新型固载酶生物传感器制备及食品中有机磷农药检测

以微晶纤维素为原料,采用新型水热氧化合成方法,制备固载酶基质材料-双醛纤维素。通过固定化酶化学键联技术制备固定化乙酰胆碱酯酶。利用扫描电镜和红外光谱对产物进行结构表征,固载酶修饰玻碳电极制备ACh E生物传感器。以O,O-二甲基-O-(2,2-二氯乙烯基)磷酸酯为反应物,碘化乙酰硫代胆碱为底物,采用循环伏安和差分脉冲伏安法考察新型传感器对农残的检测性能。结果表明:成功制备了新型固载酶电化学生物传感器,其对DDVP检测的线性范围为0.01~50μg/m L,检出限为6.99 ng/m L,具有很好的重现性和稳定性。本结果可为食品有机磷农药残留的检测提供一种新的分析方法。     

基于多孔碳球修饰硼掺杂金刚石电极的甲基对硫磷检测

目的研究甲基对硫磷检测的快速检测方法。方法 用多孔碳球材料修饰硼掺杂金刚石(BDD)电极,将乙酰胆碱酯酶(ACh E)固定在修饰电极表面制得酶传感器,用于甲基对硫磷的检测。对所制备的酶传感器进行电化学表征,对实验参数进行优化。结果 底物氯化乙酰胆碱的产物在ACh E/BDD电极和ACh E/多孔碳球/BDD电极上的氧化峰电流分别为0.4930μA和1.102μA,峰电流提高了123.53%。在ACh E/多孔碳球/BDD电极上,建立抑制率与甲基对硫磷浓度的负对数的对应关系,在浓度范围10-10~10-7g/L内呈良好的线性关系,线性方程为Y=-13.20297X+148.375,相关系数R2为0.9985。按抑制率为10%计算检出限为3.02×10-12g/L。对实际样品黄瓜汁进行检测,样品回收率为92.60%~102.00%。结论 此方法简单、快速、灵敏度高,适合用于实际样品中的甲基对硫磷残留分析。     

丝网印刷电极法快速检测纺织品中有机磷

利用丝网印刷电极,构建一种壳聚糖/乙酰胆碱酯酶的生物传感器。壳聚糖作为酶的载体,可为乙酰胆碱酯酶提供一个理想的微观环境以保持酶的活性,固载的乙酰胆碱酯酶能快速灵敏地催化溶液中的底物氯化硫代乙酰胆碱(ATCI),实现对纺织品中有机磷农药的快速筛选。该方法灵敏度高,检测速度快,成本低。     

基于新型乙酰胆碱酯酶传感器的有机磷农药检测

制备了空心碳球—离子液体1-丁基-3-甲基咪唑磷钨酸盐(HCS-[Bmim]3PW12O40)修饰玻碳(GC)电极,采用交联法将乙酰胆碱酯酶(ACh E)固定在修饰电极表面,获得ACh E/HCS-[Bmim]3PW12O40/GC传感器。在最优条件下,用该传感器对有机磷农药久效磷、克百威标准品分别进行检测,久效磷的线性范围为8.00×10-10~8.00×10-6g/L,克百威的线性范围是1.00×10-10~1.00×10-7g/L。按抑制率10%计算,久效磷和克百威的检出限分别为7.58×10-11g/L和6.76×10-11g/L。所制备的ACh E/HCS-[Bmim]3PW12O40/GC传感器灵敏度高和稳定性好,为有机磷农药的分析提供了一种有前景的工具。     

新型乙酰胆碱酯酶生物传感器快速检测敌敌畏

以氮掺杂碳球修饰玻碳电极(N-Cs/GCE)为工作电极,采用交联法固定氯化乙酰胆碱酯酶(ACh E),制备了ACh E/N-Cs/GCE传感器,对有机磷农药敌敌畏进行检测分析。结果表明:在(6.5×10~(-11)~6.5×10~(-6))g/L范围内,敌敌畏抑制率与其浓度负对数呈良好的线性关系,线性方程为Y(%)=62.488-5.149X(X为敌敌畏浓度负对数),R~2=0.993,相关性较好。按抑制率10%计算,检出限为6.46×10~(-11) g/L。用该方法对苹果皮中敌敌畏进行检测,回收率在89.32%~93.55%范围内,回收效果较好,准确度较高,具有较强的实用价值。     

聚硫堇修饰的一次性酶传感器检测 辛硫磷农药残留

目的研制一种用于蔬菜中有机磷农药残留快速检测的电化学酶传感器。方法通过循环伏安法将电子媒介体硫堇电聚合在丝网印刷电极上作为电子传递体,用壳聚糖凝胶将乙酰胆碱酯酶固定于聚硫堇电极表面,制成一种新型的有机磷农药生物传感器。结果在有机磷农药辛硫磷浓度为0.01~500μg/m L范围内,酶电极抑制率(%)与辛硫磷的浓度(c)的对数呈良好的线性关系,相关系数为0.9886,检出限以抑制率10%的农药浓度计算为0.006μg/m L。结论研制出成本低廉,使用方便,具有响应快、灵敏度高的有机磷农药生物传感器,可应用于果蔬中有机磷农药的快速检测。     

中华蜜蜂乙酰胆碱酯酶基因的克隆及其在毕赤酵母中的表达鉴定

有机磷污染是食品安全的重大隐患,乙酰胆碱酯酶(Acetyl cholinesterase,ACh E)可用于有机磷农药残留的检测。中华蜜蜂是我国独有的蜜蜂当家品种,蜜蜂对农药的敏感性很高,中华蜜蜂来源的乙酰胆碱酯酶基因尚未被克隆并应用于食品安全监测。以中华蜜蜂头部组织总RNA为模板,经RACE方法获得了ACh E基因c DNA全序列,全长约1.8 kb。构建重组表达载体p PIC3.5K-ACh E,将目的基因表达盒整合入毕赤酵母GS115的基因组中获得重组菌株,0.5%甲醇诱导重组菌株表达ACh E蛋白,经SDS-PAGE、酶活性分析表明,中华蜜蜂ACh E基因首次克隆成功并首次在毕赤酵母中获得成功表达,筛选出一株酶活性较高的菌株,纯化的重组乙酰胆碱酯酶其活性为10 568 U/mg,可以用于有机磷农药残留的检测。     

电沉积法构建AChE/COD@AuNPs共固定化酶生物传感器差分脉冲伏安法快速检测有机磷农药

有机磷农药残留危害环境和人类健康。本文采用电沉积法将乙酰胆碱酯酶(AChE)与胆碱氧化酶(COD)固定化,共修饰于裸金电极表面,构建新型固定化酶生物传感器。通过电子显微镜对固定化酶形貌进行表征,结果表明：当固定化酶生物传感器上,乙酰胆碱酯酶与乙酰胆碱氧化酶的共修饰层数为2层,修饰圈数为35圈,在氯化乙酰胆碱浓度为2 mmol/L,pH 7.8的检测体系中,该生物传感器能够识别有机磷农药信号,且检测性能尚佳。传感器在有机磷农药质量浓度为10^-9～10-7mg/L时电流响应良好,工作曲线方程为y=0.0468x+1.2124(R²=0.9985),最低检出限1.15×10^-11 mg/L(S/N=3)。本研究为现场快速检测有机磷农药提供了新的研究方法。     

鲫鱼乙酰胆碱酯酶性质及对有机磷农药的敏感性研究

以鲫鱼为原料,分别提取鱼脑、肌肉和肝脏乙酰胆碱酯酶(acetylcholinesterase,ACh E),研究其酶学性质及对有机磷农药的敏感性。结果表明,鲫鱼脑ACh E最适反应温度为30℃,最适p H为8.0,米氏常数(Michaelis constant,Km)为0.548 mmol/L;鲫鱼肌肉ACh E最适反应温度为35℃,最适p H为7.5,Km为1.75 mmol/L;鲫鱼肝脏ACh E最适反应温度为30℃,最适p H为8.0,Km为0.406 mmol/L。敌敌畏对鱼脑、肌肉和肝脏ACh E的半数抑制质量浓度(50%inhibition concentration,IC50)分别为1.70,3.55和4.52μg/m L,双分子速率常数(bimolecular rate constant,Ki)分别为7.40、7.21、5.94 L/mg·min;辛硫磷对鱼脑、肌肉和肝脏ACh E的IC50分别为2.45,4.05和9.46μg/m L,Ki分别为10.54,10.11和8.32 L/mg·min;毒死蜱对鱼脑、肌肉和肝脏ACh E的IC50分别为10.89、14.33和17.00μg/m L,Ki分别为7.81,6.85和4.99 L/mg·min。鲫鱼脑ACh E对常用有机磷农药表现出了较强的敏感性,可作为环境中有机磷农药残留监测的指示酶。     

鲤鱼来源乙酰胆碱酯酶的高效表达及分子对接模拟

乙酰胆碱酯酶介导的农药残留检测方法在食品安全中发挥着越来越重要的作用。本研究以鲤鱼来源的乙酰胆碱酯酶基因(Cp ACh E)为试验材料,通过基因密码子优化来提高酶的表达量;通过表征鲤鱼乙酰胆碱酯酶的酶学性质探究其在农药残留检测中的应用潜力。采用计算机模拟技术寻找有机磷农药克百威与酶结合的氨基酸残基位点。结果表明:经密码子优化的重组酶乙酰胆碱酯酶的酶活力为7.4 U/m L,表达量提高约40倍,最适反应温度25℃,最适p H 8.0。动力学参数K_m和V_(max)分别是1.639 mg/m L和8.071μmol/min/mg。不同有机溶剂和金属离子对酶活力的影响存在差异,大多数有机试剂对乙酰胆碱酯酶活性有抑制作用,而Mg~(2+)、K~+、Ca~(2+)和Li~+离子对该酶活性有促进作用,其中Mg~(2+)(5 mmol/L)的促进最为明显,使该酶活力提高约1.7倍。重组酶对8种有机磷和8种氨基甲酸酯类农药都表现出较强的敏感性,其中,克百威抑制作用最强,最低检测限为0.044μg/m L。此外,分子对接发现,克百威与重组酶中的19个氨基酸之间形成氢键和疏水作用,其中氨基酸Tyr146、Ser147和Tyr355很可能是与克百威结合的靶位点。本研究成功实现了乙酰胆碱酯酶的高效表达,探究了乙酰胆碱酯酶与克百威的结合位点,为该酶在食品中有机磷和氨基甲酸类农药残留检测的应用及理性改造提供了一定的理论指导。     

Reliable identification of grapevine rootstock varieties using RAPD PCR on woody samples

Since grapevine ( Vitis spp .) rootstock material is being traded increasingly as disbudded woody material a lack of distinctive morphological features on such material necessitates an alternative and reliable means of identification. Methods described here were developed for rapid and efficient extraction of DNA from woody samples rich in phenolic compounds and polysaccharides, and for subsequent identification of varieties by RAPD PCR. Using these methods, and with the application of only one selected RAPD primer, we were able to differentiate sixteen rootstock varieties, including the seven varieties most commonly used in Germany. Problems commonly encountered with reproducibility of RAPD patterns were avoided by choosing primers with a dinucleotide sequence and a high G/C content that allowed a rather high annealing temperature of 45°C. Methods described here should also be useful for other horticultural crops, especially those with woody tissues rich in phenolic compounds and polysaccharides.     

An internet compendium of analytical methods and spectroscopic information for monomers and additives used in food packaging plastics

An internet website (http://cpf.jrc.it/smt/) has been produced as a means of dissemination of methods of analysis and supporting spectroscopic information on monomers and additives used for food contact materials (principally packaging). The site which is aimed primarily at assisting food control laboratories in the European Union contains analytical information on monomers, starting substances and additives used in the manufacture of plastics materials. A searchable index is provided giving PM and CAS numbers for each of 255 substances. For each substance a data sheet gives regulatory information, chemical structures, physico-chemical information and background information on the use of the substance in particular plastics, and the food packaging applications. For monomers and starting substances (155 compounds) the infra-red and mass spectra are provided, and for additives (100 compounds); additionally proton NMR are available for about 50% of the entries. Where analytical methods have been developed for determining these substances as residual amounts in plastics or as trace amounts in food simulants these methods are also on the website. All information is provided in portable document file (PDF) format which means that high quality copies can be readily printed, using freely available Adobe Acrobat Reader software. The website will in future be maintained and up-dated by the European Commission's Joint Research Centre (JRC) as new substances are authorized for use by the European Commission (DG-ENTR formerly DGIII). Where analytical laboratories (food control or other) require reference substances these can be obtained free-ofcharge from a reference collection housed at the JRC and maintained in conjunction with this website compendium.     

Aroma characterization of various apricot varieties using headspace–solid phase microextraction combined with gas chromatography–mass spectrometry and gas chromatography–olfactometry

The characterization of the aromatic profile of several apricot cultivars with molecular tracers in order to obtain objective data concerning the aromatic quality of this fruit was undertaken using headspace–solid phase microextraction (HS–SPME). Six apricot cultivars were selected according to their organoleptic characteristics: Iranien, Orangered, Goldrich, Hargrand, Rouge du Roussillon and A4025. The aromatic intensity of these varieties measured by HS–SPME–Olfactometry were defined and classified according to the presence and the intensity of grassy, fruity and apricot like notes. In the six varieties, 23 common volatile compounds were identified by HS–SPME–GC–MS. Finally, 10 compounds, ethyl acetate, hexyl acetate, limonene, β-cyclocitral, γ-decalactone, 6-methyl-5-hepten-2-one, linalool, β-ionone, menthone and (E)-hexen-2-al were recognized by HS–SPME–GC–O as responsible of the aromatic notes involved in apricot aroma and considered as molecular tracers of apricot aromatic quality which could be utilized to discriminate apricot varieties.     

Tests of potential functional barriers for laminated multilayer food packages. Part I: Low molecular weight permeants

The advent of the functional barrier concept in food packaging has brought with it a requirement for fast tests of permeation through potential barrier materials. In such tests it would be convenient for both foodstuffs and materials below the functional barrier (sub-barrier materials) to be represented by standard simulants. By means of inverse gas chromatography, liquid paraffin spiked with appropriate permeants was considered as a potential simulant of sub-barrier materials based on polypropylene (PP) or similar polyolefins. Experiments were performed to characterize the kinetics of the permeation of low molecular weight model permeants (octene, toluene and isopropanol) from liquid paraffin, through a surrogate potential functional barrier (25 μm-thick oriented PP) into the food simulants olive oil and 3% (w/v) acetic acid. These permeation results were interpreted in terms of three permeation kinetic models regarding the solubility of a particular model permeant in the post-barrier medium (i.e. the food simulant). The results obtained justify the development and evaluation of liquid sub-barrier simulants that would allow flexible yet rigorous testing of new laminated multilayer packaging materials.     

Textural Properties of Cold-set Gels Induced from Heat-denatured Whey Protein Isolates

A 9% whey protein (WP) isolate solution at pH 7.0 was heat-denatured at 80°C for 30 min. Size-exclusion HPLC showed that native WP formed soluble aggregates after heat-treatment. Additions of CaCl2 (10–40 mM), NaCl (50–400 mM) or glucono-delta-lactone (GDL, 0.4–2.0%, w/v) or hydrolysis by a protease from Bacillus licheniformis caused gelation of the denatured solution at 45°C. Textural parameters, hardness, adhesiveness, and cohesiveness of the gels so formed changed markedly with concentration of added salts or pH by added GDL. Maximum gel hardness occurred at 200 mM NaCl or pH 4.7. Increasing CaCl2 concentration continuously increased gel hardness. Generally, GDL-induced gels were harder than salt-induced gels, and much harder than the protease-induced gel.     

Occurrence of bisphenol-F-diglycidyl ether (BFDGE) in fish canned in oil

The levels of bisphenol-F-diglycidyl ether (BFDGE) were quantified as part of a European survey on the migration of residues of epoxy resins into oil from canned fish. The contents of BFDGE in cans, lids and fish collected from all 15 Member States of the European Union and Switzerland were analysed in 382 samples. Cans and lids were separately extracted with acetonitrile. The extraction from fish was carried out with hexane followed by re-extraction with acetonitrile. The analysis was performed by reverse phase HPL C with fluorescence detection. BFDGE could be detected in 12% of the fish, 24% of the cans and 18% of the lids. Only 3% of the fish contained BFDGE in concentrations considerably above 1mg/kg. In addition to the presented data, a comparison was made with the levels of BADGE (bisphenol-A-diglycidyl ether)analysed in the same products in the context of a previous study.     

Analysis of benzo[a]pyrene in spiked fatty foods by second derivative synchronous spectrofluorimetry after microwave-assisted treatment of samples

A simple, rapid and inexpensive method has been developed for the determination of benzo[a     

The development of reference materials for paralytic shellfish poisoning toxins in lyophilized mussel. II: Certification study

This paper describes the second part of a project undertaken to develop certified mussel reference materials for paralytic shellfish poisoning toxins. In the first part two interlaboratory studies were undertaken to investigate the performance of the analytical methodology for several PSP toxins, in particular saxitoxin and decarbamoyl-saxitoxin in lyophilized mussels, and to set criteria for the acceptance of results to be applied during the certification exercise. Fifteen laboratories participated in this certification study and were asked to measure saxitoxin and decarbamoyl-saxitoxin in rehydrated lyophilized mussel material and in a saxitoxin-enriched mussel material. The participants were allowed to use a method of their choice but with an extraction procedure to be strictly followed. The study included extra experiments to verify the detection limits for both saxitoxin and decarbamoyl-saxitoxin. Most participants (13 of 15) were able to meet all the criteria set for the certification study. Results for saxitoxin.2HCl yielded a certified mass fraction of <0.07 mg/kg in the rehydrated lyophilized mussels. Results obtained for decarbamoyl-saxitoxin.2HCl yielded a certified mass fraction of 1.59+/-0.20 mg/kg. The results for saxitoxin.2HCl in enriched blank mussel yielded a certified mass fraction of 0.48 +/- 0.06 mg/kg. These certified reference materials for paralytic shellfish poisoning toxins in lyophilized mussel material are the first available for laboratories to test their method for accuracy and performance.     

Contribution of European research to risk analysis

The European Commission's, Quality of Life Research Programme, Key Action 1—Health, Food & Nutrition is mission-oriented and aims, amongst other things, at providing a healthy, safe and high-quality food supply leading to reinforced consumer confidence in the safety of European food. Its objectives also include the enhancing of the competitiveness of the European food supply. Key Action 1 is currently supporting a number of different types of European collaborative projects in the area of risk analysis. The objectives of these projects range from the development and validation of prevention strategies including the reduction of consumers risks; development and validation of new modelling approaches; harmonization of risk assessment principles, methodologies, and terminology; standardization of methods and systems used for the safety evaluation of transgenic food; providing of tools for the evaluation of human viral contamination of shellfish and quality control; new methodologies for assessing the potential of unintended effects of genetically modified (genetically modified) foods; development of a risk assessment model for Cryptosporidium parvum related to the food and water industries; to the development of a communication platform for genetically modified organism, producers, retailers, regulatory authorities and consumer groups to improve safety assessment procedures, risk management strategies and risk communication; development and validation of new methods for safety testing of transgenic food; evaluation of the safety and efficacy of iron supplementation in pregnant women; evaluation of the potential cancer-preventing activity of pro- and pre-biotic ('synbiotic') combinations in human volunteers. An overview of these projects is presented here.