time was 36 hours; in the later stage of fermentation, the culture temperature was 32 °C, the aeration rate was 5 m 3 / h, the stirring speed was 300 rpm, and the culture time was 84 hours;

[0026] Total sugar control: At 48 hours, when the total sugar concentration was detected to drop to 0.75%, 40% maltose syrup was added to control the total sugar concentration at (0.75 ± 0.1)%;

[0027] Amino nitrogen control: At 40 hours, when the amino nitrogen concentration was detected to drop to 98 mg / L, peanut powder was added in batches to control the amino nitrogen concentration at (98 ± 14) mg / L;

[0028] Detection of the titer of gibberellin GA4 in the fermentation broth:

[0029] After 120 hours of fermentation, the fermentation was completed to obtain the fermentation broth. After detection, the yield of gibberellin GA4 was 1620 mg / L.

[0030] Example 2:

[0031] Seed activation: The same as Example 1.

[0032] Seed cultivation: The same as Example 1.

[0033] Fermentation culture:

[0034] The composition of the fermentation medium is: starch liquefied liquid 5.0%, peanut powder 2.0%, soybean powder 1.5%, soybean oil 0.15%, MgSO 4 .7H 2 O 0.15%, (NH 4 )2SO 4 0.025%, KH 2 PO 4 0.20%, ZnSO 4 0.005%, MnSO 4 0.0075%;

[0035] Seeding: Inoculate 13% (9 L) of the seed liquid into a 100 L fermenter containing the fermentation medium (70 L / 100 L);

[0036] Fermentation parameter control: In the early stage of fermentation, the culture temperature is 29°C, the ventilation rate is 7 m 3 / h, the stirring speed is 350 rpm, and the culture time is 30 hours; in the later stage of fermentation, the culture temperature is 32°C, the ventilation rate is 5 m 3 / h, the stirring speed is 300 rpm, and the culture time is 90 hours;

[0037] Total sugar control: At 48 hours, when the total sugar concentration is detected to drop to 0.75%, start to supplement 40% maltose syrup to control the total sugar concentration at (0.75 ± 0.1)%;

[0038] Amino nitrogen control: At 45 hours, when the amino nitrogen concentration is detected to drop to 98 mg / L, start to supplement 20% ammonia water to control the amino nitrogen concentration at (98 ± 14) mg / L;

[0039] Detection of the titer of gibberellin GA4 in the fermentation broth:

[0040] After 120 hours of fermentation, the fermentation is completed to obtain the fermentation broth. After detection, the yield of gibberellin GA4 is 1650 mg / L.

[0041] Example 3:

[0042] Seed activation: The same as Example 1.

[0043] Seed cultivation: The same as Example 1.

[0044] Fermentation culture:

[0045] The composition of the fermentation medium is as follows: starch liquefied liquid 5.0%, peanut powder 2.0%, soybean powder 1.5%, soybean oil 0.15%, MgSO 4 .7H 2 O 0.15%, (NH4)2SO 4 0.025%, KH 2 PO 4 0.20%, ZnSO 4 0.005%, MnSO 4 0.0075%;

[0046] Inoculation: Inoculate 13% (9 L) of the seed liquid into a 100 L fermenter containing the fermentation medium (70 L / 100 L);

[0047] Fermentation parameter control: In the early stage of fermentation, the culture temperature is 29 °C, the ventilation rate is 7 m 3 / h, the stirring speed is 400 rpm, and the culture time is 30 hours; in the later stage of fermentation, the culture temperature is 31 °C, the ventilation rate is 5 m 3 / h, the stirring speed is 350 rpm, and the culture time is 90 hours;

[0048] Total sugar control: At 50 hours, when the total sugar concentration is detected to drop to 0.65%, start to supplement 24% lactose to control the total sugar concentration at (0.65 ± 0.1)%;

[0049] Amino nitrogen control: At 47 hours, when the amino nitrogen concentration is detected to drop to 84 mg / L, start to supplement 20% ammonia water to control the amino nitrogen concentration at (84 ± 14) mg / L;

[0050] Detection of the titer of gibberellin GA4 in the fermentation broth:

[0051] After 120 hours of fermentation, the fermentation is completed to obtain the fermentation broth. After detection, the yield of gibberellin GA4 is 1728 mg / L.

[0052] Example 4:

[0053] Seed activation: The same as Example 1.

[0054] Seed culture: The same as Example 1.

[0055] Fermentation culture:

[0056] The composition of the fermentation medium is as follows: starch liquefied liquid 6.0%, peanut powder 2.5%, soybean powder 1.5%, soybean oil 0.15%, MgSO 4 .7H 2 O 0.15%, (NH4)2SO 4 0.025%, KH 2 PO4 0.20%, ZnSO 4 0.005%, MnSO 4 0.0075%;

[0057] Inoculation: Inoculate 13% (9 L) of the seed liquid into a 100 L fermenter containing the fermentation medium (70 L / 100 L);

[0058] Fermentation parameter control: In the early stage of fermentation, the culture temperature is 29 °C, the aeration rate is 7 m 3 / h, the stirring speed is 400 rpm, and the culture time is 36 hours; In the later stage of fermentation, the culture temperature is 32 °C, the aeration rate is 5 m 3 / h, the stirring speed is 300 rpm, and the culture time is 108 hours;

[0059] Total sugar control: At 50 hours, when the total sugar concentration is detected to drop to 0.75%, start adding 40% maltose syrup to control the total sugar concentration at (0.65 ± 0.15)%;

[0060] Amino nitrogen control: At 50 hours, when the amino nitrogen concentration is detected to drop to 84 mg / L, start adding 20% ammonia water to control the amino nitrogen concentration at (84 ± 14) mg / L;

[0061] Detection of gibberellin GA4 titer in the fermentation broth:

[0062] After 144 hours of fermentation, the fermentation is completed to obtain the fermentation broth. After detection, the yield of gibberellin GA4 is 1945 mg / L.

[0063] Example 5:

[0064] Seed activation: The same as Example 1.

[0065] Seed culture: The same as Example 1.

[0066] Fermentation culture:

[0067] The composition of the fermentation medium is: starch liquefied liquid 5.0%, peanut powder 2.0%, soybean powder 1.5%, soybean oil 0.15%, MgSO 4 .7H 2 O 0.15%, (NH4)2SO 4 0.025%, KH 2 PO 4 0.20%, ZnSO 4 0.005%, MnSO 4 0.0075%;

[0068] Inoculation: Inoculate 12% (8.5 L) of the seed liquid into a 100 L fermenter containing the fermentation medium (70 L / 100 L);

[0069] Fermentation parameter control: In the early stage of fermentation, the culture temperature is 29°C, the aeration rate is 7 m 3 / h, the stirring speed is 400 rpm, and the culture time is 48 hours; in the late stage of fermentation, the culture temperature is 32°C, the aeration rate is 5 m 3 / h, the stirring speed is 300 rpm, and the culture time is 120 hours;

[0070] Total sugar control: At 46 hours, when the total sugar concentration is detected to drop to 0.75%, start to supplement 40% maltose syrup to control the total sugar concentration at (0.65 ± 0.15)%;

[0071] Amino nitrogen control: At 42 hours, when the amino nitrogen concentration is detected to drop to 98 mg / L, start to supplement 20% ammonia water to control the amino nitrogen concentration at (98 ± 14) mg / L;

[0072] Detection of gibberellin GA4 titer in the fermentation broth:

[0073] After 168 hours of fermentation, the fermentation ends to obtain the fermentation broth. After detection, the gibberellin GA4 yield is 2186 mg / L.

[0074] Analysis method of gibberellin GA4: GA4 is analyzed by Shimadzu LC-20AT (150 mm × 4.6 mm stainless steel column; ODS-C18 chromatographic column). The fermentation broth is adjusted to a pH of about 7.0 with 2% NaOH solution, filtered through filter paper, accurately pipette 5.0 mL of the filtrate into a 25 mL volumetric flask, diluted to the mark with the mobile phase, then centrifuged to take the supernatant, and filtered through a 0.45 μm filter membrane for injection detection. The mobile phase components include methanol / water / formic acid (67 / 33 / 0.05), and the sample is analyzed at a flow rate of 0.7 ml / min. The system detection wavelength is set at 210 nm, the injection volume is 20 μL, and the retention time of GA4 is about 11.0 min.

[0075] Total sugar detection method: Pipette 0.5 mL of the filtered fermentation broth into a 150 mL Erlenmeyer flask, add 10 mL of distilled water and 5 mL of 6 mol / L hydrochloric acid solution, heat and boil on an 800 W electric furnace for 3 min, remove and cool to room temperature, add 5 mL of 6 mol / L sodium hydroxide solution to neutralize the hydrochloric acid. Accurately add 20.0 mL of Fehling's mixture, shake well, heat and boil on the electric furnace for 3 min, cool to room temperature, add 15 mL of 2 mol / L sulfuric acid solution, immediately titrate with 0.1 mol / L sodium thiosulfate standard solution. When it turns light yellow, add 2 mL of 5 g / L starch indicator, and continue titrating until the blue color just fades to the end point. At the same time, perform a blank test and record the consumption volume of the sodium thiosulfate standard solution respectively.

[0076] Amino nitrogen detection method: Pipette 1.0 mL of the filtered fermentation broth into a 150 mL Erlenmeyer flask, add 10 mL of distilled water and one drop of methyl red indicator, adjust the solution to a faint red color with 0.15 mol / L sulfuric acid solution (if the solution is already red, do not add sulfuric acid), then add 0.01000 mol / L sodium hydroxide solution to adjust to an orange color. Continue to add 4 mL of 18% formaldehyde solution, shake well, and let stand for 10 min. Add 2 drops of 10 g / L phenolphthalein indicator, and titrate with 0.01000 mol / L sodium hydroxide standard solution until a faint red color appears as the end point. At the same time, perform a blank test.

[0077] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention. Any reference signs in the claims should not be construed as limiting the claimed claim.

[0078] In the description of the present invention, unless otherwise specified, "a plurality of" means two or more; the terms "upper", "lower", "left", "right", "inner", "outer", "front end", "rear end", "head", "tail", etc. indicate the orientation or positional relationship, which is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be construed as a limitation of the present invention. In addition, the terms "first", "second", "third", etc. are only used for descriptive purposes and cannot be construed as indicating or implying relative importance.

[0079] In the description of the present invention, it should be noted that unless otherwise clearly specified and limited, the terms "connected" and "connected" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

Claims

1. A fermentation process for gibberellin GA4, which involves a strain producing gibberellin GA4. The strain is classified and named as Fusarium fujikuroi NRF01, which was deposited at the China Center for Type Culture Collection on May 19, 2022, with the deposit number CCTCC NO. M2022683). It is characterized in that: During the fermentation process, the fermentation conditions are controlled in stages. The total sugar concentration in the fermentation broth is controlled at 0.5 - 1.0% by feeding and supplementing sugar, and the amino nitrogen concentration in the fermentation broth is controlled at 70 - 126 mg / L by feeding and supplementing nitrogen source. After fermenting for 5 - 7 days, the highest fermentation level of gibberellin GA4 can reach 2186 mg / L. For the fermentation, the composition of the fermentation medium is as follows: starch liquefied solution 4.0%-12.0%, initial nitrogen source 2.5-4.0%, soybean oil 0.08-0.16%, MgSO4.7H2O 0.08-0.16%, (NH 4 ) 2 SO 4 0.02-0.05%, KH2PO4 0.15-0.40%, ZnSO4 0.005-0.01%, MnSO4 0.005-0.01%; The staged control of fermentation conditions is as follows: in the early stage of fermentation, the culture temperature is 28-30°C, the ventilation volume is 7-8 m 3 / h, the stirring speed is 350-450 rpm, and the culture time is 24-48 hours; in the later stage of fermentation, the culture temperature is 31-33°C, the ventilation volume is 5-6 m 3 / h, the stirring speed is 200-350 rpm, and the culture time is 120-144 hours.

2. The fermentation process for gibberellin GA4 according to claim 1, It is characterized in that, It includes the following steps: (1) Use an inoculation knife to scrape a mycelium block of Fusarium fujikuroi NRF01 about 1 cm long and 1 cm wide on the inclined plane, and inoculate it into a shake flask containing a seed medium. The composition of the seed medium is as follows: glucose 2.0 - 5.0%, soybean cake powder 1.0 - 3.0%, peanut cake powder 0.8 - 1.5%, dextrin 2.0 - 4.0%, KH 2 PO 4 0.08 - 0.16%, (NH 4 ) 2 SO 4 0.01 - 0.03%, MgSO 4 .7H 2 O 0.05 - 0.15%, temperature 28 - 30 °C, shaker speed 300 - 400 rpm, culture for 24 - 48 hours to obtain 150 mL of activated seed liquid of Fusarium fujikuroi NRF01; (2) Inoculate 150 mL of the activated seed liquid into a 50 L fermenter containing the seed culture medium. The composition of the seed culture medium is as follows: glucose 2.0 - 4.0%, soybean cake powder 1.5 - 2.5%, peanut cake powder 1.0 - 2.0%, dextrin 2.0 - 4.0%, KH2PO4 0.10 - 0.20%, (NH4) 2 SO4 0.02 - 0.04%, MgSO4·7H2O 0.05 - 0.15%, temperature 28 - 30 °C, stirring speed 250 - 350 rpm, ventilation volume 1.5 - 3.0 m 3 / h, and culture for 18 - 36 hours to obtain the seed liquid; (3) Inoculate 8 - 15% (V / V) of the seed liquid into a 100 L fermenter containing the fermentation medium.

3. The fermentation process for gibberellin GA4 according to claim 1, It is characterized in that, The sugar supplemented during the fermentation process is one or a combination of maltose syrup, maltose, lactose, sucrose, dextrin, and starch liquefied liquid.

4. The fermentation process for gibberellin GA4 according to claim 1, It is characterized in that, The initial nitrogen source in the fermentation medium is one or a combination of soybean cake powder, peanut cake powder, corn protein powder, rice protein powder, defatted cottonseed protein, (NH 4 ) 2 SO 4 , NaNO 3 .

5. The fermentation process for gibberellin GA4 according to claim 1, It is characterized in that, The nitrogen source supplemented during the fermentation process can be one or a combination of peanut powder, soybean powder, ammonium sulfate, ammonia water, and ammonium acetate.

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