Study On A Zearalenone-Degrading Bacterium

Zearalenone (ZEN) is one of the major mycotoxin in foods and feeds. ZEN is a mycotoxin produced mainly by fungi belonging to the genus Fusarium, and a nonsteroidal oestrogenic mycotoxin. Foods and feeds contaminated with ZEN cause seriously economic losses in livestock and threat to human safe and health. Based on the pollution and harm of ZEN, how to control ZEN contamination in foods and feeds is an urgent problem. Currently ZEN reduction techniques can be divided into physical, chemical and biological methods. Although physical and chemical methods can remove part of ZEN, but there are inevitable shortcomings such as:destruction of nutrients, unknown the toxicity of decomposition product of ZEN, secondary pollution. Using of microbial metabolism will degradate ZEN to non-toxic small molecules is a kind of clean and no secondary pollution reduction method. The aim of this study is isolation a bacterium which can degrades ZEN efficiency. Identification, degradation characteristics, degradation mechanism were investigated and analyzed. Degrading gene of Zearalenone was investigated by using Tn5 transposon.In this study, with ZEN as the sole carbon source, the bacteria ASAG16, ASAG17 isolated from methanogenic sewage sludge and mire by enrichment culture degrade ZEN efficiently. They were identified as Pseudomonas citronelloli and Pseudomonas stutzeri respectively with physiological detection and 16S rDNA sequence analysis. ASAG16 was further studied as its better degradation.The factors influencing degradation efficiency of ASAG16 was further evaluated. ASAG16 degrades 91.59% ZEN (20 mg·L-1) in LB medium incubated for 6 days whose degradation efficiency reach the peak. At prophase of cultivation, the degradation rate is rapidly increasing with growth of time, but the degradation stay on a certain level at anaphase of cultivation. In LB medium of initial pH 4, the ZEN removal by ASAG 16 was 20.83%, while initial pH 5-9, the removals were all above75%, which indicates the degradation efficiency increased with the rise of pH in the midium. Mg2+, Ca2+and Mn2+ have less influence on the degradation of ZEN, whereas Cu2+, Zn2+, Co2+, Ba2+and EDTA reduced significantly.The degradation rate of ZEN was 12.06% and 47.66% by cell culture supernatant and bacteria cracked solution of ASAG 16, respectively. But inactivated supernatant almost have no degradation effect. It was proved that the decrease of ZEN is not the adsorption of bacteria, but rather ASAG16 can secrete active substance that can degrade ZEN. Mass spectrometric detection showed that new substances m/z=319.1547 and m/z=325.070 were produced with decreasing ZEN. The second order fragment ions of ZEN and m/z=319.1547 are similar, proved that m/z=319.1547 was one metabolites of ZEN. It was speculated that two H were added to ZEN, hydrogenation of carboxide or Carbon-Carbon Double Bond.Tn5-based transposon mutagenesis to Pseudomonas citronelloli ASAG16 was performed. Approximately 15000 kanamycin-resistant transposon insertion mutants were obtained. A mutant reduced the ability to degrade ZEN was screened, and named zen-1. The degradation rate of zen-1 decreased 30% than wild strain. Target genomic DNA with origin of replication p15A was digested with restriction enzyme digestion Sau 3A I, self-ligated, transformed to clone flanking Sequences of Tn5. The flanking Sequences were about degrading gene. Literature searches in NCBI, the sequence homologous rates of flanking Sequences and Nitric oxide reductase transcription regulator is 89%.