导　　师： 张海彬

学科专业： 090603

授予学位： 硕士

作　　者： ;

机构地区： 南京农业大学

摘　　要： 真菌毒素/(Mycotoxin/)是由某些真菌产生的有毒代谢产物或二级代谢产物。真菌毒素一般存在于霉变的农作物中,分布范围广且危害大,一旦被动物食入就有可能致畸,致突变和致癌,影响畜牧生产,进而导致人类生活品质下降。其中又以玉米赤霉烯酮/(ZEN/)、单端孢霉烯族T-2毒素/(T-2/)以及伏马菌素B1/(FB1/)污染较为广泛,毒性高。因此,加强真菌毒素检测技术的研究显得尤为重要。 ZEN主要影响动物的繁殖机能。T-2属于单端孢酶烯族毒素,T-2具有肠毒性、肝毒性。FB1具有神经毒性、肺毒性、免疫毒性、致癌性等多种毒性作用。 试验ⅠZEN的高效液相色谱检测方法的优化 本文优化了高效液相色谱定量检测饲料中ZEN含量的方法。该法采用免疫亲和柱纯化提取ZEN,优选确定了乙腈+水=75+25/(v+v/)为最佳提取溶液。优化了色谱条件：流动相为甲醇+水+乙腈=10+46+44/(v+v+v/)；检测波长/(激发波长Ex=274nm,发射波长Em=440nm/)；柱温25℃；流速0.6mL·min-1.该方法检测限为5.7μg·kg-1,定量限为19μg·kg-1。 ZEN在0.025～0.8μg·mL-1浓度范围内线性关系良好。线性方程为：y=629600χ+127.49,R2=0.9997。加标回收率高达91.3/%,相对标准偏差低于3.10/%。 试验ⅡT-2的高效液相色谱检测方法的优化 本文优化了T-2的高效液相色谱检测方法,用2-萘酰氯/(2-NC/)和4-甲基氨基吡啶/(DMAP/)做为衍生剂。本法确立的提取溶液为甲醇+水=90+10/(v+v/),优化色谱条件为：流动相是乙腈+水=75+25/(v+v/)；检测波长/(激发波长Ex=381nm,发射波长Em=470nm/);流速0.6mL·min-1,柱温30℃。该方法检测限为1.8μg·kg-1,定量限为6μg·kg-1。T-2在0.025～0.8μg·mL-1浓度范围内线性关系良好。线性方程为：y=2186.2χ-31953,R2=0.9992。加标回收率高达91.92/%,相对标准偏差低于3.13/%。 试验ⅢFB1的高效液相色谱检测方法的优化 本文优化了伏马菌素B1/(FB1/)的高效液相色谱检测方法,衍生剂为邻苯二甲醛/(OPA/)。以乙腈+甲醇+水=25+25+50/(v+v+v/)为提取溶液,优化色谱条件为：流动相：甲醇+0.1mol·L-1磷酸二氢钠=77+23/(v+v/),用磷酸调至pH3.3；检测波长/(激发波长Ex=333nm,发射波长Em=440nm/),流速0.6mL·min-1;柱温30℃。该方法检测限为2.16μg·kg-1,定量限为7.2μg-kg-1。FB1毒素在25～1000ng·mL-1浓度范围内线性关系良好。线性方程为：y=20257χ-104237,相关系数R2=0.9996。加标回收率高达90.91/%,相对标准偏差低于2.86/%。 试验Ⅳ饲料中ZEN、T-2、FB1的污染调查 试验检测了来自全国17个省共270份饲料原料和300份畜禽饲料中ZEN、T-2、 FB1的含量,结果显示：总体来看,ZEN、T-2、FB1的检出率都处于一个较高水平,其中FB1的污染情况严重,超标率最高,T-2超标率较低。 Mycotoxin is the toxic metabolite or the secondary metabolic produced by certain fungus, Generally existing in the moldy crops. It covers a wide distribution and is of great hazard. Once entering an animal's body, mycotoxin may causes serious danger, such as deformity, mutation and cancer and so on. Futhermore it will also affect livestock's production, which then leads to the decline in the quality of human life. With extensive pollution and high toxicity, ZEN, T-2and FB1are more serious of all. Therefore it is crucial to study on the method of detection about fungal toxins. ZEN toxin mainly affects the reproductive function of animals. T-2toxin, belongings to trichothecenes mycotoxins. T-2toxin is of enterotoxigenic and hepatotoxicity. Fumonisin/(FB1/) is full of neurotoxicity, pulmonary toxicity, immunotoxicity, carcinogenicity, embryotoxicity, phytotoxicity and some other toxic effects. Test I Optimization of HPLC for ZEN The method has been established and improved for quantitative detection of ZEN toxin in feedstuffs with HPLC. The clean-up procedure for extracts is immunoaffinity columns. Feedstuff samples are extracted with acetonitrile+water=75+25/(v+v/). Analysis is carried out by using a mobile phase composed of methylalcohol+acetonitrile+water=10+46+44/(v+v+v/), with a flow speed of0.6mL·min-1, a column temperature of25℃and fluorescence detection /(Ex274nm, Em440nm/). The detection limit is5.7μg·kg-1, and the limit of quantitation is19μg·kg-1. It is showed that a good linear relationship at a ranger of0.025～0.8μg·mL-1. The linear equation is y=629600χ+127.49, R2=0.9997. The coefficient of recovery is up to91.3/%, and the relative standard deviation is generally lower than3.10/%. Test Ⅱ Optimization of HPLC for T-2 The method has been established and improved for quantitative detection of T-2toxin in feedstuffs with HPLC and2-naphtoyl chloride /(2-NC/) as ladeling reagent and4-Dimethylamino pyridine as reaction accelerator. Feedstuff samples are extracted with methylalcohol+water=90+10/(v+v/). Analysis is carried out by using a mobile phase composed of acetonitrile+water=75+25/(v+v/), with a flow speed of0.6mL·min-1, a column temperature of30℃and fluorescence detection /(Ex381nm, Em440nm/). The detection limit is1.8μg·kg-1, and the limit of quantitation is6μg·kg-1. It is revealed that a good linear relationship at a range of0.025-0.8μg·mL-1. The linear equation is y=2186.2χ-31953, R2=0.9992. The coefficient of recovery reaches91.92/%, relative standard deviation is generally lower than3.13/%. Test Ⅲ Optimization of HPLC for FB1 The method has been established and improved for quantitative detection of FB1toxin in feedstuffs with HPLC and O-phthalaldehyde/(OPA/) as ladeling reagent. Feedstuff samples are extracted with acetonitrile+water+methylalcohol=25+25+50/(v+v+v/). Analysis is carried out by using a mobile phase composed of methylalcohol+0.1mol·L-1/(pH3.3/)=77+23/(v+v/), with a flow speed of0.6mL·min-1, a column temperature of30℃and fluorescence detection /(Ex333nm, Em440nm/). The detection limit is2.16μg-kg-1, and the limit of quantitation is7.2μg·kg-1. It is presented that a good linear relationship at a range of25-1000ng·mL-1. The linear equation is y=20257χ-104237, R2=0.9996. The coefficient of recovery is90.91/%, relative standard deviation is generally lower than2.86/%. Test IV Pollution situation of ZEN, T-2, FB1in feed samples Test for detection of the content of ZEN, T-2, FB1is from17provinces, with a total of270copies of feed raw materials and300copies of nutritional feed. The results showed that the detection rates of ZEN, T-2and FB1are at a relatively high level. FB1is polluted seriously whose over-standard rate is the highest. Moreover, the over-standard rate of T-2is lower.

关 键 词： 玉米赤霉烯酮 单端孢霉烯族 伏马菌素 高效液相色谱 检测 优化

分 类 号： [S816.17 O657.72]

领　　域： [农业科学] [农业科学] [农业科学] [理学] [理学]