| Zearalenone（Zearalenone）,also known as F-2 toxin,is a common mycotoxin in cereals and has significant toxic effects on humans and animals.The rapid and accurate detection of F-2 toxin in food and raw materials is of great significance to effectively control the harm of F-2 toxin.The whole process of prevention and control of toxins is also of positive significance.In this study,in view of the complex interference of food matrix and difficult sample preparation in the field of F-2 toxin detection,the preparation of F-2 toxin immunoaffinity chromatography column was carried out,in order to provide efficient detection of F-2 toxin.On the basis of effective sample pretreatment methods,a preliminary study on the degradation and transformation of F-2 toxin under typical pasta food processing conditions such as heat treatment,extrusion puffing,and enzyme treatment was carried out.The main research contents and results are as follows:1)The F-2 toxin whole antigen was prepared by chemical synthesis,and Balb/c mice were immunized.According to the polyclonal antibody serum ELISA test results,the mice with the best immune effect were selected for cell fusion.Based on hybridoma cell fusion technology and subcloning,four hybridoma cells that can specifically secrete F-2 toxin monoclonal antibody were obtained,named8H6-A4-E5-E12,8H6-H3-E5-A11,8H6-D5-A4-A11-H12 and 8H6-D5-A4-A11-E11-F1.8H6-D5-A4-A11-E11-F1 cells were selected for antibody ascites preparation.After purification by octanoic acid-ammonium sulfate precipitation method,the titer of the antibody measured by indirect and indirect competitive ELISA was 1:64000,and the IC50 value was 0.489 ng/m L,the prepared F-2 toxin monoclonal antibody does not cross-react with other mycotoxins.2)The ascites fluid of the homemade F-2 toxin monoclonal antibody was purified by the ammonium octanoate sulfate method,and the purified F-2 toxin monoclonal antibody was coupled with hydrogen bromide-activated agarose gel,blocked,and packed into a column.Coupling conditions p H,loading buffer and methanol concentration in elution buffer were calculated.The results showed that when the p H of the coupling buffer was 8.4,the coupling rate of the antibody was89%.When the methanol concentrations in the loading buffer and the elution buffer were 10%and 100%,the recovery rates of F-2 toxin were 89%,86%.The performance of the prepared immunoaffinity chromatography column was evaluated by the established UPLC-Qq Q-MS/MS detection method.Loading conditions:p H=7.0,flow rate:2.0 m L/min,elution conditions:100%methanol solution,flow rate:2.0 m L/min,the column capacity of the immunoaffinity chromatography column is1278 ng,and the number of times of use is Second,when the spiked amount was between 50～100 ng,the average recovery of the immunoaffinity chromatography column was 87%.3)A preliminary study on the degradation of F-2 toxin under typical pasta food processing conditions was carried out.The effects of temperature and extrusion mechanical treatment conditions on F-2 toxin were studied.The results of HPLC showed that the degradation rates of F-2 toxin were 15.27±0.95%,15.27±0.95%and 46.55±1.23%and 52.58±0.43%.The F-2 toxin was added to millet and black rice for extrusion treatment,and the results showed that the degradation rates of F-2toxin were 34.27±1.05%and 16.85±0.95%,respectively.The effect of four common enzyme preparations in flour on the degradation of F-2 toxin was also studied,and the results showed thatα-amylase（5000 U）,xylanase（2500 U）,hemicellulase（1000 U）and glucose oxidase The degradation rate of F-2 toxin（0.24U）was 30.93±1.94%,26.21±1.29%,35.85±1.72%and 36.20±108%,respectively.The samples were qualitatively analyzed by UPLC-Q-TOF/MS,and a total of seven new products were found.In addition,the F-2 toxin was treated with edible alkali,and a new degradation product was found at 3 min by HPLC detection,and the possible molecular formula was resolved by high-resolution UPLC-Q-TOF/MS to be C17H24O4,m/z=291.157. |
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