A fermentation method for simultaneously inhibiting ISO-GA3, dienolic acid and epoxy GA in gibberellin acid fermentation liquor to reduce the total impurity content

Abstract

The application discloses a fermentation method for simultaneously inhibiting ISO-GA3, dihydrogibberellic acid and gibberellic acid epoxide in gibberellic acid fermentation liquor to reduce the total impurity content, and belongs to the field of gibberellic acid production. In the fermentation process, in addition to the conventional feeding, the method also performs the cooperative control of the impurity-reducing feeding. The application reduces the total impurity content of the fermentation liquor.

Description

Technical Field

[0001] This invention relates to the field of gibberellic acid production, and particularly to a fermentation method that simultaneously inhibits ISO-GA3, gibberellic acid and epoxygibacic acid in gibberellic acid fermentation broth to reduce the total impurity content. Background Technology

[0002] Gibberellic Acid 3 (GA3) is a plant growth regulator produced by Gibberella fuciformis. It plays a crucial role in plant growth and development and is used in many fields, including agriculture, forestry, and brewing, demonstrating significant economic and social benefits. Traditional methods for producing GA3 mainly include plant extraction, chemical synthesis, and microbial fermentation. However, GA3 extraction from plants yields low output, generates substantial waste, and is costly. Chemical synthesis methods are also limited by cumbersome processes and the resulting pollution. With the rapid development of synthetic biology, microbial production of GA3 is attracting increasing attention.

[0003] CN108285915A discloses a fermentation method for GA3. This method uses *Fujikura scab*, inoculating a pre-inoculated seed culture medium into a fermentation tank. During fermentation, the air flow rate into the fermentation tank is 2000-3000 Nm³ / h. When dissolved oxygen rebounds to 20%, an automatic flow of glucose solution is added to control dissolved oxygen at 20%-30%. Although the fermentation broth of CN108285915A has a high GA3 content, the GA3 content needs to be further increased to improve economic efficiency.

[0004] CN19242756A discloses a method for increasing the content of gibberellic acid (GA3) in fermentation broth by reducing the byproduct ISO-GA3. The process involves introducing refrigerated feed into the fermenter along with air during the feeding process. After adopting this process, the content of ISO-GA3 in the fermentation broth decreases significantly, and the content of GA3 increases accordingly.

[0005] In the fermentation process for producing GA3, the target product is GA3, and all other components are considered non-target products. Therefore, the industry generally strives for the highest possible GA3 content and the lowest possible content of other impurities; however, CN19242756A only controls the content of ISO-GA3, a single impurity. In addition to ISO-GA3, the fermentation broth obtained from the fermentation method also contains other impurities such as gibberellic acid and epoxygibacic acid; typically, these three impurities account for 20% to 30% of the total GA3 and impurities.

[0006] Therefore, how to simultaneously achieve synergistic inhibition of the three main impurities—ISO-GA3, gibberellic acid, and epoxygibacic acid—and reduce the total impurity content has become an important direction for further optimizing fermentation processes in this field. Summary of the Invention

[0007] To address the aforementioned deficiencies, this invention provides a fermentation method that simultaneously inhibits ISO-GA3, gibberellic acid, and epoxygibacic acid in gibberellic acid fermentation broth to reduce the total impurity content, thereby reducing the total impurity content of the fermentation broth.

[0008] The technical solution is: a fermentation method that simultaneously inhibits ISO-GA3, gibberellic acid and epoxygibacic acid in gibberellic acid fermentation broth to reduce the total impurity content. The synergistic control of impurity reduction feeding involves adding curcumin during the logarithmic growth phase and adding N-acetylcysteine ​​and borax during the initial stage of product synthesis.

[0009] Furthermore, the synergistic control of impurity reduction feeding involves adding curcumin during the logarithmic growth phase and adding N-acetylcysteine ​​and borax during the initial vigorous growth phase of product synthesis.

[0010] Furthermore, this method also controls the pH at 3.8–4.2 during the later stages of product synthesis.

[0011] Furthermore, it includes the following steps: S1, take the seed culture and fermentation broth; S2, Add the seed culture and fermentation broth into the fermenter; S3 undergoes a fermentation reaction; In S3, routine feeding and collaborative control for impurity reduction feeding are performed; The synergistic control of impurity reduction feeding is as follows: when fermentation reaches 48h, add curcumin at a rate of 50 mg to 80 mg per liter; when fermentation reaches 120h, add N-acetylcysteine ​​and borax at a rate of 100 to 150 mg per liter and 1.3 mmol to 1.6 mmol per liter.

[0012] Furthermore, in S3, the fermentation temperature is 28~30℃; the air flow rate is 1500~4600 Nm. 3 / h, pressure is 0.01~0.05MPa.

[0013] Furthermore, in S3, the routine feeding procedure is as follows: when fermentation reaches a dissolved oxygen rebound of 60%, glucose solution and salad oil are added. The initial feeding rate is 100 L / h for glucose solution and 40 L / h for salad oil. In the subsequent process, when the dissolved oxygen is below 20%, the feeding rate is reduced, and when the dissolved oxygen is above 30%, the feeding rate is increased.

[0014] Furthermore, in S3, the pH is controlled as follows: the pH is controlled to be 5.0-5.2 by adding ammonia water.

[0015] Furthermore, in S3, the pH is controlled as follows: ammonia water is added to control the pH to 5.0-5.2. When fermentation reaches 168h, in addition to adding ammonia water, 10 wt%-20 wt% citric acid aqueous solution is also added to control the pH to 3.8-4.2.

[0016] Furthermore, in S1, the liquid culture medium is: Corn gluten meal 10~30 g / L Sucrose 1~10g / L Potassium dihydrogen phosphate 1~10g / L Glucose 1~10g / L MgSO4·7H2O 0.5~1.5 g / L Salad oil 1~3 g / L Ammonium sulfate 0.5~1.5 g / L FeSO4·7H2O 0.005~0.015 g / L ZnSO4·7H2O 0.005~0.015 g / L MnSO4·H2O 0.005~0.015 g / L NaMoO4·2H2O 0.001~0.006 g / L CuSO4·5H2O 0.001~0.006 g / L CoCl2·6H2O 0.001~0.006 g / L; and The solvent is water.

[0017] Furthermore, the seed liquid is a seed liquid cultured with Fusarium oxysporum, the preservation number of which is CGMCC No. 23265.

[0018] Compared with the prior art, the inventive principle and beneficial effects of this invention are as follows: This invention employs synergistic feeding control during the logarithmic growth phase and the early stage of product synthesis to reduce impurities, while simultaneously inhibiting ISO-GA3, gibberellic acid, and epoxygibacic acid in the gibberellic acid fermentation broth. Specifically, feeding during the logarithmic growth phase inhibits PKS4 polyketide synthase activity, blocking gibberellic acid synthesis at its source, clearing intracellular ROS, and reducing epoxygibacic acid production. Feeding during the early stage of product synthesis forms a complex with the ortho-dihydroxyl group of GA3, protecting the 3-position hydroxyl group from isomerization and effectively reducing the total impurity content in the fermentation broth. Furthermore, this invention further avoids GA3 degradation caused by pH fluctuations by controlling the pH at 3.8~4.2 during the later stage of product synthesis. Detailed Implementation

[0019] As used in this article: "Prepared from" is synonymous with "comprising". The terms "comprising", "including", "having", "containing", or any other variations thereof as used herein are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0020] The conjunction "composed of..." excludes any unspecified elements, steps, or components. If used in a claim, this phrase makes the claim closed, excluding materials other than those described, except for associated conventional impurities. When the phrase "composed of..." appears in a clause of the body of a claim rather than immediately following it, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.

[0021] When a quantity, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1–5” is disclosed, the described range should be interpreted as including ranges “1–4”, “1–3”, “1–2”, “1–2 and 4–5”, “1–3 and 5”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0022] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of this application. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0024] In these embodiments, unless otherwise specified, the portions and percentages are all by weight.

[0025] "Parts by mass" refers to the basic unit of measurement that expresses the mass ratio of multiple components. One part can represent any unit mass, such as 1g or 2.689g. If we say that component A has "a" parts by mass and component B has "b" parts by mass, it means the ratio of the mass of component A to the mass of component B is a:b. Alternatively, it can mean that the mass of component A is aK and the mass of component B is bK (K is any number representing a multiplier). It is important to understand that, unlike the number of parts by mass, the sum of the mass parts of all components is not limited to 100 parts.

[0026] "And / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0027] Example 1: Preparation of Seed Liquid Before Fermentation The seed culture before fermentation was prepared according to the process in 1.1-1.3 of Example 1 in CN108285915A, with the GA3 strain, process and culture medium being exactly the same. The GA3 strain is Fusarium oxysporum, with the preservation number CGMCC No. 23265 (disclosed in CN114231421A).

[0028] The seed liquid test data are shown in Table 1 below.

[0029] Table 1. Seed liquor test data before fermentation The seed liquid prepared in Example 1 was used to prepare fermentation broths for Examples 2-5 and Comparative Examples 1-7.

[0030] In Examples 2-5 and Comparative Examples 1-7, the concentration and amount of seed liquid were the same.

[0031] Example 2 A fermentation method for simultaneously inhibiting ISO-GA3, gibberellic acid, and epoxygibacic acid in gibberellic acid fermentation broth to reduce the total impurity content includes the following steps: S1. Take the seed liquid and fermentation culture medium from Example 1. The mass of the seed liquid is 10% of the mass of the fermentation culture medium. The fermentation culture medium is shown in Table 2 below.

[0032] S2, add the seed culture and fermentation broth into the fermenter.

[0033] S3 undergoes a fermentation reaction.

[0034] In this step, the fermentation reaction conditions are as follows: Fermentation volume: 82t for fixed volume, 90t for volume after sterilization, and 100t for volume after conversion.

[0035] Fermentation temperature: 29±0.2℃.

[0036] Operating tank pressure: 0.035 MPa.

[0037] Airflow rate: 2200 Nm 3 / h.

[0038] Fermentation cycle: 198 hours.

[0039] pH: The pH is controlled at 5.0-5.2 by adding ammonia water. The ammonia water enters from the feed port of the fermenter at room temperature and has a concentration of 25wt% (before 168h, the pH is controlled at 5.0-5.2). When fermentation reaches 168h, in addition to adding ammonia water, a 15% citric acid aqueous solution is also added to control the pH at 3.8-4.2 (that is, after 168h, the pH is controlled at 3.8-4.2).

[0040] Regular feeding: When the dissolved oxygen level rebounds to 60%, feeding begins (i.e., adding 45wt% glucose solution and salad oil). The initial feeding rate is 100L / h for glucose solution and 40L / h for salad oil. Subsequently, when the dissolved oxygen level is below 20%, the feeding rate is reduced, and when the dissolved oxygen level is above 30%, the feeding rate is increased. The feed enters the airflow channel through the feeding pipe and is carried into the fermenter by the airflow. The regular feeding temperature is room temperature.

[0041] Collaborative control of impurity reduction feeding: When fermentation reaches 48h, curcumin is added at a rate of 65mg / L (i.e., 5.85kg – based on a base of 90T, the same below); when fermentation reaches 120h, N-acetylcysteine ​​and borax are added. The amount of N-acetylcysteine ​​added is 125mg / L (i.e., 11.25kg), and the amount of borax added is 1.5mM (equivalent to 0.572g / L, i.e., 51.48kg). The feeding is done at room temperature and enters the airflow channel through the feeding pipe, following the airflow into the fermenter. After fermentation, the product was sterilized and placed in a tank. Samples were taken for testing, and the results are shown in Table 3 below.

[0042] Table 2 Fermentation broth Example 3 The difference between this embodiment and embodiment 2 is as follows: pH: The added citric acid aqueous solution is 12%.

[0043] Synergistic control of impurity reduction feeding: Curcumin supplementation amount is 50 mg / L; N-acetylcysteine ​​supplementation amount is 100 mg / L.

[0044] After fermentation, the product was sterilized and placed in a tank. Samples were taken for testing, and the results are shown in Table 3 below.

[0045] Example 4 The difference between this embodiment and embodiment 2 is as follows: pH: The added citric acid aqueous solution is 20%.

[0046] Synergistic control of impurity reduction feeding: Curcumin supplementation amount is 80 mg / L; N-acetylcysteine ​​supplementation amount is 150 mg / L.

[0047] After fermentation, the product was sterilized and placed in a tank. Samples were taken for testing, and the results are shown in Table 3 below.

[0048] Example 5 The difference between this embodiment and embodiment 2 is as follows: pH: No citric acid was added during the entire fermentation process. The pH was controlled at 5.0-5.2 by adding ammonia water.

[0049] After fermentation, the product was sterilized and placed in a tank. Samples were taken for testing, and the results are shown in Table 3 below.

[0050] Comparative Example 1 The difference between this comparative example and Example 2 is as follows: The synergistic control of impurity reduction feeding is as follows: when fermentation reaches 48 hours, curcumin is added at a dosage of 65 mg / L.

[0051] After fermentation, the product was sterilized and placed in a tank. Samples were taken for testing, and the results are shown in Table 3 below.

[0052] Comparative Example 2 The difference between this comparative example and Example 2 is as follows: The synergistic control of impurity reduction feeding was as follows: when fermentation reached 120h, N-acetylcysteine ​​and borax were added, with N-acetylcysteine ​​added at 75mg / L and borax added at 1.5mM.

[0053] After fermentation, the product was sterilized and placed in a tank. Samples were taken for testing, and the results are shown in Table 3 below.

[0054] Comparative Example 3 The difference between this comparative example and Example 2 is as follows: The synergistic control of impurity reduction feeding is as follows: when fermentation reaches 120h, N-acetylcysteine ​​is added at a dosage of 75mg / L.

[0055] After fermentation, the product was sterilized and placed in a tank. Samples were taken for testing, and the results are shown in Table 3 below.

[0056] Comparative Example 4 The difference between this comparative example and Example 2 is as follows: The method for synergistic control of impurity reduction feeding is as follows: when fermentation reaches 120h, borax is added, and the amount of borax added is 1.5mM.

[0057] After fermentation, the product was sterilized and placed in a tank. Samples were taken for testing, and the results are shown in Table 3 below.

[0058] Comparative Example 5 The difference between this comparative example and Example 2 is as follows: The synergistic control of impurity reduction feeding was as follows: when fermentation reached 48h, curcumin, N-acetylcysteine ​​and borax were added at a dosage of 65mg / L; the dosage of N-acetylcysteine ​​was 75mg / L and the dosage of borax was 1.5mM.

[0059] After fermentation, the product was sterilized and placed in a tank. Samples were taken for testing, and the results are shown in Table 3 below.

[0060] Comparative Example 6 The difference between this comparative example and Example 2 is as follows: The synergistic control of impurity reduction feeding was as follows: when fermentation reached 120h, curcumin, N-acetylcysteine, and borax were added at a dosage of 65mg / L; the dosage of N-acetylcysteine ​​was 75mg / L, and the dosage of borax was 1.5mM.

[0061] After fermentation, the product was sterilized and placed in a tank. Samples were taken for testing, and the results are shown in Table 3 below.

[0062] Comparative Example 7 The difference between this comparative example and Example 2 is as follows: pH: During the entire fermentation process, there is no citric acid fed in; the pH is controlled at 5.0–5.2 by feeding in ammonia.

[0063] Throughout the entire fermentation process, there was no coordinated control of the addition of impurity-reducing feed.

[0064] After fermentation, the product was sterilized and placed in a tank. Samples were taken for testing, and the results are shown in Table 3 below.

[0065] Table 3 Fermentation Broth The results show that the most significant factor affecting the impurity content is borax, followed by N-acetylcysteine ​​and curcumin. Comparative Example 4 shows that adding borax alone is also effective in controlling impurities, but the acid production is significantly reduced compared to Example 2. The synergistic effect of the three factors, combined with pH control, can achieve the goal of controlling impurities without a significant decrease in GA3.

[0066] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A fermentation method for simultaneously inhibiting ISO-GA3, gibberellic acid, and epoxygibacic acid in gibberellic acid fermentation broth to reduce the total impurity content, characterized in that, In addition to conventional feeding, this method also incorporates synergistic control of impurity reduction feeding during fermentation.

2. The fermentation method according to claim 1, which simultaneously inhibits ISO-GA3, gibberellic acid, and epoxygibacic acid in gibberellic acid fermentation broth to reduce the total impurity content, is characterized in that, The synergistic control of impurity reduction feeding involves adding curcumin during the logarithmic growth phase and adding N-acetylcysteine ​​and borax during the initial vigorous growth phase of product synthesis.

3. The fermentation method according to any one of claims 1 or 2, which simultaneously inhibits ISO-GA3, gibberellic acid, and epoxygibacic acid in gibberellic acid fermentation broth to reduce the total impurity content, is characterized in that... This method also controls the pH at 3.8-4.2 in the later stages of product synthesis.

4. The fermentation method according to claim 1, which simultaneously inhibits ISO-GA3, gibberellic acid, and epoxygibacic acid in gibberellic acid fermentation broth to reduce the total impurity content, is characterized in that, Includes the following steps: S1, take the seed culture and fermentation broth; S2, Add the seed culture and fermentation broth into the fermenter; S3 undergoes a fermentation reaction; In S3, routine feeding and collaborative control for impurity reduction feeding are performed; The synergistic control of impurity reduction feeding is as follows: when fermentation reaches 48h, add curcumin at a rate of 50 mg to 80 mg per liter; when fermentation reaches 120h, add N-acetylcysteine ​​and borax at a rate of 100 to 150 mg per liter and 1.3 mmol to 1.6 mmol per liter.

5. The fermentation method according to claim 4, which simultaneously inhibits ISO-GA3, gibberellic acid, and epoxygibacic acid in gibberellic acid fermentation broth to reduce the total impurity content, is characterized in that... In S3, the fermentation temperature is 28~30℃; the air flow rate is 1500~4600 Nm. 3 Fermentation at a pressure of 0.01–0.05 MPa per hour.

6. The fermentation method according to claim 5, which simultaneously inhibits ISO-GA3, gibberellic acid, and epoxygibacic acid in gibberellic acid fermentation broth to reduce the total impurity content, is characterized in that... In S3, the routine feeding procedure is as follows: when the dissolved oxygen rebounds to 60%, glucose solution and salad oil are added. The initial feeding rate is 100 L / h for glucose solution and 40 L / h for salad oil. In the subsequent process, when the dissolved oxygen is below 20%, the feeding rate is reduced, and when the dissolved oxygen is above 30%, the feeding rate is increased.

7. The fermentation method according to claim 5, which simultaneously inhibits ISO-GA3, gibberellic acid, and epoxygibacic acid in gibberellic acid fermentation broth to reduce the total impurity content, is characterized in that... In S3, the pH is controlled by adding ammonia water to maintain a pH of 5.0 to 5.

2.

8. The fermentation method according to claim 5, which simultaneously inhibits ISO-GA3, gibberellic acid, and epoxygibacic acid in gibberellic acid fermentation broth to reduce the total impurity content, is characterized in that... In S3, the pH is controlled as follows: ammonia water is added to control the pH to 5.0-5.

2. When fermentation reaches 168h, in addition to adding ammonia water, 10 wt%-20 wt% citric acid aqueous solution is added to control the pH to 3.8-4.

2.

9. The fermentation method according to claim 4, which simultaneously inhibits ISO-GA3, gibberellic acid, and epoxygibacic acid in gibberellic acid fermentation broth to reduce the total impurity content, is characterized in that... The liquid culture medium is: Corn gluten meal 10~30 g / L Sucrose 1~10g / L Potassium dihydrogen phosphate 1~10g / L Glucose 1~10g / L MgSO4·7H2O 0.5~1.5 g / L Salad oil 1~3 g / L Ammonium sulfate 0.5~1.5 g / L FeSO4·7H2O 0.005~0.015 g / L ZnSO4·7H2O 0.005~0.015 g / L MnSO4·H2O 0.005~0.015 g / L NaMoO4·2H2O 0.001~0.006 g / L CuSO4·5H2O 0.001~0.006 g / L CoCl2·6H2O 0.001~0.006 g / L and The solvent is water.

10. The fermentation method according to claim 4, which simultaneously inhibits ISO-GA3, gibberellic acid, and epoxygibacic acid in gibberellic acid fermentation broth to reduce the total impurity content, is characterized in that... The seed culture was cultured from Fusarium graminearum, whose preservation number was CGMCC No. 23265.

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