Isolation And Characteristics Of An Aflatoxin B₁-degrading Bacterium

Aflatoxin (AF) is a group of toxic and carcinogenic fungal metabolites predominantly produced by the filamentous fungi Aspergillus flavus and Aspergillus parasiticus. Aflatoxin contamination not only leads to serious economic losses, but also is detrimental to human and animal health. Much attention has been paid to the biodegradation of mycotoxins in recent years, especially using microbes. It was reported that the microorganisms could bioconvert aflatoxin into non-toxicity or low toxicity products under mild conditions and without using chemicals and losing nutritive value. In this thesis, an aflatoxin-degrading strain was screened and identified, and its degradation characteristics were studied. The main results were as follows:1. Samples from various sources were screened by using coumarin as the sole carbon source, which is a structural analog of aflatoxin. The strains that could grow in the medium with coumarin carbon source were detected for their AFB1-degrading ability by addition of AFB1(2.5μg/mL) to the cultured broth. Ten isolates showed good AFB1reduction activity and they could grow well on coumarin carbon source. Strain F4isolated from Budorcas taxicolor feces sample could reduce AFB1by90.03%after incubation at37℃for72h. A method for analysis of AFB1in the cultured broth with HPLC was established. AFB1was extracted from the cultured broth with dichloromethane, separated with RP-C18separation column, and detected at362nm. Under the conditions above, HPLC gave a well-resolved and symmetrical AFB1peak. Peak area values showed good reproducibility with relative standard deviation (RSD) values at2.39%, revealed good degree of accurancy with average recovery rate of AFB1at99.17%as well. The calibration curve for AFB1was calculated by linear least-squares regression. The regression equation was y=67.3368x-8.5095(R2=0.9997) when the initial AFB1concentration in the range0.01～20.00μg/mL. The result indicated that the method described was relatively simple, accurate and reliable for the determination of AFB1in the cultured broth. 2. For investigation of microbial detoxification, however, a reliable method has to be needed to decide whether the toxin is degraded or reversibly bound. In this section, several methods were applied to differentiate the degrading AFB1from adsorbing during the screening of AFB1-degrading microbes. Through comparing the AFB1detoxification differences between living cells and dead cells, analyzing the residual AFB1from microbial cells by aqueous washing and organic solvent extracting, and maintaining a stable pH of medium, we had effectively excluded the biological adsorbing and binding and pH interferences, and distinguished the degradation from the reversible adsorbing. It was demonstrated that F4and F7strains have high AFB1degrading activity.3. Among the positive strains, F4strain with the highest activity was identified according to its morphological, physiological and biochemical properties together with phylogenetic analysis of its16S rRNA sequence. The fragments of1498bp of the partial16S rRNA gene of F4was amplified by PCR and sequenced. The phylogenetic tree was constructed after comparing the16S rRNA gene of strain F4and related taxa. The result indicated that the16S rRNA sequence of F4shared99%identity with Pseudomonas stutzeri (Accession No. JN712254). Hence, F4was preliminary identified to be Pseudomonas stutzeri with morphology, physiological and biochemical properties and16S rRNA gene sequence.4. The factors that affect AFB1degradation, such as bacterial cell concentration, pH, temperature, metal ions, were investigated. It was found that the degrading activity component existed in the cell of F4, and the degrading activity was interrelated with cell concentration, temperature, pH and metal ions. Both pH and temperature had a significant influence on the degrading AFB1by strain F4. Ions Mg2+was an activator for AFB1degradation, increasing by7.68%, however, ion Cu2+was a strong inhibitor, decreasing by51.1%. The results indicated the bioactive component for AFB1degradation was probably an enzyme. Moreover, the F4cell presented the strongest AFB1degradation activity under temperature35℃, pH7.0, and Mg2+concentration1.00mM. Based on the single factor experiments, the reaction condition for P. stutzeri F4was optimized using response surface methodology. The result showed the optimum degradation condition was temperature 35.49℃, pH7.12and Mg2+concentration1.07mM. Under this condition, the AFB1degradation ratio of F4cell reached to97.34%.