几种天然食用成分对副溶血性弧菌抑菌作用研究

副溶血性弧菌是引起我国特别是沿海地区细菌性食物中毒危害的首要食源性致病菌。本实验通过单因素试验和正交试验研究醋酸、酒精与茶多酚3 种天然食用成分对菌悬液中副溶血性弧菌存活率的影响。单因素试验结果表明：经体积分数高于50%酒精或质量浓度为0.5mg/mL的茶多酚处理5min能完全杀灭菌悬液中的副溶血性弧菌;经pH2 的醋酸溶液处理也能使菌悬液中副溶血性弧菌降低4.79lg(CFU/mL)。正交试验结果表明：酒精体积分数、茶多酚质量浓度和酸度对副溶血性弧菌的抑制效果具有明显的协同作用;pH2.8、酒精体积分数20% 及茶多酚质量浓度0.125mg/mL 或pH2.4、酒精体积分数12% 及茶多酚质量浓度0.125mg/mL 的混合体系均能有效抑制副溶血性弧菌的生长。这些研究结果说明食用酒精、醋酸和茶叶提取物的适当配合将有可能作为复合杀菌剂用于水产食品中,从而降低副溶血性弧菌感染的风险。     

副溶血性弧菌微滴数字PCR定量方法的建立

目的:采用微滴式数字PCR技术(Droplet digital PCR,ddPCR),建立副溶血性弧菌快速定量检测。方法:以副溶血性弧菌TLH基因为靶基因,验证确定ddPCR方法的特异性及定量检测的线性范围,以常检出副溶血性弧菌的海产品鳕鱼为样品进行阳性添加,确定方法的可行性。结果:本试验中采用ddPCR检测得到副溶血性弧菌特异性好,有效基因组DNA浓度范围为2~19440拷贝/20μL,分析得菌悬液浓度为50~4.86×105 CFU/mL,与3M测试片所得菌悬液浓度无显著性差异(p>0.05),与荧光PCR相比,可以进行更低浓度检测且能准确定量。结论:副溶血性弧菌ddPCR定量检测技术特异性良好、灵敏度高、结果准确,具有实际应用价值。     

副溶血性弧菌实时荧光单引物等温扩增方法的建立

以副溶血性弧菌(Vibrio parahaemolyticus)gyr B基因特异序列为靶序列,设计RNA-DNA组合引物和链终止序列,优化反应体系,建立实时荧光单引物等温扩增(Real-time fluorescence single primer isothermal amplification,实时荧光SPIA)检测副溶血性弧菌的方法。实时荧光SPIA在40 min反应时间内,对3株副溶血性弧菌和16株其他食源性致病菌进行实时荧光SPIA检测,结果表明除3株副溶血性弧菌外,其他细菌均未扩增出荧光曲线。进一步研究表明,实时荧光SPIA检测副溶血性弧菌纯培养DNA的灵敏度为8.2 fg/μL,对副溶血性弧菌菌悬液的检测灵敏度为13.5 CFU/m L;对鳕鱼、海蟹、牡蛎和咸鸭蛋等4种模拟样品中副溶血性弧菌的检出限均为14.7 CFU/g。研究结果表明,实时荧光SPIA检测副溶血性弧菌灵敏度高,特异性强,耗时短,方法简便。     

水产品中副溶血性弧菌LAMP检测方法的优化

本文用副溶血性弧菌不耐热溶血素（tlh）基因作靶标,优化环介导等温扩增技术检测水产品副溶血性弧菌的方法。体系用Bst DNA聚合酶催化,恒温反应60 min,产物分别用2%琼脂糖凝胶电泳和SYBR Green I染色鉴定,对各项反应参数进行优化;将新鲜菌液作10倍梯度稀释后进行LAMP和PCR反应,比较二者的敏感度;对32株食源性病原菌（包括分离于水产品的25株副溶血弧菌,1株副溶血性弧菌ATCC17802标准株,大肠杆菌D5、金黄色葡萄球菌CMCC26003、志贺氏菌B4、蜡样芽胞杆菌63302、单核细胞增生李斯特菌54001和沙门氏菌50041各1株）进行LAMP扩增,验证其特异性;虾样品用菌液进行模拟污染,分析LAMP的可靠性。各参数最佳条件为：Mg2+浓度为3.6 mmol/L,dNTPs浓度为0.96 mmol/L,Bst DNA聚合酶用量为4.8 U,内外引物浓度比为8:1,最佳反应温度为63 ℃,时间为60 min;LAMP的检测限为1 CFU/mL,低于PCR方法的最低检测限;特异性试验检测26株副溶血性弧菌均为阳性,6株非副溶血性弧菌为阴性;人工污染试验中检测限达1 CFU/mL,无假阳性。建立的LAMP方法操作简单且灵敏度高,适用于水产食品中副溶血弧菌的现场快速检测。     

青芥辣对食源性致病菌的抗菌作用初步研究

采用伤寒沙门氏菌(Salmonella.typhi)、大肠杆菌(Escherichia.coil)、普通变形杆菌(Proteusrulgaris)、副溶血性弧菌(Vibrioparahaemolyticus)等食源性致病菌作为指示菌株测试青芥辣的抗菌作用。实验条件为加入5×105-5×107cfu／mL的菌悬液到20g样品中,作用3min后测试残留菌量,实验结果表明,不同种类青芥辣对伤寒沙门氏菌(Salmonella.typhi)、致病性大肠杆菌(Escherichia.coil)、普通变形杆菌(Proteusrulgaris)、副溶血性弧菌(Vibrioparahaemolyticus)等食源性致病菌有不同程度的抗菌作用。     

海产品中副溶血性弧菌免疫磁分离和环介导等温扩增快速检测方法的建立

采用纳米免疫磁珠分离副溶血性弧菌,建立副溶血性弧菌环介导等温扩增检测方法。方法 采用副溶血性弧菌单克隆抗体,制备纳米免疫磁珠,特异性吸附副溶血性弧菌,结合环介导等温扩增技术,建立副溶血性弧菌快速检测方法。结果 副溶血性弧菌纳米免疫磁珠在菌体浓度为10³cfu/ml水平时,对副溶血性弧菌的捕获率达到74%。免疫磁分离结合环介导等温扩增技术,在纯培养、无需增菌情况下,检测灵敏度达到140cfu/ml增菌液;通过对134株副溶血性弧菌和74株非目标菌的测试,环介导等温扩增技术具有良好的特异性;食品基质添加试验中,在增菌时间缩短至8h的条件下,其检测限为2cfu/25g样品。结论 副溶血性弧菌免疫纳米磁珠结合环介导等温扩增技术,有效缩短了增菌时间,适用于副溶血性弧菌的快速检测。     

水产品中副溶血性弧菌PMA-ddPCR活菌定量检测方法的研究

本研究旨在针对水产品中活的副溶血性弧菌建立一种快速定量的PCR检测方法。基于叠氮溴化丙锭(PMA)在一定光照条件下能抑制死亡菌DNA扩增,以及微滴式数字PCR技术能将检测精度扩展至单分子目标基因,并实现绝对定量的特点,以副溶血性弧菌tlh基因为目的片段设计及筛选适合的特异性引物与探针,优化反应体系,通过对PMA浓度及曝光条件等优化,建立了一种联用PMA-dd PCR技术快速检测水产品中活的副溶血性弧菌的定量检测方法。研究结果显示:选择16μg/m L作为PMA工作浓度,曝光时间为8 min,此条件下能够完全抑制副溶血性弧菌死菌的DNA扩增并对活菌扩增无影响。通过对比PMA-dd PCR和PMA-q PCR的检测低限分别为2×10~1 cfu/mL、2×10~2 cfu/mL,PMA-dd PCR法的灵敏度比PMA-q PCR法的高。应用PMA-dd PCR定量方法检测人工污染的基围虾和小帆立贝这两种海产品,在基围虾中最低可检出1.9×10~1 cfu/g的副溶血性弧菌、在小帆立贝中最低可检出8.9 cfu/g的副溶血性弧菌。该研究为将PCR技术实际应用于水产品中低量污染、活的副溶血性弧菌的定量检测奠定了基础。     

脉冲高电压对副溶血性弧菌病原性的影响

本实验运用布鲁姆林式脉冲成形网络系统对副溶血性弧菌菌悬液进行处理,发现该系统对副溶血性弧菌有较强的灭菌效果,同时细胞毒性有所下降,且随电压升高而降低;而三型分泌系统相关的基因表达随电压升高而增强,表明脉冲高压电场对细菌病原性有一定的影响,可为液体食品灭菌、生物制品以及疫苗制备过程中抗原灭活提供一定的理论依据。     

提升副溶血性弧菌检验前增菌速度

为有效缩短前增菌时间并打破副溶血性弧菌快检的技术瓶颈,研究通过优化培养基配方和培养条件,探索提升副溶血性弧菌前增菌速度的技术方法。通过单因素和响应面分析确定最适培养基配方为：大豆蛋白胨7.4 g/L、酵母浸粉9.8 g/L、牛心浸粉20.6 g/L,Na Cl 30.0 g/L,p H8.0。最佳培养条件为：温度36℃,转速180 r/min。应用优化的增菌培养方法,对副溶血性弧菌增菌培养6h后,即可获得满足荧光聚合酶链式反应(Polymerase chain reaction, PCR)检测灵敏度要求的增菌液。在添加食品基质的样本中,与GB 4789.7增菌方法相比,优化的增菌培养方法表现出了较高的增菌效率。研究结果表明,改良培养基配方和改善培养条件均可以有效缩短副溶血性弧菌检测的前增菌时间。大幅缩短增菌时间可以为实现食源性致病菌快检奠定基础。     

双重LAMP技术快速检测水产品中副溶血性弧菌和霍乱弧菌的方法学研究

建立环介导等温扩增技术(LAMP)同时快速检测水产品中副溶血性弧菌和霍乱弧菌的方法。针对副溶血性弧菌tox R和霍乱弧菌omp W基因设计特异性引物,优化反应条件,建立水产品中副溶血性弧菌和霍乱弧菌的检测方法,并同时应用双重LAMP技术和PCR技术对实验菌株进行副溶血性弧菌和霍乱弧菌检测,比较两种方法的特异性和灵敏度。LAMP的最佳反应温度为61℃,在此条件下,双重LAMP检测技术检测副溶血性弧菌和霍乱弧菌DNA的敏感度可达3.12 fg,且与其他常见的细菌株无交叉反应,特异性为100%;对模拟食品样品进行直接检测时检测限为50 cfu/m L;对60份水产样品进行检测时,6份样品出现LAMP及PCR阳性,而传统培养方法检测出4份阳性。实验结果表明所建立的双重LAMP技术在检测水产品中副溶血性弧菌和霍乱弧菌时灵敏度、特异性高,时间成本低,适合于水产品中副溶血性弧菌和霍乱弧菌的快速检测。     

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.     

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.     

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.     

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.     

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.