不同水源中GⅡ型诺如病毒实时荧光逆转录聚合酶链式反应检测方法建立及应用

目的建立不同水源中GⅡ型诺如病毒(NoV GⅡ)实时荧光逆转录聚合酶链式反应(RT-PCR)检测方法 ,对某市疾病预防控制中心送检的10份疑似引起诺如病毒中毒的水样进行NoV GⅡ检测。方法以瓶装水、河水和生活污水为研究对象,采用硝酸纤维素膜-聚乙二醇(PEG)沉淀法富集病毒,对取样体积、病毒洗脱条件、PEG终浓度及PEG沉淀条件等进行了优化,提取病毒RNA、建立实时荧光RT-PCR检测方法 ;通过外加MS2计算方法的回收率,评价所建方法对水样中诺如病毒的回收效果。结果建立的方法对瓶装水、河水和生活污水中NoV GⅡ的平均回收率分别为(60.1±8.0)%、(22.0±6.5)%和(35.7±8.1)%,10份送检水样中3份检出NoV GⅡ。结论建立的实时荧光RT-PCR方法适用于瓶装水、河水和生活污水中NoV GⅡ的检测,饮用水被NoV GⅡ是引发某市人员中毒的原因之一。     

扇贝蛋白酶解液脱腥脱苦工艺研究

目的:研究扇贝蛋白酶解液的脱腥脱苦工艺。方法:通过正交试验优化活性炭吸附法、掩蔽法、超滤法、超滤和掩蔽联合法的最佳工艺条件,比较所选方法的脱腥脱苦效果。结果:活性炭吸附法脱腥脱苦的最佳工艺条件:活性炭用量为2.0%,时间为60min,温度为20℃,按此条件进行实验,蛋白质回收率为81.6%,腥味值2分,苦味值2分,蛋白质损失较大;掩蔽法脱腥脱苦的最佳工艺条件为0.02%乙基麦芽酚、0.008%乳酸乙酯、0.3%料酒、0.5%食盐,按此组合进行实验,腥味值2分,苦味值2分,蛋白质无损失,但处理后的水解液色泽和透明度较差;超滤法蛋白质回收率为92.0%,处理后的水解液较活性炭脱腥后色泽好,与超滤前相比腥苦味明显降低,但不及活性炭吸附和掩蔽法脱腥脱苦效果好;超滤和掩蔽联合法联合应用时,掩蔽剂最佳用量为0.005%乙基麦芽酚、0.002%乳酸乙酯、0.1%料酒、0.5%食盐,在此条件下进行实验,腥味值0分,苦味值0分,蛋白质回收率为92.0%。结论:采用超滤和掩蔽联合法处理扇贝蛋白酶解液具有较好的脱腥脱苦效果。     

反转录PCR技术检测贝类中诺瓦克样病毒的研究

目的:从病毒富集和核酸提取两方面进行探索,旨在建立一个反转录(RT)PCR技术检测贝类中诺瓦克样病毒(NLVs)的方法.方法:利用脊髓灰质炎病毒作为参照毒株,优化甘氨酸缓冲液一聚乙二醇(PEG)病毒浓缩方法;同时比较异硫氰酸胍法、SDS一蛋白酶K法、Trizol-异丙醇法、试剂盒法4种RNA提取方法:对市售贝类样品进行了检测,并利用基因测序对阳性样品进行验证.结果:采用pH 9.5甘氨酸缓冲液-16%聚乙二醇病毒浓缩法,病毒的回收率为16.8%;利用Trizol-异丙醇法提取RNA,检出限为8.1×102 RT-PCR50/5gg贝肉;实际检测贝类样品25件,其中3件样品为阳性,基因测序结果亦证实为阳性.结论:建立了一个灵敏度较高的RT-PCR检测贝类中诺瓦克样病毒的方法.     

RT-PCR方法检测贝类中诺沃克样病毒的研究

目的:本研究从病毒富集和核酸提取两方面进行探索,旨在建立一个反转录(RT)PCR技术检测贝类中诺沃克样病毒(NLVs)的方法.方法:利用脊髓灰质炎病毒作为参照毒株,优化了甘氨酸缓冲液-聚乙二醇(PEG)病毒浓缩方法;同时比较了异硫氰酸胍法、SDS-蛋白酶K法、Trizol-异丙醇法、试剂盒法四种RNA提取方法;对市售贝类样品进行了检测,并利用基因测序对阳性样品进行验证.结果:本研究采用的pH9.5甘氨酸缓冲液 -16%聚乙二醇病毒浓缩法,病毒的回收率为16.8%,利用Trizol-异丙醇法提取RNA,检出限为8.1×10 2 RT-PCR50/5g贝肉,实际检测贝类样品25件,其中3件样品为阳性,基因测序结果亦证实为阳性.结论:本研究建立了一个灵敏度较高、较为有效的RT-PCR技术检测贝类中诺沃克样病毒的方法.     

牛乳中α-乳白蛋白的分离纯化工艺研究

本文以牛乳为原料,离心脱脂后采用酸沉法分离酪蛋白,微滤除菌后得到乳清蛋白溶液,结合膜分离法和离子交换色谱法分离纯化乳清蛋白溶液中的α-乳白蛋白,并利用高效液相色谱法检测其纯度。结果表明,分离酪蛋白沉淀得到的上清溶液,经0.45μm的陶瓷膜微滤除菌后,除菌率可达99%以上,微滤得到的乳清蛋白溶液蛋白质含量可以达到6.53±0.22 mg/m L。乳清蛋白溶液经卷式聚砜膜超滤,富集α-乳白蛋白,并优化超滤条件,在35℃、出口压力0.3 MPa的条件下,得到的溶液中α-乳白蛋白含量可达24.28±0.32%。使用DEAE Sepharose Fast Flow对蛋白粗品进一步纯化,所获得的α-乳白蛋白纯度为90.89±0.31%,乳清中α-乳白蛋白的回收率可达79.02±0.13%。     

超滤法去除食源性低聚肽中的细菌内毒素

目的:探讨超滤法去除食源性低聚肽中细菌内毒素的效果。方法:采用凝胶法半定量检测5种食源性低聚肽中细菌内毒素的含量;利用膜材料为聚醚砜、截留分子质量为3000、5000、10000、30000、50000的超滤离心管去除样品溶液中的内毒素,评价不同截留分子质量超滤膜的内毒素清除效果;综合考虑内毒素清除率、超滤速度、生产效率、产品回收率等因素,选定截留分子质量为13000的卷式超滤膜进行工艺放大。结果:截留分子质量为3000、5000、10000的超滤膜可将内毒素控制在50 EU/g以内,截留分子质量为30000和50000的超滤膜可分别将内毒素控制在50~200 EU/g、200~400 EU/g范围内;经截留分子质量为13000的卷式超滤膜超滤后,食源性低聚肽样品中细菌内毒素<50 EU/g,清除率>97.5%,产品回收率>75%。结论:超滤法可有效去除食源性低聚肽中细菌内毒素,并且具有可重复利用、生产成本低等优点,适合用于工业化生产中。     

γ-氯丙基三乙氧基硅烷改性纳米Si-HAP分离富集-火焰原子吸收光谱测定水样中痕量的锰、钴、镍

目的建立γ-氯丙基三乙氧基硅烷改性纳米Si-HAP分离富集-火焰原子吸收法测定痕量锰、钴、镍的新方法。方法研究γ-氯丙基三乙氧基硅烷改性纳米Si-HAP在不同p H、吸附温度和时间、洗脱条件及共存离子对锰、钴、镍分离富集的影响。结果在最佳吸附条件下,该方法检出限分别为锰0.21μg/L,钴0.18μg/L,镍0.24μg/L,相对标准偏差分别为锰0.90%(1μg/mL),钴0.78%(2μg/mL),镍0.18%(5μg/mL)(n=11),样品加标回收率在95.0%~108.0%之间。结论该方法适用于水样中痕量锰、钴、镍的测定。     

猪胃黏蛋白偶联磁珠和聚乙二醇富集检测青葱和葡萄中诺如病毒的比较研究

目的:以青葱与葡萄为材料,建立以猪胃黏蛋白偶联磁珠(PGM-MB)和聚乙二醇8000(PEG8000)富集检测水果、蔬菜中诺如病毒的方法。方法:确定病毒原液中的相对病毒量,梯度稀释病毒原液并进行实时荧光-聚合酶链式反应检测,以每个反应管内的荧光信号达到设定的域值时所经历的循环数值与病毒量(实时荧光-聚合酶链式反应单位数)的常用对数值绘制标准曲线和线性方程;人工接种诺如病毒于青葱与葡萄表面,洗脱后,分别用PEG8000和PGM-MB富集诺如病毒,实时荧光-聚合酶链式反应扩增,用标准曲线对回收的病毒进行相对定量。结果:基质为青葱时,高接种量条件下,两种富集方法的病毒回收效果相当,低接种量下,PGM-MB法的富集回收率高于PEG8000法,且PGM-MB的检测下限更低;基质为葡萄时,PGM-MB法的富集回收率均高于PEG8000法,且检测下限更低。结论:PGM-MB富集效果良好,快速方便,适合应用于水果和蔬菜中的诺如病毒的富集检测。     

噬菌体制剂中内毒素脱除方法的比较研究

目的 建立动态浊度法检测噬菌体制剂中内毒素含量, 并探索不同方法对制剂中内毒素的脱除效果。方法 采用动态浊度法检测噬菌体制剂中内毒素含量, 然后采用凝胶层析、超滤及活性炭吸附法对噬菌体制剂进行处理, 检测处理前后噬菌体制剂中内毒素含量及噬菌体效价的变化。结果 基于动态浊度法,建立了内毒素标准曲线回归方程lgT=-0.2732 lgC+3.149, 其线性浓度范围为0.05-10.0 EU/mL,相关系数│r│=0.9986, 满足可靠性分析要求;凝胶层析、超滤法和活性炭吸附法均能有效脱除噬菌体制剂中的内毒素, 脱除率分别为92.6%, 97.1%和99.1%, 但活性炭吸附法选择性差, 噬菌体效价显著降低。结论 动态浊度法能灵敏检测噬菌体制剂中的内毒素含量,凝胶层析和超滤法可作为噬菌体制剂内毒素脱除的可行策略。     

豆制品废水中蛋白质回收研究

以蛋白质回收率、总糖透过率、异黄酮透过率和超滤膜通量为评价指标,采用絮凝-超滤联用的技术初步分离废水中的蛋白质、低聚糖和异黄酮。最佳试验条件为用0.3 mg/mL壳聚糖对废水进行絮凝处理,并用10 k Da的聚醚砜膜进行超滤处理。蛋白质回收率达到76.1%,总糖透过率达到76.0%,异黄酮透过率达到77.2%,平均膜通量18.9~20.0 L/(m~2·h)。絮凝-超滤后蛋白质含量为50.7%。     

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.     

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.     

Influence des ions sur le pouvoir hydratant de l'ure e: e tude sur peau de porc ex vivo

This study deals with the influence of ions (NaCl and MgSO4) in a W/O emulsion containing 10% urea. Moisturization kinetics are assessed by corneometry on pig skin ex vivo. The formula's influence on urea penetration is measured by infrared spectrometry with an ATR device and the stripping method. Corneometry and spectroscopy were chosen to record simultaneously the hydratation levels and urea localization into superficial cell layers. Urea crystallization after evaporation of emulsions and aqueous solutions is described. Results show that urea does not hydrate nor penetrate when applied to the skin through an aqueous gel. In a W/O emulsion, sodium chloride increases the ability of urea to moisturize without improving penetration. In vitro urea crystallization is disturbed by sodium chloride or magnesium sulphate for solutions and emulsions. This stabilization by ions is correlated with good moisturization values. The stabilization of urea in the solute state provided by ions increases its water epidermal binding capacity without enhancing penetration.     

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.