【作者】 田园；

【导师】 李建科；

【作者基本信息】 陕西师范大学 ， 食品科学， 2007， 硕士

【摘要】 近年来，农药残留问题越来越多的受到人们的关注。尽管国家有关部门三令五申禁止将有机磷和氨基甲酸酯类农药中的高毒、剧毒品种用于蔬菜果品的生产，但全国不少地区仍在使用像甲胺磷、对硫磷、氧化乐果和呋喃丹等高毒高残留农药，而且因食用含大量农残的蔬菜所引起的人畜中毒事件也时有发生，带来了严重的食品安全问题。因此，针对我国农产品小规模、分散式的产销特点，研究和开发适合我国农产品产销特点的简便、快速、灵敏、实用的检测技术是控制当前农药残留的当务之急。酶生物传感器法用于农药残留检测是目前国内外研究的热点之一。如何能使传感器具有令人满意的灵敏度，关键就是保证固定化的膜片上有足够量高活性酶。本研究首次采用了我国特产的魔芋中提取的魔芋葡甘聚糖作为植物酯酶的固定化载体，利用魔芋葡甘聚糖良好的生物相容性和成膜性制备出一种功能性酶膜，并作为生物传感器的敏感元件，通过待测体系pH值的变化就可对样品中的农药残留进行定量分析。研究结果和内容如下：（1）设计了一种简单易行的KGM的纯化方法，采用的试剂主要是水和乙醇，毒性小、无污染、易回收，提取出的KGM产品纯度可达89.61％，完全能满足进一步制备功能性酶膜的要求。（2）采用添加Na₂CO₃改性，能有效去除KGM分子中的乙酰基，当KGM溶胶浓度为1％，pH为10，增塑剂添加量为1％时，成膜效果最佳。与未改性膜相比，抗水性及耐洗刷性能得到明显改善，且膜表面均匀度高于未改性膜。（3）首次采用魔芋葡甘聚糖固定化植物酯酶，通过正交试验确定了KGM植物酯酶膜的最佳制备条件为：干燥温度为25℃，体系pH为9.0，BSA添加量为20μL，干燥时间为7小时。酶活回收率可达到50％左右，明显优于采用硝酸纤维素膜固定化植物酯酶。（4）建立了植物酯酶生物传感器对农药残留的检测方法，即：5mL待测样品于自制平底试管中，插入传感器电极反应20min后加入250μLa-乙酸萘酯，立即测定pH值，再将平底试管放入40℃水浴中反应20min，降至室温并测定此时pH值，同时以参比电极作对照。（5）对11种农药的灵敏性和最低检测限研究表明，本试验制备的植物酯酶生物传感器对被检测的大多数农药敏感性较好，除久效磷以外对辛硫磷、乐果、甲拌磷、马拉硫磷、氧化乐果、敌敌畏、叶蝉散、残杀威、速灭威、抗蚜威，10种农药的最低检测限都远远低于其MRL值。因此，本研究制备的生物传感器完全可以满足除久效磷以外的其他10种有机磷及氨基甲酸酯类农药残留检测的最低限值要求。更多还原

【Abstract】 Recent years, the issue of pesticide residues in food have caused more attention over the world. Some virulent kinds of orgnaophosphorus and carbamate pesticides such as Tamaron, DNTP, Folimat and Carbofuran are still used to vegetables and fruits in some regions although these have forbidden by the government, the toxicosis incidents of human and animal resulted from taking food which contains high level of pesticide residues are happened usually, which are harming to food security. Therefore, research and development a convenient, rapid, sensitive and practical technique to adapt to the produce and sale status of agriculture product for inspection pesticide residues are urgent demands in our country today.At present, detection of pesticide residues by enzyme-biosensor is a hot spot. High activated enzyme of immobilized enzyme membrane is key technique which ensures good sensitivity of biosensor. This study applied Konjac Glucomannan as carrier immobilized phytoesterase firstly. Then prepared biosensor by this neotype immobilized enzyme membrane, which can analyse quantitatively pesticide residues in food rapidly.The results are as follows:（1） The method of purify KGM is easy to operate and the purity reached 89.61%. The major agent is Alcohol, which toxicity is lower, pollution is smaller and easy to recovery. The production can be produced membrane to immobilization phytoesterase.（2） Konjac Glucomannan was deacetyl for membranes by Na₂CO₃. The membranes made from denatured KGM showed better water resistance capability, uniformity and scrub resistance than that from undenatured KGM.（3） This study applied KGM as carrier immobilized phytoesterase firstly. The optimized parameters of produce enzyme membrane was as follows: optimal pH9.0, and the quantity of bovine serum albumin（BSA） is 20μL, drying temperature and time is 25℃and 7h respectively. The enzyme activity recoveries ratio was reached 50%.（4） The method of rapid detection pesticide residues using enzyme-biosensor has been established. The operation process is: put the biosensor into 5mL sample solution, react for 20min, then add 0.5mL of a-Naphthyl acetate and detect pH immediately, and then react at 40℃for 20min, detect pH at room temperature. Meanwhile reference electrode is to contrast.（5） The sensitivities tests were showed that the enzyme-biosensor was sensitive to most of the organophosphate pesticides and all of the carbamate pesticides which we have studied. The LDCs are lower than their MRLs except monocrotophos. So, we can conclude that the enzyme-biosensor can fulfill the detection demands of organophosphate and carbamate pesticides which we have studied.更多还原