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Isolation,Identification and Degradation Mechanism of Zearalenone--Degrading Strain

Author: WangHongJie
Tutor: ShiJianRong
School: Nanjing Agricultural College
Course: Plant Pathology
Keywords: zearalenone purification HPLC detection ZDS degradation
CLC: S432.44
Type: Master's thesis
Year: 2011
Downloads: 19
Quote: 0
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Abstract


Zearalenone (ZEN) also known as F-2toxin is one of estrogen-like fungal toxins by Fusarium spp.It was widespread in moldy seeds of corn, sorghum, wheat, oats, barley and other cereal crops or in milk. Zearalenone affected food safety and hazarded human and animal health while zearalenone was accumulated in human and animal body by food chain. Some physical, chemical and biological means can destroy or reduce its toxicity. As traditional physical and chemical methods are always unable to detoxify the ZEN, biological degradation becomes research hotspot.In this paper, we successfully identified a bacterial isolate which has capability to detoxify ZEN. Then we elucidated the detoxifying capability, detoxifying mechanisms and factors affecting detoxifying efficiency of the bacterial isolate.This study can be divided into four parts. Firstly, zearalenone extraction condition and HPLC detection was optimized for providing pure substrate and optimum detection condition to isolate zearalenone degrading bacteria. The purification conditions were as follows:the samples was extracted by methanol and NaCl solution and further purified by silica gel column, which was eluted by6ml methanol solution. The target compound was assayed by HPLC. The mobile phase was methanol/water at the proportion of80/20(v/v), the flow rate was0.6ml·min-1and the ultraviolet detection was at236nm.The detection limit of this method was5μg·kg-1.The zearalenone concentration and the peak area exhibited linear relationship in the range of1.25～40.00μg·ml-1(r=0.9991), and the recovery rate was96.3%.Secondly, it was screening for bacteria which has capability to detoxify ZEN. One of the obtained bacterial isolates exhibited detoxifying capability. The molecular evidence with PCR-mediated amplification of the16S rDNA, purification and sequencing of the PCR products, it was revealed that the isolate belonged to the genus Bacillus amyloliquefaciens with the help of its morphological, cultural and biochemical characteristics of ZDS. The isolate was named as zearalenone degradation bacterium (ZDS) by our lab.Thirdly, many of factors were studied for the effects of degradation efficiency, such as incubation time, bacterial concentration, temperature, pH value and ZEN analogues. The result demonstrated that the favorable degradation condition was at30℃of temperature, pH7.0,5%of inoculation quantity and180rpm of rotation speed.50ppm zearalenone was completely degradated in a week with this condition. However the highest ZEN concentration was100ppm which can be detoxified.Lastly, the detoxifying mechanism was investigated. The crude degradation substance could be separated from the liquid culture primarily by ammonium sulphate precipitations. The result showed that the active substances had the strongest detoxification activity when the saturation of ammonium sulphate was at40%. The active substance was against heat and sensitive to pH.In summary, ZDS had significantly detoxifying capability of zearalenone. ZDS strains can serve as a potential biological detoxification agent.

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CLC: > Agricultural Sciences > Plant Protection > Pest and Disease Control > Plant diseases and their prevention > Transgression ( pass ) an infectious agent harmful > Fungi
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