鸡肝酯酶在有机磷和氨基甲酸酯类农药检测中的应用研究

利用农药对酯酶活力的抑制反应,分别采用游离的和固定化的鸡肝酯酶,对不同浓度的敌敌畏、敌百虫、马拉硫磷和西维因进行了测定。结果发现,在低浓度时,农药浓度的对数值与其对应的酶活抑制率之间具有较好的线性关系;并且与游离鸡肝酯酶相比,固定化酶不同程度地降低了对农药检测的下限值。敌敌畏、马拉硫磷和西维因的检测下限值依次从游离酶检测时的0．001、4．34和2．52mg/L,降低到了用固定化酶检测时的0．00004、0．0004和0．78mg/L。固定化的鸡肝酯酶对敌百虫的检测下限改善不大。     

小麦酯酶提取及其对有机磷农药检测初探

研究从小麦面粉提取小麦酯酶提取条件及其粗酶液对4种有机磷农药最低检测限和灵敏度。运用单因素实验和正交实验对小麦酯酶提取条件进行优化,确定适宜提取条件,提取液为0.15 mol/L NaCl溶液、料液比1∶4、40℃振荡提取60 min,提取粗酶液酶活为1.675 U/mL;并确定粗酶液对敌敌畏、敌百虫、乐果和毒死蜱等4种有机磷农药最低检测限分别为0.05 mg/kg、0.04 mg/kg、0.05 mg/kg和0.07 mg/kg,均低于国家规定农药最大残留量,且粗酶液对敌敌畏灵敏度最佳。     

酶标甲胺磷交联反应的pH及缓冲体系优化

采用碳二亚胺法制备酶标农药(过氧化氢酶标记甲胺磷),以酶标农药对鸡肝酯酶的抑制率和抑制后酶标农药-酯酶复合物中标记酶酶活为指标,对制备酶标甲胺磷交联反应的pH和所用缓冲液类型进行优化,并对其偶联率进行了分析。结果发现,pH增加有效促进了甲胺磷和标记酶分子之间的交联,而缓冲体系类型对此交联反应的影响相对较小。然而随之持续上升的偶联率带来的空间位阻效应也逐渐明显。随着pH增加,标记物抑制率和抑制后的标记酶活均呈现先增加后略下降的趋势,且分别在pH为6.0和5.5时达到峰值,其峰值时偶联率分别为24和22。综合考量,采用pH 6.0的2-(N-吗啉)乙磺酸一水合物(MES)缓冲液制备的酶标农药,其抑制率和标记酶活均达到较优值,分别为45.29%和47.67U,此时其偶联率约为24。     

流动注射式酶生物传感器检测蔬菜中农药残留的定量标准的研究

本文基于酶抑制原理,采用固定化鸡肝酯酶和自行研制的流动注射式酶生物传感器,研究了不同农药对酶的抑制程度,确定了甲胺磷作为农药检测的定量标准。同时,针对不同的抑制前的初始酶活,绘制了相应的抑制率--甲胺磷含量的标准曲线。并在此基础上,选用了甲胺磷、氧化乐果、乙酰甲胺磷、毒死蜱、西维因和灭多威作为强化农药,对添加了一定强化农药量的黄瓜、青菜进行了定量测定,发现通过标准的建立可以较真实地反映样品中的农药残留量。     

固定化小麦酯酶生物传感器的制备及应用

将固定化酶膜与精密酸度计的玻璃电极连接制备出了生物传感电极探头,并对两种常见的有机磷农药乐果和敌百虫进行测试,结果:乐果浓度在1×10-8～1×10-3mol/L内与小麦酯酶的抑制率呈线性关系,回归方程为y=13.02x+118.7,相关系数为0.995,最低检出限(LDC)为1.7×10-9mol/L;敌百虫浓度在1×10-8～1×10-3mol/L内与小麦酯酶的抑制率呈线性关系,回归方程为y=13.08x+112.7,相关系数为0.997,LDC为1.1×10-8mol/L。以国家标准方法测得结果为对照,进行实际样品检测,检测方程得出的结果回收率为真实值的87.9%～92.3%,而生物敏感膜重复使用20次以内其酶活保存率为初始酶活的90%左右。因此,此传感器可以应用于现场有机磷农药的实验检测等。     

分离纯化提高鸡肝酯酶对农药抑制响应的研究

本文对鸡肝酯酶粗酶液分别进行了硫酸铵分级盐析、硫酸铵分级盐析+透析和硫酸铵分级盐析+排阻色谱三种分离纯化操作,并采用四种结构不同的有机磷、氨基甲酸酯类农药,考察了不同的分离纯化操作对鸡肝酯酶对农药抑制响应的影响。发现经过这三种分离操作后,酯酶对农药抑制的响应程度显著提高,其被四种农药抑制的程度分别提高了6.49~11.30%、14.30~26.26%和10.95~39.87%,而敏感酯酶回收率分别为86.07~89.96%、66.60~73.57%和33.01~41.62%。由于分级盐析+排阻色谱的回收率过低,分级盐析+透析操作对于大规模的检测用酶制备更为实用。此外,研究还发现对这四种农药较敏感的酯酶均集中地分布在相同的分离部分,因此推测鸡肝酯酶粗酶液中对农药抑制敏感的酯酶可能是一种酶或几种溶解性质及分子量都比较接近的酶的混合。     

固定化对动植物酯酶性质的影响

比较了离子交换法得到的固定化小麦酯酶和固定化鸡肝酯酶的主要性质与相应的游离酶的性质。结果表明 ,固定化小麦酯酶的最适pH在 5 5附近 ,比游离小麦酶的最适 pH增加了0 8个 pH单位 ;固定化鸡肝酯酶的最适pH在 8 0附近 ,比游离鸡肝酯酶的最适 pH增加了 0 5个pH单位 ;固定化小麦酯酶的最适温度与游离酶的相当 ,但酶活力 温度曲线的峰形变小 ;固定化鸡肝酯酶的最适温度比游离鸡肝酯酶的下降了 5℃ ,酶活力 温度曲线的峰形也略变陡峭。这 2种固定化酯酶对有机磷类农药 (敌敌畏 )抑制的响应明显高于游离酶对农药抑制的响应     

小麦酯酶的纯化、冻干及对有机磷农药的检测

该文研究从小麦面粉提取的小麦酯酶粗酶液的纯化、冻干和其有机磷农药的最低检测限。确定了用饱和度为30%和60%的硫酸铵对小麦酯酶粗酶液进行分段盐析,纯化后酶液的比酶活是纯化前的2.4倍;最佳保护剂为5%的聚乙二醇,它不仅可以有效保护纯化后的小麦酯酶在冷冻干燥过程中不失活,而且还具有良好的稳定性;最后确定了冻干小麦酯酶酶粉中敌敌畏、敌百虫、毒死蜱、乐果和辛硫磷等5种有机磷农药最低检测限分别为0.01 mg/kg、0.02 mg/kg、0.05 mg/kg、0.07 mg/kg和0.03 mg/kg,均低于国家规定农药最大残留量,冻干酶粉对敌敌畏灵敏度最佳。     

茶叶中甲胺磷农药快速测定研究

利用有机磷农药能抑制植物酯酶酶促反应的机理,建立了快速测定茶叶中甲胺磷农药残留的方法。用70%饱和硫酸铵对面粉中植物酯酶进行纯化,在45℃、pH4.5～5.5时制备的酶催化反应活性最大,酶催化反应动力学显示10min内反应呈线性关系。测定甲胺磷农药残留反应体系最大吸收峰是在480nm之间,最适乙酸-β-萘酯用量为1.0ml;在甲胺磷浓度在0.1～1.0mg/kg之间时,反应体系的吸光值与甲胺磷浓度呈线性相关;在茶叶汁提取液回收率为84%。     

小麦粉中植物酯酶提取、纯化及应用研究

从小麦粉中提取植物酯酶,用70%饱和硫酸铵对植物酯酶进行纯化,酶催化反应温度在45℃、pH4.5~5.5时酶活性最大,酶催化反应动力学显示10m in内反应呈线性关系。利用有机磷农药能抑制植物酯酶酶促反应的机理,建立了快速测定粮食中有机磷农药残留的方法。测定有机磷农药残留反应体系最大吸收峰是在480nm之间,最适乙酸-β-萘酯用量为1.0mL;在农药浓度在0.1~1.0mg/kg之间时,所到之处得到的吸光值与农药浓度呈线性相关;在青菜汁、茶叶汁和大米提取液回收率分别为89%、84%和93%。     

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.     

Chlorohydrins of bisphenol A diglycidyl ether (BADGE) and of bisphenol F diglycidyl ether (BFDGE) in canned foods and ready-to-drink coffees from the Japanese market

BADGE.2HCl and BFDGE.2HCl were determined in 28 samples of ready-to-drink canned coffee and 18 samples of canned vegetables (10 corn, 5 tomatoes and 3 others), all from the Japanese market. HPLC was used as the principal analytical method and GCMS for confirmation of relevant LC fractions. BADGE.2HCl was found to be present in one canned coffee and five samples of corn, BFDGE.2HCl in four samples of canned tomatoes and in one canned corn. No sample was found which exceeded the 1mg/kg limit of the EU for the BADGE chlorohydrins. However the highest concentration was found for the sum of BFDGE.2HCl and BFDGE.HCl.H₂O at a level of 1.5mg/kg. A Beilstein test confirmed that all cans containing foods contaminated with BADGE.2HCl or BFDGE.2HCl had at lest one part coated with a PVC organosol.     

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.     

European priorities for research to support legislation in the area of food contact materials and articles

A strong science base is required to underpin the planning and decision-making process involved in determining future European community legislation on materials and articles in contact with food. Significant progress has been made in the past 5 years in European funded work in this area, with many developments contributing to a much better understanding of the migration process, and better and simpler approaches to food control. In this paper this progress is reviewed against previously identified work-areas (identified in 1994) and conclusions are reached about future requirements for R&D to support legislation on food contact materials and articles over the next 5 or so years.     

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.     

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.     

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.     

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.