Bacillus cereus and isolated culture method and application thereof
By screening and isolating the Bacillus cereus strain WMCC10515, the problem of difficulty in effectively degrading vomit toxins in grains in the prior art is solved, and efficient vomit toxin degradation and fungal disease prevention and control are achieved.
Patent Information
- Application Number
- CN202411877258.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-19
- Publication Date
- 2025-02-14
AI Technical Summary
The prior art is difficult to effectively degrade vomiting toxins present in grains, resulting in food safety problems.
A Bacillus cereus strain WMCC10515 was screened and isolated, which had significant vomittoxin degradation ability and was identified as Bacillus cereus by the 16S rRNA sequence.
This strain can achieve a degradation rate of vomit toxin in the feed to 82.68% within 3 to 5 days under constant temperature of 37±1℃, and also has the potential to prevent and treat fungal diseases.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a Bacillus cereus and a separation and cultivation method and application thereof. Background Art
[0002] Mycotoxins are harmful secondary metabolites produced by fungi during their growth process. They have significant toxic effects on humans and animals, and can cause severe poisoning reactions if ingested. Since mycotoxins mainly contaminate grains, oils, and related products, the nutritional value and commercial value of these foods are greatly damaged. Therefore, mycotoxins have become one of the pollutants that have attracted much attention in the field of food safety.
[0003] Deoxynivalenol (DON), also known as deoxynivalenol (DON), has a chemical name of 3α,7α,15-trihydroxy-12,13-epoxytrichothecenes-9-ene-8-one. It belongs to the B-type trichothecenes family and is a fungal toxin produced by the secondary metabolism of Fusarium. As one of the common fungal toxins, DON is widely present in grains and their products. After ingesting DON, humans and animals will experience vomiting, nausea, and even death, causing serious harm to health. In order to respond to the global pollution of DON, reduce the intake of DON by humans, and prevent food safety problems, many countries have established limit standards for the content of DON in imported and exported grains and grain products: GB2761-2011 stipulates that the content of DON in grains and grain products cannot exceed 1 mg / kg, and the limit standards of the United States and the European Union are 1 mg / kg and 0.75 mg / kg respectively. Traditional physical and chemical degradation methods have problems such as incomplete detoxification, low efficiency, high cost, harsh conditions, and affecting crop nutrition and taste. Biodegradation has the advantages of high degradation rate, low cost, and no impact on crop nutritional value, and has become an emerging method for detoxification of vomitoxin. Therefore, studying microorganisms that can degrade vomitoxin will play an important guiding role in analyzing the degradation of fungal toxin environmental pollutants and controlling environmental pollution.
[0004] As a widely used biocontrol bacteria, Bacillus not only has strong survival ability and environmental adaptability, but also shows remarkable effects in preventing and controlling various animal and plant diseases. Bacillus can produce a variety of antibacterial substances, which can effectively inhibit the growth and reproduction of pathogens, thereby reducing the incidence of crop diseases. In addition, Bacillus can also degrade organic pollutants in the environment and reduce the degree of environmental pollution. Therefore, it has broad application prospects in agricultural production and environmental protection. Bacillus cereus can produce secondary metabolites such as antimicrobial proteins, bacteriocins, reactive oxygen species, organic acids, etc. to destroy the cell membrane of pathogens, inhibit the metabolic activities of pathogens, or interfere with the cell signal transduction of pathogens, thereby achieving the degradation of fungal toxins. The Bacillus cereus B.JG05 obtained by screening and isolation using vomitoxin as the sole carbon source has a degradation rate of up to 82.68% for vomitoxin in feed; the Bacillus ODS-1 obtained by domestication and enrichment using sow manure as the material can simultaneously degrade aflatoxin B1, zearalenone and vomitoxin.
[0005] Therefore, screening out new Bacillus cereus that can degrade vomitoxin can provide new microbial resources and prevention and control ideas for the degradation of vomitoxin in grains and their fermented products, ensure food safety, and promote the healthy and sustainable development of related industries. Summary of the invention
[0006] The object of the present invention is to provide a strain of Bacillus cereus. The Bacillus cereus obtained by separation and screening of the present invention has good environmental adaptability and plays an important role in degrading vomitoxin.
[0007] To achieve the above-mentioned purpose, in the first aspect, the present invention provides a Bacillus cereus, whose strain is named Bacillus cereus (Bacillus cereus) WMCC10515, which has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on March 28, 2024, and its deposit number is CGMCC NO.30176.
[0008] The invention analyzes the strain through 16S rRNA sequence, and the strain is identified as Bacillus cereus, the length of the 16S rRNA sequence is 1543 bp, and the strain is named as Bacillus cereus WMCC10515.
[0009] The 16S rDNA sequence of Bacillus cereus WMCC10515 of the present invention is shown as SEQ ID NO:1.
[0010] SEQ ID NO:1 The 16S rDNA sequence of Bacillus cereus WMCC10515 is as follows:
[0011]
[0012] The Bacillus cereus WMCC10515 of the present invention is a Gram-positive bacterium, and its colony characteristics are as follows: in an LB solid culture medium, the colonies are off-white, round, moist and opaque, with raised surfaces, neat edges and low viscosity; under a 100-fold optical microscope, the strain cells are rod-shaped and have spores.
[0013] The present invention also provides a method for isolating and culturing the above-mentioned Bacillus cereus, which comprises the following steps:
[0014] A. Preparation of bacterial suspension: Mix the Baobaoqu and PBS buffer evenly, let it stand, and take the supernatant; dilute the supernatant with PBS buffer to 10 -5 , obtaining a koji fungus suspension;
[0015] B. Screening of strains: The Koji fungus suspension was inoculated into LB solid culture medium containing vomitoxin, and initially screened and cultured to obtain single colonies with different morphologies, and then purified and cultured to obtain single colonies with consistent morphologies. Finally, the purified strain was inoculated into LB liquid culture medium containing vomitoxin, and rescreened and cultured to obtain Bacillus cereus WMCC10515.
[0016] Among them, in the above-mentioned separation and culture method, in step A, the Baobaoqu and PBS buffer are evenly mixed in a mass volume ratio of 1:5 to 20.
[0017] Among them, in the above-mentioned separation and culture method, in step B, the composition and preparation method of the LB solid culture medium containing vomitoxin are: 5.0g yeast powder, 10.0g peptone, 10.0g sodium chloride, 18.0g agar powder, add water to 1L, use NaOH or HCl to adjust the pH to 7.0-7.2, and sterilize at 121°C for 20min; add vomitoxin before use to make the final concentration of vomitoxin 0.5-3.0ug / ml.
[0018] Among them, in the above-mentioned separation and culture method, in step B, the composition and preparation method of the LB liquid culture medium containing vomitoxin are: 5.0g yeast powder, 10.0g peptone, 10.0g sodium chloride, add water to 1L, use NaOH or HCl to adjust the pH to 7.0-7.2, and sterilize at 121°C for 20min; before use, add vomitoxin to make the final concentration of vomitoxin 0.5-3.0ug / ml.
[0019] Wherein, in the above-mentioned separation and culture method, in step B, the conditions for the initial screening culture are: constant temperature inversion culture at 37±1°C for 12 to 36 hours.
[0020] Wherein, in the above separation and culture method, in step B, the purification culture conditions are: constant temperature inversion culture at 37±1°C for 12 to 36 hours.
[0021] Wherein, in the above separation and culture method, in step B, the purification culture is performed 3 to 5 times.
[0022] Among them, in the above-mentioned separation and culture method, in step B, the conditions for the re-screening culture are: culturing on a constant temperature shaker at 37±1°C, the shaker speed is 100-250rpm / min, and culturing until the bacteria are turbid.
[0023] The present invention also provides a microbial agent capable of degrading mycotoxins, which comprises the above-mentioned Bacillus cereus.
[0024] Among them, the bacterial concentration of Bacillus cereus in the above-mentioned microbial agent is OD 600 =0.6~0.7.
[0025] Among them, in the above-mentioned microbial agent, the fungal toxin is vomitoxin.
[0026] The present invention also provides use of the Bacillus cereus or the microbial agent in preparing a medicine for degrading mycotoxins.
[0027] Wherein, in the above use, the fungal toxin is vomitoxin.
[0028] The present invention also provides a method for degrading vomitoxin by using the above-mentioned Bacillus cereus, which comprises the following steps: preparing Bacillus cereus WMCC10515 with a bacterial concentration of OD 600 =0.6-0.7 fermentation broth, add 0.5-3.0ug / ml vomitoxin acetonitrile solution thereto, and culture at 37±1°C constant temperature for 3-5 days.
[0029] The present invention also provides the use of the Bacillus cereus in preparing koji medicine.
[0030] Beneficial effects of the present invention:
[0031] The present invention screened and isolated a strain of Bacillus cereus WMCC10515 from Baobaoqu, which has a significant ability to degrade vomitoxin and has the biocontrol potential for preventing and controlling fungal diseases. It also has a wide range of metabolic capabilities and can effectively utilize mannitol, glucose, starch, nitrate and other substances for various life activities such as growth and metabolism, and has good environmental adaptability. GO analysis results show that it also has certain detoxification and antibacterial properties, providing new microbial resources and ideas for the biodegradation of fungal toxins and the prevention and control of fungal toxin infection in grains.
[0032] The deposit number of the Bacillus cereus of the present invention is CGMCC NO.30176. The deposit time is March 28, 2024, and the deposit center is: China General Microbiological Culture Collection Center (CGMCC), the address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, zip code 100101. The classification name is Bacillus cereus WMCC10515. BRIEF DESCRIPTION OF THE DRAWINGS
[0033] Figure 1 This is a graph showing the degradation rate changes of Bacillus cereus WMCC10515 under different vomitoxin concentrations in Example 1 of the present invention.
[0034] Figure 2 The morphological diagram of Bacillus cereus WMCC10515 isolated in Example 2 of the present invention; wherein (a) is the colony morphology of the strain on LB solid culture medium; and (b) is the microscopic morphology of the strain under a 100x optical microscope after Gram staining.
[0035] Figure 3 This is a diagram of the PCR reaction system in Example 2 of the present invention.
[0036] Figure 4 This is a phylogenetic tree diagram of Bacillus cereus WMCC10515 based on 16SrRNA in Example 2 of the present invention.
[0037] Figure 5 This is the genome circle map of Bacillus cereus WMCC10515 in Example 3 of the present invention.
[0038] Figure 6 This is a diagram of the homologous clustering gene cluster analysis of Bacillus cereus WMCC10515 in Example 3 of the present invention.
[0039] Figure 7 This is the GO analysis diagram of Bacillus cereus WMCC10515 in Example 3 of the present invention. DETAILED DESCRIPTION
[0040] The following will be combined with the embodiments of the present invention and the accompanying drawings to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0041] In the following examples, the preparation and concentration of the vomitoxin working solution: 5 mg of the vomitoxin standard was weighed into a 100 ml volumetric flask and dissolved to the mark with acetonitrile to prepare a vomitoxin working solution with a concentration of 50 ug / ml.
[0042] In the following examples, the composition and preparation method of LB liquid culture medium (containing vomitoxin) are as follows: 5 g yeast powder, 10 g peptone, 10 g sodium chloride, distilled water is added to 1 L, pH is adjusted to 7.0-7.2 with NaOH or HCl, and sterilized at 121°C for 20 min. Before use, vomitoxin working solution is added to a final concentration of 1.5 ug / ml or 2.5 ug / ml.
[0043] In the following examples, the composition and preparation method of LB solid medium (containing vomitoxin) are as follows: 5 g yeast powder, 10 g peptone, 10 g sodium chloride, 18 g agar powder, distilled water is added to 1 L, pH is adjusted to 7.0-7.2 with NaOH or HCl, and sterilized at 121°C for 20 min. Before use, vomitoxin working solution is added to a final concentration of 2.0 ug / ml.
[0044] In the following examples, the composition and preparation method of PBS buffer solution are as follows: 8.0 g sodium chloride, 1.2 g disodium hydrogen phosphate, 0.2 g potassium dihydrogen phosphate, 0.2 g potassium chloride, and distilled water is added to make up to 1 L.
[0045] Example 1: Screening and Isolation of Bacillus cereus WMCC10515
[0046] (1) Sample collection
[0047] Wuliangye Baobaoqu: Collected from the Wuliangye Qu-making workshop in Yibin, Sichuan Province, China.
[0048] (2) Preparation of bacterial suspension
[0049] Accurately weigh 2.5g of crushed Wuliangye Baobaoqu, place it in a 50ml centrifuge tube, add 25ml of sterile PBS buffer to the centrifuge tube, and use a vortexer to fully vortex the mixture for 5 minutes to ensure that the bacteria can be evenly dissolved in the sterile PBS buffer. After the shaking is completed, let the centrifuge tube stand for 5 minutes. After the solution is clarified, take 1ml of the supernatant and dilute it in 9ml of sterile PBS buffer to 10 -5 , make the required koji suspension.
[0050] (3) Preliminary screening of vomitoxin-degrading strains
[0051] Perform aseptic operation in the clean bench, and use the plate streak method to isolate and screen the target strain. Use a sterile inoculation loop to dip the prepared gradient diluted koji suspension, and streak inoculate on the surface of the LB solid culture medium containing vomitoxin. After the inoculation is completed, place the culture medium plate in a 37±1℃ constant temperature incubator and invert it for 24 hours. During the culture process, observe the single colonies of various shapes growing on the culture medium, and use a sterile inoculation loop to select these single colonies for further pure culture to ensure that a pure strain is obtained.
[0052] (4) Purification of vomitoxin-degrading strain
[0053] For the single colonies with different morphologies obtained in the preliminary screening, further purification steps are adopted to ensure the purity of the strain. These single colonies are streaked onto new LB solid culture medium containing vomitoxin, and then the culture medium is placed in a constant temperature incubator at 37±1℃ for inverted culture. To ensure the purity of the strain, the above purification process is repeated 3 to 4 times, and each time single colonies are screened and isolated on a new culture medium by streaking. When the morphology of the colonies grown on the plate is consistent, it means that the strain has been fully purified. At this time, pick a single colony with a sterile inoculation loop and store it in a preservative solution containing 80% glycerol, and then place the preservative solution at a low temperature of 4℃ for long-term storage for subsequent experiments or applications.
[0054] (5) Rescreening of DON-degrading strains
[0055] The above-mentioned preservation solution containing the purified single colony was activated (cultured at 37±1℃, constant temperature shaker with a rotation speed of 180-200rpm / min for 12h), and inoculated into 30ml LB liquid culture medium containing 1.5ug / ml and 2.5ug / ml vomitoxin respectively and cultured in a constant temperature shaker at 37±1℃ with a rotation speed of 135rpm / min until the bacteria became turbid. A 30ml LB liquid culture medium containing 1.5ug / ml and 2.5ug / ml vomitoxin without the strain was set as a blank control. The content of vomitoxin in the blank control and the bacterial solution was accurately detected by high performance liquid chromatography, and the initially screened strains were rescreened to screen out and preserve Bacillus cereus WMCC10515 with the ability to degrade vomitoxin. The test results of the strain's ability to degrade vomitoxin are as follows: Figure 1 As shown in the figure, A indicates that the initial concentration of vomitoxin is 1.5ug / ml, and B indicates that the initial concentration of vomitoxin is 2.5ug / ml. Figure 1It can be seen from the results that when the concentration of vomitoxin in the LB liquid culture medium is 1.5ug / ml, the degradation rate of vomitoxin by Bacillus cereus WMCC10515 is 24.82%; when the concentration of vomitoxin in the LB liquid culture medium is 2.5ug / ml, the degradation rate of vomitoxin by Bacillus cereus WMCC10515 is 37.73%.
[0056] Example 2: Identification of Bacillus cereus WMCC10515
[0057] (1) Morphological identification
[0058] The Bacillus cereus WMCC10515 isolated in Example 1 was subjected to morphological examination. Figure 2 shown. Figure 2 (a) is a photo of the colony morphology of Bacillus cereus WMCC10515 on LB solid medium. Figure 2 As can be seen in Figure (a), the colonies are off-white, round, moist and opaque, with raised surfaces and neat edges, indicating that they grow evenly and stably. In addition, Bacillus cereus WMCC10515 has a low viscosity, is easily picked up, and has a rod-shaped cell shape. Figure 2 (b) is a microscopic photo of Bacillus cereus WMCC10515 under a 100x optical microscope. Figure 2 (b) It can be seen that the cells of this strain are rod-shaped, about 2 μm in size, and have spores.
[0059] (2) Molecular identification
[0060] The Bacillus cereus WMCC10515 isolated in Example 1 was subjected to 16sDNA identification. The genomic DNA of the strain was extracted using a bacterial DNA extraction kit, and the DNA sequence of the target region was amplified by PCR using the specific primers 27F and 1492R of the bacterial 16S rDNA gene.
[0061] The DNA sequence of the specific primer 27F is shown in SEQ ID NO: 2: 5'-AGAGTTTGATCCTGGCTCAG-3'.
[0062] The DNA sequence of the specific primer 1492R is shown in SEQ ID NO: 3: 5'-GGTTACCTTGTTACGACTT-3'. The PCR reaction conditions are shown in Table 1.
[0063] Table 1 PCR reaction conditions
[0064] Components volume 10×Ex Taq Buffer 2.0μl 5u Ex Taq 0.2μl 2.5mM dNTP mixed solution 1.6μl 5p Primer 27F 1μl 5p Primer 1492R 1μl DNA 0.5μl Ultrapure water 13.7μl Total volume 20μl
[0065] PCR reaction system Figure 3 As shown.
[0066] The 16sRNA sequence of the strain is shown in SEQ ID NO: 1. By comparing this sequence with the known sequence in genebank, it was found that it was highly similar to Bacillus cereus, so the bacterium was confirmed to be Bacillus cereus. Bacillus cereus WMCC10515 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on March 28, 2024, with the deposit number CGMCC NO.30176, and the classification name Bacillus cereus.
[0067] The BLAST function of the NCBI website was used to perform homology analysis on the measured 16S rRNA sequences to clarify the relationship of the strain in the microbial kingdom, and the Neighbor-Joining method of MEGA6.0 software was used to construct a phylogenetic tree to further reveal its evolutionary relationship. Figure 4 As shown. Figure 4 It can be seen that the strain has the highest similarity with Bacillus cereus ATCC14579 (GCF_000007825.1) and is on the same branch of the phylogenetic tree. Combining the bacterial morphological characteristics, physiological and biochemical characteristics and 16S rRNA sequence comparative analysis, the strain was identified as Bacillus cereus.
[0068] (3) Identification of physiological and biochemical characteristics
[0069] Gram staining: Gram staining of Bacillus cereus WMCC10515 showed the following morphology: Figure 2 as shown in (b). Figure 2 (b) is a photo of the colony morphology of Bacillus cereus WMCC10515 under a 100x optical microscope after Gram staining. Figure 2 (b) It can be seen that the bacterial morphology of Bacillus cereus WMCC10515 is rod-shaped, and the cells are blue-purple after staining, which is a Gram-positive bacterium. Figure 2 In (b), it can be clearly seen that the strain produces spores and belongs to Bacillus.
[0070] Analysis of physiological and biochemical characteristics: Based on the precise detection of the Bergey's Manual of Bacterial Identification (8th edition) and the HKI003 EasyID Bacillus cereus biochemical identification kit, the physiological and biochemical characteristics of Bacillus cereus WMCC10515 were analyzed in detail. The results are shown in Table 2 ("+" indicates a positive reaction, and "-" indicates a negative reaction).
[0071] As can be seen from Table 2, Bacillus cereus WMCC10515 has a wide range of metabolic capabilities and can effectively use mannitol, glucose, starch and other substances for growth and metabolism and other life activities. In addition, the strain can also use nitrate for nitrogen source metabolism. These physiological and biochemical characteristics further verify that Bacillus cereus WMCC10515 belongs to the classification of Bacillus cereus.
[0072] Table 2 Physiological and biochemical characteristics of Bacillus cereus WMCC10515
[0073] index feature Lysozyme resistance + Mannitol acid production - Glucose utilization (anaerobic) + starch + Nitrate reduction + VP test + power - Citrate - gelatin + Catalase + Hemolysis (sheep red blood cells) +
[0074] Example 3: Whole genome sequencing of Bacillus cereus WMCC10515
[0075] A single colony of Bacillus cereus WMCC10515 was inoculated into LB liquid culture medium and cultured at 37°C and 135rpm until the logarithmic growth phase. The bacteria were successfully collected by centrifugation at 6000r / min for 5min. Subsequently, a DNA extraction kit was used to extract the genomic DNA of Bacillus cereus WMCC10515 strain to ensure that the quality of the extracted genomic DNA was qualified. The qualified genomic DNA was sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for whole genome sequencing. The results are as follows Figure 5 , Figure 6 and Figure 7 As shown. The sequencing results showed that the chromosome size of Bacillus cereus WMCC10515 was 6.080726Mb, the GC content was 35.02%, the number of CDS (Protein-coding sequences) was 6084, and it contained 4 plasmids. Among them, the size of plasmid A was 441248bp, the GC content was 32.69%; the size of plasmid B was 144353bp, the GC content was 32.65%; the size of plasmid C was 43156bp, the GC content was 41.10%; the size of plasmid D was 20672bp, the GC content was 32.24%.
[0076] Figure 5This is the circle map of the genome of Bacillus cereus WMCC10515. The first and fourth circles from the outside to the inside of the circle map are CDS on the positive and negative chains, respectively, and different colors represent different COG functional classifications; the second and third circles are CDS, tRNA, and rRNA on the positive and negative chains, respectively; the fifth circle is the GC content, the outward part indicates that the GC content of this region is higher than the average GC content of the whole genome, and the higher the peak value, the greater the difference with the average GC content, and the inward part indicates that the GC content of this region is lower than the average GC content of the whole genome, and the higher the peak value, the greater the difference with the average GC content; the sixth circle is the GC-Skew value, and the specific algorithm is GC / G+C, which can assist in judging the leading chain and the lagging chain; the innermost circle is the genome size marker.
[0077] Figure 6 This is a homologous clustering gene cluster (COG) analysis diagram of Bacillus cereus WMCC10515. Among them, A represents RNA processing and modification; B represents chromatin structure and dynamics; C represents energy generation and conversion; D represents cell cycle control, cell division, chromosome segmentation; E represents amino acid transport and metabolism; F represents nucleotide transport and metabolism; G represents carbohydrate transport and metabolism; H represents coenzyme transport and metabolism; I represents lipid transport and metabolism; J represents translation, ribosome structure and biogenesis; K represents transcription; L represents replication, recombination and repair; M represents cell wall / membrane / envelope biogenesis; N represents cell movement; O represents post-translational modification, protein turnover, molecular chaperone; P represents inorganic ion transport and metabolism; Q represents biosynthesis, transport and catabolism of secondary metabolites; R represents only general function prediction; S represents unknown function; T represents signal transduction mechanism; U represents intracellular transport, secretion and vesicle transport; V represents defense mechanism; W represents extracellular structure; X represents mobile group represents phage, transposon; Y represents nuclear structure; Z represents cytoskeleton.
[0078] Figure 7 This is the GO analysis diagram of Bacillus cereus WMCC10515. GO annotation includes three aspects: BP (Biological Process): describes the biological process in which the gene product is involved, such as cell growth, metabolism, etc.; CC (Cellular Component): describes the location of the gene product in the cell, such as cell membrane, cell nucleus, etc.; MF (Molecular Function): describes the activity of the gene product at the molecular level, such as catalysis, binding, etc.
[0079] Figure 6COG (Clusters of Orthologous Groups of proteins) analysis showed that the most abundant genes were those with common function predictions, with 442 genes; followed by genes involved in transcription, with 426 genes; the numbers of genes involved in amino acid transport and metabolism, cell wall / membrane / envelope biogenesis, translation, and carbohydrate transport and metabolism were 425, 323, 322, and 302, respectively.
[0080] Figure 7 In the results of GO (Gene Onotology) analysis, Bacillus cereus WMCC10515 contained genes related to oxidative stress response (GO:0006979), salt stress response (GO:0009651), heat stress response (GO:0009408), and cold stress response (GO:0009409), indicating that Bacillus cereus WMCC10515 has certain environmental stress resistance characteristics. In addition, Bacillus cereus WMCC10515 also contains genes related to detoxification (GO:1990961) and antioxidant activity (GO:0016209), indicating that Bacillus cereus WMCC10515 has certain detoxification and antibacterial properties.
[0081] The above results show that Bacillus cereus WMCC10515 has significant ability to degrade vomitoxin and has the potential to prevent and control fungal diseases. It provides a new prevention and control strategy for fungal toxin infection of grain and also provides valuable Bacillus cereus strain resources for the agricultural field.
Claims
1. Bacillus cereus, characterized in that: The strain of Bacillus cereus is named Bacillus cereus WMCC10515, and is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, and its deposit number is CGMCC NO.30176.
2. The method for isolating and culturing Bacillus cereus according to claim 1, characterized in that: The following steps are involved: A. Preparation of bacterial suspension: Mix the Baobaoqu and PBS buffer evenly, let it stand, and take the supernatant; dilute the supernatant with PBS buffer to 10 -5 , obtaining a koji fungus suspension; B. Screening of strains: The Koji fungus suspension was inoculated into LB solid culture medium containing vomitoxin, and initially screened and cultured to obtain single colonies with different morphologies, and then purified and cultured to obtain single colonies with consistent morphologies. Finally, the purified strain was inoculated into LB liquid culture medium containing vomitoxin, and rescreened and cultured to obtain Bacillus cereus WMCC10515.
3. The method for isolating and culturing Bacillus cereus according to claim 2, wherein: In step A, the Baobaoqu and PBS buffer are mixed evenly at a mass volume ratio of 1:5-20.
4. The method for isolating and culturing Bacillus cereus according to claim 2, wherein: At least one of the following is met: In step B, the composition and preparation method of the LB solid culture medium containing vomitoxin are as follows: 5.0 g of yeast powder, 10.0 g of peptone, 10.0 g of sodium chloride, 18.0 g of agar powder, add water to 1 L, adjust the pH to 7.0-7.2 with NaOH or HCl, and sterilize at 121° C. for 20 min; add vomitoxin before use to make the final concentration of vomitoxin 0.5-3.0 ug / ml; In step B, the composition and preparation method of the LB liquid culture medium containing vomitoxin are as follows: 5.0 g of yeast powder, 10.0 g of peptone, 10.0 g of sodium chloride, add water to 1 L, adjust the pH to 7.0-7.2 with NaOH or HCl, and sterilize at 121° C. for 20 min; before use, add vomitoxin to make the final concentration of vomitoxin 0.5-3.0 ug / ml; In step B, the conditions for the initial screening culture are: constant temperature inversion culture at 37±1°C for 12 to 36 hours; In step B, the purification culture conditions are: constant temperature inversion culture at 37±1°C for 12 to 36 hours; In step B, the purification culture is performed 3 to 5 times; In step B, the conditions for the rescreening culture are: culturing in a constant temperature shaker at 37±1°C, with a shaker speed of 100-250 rpm / min, and culturing until the bacteria become turbid.
5. A microbial agent capable of degrading mycotoxins, characterized in that: The microbial agent comprises the Bacillus cereus according to claim 1.
6. The microbial agent according to claim 5, characterized in that: At least one of the following is met: In the microbial agent, the bacterial concentration of Bacillus cereus described in claim 1 is OD 600 =0.6~0.7; The fungal toxin is vomitoxin.
7. Use of the Bacillus cereus according to claim 1 or the microbial agent according to claims 5 to 6 in the preparation of a medicament for degrading mycotoxins.
8. The use according to claim 7, characterized in that: The fungal toxin is vomitoxin.
9. The method for degrading vomitoxin using the Bacillus cereus according to claim 1, characterized in that: The following steps are involved: Bacillus cereus WMCC10515 was prepared into a bacterial concentration of OD 600 =0.6-0.7 fermentation broth, add 0.5-3.0ug / ml vomitoxin acetonitrile solution thereto, and culture at 37±1°C constant temperature for 3-5 days.
10. Use of the Bacillus cereus according to claim 1 in preparing koji medicine.