A kind of Aspergillus flavus antagonistic bacteria and its application in preparing feed mildew inhibitor

Abstract

The invention belongs to the field of biotechnology, and specifically discloses an antagonistic bacterium of Aspergillus flavus and its application in preparing a feed mildew inhibitor. The antagonistic bacterium of Aspergillus flavus is a methylotrophic Bacillus sp. Bacillus methylotrophicus 1-11-2, deposited in the General Microbiological Center of China Microbiological Culture Collection Committee, with the deposit number CGMCC No: 32408, the deposit date is October 30, 2024, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. Compared with the prior art, the methylotrophic Bacillus of the present invention Bacillus methylotrophicus The lipopeptide active substances in the metabolites of 1‑11‑2 can effectively inhibit the germination of Aspergillus flavus spores and the growth of mycelium; they have a significant inhibitory effect on the production of aflatoxin B1, and can still maintain antifungal activity under extreme conditions such as strong acid, strong alkali, and high temperature. This fungus can be used as a reserve resource for the development of new antifungal agents.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to an antagonistic bacterium of Aspergillus flavus and application thereof in the preparation of a feed mildew inhibitor. Background Art

[0002] Aflatoxins are mainly toxic secondary metabolites produced by two types of filamentous fungi, Aspergillus flavus and Aspergillus parasiticus, which are highly carcinogenic, teratogenic, and mutagenic. Compared with other mold pollution, the biggest feature of Aspergillus flavus pollution is mainly reflected in its metabolites, namely aflatoxins (a kind of fungal toxin), which are highly toxic substances. Aflatoxins are mainly of types B1, B2, G1 and G2. Due to the extremely strong toxicity of aflatoxin B1 (AFB1), it is classified as a Class I carcinogen by the International Cancer Organization. It is considered to be the most toxic substance in nature, much more toxic than arsenic and ten times more toxic than potassium cyanide. At the same time, there are reports that trace amounts of aflatoxins (1μg / kg) in food can induce cancer. Aflatoxin contamination is common around the world, usually occurring in corn, peanuts, nuts, etc. This is a common problem around the world. Food contamination by aflatoxin, on the one hand, leads to food discarding, resulting in economic losses, and on the other hand, due to insufficient safety supervision, it is consumed, causing harm to health. How to remove or degrade toxins in food that has been contaminated with aflatoxin is both a social reality and a scientific issue.

[0003] Chinese invention patents CN114644995B and CN110878265B use Bacillus amyloliquefaciens and Bacillus subtilis to degrade aflatoxin B1 respectively. In addition, the removal or degradation of aflatoxin by microorganisms is still not efficient enough, and the removal rate of aflatoxin is usually around 70-90%. Finding a suitable microorganism is the key to solving the above problems.

[0004] Currently available antifungal agent development reserve resources are limited, therefore, new aflatoxin antagonists need to be developed. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a flavus antagonistic bacterium and its application in the preparation of a feed mildew inhibitor.

[0006] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0007] One of the purposes of the present invention is to provide an antagonistic bacterium to Aspergillus flavus, wherein the antagonistic bacterium to Aspergillus flavus is a methylotrophic Bacillus sp. 甲基营养型芽孢杆菌1-11-2, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No: 32408, the deposit date is October 30, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The second object of the present invention is to provide an application of aflatoxin antagonistic bacteria in the preparation of feed mildew inhibitors, wherein methylotrophic Bacillus 甲基营养型芽孢杆菌 1-11-2 After continuous shaking culture in LB broth medium for 72 hours, the fermentation supernatant was collected and used to prepare feed mildew inhibitor.

[0009] Compared with the prior art, the methylotrophic Bacillus sp. 甲基营养型芽孢杆菌 The lipopeptide active substances in the metabolites of 1-11-2 can effectively inhibit the germination of Aspergillus flavus spores and the growth of mycelium; they have a significant inhibitory effect on the production of aflatoxin B1, and can still maintain antifungal activity under extreme conditions such as strong acid, strong alkali, and high temperature. This fungus can be used as a reserve resource for the development of new antifungal agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Methylotrophic Bacillus 甲基营养型芽孢杆菌 Screening results of 1-11-2: (a) Methylotrophic Bacillus 甲基营养型芽孢杆菌 The antibacterial effect of 1-11-2; (b) is methylotrophic Bacillus 甲基营养型芽孢杆菌 The antibacterial effect of the fermentation broth of 1-11-2.

[0011] Figure 2 Methylotrophic Bacillus 甲基营养型芽孢杆菌 1-11-2 Observation of colony morphology and Gram staining results: (a) is the colony morphology; (b) is the Gram staining result.

[0012] Figure 3 Methylotrophic Bacillus 甲基营养型芽孢杆菌 1-11-2 The 16S rDNA sequencing results were BLASTed with the NCBI database and a phylogenetic tree was constructed.

[0013] Figure 4 Methylotrophic Bacillus 甲基营养型芽孢杆菌 1-11-2 Whole genome sequencing and analysis results.

[0014] Figure 5 Methylotrophic Bacillus 甲基营养型芽孢杆菌 s 1-11-2 HPLC purification results of fermentation broth: A1 represents methylotrophic Bacillus 甲基营养型芽孢杆菌 The first antimicrobial lipopeptide among the metabolites of 1-11-2; A2 represents methylotrophic Bacillus甲基营养型芽孢杆菌 The second antimicrobial lipopeptide among the metabolites of 1-11-2.

[0015] Figure 6 The results of the qualitative analysis of the active single peak TLC: A1 represents methylotrophic Bacillus 芽孢杆菌属 甲基营养型 The first antimicrobial lipopeptide among the metabolites of 1-11-2; A2 represents methylotrophic Bacillus 甲基营养型芽孢杆菌 The second antimicrobial lipopeptide among the metabolites of 1-11-2.

[0016] Figure 7 The results of the single peak activity test for the ability to inhibit the germination of Aspergillus flavus spores: A1 represents methylotrophic Bacillus 甲基营养型芽孢杆菌 The first antimicrobial lipopeptide among the metabolites of 1-11-2; A2 represents methylotrophic Bacillus 甲基营养型芽孢杆菌 The second antimicrobial lipopeptide among the metabolites of 1-11-2.

[0017] Figure 8 IC of lipopeptide A1 on the growth of Aspergillus flavus 50 Measurement results.

[0018] Figure 9 This is the effect of lipopeptide A1 on the germination rate of Aspergillus flavus spores.

[0019] Figure 10 The results of the acid-base stability test of lipopeptide A1: +A1 indicates the addition of lipopeptide A1 treatment.

[0020] Figure 11 These are the results of metal ion stability test of lipopeptide A1.

[0021] Figure 12 This is the temperature stability test result of lipopeptide A1.

[0022] Figure 13 This is the test result of anti-aflatoxin activity of lipopeptide A1.

[0023] Figure 14 These are the results of the test on the ability of lipopeptide A1 to inhibit aflatoxin.

[0024] Figure 15 The results of observation on the protective ability of lipopeptide A1 against corn infected with Aspergillus flavus: (a) The growth of Aspergillus flavus in corn seeds sprayed with Aspergillus flavus spore solution; (b) The growth of Aspergillus flavus in corn seeds sprayed with Aspergillus flavus spore solution and lipopeptide A1. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0026] Example 1. Screening and identification of antagonistic bacteria to Aspergillus flavus

[0027] Antagonistic bacteria screening: Collect samples of plant roots, weed roots, plant rhizosphere soil and marine sediments from different regions. Weigh 4g of plant tissue or soil sample, add it to 36mL of sterile water, and shake it for 20 minutes. Then, dilute 100µL of the suspension to 10 -1 , 10 -2 , 10 -3 , 10 -4 , and placed on LB agar plates (LB solid culture medium includes the following ingredients: 6g yeast extract, 10g tryptone, 10g sodium chloride, 1L deionized water, 20g agar powder) at 28°C for 16-18 hours. Pick a single colony into 1 mL LB liquid culture medium and culture overnight at 28°C and 180 rpm. Inoculate the fresh mycelium cake of Aspergillus flavus (purchased from Beina Biological, No.: 142803) in the center of the solid plate made of PDA medium (200.0 g potato, 20.0 g glucose, 20.0 g agar, 1.0 L distilled water; natural pH, sterilized at 115°C for 30 min), and then drop 2µL of the strain to be screened at 2cm from the center, and repeat 3 times for each strain. The PDA culture dish with only Aspergillus flavus mycelium cake in the center was used as a blank control. After culturing at 28°C for 5 days, the diameter of the Aspergillus flavus colony was measured and the inhibition rate was calculated. The strains with an inhibition rate higher than 50% were selected as the preliminary screening strains for subsequent biological control research. The inhibition rate was calculated as follows:

[0028] Inhibition rate (%) = (control colony diameter - treated colony diameter) / control colony diameter × 100

[0029] The antagonistic bacteria with antibacterial effect were purified and stored in a -80°C refrigerator with 16% glycerol.

[0030] The results showed that one strain was screened out, which had a significant inhibitory effect on the growth of Aspergillus flavus. The inhibitory rate of the strain was 84%. At the same time, its fermentation supernatant was detected to have a significant anti-aflatoxin effect (see Figure 1 ). The colony morphology was observed to be white and wrinkled, with a rough surface and irregular edges. Gram staining results showed that the strain could be stained purple, and was identified as a Gram-positive bacterium (see Figure 2 ).

[0031] Identification method: By using molecular biological techniques, the strain was sequenced for 16S rDNA, and the 16S gene sequence was:

[0032]

[0033] The sequence was compared with the NCBI database by BLAST. Figure 3 As shown, the strain was identified as methylotrophic Bacillus 甲基营养型芽孢杆菌 1-11-2, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No: 32408, the deposit date is October 30, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0034] Example 2: Preparation of metabolites and detection of their antibacterial effects

[0035] Fermentation broth preparation: Methylotrophic Bacillus 甲基营养型芽孢杆菌 1-11-2 was inoculated in LB broth (10.0 g tryptone, 5.0 g yeast extract, 10.0 g NaCl, 1.0 L distilled water; pH 7.0, sterilized at 121°C for 20 min) with an inoculum size of 1%. After continuous shaking culture at 37°C for 72 h, the fermentation supernatant was collected and the antibacterial activity was tested.

[0036] Whole-genome sequencing and analysis: Genomic DNA was quantified using a Qubit 4 Fluorometer (LifeTechnologies, USA), and libraries were prepared using the SQK-LSK109 Ligation Sequencing kit (Oxford Nanopore Technologies, UK). The experimental procedures followed the standard protocol provided by Oxford NanoporeTechnologies (ONT), including sample quality testing, library construction, library quality testing, and library sequencing. In terms of genome assembly, the filtered reads were assembled using Canu v1.5 software and then circularized using Circlator v1.5.5. In terms of genome annotation, Prodigal v2.6.3 was used to predict coding genes, and the GenBlastA v1.0.4 program was used to scan the entire genome after predicting functional genes. AntiSMASH (V6.0.1) was applied to discover and identify secondary metabolite biosynthesis gene clusters.

[0037] The whole genome sequencing results are as follows Figure 4 As shown, methylotrophic Bacillus 甲基营养型芽孢杆菌The whole genome sequence of 1-11-2 consists of a circular chromosome of 3,594,972 bp, containing 3837 coding sequences (CDS) with an average length of 936 bp. In addition, the chromosome also contains 21 rRNAs and 72 tRNAs. The antiSMASH website was used to predict the secondary metabolite biosynthesis gene clusters in the genome. The results are shown in Table 1. It was found that lipopeptides exist in the genome. According to relevant reports in the literature, lipopeptides are the main antifungal substances synthesized by methylotrophic Bacillus.

[0038] Table 1 Methylotrophic Bacillus 甲基营养型芽孢杆菌 Statistics of gene clusters for biosynthesis of secondary metabolites in 1-11-2 genome

[0039] 基因簇 类型 基因组位置 最相似的已知簇 相似性 簇1 非核糖体肽合成酶 240,774-305,861 表面活性素 78% 簇2 I型聚酮合酶,反式-AT聚酮合酶 584,968-695,722 粘病毒素A1 35% 簇3 类聚酮合酶 915,924-957,168 丁酰苷菌素A / 丁酰苷菌素B 7% 簇4 萜烯 1,042,770-1,060,163 簇5 反式-AT聚酮合酶 1,339,672-1,427,489 大环内酯H 100% 簇6 反式-AT聚酮合酶,III型聚酮合酶,非核糖体肽合成酶 1,646,997-1,747,541 杆菌烯 100% 簇7 非核糖体肽合成酶,反式-AT聚酮合酶, 1,819,492-1,956,798 丰原素 100% 簇8 萜烯 1,979,830-2,001,713 簇9 III型聚酮合酶 2,052,136-2,093,236 簇10 反式-AT聚酮合酶 2,264,133-2,357,925 艰难梭菌素 100% 簇11 非核糖体肽-金属载体,非核糖体肽合成酶,核糖体合成和翻译后修饰肽样 2,974,396-3,026,186 杆菌铁载体 100% 簇12 其他 3,546,822-3,588,240 杆菌溶素 100%

[0040] Separation, purification and characterization of lipopeptide antibacterial active substances:

[0041] 1) First, the fermentation supernatant was treated with 6 M HCl to precipitate protein substances, and then methanol was used for repeated extraction to obtain crude lipopeptides, which were then purified by high-performance liquid chromatography. The results are as follows: Figure 5 As shown in the figure, two single peaks, namely lipopeptides A1 and A2, were purified from the metabolites. The purified samples were qualitatively analyzed by thin layer chromatography combined with literature reports, and the thin layer chromatography plates were stained with iodine vapor. The results are shown in the figure. Figure 6 As shown, both substances can be stained with elemental iodine, indicating that there are fatty acid structures in the structure. Then, the mobility is 0.75, which is judged to be a surfactin lipopeptide.

[0042] 2) Evaluation of the antibacterial activity of lipopeptides:

[0043] Preparation of Aspergillus flavus spores: Aspergillus flavus (purchased from Beina Biotechnology, No. 142803) was inoculated into a 250 mL bottle containing 100 mL CMC medium (CMC-Na 20.0 g; Na2HPO4 2.5 g; KH2PO4 1.5 g; peptone 2.5 g; distilled water to 1.0 L; pH 7.0-7.5, sterilized at 121°C for 20 min), and cultured with shaking at 180 rpm and 25°C for 7 days. The culture was filtered with two layers of sterile gauze to remove the mycelium and prepare a large conidia suspension. The spores were collected after centrifugation at 4°C and 10,000 rpm for 20 min, washed twice with distilled water, and the concentration of the suspension was adjusted to 1×10 using 0.1% (v / v) Tween 20 solution. 5 CFU / mL.

[0044] The minimum inhibitory concentration (MIC) of two lipopeptides A1 and A2 was determined by the microbroth method. First, a 96-well plate was prepared. PDB and lipopeptides A1 and A2 were added to the first row, respectively, with a total volume of 190 μL and an initial concentration of 512 μg / mL. Then, the sample wells on the same column were diluted 2 times. The final concentration of each sample well was 512 μg / mL, 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, and 4 μg / mL. Finally, 10 μL of spore solution was added to make the final spore concentration of 1×10 5 CFU / ml. Place it in a 28°C temperature box for 12 hours, and use enzyme markers to detect the light absorption value of each well when the OD value is 600. Figure 7 As shown, after testing, lipopeptide A1 had the best anti-aflatoxin effect, with a minimum inhibitory concentration (MIC) of 64 μg / mL.

[0045] 3) Detection of the inhibitory effect of lipopeptides on the mycelial growth of Fusarium solani:

[0046] Lipopeptides were diluted to different concentrations with methanol, and then 0.2 mL of the diluted lipopeptides was added to 14.8 mL of the prepared PDA medium, cooled to 40-50 °C, and poured into 60 mm culture dishes, 5 ml per plate. The final concentrations of lipopeptides were 80 μg / mL, 40 μg / mL, 20 μg / mL, 10 μg / mL, and 5 μg / mL, respectively. Methanol was used instead of antifungal peptide solution in the control group. A mycelial disk with a diameter of 5 mm was inoculated in the center of the plate in the treatment group and the control group, respectively, and then cultured at 28 °C. When the mycelial colony of the control group grew to the edge of the culture dish (about 96 h), the colony diameter of each treatment group and the control group was determined by the cross method. The rate of inhibition of mycelial growth was calculated using the following formula: Growth inhibition rate = (mycelial colony diameter control group − mycelial colony diameter treatment group) / mycelial colony diameter control group × 100%. Half inhibitory concentration (IC 50 ) were calculated using GraphPad 9.5 software. The results showed that the IC 50 23.91 μg / mL (see Figure 8 )

[0047] 4) Determination of spore germination rate by lipopeptide:

[0048] Lipopeptide was mixed with 1×10 5The CFU / mL spore suspension was fully mixed, and the final concentrations of lipopeptides were 64μg / mL, 32μg / mL, 16μg / mL, 8μg / mL, and 4μg / mL, respectively. Then, they were dropped onto the concave glass slides, and each treatment was repeated 3 times. The spore suspensions mixed with water and an equal volume of methanol were used as controls. They were placed at a constant temperature of 26°C. After 12 hours, the germination of conidia was examined under a microscope, and the inhibition rate of spore germination of each treatment was compared with that of the control.

[0049] The inhibition rate calculation formula is:

[0050] (number of spores germinated in the control group - number of spores germinated in the treatment group) / number of spores germinated in the control group × 100%,

[0051] The results are as follows Figure 9 As shown, 64 μg / mL of lipopeptide A1 could completely inhibit the germination of Aspergillus flavus spores.

[0052] 5) Stability testing of lipopeptide antibacterial substances

[0053] The antifungal effect of the lipopeptide solution under different treatments was determined by the filter paper method. Briefly, a 6 mm diameter filter paper was placed on the PDA plate, and then 7 μL of 2.5 mg / mL lipopeptide solution was added to the center of the filter paper. After it was completely absorbed, 5 μL of Aspergillus flavus spore suspension (final spore concentration was 1×10 5 CFU / mL) was added to the center of the filter paper, and then placed in a 28°C temperature box for 24 hours, and the colony diameters of the treated and control groups on the filter paper were measured. The specific treatment method is as follows:

[0054] Acid-base stability: The lipopeptide was prepared into a solution with a concentration of 2.5 mg / mL, and its pH value was adjusted to 2-14 with sodium hydroxide and hydrochloric acid, respectively, and the antibacterial effect of each treatment group was detected by the above-mentioned detection method.

[0055] Metal ion stability: Na+, K + , Ca 2+ Mg 2+ , Cu 2+ , Fe 2+ , Fe 3+ The final concentration of the ion solution was 10 μM, and the lipopeptide powder was dissolved in the metal solution to a final concentration of 2.5 mg / mL. The antibacterial effect of each treatment group was detected using the above detection method.

[0056] Temperature stability: The lipopeptide solution was prepared at 2.5 mg / ml and treated at 25 ℃, 50 ℃ and 100 ℃ for 30 min respectively. The antibacterial effect was detected using the above detection method.

[0057] The results showed that lipopeptide A1 can maintain antifungal activity over a wide pH range (seeFigure 10 ), and its activity is not interfered by metal ions (see Figure 11 In addition, the lipopeptide has high temperature stability and can still maintain anti-aflatoxin activity after being treated at 100°C (see Figure 12 ).

[0058] Application Example 1: Detection of the actual inhibitory ability of lipopeptide A1 on Aspergillus flavus:

[0059] 1) Toxin content, mycelium dry weight and spore yield detection:

[0060] Prepare PDB, put it into test tubes, 5 mL per tube, and add Aspergillus flavus spores to a final concentration of 1×10 5 CFU / mL. The final concentrations of lipopeptides added to the test tubes were 64μg / mL, 32μg / mL, 16μg / mL, 8μg / mL, and 4μg / mL, respectively. Methanol was used as a negative control, and PDB culture medium was used as a blank control. After 14 days of shaking culture at 28°C, the toxin content under each treatment was detected using the AFB1 toxin detection kit, and the detection value in the blank culture medium without any treatment was used as zero correction. The mycelium was dried and weighed to detect the inhibitory ability of lipopeptides on the biomass of Aspergillus flavus. The number of Aspergillus flavus spores in the culture medium was calculated using a hemocytometer to evaluate the effect of lipopeptides on the yield of Aspergillus flavus spores. The results are shown in Fig.13 , Fig.14 Methylotrophic Bacillus Bacillus methylotrophicus The lipopeptide substance A1 produced by 1-11-2 metabolism has a significant inhibitory effect on the spore production, toxin synthesis and biomass of Aspergillus flavus.

[0061] 2) Detection of lipopeptide's ability to protect corn seeds:

[0062] In order to evaluate the protective effect of lipopeptides on grains in actual use, lipopeptides were dissolved in 10% methanol solution with a concentration of 64 μg / mL to prepare a spray preparation. The corn seeds were treated with spray to evenly cover the seed surface with lipopeptides. After the seeds were naturally dried, the final concentration of 1×10 5 A solution of Aspergillus flavus spores at CFU / mL was sprayed onto the surface of the seeds. The seeds not treated with lipopeptide were used as a control and were also sprayed with Aspergillus flavus spores. The seeds were then placed in a culture dish and cultured at room temperature to observe the growth of Aspergillus flavus in each group.

[0063] The results showed that lipopeptides have a good protective effect on corn contaminated by Aspergillus flavus and can effectively prevent and control the contamination of Aspergillus flavus and its toxins (see Fig.15 ).

[0064] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A flavus antagonistic bacterium, characterized in that: The aflatoxin antagonistic bacteria is a methylotrophic Bacillus ( Bacillus methylotrophicus )1-11-2, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No: 32408, the deposit date is October 30, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of the antagonistic bacteria of Aspergillus flavus as claimed in claim 1 in the preparation of a feed mildew inhibitor for inhibiting the growth of Aspergillus flavus.

3. The use of the antagonistic bacteria of Aspergillus flavus according to claim 2 in the preparation of a feed mildew inhibitor for inhibiting the growth of Aspergillus flavus, characterized in that: Methylotrophic Bacillus Bacillus methylotrophicus )1-11-2 After continuous shaking culture in LB broth medium for 72 hours, the fermentation supernatant was collected and used to prepare a feed mildew inhibitor for inhibiting the growth of Aspergillus flavus.

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