High-throughput screening method of bacillus subtilis strain for inhibiting aspergillus flavus

Abstract

The invention provides a high-throughput screening method of a bacillus subtilis strain for inhibiting aspergillus flavus. The method comprises the following steps: activating bacillus subtilis and then carrying out single colony separation; numbering the obtained single colonies, respectively inoculating the single colonies into 96 deep-well plates for fermentation, centrifuging fermentation liquor, taking supernate, and passing the supernate through a membrane to obtain fermentation supernate; respectively adding each fermentation supernatant into a 96-well plate, and adding a developing working solution for incubation; the developing working solution is a mixed solution of an aspergillus flavus spore suspension and a resazurin solution; screening strains corresponding to holes with fluorescence intensity lower than that of the blank control group and color close to blue as primary screening strains; and carrying out secondary screening on the fermentation supernatant of the primary screening strain by using the developing working solution, and screening the bacillus subtilis strain with high aspergillus flavus inhibition activity according to the fluorescence intensity. Therefore, the bacillus subtilis single strain with high inhibitory activity on aspergillus flavus can be efficiently screened from a large number of samples.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a high-throughput screening method for Bacillus subtilis strains that inhibit Aspergillus flavus. Background Technology

[0002] Bacillus subtilis belongs to the genus Bacillus. It is an aerobic or facultative anaerobic, mesophilic, Gram-positive rod-shaped bacterium with the ability to produce spores. It has significant antifungal activity and stress resistance, and has important application value in the fields of agricultural disease control and food preservation.

[0003] Aspergillus flavus is a common foodborne pathogenic fungus that produces numerous conidia during its growth, and its easy spread can cause widespread crop infection. Aflatoxins, particularly B1, B2, G1, and G2, are known potent carcinogens. Aflatoxin B1 (AFB1) easily accumulates in crops and cannot be degraded even after high-temperature sterilization. These toxins are not only toxic to the liver, potentially causing liver cancer with long-term exposure, but also cause irreversible damage to the immune system, cardiovascular system, and gastrointestinal tract. Currently, research on the control of Aspergillus flavus mainly focuses on toxin detection and the development of chemical antibacterial agents, but screening for highly effective Aspergillus flavus-inhibiting strains from microbial resources remains a research hotspot. Summary of the Invention

[0004] The present invention aims to at least partially solve one of the technical problems in the above-mentioned technology, namely, to provide a method for screening Bacillus subtilis strains that inhibit Aspergillus flavus, which can efficiently screen individuals with high inhibitory activity against Aspergillus flavus from a large number of Bacillus subtilis single strain samples.

[0005] Therefore, this invention proposes a high-throughput screening method for Bacillus subtilis strains that inhibit Aspergillus flavus, which includes the following steps:

[0006] Single colonies of Bacillus subtilis were isolated after activation.

[0007] The obtained single colonies were numbered and inoculated into 96-well plates for fermentation. The fermentation broth was centrifuged, and the supernatant was obtained by sieving through a membrane to obtain the fermentation supernatant.

[0008] Each of the fermentation supernatants was added to a 96-well plate and incubated with a colorimetric working solution; the colorimetric working solution was a mixture of Aspergillus flavus spore suspension and Resveratrol solution.

[0009] Strains with fluorescence intensity lower than the blank control group and color close to blue were selected as the first round of initial screening strains.

[0010] The fermentation supernatant of the strains from the initial screening was re-screened using the aforementioned colorimetric working solution, and Bacillus subtilis strains with high inhibitory activity against Aspergillus flavus were selected based on fluorescence intensity.

[0011] This invention discloses a high-throughput screening method for Bacillus subtilis strains that inhibit Aspergillus flavus. This method utilizes the sporulation characteristics of Aspergillus flavus and the redox properties of resazurin. An Aspergillus flavus spore suspension is mixed with a resazurin solution to prepare a chromogenic working solution. This chromogenic solution and the fermentation supernatant of a single Bacillus subtilis strain are simultaneously added to a 96-well plate. The color change of resazurin indicates the degree of inhibition of Aspergillus flavus spore growth and metabolism. Relying on the sensitivity of resazurin reduction and the high throughput of the 96-well plate, individuals with high inhibitory activity against Aspergillus flavus can be efficiently screened from a large number of Bacillus subtilis strain samples. Single colonies are obtained by diluting and plating the bacterial suspension. Single Bacillus subtilis strains that significantly inhibit Aspergillus flavus spore germination can be obtained through resazurin chromogenic analysis combined with high-throughput screening using 96-well plates. This method has advantages such as simple operation, high throughput, and strong specificity, and can be widely applied to the development of agricultural microbial resources.

[0012] Optionally, the fermentation time in the 96-well plate is 60 hours.

[0013] Optionally, the concentration of the Aspergillus flavus spore suspension is 1×10⁻⁶. 6 The concentration of the resazurin solution is 0.1 mg / mL, and the volume ratio of the Aspergillus flavus spore suspension to the resazurin solution in the colorimetric working solution is 9:1.

[0014] Optionally, in each well of the 96-well plate, the volume ratio of the colorimetric working solution to the fermentation supernatant is 9:1.

[0015] Optionally, the color of the sample and the fluorescence intensity are observed after the 96-well plate is incubated at 30°C for 24 hours.

[0016] Furthermore, the fluorescence intensity of the sample was measured at an emission wavelength of 530 nm and an excitation wavelength of 590 nm.

[0017] Optionally, after activating the Bacillus subtilis in LB liquid medium, the bacterial solution is diluted and spread to separate single colonies, and single colonies with larger diameter and better morphology are selected.

[0018] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0019] Figure 1 The image shows the color changes of *Razoria repens* under the action of different concentrations of *Razoria repens* and spore suspension according to an embodiment of the present invention.

[0020] Figure 2 This is a graph showing the changes in fluorescence intensity under the action of different concentrations of *Razoria repens* and spore suspension according to an embodiment of the present invention;

[0021] Figure 3 This is a diagram showing the optimal fermentation time for a single strain of Bacillus subtilis in a deep-well plate according to an embodiment of the present invention.

[0022] Figure 4 This is a high-throughput screening plate colorimetric image of 120 Bacillus subtilis strains with Resveratrol staining according to an embodiment of the present invention.

[0023] Figure 5 The fluorescence intensity diagram of 120 Bacillus subtilis strains selected for high-throughput screening using the azurite staining method according to an embodiment of the present invention;

[0024] Figure 6 This is a colorimetric image of a high-throughput screening plate for 10 strains of Bacillus subtilis using the azurite staining method according to an embodiment of the present invention.

[0025] Figure 7 The fluorescence intensity diagram is shown for high-throughput screening of 10 Bacillus subtilis strains using the azuril staining method according to an embodiment of the present invention. Detailed Implementation

[0026] The technical solution of the present invention is illustrated below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or defining the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0027] To better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

[0028] Traditional Bacillus subtilis screening methods rely on plate confrontation or shaking flask fermentation followed by detection of inhibition zones, which suffer from low throughput, long cycle time, and high subjectivity, making it difficult to meet the rapid screening needs of large strain libraries.

[0029] To address the issues of low screening efficiency and complex operation in Bacillus subtilis screening, this invention provides a high-throughput screening method for Bacillus subtilis that inhibits Aspergillus flavus, enabling rapid screening from a large number of Bacillus subtilis strains to identify single strains with significant inhibitory activity against Aspergillus flavus spore germination. Resazurin, as a redox indicator, reflects the metabolic activity of organisms through color changes (blue → pink) and fluorescence intensity changes, and has been applied to cell viability detection and bacterial contamination assessment; however, there are no reports on its use for high-throughput screening to inhibit Aspergillus flavus spore germination.

[0030] Therefore, this invention proposes a high-throughput screening method for Bacillus subtilis strains that inhibit Aspergillus flavus, which includes the following steps:

[0031] Single colonies of Bacillus subtilis were isolated after activation.

[0032] The obtained single colonies were numbered and inoculated into 96-well plates for fermentation. The fermentation broth was centrifuged, and the supernatant was obtained by sieving through a membrane to obtain the fermentation supernatant.

[0033] Each of the fermentation supernatants was added to a 96-well plate and incubated with a colorimetric working solution; the colorimetric working solution was a mixture of Aspergillus flavus spore suspension and Resveratrol solution.

[0034] Strains with fluorescence intensity lower than the blank control group and color close to blue were selected as the first round of initial screening strains.

[0035] The fermentation supernatant of the strains from the initial screening was re-screened using the aforementioned colorimetric working solution, and Bacillus subtilis strains with high inhibitory activity against Aspergillus flavus were selected based on fluorescence intensity.

[0036] This invention discloses a high-throughput screening method for Bacillus subtilis strains that inhibit Aspergillus flavus. This method utilizes the sporulation characteristics of Aspergillus flavus and the redox properties of resazurin. An Aspergillus flavus spore suspension is mixed with a resazurin solution to prepare a chromogenic working solution. This chromogenic solution and the fermentation supernatant of a single Bacillus subtilis strain are simultaneously added to a 96-well plate. The color change of resazurin indicates the degree of inhibition of Aspergillus flavus spore growth and metabolism. Relying on the sensitivity of resazurin reduction and the high throughput of the 96-well plate, individuals with high inhibitory activity against Aspergillus flavus can be efficiently screened from a large number of Bacillus subtilis strain samples. Single colonies are obtained by diluting and plating the bacterial suspension. Single Bacillus subtilis strains that significantly inhibit Aspergillus flavus spore germination can be obtained through resazurin chromogenic analysis combined with high-throughput screening using 96-well plates. This method has advantages such as simple operation, high throughput, and strong specificity, and can be widely applied to the development of agricultural microbial resources.

[0037] It should be noted that all test materials used in this invention are commercially available products. Specifically, Bacillus subtilis 168 was purchased from Shanghai Chunshi Biotechnology Co., Ltd., and the 120 strains of Bacillus subtilis used in the examples were all isolated from Bacillus subtilis 168 culture medium and preserved at the College of Marine Food and Bioengineering, Jimei University.

[0038] Aspergillus flavus (BNCC 336678) was purchased from Beina Biotechnology.

[0039] The LB medium consists of 10g tryptone, 5g yeast extract, and 5g sodium chloride, dissolved in 1L of distilled water, and the pH is adjusted to 7.0.

[0040] The PDA medium consists of 22.5g of potato (from which extract powder is obtained) and 1.5g of glucose, dissolved in 1L of distilled water.

[0041] The RPMI 1640 medium consisted of 2000 mg / L D-glucose, 2000 mg / L NaHCO3, and 300 mg / L L-glutamine, dissolved in 500 mL of distilled water.

[0042] Landy medium consists of 20g glucose, 10g peptone, 5g monosodium glutamate, 0.5g magnesium sulfate heptahydrate, 0.5g potassium chloride, 1g potassium dihydrogen phosphate, 0.00015g ferrous sulfate, 0.005g manganese sulfate, and 0.00016g copper sulfate, dissolved in 1L distilled water and adjusted to pH 7.1.

[0043] Sodium razorazine was purchased from Xilong Scientific Co., Ltd.

[0044] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0045] Example 1: Preparation of Aspergillus flavus spore suspension

[0046] Inoculate *Aspergillus flavus* onto PDA medium and incubate at 28°C for 5-7 days. When obvious sporulation is observed, inject 5 mL of physiological saline containing 0.5% Tween 80 onto the plate surface, gently scrape the spores off with an inoculation loop, and vortex thoroughly to obtain a spore suspension. After removing the mycelium from the spore suspension with sterile cotton, count the spores using a hemocytometer. Using a hemocytometer with 25 medium squares × 16 small squares, add 10 μL of the mycelium-free spore suspension from the edge of the coverslip to the upper and lower chambers of the hemocytometer, respectively. After the suspension has fully soaked the counting chambers, observe under a 400X optical microscope. Select the five central squares at the four corners and the center, and count the spores in each square according to the rule of "counting the top but not the bottom, counting the left but not the right," and calculate the concentration of the spore suspension added in a single counting chamber using the following formula:

[0047]

[0048] The prepared spore suspension was stored at 4°C for later use.

[0049] Example 2: Determination of Optimal Experimental Conditions for the Azure Colorimetric Method

[0050] The Aspergillus flavus spore suspension obtained in Example 1 was diluted to 1×10⁻⁶ with RPMI 1640 medium. 6 cfu / mL, 5×10 5 cfu / mL, 2×10 5 cfu / mL, 1×10 5 cfu / mL.

[0051] The sodium salicylate stock solution was diluted to 1 mg / mL, 0.5 mg / mL, 0.2 mg / mL, and 0.1 mg / mL, respectively.

[0052] With a concentration of 1×10 6 A CFU / mL Aspergillus flavus spore suspension was mixed with four different concentrations of resazurin sodium solution at a volume ratio of 9:1, and 200 μL was added to each well of a 96-well plate. The same procedure was repeated for the other three Aspergillus flavus spore suspension concentrations. A positive control was set up with 20 μL of resazurin sodium solution and 180 μL of RPMI 1640 medium, and a blank control was set up with 200 μL of Aspergillus flavus spore suspension. All sample addition operations were repeated three times. The 96-well plates were incubated at 28°C for 24 hours, and color changes were recorded every 2 hours. Fluorescence intensity was measured at an emission wavelength of 530 nm and an excitation wavelength of 590 nm.

[0053] During the interaction between respora sinensis and spores, if the spore suspension concentration is too high or the respora sinensis solution concentration is too low, respora sinensis is easily and rapidly reduced to colorless dihydrohalogenin. This may result in the failure to detect strains with strong inhibitory effects, i.e., false negatives. Conversely, if the spore suspension concentration is too low or the respora sinensis solution concentration is too high, the reduction rate of respora sinensis is significantly reduced, which may lead to the detection of single strains with weak inhibitory effects, i.e., false positives. Therefore, the optimal reaction conditions were set at the concentration combination with the highest fluorescence intensity and a continuous increase within a 24-hour reaction time. Under these conditions, all samples were in the state where respora sinensis changed from blue (non-fluorescent) to pink (fluorescent). At this point, the degree of inhibition of spore germination could be judged based on the sample color, i.e., the fluorescence intensity, effectively avoiding the detection of false positives and false negatives. The results are as follows: Figure 1 and Figure 2 As shown, when the spore suspension concentration is 1×10 6 When the cfu / mL concentration of the resazurin solution was 0.1 mg / mL, the solution in the well plate exhibited the brightest pink color and the highest fluorescence intensity. Therefore, these two concentrations were selected as the working concentrations for subsequent screening.

[0054] Example 3: Determination of the optimal fermentation time for Bacillus subtilis in deep-well plates

[0055] 1 mL of preserved Bacillus subtilis 168 bacterial culture was inoculated into 5 mL of LB broth and incubated overnight at 37°C (180 rpm) for activation. The activated bacterial culture was then diluted 10... 5～6 After the initial fermentation, 100 μL of the solution was spread onto LB agar plates, and single colonies with larger diameters and good morphology were selected and numbered. 1 mL of Landy medium was added to each well of a 96-well plate, and 36 single colonies were randomly selected using a sterile toothpick and inoculated into the fermentation medium. The plates were sealed with a breathable sealing film and incubated at 37°C at 180 rpm. Samples were taken at 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h, with three replicates at each time point. After sampling, all samples were centrifuged at 12000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm microporous membrane and stored at 4°C for later use. Following the optimal conditions for the rezin chromogenic method determined in Example 2, 20 μL of the fermentation supernatant was added to a 96-well plate. Then, 180 μL of rezin chromogenic working solution was added, with amphotericin B as a positive control and Landy medium as a negative control, and the plates were incubated at 30°C. The effect of supernatant at different fermentation times on the color development of razor violet was observed.

[0056] The results are as follows Figure 3As shown, during the first 48 hours of fermentation in the deep-well plate, the fluorescence intensity of the treatment group was not significantly different from that of the negative control group (BC). However, when the fermentation time reached 60 hours and 72 hours, the fluorescence intensity of the treatment group was significantly lower than that of the negative control group, indicating that an antibacterial substance capable of inhibiting the germination of Aspergillus flavus spores was produced in the fermentation broth at this time. To shorten the screening time, 60 hours was selected as the optimal fermentation time for a single strain of Bacillus subtilis in the deep-well plate.

[0057] Example 3: High-throughput screening of single colonies of Bacillus subtilis to inhibit Aspergillus flavus spore germination.

[0058] The supernatant from the single-colony fermentation of Bacillus subtilis from Example 2, fermented for 60 h, was added to a 96-well plate at a rate of 20 μL per well. Amphotericin B was used as a positive control, and Landy's medium was used as a negative control. Each sample was added in triplicate. 1×10 6 A chromogenic working solution was prepared by mixing a CFU / mL Aspergillus flavus spore suspension and a 0.1 mg / mL resazurin solution at a volume ratio of 9:1. 180 μL of this solution was added to each well of a 96-well plate. The 96-well plates were incubated at 30°C for 24 h. Subsequently, the fluorescence intensity of the samples was measured at an emission wavelength of 530 nm and an excitation wavelength of 590 nm. The strains corresponding to the wells with the lowest fluorescence intensity that still showed a bluish-purple color after 24 h were labeled as the first round of initial screening strains.

[0059] The fermentation samples of the strains obtained from the first round of initial screening were then re-screened using the resazurin staining method. The results of the first round of initial screening are as follows: Figure 4 and Figure 5 As shown, among the 120 strains, strains 4, 5, 11, 13, 18, 19, 25, 33, 34, 35, 37, 38, 41, 44, 46, 48, 49, 54, 55, 57, 59, 60, and 86 showed significantly lower fluorescence intensity than the control group when the color was blue-purple. Based on the fluorescence intensity, strains 4, 18, 25, 35, 38, 46, 48, 57, 59, and 86 were selected as the first round of initial screening strains. After a second screening using rezamidocyanine staining, the results were as follows... Figure 6 and Figure 7 As shown, except for strains 4 and 48, the fluorescence intensity of the other strains was significantly lower than that of the blank control group. This indicates that the screening method can effectively and quickly screen out single strains with high antibacterial activity from a large number of samples. Based on the fluorescence intensity, strain 18 with the lowest fluorescence intensity was determined as the optimal screening strain and named JMU-rBS18.

[0060] In summary, according to embodiments of the present invention, high-throughput screening using resazurin colorimetry can efficiently screen out Bacillus subtilis individuals with high inhibitory activity against Aspergillus flavus from a large number of bacterial strain samples; the color change of resazurin can intuitively reflect the antibacterial effect, and fluorescence intensity measurement enables quantitative analysis, solving the subjective problem of traditional methods relying on visual observation; by optimizing the spore suspension concentration (1×10⁻⁶), 6 The concentration of resazurin (0.1 mg / mL) and cfu / mL is used to avoid false negatives (resazurin is reduced to colorless too quickly) and false positives (insufficient reduction), thus improving the accuracy of screening. Furthermore, by combining 96-well plate fermentation and 96-well plate colorimetric detection, 96 strains can be screened in a single run, which is much more efficient than the traditional plate method. The reliability of the screening results is ensured by a second round of screening.

[0061] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. The illustrative expressions of the above terms in this specification should not be construed as necessarily referring to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

[0062] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A high-throughput screening method for Bacillus subtilis strains that inhibit Aspergillus flavus, characterized in that, Includes the following steps: Single colonies of Bacillus subtilis were isolated after activation. The obtained single colonies were numbered and inoculated into 96-well plates for fermentation. The fermentation broth was centrifuged, and the supernatant was obtained by sieving through a membrane to obtain the fermentation supernatant. Each of the fermentation supernatants was added to a 96-well plate and incubated with a colorimetric working solution; the colorimetric working solution was a mixture of Aspergillus flavus spore suspension and Resveratrol solution. Strains with fluorescence intensity lower than the blank control group and color close to blue were selected as the first round of initial screening strains. The fermentation supernatant of the strains from the initial screening was re-screened using the aforementioned colorimetric working solution, and Bacillus subtilis strains with high inhibitory activity against Aspergillus flavus were selected based on fluorescence intensity.

2. The high-throughput screening method as described in claim 1, characterized in that, The fermentation time in the 96-well plate was 60 hours.

3. The high-throughput screening method as described in claim 1, characterized in that, The concentration of the Aspergillus flavus spore suspension was 1×10⁻⁶. 6 The concentration of the resazurin solution is 0.1 mg / mL, and the volume ratio of the Aspergillus flavus spore suspension to the resazurin solution in the colorimetric working solution is 9:

1.

4. The high-throughput screening method as described in claim 1, characterized in that, In each well of the 96-well plate, the volume ratio of the colorimetric working solution to the fermentation supernatant is 9:

1.

5. The high-throughput screening method as described in claim 1, characterized in that, The 96-well plate was incubated at 30 °C for 24 h, and the sample color and fluorescence intensity were observed and measured.

6. The high-throughput screening method as described in claim 5, characterized in that, The fluorescence intensity of the sample was measured at an emission wavelength of 530 nm and an excitation wavelength of 590 nm.

7. The high-throughput screening method as described in claim 1, characterized in that, After activating the Bacillus subtilis in LB liquid medium, the bacterial solution was diluted and spread to separate single colonies. Single colonies with larger diameters and better morphology were selected.

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