Application of non-toxigenic Aspergillus flavus in degrading aflatoxin
A technology of aflatoxin and Aspergillus flavus, applied in the field of microorganisms, can solve the problems of unseen research and reports on Aspergillus aflatoxin, and achieve high-efficiency degradation and good application prospects
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Embodiment 1
[0034] Embodiment 1, the collection, separation and identification of non-toxin-producing Aspergillus flavus
[0035] 1. Isolation of Aspergillus flavus strain GZ-17 from peanut planting soil
[0036] Isolation of Aspergillus flavus strains from Guangdong peanut planting soil with DG18 medium. The specific operation is as follows:
[0037] 1. Preparation of soil sample bacterial suspension
[0038] Take 10g soil sample, add 90mL 0.1% peptone sterile water (w / v), shake at room temperature for 30min, and make 10 -1 bacterial suspension; take 0.5mL10 -1 Add 4.5mL0.1% peptone sterile water to the bacterial suspension to prepare 10 -2 Dilution bacterial suspension; prepare 10 as above -3 Dilution bacterial suspension.
[0039] 2. Isolation and purification of strains
[0040] Take 0.1mL bacterial solution for each dilution, smear it on DG18 medium, incubate in the dark at 30°C for 5 days, repeat each dilution 3 times, pick the strains with yellow spores, and perform secondar...
Embodiment 2
[0067] Embodiment 2, non-toxin-producing Aspergillus flavus GZ-17 to aflatoxin B 1 Analysis of degradability
[0068] 1. Aflatoxin B 1 Preparation of Standards
[0069] 1 mg aflatoxin B 1 (AFB 1 ) was dissolved in 2mL of chromatographically pure methanol to obtain a concentration of aflatoxin B of 500ppm 1 solution.
[0070] 2. Culture of strains
[0071] Inoculate non-toxigenic Aspergillus flavus GZ-17 on MEA slant test tube culture medium, culture at 28°C for 5 days, then use cotton swab to dip the spores on the medium into sterile 0.1% Tween-80, shake with a shaker Evenly, and then adjust the spore concentration to 1×10 by using a hemocytometer 6 cfu / mL.
[0072] 3. Non-toxin-producing Aspergillus flavus GZ-17 to aflatoxin B 1 Degradation
[0073] Take the spore liquid of strain GZ-17 (1×10 6 cfu / mL) 1ml into sterilized 50mL PDB medium, add filter-sterilized aflatoxin to make the concentration in the culture solution 500ppb, mix thoroughly and culture in a shaker...
Embodiment 3
[0080] Embodiment 3, non-toxin-producing Aspergillus flavus GZ-17 degrades aflatoxin B 1 product analysis
[0081] Analysis of Aflatoxin B by LC-qTOF / MS 1 degradation products. Aflatoxin B 1 The degradation products are first separated by a separation column, and the separated components are ionized by an MS ion source; after passing through the first-stage mass spectrometer, they reach the TOF through mass spectrometry analysis without CID, and the CID mode is used for MS / MS analysis, and the parent ion is extracted in the CID Cleavage to get the cleavage path.
[0082] LC detection conditions are mobile phase acetonitrile (chromatographic grade): 0.1% formic acid aqueous solution = 70:30, ultrasonic degassing for 5 minutes after ultrafiltration; flow rate: 0.4mL / min, time 12 minutes; Agilent enhanced C 18 Chromatographic column (2.1 mm×150 mm, 5 μm); injection volume: 2 μL.
[0083] Mass spectrometry conditions: compound analysis mode is positive ion mode; capillary and...
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