Degradation Mechanism And Safety Evaluation Of Deoxynivalenol By Ozone

Deoxynivalenol is one of the most prevalent type B trichothecenes,which is mainly produced by Fusarium graminearum and Fusarium culmorum.It has an acute toxicity,teratogenesis,mutagenesis and other toxic effects on human and test animals.It is usually found in wheat,corn,barley and other crops,especially in wheat and its products.It serious threats the health of consumers and affects the development of wheat processing industry.In recent years,ozone technology has been widely used to degrade mycotoxins,however,variety of studies on the degradation of DON only focus on the degradation effect.In this study,the factors affecting the ozone degradation of DON in wheat bran were explored,and analysed the kinetics of DON degradation.At the same time,ozone was used to degrade DON in pure water model system,and the ozone degradation products of DON in the model system were determined by UPLC-QTOF-MS/MS,meanwhile the ozonolysis pathway was also elucidated.Finally,the cytotoxicity experiment of DON before and after being detoxified by ozone was evaluated.These results would provide theoretical basis and technical support for the application of ozone technology in removing DON in wheat flour and grain-based food products.The main results are as followed:?1?Factors influencing the ozone degradation of DON and condition optimizationThe effects of wheat bran moisture content,ozone concentration,ozone treatment time and ozone state on the degradation of DON were studied.The optimized treatment condition of DON degradation was 40%moisture content of wheat bran,14 g/m³ of ozone gas concentration and 180 min of ozone exposure time,on this treatment condition,the DON concentration evidently decreased from 2137.96?g/kg for the control to 508.13?g/kg for the one treated with ozone for 180 min,the degradation rate reached 76.23%.The degradation kinetics of DON in wheat bran results showed that the moisture content,ozone treatment time,ozone concentration and ozone aqueous solution treatment time were all coincided with the pseudo-first order kinetics models.Among the four factors,the degradation rate constant of DON was the highest in ozone concentration,it indicated that ozone concentration had the greatest effect on the ozone degradation efficiency of DON.?2?Kinetics of ozone degradation of DON in pure water systemOzone could effectively degrade DON solutions,DON was treated by ozone at a concentration of 10.84 g/m³ and a flow rate of 80 mL/min for the times ranging from 0-9 min.With the increased of ozone exposure time,the concentration of DON was significantly decreased?P<0.05?.DON concentration was reduced rapidly from 51.11 mg/L to 14.97 mg/L within 3 min of ozone exposure,and reduced by 70.60%.At the 6 min of exposure time,the degradation rate of DON reached to 92.64%,and DON was degraded at 9 min with 98.30%of degradation rate.The ozonolysis of DON followed the first-order kinetic model?y=0.456x-0.0664?with a correlation coefficient?R²?of 0.9983.?3?Ozone degradation products and pathway of DON in model systemIn the model system,DON standard solution(C₁₅H₂₀O₆,m/z=297.1333)was treated by ozone,based on the separation and analysis of HPLC-QTOF/MS/MS,four products were produced during the ozone degradation of DON,i.e.Product 1,2,3 and 4.We deduced the molecular formula and the mass-to-nuclear ratio of the four products,i.e.(C₁₅H₁₈O₇,m/z=311.1129),(C₁₃H₁₄O₁₀,m/z=331.1022),(C₁₃H₁₄O₁₁,m/z=347.0611)and(C₁₃H₁₄O₁₂,m/z=363.0555).Their molecular structures were very similar to that of DON.The ozonolysis of DON abides by the Criegee mechanism,DON(C₁₅H₂₀O₆,m/z=297.1333)was degraded by ozone,the double bond at the at the C9-C10 was firstly oxidized to 1,2,3-trioxolane(C₁₅H₂₀O₉),and then it transformed to a carbonyl compound spontaneously.The carbonyl compound of DON was oxidized to 1,2,4-trioxolane(C₁₅H₂₀O₉)by 1,3-dipolar cycloaddition.1,2,4-trioxolane of DON continued to be oxidized to aldehyde/ketone of DON by ozone.After the Criegee reaction of DON,the hydroxyl group at the C7 and the 12,13-epoxide ring of aldehyde/ketone of DON were transformed to the carbonyl group and double bond,respectively,and formed the product 1(C₁₅H₁₈O₇,m/z=311.1129).The aldehyde group at the C10 of product 1 was oxidized to carboxyl group and formed intermediate C₁₅H₁₈O₈,the intermediate(C₁₅H₁₈O₈)by releasing formic acid?HCOOH?to form the product 2(C₁₃H₁₄O₁₀,m/z=331.1022).The product 2 was transformed to the product 3(C₁₃H₁₄O₁₁,m/z=347.0611)through an intermediate(C₁₃H₁₄O₁₀).The single bond between O1 and C11 at the role of ozone to form the product 4(C₁₃H₁₄O₁₂,m/z=363.0555)via an intermediate(C₁₃H₁₄O₁₁).?4?Safety evaluation of DON after ozone detoxificationHepG2 and Hela cells were exposed to DON medium with different concentrations ranging from 0 to 4.0 mg/L and from 0 to 3.0 mg/L,The IC₅₀0 values of HepG2 and Hela cells were 2.10 mg/L and 1.33 mg/L after 48 h of exposure,respectively,and presented a dose-dependent relation between them.The ozone exposure significantly reduced the cytotoxicity of DON on Hela and HepG2 cells?P<0.05?,and it was dependent on the ozone exposure time.For HepG2 cells,the cell viability evidently increased from 53.95%for the control without ozone treatment to 96.75%for the one treated with ozone for 15 min?P<0.05?.Similarly,for Hela cells,the cell viability rapidly increased from 37.86%for the control without ozone treatment to 91.31%?P<0.05?for the one treated with ozone for 15 min.There were no obvious differences in the cell viability between the negative control?100%,without DON exposure?the one with DON treatment after ozone exposure for 20 min?P>0.05?.Cell morphology observation indicated that with the increased of ozone treatment time,the morphology and number of Hela and HepG2 cells tended to be normal,the toxicity of DON to Hela and HepG2cells was decreased and the effect on cell growth was significantly reduced.