Method for improving fermentation efficiency and microbial agent activity of bacillus subtilis based on 2,6-pyridinedicarboxylic acid

Abstract

This invention discloses a method for improving the fermentation efficiency and inoculum activity of Bacillus subtilis based on 2,6-pyridine dicarboxylic acid, belonging to the field of biotechnology. The method involves using Bacillus subtilis (… Bacillus subtilis During the fermentation process of Bacillus subtilis, a low concentration of 2,6-pyridine dicarboxylic acid (DPA) is added to the fermentation medium. This invention is the first to discover and utilize the promoting effect of low concentration DPA on the growth of Bacillus subtilis. Furthermore, as an additive, it can simultaneously enhance the biofilm formation ability of Bacillus subtilis, thereby significantly improving the colonization competitiveness and biocontrol effect of the agent. This method is simple, low-cost, and easy to scale up for production, making it suitable for the industrial preparation of highly active Bacillus subtilis biocontrol agents.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for improving the fermentation efficiency and activity of Bacillus subtilis based on 2,6-pyridinedicarboxylic acid. Background Technology

[0002] Bacillus subtilis ( Bacillus subtilis As an important biocontrol agent, the industrial production and field colonization efficiency of Bacillus subtilis are crucial to its disease control effectiveness. In fermentation production, high-density cell biomass is typically pursued; however, high-density fermentation can sometimes lead to decreased cell viability and stress-response gene expression, thus affecting its competitive colonization ability in complex soil environments. Biofilm formation ability is one of the core traits for Bacillus subtilis to establish ecological niche dominance. Therefore, developing processes that can simultaneously improve fermentation cell yield and agent colonization activity (such as biofilm formation ability) is a common industry need to improve the actual control efficacy of biocontrol agents and reduce application costs.

[0003] 2,6-Pyridinedicarboxylic acid (DPA) is a key component of Bacillus subtilis spores and has also been reported as a secondary metabolite with broad-spectrum antifungal activity. Some studies have used genetic modification (such as replacing...) spoVF Operators and promoters are used to increase bacterial DPA production, aiming to obtain higher concentrations of antimicrobial substances. However, the core logic of these works is to treat DPA as an outward-facing antimicrobial weapon.

[0004] The inventors discovered that low concentrations of DPA do not inhibit Bacillus subtilis itself, but rather exhibit a significant promoting effect. This phenomenon suggests that DPA may possess an inward regulatory function that has not yet been recognized. Currently, there are no reports, either domestically or internationally, on the use of DPA as a fermentation additive to regulate the physiological state of biocontrol bacteria and simultaneously improve their production efficiency and product performance. Summary of the Invention

[0005] The purpose of this invention is to provide a new method that can simultaneously improve the fermentation yield of Bacillus subtilis and its colonization activity, so as to overcome the contradiction between high density and high activity in existing fermentation technologies.

[0006] The first aspect of the present invention is to provide a method for improving the fermentation efficiency of Bacillus subtilis, wherein 2,6-pyridinedicarboxylic acid is added to the fermentation medium during the fermentation process of Bacillus subtilis.

[0007] Furthermore, the Bacillus subtilis is Bacillus subtilis 168 or... spoVF Operator expression enhances engineered Bacillus subtilis strain.

[0008] Furthermore, the concentration of the 2,6-pyridinedicarboxylic acid is from 50 μM to 500 μM.

[0009] Furthermore, the fermentation medium is LB medium supplemented with anhydrous glucose.

[0010] Furthermore, the fermentation conditions are 37°C and 200 rpm shaking culture.

[0011] The second aspect of the present invention is to provide a biocontrol agent, which is prepared by the following method: Bacillus subtilis is added to a fermentation medium supplemented with 2,6-pyridine dicarboxylic acid for fermentation culture. After fermentation, the bacterial sludge is collected by centrifugation and resuspended in sterile water to obtain the bacterial agent.

[0012] Furthermore, the Bacillus subtilis is Bacillus subtilis 168 or... spoVF Operator expression enhances engineered Bacillus subtilis strain.

[0013] Furthermore, the concentration of the 2,6-pyridinedicarboxylic acid is from 50 μM to 500 μM.

[0014] Furthermore, the fermentation medium is LB medium supplemented with anhydrous glucose.

[0015] Furthermore, the fermentation conditions are 37°C, 200 rpm shaking culture for 16 h.

[0016] A third aspect of the present invention is to provide the application of the above-mentioned biocontrol agent in the control of plant pathogenic fungi.

[0017] In one specific embodiment of the present invention, the plant pathogenic fungus is *Fusarium solani* (…). Fusarium ipomoeae ).

[0018] This invention is the first to discover and utilize the promoting effect of low-concentration DPA on the growth of Bacillus subtilis. Furthermore, using it as an additive can simultaneously enhance the biofilm-forming ability of Bacillus subtilis, thereby significantly improving the colonization competitiveness and biocontrol efficacy of the bacterial agent. This method is simple, low-cost, and easy to scale up for production, making it suitable for the industrial-scale preparation of highly active Bacillus subtilis biocontrol agents.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Synergistic effects: For the first time, the beneficial effects of low-concentration DPA on Bacillus subtilis are utilized to achieve the dual goals of simultaneously increasing bacterial biomass (yield) and biofilm formation ability (activity), breaking through the limitations of traditional fermentation processes.

[0020] 2. Novel Mechanism: DPA is redefined from the traditional "antibacterial substance" or "spore component" as a "fermentation promoter" and "colony activity inducer", opening up a completely new application field for DPA.

[0021] 3. Simple operation and low cost: This method does not require complex genetic modification and can be achieved simply by adding trace amounts of DPA exogenously. The process is simple and easy to implement and scale up in existing fermentation systems, and has significant economic and application promotion value.

[0022] 4. Significant effects: The microbial agents prepared using this method are expected to have better colonization competitiveness and disease control effects on plant surfaces or rhizospheres than those prepared using conventional fermentation methods, due to the high density and high biofilm formation capacity of the microbial cells. Attached Figure Description

[0023] Figure 1 In Example 1 of this invention, different concentrations of DPA were used to study the effects of Bacillus subtilis 168 (WT) and... spoVF Graphs showing the effect of operon expression on the growth of engineered bacteria (MT). Where: a represents the growth effect on WT; b represents the growth effect on MT.

[0024] Figure 2 This is a graph illustrating the effect of adding the optimal concentration of DPA during shake-flask fermentation on the fermentation performance of Bacillus subtilis WT and MT engineered bacteria, as shown in Example 2 of this invention. Specifically, it shows the cell density (OD) during fermentation. 600 ) Change curve.

[0025] Figure 3 This is a graph illustrating the effect of DPA on the biofilm formation ability of Bacillus subtilis in Example 3 of the present invention. Wherein: a is a visual representation of the biofilm stained with crystal violet; b is a graph showing the effect of DPA on the biofilm formation ability of Bacillus subtilis at absorbance OD. 575 Quantitative results of the corresponding biofilm at the location.

[0026] Figure 4 This is a diagram illustrating the preventive and therapeutic effects of different Bacillus subtilis agents on Fusarium rot in soybean pods in Example 4 of this invention. In diagram a, the preventive effect is shown, and from left to right: a blank control group sprayed with sterile water, a treatment group sprayed with WT, a treatment group sprayed with WT containing 200 μM DPA, a treatment group sprayed with MT, and a treatment group sprayed with MT containing 500 μM DPA. Diagram b, the therapeutic effect, is also shown, and from left to right: a blank control group sprayed with sterile water, a treatment group sprayed with WT, a treatment group sprayed with WT containing 200 μM DPA, a treatment group sprayed with MT, and a treatment group sprayed with MT containing 500 μM DPA. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. The following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. All technologies implemented based on the above content of this invention fall within the scope of this invention.

[0028] Bacillus subtilis in the present invention and embodiments ( Bacillus subtilis Strain 168 has been extensively disclosed in existing technologies, such as in the literature "Sequencing and functional analysis of the genome of Bacillus subtilis It has been disclosed in strain 168 and is a commonly used model bacterium in the field of biotechnology. The accession number of the CMCC Culture Collection Center is ZKCC153891.

[0029] Bacillus subtilis in the present invention and embodiments ( Bacillus sublitis MT strain is spoVF The construction method of the engineered strain with promoter replacement has been described in the literature "(1) Toya Y, Hirasawa T, Ishikawa S, Chumsakul O, Morimoto T, Liu S, Masuda K, Kageyama Y, Ozaki K, Ogasawara N, Shimizu H. Enhanced dipicolinic acid production during the stationary phasein Bacillus subtilis by blocking acetoin synthesis[J]. Bioscience Biotechnologyand Biochemistry, 2015, 79: 2073-2080. (2) Wang T, Wang XC, Han MH, Song XG, Yang DJ, Wang SY, Laborda P, Shi XC. Enhanced spoVF operon increases hostattachment and biocontrol ability of Bacillus subtilis for the managementof Ceratocystis fimbriataThe method of constructing the sweet potato is traditional homologous recombination, which is simple to operate and can be obtained by the public from the Agricultural Microbial Application Technology Team of Nantong University. This was reported in the paper "in sweet potato[J]. Biological Control, 2021, 161:104651." and patent CN112322662A. Example 1

[0030] Determination of the effect of DPA on the growth of Bacillus subtilis Strains and culture media: Bacillus subtilis 168 (WT) and spoVF Operant expression was enhanced using engineered Bacillus subtilis strain (MT). M9 basal medium was used to avoid interference from complex components. The formulation of M9 medium is shown in Table 1.

[0031] Table 1 M9 culture medium formulation (1 L)

[0032] Bacterial culture preparation: Using a 50 μL sterile pipette tip, take a small amount of Bacillus subtilis 168 (WT) stored in a glycerol tube at -80℃ and spoVF The operon expression enhanced Bacillus subtilis engineered strain (MT) was inoculated into two 50 mL LB media and cultured overnight at 37°C and 200 rpm.

[0033] Growth assay: OD was measured using 30 mL of overnight WT and MT bacterial cultures. 600 The bacterial cells were collected by centrifugation at 5000 rpm for 8 minutes at 4°C, then resuspended in an equal volume of M9 medium and used as OD. 600 =0.1 inoculum was transferred to M9 medium containing serial concentrations of DPA (0, 25, 50, 100, 200, 500, 1000 μM). The medium was then incubated in 250 mL Erlenmeyer flasks containing 50 mL of M9 medium at 37°C with shaking at 200 rpm. OD was measured every 4 h. 600 A 16-hour growth curve was plotted. In this example, the OD... 600 All measurements were taken using a spectrophotometer.

[0034] The results are as follows Figure 1 As shown. According to Figure 1 It was found that low concentrations of DPA (25-500 μM) significantly promoted the growth of both WT and MT strains, with the MT strain showing a more sensitive response, reaching a peak promotion effect at 500 μM DPA; while high concentrations of DPA (>500 μM) exhibited an inhibitory effect. This experiment determined the optimal concentration window for DPA to exert its probiotic effect to be 20-500 μM, and provided a basis for the selection of optimal MT engineered strains. Example 2

[0035] Validation of the application of DPA as a fermentation additive in shake flask fermentation Preparation of bacterial culture: Using a 50 μL pipette tip, take a small amount of Bacillus subtilis 168 (WT) stored in a glycerol tube at -80℃ and... spoVF The operon expression enhanced Bacillus subtilis engineered strain (MT) was inoculated into two 50 mL LB media and cultured overnight at 37 °C and 200 rpm to form seeds.

[0036] Fermentation culture: WT and MT engineered bacteria were used as fermentation strains. The seed culture was appropriately diluted with sterile ddH2O and then... 600 A 0.1 g inoculum was transferred to a 250 mL shake flask containing 50 mL of LB fermentation medium supplemented with 1 g of anhydrous glucose. Two groups were set up: a control group (WT and MT without DPA); and an experimental group (WT with DPA supplemented to a final concentration of 200 μM, and MT with DPA supplemented to a final concentration of 500 μM). The cultures were incubated at 37 °C and 200 rpm with shaking for 16 h.

[0037] Table 2 LB medium formulation (50 mL)

[0038] Index detection: OD is measured every 4 hours. 600 Growth curves were plotted to assess the effect of exogenous DPA addition on bacterial cell growth.

[0039] The results are as follows Figure 2 As shown. According to Figure 2 It can be observed that, compared with the control group, the OD of the experimental group (WT with 200 μM DPA added, MT with 500 μM DPA added) was significantly higher. 600 The value was significantly higher than that of the control group, indicating that the addition of DPA promoted the growth of Bacillus subtilis and improved fermentation efficiency. Example 3

[0040] The effect of DPA-enhanced fermentation on the biofilm formation ability of Bacillus subtilis Preparation of bacterial culture: The WT and MT bacterial cultures obtained after fermentation in Example 2 were collected by centrifugation and resuspended in sterile ddH2O to OD200. 600 = 1.2, spare.

[0041] Biofilm formation experiment: Take 5 mL of the bacterial suspension obtained above and place it in a 10 mL centrifuge tube. Collect the cells by centrifuging at 5000 rpm and 4℃ for 8 minutes, and then resuspend them in an equal volume of sterile ddH2O. Then, inoculate 30 µL aliquots of the cell suspension into glass tubes containing different DPA concentrations, and add LB medium to the tubes to a final volume of 3 mL. Incubate at 37℃ for 3 days.

[0042] Quantitative determination of biofilm: After removing the aqueous solution, add 1 mL of 10% crystal violet solution (dissolved in 95% ethanol) to the glass tube and treat the biofilm for 1 h. Then, wash the test tube repeatedly with 10 mL of ddH2O until the purple color disappears, and dry the test tube with an oven to remove excess water. Finally, add 3 mL of 40% methanol and 10% glacial acetic acid aqueous solution to dissolve the crystal violet staining. Quantitative determination of biofilm using Nanodrop One at OD 575 The absorbance of the dissolved crystal violet was measured.

[0043] The results are as follows Figure 3 As shown in Table 3.

[0044] Table 3 Biomembrane OD 575 numerical values

[0045] Enhanced biofilm formation capacity and OD 575 The absorbance of the sample was significantly higher than that of the control group, indicating that the addition of DPA significantly enhanced the biofilm formation ability of Bacillus subtilis and improved the colonization potential of the inoculant. Example 4

[0046] Verification of the disease prevention effect of microbial agents prepared by DPA-enhanced fermentation method (using Fusarium solani as an example) Fusarium ipomoeae (Taking the edamame pod system as an example) Preparation of inoculum: WT and MT fermentation broths were prepared using fermentation without DPA (control group, method as in Example 2) and DPA-enhanced fermentation (experimental group, method as in Example 2), respectively. After fermentation, the inoculum was collected by centrifugation and resuspended in sterile water to the same viable cell concentration (e.g., 1×10⁻⁶). 8 (CFU / mL) was used to prepare the test bacterial agent.

[0047] In vivo prevention experiment: Healthy soybean pods of approximately equal size were selected, and their surfaces were disinfected with 4% 84 disinfectant and then air-dried. The following treatments were set up: ① Blank control (sprayed with sterile water); ② Conventional fermentation agent treatment; ③ DPA-enhanced fermentation agent treatment. Each treatment was replicated with at least three pods. Wounds of approximately 0.2 cm² were then created on the surface of the soybean pods using a sterile scraper. The experimental groups were then sprayed with biocontrols: Bacillus subtilis (WT and MT) without DPA was sprayed onto the soybean pods (approximately 20 μL of bacterial solution was sprayed onto the wound per pod); then, bacterial solution with DPA was sprayed onto other pods, following the same procedure as the control group. The control group was sprayed with an equal volume of sterile water. After spraying, the pods were dried at room temperature for 1 h. Then, they were inoculated with a pathogen spore suspension (1×10⁻⁶). 6 Apply spores (20 µL / mL) to the wound. Incubate at 28°C and 80% relative humidity for 5 days. Observe and evaluate the preventive effect.

[0048] In vivo treatment experiment: Healthy soybean pods were used, similar to those used in the in vivo prevention experiment. Wounds (approximately 0.2 cm²) were created using a sterile scraper. The pods were immediately inoculated with a suspension of pathogenic spores (1 × 10⁻⁶). 6 Apply spores (20 µL / mL, spores / mL) to the wound. Incubate at 28°C and 80% relative humidity for 24 h to allow the pathogen to establish infection. After 24 h, spray with Bacillus subtilis agent (method as in the prevention experiment). The control group is sprayed with an equal volume of sterile water. Continue incubation under the same conditions for 5 days. Finally, observe and evaluate the treatment effect.

[0049] The results are as follows Figure 4 As shown. According to Figure 4 It was found that in both prevention and treatment experiments, the microbial agent prepared by the DPA-enhanced fermentation method was significantly more effective than the microbial agent prepared by the conventional fermentation method and the control group in treating Fusarium rot of edamame pods. This proves that the method of the present invention can effectively improve the final biocontrol efficacy of the microbial agent.

Claims

1. A method for improving the fermentation efficiency of Bacillus subtilis, characterized by, In the fermentation process of Bacillus subtilis, 2,6-pyridine dicarboxylic acid is added to the fermentation medium to improve the fermentation efficiency of Bacillus subtilis.

2. The method according to claim 1, characterized in that, The B. subtilis is B. subtilis 168 or spoVF Enhanced operon expression in engineered B. subtilis 3. The method according to claim 1, characterized in that, The concentration of the 2,6-pyridinedicarboxylic acid is from 50 μM to 500 μM.

4. The method according to claim 1, characterized in that, The fermentation medium is LB medium supplemented with anhydrous glucose.

5. A biocontrol agent, characterized in that, The bacterial agent was prepared by adding Bacillus subtilis to a fermentation medium supplemented with 2,6-pyridinedicarboxylic acid for fermentation culture. After fermentation, the bacterial sludge was collected by centrifugation and resuspended in sterile water to obtain the bacterial agent.

6. The biocontrol agent according to claim 5, characterized in that, The Bacillus subtilis is Bacillus subtilis 168 or... spoVF Operator expression enhances engineered Bacillus subtilis strain.

7. The biocontrol agent according to claim 5, characterized in that, The concentration of the 2,6-pyridinedicarboxylic acid is from 50 μM to 500 μM.

8. The biocontrol agent according to claim 5, characterized in that, The fermentation medium is LB medium supplemented with anhydrous glucose.

9. The biocontrol agent according to claim 5, characterized in that, The fermentation conditions were 37°C and 200 rpm for 16 hours with shaking.

10. The application of the biocontrol agent according to any one of claims 5-9 in the control of plant pathogenic fungi.

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