Optimization Of Degradation Ability And Degradation Mechanism Of Zearalenone By Aspergillus Niger FS10

Zearalenone（Zearalenone,ZEN）is a mycotoxin that exists in food crops,and its pollution range is very wide,mainly polluting common food crops such as wheat and corn.After food crops are contaminated by ZEN,they will no longer be able to be eaten,resulting in a large amount of food waste.In addition,since the estrogen-like toxicity of ZEN can seriously endanger the health of humans and animals,the establishment of a ZEN removal and degradation method that can be widely used in food crops has become a research hotspot in the field of food safety.Although many researchers have proposed a variety of ZEN degradation methods,such as physical degradation and chemical degradation,there are problems such as low degradation efficiency or questionable degradation safety.Therefore,on the basis of the known Aspergillus niger strain FS10 that can be used for ZEN degradation,this paper improved the ability of the strain to degrade ZEN by stress method,purified and identified an extracellular enzyme FSZ with ZEN degradation effect,and explored the Two main degradation products were proposed for its degradation mechanism.The in vitro toxicity was analyzed according to the cytotoxicity experiment,which provided a theoretical basis for the efficient and safe degradation of ZEN.The main research contents of this paper can be summarized into the following three points:1.The optimization and metabolic mechanism of ZEN degradation ability of Aspergillus niger FS10.ZEN（1.0μg/m L）was used to stress Aspergillus niger FS10 for 24 hours,and the stress cycle was repeated 5 times.The degradation rate of ZEN/m L reached 96.00%,which was about 11.44%higher than that of the original strain FS10（84.56%）.At the same time,according to the 18s r DNA sequencing results,there was no significant change at the gene level before and after stress.According to the analysis of the metabolomics data of the strains before and after stress,it was found that the difference in the degradation ability of the two strains was that the amino acid synthesis represented by DL-asparagine and the sugar metabolism represented by melibiose were significantly up-regulated.This led to an improvement in the ability of the strain to metabolize ZEN.This may be a factor that promotes the degradation rate of ZEN-S-FS10.In the investigation of the properties of the strain ZEN-S-FS10 after stress,it was found that the degradation ability of ZEN-S-FS10 to ZEN remained between 95%and 97%after multiple passages,and it could exist stably.And when the spore concentration was 10⁴CFU/m L to degrade 1.0μg/m L ZEN,the degradation rate in 28h could reach more than 96.00%.2.Identification and structural information analysis of ZEN-degrading enzymes of Aspergillus niger ZEN-S-FS10 after stress.By exploring the degradation site of ZEN-S-FS10,it was found that the main site with ZEN degradation ability was the extracellular enzyme secreted during the growth process,and the extracellular enzyme liquid was separated and purified by using（?）KTA pure protein purification system,obtained an Aspergillus niger extracellular enzyme FSZ with the ability to degrade ZEN,and the molecular weight of the enzyme was 62.4 k Da.The related properties of FSZ were also explored.FSZ has a degradation rate of about 80%to 1.0μg/m L ZEN in the temperature range of 28-38°C.In addition,the degradation rate of ZEN by FSZ was not affected when the culture p H≤7.Moreover,the presence of metal ions such as Cd²⁺and Mn²⁺also affects the degradation of ZEN by FSZ.However,the presence of Ca²⁺can improve the degradation rate of ZEN（ZEN degradation rate>80%）.The kinetic parameters of the enzyme were found to be V_max=6.52μg·m L^-1·h^-1and K_m=0.85μg/m L,which were better than known ZEN-degrading enzymes.And the three-dimensional structure of FSZ protein was predicted,and the molecular docking between FSZ and ZEN was carried out,and 3 binding sites（PHE307,THR55,GLU129）were found to bind to ZEN,forming a triangular binding domain,so that ZEN and FSZ could chemically interact.combine.3.Identification of ZEN degradation products and their in vitro toxicity studies.The extracellular crude enzyme solution of ZEN-S-FS10 and the ZEN degradation products of purified FSZ were identified by high-resolution UPLC-MSMS,and a major ZEN degradation product was found:product A:C₁₈H₂₆O₄,relative molecular weight 306.18 g/mo L,a major low-toxicity transformation product:Product B:Zearalenone 4-sulfate,(C₁₈H₂₂O₈S),relative molecular weight 398.4 g/mo L;the in vitro toxicity of ZEN and its products was evaluated by HepG2 cells,and it was found that ZEN invaded Under staining,it is difficult for the cells to maintain the normal shape,but after the product is treated,the cell shape tends to be normal.And under ZEN treatment,the cell viability remained at a low level,while the cell viability in the product-treated group was significantly higher than that in the ZEN-treated group.In addition,we also found that the cell viability of ZEN product was significantly higher than that of ZEN standard product under different concentrations of ZEN and ZEN product treatment,and for different time of cell treatment,so the in vitro toxicity of ZEN product was significantly lower than that of ZEN standard product.In summary,by improving the degradation ability of FS10,we obtained a degrading enzyme that can be used for the degradation of ZEN,and verified the structure and toxicity of its degradation group products.This provides a new idea for the safe degradation of ZEN in food.