鼠李糖乳酸杆菌对河豚肝脏中河豚毒素毒性的影响

以野生河豚肝脏中的河豚毒素（tetrodotoxin,TTX）为研究对象,研究活化与热灭活处理的鼠李糖乳杆菌对其毒性的消减作用。将活化的与热灭活处理的鼠李糖乳杆菌作为材料对TTX进行脱除,采用酶联免疫吸附剂测定（enzyme-linked immunosorbnent assay,ELISA）、层析试纸检测和小鼠生物法分析TTX消减率的变化,采用气相色谱-质谱分析发酵处理后的肝脏二十碳五烯酸（eicosapentaenoic acid,EPA）与二十二碳六烯酸（docosahexaenoic acid,DHA）含量变化。竞争性ELISA结果显示加热灭活后的鼠李糖乳杆菌对TTX的消减率高达82.16%;而未经灭活处理的鼠李糖乳杆菌对TTX的消减率为70.05%,表明热灭活处理的鼠李糖乳杆菌消减效果更佳。此外,经鼠李糖乳杆菌发酵7 d后野生河豚肝脏的TTX消减率为93.27%,气相色谱-质谱检测结果显示发酵后肝脏内的EPA与DHA含量分别减少了11.93%、22.50%。综上所述,鼠李糖乳杆菌能消减河豚肝脏中的TTX,经鼠李糖乳杆菌发酵处理后消减效果更佳,本研究结果可为河豚内脏组织的毒性消减提供参考。     

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。     

鲀毒鱼毒素检测及物种鉴定技术研究进展

鲀毒鱼引起的河鲀毒素(tetrodotoxin,TTX)中毒是中国沿海地区食物中毒致死的主要原因之一.河鲀毒素中毒发病迅速,至今尚无特效药,因此,通过检测TTX含量或鉴定携带TTX的物种可以更好地进行TTX中毒的风险分析、管理和控制.本文综述了鲀毒鱼TTX的检测技术以及鲀毒鱼物种鉴定的方法,以期能预警并减少鲀毒鱼引起的...     

用昆明小鼠定量测试河豚毒素的研究

为建立昆明小鼠定量测定河豚毒素(TTX)方法,选用雄性昆明小鼠进行研究,结果显示TTX致昆明小鼠的LD50为0165μg,TTX剂量在045～256μg之间与昆明小鼠平均死亡时间成线性关系;当河鱼样品提取液TTX浓度低于045μgml时,腹腔注射1ml提取液致小鼠平均死亡时间大于4min,在此范围内与ICR小鼠进行比较,毒素检测结果基本一致。此外对该传统实验方法进行了无需小鼠体重校正、利用平均数计算等项改进。用昆明小鼠定量测试河豚毒素既方便又可靠。     

共培养对己酸菌生长代谢影响的初步研究

己酸菌是浓香型大曲酒发酵产生己酸及己酸乙酯起决定作用的窖泥功能菌,为了掌握其生长代谢规律,研究了己酸菌与醋酸菌、放线菌和窖泥共培养时,发酵液中己酸产量和蛋白质含量的变化规律。     

河豚毒素在暗纹东方鲀体内的转移分布规律及相关代谢酶活性分析

为了研究河豚毒素(TTX)在暗纹东方鲀体内的转移分布规律及其对肝脏代谢酶活性的影响,实验选择口服灌喂的给药方式,通过超高效液相色谱串联质谱法测定不同组织中TTX的浓度变化,同时测定肝脏代谢酶活力。结果如下,在灌喂TTX后的短期转移分布期间,性腺中TTX的浓度从19.00 ng/g升至42.02 ng/g,蓄积率为121%。肝脏中TTX浓度呈波动性变化,在4 h时,TTX浓度最高为11.43 ng/g,在48 h时,TTX浓度最低为1.27 ng/mL。皮肤、肌肉、血液、肠道、鳃组织中TTX含量在2 h时最高,分别为7.43 ng/g、1.15 ng/g、11.44 ng/g、9.77 ng/g、31.75 ng/g。脾脏、肾脏、胆组织中TTX浓度分别在2 h、4 h、6 h时最高为5.05 ng/g、6.54 ng/g、9.06 ng/g。此外,肝脏代谢酶活力的检测结果显示,TTX显著提高了乳酸脱氢酶、谷草转氨酶和谷丙转氨酶的活性(p0.05),在前期对两种磷酸酶活性具有明显的抑制效应(p0.05),而后恢复至正常水平。实验结果表明,TTX在暗纹东方鲀中的转移分布具有组织特异性,并且对肝脏中不同代谢酶活性具有促进和抑制作用。     

鼠李糖乳杆菌对河豚毒素的免疫活性消减作用方式

为探究鼠李糖乳杆菌（Lactobacillus rhamnosus）对河豚毒素（tetrodotoxin，TTX）免疫活性的消减作用方式和可能作用位点，分别采用活化、热灭活和破碎的鼠李糖乳杆菌以及去除胞外多糖、原生质体、细胞壁和肽聚糖的分离样品，与TTX标准品在37 ℃孵育1 h，使用竞争性酶联免疫吸附法分析作用前后TTX的免疫活性变化。通过化学处理掩蔽鼠李糖乳杆菌表面的羧基和氨基，采用傅里叶变换红外光谱（Fourier transform infrared spectroscopy，FTIR）分析菌体表面的化学键振动类型和官能团变化情况。结果发现，活化、热灭活和破碎后的鼠李糖乳杆菌均可消减TTX的免疫活性，因此TTX免疫活性的变化和鼠李糖乳杆菌的活性无关。各分离样品对TTX的消减作用表明肽聚糖成分对TTX免疫活性的消减率最高可达到47%。FTIR分析发现鼠李糖乳杆菌表面羧基掩蔽后可显著降低TTX免疫活性的消减率至18%，而掩蔽氨基则对作用前后的TTX消减率影响无显著差异（P＞0.05）。实验表明，消减TTX免疫活性的原因可能为TTX与鼠李糖乳杆菌表面肽聚糖上的羧基结合。     

组合菌发酵葡萄糖生产V_C前体——2-酮基-古龙酸的研究进展

利用组合菌从葡萄糖发酵生产 2 酮基 L 古龙酸 ,进而产生VC,为改变化学合成VC及降低成本 ,提供了一条新途径。组合菌有特殊的生理功能和多种组合方式 ,其应用较为复杂。随着分子生物学技术的发展 ,VC 发酵有利用组合菌向单一菌发展的趋势。文中从研究菌种组合、发酵途径、菌种选育、构建基因工程菌等方面对国内外VC 发酵的研究概况作一综述 ,并提出进一步研究和探索的方向     

镰刀菌毒素生殖发育毒性研究进展

镰刀菌毒素是指镰刀菌属的毒性代谢产物,其中单端孢霉烯族毒素是粮食中最常见的一类污染性镰刀菌毒素,可以引起人或动物急性、慢性毒性作用,部分霉菌毒素已证明具有致突变性及致癌性。流行病学及体内、体外实验研究结果表明,部分镰刀菌毒素对人或动物生殖细胞、受孕、妊娠、分娩、哺乳等亲代生殖机能以及对其子代胚胎-胎儿发育、出生后发育产生不良影响。本文对镰刀菌毒素生殖、发育毒性研究及其进展进行综述。     

玉米赤霉烯酮生物脱毒及关键酶作用机理的研究进展

玉米赤霉烯酮(Zearalenone,ZEN,ZEA)是由镰刀菌属产生的一类具有类雌激素生物活性的霉菌毒素,广泛存在于玉米、小麦等谷物及其制品中。本文综述了各类微生物降解ZEN的研究现状,重点比较其脱毒效率及产物毒性,尤以不动杆菌(Acinetobacter sp.SM04)、粉红粘帚菌(Gliocladium roseum IFO 7063)、毛孢子菌(Trichosporon mycotoxinivorans)和假单胞菌(Pseudomonas sp.ZE-1)研究较为全面,进一步总结了ZEN降解的关键酶及其作用机制,为微生物降解玉米赤霉烯酮的研究提供依据,也为大环内酯化合物和芳香族化合物的降解开拓更广泛的途径。     

Liquid chromatography-mass spectrometry method to detect Tetrodotoxin and Its analogues in the puffer fish Lagocephalus sceleratus (Gmelin, 1789) from European waters

The presence of tetrodotoxin (TTX) and its analogues, 4-epiTTX, 4,9-anhydroTTX, 5-deoxyTTX, 11-deoxyTTX, 5,6,11-trideoxyTTX, 11-norTTX-6(S)-ol, and 11-norTTX-6(R)-ol was investigated for the first time in five different tissues (liver, gonads, gastrointestinal tract, muscle, and skin) of six specimens of the marine puffer fish Lagocephalus sceleratus from European waters (Aegean Sea, Greece) by using liquid chromatography coupled to electrospray ionisation mass spectrometry operating in the conventional mode in addition to low-energy collision dissociation tandem mass spectrometry (CID-MS/MS).Two isomers of 5,6,11-trideoxyTTX were detected in all specimens as the major TTX analogues, followed by 11-deoxyTTX, 11-norTTX-6(S)-ol, and TTX. However, minor amounts of 4-epiTTX, 4,9-anhydroTTX, 5-deoxyTTX, and 11-norTTX-6(R)-ol were also found in most of the tested tissues. In all puffer fish specimens, gonads, gastrointestinal tract, and liver contained the highest toxin levels, whereas muscle and skin contained lower amounts. Toxin distribution within the tissues of the six L. sceleratus specimens was different depending on fish size, area, and season where fish were caught. The LC-ESI-CID-MS/MS analysis employed is proposed as a suitable technique for determination of TTX and its analogues with a low detection limit (0.08 μg/g).     

河鲀中河鲀毒素检测研究进展

河鲀毒素(tetrodotoxin,TTX)是一种毒性极强的天然小分子生物碱,无特效解毒剂,中毒潜伏期短,在水产品质量安全领域受到极大关注。本文总结了河鲀毒素的毒性、相关法规、化学性质和反应特点,并对河鲀毒素检测过程中样品前处理以及检测方法进行了总结,并详细综述了河鲀毒素常用的生物检测法、液相色谱荧光检测法、气相色谱串联质谱法、液相色谱串联质谱检测法和免疫及电化学检测方法应用情况,尤其对免疫检测与电化学技术结合的方法进行了分类分析。最后,本文分析了免疫分析过程中竞争型和非竞争型免疫的区别与优缺点,并对新型电化学免疫快速检测的方法开发进行了展望,以期为检测TTX等小分子化合物的新型检测技术的开发提供参考。     

High-resolution mass spectrometry analysis of tetrodotoxin (TTX) and its analogues in puffer fish and shellfish

Tetrodotoxin (TTX) is an emerging toxin in the European marine environment. It has various known structural analogues. It acts as a sodium channel blocker; the ability of each analogue to bind to the sodium channel varies with the particular structure of each analogue. Thus, each analogue will vary in its toxic potential. TTX analogues co-occur in food samples at variable concentrations. An LC-MS method was developed for the identification and quantitation of several analogues of TTX using an LTQ-Orbitrap XL mass spectrometer. The LTQ-Orbitrap XL mass spectrometer facilitates high mass accuracy measurement up to 100,000 full width at half maximum (FWHM). Using high resolution at 100,000 FWHM allows for the identification of TTX and its analogues in various matrices, including puffer fish and molluscan shellfish samples (Δ ppm = 0.28–3.38). The confirmation of characteristic fragment ions of TTX and its analogues was achieved by determining their elemental formulae via high mass accuracy. A quantitative method was then developed and optimised using these characteristic fragment ions. The limit of quantitation (LOQ) of the method was 0.136 µg g^–1 (S/N = 10) and the limit of detection (LOD) was 0.041 µg g^–1 (S/N = 3) spiking TTX standard into TTX-free mackerel fish extracts. The method was applied to naturally contaminated puffer fish and molluscan shellfish samples to confirm the presence of TTX and its analogues.     

昆明系小鼠生物法定量测定水产品中河豚毒素

为建立昆明系小鼠定量测定河豚毒素(TTX)方法,选用体质量19～21g的昆明系小鼠作为实验对象,用不同质量浓度TTX溶液腹腔注射小鼠。结果显示：TTX致昆明系小鼠的半数致死量(LD50)为0.18μg,小鼠死亡时间与TTX注射剂量满足方程Y=e(0.357/X＋1.005)。此外,采用体积分数0.5%乙酸溶液提取TTX,回收率达到90%以上,对样品的最低检测限为0.56μg/g。昆明系小鼠定量测定河豚毒素,准确、稳定、方便快捷,可用于水产品安全检测。     

Comparison of sample preparation methods,validation of an UPLC–MS/MS procedure for the quantification of tetrodotoxin present in marine gastropods and analysis of pufferfish

Tetrodotoxin (TTX) is one of the most potent marine neurotoxins reported. The global distribution of this toxin is spreading with the European Atlantic coastline now being affected. Climate change and increasing pollution have been suggested as underlying causes for this. In the present study, two different sample preparation techniques were used to extract TTX from Trumpet shells and pufferfish samples. Both extraction procedures (accelerated solvent extraction (ASE) and a simple solvent extraction) were shown to provide good recoveries (80–92%). A UPLC–MS/MS method was developed for the analysis of TTX and validated following the guidelines contained in the Commission Decision 2002/657/EC for chemical contaminant analysis. The performance of this procedure was demonstrated to be fit for purpose. This study is the first report on the use of ASE as a mean for TTX extraction, the use of UPLC–MS/MS for TTX analysis, and the validation of this method for TTX in gastropods.     

LC-MS/MS method for the determination of tetrodotoxin (TTX) on a triple quadruple mass spectrometer

Tetrodotoxin (TTX), often referred to as the ‘puffer fish’ poison, is a marine toxin and it has been identified as the agent responsible for many food poisoning incidents around the world. It is a neurotoxin that blocks voltage-gated sodium channels, resulting in respiratory paralysis and even death in severe cases. It is known to occur in many different species of fish and other organisms. The toxin is mainly found in the Southeast Asia region. Worryingly, TTX is starting to appear in European waters. It is suspected that this is a consequence of Lessepsian migration, also known as the Erythrean invasion. Therefore, straightforward and reliable extraction and analytical methods are now urgently required to monitor seafood of European origin for TTX. This paper provides a versatile, dependable and robust method for the analysis of TTX in puffer fish and trumpet shellfish using LC-MS/MS. A three-stage approach was implemented involving: (1) the screening of samples using fast multiple reaction monitoring (MRM) mass spectral analysis to identify quickly positive samples on a triple quadrupole mass spectrometer (QqQMS/MS), the API 3000; (2) a Fourier-transform (FT)-MS full-scan analysis of positive samples to collect qualitative data; and (3) a method with a longer chromatography run to identify and quantitate the positive samples using the QqQMS. The quantitative LC-QqQMS method delivered excellent linearity for solvent-based standards (0.01–7.5 µg ml^–1; R² ≥ 0.9968) as well as for matrix-matched standards (0.05–37.50 µg g^–1; R² ≥ 0.9869). Good inter-day repeatability was achieved for all the relevant analytes with %RSD values (n = 9) ranging from 1.11% to 4.97% over a concentration range of 0.01–7.5 µg ml^–1. A sample clean-up procedure for the puffer fish and trumpet shellfish was developed to ensure acceptable and reproducible recoveries to enable accurate and precise determination of TTX in a myriad of tissues types. Blank mackerel matrix was used for the TTX standard spiking studies in order to calculate the recoveries of the toxin during the extraction procedure. The recovery was 61.17% ± 5.42% for the extraction protocol. MS/MS studies were performed on a linear-trap quadruple-Orbitrap mass spectrometer (LTQ-Orbitrap) to obtain high-mass-accuracy data of the target analytes and their characteristic fragment ions in the puffer fish and trumpet shellfish samples. This facilitated identification of TTX and its associated analogues. These high-mass-accuracy studies facilitated the development of a rapid MRM-based quantitative method for TTX determination on the LC-QqQMS.     

河鲀毒素生物传感器的研究

基于河鲀毒素(TTX)对细胞膜Na+通道的阻断作用,建立一种检测TTX的简易组织生物传感器。组织生物传感器由pNa电极、青蛙膀胱膜、电位计等构成。在pNa电极顶端用两层醋酸纤维薄膜包裹,中间夹入青蛙膀胱膜,构成感受器。将电极插入pNa=4的NaCl溶液中,使电极输出信号趋于稳定。将TTX被测液注入感受器系统,测定感受器输出量的抑制值。每次测定只需5min。最低检出限为0.002MU/ml(3.56×10-3μg/ml)。用0.003%Na3N保存,可连续使用250h。以生物传感器和小鼠生物试验法比较,建立了工作曲线,便于在餐饮企业对原料鱼进行检测和应用。     

Transfer profile of orally and intramuscularly administered tetrodotoxin to artificial hybrid specimens of the pufferfish Takifugu rubripes and Takifugu porphyreus

Tetrodotoxin (TTX) was administered to artificially hybridized specimens of the pufferfish Takifugu rubripes and Takifugu porphyreus to investigate toxin accumulation in hybrids and TTX transfer/accumulation profiles in the pufferfish body. In test fish administered TTX-containing feed homogenate at a dose of ～400 MU/fish by oral gavage using a syringe (OGA group), the toxin content (MU/g tissue) of the digestive tract rapidly decreased and that of the liver increased from 1 to 24 h after administration. From 24 to 120 h, the toxin content of the liver decreased gradually, and the toxin appeared in the skin. On the other hand, intramuscularly administered TTX (400 MU/fish) was rapidly transferred to the liver and skin via the blood, and only a little toxin remained in the muscle even at 1 h (IMA group). The total amount of toxin remaining in the whole body (% of administered toxin) was 31-45% in the OGA group, and 42-74% in the IMA group; the scores in the OGA group were generally lower than those in the IMA group. In both OGA and IMA groups, the greatest amount of toxin accumulated in the liver (23-52%) after 8 h, followed by the skin (11-21%) after 72 h. The TTX administration experiment, especially using the oral gavage administration method, revealed that skins and livers of 'torama' pufferfish hybrid are endowed with TTX-accumulating ability, but the muscles are not, and that TTX taken up from toxic feed to the pufferfish body is transferred first to the liver and then to the skin via the blood.