| Zearalenone(ZEN),also known as F-2 toxin,is an estrogenic mycotoxin produced by Fusarium graminearum,Fusarium equiseti,Fusarium roseum and Fusarium moniliforme.ZEN is stable and does not degrade under storage,processing and cooking at high temperatures.It could be absorbed through food and harm human and animal health.The detoxification of ZEN thus is important.At present,the commonly used physical and chemical detoxification have obvious defects,when they remove toxins,nutrients in grains are also deceased or changed.Therefore,many researchers believe that the best method for detoxification of ZEN is biological detoxification.In this dissertation,a highly efficient degrading strain-7D3-2 was isolated by enrichment and cultivation methods from the long-term polluted wheat field by Fusarium toxin.It was identified as Bacillus amyloliquefaciens and the growth characteristics,degradation characteristics and degradation mechanism were studied.This paper is divided into four parts.The first part describes the screening and identification of ZEN-degrading bacteria.A highly efficient degrading bacteria strain 7D3-2 was isolated from the long-term polluted wheat field by Fusarium toxin.Through the comprehensive analysis of the strain,s growth morphology,physiological and biochemical characteristics and phylogenetic tree of 16S rDNA gene and gyrA gene,it is finally determined as Bacillus amyloliquefaciens.In the second part,we studied the effects of various environmental conditions on the growth of 7D3-2 and the degradation properties of ZEN,to determine the best conditions for 7D3-2 growth and ZEN degradation,the main influencing factors including culture temperature,initial pH value,inoculum size,liquid volume were studied.The results showed that the optimal growth conditions for 7D3-2 degrading bacteria were 30?,pH 7.0,NaCl concentration 10.0 g/L,and good ventilation.When the temperature is 30?,the pH is 7.0,and the inoculation amount is greater than 10%.,the degradation efficiency of ZEN is higher.And when the concentration of ZEN is less than 25.0 mg/L,the degradation rate is faster,but the absolute degradation rate of ZEN increases with the increase of the initial concentration of ZEN.In the third part,preliminary studies were conducted on the degrading enzymes in the degrading bacteria 7D3-2.The active degradation material was obtained by precipitation with ammonium sulfate,and the enzymatic properties of the crude extract with the most active part were studied.The results showed that the most active part of the degrading enzyme were precipitated when the ammonium sulfate saturation was 60%.The crude enzyme fluid extracted from bacteria 7D3-2 can exert its maximum enzyme activity under the conditions of 30? and pH 7.0.The enzyme can maintain more than 80%of the activity in the range of 20?40? and pH of 6?9.Most metal ions promote degradative enzyme activity at concentrations from 0.2 mmol/L to 2 mmo/L,and EDTA inhibits the enzyme activity.The localization of the enzyme is extracellular.The last part is a preliminary study of the metabolic pathways of degrading bacteria and the cloning of degradation genes.ZEN formed the intermediate compound 1-(3,5-dihydroxyphenyl)-6'-hydroxy-1'-undecene-10'-one under the degradation by 7D3-2,it is speculated that during the degradation of ZEN by 7D3-2,it undergoes cleavage of the lactone ring,followed by a decarboxylation reaction,demonstrates that esterase catalysis occurs during the degradation of ZEN.Using the shuttle vector pMarA carrying the transposon TnYLB-1 to electroporate into the zearalenone degrading bacteria 7D3-2,under the condition of 50 ? mutagenesis,the transposon TnYLB-1 on the shuttle vector pMarA was randomly inserted into the genome of the strain,a mutant library of strain 7D3-2 was constructed.The ZEN degradation performance of the mutants in the library was tested.At present,no effective mutations in the degradation function of ZEN has been found. |
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