Isolation Of Aflatoxin B1-degrading Strains In Distilled Grains And Study Of The Fermentation Process

Aflatoxin B1 (AFB1) is a kind of fungal secondary metabolites which is highly toxic and carcinogenic. It can contaminate grain, feed, spices and so on depending on weather conditions, which caused great economic losses because of the larde range of pollution. Distilled grains is a kind of high quality animal feed, but improper storage or for too long will cause AFB1 contamination. There are many methods currently to detect AFB1 in which HPLC is considered the most stable, reliable, sophisticated method. This paper established a HPLC method for the detectoin of AFB1 in distilled grains, screened out 7 strains which have AFB1 degradation capability, and initially applied to the AFB1 degradation in distilled grains. The main results are as follows:The chromatographic condition of AFB1 detected by HPLC were determined: fluorescence detector wavelength:λex= 360 nm, λem= 440 nm, the mobile phase is methanol-acetonitrile-water, three-phase ratio is:methanol:acetonitrile:water (v/v)= 10:30:60, flowing rate is 0.6 mL/min, column temperature is 30 ℃, the injection volume is 20 μL. Under this chromatographic condition, the obtained standard peaks is sharp and â†'symmetrical, the response value is higher, and the analysis time is 22 min. From the concentraton of 10 μg/L to 10000 μg/L, the linearity is good, the correlation coefficient is 1.0000. The LOD is 1.0 μg/L calculated by three times the signal to noise ratio.The HPLC Method to detect the AFB1 in distilled grains were established:Putting 20 g of distilled grains powder into 100 mL 60% methanol extract solution in a 250 mL erlenmeyer flask, adding 4 g NaCl, shaking 30 min in a shaker, filtered, adjusting pH to 7, taking 10 mL filtrate and diluted with 10 mL of distilled water.Pass the diluent from the immunoaffinity column, respectively wash it with 10 mL PBS-T buffer and 10 mL distilled water, and finally elute AFB1 from the immunoaffinity column with 1 mL methanol. Pass it through the 0.22μm membrane, testing by HPLC. Spiked recovery experiments proved that the recoveries of this method are higher than 80% with high spiking level,which is opposite with low spiking level.Screened out 7 strains which were capable of degrading AFB1 and degradation rate are all over 70%. The highest degradation rate is 4# which can reach 93.1%.The identification result is:seven bacteria are Staphylococcus warneri, Bacillus methylotrophicus, Brevibacterium sp. MN3-3, Bacillus sp. Dca24 and Arthrobacter sp. 3MW2b. Applied the four strains whose degradation rate are above 80% to the distilled grains,6# has the best AFB1 degradation ability. It’s best degradation condition is:pH=7,culturing temperature is 30℃, culturing time is 72h, the degradation rate is 52.83%. This is the first time that appling the AFB1-degrading microorganism to the distilled grains, and the AFB1 degrading effect is good.