Fermentation method for reducing ISO-GA3 for producing gibberellic acid

Abstract

The invention discloses a fermentation method for producing gibberellic acid by reducing ISO-GA3 (International Standardization Organization-GA3), which comprises the following steps of: in the material supplementing process of fermentation, feeding refrigerated supplemented materials into a fermentation tank along with air, and controlling the temperature of the refrigerated supplemented materials to be 1t; 25 DEG C. According to the method, the content of ISO-GA3 in the fermentation liquor after fermentation can be reduced, and the content of GA3 can be increased.

Description

Technical Field

[0001] The invention relates to gibberellic acid production, and in particular to a fermentation method for producing gibberellic acid by reducing ISO-GA3. Background Art

[0002] Gibberellic Acid 3 (GA3) is a plant growth regulating hormone produced by Gibberellic fungi. It plays an important regulatory role in plant growth and development and is used in many fields, including agriculture, forestry and brewing, with good economic value and social benefits. The traditional production methods of GA3 mainly include plant extraction, chemical synthesis and microbial fermentation; however, the yield of GA3 extracted from plants is low, it is easy to produce a lot of waste and the cost is high, and the chemical synthesis method is also limited by cumbersome process steps and the resulting pollution; with the rapid development of synthetic biology, microbial production of GA3 has attracted more and more attention.

[0003] At present, GA3 can be produced by fermentation of a variety of fungi, such as F.fujikuroi, Aspergillus niger and Fusarium oxysporum; the strain of the same cluster, F.moniliforme, has a higher yield, but requires solid-state fermentation culture and is not suitable for industrial production. Therefore, the main one used in the industrial production of gibberellin GA3 is F.fujikuroi, which has a higher yield and can be fermented in a controlled manner.

[0004] In recent years, due to its wide application in agriculture and horticulture, the GA3 market has significant potential and market demand, and the industrial production of GA3 has been achieved using Fusarium fusogenum.

[0005] CN108285915A discloses a GA3 fermentation method, which uses Fusarium fusogenum, inoculates the seed liquid into the fermentation medium, and ferments in a fermenter. During the fermentation process, the air flow rate into the fermenter is 2000~3000Nm3 / h. When the dissolved oxygen rebounds by 20%, the automatic flow is turned on to add glucose solution to control the dissolved oxygen at 20%~30%. Although the GA3 content in the fermentation liquid of CN108285915A is high, the GA3 content in the fermentation liquid needs to be further increased to improve the economy.

[0006] Iso-GA3 is an impurity detected in the fermentation liquid in the fermentation tank. How to reduce the iso-GA3 content and increase the GA3 content has become the pursuit of industry technicians.

[0007] The above background technology is for facilitating the understanding of the present invention and is not the known technology disclosed to the general public before the application of the present invention. Summary of the invention

[0008] In view of the above defects, the present invention provides a fermentation method for producing gibberellic acid by reducing ISO-GA3, which can reduce the content of ISO-GA3 in the fermentation liquid after fermentation and increase the content of GA3.

[0009] The technical solution is: a fermentation method for producing gibberellic acid by reducing ISO-GA3, in which during the feeding process of fermentation, the refrigerated feed is introduced into the fermentation tank along with the air, and the refrigerated feed temperature is less than 25°C. Furthermore, the refrigerated feed temperature is 5-20°C.

[0010] Further, the method comprises the following steps: S1, take seed solution and fermentation culture medium, the mass of seed solution is 5-15% of the mass of fermentation culture medium; S2, adding seed solution and fermentation culture solution into the fermentation tank; S3, performing a fermentation reaction to obtain a GA3 fermentation liquid with a reduced ISO-GA3 content.

[0011] Further, in S3, the fermentation reaction conditions are: Fermentation temperature is 28~30℃; air flow is 1500~4600 Nm 3 / h, PH 5.0~5.2, pressure 0.01~0 .05MPa; When the dissolved oxygen rebounds to 50~80%, start feeding at the same time. The initial feeding rate is: 20~200L / h for glucose solution and 5~65L / h for salad oil. When the dissolved oxygen is lower than 15~25%, reduce the feeding rate. When the dissolved oxygen is higher than 28~38%, increase the feeding rate.

[0012] Furthermore, the feed is glucose solution and salad oil.

[0013] Furthermore, the concentration of the glucose solution is 45wt%.

[0014] Furthermore, the liquid culture medium is: Corn gluten meal 5~7.5 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 Soy protein 1~10 g / L and The solvent is water.

[0015] Furthermore, 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.

[0016] Furthermore, the seed liquid is seed liquid cultured with Gibberella fujikura, and the preservation number of Gibberella fujikura is CGMCC No.23265.

[0017] Compared with the prior art, the inventive principle and beneficial effects of the present invention are: The inventors of the present invention surprisingly discovered by accident that during the fermentation process, when feeding and carbon source supplementation are performed, cooling and lowering the temperature can reduce ISO-GA3 in the fermentation liquid in the lower tank, while increasing the content of GA3. DETAILED DESCRIPTION

[0018] As used herein: "Prepared from" is synonymous with "comprising." As used herein, the terms "comprising," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0019] The conjunction "consisting of excludes any unspecified element, step, or component. If used in a claim, this phrase renders the claim closed-ended so that it does not include materials other than those described, except for conventional impurities associated therewith. When the phrase "consisting of" appears in a clause of the body of a claim rather than immediately following the subject matter, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.

[0020] When an amount, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted as including ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described in this article, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range.

[0021] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are 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 ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Those skilled in the art will appreciate that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. If specific conditions are not specified in the examples, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0023] In these examples, parts and percentages are by mass unless otherwise indicated.

[0024] "Parts by mass" refers to the basic unit of measurement for expressing the mass ratio of multiple components. 1 part can represent any unit mass, such as 1g or 2.689g. If we say that the mass of component A is a parts and the mass of component B is b parts, it means the ratio of the mass of component A to the mass of component B is a:b. Alternatively, it means that the mass of component A is aK and the mass of component B is bK (K is an arbitrary number, indicating a multiple factor). It should not be misunderstood that, unlike the mass parts, the sum of the mass of all components is not limited to 100 parts.

[0025] "And / or" is used to indicate that one or both of the stated situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0026] Example 1 Preparation of Seed Liquid before Fermentation 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, with a deposit number of CGMCC No. 23265 (disclosed in CN114231421A).

[0027] The seed liquid test data is shown in Table 1.

[0028] Table 1 Seed liquid test data before fermentation The seed liquid prepared in Example 1 was used to prepare the fermentation liquids of the following Examples 2 to 5 and Comparative Examples 1 to 5, respectively.

[0029] In Examples 2 to 5 and Comparative Examples 1 to 5, the concentration and dosage of the seed solution are equal.

[0030] Example 2 A gibberellic acid fermentation method comprises the following steps: S1, take the seed solution and fermentation culture medium of Example 1, the mass of the seed solution is 10% of the mass of the fermentation culture medium, and the fermentation culture medium is as shown in Table 2.

[0031] S2, adding seed solution and fermentation culture medium into the fermentation tank.

[0032] S3, fermentation reaction.

[0033] In this step, the fermentation reaction conditions are: Fermentation volume: fixed volume 82t, volume after digestion 90t, volume after transfer 100t.

[0034] Fermentation temperature: 29±0.2℃.

[0035] Operating tank pressure: 0.035MPa.

[0036] Air flow: 2200Nm 3 / h.

[0037] PH: pH was controlled at 5.0-5.2 by adding ammonia water. Ammonia water entered from the feed port of the fermentation tank. The ammonia water was at room temperature of 25°C and the concentration of ammonia water was 25wt%.

[0038] Feeding: When the dissolved oxygen rebounds to 60%, feed (i.e., add 45wt% glucose solution and salad oil) is started. The initial feed rate is 100L / h for glucose solution and 40L / h for salad oil. In the subsequent process, when the dissolved oxygen is lower than 20%, the feed rate is reduced, and when the dissolved oxygen is higher than 30%, the feed rate is increased. The feed enters the airflow channel from the feed pipe and enters the fermenter with the airflow. The feed is a cooled liquid, and the feed temperature is 5°C.

[0039] Fermentation period: 198 hours.

[0040] After fermentation, the tanks were sterilized and samples were taken for testing. The results are shown in Table 4.

[0041] Table 2 Fermentation broth of Example 2 Example 3 The difference between this embodiment and embodiment 2 is that the cooling degree before adding the feed is different. In this embodiment, the feed temperature is 10°C.

[0042] After fermentation, the tanks were sterilized and samples were taken for testing. The results are shown in Table 4.

[0043] Example 4 The difference between this embodiment and embodiment 2 is that the cooling degree before adding the feed is different. In this embodiment, the feed temperature is 20°C.

[0044] After fermentation, the tanks were sterilized and samples were taken for testing. The results are shown in Table 4.

[0045] Example 5 The difference between this embodiment and embodiment 2 is that the fermentation broth is different. The fermentation broth of this embodiment is shown in Table 3.

[0046] Table 3 Fermentation broth of Example 5 After fermentation, the tanks were sterilized and samples were taken for testing. The results are shown in Table 4.

[0047] Comparative Example 1 The only difference between this comparative example and Example 2 is the degree of cooling before adding the feed. In this example, the feed temperature is 0°C.

[0048] After fermentation, the tanks were sterilized and samples were taken for testing. The results are shown in Table 4.

[0049] Comparative Example 2 The only difference between this comparative example and Example 2 is that the feed was not cooled before addition, but added at room temperature. The feed temperature was 25°C.

[0050] After fermentation, the tanks were sterilized and samples were taken for testing. The results are shown in Table 4.

[0051] Comparative Example 3 The only difference between this comparative example and Example 2 is that the feed is heated to the reaction temperature before adding the feed. The feed temperature is 29°C.

[0052] After fermentation, the tanks were sterilized and samples were taken for testing. The results are shown in Table 4.

[0053] Comparative Example 4 The difference between this comparative example and Example 2 is that the position of adding supplementary material is different. In this comparative example, the position of adding supplementary material is the feed inlet, which is the same as the position of adding ammonia water.

[0054] After fermentation, the tanks were sterilized and samples were taken for testing. The results are shown in Table 4.

[0055] Comparative Example 5 The only difference between this comparative example and Example 5 is that the feeding temperature is different, and the feeding temperature is room temperature 25°C.

[0056] After fermentation, the tanks were sterilized and samples were taken for testing. The results are shown in Table 4.

[0057] Table 4 Fermentation broth test data Comparing the GA3 and ISO-GA3 contents of the fermentation broth of Examples 2-4 with those of Comparative Examples 1-3, adding cooled glucose solution and salad oil to the fermentation tank during feeding can reduce the ISO-GA3 of the final fermentation broth and increase the GA3 content, but the cooling effect is only effective within a certain range. When the temperature is reduced to 0°C, the effect decreases instead of increasing; when the feeding temperature is close to the reaction temperature, the ISO-GA3 content increases and the GA3 content decreases compared with room temperature. Comparing Example 2 with Comparative Example 4, the cooled glucose solution and salad oil can enter the fermentation tank together with the airflow to obtain better results. By comparing Example 5 with Comparative Example 5, it can be seen that after changing the fermentation broth, lowering the temperature of the feeding liquid also has the effect of reducing the ISO-GA3 of the final fermentation broth and increasing the GA3 content.

[0058] 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 fermentation method for producing gibberellic acid by reducing ISO-GA3, characterized in that: In the method, during the feeding process of fermentation, the refrigerated feed is introduced into the fermentation tank along with the air, and the temperature of the refrigerated feed is less than 25°C.

2. The fermentation method for producing gibberellic acid by reducing ISO-GA3 according to claim 1, characterized in that The refrigerated feed temperature is 5-20°C.

3. The fermentation method for producing gibberellic acid by reducing ISO-GA3 according to any one of claims 1 or 2, characterized in that, The method comprises the following steps: S1, take seed solution and fermentation culture medium, the mass of seed solution is 5-15% of the mass of fermentation culture medium; S2, adding seed solution and fermentation culture solution into the fermentation tank; S3, performing a fermentation reaction to obtain a GA3 fermentation liquid with a reduced ISO-GA3 content.

4. The fermentation method for producing gibberellic acid by reducing ISO-GA3 according to claim 3, characterized in that In S3, the fermentation reaction conditions are: Fermentation temperature is 28~30℃; air flow is 1500~4600 Nm 3 / h, PH 5.0~5.2, pressure 0.01~0.05MPa; When the dissolved oxygen rebounds to 50~80%, start feeding at the same time. The initial feeding rate is: 20~200L / h for glucose solution and 5~65 L / h for salad oil. When the dissolved oxygen is lower than 15~25%, reduce the feeding rate. When the dissolved oxygen is higher than 28~38%, increase the feeding rate.

5. The fermentation method for producing gibberellic acid by reducing ISO-GA3 according to claim 4, characterized in that The supplementary feed is glucose solution and salad oil.

6. The fermentation method for producing gibberellic acid by reducing ISO-GA3 according to claim 5, characterized in that The concentration of the glucose solution is 45wt%.

7. The fermentation method for producing gibberellic acid by reducing ISO-GA3 according to claim 4, characterized in that The liquid culture medium is: Corn gluten meal 5~7.5 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 Soy protein 1~10 g / L and The solvent is water.

8. The fermentation method for producing gibberellic acid by reducing ISO-GA3 according to claim 4, 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.

9. The fermentation method for producing gibberellic acid by reducing ISO-GA3 according to claim 4, characterized in that The seed liquid is the seed liquid cultured from Gibberella fujikura, and the preservation number of Gibberella fujikura is CGMCC No.23265.

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