Production Patterns, Isolation And Purification Of Masked Deoxynivalenol Mycotoxin In Wheat

In C hina, the cultivated area of wheat rank only second to r ice and remain stable. The cultivated area of wheat, which is the main source of food for more than half of the population, was 24 million hectares and the output was 122 million tons. Therefore, the wheat quality safety is directly related to the wheat products and the safety of people’s lives. When the cereal crops such as wheat, barley are infected by Fusarium graminearum, the grain may be contaminated with several Fusarium mycotoxins. Among them, deoxynivalenol(DON, vomitoxin) is the most common mycoto xins. DON has been evaluated by the Joint FAO/WHO Expert Committee on Food Additives(JECFA) as one of the most dangerous mycotoxins in food natural mycotoxins. DON is deleterious to the healthiness of mankind and livestock. However, when grain is infected by Fusarium mycotoxins, there exists not only free DON but also some masked DON, such as actylation of DON(3ADON, 15ADON) and DON-β-D-glucoside(D3G). These toxins are easy to be undetected in the normal extraction process. But when the toxins enter the digestive tract of person and animal, they will release the toxic precursor and become potentially ha zardous to the health of human and animals. Since the toxicity of masked DON after digestion is still uncertainty and the separation and purification of D3 G toxin is mainly obtained by chemical synthesis which is more expensive and complicated. Therefore, the purpose of our research is to explore the production patterns of DON, D3 G, 3ADON and 15 ADON and a new method of separation and purification of D3 G toxin.In this study, firstly we inoculated wheat in different states(sterilization, 8k Gy irradiation, 15 k Gy irradiation) with three different Fusarium graminearum strains to find out the production condition of the hidden DON and to determine what state of wheat inoculated with Fusarium graminearum could generate the masked mycotoxin D3 G. Secondly, the field experiment indoor and outdoor inoculation experiments explored whether the masked DON production was related to the varieties of wheat, Fusarium graminearum species or other factors. Finally, to separate and purify D3 G toxin, we optimized the cultivatio n and extraction conditions of inoculated wheat. As convinced, our results could provide the basis for subsequent studies on the regulatory mechanism of D3 G biosynthesis. The main research contents and results are as follows:1. To find out the content of D3 G accumulation and variation in wheat grain and its relationship with Fusarium isolate, the activity of wheat grain and incubation time was determined. The sterilized wheat grain, 8k Gy irradiated and 15 k Gy irradiated wheat grain was inoculated artificially with one of Fusarium graminearum strain and two of Fusarium asiaticum strains respectively, for 7, 14, 21, 28 and 35 days. And then the contents of masked DON were determined by LC-MS/MS. The results showed that the content of D3 G in sterilized and 15 k Gy irradiated wheat grain inoculated with three strains were blow limit of detection; DON content increased gradually with time in 8k Gy irradiated wheat grain while the content of D3 G decreased with time. The ratio of D3 G and total DON dropped exponentially over the incubation time. The results showed that the content of D3 G in wheat grain inoculated with F-1 was significantly(p<0.05) higher than that of PH-1 and 5035. The highest D3 G content was 15923.43μg/kg in wheat grain inoculated with F-1 for 7 days. Wheat germ were damaged due to high pressure sterilization which can not germinate. The 15 k Gy irradiation wheat could not germinate. But 8k Gy irradiation wheat still retained the germination rate about 75%-80%. The dose of 8k Gy irradiation can effectively remove bacteria in wheat. As convinced, our results could provide the basis for subsequent studies on the regulatory mechanism of D3 G biosynthesis.2. The above research showed that only when activity wheat inoculated with Fusarium graminearum, the masked mycotoxin D3 G could generate. In order to explore the differences between the different resistant wheat and the content of D3 G after infection, the experiment of this chapter was divided into two parts- coleoptile inoculation and field inoculation outdoor. Studying these two prats will be beneficial to explore the relationship between toxin accumulation and different growth periods. Yangmai 15, Yangmai 16, N ingmai 13 and Sumai 3 were inoculated with three different strains of Fusarium graminearum, and the contents of mycotoxin accumulation after infection were also analyzed. The results of coleoptile and field inoculation experiments were consistent. The analysis showed that DON accumulation in Yangmai 15 was significantly(p<0.05) higher than that in Sumai 3 while the content of D3 G accumulation was contrary. The results of field experiment showed that the infected spikelets had a close relationship with D3G/total DON. R2 of PH-1 exponential model regarding the ratio of D3G/total DON and infected spikelets was 0.91. There is a certain correlation between D3 G accumulation and resistance of wheat.3. Deoxynivalenol-3-β-D-glucopyranoside(D3G), a conjugated derivative of deoxynivalenol(DON), can escape from the routine detection. Moreover, there is very little toxicological data of DON-3-glucoside but it may be hydrolysed back to its toxic precursors in the mammalian gastrointestinal tract digestion. In order to attain pure DON-3-glucoside for food safety monitoring and toxicological research, an economical and reproducible purification method was developed for production of DON-3-glucoside from irradiated wheat culture inoculated with a Fusarium graminearum strain PH-1. Firstly, the inoculated wheat culture was extracted with acetonitrile/water/acetic acid(79/20/1, v/v/v) followed by evaporating to dryness on a rotary evaporator. Then the remaining liquids were extracted by ethyl acetate and dissolved in water after vacuum freeze drying. Finally, the liquids contained DON-3-glucoside were collected by the final purification procedure through preparative high performance liquid chromatography. A combined approach of ultra high performance liquid chromatography(UHPLC) and mass spectrometry(MS) was applied for the multi-dimensional characterization of the target compound. As a result, the relative recovery of DON-3-glucoside from the crude wheat extract to the final product was up to 85%. The new preparative route makes it possible to obtain DON-3-glucoside at mg scale. Compared with other described approaches, this is the first time that the DON-3-glucoside was extracted from irradiated wheat culture. This proposed strategy might act as a valuable reference to obtain rather expensive compounds in a straightforward way.These results above mentioned would provide useful aids to the resistance mechanismof wheat, production patterns of masked DON. And we provided a new method for separation and purification of D3 G standards. The D3 G standards can be used for the further toxin toxicology.