Screening Research Of Zearalenone–degrading Bacillus

Zearalenone（ZEN） is a nonsteroid oestrogenic mycotoxin produced by Fusarium, which is distributed widely in the nature. People and animals could get infected through ZEN contaminated grains. Now taking advantage of microorganisms for detoxification of ZEN is the research hotspot. In this study, ZEN-degrading strains were screened from 617 strains of Bacillus using the co-culture with ZEN, through the high throughput microculture method. Then the obtained strains were identified, and their ZEN degradation characteristics were studied, and a Fosmid library of one ZEN-degrading Bacillus was constructed. The conclusions of this study were described as below.（1） Twelve Bacillus capable of removing 10.0 μg/m L ZEN were screened from 617 strains by high throughput microculture method and co-culture with ZEN. Then four Bacillus which could elimilate 15.0 μg/m L of ZEN were obtained from the twelve strains.（2） These four strains were analyzed through morphological observation and 16 S rDNA sequences. Strain T-246 was identified as Bacillus stratosphericus; T-420 was Bacillus safensis; 373-2 was a new Bacillus species and 411-1 was Bacillus subtilis.（3） Strain T-246, T-420, 373-2 and 411-1 could eliminate 15.0 μg/mL ZEN completely within 6 h, 9 h, 8 h and 6 h respectively. Moreover the strains were able to degrade and bind ZEN. Native PAGE of extracellular extracts from four Bacillus demonstrated that the main effect of ZEN scavenging was due to the degradation activity from exoenzyme.（4） Effect of external factors on ZEN degradation efficiency were investigated. Results showed that the optimum pH range for ZEN degradation of strain T-246 was 7.0-8.0. ZEN-degrading efficiency could be inhibited by low concentration of EDTA, and in the range of 0.5-2.0 mM, degradation efficiency could be also inhibited by Ni²⁺, Zn²⁺ and Co²⁺ with the effect of Zn²⁺>Co²⁺>Ni²⁺, while Fe²⁺, Cu²⁺ and Mn²⁺ treatment made no difference in the degradation efficiency of strain T-246. At the same time ZEN-degrading efficiency of strain T-246 was improved by ZEN induction. Otherwise, ZEN-degrading efficiency was improved after that the strain T-246 were inducd by ZEN.（5） The Fosmid library of strain T-246 was constructed with an average insert fragement of 38 kb; The inserting rate of exogenous fragment on recombinant plasmid was 100% and library titer was 4.6×105 CFU/mL. Ten clone pools of 96-well plates were maded with 70-100 clone in each well.