| Aflatoxin(AFT)is a natural pollutant with strong toxicity and high carcinogenicity.It is easy to contaminate crops such as corn,peanuts as well as their processed food and feed,posing a huge threat to human and animal health.A technology that controls aflatoxin pollution with high efficiency,strong specificity and no pollution to feed and environment is in urgent need.This study aimed to screen out strains that can efficiently degrade aflatoxin B1(AFB1),optimize its culture conditions,and analyze the active components of AFB1 degradation produced by the strains.The research contents included the following aspects:Experiment 1:Screening and identification of aflatoxin B1 degrading strainsUsing coumarin as the sole carbon source,we initially screened 24 strains that can grow well on the primary screening medium from the collected samples and laboratory strains,and further screened out 12 strains that can degrade AFB1.Among them,strain Y1-B1 had the highest degradation rate of AFB1,reaching 73.18%.Through morphological observation,physiological and biochemical tests and 16S rRNA sequence alignment analysis,the strain Y1-B1 was identified as Bacillus amylolyticus.Experiment 2:Optimization of culture conditions of Bacillus amyloliquefaciens Yl-B1Taking the degradation rate of AFB1 as the indicator,LB medium was selected as the basic medium of strain Y1-B1.The fermentation conditions of strain Y1-B1 were optimized as follows:strain inoculation volume 2%,liquid fermentation medium volume 25 mL,rotation speed 180 r/min,initial medium pH 7.5,fermentation time 72 h,fermentation temperature 30℃.After optimization,the degradation rate of AFB1 increased from 76.14%to 85.96%.The degradation efficiency of AFB1 by strain Y1-B1 increased gradually with the extension of the degradation time,and stopped increasing after 72 h.Experiment 3:Analysis of active products produced by Bacillus amyloliquefaciens for AFB1 degradationBased on the optimization of the culture conditions of Bacillus amylobacillus Y1-B1,the active substances of AFB1 degradation were analyzed.The supernatant of cell culture of strain Yl-B1 showed strong AFB1 degradation activity,and its degradation rate of AFB1 was 87.8%.The active AFB1 degrading components in the supernatant of cell culture had good stability and strong resistance to high temperature and ultraviolet radiation treatment.Three active substances with high AFB1 degradation ability were separated from the supernatant of cell culture by acid precipitation,methanol extraction and reverse-high performance liquid chromatography(RP-HPLC).Combined with PCR identification and liquid chromatography-mass spectrometry system identification results,the three substances were identified as iturin family lipopeptide homologues.After the degradation of AFB1 by the supernatant of cell culture,the toxic effect of AFB1 on LMH cell was significantly decreased,and the effect of apoptosis was weakened. |
