Solid state fermentation optimization method of GA4+7 mutant strain

Abstract

The invention provides a solid state fermentation optimization method of a GA4 + 7 mutant strain. According to the method, a solid-state fermentation culture medium and solid-state fermentation conditions for producing gibberellin GA4 + 7 are explored and optimized, bran is selected as a solid-state culture medium structure support, meanwhile, the initial water content of the solid culture medium is optimized, the optimal water content is determined to be 60%, the yield of gibberellin GA4 + 7 is increased, the yield of gibberellin GA4 + 7 is increased, and the yield of gibberellin GA4 + 7 is increased. The gibberellin can be conveniently extracted after fermentation is finished. In the extraction stage, 20% methanol is added in the extraction stage, so that the extraction amount of gibberellin can be increased, and the use amount of an organic solvent is also reduced. Finally, under the cooperation of various factors such as a structural support, the initial water content of a solid culture medium and a solid-state fermentation product extraction method, after solid-state fermentation and extraction are carried out on the gibberellic gibberellic mutant strain, the yield of the GA4 + 7 reaches 6.22 g / kg, and the liquid submerged fermentation level is greatly improved.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically relating to the bio-fermentation of gibberellin, and more specifically to a GA 4+7 Solid-state fermentation medium for mutant strains and optimization methods for solid-state fermentation. Background Technology

[0002] Gibberellins are endogenous growth regulators widely found in plants, participating in multiple biological processes of plant growth and development, and playing an important regulatory role in plant growth and development. In recent years, GA... 4+7 Its applications in flower and fruit preservation and dormancy breaking have attracted attention. In promoting stem and leaf growth in dwarf crops, GA4 showed superior activity compared to GA3; when used to adjust fruit shape, GA... 4+7 Because of its higher activity and more direct effects, it is increasingly widely used as a plant growth regulator in agricultural and forestry production processes.

[0003] Currently, gibberellin GA 4+7 Industrial production of gibberellin still primarily relies on liquid submerged fermentation because the fermentation process is easy to control and scale up. However, liquid fermentation also presents challenges such as high equipment investment, complex process control, and relatively high production costs, with both fermentation potency and cost control nearing their limits. Solid-state fermentation, on the other hand, requires less investment and is simpler to manage and operate, offering certain production advantages. Therefore, solid-state fermentation technology is gaining increasing attention. However, achieving large-scale industrial solid-state fermentation of gibberellin in the short term still faces numerous difficulties.

[0004] Solid-state fermentation offers a wide range of culture medium options, including conventional liquid culture medium formulations and alternatives such as agricultural residues like straw and corn cobs to reduce production costs. While the solid-state fermentation substrate can be directly used in agricultural production after drying and pulverizing, it contains a relatively low dose of gibberellin, resulting in poor efficacy. Furthermore, the presence of agricultural residues in the solid-state fermentation substrate makes gibberellin extraction more difficult than with liquid fermentation, and the recovery rates vary significantly depending on the extraction method. Summary of the Invention

[0005] To address the challenges of existing solid-state fermentation technology for gibberellin (GA) 4+7 The present invention addresses the problems of low yield and difficult extraction, and provides a method suitable for GA. 4+7 Solid-state fermentation medium and optimization methods for mutant strains to improve GA 4+7 Yield and extraction rate.

[0006] The technical solution adopted in this invention is: a GA 4+7The solid-state fermentation medium for the mutant strain comprises 8-12 parts by weight of structural support, 8-12 parts by weight of carbon source, 0.01-0.02 parts by weight of MgSO4·7H2O, 0.01-0.02 parts by weight of KH2PO4, and 10-95 parts by weight of H2O; the structural support comprises at least one of wheat bran, sawdust, and rice husk.

[0007] Different culture medium structures and support materials, due to variations in particle size, can significantly impact oxygen, moisture, and cell adhesion during fermentation, thereby affecting gibberellin yield. This invention utilizes bran, sawdust, and rice husks—materials that facilitate subsequent product extraction and separation—as support materials for the gibberellin GA strain of *Gibberella fuciformis*. 4+7 The effects of different substrate components on gibberellin yield were investigated. The experimental results show that different substrate components have a significant impact on gibberellin yield; when bran is used as the substrate structural support, GA… 4+7 The highest yield was achieved, reaching 3.6 g / kg. A control group without a substrate structure was also established. The experiment revealed that without a substrate support, the culture medium easily formed large clumps, leaving almost no space for bacterial growth, resulting in lower gibberellin yields. This also demonstrates the effectiveness of gibberellin production in *Gibberella fuciformis* GA. 4+7 In the solid-state fermentation of mutant strains, the importance of the substrate support structure is not only related to gibberellin GA 4+7 Yield will also affect the extraction of gibberellin after fermentation.

[0008] Preferably, the initial moisture content of the solid fermentation medium is 40% to 80%, more preferably 40% to 60%, and even more preferably 60%.

[0009] Preferably, the carbon source includes at least one of rice flour and corn starch.

[0010] Preferably, the carbon source comprises 4-6 parts by weight of rice flour and 4-6 parts by weight of corn starch.

[0011] Preferably, the solid fermentation culture medium comprises 10 parts by weight of wheat bran, 5 parts by weight of rice flour, 5 parts by weight of corn starch, 0.01 parts by weight of MgSO4·7H2O, 0.01 parts by weight of KH2PO4, and 30 parts by weight of H2O.

[0012] The present invention also provides a GA 4+7 The method for optimizing the solid-state fermentation of mutant strains includes the following steps:

[0013] S1. Will GA 4+7 The mutant strain was inoculated into the solid-state fermentation medium and fermented.

[0014] S2. After fermentation, the solid-state fermentation culture was extracted in an acidic solution containing organic solvent. The supernatant was extracted with ethyl acetate, concentrated, and evaporated to dryness to obtain the product GA. 4+7 .

[0015] Preferably, the fermentation culture conditions include: culturing at 25~30℃ for 5~10 days.

[0016] Preferably, the extraction time is 2-3 hours.

[0017] Preferably, the mixture includes at least one of methanol and ethanol, with methanol being the most preferred.

[0018] Preferably, the concentration of the organic solvent in the acid solution is 10% to 40%, more preferably 10% to 20%, and even more preferably 20%.

[0019] Preferably, the pH of the acid solution is 2-3, and the acid solution is a hydrochloric acid solution.

[0020] Preferably, the method includes the following steps:

[0021] S1. Will GA 4+7 The mutant strain was inoculated into the solid fermentation medium described above and cultured at 25-30°C for 5-10 days.

[0022] S2. After fermentation, the solid-state fermentation culture was extracted in an acidic solution with a pH of 2-3 for 2-3 hours. The supernatant was extracted with ethyl acetate, concentrated, and evaporated to dryness to obtain the product GA. 4+7 The acid solution contains 20% methanol.

[0023] Beneficial effects of the present invention: The present invention explores and optimizes the production of gibberellin (GA). 4+7 Solid-state fermentation medium and conditions for the Fujikura gibberellin mutant strain were determined. Wheat bran was selected as the structural support for the solid-state medium, and the initial moisture content of the solid-state medium was optimized, with an optimal moisture content of 60%. This not only improved the yield of gibberellin GA... 4+7 This increases yield and facilitates gibberellin extraction after fermentation. During the extraction stage, adding 20% ​​methanol not only increases the gibberellin extraction rate but also reduces the amount of organic solvent used. Ultimately, through the synergistic effect of various factors such as structural support, initial moisture content of the solid culture medium, and solid-state fermentation product extraction methods, the Fujikura gibberellin mutant strain, after solid-state fermentation and extraction, yielded GA... 4+7 The yield reached 6.22 g / kg, which is a significant improvement over the liquid deep fermentation level (generally 2.5 g / L). Attached Figure Description

[0024] Figure 1 Gibberellin (GA) 4+7Solid-state fermentation process diagram.

[0025] Figure 2 Examples of different structural supports for GA in Example 1 4+7 Impact on production.

[0026] Figure 3 Example 2: Effects of different initial moisture contents on GA 4+7 Impact on production.

[0027] Figure 4 Example 3: Different organic solvents for GA 4+7 The impact of extraction rate. Detailed Implementation

[0028] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0029] Strain selection: The OEthmgr-ggs2-cps / ks strain was used for fermentation culture in this embodiment. The construction method of strain OEthmgr-ggs2-cps / ks included: using *Fujikura gibberellinii* with the P450-3 gene knocked out as the substrate strain; constructing a vector co-expressing the truncated hydroxymethylglutaryl-CoA reductase gene *thmgr*, the geranyl diphosphate synthase gene *ggs2*, and the diterpenoid cyclase gene *cps / ks*; and integrating the vector into the substrate strain genome via protoplast transformation to construct the resulting gibberellin GA. 4+7 Highly efficient production strain OE thmgr-ggs2-cps / ks.

[0030] Strain activation: Using a sterile pipette, 10 μL of bacterial culture from a glycerol tube stored at -80℃ was inoculated into seed culture medium. The culture was incubated at 250 rpm and 28℃ for 2 days. Then, 1 mL of the culture was transferred to MYG slant culture medium in an eggplant bottle and spread evenly. The culture was incubated at 28℃ for 2-3 days and then stored at 4℃ for later use.

[0031] Seed culture preparation: Using a sterile inoculation spatula or toothpick, pick a single colony from MYG medium and transfer it to a 250 mL Erlenmeyer flask containing 25 mL of seed culture medium. Incubate at 250 rpm and 28°C for 2 days to obtain the seed culture. Seed culture medium composition: corn starch 20 g / L, sucrose 15 g / L, peanut powder 15 g / L, soybean powder 3 g / L, KH₂PO₄ 1 g / L, MgSO₄ 1 g / L, natural pH.

[0032] Example 1: The effect of different structural supports on GA 4+7 Impact on production

[0033] Three solid-state fermentation media (250 mL Erlenmeyer flasks) were prepared by selecting wheat bran, wood chips, and rice husks as structural supports.

[0034] Solid-state fermentation medium A: 10 g wheat bran, 5 g rice flour, 5 g corn starch, 0.01 g MgSO4·7H2O, 0.01 g KH2PO4, 20 mL H2O, pH natural.

[0035] Solid-state fermentation medium B: 10 g sawdust, 5 g rice flour, 5 g corn starch, 0.01 g MgSO4·7H2O, 0.01 g KH2PO4, 20 mL H2O, pH natural.

[0036] Solid-state fermentation medium C: 10 g rice husks, 5 g rice flour, 5 g corn starch, 0.01 g MgSO4·7H2O, 0.01 g KH2PO4, 20 mL H2O, pH natural.

[0037] Seed culture medium was used as a control group.

[0038] Two mL of seed culture was evenly inoculated onto the surfaces of solid-state fermentation media A, B, C, and the control group using a sterile pipette. After incubation at 28°C and 100 rpm for 7 days, gibberellin yield was measured. Results are as follows: Figure 2 As shown. Different substrate structures, due to differences in particle size, significantly affect oxygen, moisture, and cell adhesion during fermentation, thus impacting gibberellin yield. Experimental results indicate that different substrate components have a substantial impact on gibberellin yield; when wheat bran is used as the substrate structure support, GA… 4+7 The highest yield was achieved, reaching 3.6 g / kg. A control group without a substrate structure was also established. The experiment revealed that without a substrate support, the culture medium easily formed large clumps, leaving almost no space for bacterial growth, resulting in lower gibberellin yields. This also demonstrates the effectiveness of gibberellin production in *Gibberella fuciformis* GA. 4+7In the solid-state fermentation of mutant strains, the importance of the substrate support structure is not only related to gibberellin GA 4+7 Yield will also affect the extraction of gibberellin after fermentation.

[0039] Example 2: Effects of different initial moisture contents on GA 4+7 Impact on production

[0040] Based on solid-state fermentation medium A in Example 1, the effect of initial moisture content of the medium on gibberellin synthesis was investigated. With other conditions remaining constant in solid-state medium A, solid-state mediums with initial moisture contents of 40%, 50%, 60%, 70%, and 80% were prepared (i.e., 13 mL, 20 mL, 30 mL, 47 mL, and 80 mL of water were added to the solid-state medium, respectively). After culturing the seed culture for two days, 2 mL of seed culture was evenly inoculated onto the surface of the solid-state medium using a sterile pipette. The medium was incubated at 28°C and 100 rpm for 7 days, and then the gibberellin yield was measured. The results are as follows: Figure 3 As shown. Microbial growth and reproduction cannot occur without water. In solid-state fermentation, the moisture content of the microbial cells mainly comes from the initial moisture content of the culture medium. Therefore, different initial moisture contents have a significant impact on cell growth and production. Using wheat bran as the structural support of the culture medium, when the initial moisture content is 60%, gibberellin GA... 4+7 The highest yield was 4.44 g / kg, indicating that too little or too much moisture can inhibit gibberellin production. Excessive moisture leads to bran clumping, causing the culture medium to spread flat and difficult to turn, thus limiting the growth space and air contact of the cells. On the other hand, an appropriate moisture content ensures cell growth while allowing the culture medium to be easily turned, increasing the gaps between cell growth and the surface area for air contact. Therefore, the gibberellin yield is higher, and the softer bran matrix also facilitates subsequent gibberellin extraction.

[0041] Example 3: Effects of different organic solvents on GA 4+7 Effect of extraction rate

[0042] Based on the solid-state fermentation medium A with an initial moisture content of 60% in Example 2, the effect of extraction with different organic solvents on the recovery of gibberellin was investigated. Methanol and ethanol were added to 100 mL of hydrochloric acid solution to concentrations of 10%, 20%, 30%, and 40%, respectively, to prepare different acid solutions (pH=2). The solid-state fermentation medium after fermentation was extracted in each of the different acid solutions for 2 h. The supernatant was extracted with ethyl acetate, concentrated under low temperature and vacuum, and evaporated to dryness to obtain the product GA. 4+7 And the yield was measured. Results are shown below. Figure 3It can be seen that the effect of adding methanol is better than that of adding ethanol. When the methanol concentration is 20%, the gibberellin extraction reaches the maximum of 6.22 g / kg. After that, as the amount of organic solvent increases, the gibberellin extraction decreases. The reason for this phenomenon may be that the excessive addition of organic solvent causes the coagulation and precipitation of proteins and polysaccharides in the solid matrix, resulting in a reduction in the leaching amount of gibberellin.

[0043] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope of the present invention.

Claims

1. A type of GA 4+7 The solid-state fermentation medium for the mutant strain is characterized by, The solid fermentation medium comprises 8-12 parts by weight of structural support, 8-12 parts by weight of carbon source, 0.01-0.02 parts by weight of MgSO4·7H2O, 0.01-0.02 parts by weight of KH2PO4, and 10-95 parts by weight of H2O. The structural support includes at least one of bran, wood chips, and rice husks.

2. The solid-state fermentation culture medium as described in claim 1, characterized in that, The initial moisture content of the solid fermentation medium is 40%~80%.

3. The solid-state fermentation culture medium as described in claim 1, characterized in that, The carbon source includes at least one of rice flour and corn starch.

4. The solid-state fermentation culture medium as described in claim 1, characterized in that, The carbon source includes 4-6 parts by weight of rice flour and 4-6 parts by weight of corn starch.

5. The solid-state fermentation culture medium as described in claim 1, characterized in that, The solid-state fermentation medium comprises 10 parts by weight of wheat bran, 5 parts by weight of rice flour, 5 parts by weight of corn starch, 0.01 parts by weight of MgSO4·7H2O, 0.01 parts by weight of KH2PO4, and 30 parts by weight of H2O.

6. A type of GA 4+7 The method for optimizing solid-state fermentation of mutant strains is characterized by, Includes the following steps: S1. Will GA 4+7 The mutant strain was inoculated into the solid fermentation medium according to any one of claims 1 to 5 and fermented. S2. After fermentation, the solid-state fermentation culture was extracted in an acidic solution containing organic solvent. The supernatant was extracted with ethyl acetate, concentrated, and evaporated to dryness to obtain the product GA. 4+7 .

7. The method as described in claim 6, characterized in that, The fermentation conditions include: culturing at 25-30℃ for 5-10 days; The pH of the acid solution is 2-3; The extraction time is 2-3 hours.

8. The method as described in claim 6, characterized in that, The term includes at least one of methanol and ethanol.

9. The method as described in claim 6, characterized in that, The concentration of organic solvent in the acid solution is 10% to 40%.

10. The method as described in claim 6, characterized in that, Includes the following steps: S1. Will GA 4+7 The mutant strain was inoculated into the solid fermentation medium according to any one of claims 1 to 5 and cultured at 25 to 30°C for 5 to 10 days; S2. After fermentation, the solid-state fermentation culture was extracted in an acidic solution with a pH of 2-3 for 2-3 hours. The supernatant was extracted with ethyl acetate, concentrated, and evaporated to dryness to obtain the product GA. 4+7 The acid solution contains 20% methanol.

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