河豚毒素中毒机制和防治的研究进展

河豚毒素(TTX)是一种强神经毒素。它的化学结构、作用机理、中毒防治方法以及在医学和化工上应用受到关注，成为海洋生物资源研究开发的重要课题。本文综述了近年来有关河豚毒素的化学结构、中毒机制、检测技术、中毒防治等方面的最新研究进展，并提出了河豚毒素研究要突变的科技问题以及制备高纯度河豚毒素在医学上作为抗肿瘤、抗神经痛等高效制剂。     

水产品中河豚毒素检测技术研究进展

河豚毒素是河豚鱼及其他生物体内含有的一种内源性生物碱, 是自然界中所发现的毒性最大的神经毒素之一, 被认为是自然界中毒性最强的非蛋白类毒素。自从2016年9月有条件放开养殖红鳍东方鲀和养殖暗纹东方鲀加工经营后, 水产品中河豚毒素含量的准确测定, 已经受到了越来越多的关注。本文综述了水产品中河豚毒素含量色谱、质谱检测技术的最新研究进展, 着重总结了样品前处理技术和分析检测技术, 并对各种样品前处理技术和检测技术的优缺点进行了探讨。     

织纹螺中河豚毒素限量研究

目的提出织纹螺中河豚毒素的限量建议值,为日常监控提供判定依据。方法对织纹螺中河豚毒素的来源、含量情况、毒性、检测标准以及国内外有关河豚毒素、麻痹性贝类毒素的规定等文献资料进行分析比较,探讨织纹螺中河豚毒素的限量要求。结果织纹螺可分为有毒织纹螺、无毒织纹螺、季节性有毒织纹螺,织纹螺中河豚毒素的浓度范围为0～177 mg/kg,而且河豚毒素毒性是麻痹性贝类毒素的30倍以上(以两者对小鼠的经口LD50比较)。对光织纹螺、正织纹螺等常年带毒且毒素含量高的有毒织纹螺应有效识别并禁止采捕、销售、食用,建议无毒织纹螺和季节性有毒织纹螺中河豚毒素的限量为0.05 mg/kg,测定方法采用GB/T 23217—2008《水产品中河豚毒素的测定》。结论提出织纹螺中河豚毒素的限量建议值应合理可行,有效地完善了织纹螺中河豚毒素的监控体系,保障广大公众的身体健康与生命安全。     

河豚毒素抗血清的制备与纯化

研究中利用甲醛作为交联剂,将河豚毒素(Tetrodotoxin,TTX)与匙孔槭血兰蛋白(Keyhole limpethemocyanin,KLH)连接起来作为免疫抗原免疫新西兰大白兔,以牛血清白蛋白(Bovine serum albumin,BSA)与河豚毒素的交联物作为包被抗原,成功的制备了兔抗河豚毒素抗血清,并用DEAE-纤维素柱将抗血清进行了纯化.     

河豚毒素DNA适配子的筛选与结构分析

人工合成113个碱基的随机ssDNA文库,以河豚毒素为靶物质,采用SELEX技术进行12轮筛选,测定各轮富集文库与河豚毒素的亲和力,其中第10轮富集文库与河豚毒素的亲和力最高。将第10轮富集文库进行克隆、测序,用DNAMAN软件分析克隆子的一级和二级结构,并测定克隆子与河豚毒素的亲和力。结果表明:与河豚毒素结合力高的DNA适配子经过10轮筛选后得到富集,成功筛选到最高亲和力的DNA适配子G10。     

养殖河豚鱼的加工及河豚毒素消减技术研究概述

河豚鱼肉质鲜嫩,味道鲜美,享誉古今中外。我国东南沿海地区、日本等许多国家和地区均有吃河豚的习惯,但河豚鱼的内脏、表皮、卵巢等部位含有河豚毒素,因食用河豚鱼导致的中毒事件时有发生。因此,有效消减控制河豚毒素对河豚鱼产业发展有重要意义。该文主要介绍了我国河豚鱼食用历史和消费现状,对养殖河豚鱼深加工制品种类做详细阐述,重点探讨了河豚毒素消减方法和消减机理的研究情况,以期对河豚毒素的消减控制提供参考。     

河豚毒素快速检测技术在养殖河豚鱼 监管中的应用

河豚毒素(tetrodotoxin,TTX)是一种分子量约为319的含氮生物碱,具有极高的毒性,食用河豚毒素含量高的河豚鱼易发生中毒事件。TTX含量的多少因河豚鱼的种类、部位及季节等而有差异,TTX主要集中于河豚的卵巢和肝脏,且在卵巢孕育阶段毒性最强,河豚鱼肉(皮)、精囊无毒。日、韩等国对河豚鱼消费实行科学分类管理,严格区分了"可食用"和"不可食用"的河豚鱼品种、部位等。我国对河豚鱼一直实行"禁食"管理;然而,我国河豚鱼产业却处于"禁食而不禁养"状况,且在国内已形成了较大的"灰色"消费市场。本文梳理了近年来TTX的快速检测技术发展现状,并对快速检测技术在养殖河豚鱼监管中的应用发展前景进行探讨,以期为从事食品安全监管及快速检测技术研究工作的人员提供参考。     

超高效液相色谱-串联质谱法测定闽东地区织纹螺中河豚毒素

建立了快速检测闽东地区织纹螺中河豚毒素含量的高效液相色谱-串联质谱方法。织纹螺均质样品用1%乙酸水溶液提取,0.5%乙酸甲醇溶液稀释,冷冻10min后离心除蛋白,亲水作用色谱柱分离,乙腈-0.1%甲酸水溶液作为流动相梯度洗脱,UPLC-MS/MS分析。基质匹配标准曲线外标法定量。方法的检出限为10μg/kg,定量限为25μg/kg,满足欧洲食品安全委员会对双壳贝类和腹足类中河豚毒素安全限量值44μg/kg的检测要求。在织纹螺中添加50、100、250μg/kg 河豚毒素进行加标回收试验,河豚毒素回收率为79.6%~118.1%,相对标准偏差为(RSD,n=6)7.7%~10.6%。采用该方法测定闽东地区14份织纹螺样品,最高检出河豚毒素 25718.2μg/kg。该研究方法准确性高、成本低、操作性强,能够满足当前快速定量检测织纹螺中河豚毒素含量的技术要求。     

假睛东方鲀肝脏中河豚毒素的分离纯化

本文主要研究分子层析柱对河鲀鱼肝脏中河豚毒素分离纯化效果,为河豚毒素分离纯化工艺提供参考。本实验以假睛东方鲀肝脏为原料,对肝脏中的TTX进行提取,然后利用柱层析技术,确定了中性氧化铝柱、活性炭柱、CM HF和D152弱酸性阳离子交换树脂柱的上样液最适pH、吸附率、回收率。并根据四种柱子的吸附率和净化回收率的结果综合评定,其中D152弱酸性阳离子交换柱吸附率和回收率分别为91.97%和93.98%,且该柱子纯化河豚毒素的效果好,损失率低,最后得到产物多,因此选择D152柱作为分离纯化河豚毒素粗提液纯化柱。最后以假睛东方鲀肝脏为原料,将肝脏匀浆得到的粗提液经D152柱纯化分离后,结晶析出得到河豚毒素粗品,纯度为80%。此研究为河豚毒素分离纯化工艺提供有益参考。     

河豚毒素DNA适配子核心区域的识别

用DNA folding form在线软件分析河豚毒素DNA适配子A3、G10的二级结构,根据二级结构,分别合成适配子A3、G10的各4个区域的5’端地高辛标记的序列。用丙烯酸-环氧乙烷珠子固定河豚毒素,测定抗地高辛碱性磷酸酶的适宜稀释倍数,用地高辛-抗地高辛碱性磷酸酶系统检测各区域与河豚毒素的亲和力,识别出适配子的核心区域。结果表明,抗地高辛碱性磷酸酶的适宜工作浓度为2×104倍稀释;合成的适配子A3的4个区域中,A3-2区域与河豚毒素的亲和力最强,即适配子A3的核心区域为A3-2区域,实际上含有47个核苷酸;合成的适配子G10的4个区域中,G10-4区域与河豚毒素的亲和力最强,即适配子G10的核心区域为G10-4区域,实际上含有56个核苷酸。     

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.