| Ochratoxin A(OTA)is mainly produced by fungi such as Penicillium genus and Aspergillus genus,and is one of the most important mycotoxin contaminants in grapes and their products,which caused great threat to humans and animals.Therefore,it is particularly important to seek a strategy to control OTA in grapes and their products.The use of antagonistic yeasts to control and degrade the OTA in grapes and their products have gradually drawn the attention of researchers at home and abroad for the advantages of safety and high efficiency and so on.However,the mechanisms involved in OTA degradation,especially the molecular mechanisms are not yet clear and further research is needed.A strain of antagonistic yeast(Yarrowia lipolytica Y-2)which was isolated from the grapes of orchard by our research team,has good control efficacy on postharvest diseases of grapes caused by Talaromyces rugulosus O1,and could degrade OTA in vitro.Based on experimental results,it was speculated that OTA degradation by Y.lipolytica Y-2 is attributed to the action of intracellular enzyme.However,the control efficacy of Y.lipolytica Y-2 on OTA accumulation in the wounds of different varieties of grapes and the molecular mechanisms of OTA degradation are still unclear.In this thesis,the biocontrol efficacy of Y.lipolytica Y-2 on OTA accumulation in the wounds of different varieties of grapes and the role of intracellular enzymes of Y.lipolytica Y-2 in OTA degradation were investigated.Then,the proteins in the intracellular enzymes of Y.lipolytica Y-2 precipitated with 55-65% saturated ammonium sulfate were identified and analyzed.Furthermore,degradation products were identified and the toxicity of products were studied,to reveal the mechanisms involved in OTA degradation by Y.lipolytica Y-2.The main results of the paper are as follows:1.The control efficacy of Y.lipolytica Y-2 on OTA accumulation in the wounds of different varieties of grapes were studied in vivo.The results showed that Y.lipolytica Y-2 significantly inhibited the blue mold decay caused by T.rugulosus O1 in different varieties of grapes,thereby reducing the content of OTA in the wound of different varieties of grapes.Additionally,the biocontrol efficacy increased enhancement of yeast concentrations.Y.lipolytica Y-2 has a significant control effect on OTA accumulation in the wounds of grapes during storage.2.The degradation efficacy of intracellular enzymes of Y.lipolytica Y-2 on OTA in vitro was studied.The results showed that the intracellular enzymes of Y.lipolytica Y-2 cultured with OTA or not were both able to degrade OTA.The degradation rate of the fraction precipitated with 55%-65% saturation of ammonium sulfate was the highest,and it was up to 91% at 2 h.3.The proteins in the intracellular enzymes of Y.lipolytica Y-2 and Y.lipolytica Polh(control,without the ability to degrade OTA)precipitated with 55%-65% saturated of ammonium sulfate were identified by LC-MS/MS and comparatively analyzed with bioinformatics technology.It was speculated that carboxypeptidase in Y.lipolytica Y-2 is directly involved in the degradation of OTA.In addition,energy metabolism related proteins such as succinyl-CoA ligase,adenosine triphosphate synthase,oxidative stress response related proteins such as peroxidase,Cu/Zn-superoxide dismutase,flavoprotein,glutathione,glutathione synthetase,glutathione-S-transferase,glutathione peroxidase were also play an important role in response to oxidative stress caused by OTA.4.The degradation products of OTA by the intracellular enzymes of Y.lipolytica Y-2 were identified by the thin layer chromatographic(TLC),high performance liquid chromatography equipped with fluorescence detector(HPLC-FLD)and high performance liquid chromatography coupled to photodiode array and mass spectrometry detectors(HPLC-PDA-MS),and the toxicity of the degradation products to HEK293 cells was also studied.The results showed that the degradation product by the intracellular enzymes of Y.lipolytica Y-2 was OTα,which had a lower toxicity effect on HEK293 cells than OTA. |
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