Preliminary Studies On The Detection Technology And Distribution Of Five Alternaria Mycotoxins Pollution In Citrus

Alternaria mycotoxins are naturally occurring secondary metabolites produced by toxigenic microfungi of Alternaria species, these toxins have been shown to have harmful effects in animals, including mutagenic, genotoxicity, fetotoxic and teratogenic and acute toxicity. Among them, alternariol(AOH),alternariol monomethyl ether(AME), altenuene(ALT),altertoxins(ATX-I,ATX-II,ATX-III),tenuazonic acid(TeA)and tentoxin(TEN) are considered the most important Alternaria toxins. Citrus is one of the most important agricultural products and economic crops. China is a big country of citrus plantation and production, its current planting area and yield are the top of the world. Mold fungi can susceptible contaminated citrus in the citrus growing, harvesting, storage, transportation and marketing of various processes, then produce and accumulate mycotoxins, such as Patulin, Alternaria toxins. However, if these mycotoxins polluted in citrus, it is a significant security risks and serious impact on safe to eat for fresh citrus, processing industry of citrus fruit juice and citrus peel slag utilization, it also harmful to human being and animals.Near two years, various countries of researchers pay attention to serious diseases caused by Alternaria species on citrus: Alternaria brown spot, Alternaria black rot. And they also pay more attention to citrus food safety hazards by Alternaria toxins. Now, the existing national food safety standards also does not specify the maximum levels of Alternaria mycotoxins, nor have systematic studies. Therefore, it is meaningful and important for formulating the maximum levels of Alternaria mycotoxins and risk assessment to study on five Alternaria mycotoxins in citrus fruits.In this paper, the method of rapid detection alternariol, alternariol monomethyl ether, altenuene, tenuazonic acid and tentoxin five Alternaria toxin in Citrus using QuEChERS combined with Ultra Performance Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry were studied, meanwhile investigate to five Alternaria toxins of pollution and monitor analysis in Chongqing citrus. And studied on the production and distribution of five Alternaria mycotoxins in varieties of citrus inoculated by Alternaria alternate.The detail and results are follows:1. A novel method was developed for the rapid determination of Ten, ALT, AOH, AME and TeA total five alternaria mycotoxins in citrus using Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry combined with optimized QuEChERS. The sample preparation using the modified QuEChERS method completed the extraction and clean-up steps in one procedure. In this QuEChERS method, sample was extracted with acetonitrile(1.5% formic acid), then salted out with anhydrous MgSO4 and NaCl, performed on an ACQUITY UPLC BEH C18, used acetonitrile and 0.1% formic acid aqueous solution in gradient elution, the MS condition used were negative(ESI+) electrospray ionization and MRM models, liquid chromatography column analysis on UPLC-MS/MS. Under this conditions, five alternaria mycotoxins and have good separation, this method made a good linarity in the 2.0~100 μg/L with more than 0.99(R2), the limits of detection were in the range of 0.11~0.91 μg/kg. Three fortified levels at 5, 20 and 100 μg/kg were spiked into sample, the recoveries ranged from 71% to 112% with the relative standard deviations varying from 1.08% to 9.87%, which met requirement of mycotoxins determination.2. According to the above, we carried out research about the pollution level of five different Alternaria mycotoxins in varieties of citrus in Chongqing major supermarkets and major area of the plant. In this experiment, 113 batches of citrus samples were selected at random. The major results were list as follows:(1) The level of total five different Alternaria mycotoxins in all citrus was the detection rate of 27.43%, range from 2.14 to 66.55 μg/kg, the average concentration of 12.70 μg/kg. Among these five Alternaria mycotoxins, the detection rate and the concentration of TeA, Ten and AOH was higher than others, the maximum detection rate of Ten to 15.93%,TeA check out the highest concentration up to 66.55 μg/kg.(2) Some part of citrus samples in Chongqing major supermarkets and major area of the plant all can be detected some Alternaria mycotoxins.While the detection rate of Citrus reticulate Blanco in market sample higher than base sample, and detection rate and check the concentration of the other varieties of citrus in base sample higher than the market.(3) Among these varieties of citrus, the concentration of total Alternaria mycotoxins is the higher in Citrus reticulate Blanco and Citrus grandis Osbeck, only in Citrus reticulate Blanco detected all five Alternaria mycotoxins, the detection rate of 34.88%, the concentration was between from 2.24 to 6.55 μg/kg, the average concentration of 11.54 g/kg.3. Researches about the variation and distribution of five Alternaria mycotoxins in infected whole fruits, health pulp and peel of the six varieties of citrus which were inoculated by Alternaria alternate. The results demonstrated that:(1) Ten, ALT, AME, AOH and TeA all can be detected in five day at varieties of citrus after inoculated by Alternaria alternate. Among these five Alternaria mycotoxins, the concentration of Te A is the highest, which up to 55880.71 μg/kg. The concentration of total Alternaria mycotoxins increased with increasing diameter of the lesion, and the concentration of different kind of mycotoxins is different. Therefore, the concentration of Alternaria mycotoxins in different varieties of the citrus has a tendency of: Satsuma Orange > Red Tangerine > Glorious Oranges > lemon > Citrus ichangensis Swingle > grapefruit.(2) The concentration of five Alternaria mycotoxins increased with increasing diameter of the lesion location in infected whole fruits, health pulp and peel of the six varieties of citrus which were inoculated by Alternaria alternate, among in infected whole fruits, which vary from 0 to 55880.71 μg/kg, while in health peel location range from 0 to 26336.65 μg/kg, while in health pulp location range from 0 to 40677.5 μg/kg. Also found that the peel of orange and pomelo have strong antibacterial ability, Citrus reticulate Blanco weak resistance. Our results indicate that the citrus infected with toxigenic fungi will produce mycotoxins, and Alternaria mycotoxins will spread from the infected parts to the healthy parts with a high speed, resulting a large amount of mycotoxins accumulating in citrus. Therefore, we should pay more attention to the fresh processing and risk assessment to ensure people’s consumption safety.