CN110055186B - Microbial preparation for degrading vomitoxin and application - Google Patents
Microbial preparation for degrading vomitoxin and application Download PDFInfo
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Abstract
The invention discloses a microbial preparation for degrading vomitoxin and application thereof, belonging to the technical field of vomitoxin detoxification. The effective component of the microbial preparation for degrading vomit is Siamese bacillus full culture solution culture or Siamese bacillus suspension or crude extract of Siamese bacillus or extracellular metabolite of Siamese bacillus; the Siamese bacillus is Siamese bacillus A2025, and is stored in the Siamese bacillus A2025 in 2018 in the 12 th month 04: china general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC NO:15611 and an address of: west road No. 1, north zhou yang ward, beijing, the requested depository is peanut institute of shandong province. The Siamese bacillus A2025 disclosed by the invention has a degradation effect on vomitoxin.
Description
Technical Field
The invention belongs to the technical field of vomitoxin detoxification, and particularly relates to a microbial preparation for degrading vomitoxin and application thereof.
Background
Vomitoxin is also called deoxynivalenol toxin (DON), is a type B trichothecene toxin, is a secondary metabolite mainly produced by Fusarium graminearum (Fusarium graminearum) and Fusarium flavum (Fusarium culmorum), and is widely present in polluted grains, feeds and foods. The vomitoxin has higher cytotoxicity and immunosuppressive property, enters human bodies and animal bodies to influence digestive systems to cause symptoms such as vomit and the like, reduces the immunity of the organisms, and causes serious harm to the health of the human bodies and the animals.
At present, the vomitoxin is removed by mainly adopting a physical method, a chemical method and a biological method at home and abroad. Physical and chemical methods have the disadvantages of non-ideal detoxification effect, introduction of other harmful substances and the like. Compared with the traditional physical and chemical methods, the biological method can convert the vomitoxin into nontoxic or low-toxic substances without causing the nutrient loss of agricultural products, so that the method is a green and safe detoxification method and is considered as the best method for removing the vomitoxin.
Disclosure of Invention
In view of the problems in the prior art, the invention aims to provide a microbial preparation for degrading vomitoxin and a method for detoxifying an agricultural product polluted by the vomitoxin by using the microbial preparation.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a microbial preparation for degrading vomitoxin comprises the effective components of a Siamese bacillus full culture solution culture or Siamese bacillus suspension or a crude extract of Siamese bacillus or an extracellular metabolite of Siamese bacillus;
the Siamese Bacillus is Siamese Bacillus (Bacillus siamensis) A2025, and is preserved in the culture medium in 12 months 04 in 2018: china general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC NO:15611 and an address of: west road No. 1, north zhou yang ward, beijing, the requested depository is peanut institute of shandong province.
On the basis of the scheme, the preparation method of the Siamese bacillus A2025 full culture solution culture comprises the following steps:
inoculating 200 mu L of frozen Siamese bacillus A2025 bacterial liquid into a triangular flask filled with 200mL of TSB liquid culture medium, and performing shake culture at 37 ℃ for 96h, wherein the concentration of the bacterial liquid is 2.1 multiplied by 108cfu/mL。
On the basis of the scheme, the preparation method of the Siamese bacillus A2025 bacterial suspension comprises the following steps:
taking 20mL Siamese bacillus A2025 full culture solution culture, wherein the concentration of the bacterial solution is 2.1 multiplied by 108cfu/mL, centrifuging for 20min at 8000r/min, separating to obtain thalli and supernatant, washing the prepared thalli with sterile water, centrifuging, adding sterile water to make up to 20mL, and suspending the thalli to obtain Siamese bacillus A2025 bacterial suspension.
On the basis of the scheme, the preparation method of the extracellular metabolite of the Siamese bacillus A2025 comprises the following steps:
taking 20mL Siamese bacillus A2025 full culture solution culture, wherein the concentration of the bacterial solution is 2.1 multiplied by 108cfu/mL, centrifuging at 8000r/min for 20min, separating to obtain thallus and supernatant, wherein the supernatant is Siamese bacillus A2025 extracellular metabolite.
On the basis of the scheme, the preparation method of the Siamese bacillus A2025 crude extract comprises the following steps:
after the Siamese bacillus A2025 bacterial suspension is subjected to low-temperature ultrasonic crushing, the Siamese bacillus A2025 bacterial suspension is centrifuged at 8000r/min for 20min to obtain liquid, and the liquid is filtered by a 0.22 mu m filter membrane to prepare a crude extract of the Siamese bacillus A2025.
The application of the microbial preparation for degrading vomitoxin in detoxification of agricultural products polluted by vomitoxin or products further processed from the agricultural products.
A method for detoxifying vomitoxin comprises incubating the microbial preparation and vomitoxin-containing sample at a ratio of 1% -10% at 37 deg.C for 24 h.
Drawings
FIG. 1A2025 colony morphology;
FIG. 2A2025 Strain 16S rRNA agarose gel electrophoresis, on the left panel of Marker, and on the right panel of A2025 16S rRNA electrophoresis;
FIG. 3 degradation rate of Siamese Bacillus A2025 on vomitoxin.
Detailed Description
Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified.
The present invention will be described in further detail with reference to the following data in conjunction with specific examples. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
The Siamese Bacillus is Siamese Bacillus (Bacillus simensis) A2025, and is stored in the following storage room in 12 months 04 in 2018: china general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC NO:15611 and an address of: west road No. 1, north zhou yang ward, beijing, the requested depository is peanut institute of shandong province.
Example 1 sample Collection, Strain isolation and screening
1. Time and place of sample collection
Peanut field soil samples were collected from the experimental base of peanut institute in Shandong province (Wangxi city town, Qingdao city) 5.5.5.5.2017 at morning.
2. Sample processing, strain isolation and screening
Taking 1g of collected soil sample in a super clean bench to suspend in 10mL of sterile water, shaking and diluting to prepare a soil suspension, and then diluting by 100 times with sterile distilled water. 100 μ L of the suspension was spread on an LB solid plate, and cultured at 37 ℃ for 3 days, and the colonies grown were streaked on an LB solid plate and purified 3 times to obtain a single strain, No. A2025.
Example 2 identification of Strain A2025
The morphological characteristics and the physiological and biochemical characteristics of the strain A2025 were identified according to the methods described in Bergey's Manual of bacteriological identification (eighth edition), with the following results:
1. morphological characteristics: the single colony of the strain A2025 is raised, milky white and opaque on an LB culture medium (figure 1), the colony is about 4-6mm after being cultured for 2 days at 37 ℃, and wrinkles appear on the surface of the colony after being cultured for 3 days.
2. Physiological and biochemical characteristics: oxidase activity is negative, can grow at 4-54 deg.C, gram stain is positive, can hydrolyze and utilize casein, gelatin and starch, and cannot utilize Tween 40 and Tween 80.
3. 16S rRNA Gene analysis
Extracting the bacterial genome DNA of A2025, performing PCR amplification by using a 16S rRNA gene universal primer, connecting an amplification product to a pMD19-T vector, transforming a recombinant plasmid to escherichia coli, and sequencing to obtain a 1508bp gene sequence. According to the homology comparison of standard strain sequences in an EzTaxon-e server database, the 16S rRNA gene of the strain A2025 is compared with the standard strain Bacillus siamensis KCTC 13613TThe 16S rRNA gene homology of the strain is 100 percent, and the gene analysis shows that the strain is Siamese Bacillus (Bacillus simensis).
By integrating morphological characteristics, physiological and biochemical identification, 16S rDNA sequencing and homology analysis results, the strain A2025 is identified to be Siamese Bacillus (Bacillus siamensis). Named as Siamese Bacillus (Bacillus siamensis) A2025, which is preserved in China general microbiological culture Collection center at 12.04.2018, and the addresses are as follows: western road No. 1, north chen, chaoyang district, beijing, ministry of sciences, china, institute of microbiology, zip code 100101; the preservation unit is Shandong province peanut research institute, and the preservation number is CGMCC NO: 15611.
Example 3 degradation of vomitoxin by Siamese Bacillus A2025
1. Siamese bacillus A2025 culture for degrading vomitoxin
The culture medium of Siamese bacillus A2025 for degrading vomitoxin is TSB culture medium: 15g tryptone, 5g Soy peptone, 5g sodium chloride, 1000mL water, pH 7.2.
Frozen Siamese bacillus A2025 is taken out from a refrigerator at the temperature of minus 80 ℃, 50 mu L of Siamese bacillus A2025 bacterial liquid is taken out from a clean bench and inoculated into a test tube filled with 5mL of TSB liquid culture medium, and 12h of activated bacterial strain is cultured. Inoculating activated 500 μ L bacterial liquid into 100mL TSB liquid culture medium, shake culturing at 37 deg.C for 96 hr to obtain bacterial liquid with concentration of 2.1 × 108cfu/mL。
2. Degradation of vomitoxin by Siamese bacillus A2025
Taking 1.98mL of Siamese bacillus A2025 bacterial liquid (the concentration of the bacterial liquid is 2.1 multiplied by 10)8cfu/mL) was placed in a 10mL sample tube, 20. mu.L of 200ppm vomitoxin working stock solution was added to a final concentration of 2ppm, and a control sample was given a blank TSReplacing the bacterial liquid with a liquid culture medium, incubating for 24h at 37 ℃, centrifuging for 5min at 8000rpm to obtain a supernatant, and recording the supernatant as a test solution; mu.L of 200ppm vomitoxin working stock solution was added to 1.98mL of non-inoculated medium as a control, and the control solution was recorded.
3. Analysis of degradation capability of Siamese bacillus A2025 on vomitoxin
1mL of the sample was centrifuged at 12000rpm at 4 ℃ for 20min, and the supernatant was filtered through a 0.22 μm filter and then the remaining vomitoxin content in the medium was measured by HPLC. The following conditions for detecting vomitoxin by HPLC are adopted, and the chromatographic column comprises: agilent ZORBAX SB-C18 column (150 mm. times.4.6 mm, 5 μm); mobile phase: methanol-water (30:70, v/v); the detection wavelength is 218 nm; column temperature: 30 ℃; flow rate: 1.0 mL/min; sample injection amount: 10 μ L.
Vomitoxin degradation rate (%) (content of vomitoxin in control group-content of vomitoxin in experimental group)/content of vomitoxin in control group × 100.
The result is shown in fig. 3, and the result shows that the degradation effect of Siamese bacillus A2025 on vomitoxin is better under the condition of 37 ℃, and the degradation rate is 71.2%.
Example 4 application of Bacillus siamensis A2025 in degradation of vomit toxin of putrid corn
Fusarium graminearum is inoculated into 100g of sterile corn, the corn is cultured for 10 days at 28 ℃, polluted corn is crushed, and 200mL of sterile water is added to prepare corn steep liquor. Adjusting the pH value of the corn steep liquor to 7.0, mixing the corn steep liquor and Siamese bacillus A2025 in a conical flask according to the volume ratio of 9:1, mixing and stirring at 37 ℃ and 150rpm for 24 hours; in the blank control, isometric sterile water is used for replacing Siamese bacillus A2025 bacterial liquid. Vomitoxin content was determined by HPLC.
The result shows that the Siamese bacillus A2025 bacterial solution can reduce the vomitoxin in the corn steep liquor from 1450ppb to 480ppb, and the degradation rate is 66.9%.
Sequence listing
<110> institute for peanut research in Shandong province
<120> microbial preparation for degrading aflatoxin and application thereof
<130> 2019
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1508
<212> DNA
<213> 16S rRNA(Bacillus siamensis)
<400> 1
agagtttgat cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60
ggacagatgg gagcttgctc cctgatgtta gcggcggacg ggtgagtaac acgtgggtaa 120
cctgcctgta agactgggat aactccggga aaccggggct aataccggat ggttgtttga 180
accgcatggt tcagacataa aaggtggctt cggctaccac ttacagatgg acccgcggcg 240
cattagctag ttggtgaggt aacggctcac caaggcgacg atgcgtagcc gacctgagag 300
ggtgatcggc cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtagg 360
gaatcttccg caatggacga aagtctgacg gagcaacgcc gcgtgagtga tgaaggtttt 420
cggatcgtaa agctctgttg ttagggaaga acaagtgccg ttcaaatagg gcggcacctt 480
gacggtacct aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg taatacgtag 540
gtggcaagcg ttgtccggaa ttattgggcg taaagggctc gcaggcggtt tcttaagtct 600
gatgtgaaag cccccggctc aaccggggag ggtcattgga aactggggaa cttgagtgca 660
gaagaggaga gtggaattcc acgtgtagcg gtgaaatgcg tagagatgtg gaggaacacc 720
agtggcgaag gcgactctct ggtctgtaac tgacgctgag gagcgaaagc gtggggagcg 780
aacaggatta gataccctgg tagtccacgc cgtaaacgat gagtgctaag tgttaggggg 840
tttccgcccc ttagtgctgc agctaacgca ttaagcactc cgcctgggga gtacggtcgc 900
aagactgaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa 960
ttcgaagcaa cgcgaagaac cttaccaggt cttgacatcc tctgacaatc ctagagatag 1020
gacgtcccct tcgggggcag agtgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg 1080
agatgttggg ttaagtcccg caacgagcgc aacccttgat cttagttgcc agcattcagt 1140
tgggcactct aaggtgactg ccggtgacaa accggaggaa ggtggggatg acgtcaaatc 1200
atcatgcccc ttatgacctg ggctacacac gtgctacaat gggcagaaca aagggcagcg 1260
aaaccgcgag gttaagccaa tcccacaaat ctgttctcag ttcggatcgc agtctgcaac 1320
tcgactgcgt gaagctggaa tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt 1380
tcccgggcct tgtacacacc gcccgtcaca ccacgagagt ttgtaacacc cgaagtcggt 1440
gaggtaacct ttatggagcc agccgccgaa ggtgggacag atgattgggg tgaagtcgta 1500
acaaggta 1508
Claims (2)
1. Bacillus siamensis: (Bacillus siamensis) Use of a2025 for degrading emetic toxin in agricultural products;
wherein, the Siamese bacillus (B), (B) and (C)Bacillus siamensis) A2025 was deposited at 12.04.2018 in: china general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC NO:15611 and an address of: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North.
2. A method of detoxifying vomitoxin, comprising: containing Siamese bacillus (B), (B) and (C)Bacillus siamensis) Mixing the microbial preparation A2025 with vomitoxin-containing sample at a ratio of 1% -10%, and incubating at 37 deg.C for 24 hr;
wherein the content of the first and second substances,said Siamese Bacillus (B.), (C. and (C.)) and (C.), (C.) aBacillus siamensis) A2025 was deposited at 12.04.2018 in: china general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC NO:15611 and an address of: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North.
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| EP3024329A1 (en) * | 2013-07-24 | 2016-06-01 | Bayer CropScience Aktiengesellschaft | Binary fungicidal composition |
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