Bacillus tequilensis and application thereof in inhibiting aspergillus flavus and degrading aflatoxin

Abstract

The application discloses a bacillus turicatae and application thereof in inhibiting aspergillus flavus and degrading aflatoxin, and belongs to the technical field of microorganisms. The bacillus turicatae is preserved in the China General Microbiological Culture Collection Center, and has a preservation number of CGMCC No. 38386. The bacillus turicatae has significant antagonistic effect on aspergillus flavus and excellent degrading effect on aflatoxin, thereby having important application prospect and value in the prevention and control of aspergillus flavus and aflatoxin in agricultural products.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Bacillus tekirae and its application in inhibiting Aspergillus flavus and degrading aflatoxin. Background Technology

[0002] Aflatoxins are a class of secondary metabolites mainly produced by fungi such as *Aspergillus flavus* and *Aspergillus parasiticus*. Structurally, these toxins are structural analogs composed of difuran rings and coumarin. Aflatoxins are extremely teratogenic, carcinogenic, and mutagenic, and widely contaminate agricultural products and foods such as peanuts, corn, cottonseed, rice, dried fruits, and milk. More than twenty types of aflatoxins have been identified, among which aflatoxin B1 (AFB1) has the widest distribution and the strongest toxicity, and is classified as a Group IA carcinogen by the International Agency for Research on Cancer (IARC) of the World Health Organization.

[0003] Currently, many microorganisms, such as Aspergillus antagonists and non-toxin-producing Aspergillus flavus, have been isolated and purified for the biocontrol of Aspergillus flavus. However, existing microorganisms suffer from problems such as complex culture conditions and poor antagonistic effects against Aspergillus flavus. Therefore, screening for strains that effectively antagonize the growth of Aspergillus flavus is of great significance for improving the control of aflatoxin. Summary of the Invention

[0004] This invention provides a strain of Bacillus tequilensis, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38386.

[0005] This invention provides the application of the above-mentioned Bacillus tekiria in the preparation of formulations that inhibit Aspergillus flavus.

[0006] This invention provides the application of the above-mentioned Bacillus tergentii in the preparation of aflatoxin-degrading formulations; wherein the aflatoxin is preferably aflatoxin B1.

[0007] This invention provides an aflatoxin inhibitor, wherein the inhibitor contains the aforementioned *Bacillus tekirae*. The content of *Bacillus tekirae* in the inhibitor is at least 1 × 10⁻⁶. 8 cfu / mL.

[0008] This invention provides an aflatoxin-degrading formulation containing the aforementioned *Bacillus tekirae*. The content of *Bacillus tekirae* in the formulation is at least 1 × 10⁻⁶. 8 cfu / mL.

[0009] This invention provides a method for inhibiting Aspergillus flavus, comprising the following steps: The aflatoxin inhibitor was placed in the sample infected with Aspergillus flavus and incubated at 20-40 °C for 48-120 h to inhibit the Aspergillus flavus in the sample.

[0010] This invention provides a method for the degradation of aflatoxin, comprising the following steps: The aflatoxin degradation agent is placed in the sample containing aflatoxin and incubated at 20-40 °C for 48-120 h to achieve the degradation of aflatoxin in the sample.

[0011] The beneficial effects of this invention are as follows: This invention provides a strain of Bacillus tekirae that exhibits significant antagonistic activity against Aspergillus flavus and excellent degradation of aflatoxin, thus showing important application prospects and value in the prevention and control of aflatoxin and its toxins in agricultural products. Attached Figure Description

[0012] Figure 1 The colony morphology of strain S12 is shown.

[0013] Figure 2 This is a plate confrontation experiment; in the middle is Aspergillus flavus, and on both sides is strain S12.

[0014] Figure 3 This is an experiment on the antibacterial activity of corn; where A is the control group and B is the experimental group. Detailed Implementation

[0015] Other materials used in this invention, unless otherwise stated, are commercially available. Other terms used in this invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. The following embodiments are merely illustrative and not intended to limit the scope of the invention in any way.

[0016] Example 1: Isolation and Identification of Strains 1. Strains Isolation Soil samples were collected from peanut fields. 1 g of soil was suspended in 20 mL of sterile water in a clean bench, and the mixture was diluted by shaking to prepare a soil suspension. This suspension was then diluted 200-fold with sterile distilled water. 200 μL of the suspension was spread onto LB agar plates and incubated at 28°C. After 3 days, multiple colonies grew on the plates. Based on differences in color and morphology, the strains were purified by streak plating three times. The degradation efficiency of AFB1 by each strain was analyzed. Strain S12 showed the highest degradation efficiency for AFB1.

[0017] 2. Strain identification Morphological characteristics: Colony morphology of strain S12 after incubation at 37 °C for 24 h on LB plates, such as... Figure 1 As shown, the circular protrusions are milky white in color, wrinkled, and viscous, which are consistent with the morphological characteristics of Bacillus.

[0018] Biological characteristics: Gram staining is positive.

[0019] Genetic characterization: 16S rRNA gene analysis was performed. Genomic DNA was extracted from bacteria S12, and PCR amplification was performed using universal primers for the 16S rRNA gene. The resulting 1454 bp gene sequence is shown below: Based on sequence homology comparison with standard strains in the EzTaxon-e server database, the 16S rRNA gene of strain S12 is similar to that of the standard strain Bacillus tequilensis KCTC 13622. T The 16S rRNA gene showed 99.93% homology, and gene analysis indicated that the bacterium was Bacillus tequilensis.

[0020] Based on morphological, biological, physiological, and biochemical characteristics, and gene sequence features, strain S12 was identified as *Bacillus tequilensis*. This bacterium was deposited on April 24, 2026, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), with accession number CGMCC No. 38386.

[0021] Example 2: Flat Plate Standoff Test 30 μL of Bacillus tekiri S12 bacterial suspension (concentration 2.1×10⁻⁶) was added. 8 The spores of Aspergillus flavus NRRL3357 (a standard strain of Aspergillus flavus NRRL 3357, provided by Professor He Zhumei of Sun Yat-sen University) were 2.6 × 10⁻⁶ CFU / mL and 50 μL of spore solution (spore concentration 2.6 × 10⁻⁶ CFU / mL). 5 The bacteria (number per mL) were inoculated onto PDA medium solid plates and cultured at 28°C for 5 days. The inhibitory effect of Bacillus tekirae S12 on Aspergillus flavus was then observed.

[0022] The test results are as follows Figure 2 As shown: The middle colony is Aspergillus flavus, and the two colonies on the left and right are strain S12. The Aspergillus flavus colonies near Bacillus tekirae S12 are significantly inhibited.

[0023] Example 3: Antibacterial test of maize Petri dishes were labeled A and B, with 10 corn kernels placed in each dish. 2 mL of sterilized LB medium was used, and 10 μL of 5.2 × 10⁻⁶ corn kernels were added. 5 A solution of Aspergillus flavus spores per mL was thoroughly mixed and added to Petri dish A as a control group; 2 mL of Bacillus tekirae S12 culture (concentration 6.5 × 10⁻⁶) was taken. 8 Add 10 μL of a solution with a concentration of 5.2 × 10⁻⁶ CFU / mL. 5 Add a solution of Aspergillus flavus spores per mL to Petri dish B after thorough mixing. Incubate at 28°C for 5 days.

[0024] The test results are as follows Figure 3 As shown: In plate A, corn was covered with Aspergillus flavus and appeared yellowish-green; in plate B, corn was clean and shiny with no Aspergillus flavus growth, showing a normal color. This indicates that Bacillus tekirae S12 can significantly inhibit the growth of Aspergillus flavus in corn.

[0025] Example 4 Aflatoxin Degradation Test Dissolve 1 mg of aflatoxin B1 (AFB1) standard in 20 mL of chromatographic grade methanol to prepare an AFB1 stock solution with a concentration of 50 ppm. Take 0.5 mL of 50 ppm AFB1 and add it to 4.5 mL of chromatographic grade methanol to prepare an AFB1 working stock solution with a concentration of 5000 ppb.

[0026] Strain S12 was inoculated into LB liquid fermentation medium (g / L): 10 g tryptone, 5 g yeast extract, 10 g NaCl, pH 7.0; and cultured on a shaker at 37°C for 2 days. 1.96 mL of the S12 bacterial suspension (crystal concentration 4.7 × 10⁻⁶) was then collected. 8 The sample was placed in a 10 mL sample tube, and 40 μL of 5000 ppb AFB1 working stock solution was added to bring the final concentration to 100 ppb. After inverting and mixing, the sample was incubated at 37°C for 72 h. The supernatant was then obtained by centrifugation at 12000 rpm for 5 min and recorded as the test group solution. 1.96 mL of uninoculated culture medium was mixed with 40 μL of 5000 ppb AFB1 working stock solution as the control group solution.

[0027] The AFB1 content in the experimental and control groups was detected using an aflatoxin B1 ELISA kit, and the degradation effect of strain S12 on AFB1 was calculated.

[0028] The test results are as follows: The AFB1 content in the experimental group was 12.4 ppb, while the AFB1 content in the control group was 99.7 ppb. Calculations showed that strain S12 exhibited the best degradation effect on AFB1 under conditions of 37 ℃ for 72 h, with a degradation rate of 87.6%.

[0029] Application examples Peanut meal with excessive AFB1 levels was sterilized at 121℃ for 20 min and divided into two portions. The experimental group was inoculated with Bacillus tekirae S12 bacterial suspension (bacterial concentration of 4.7 × 10⁻⁶). 8 (cfu / mL), the control group was treated with sterile LB liquid fermentation medium at 37°C for 72 h.

[0030] The AFB1 content in the experimental and control groups was detected using an aflatoxin B1 ELISA kit, and the degradation effect of strain S12 on AFB1 was calculated.

[0031] The test results are as follows: The AFB1 content in the experimental group was 22.5 ppb, while that in the control group was 124.2 ppb. Calculations showed that strain S12 exhibited the best degradation effect on AFB1 in peanut meal at 37 ℃ for 72 h, achieving a degradation rate of 81.9%.

[0032] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A strain of Bacillus tequilensis, characterized in that, The Bacillus tekirae described is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38386.

2. The use of Bacillus tekirii according to claim 1 in the preparation of a formulation for inhibiting aflatoxin.

3. The use of Bacillus tekirii according to claim 1 in the preparation of aflatoxin-degrading formulations.

4. The application according to claim 3, characterized in that, The aflatoxin in question is aflatoxin B1.

5. An aflatoxin inhibitor, characterized in that, The inhibitor contains Bacillus tekirae as described in claim 1.

6. The aflatoxin inhibitor according to claim 5, characterized in that, The inhibitor contains at least 1 × 10⁻⁶ Bacillus tekirae. 8 cfu / mL.

7. An aflatoxin degradation agent, characterized in that, The preparation contains Bacillus tekirae as described in claim 1.

8. The aflatoxin degradation agent according to claim 7, characterized in that, The preparation contains at least 1 × 10⁻⁶ Bacillus tergentii. 8 cfu / mL.

9. A method for inhibiting Aspergillus flavus, characterized in that, The steps are as follows: The aflatoxin inhibitor described in claim 5 is placed in a sample infected with Aspergillus flavus and incubated at 20-40 °C for 48-120 h to achieve inhibition of Aspergillus flavus in the sample.

10. A method for degrading aflatoxin, characterized in that, The steps are as follows: The aflatoxin degradation agent according to claim 7 is placed in a sample containing aflatoxin and incubated at 20-40 °C for 48-120 h to achieve the degradation of aflatoxin in the sample.

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