| IntroductionOchratoxin A(OA) is a toxic metabolite of some Aspergillus and Penicillium. According to some animal experiments, OA has immuno-suppressive toxicity, nerve toxicity, teratogenesis, and carcinogenisis. The International Cancer Research Institute regards the OA as a possible carcinogen for human being. Intake of cereal contaminated by OA is the main resouce of OA in human being body.Biotechnic food can be made in industry of fermentation food by using some characteristics of microorganisms. However, the safety of fungi in fermentation was suspected recently. Determination of OA is critical in evaluating the safety of fungi in industry of fermentation food.There is many methods for determining the OA, At the present time, high -performance liquid chromatography is usually used.This study was designed to determine the OA in samples of wheat and paddy and different cultures. Our aims were to build up the method , basing on the method, we investigated the contamination of cereal by OA and to compare the toxicity of different strains of Aspergillus Niger in different cultures. Finally, we may provide a method formanaging fungi strains in industry of fermentation food scientifically.Materials and Mathods1. samplesPaddy (27portions) and wheat (35 portions) were all purchased from Food stockhouse of Shenyang. Different cultures with fungi of As-pergillus Niger.2. Methods2. 1 Confirmation of chromatographic conditions2. 1. 1 Wavelength A solution of OA (50ng/ml) was produced, we measured its excitation spectrum and absorption spectrum using fluorescence spectrophotometer to confirm the optimistic excitation wavelength and emission wavelength.2. 1. 2 Confirmation of mobile phase The chromatographic conditions, including the excitation wavelength ( 338nm ), emission wavelength (455 nm) , the temperature of separation column ( 301) , and speed ( 1. Oml/min ) were kept. The areas of chromatographic peak of standard OA solution (500ng/ml) were measured in different mobile phases to confirm the optimistic mobile phase.2. 1.3 Confirmation of temperature of separation column The chromatographic conditions were kept. We measured the areas of chromatographic peak of standard OA solution (50ng/ml) to confirm the optimistic temperature of separation column.2.2 Standard CurveThe 2 -grade stock solution of standard OA (5jxg/ml) were pipetted to volumetric bottles , Serial standard solutions were got with concentrations of 2,5,10,50,100,200,400,500ng/ml, We used 40ul to make standard curve.2. 3 Pre - treatment of Samples2. 3. 1 Pre - treatment of Wheat and paddy samples samples were ground using a Romer mill to obtain a 20 - mesh sieve size. The ground samples were fully mixed and 10 g of each sample were extracted with 60 ml chloroform and 5 ml 0.1M phosphoric acid by shaking for 30min,and the extracted filtered through a fluted filter paper . A 10 ml volume of filture was collected and in a 10 ml vial and evaporated under a strean of nitrogen at 60C. The residue was quickly reconstitu-ed in 400 ul of acetonitrile-0.008 M phosphoric acid by vortexing for 1 min, and the extract defatted with 1ml nhexane by vortexing again for 1 min and centrigued at 4000 rpm for 10 min. The lower phase was injected into HPLC with full technique.2. 3. 2 Pretreatment of cultures A solution containing 33. 7ml of 85% orthophosphoric acid and 118 g sodium chloride per liter was prepared and was added to a sample 2ml culture in 150 - glass bottle and mixed for 1 min . After addition of 5ml of chloroform and intensively mixing for 30 min. The mixture was centrifuged at 2500g for 15 min . A compact thin layer formed between the two phases . the clear organic phase at the bottom of the tube was carefully withdraw and tansferred to flask. The extraction was repeated with another 5ml of chloroform and the combined extracts were evaperated to dryness at 60C.2. 4 Precision of the methodStandard solutions were got with concentrations of 5 , 50, 500 ng/ ml, at the same conditio...
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