Study On Isolation And Purification And The Toxicity Of Fumonisins

In order to study the toxicity of fumonisin, the changes of toxin production over time and the purity of fumonisin for different culture were researchedin this study. Rice was the optimal culture to inoculate fusariummoniliforme for getting a lot of toxigenic culture. MAX was used to purify the extract of toxigenic matrix, following two steps to preparate FB1, FB2, FB3 though preparative liquidchromatography. To fill gaps in basic research of toxin FB1 toxicology, body weight, food intake, organ coefficient, blood index and serum biochemical parameters of mice were observed though injecting prepared FB1 to BALB/c mice. This assay aims to provide reference data and theoretical foundation for the isolation and purification of fumonisins, depth study of the mechanism of toxicity and control pollution of mycotoxins in livestock production.The main contents and conclusions of this study are as follows:(1) Fusarium moniliforme is the main fungi which produces fumonisins in nature. It was inoculated on four kinds of culture in this study, PDA, PDB were cultured at 26 ℃ for 15 d under dark conditions and sampled once every 3 days and rice and maize were cultured for 5 weeks and sampled once a week. The content of FB1, FB2 in four medium was detected by liquid chromatography-tandem mass spectrometry(LC-MS/MS). The results showed that the content of FB1 and FB2 reached maximum level in the first 9 day, 28 day, respectively. The content of FB1, FB2 in maize, rice were higher than PDA, PDB, which were 1143.40 mg/kg, 460.88 mg/kg and 817.08 mg/kg, 329.01 mg/kg, respectively. Extracts of the four cultures were full scanned by ultraviolet andit indicated that impurity peak of PDA, PDB and rice were significantly less than maize. Rice was chose as culture because of the less containing of pigments and other impurities under the premise of not affecting the production efficiency. It is not only beneficial to the separation and purification of fumonisins, but also to extend column life.(2)Fusarium moniliformewas inoculated to get a lot of toxigenic matrix with rice as a culture and MAX used as pretreatment purification column for toxigenic matrix extract. Mobile phase and gradientprocedure was optimized using unitary C18 analytical column(4.6 mm × 250 mm, 5 μm) by means of LC-MS/MS, which achieved satisfactory separation for FB1, FB2 and FB3. Then, a method of two-step was used to separate and purify FB1, FB2 and FB3 by amplification of proportion to preparative liquid chromatographic column. The fumonisins obtained were firstly qualitative identified by LC-MS/MS, the results showed that a single chromatographic peak with high peak degree were obtained in the positiveion mode(ESI+) and negative ion mode(ESI-). The precursor ions for FB1, FB2 and FB3 were 722.30 [M+H]+, 706.30 [M+H]+ and 706.30 [M+H]+ in the positive ion mode. The major product ions were 352.30 [M+H-2TCA-H2O]+, 334.30 [M+H-2TCA-2H2O]+, 704.35 [M+H-H2O]+ and 354.30 [M+H-2TCA]+, 336.30 [M+H-2TCA-H2O]+, 318.30 [M+H-2TCA-2H2O]+ for FB1, FB2 and FB3, respectively. UPLC-PDA full scan and NMR were used to demonstrate FB1, FB2 and FB3 for further qualitative analysis, the results showed that fumonisins prepared were high purity. Finally, purity of FB1, FB2 and FB3 were determined by external standard method though LC-MS/MS, which provedthe purity were 98.760±0.12%、99.009 ± 0.08% 、 98.838 ± 0.65%, respectively. This method is simple, low-cost, high-efficiency and FB1, FB2 and FB3 prepared can used to analysis and research as a high-purity fumonisin for further research and application.(3) In this test, 80 BALB/c mice were randomly divided into four groups including the control group(sterile saline), low dose group(0.5 mg/kg.bw), middle dose group(1.5 mg/kg.bw) and high dose group(4.5 mg/kg.bw), respectively. The FB1 isolated and purified was used to feed mices. Body weight, food intake, organ coefficient and biochemical parameters of blood and serum of BALB/c were observed under different doses of FB1. The results showed that the feed intake and body weight gradually decreased alongwith doses of FB1 increasing, but the ratio Sa/So had no significant difference. The index of heart and liver of mice did not exhibit changes with different doses treatment. However, when gavage doses of FB1 was greater than 1.5 mg/kg.bw, spleen, lungs and kidneys were significant swelling and liver was damaged under doses of FB1 in 4.5 mg/kg.bw. GR was increased gradually with increasing doses of FB1, it was significantly higher than control group(P<0.05) when the doses of FB1 in 1.5 mg/kg.bw, the 4.5 mg/kg.bw group significantly higher than the other three groups(P<0.05 or P<0.01). HGB and PLT index increased with the increasing concentration of FB1 and middle dose group and high dose group were significantly higher than the first two groups(P<0.05 or P<0.01). HGB, PLT, platelets and hemoglobin were significant increasegin high doses of FB1(4.5 mg/kg.bw), which indicated high doses of FB1 could cause blood viscosity and coagulation undesirable. Blood biochemical parameters of ALB, ALP, ALT, GLU, TP and UN didnot change withdifferent concentrations of FB1 treatment, butthe activity of AST in middle dose group and high dose group was significantly higher than control group and low dose group(P<0.05). TBLI concentration of high dose group was significantly higher than the other groups(P<0.01) and CRE concentration was significantly higher than control group and low dose group(P<0.05).