食品中鸡源性成分实时荧光PCR检测方法的建立

目的：建立基于实时荧光聚合酶链式反应技术的食品中鸡源性成分快速检测方法。方法：以鸡线粒体细胞 色素b为目的基因,设计特异性引物和探针,通过特异性、灵敏性实验及模拟混合肉样和市售肉制品检测,对该体 系进行验证。结果：该鸡源荧光聚合酶链式反应检测体系具有很好的特异性及灵敏性,可检测3.5 pg/μL鸡源DNA 的存在;经含鸡源成分的模拟混合肉样检测,证实体系抗干扰能力强;并且通过市售食品检测表明体系可用于定性 加工食品中的鸡源成分。结论：所建立的鸡源引物探针体系具有特异性好、灵敏度高、快速高效等优点,可用于对 食品中鸡源性成分的掺假鉴别。     

同时检测速冻食品中鸡、鸭、猪和牛源性成分实时荧光聚合酶链式反应方法的建立及应用

以鸡转化生长因子β-3(transforming growth factor beta-3,TGFB3)基因、猪朊蛋白(prion protein,PRNP)基因和鸭、牛生长激素(growth hormone,GH)基因为靶基因,设计合成特异性引物和TaqMan探针,通过对实时荧光聚合酶链式反应(polymerase chain reaction,PCR)体系和反应条件的优化,建立同时检测速冻食品中鸡、鸭、猪和牛源性成分的实时荧光PCR检测方法。结果表明:本研究所建立的方法只在鸡、鸭、猪或牛源性成分存在时才产生特异性扩增曲线,表明该方法具有良好的特异性;且对鸡、鸭、猪和牛源性成分的最低检测质量浓度分别可达到10.0、1.0、10.0、10.0 pg/μL,具有良好的灵敏性;应用建立的检测方法对市售34份速冻食品鸡、鸭、猪和牛源性成分进行同时检测,能够实现对速冻食品中4种动物源性成分的有效检测,进一步分析发现,15份速冻食品源性成分测定结果与外包装标注成分不一致。本研究所建立的同时检测速冻食品中鸡、鸭、猪和牛源性成分的实时荧光PCR检测方法具有良好的特异性和灵敏性,且操作简单、适用范围广,能够满足日常检测需求。     

实时荧光PCR技术快速检测奶制品中掺入的大米源性成分

目的:建立基于实时荧光PCR技术的奶制品中掺入大米源性成分的快速检测方法。方法:以水稻的根部表达基因(gos9)为靶基因设计特异性引物和探针,经特异性实验和灵敏度实验验证引物探针可行性,并经模拟含大米奶粉及市售奶类制品检测验证其实际检测能力。结果:该引物探针体系只针对大米DNA进行扩增,与奶类主成分牛、羊及其它谷类和植物性食物DNA均无交叉扩增;最低能检测到0.1ng的大米DNA,对含大米粉的模拟混合奶粉样品,检出限可达0.1%(W/W)。将其应用于21份市售奶制品样品检测,对含大米源性成分的奶制品扩增阳性,检测结果与食品标签相符。结论:该实时荧光PCR检测体系具有快速、特异、灵敏的优点,可以准确鉴定出奶制品中大米成分,适用于奶类中掺加大米源性成分的检测。     

普通聚合酶链式反应鉴定牛肉及其制品中的牛源性成分

依据牛种属的线粒体细胞色素b(cytochrome b,Cytb)基因上的保守序列设计牛源特异性引物对,建立一种特异性强、灵敏性高、耗时短的鉴定食品中牛源性成分的聚合酶链式反应(polymerase chain reaction,PCR)方法。选取牦牛、水牛、黄牛品种及羊、猪、鸡、鸭、鹅、兔、马、驴、鱼、大豆、玉米等动植物源成分进行特异性试验。结果显示:所建立的方法高度特异于牛源性检测,其他非牛动植物源成分均无扩增条带。采用常见的牛品种进行灵敏性试验,结果显示所建立的方法可检测到的最低牛DNA量为0. 2 pg;将牛肉分别与猪肉、驴肉、羊肉、大豆组织混合进行PCR测定,结果表明最低检测限为鲜样品含0. 01%质量分数的牛源成分,高压121℃、0. 1MPa,20 min处理样品含0. 01%质量分数的牛源成分。对市售食品中牛源性成分检测结果与现行PCR牛源性成分检测标准方法的检测结果 100%一致。     

肉制品中牛源性成分多重实时荧光PCR检测 方法的建立及初步应用

目的建立肉制品中牛源性成分的荧光PCR检测方法。方法根据牛特异性线粒体DNA片段,设计合成两对引物,以生、熟牛肉及超市牛肉加工品为材料,建立肉制品中牛源性成分的多重实时荧光PCR检测方法,并用该法与国标法同时对市售的25份肉制品同时进行检测,通过对其他种类的肉源DNA进行扩增验证方法的特异性;对含有不同比例牛肉成分的DNA样本进行检测确定检出限。结果该方法可成功检测出肉制品中的牛源性成分。在25份肉制品检测中,与国标法检测结果一致。该法的特异性为100%,灵敏度检测线为1%。结论本研究成功建立牛源性肉制品的检测方法,该方法快速简便,且具有较高的特异性和灵敏度,可用于市售肉制品中牛源性成分的鉴定。     

重组酶介导等温扩增技术快速检测牛肉及 牛肉制品中的牛源性成分

目的建立重组酶介导等温扩增技术快速检测牛肉及牛肉制品中牛源性成分的方法。方法应用重组酶聚合酶等温扩增技术(recombinase polymerase amplification,RPA),根据牛源性线粒体细胞色素b(Cytb)基因序列设计筛选了一对可用于扩增的引物,建立基于重组酶介导的等温扩增技术检测方法,并对该方法进行扩增温度、特异性和灵敏性验证。结果 40℃等温扩增条件下,所设计的引物的特异性为100%,该检测方法的灵敏性可达0.1 ng/μL。结论本研究成功建立了牛源性肉制品的等温扩增方法,该方法快速简便,具有较高的特异性和灵敏性,适用于市售生鲜肉制品及加工肉制品中牛源性成分的鉴定。     

鸭肉冒充牛羊肉的分子生物学检测

针对送检牛肉和羊肉样品,设计合成检测动物源性成分的通用引物,并对PCR产物测序后在NCBI数据库中进行比对;根据牛、羊、鸭源性成分检测的相关标准,合成引物和探针,进行送检样品中牛、羊和鸭源性成分的实时荧光PCR检测.结果表明:在7份送检样品中4份检测到鸭源性成分,2份检测到牛源性成分,1份检测到羊源性成分.实时荧光PCR方法灵敏性更高、特异性更强、结果判定更为迅速.     

一种基于实时荧光聚合酶链式反应的肉及肉制品中猪源性成分含量测定

为了建立一种基于实时荧光聚合酶链式反应的肉及肉制品猪源性成分含量测定方法,分别以猪线粒体DNA的NADH4基因和16S rRNA基因为靶位点设计猪特异性引物、探针和通用引物、探针,建立猪源性成分含量测定方法,通过特异性、灵敏性、线性、准确度及市售肉制品检测,对该方法体系进行检验和评价.结果表明:建立的猪源性成分含量实时荧光聚合酶链式反应测定方法体系具有良好的特异性及灵敏性,最低可检测到0.4pg/μL纯猪肉DNA;无论是猪特异性体系还是通用体系都呈现良好的线性关系(R2均达到0.999以上)和较宽的线性范围;通过含猪源性成分的模拟混合肉样检测,样品猪源性成分含量的回收率平均值为122.27％,说明该方法具有较高的准确度;通过市售肉制品检测表明该方法可以用来检测实际样品(包括生鲜样品和熟肉样品)中猪源性成分含量.     

食品中马源性成分的实时荧光PCR检测

针对马线粒体DNA细胞色素b基因设计特异性引物和探针,建立食品中马源性成分实时荧光PCR检测方法,并经特异性和灵敏度试验验证其可行性。结果表明:该体系可扩增马DNA片段,长度为127 bp,其他常见畜、禽肉成分均无法正常扩增。该体系的检测灵敏度为1.25 pg马DNA和质量分数0.001%马肉粉。经市售食品的检测验证,表明所建立的马引物探针体系具有特异性好、灵敏度高、快速、高效等优点,可用于对食品中马源性成分的掺假鉴别检测。     

实时荧光聚合酶链式反应法检测食品中猪源性成分

针对猪线粒体细胞色素b基因序列设计特异的引物和探针,建立食品中猪源性成分实时荧光聚合酶链式反应检测方法,并经特异性和敏感性试验验证其可行性。结果表明：该体系可扩增猪肉DNA片段,长度为98bp,其他常见畜、禽肉成分均无法正常扩增。该体系灵敏度低至1pg,且阴性样品扩增后的Ct值限制在35循环以后。对于各模拟肉类样品中掺杂的猪源性成分,其检测限均达1%,经市售加工食品检测验证,表明所建立的猪引物探针体系具有特异性好、灵敏度高、快速、高效等优点,可用于对食品中猪源性成分的掺假鉴别检测。     

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.