浙江杭嘉地区水体藻毒素及富营养化状况调研

通过对嘉兴运河干流、新塍塘等河流20个采样点藻毒素及水质富营养化指标调查、评价、发展趋势分析,得出嘉兴市主要湖泊和水系的微囊藻毒素(MC-LR)分布状况及年内变化规律,为当地监管部门监测及控制微囊藻毒素提供支持与参考。采用定位仪(GPS)对各个湖泊水体进行定位,以ELISA免疫法检测MC-LR浓度,以卡尔森指数方法评价水体营养状态。结果表明, 20个采样点中,王江泾和洛东大桥2处水体全年平均藻毒素含量最高,分别为0.53μg/L和0.49μg/L。2处采样点6月份和7月份藻毒素含量在全年最高,分别为2.83μg/L和2.44μg/L。南湖中心与湘家荡的水体相比,富营养化程度更高。ELISA法能快速、灵敏地检测MC-LR含量,嘉兴地区水体6月份和7月份的微囊藻毒素含量在某些区域已超过饮用水标准,提示当地需加强监测及防控力度。     

环太湖水域微囊藻毒素-LR污染状况检测研究

目的 了解环太湖沿岸主要城市生活饮用水中微囊藻毒素-LR的污染状况, 建立环太湖水源水及无锡、苏州和湖州自来水厂出厂水中MC-LR浓度的时间分辨荧光免疫分析方法(TRFIA)。方法 将偶联牛血清白蛋白的MC-LR包被96孔板, 密封抽真空后, 加入MC-LR标准溶液或样品, 再加入抗MC-LR兔血清后用全自动TRFIA检测仪测量, 通过标准曲线计算样品中MC-LR的含量。结果 环太湖水源水MC-LR检出率为60.0%, 平均浓度仅0.390 μg/L, 自来水厂出厂水检出率为28.8%, 其中全年检出最大量1.95 μg/L。结论 太湖水存在藻毒素污染的问题, 但是水质符合国家标准。     

表面等离子体共振免疫传感器用于快速检测微囊藻毒素的研究

针对现有微囊藻毒素(MC-LR)检测方法操作复杂、因标记而污染环境、仪器贵重,不利于现场快速检测等问题,将自行研制的表面等离子体共振(SPR)生物芯片检测仪应用于微囊藻毒素的检测,提出抑制型SPR生物芯片快速检测痕量微囊藻毒素的方法。采用该方法分别对浓度为3.5、2.5、1.5、1、0μg/L的MC-LR样品进行了检测。结果表明:该方法检测限小于1μg/L,可满足世界卫生组织(WHO)对于饮用水和我国地表水环境质量标准中MC-LR最低含量检测的需求。该方法完成一个样品检测耗时约8 min,相比于高效液相色谱法(HPLC)和酶联免疫吸附法(ELISA)等传统检测方法,快速定量是其最大的优势,可用于食品质量监控和现场实时检测。     

钼对盐藻生长与物质积累的调控作用

实验研究了不同浓度的钼对盐藻细胞生长物质积累的调控作用.结果表明,培养液中供给钼过多或过少都不利于盐藻细胞的生长与物质积累.以培养基中60μg/L的钼浓度对盐藻细胞生长、蛋白质合成与β-胡萝卜素积累的促进作用最大.这一钼浓度可用于盐藻的生产性培养.当培养液中钼浓度较高(80μg/L)或较低(20μg/L)时,单个盐藻细胞中的蛋白质与β-胡萝卜素含量较高.但此时,因培养液中细胞密度较低,盐藻细胞积累的物质总量仍然较少.在钼浓度较高或较低的逆境条件下,盐藻可能通过适应性反应形成了逆境蛋白质与胡萝卜素等.     

褪黑素对雨生红球藻虾青素积累和脂肪酸合成的影响

以雨生红球藻H.pluvialis LUGU为对象,研究了不同浓度的褪黑素(MLT)对藻细胞生长、虾青素积累、脂肪酸及脂肪酸合成相关酶活性的影响。结果显示:10μmol/L MLT诱导条件下培养13 d藻细胞中虾青素含量可达26.62 mg/g,是对照组(12.3 mg/g)的2.16倍;培养5 d脂肪酸合成量(15.32%)最大,是对照组的1.24倍,同时提高了虾青素酯含量;MLT诱导条件下,脂肪酸合成关键酶乙酰辅酶A羧化酶(ACCase)活性升高,而磷酸烯醇式丙酮酸羧化酶(PEPCase)活性降低。研究表明,外源添加适当浓度的MLT不仅可以促进雨生红球藻中虾青素的积累,而且提高了脂肪酸的合成量,改变了脂肪酸组成。     

高产虾青素的雨生红球藻胁迫条件和中试研究

探究雨生红球藻在不同培养条件和规模下积累虾青素的情况,结果表明在缺氮、光强6 000~8 000Lux、装液量100 m L/500 m L三角瓶的条件下虾青素含量可达藻细胞干重的(4.20±0.19)%,质量浓度为(59.23±2.13)mg/L。平板式光照生物反应器中试实验表明,绿色营养阶段在室内和半室外培养时,培养液中的生物量可达到0.88 g/L与1.04 g/L,进入稳定期前生产率分别为0.048 5 g/(L·d)和0.057 1 g/(L·d),红色胁迫阶段在半室外的培养条件下生物量可达到1.17 g/L,藻细胞中虾青素含量为1.49%,单位体积虾青素产量为17.49 mg/L,虾青素生产率为1.05 mg/(L·d)。     

超高效液相色谱串联四极杆飞行时间质谱法同时测定蟾蜍皮中8种蟾蜍毒素

目的 建立超高效液相色谱串联四极杆飞行时间质谱法快速定性定量检测中毒患者食用剩余蟾蜍皮中8种蟾蜍毒素。方法 样品经甲醇提取后,待测液经过A CQUITY UPLC?HSS T3色谱柱(100 mm×2.1 mm,1.8μm)分离,以0.1%甲酸水溶液为流动相A、甲醇为流动相B进行梯度洗脱,流速0.3 mL/min,柱温35℃,进样量2μL,经ESI正离子模式采集。结果 在10.0～1 000.0μg/L范围内8种毒素线性关系良好,r²均≥0.990;检出限为0.7～2.1μg/kg,定量限为2.5～7.0μg/kg;三水平加标平均回收率均≥75.79%,相对标准偏差(RSD)均≤9.84%。样品中检测出蟾毒灵、去乙酰华蟾毒精、去乙酰华蟾毒它灵、伪异沙蟾毒精、蟾毒它灵,含量分别为123.8±3.1、5 920.4±1.9、680.7±3.2、671.6±9.1、1 796.6±74.9μg/kg,和蟾蜍他灵、华蟾毒精与华蟾毒它灵均未检出。结论 该方法同时测定蟾蜍毒素中华蟾毒精、蟾毒灵、伪异沙蟾毒精、蟾毒它灵、去乙酰华蟾毒它灵、去乙酰华蟾毒精、和蟾蜍他灵、华蟾毒它灵,...     

基于巯基磷酸和巯基环糊精双修饰纳米金的修饰电极测定微囊藻毒素的研究

为了快速灵敏的检测微囊藻毒素(MC)的含量,本研究根据微囊藻毒素LR亚型(MC-LR)的结构特点对纳米金进行了巯基β-环糊精和巯基磷酸的双修饰,建立了一种基于双修饰纳米金材料的微分脉冲伏安电化学分析方法检测MC。结果表明双修饰的纳米金对MC具有良好的吸附性能,其吸附量达到2.125μg/m L,静态分布系数为5.67。利用电聚合中性红修饰的玻碳电极表面正电荷吸附双修饰纳米金材料制备电化学传感器,测定MC,结果表明该电极对MC-LR的线性范围为26 nmol/L~213μmol/L,检测限为(3σ/S)19 nmol/L,且具有良好的抗干扰能力。     

光照、氮素和高盐胁迫对雨生红球藻积累虾青素的影响研究

为摸索雨生红球藻积累虾青素的最优条件,研究光照强度、NaNO_3和NaCl浓度对雨生红球藻光合作用和积累虾青素的影响,结果表明:不同光照强度处理组最大光能转化效率(Fv/Fm)较对照组均显著降低(P 0.05);试验第24天,光照强度为236μmol/m~2/s的试验组微藻积累虾青素达最高值9.01 mg/L;NaCl浓度为10 g/L和12.5 g/L时雨生红球藻Fv/Fm和有效光能转化效率值(Yield)均低于对照组,而NaCl浓度为5 g/L时微藻Fv/Fm和Yield值均高于对照组,NaCl浓度为5、7.5、10 g/L时微藻积累虾青素水平显著高于其他试验组(P 0.05),试验第24天,NaCl浓度为7.5 g/L时,微藻积累虾青素含量达到最高,较对照组提高了34.4%;NaNO_3浓度为0、0.032、0.065 g/L时,雨生红球藻Fv/Fm整体呈先上升后缓慢降低的趋势,较对照组差异显著(P 0.05),在氮胁迫处理条件下雨生红球藻积累虾青素含量逐渐升高,试验第22天后细胞内虾青素水平渐趋稳定,当NaNO_3浓度为0时细胞内虾青素含量显著高于其他试验组(P 0.05)。     

极大螺旋藻的硒富集化培养研究

为得到大量含有机硒的极大螺旋藻(Spirulina maxima),以生物量和藻体内硒富集量作为指标,研究不同硒添加量和硒添加方式对极大螺旋藻生长及富硒效果的影响。结果表明,分8d等量(100μL)多次添加100μg/mL Na2SeO3溶液,是最佳添加量和添加方式,可得到生物量为0.903g/L,单位干藻粉有机硒含量为1413.168μg/g的富硒极大螺旋藻。     

Drop coating deposition Raman (DCDR) for microcystin-LR identification and quantitation

A drop coating deposition Raman (DCDR) method was developed for the analysis of 2-200 ng samples of microcystin-LR (MC-LR), a ubiquitous and deadly hepatotoxin secreted by cyanobacteria. Solid phase extraction (SPE) of the toxin from a water sample enabled identification of MC-LR at 5 μg/L to 100 mg/L concentrations, and the collected results suggest lower detection limits can be readily attained following DCDR substrate modification. The DCDR process was applied to aqueous sample volumes of 0.5-20 μL that generated sample deposits from which MC-LR Raman spectra could be obtained within seconds. Larger volume samples were not required to improve spectral resolution. Volumes of 2 μL were ideal, producing "coffee-ring" MC-LR deposits that displayed distinct MC-LR Raman signals with high signal-to-noise within 1 s for a 200 ng sample and 300 s for a 2 ng sample. A linear correlation between Raman signal intensity and concentration was observed for 2-100 ng MC-LR samples after signal normalization. Reproducible MC-LR Raman spectra were collected from both fresh and aged samples. The presence of dissolved organic matter (DOM) did not preclude MC-LR identification in DCDR deposits of 3 μg of DOM mixed with 0.7 μg of MC-LR. Application of DCDR to environmental samples will require sample purification such as SPE before analysis, including critical cartridge wash and toxin rinsing steps. Raman based methods may one day facilitate simpler and faster sample throughput than traditional MC-LR detection methods.     

Accumulation of paralytic shellfish poison (PSP) and biotransformation of its components in oysters, Crassostrea gigas, fed with the toxic dinoflagellate Alexandrium tamarense

As a part of our studies on the mechanism of uptake of paralytic shellfish poison (PSP) and the kinetics of its accumulation in bivalves, oysters Crassostrea gigas were experimentally contaminated with PSP by being fed with the toxic dinoflagellate Alexandrium tamarense for 2, 4, 6, 8 and 10 days. Temporal variations in the PSP contents and their profiles in oysters during the feeding experiment were monitored by high-performance liquid chromatography (HPLC) and the toxin profile of the oysters was compared with that of A. tamarense. Toxins excreted from the infested oysters into the seawater for 2 and 10 days were recovered and analyzed by HPLC. PSP toxicity rapidly appeared in the tissues of oysters and their toxicity levels reached 0.6 (0.3), 2.2 (1.1), 1.0 (0.5), 3.4 (1.6) and 1.1 (0.5) MU/g (nmol/g) shucked meat at 2, 4, 6, 8 and 10 days, respectively. The accumulation rates of toxin, calculated from the total amount (nmol) of toxins expressed by the total cell number fed during the exposure period and the toxicity of the oysters, were 14.1, 18.7, 5.1, 14.9 and 3.2% for 2, 4, 6, 8 and 10 days. During feeding experiments, the toxin profile of oysters changed substantially, showing marked differences from the proportions found in the toxigenic dinoflagellate used as food. The toxin components in this strain existed almost exclusively as beta-epimers, which accounted for 66.3 mol% of the total. This contrasts with the case of the oysters, where the beta-epimers represented 24.8, 29.8, 25.1, 27.3 and 25.2 mol% of the total at 2, 4, 6, 8 and 10 days, respectively. The amount of gonyautoxin-1 (GTX1) accumulated in oysters increased linearly and slowly for 8 days and the maximum content of GTX1 reached 51.3 mol%. The composition of GTX group compounds recovered from the seawater in which the oysters had been reared was a little different from that within the oyster tissues.     

Determination of Deoxynivalenol and Nivalenol in Wheat by Ultra-Performance Liquid Chromatography/Photodiode-Array Detector and Immunoaffinity Column Cleanup

An ultra-performance liquid chromatography (UPLC®) method has been developed for the simultaneous determination of deoxynivalenol (DON) and nivalenol (NIV) in wheat. Ground sample was extracted with water and the filtered extract was cleaned up through an immunoaffinity column containing a monoclonal antibody specific for DON and NIV. Toxins were separated and quantified by UPLC® with photodiode-array detector (λ?=?220 nm) in less than 3 min. Mean recoveries from blank wheat samples spiked with DON and NIV at levels of 100–2,000 μg/kg (each toxin) ranged from 85 to 95 % for DON and from 81 to 88 % for NIV, with relative standard deviations less than 7 %. Similar recoveries were observed from spiked samples when methanol/water (80:20, v/v) was used as extraction solvent. However, by using a wheat sample naturally contaminated with DON and NIV, the one-way analysis of variance (Student–Newman–Keuls test) between different extraction solvents and modes showed that water extraction provided a significant increase (P?<?0.001) in toxin concentrations (mean values of six replicate analyses) with respect to methanol/water (80:20, v/v). No significant difference was observed between shaking (60 min) and blending (3 min). The limit of detection (LOD) of the method was 30 μg/kg for DON and 20 μg/kg for NIV (signal-to-noise ratio 3:1). The immunoaffinity columns showed saturation of DON/NIV binding sites at levels higher than 2,000 ng in blank wheat extracts spiked with the corresponding amount of mycotoxin, as single mycotoxin or sum of DON and NIV. The range of applicability of the method was from LOD to 4,000 μg/kg, as single mycotoxin or sum of DON and NIV in wheat. The analyses of 20 naturally contaminated wheat samples showed DON contamination in all analyzed samples at level ranging from 30 to 2,700 μg/kg. NIV was detected in two samples at negligible toxin levels (up to 46 μg/kg). This is the first UPLC® method using immunoaffinity column cleanup for the simultaneous and sensitive determination of DON and NIV in wheat.     

Potential health hazards due to the occurrence of the mycotoxin tenuazonic acid in infant food

The mycotoxin tenuazonic acid (TA) was analyzed in different infant foods and beverages including tea infusions (fruit, herbal and fennel tea), puree infant food in jars (complementary food and side dishes) and infant cereals (for preparation of meals after addition of water or milk) by means of a stable isotope dilution assay (SIDA). The median content of TA in infant tea infusions (n = 12) was 2 μg/L, but values up to 20 μg/L were found in fennel tea infusions. In puree infant food in jars (n = 12), the median content of TA was 7 μg/kg, but higher values were detected in products containing tomato (25 μg/kg), banana and cherry (80 μg/kg) and sorghum (20 μg/kg). Infant cereals on the basis of wheat and/or oats, rice, spelt and barley (n = 4) did not contain TA in values higher than 30 μg/kg, but if sorghum was the major ingredient (n = 12), the mean content of TA was 550 μg/kg and the maximum level was 1,200 μg/kg. The European Food Safety Authority (EFSA) evaluated the toxicological potential of TA by following the threshold of toxicological concern (TTC) approach yielding a TTC value of 1,500 ng TA/kg body weight per day. Although long-term studies are needed to enlarge the database on TA contamination of sorghum-based infant food, our preliminary study points out to a tendency that the TTC value may be exceeded by infants consuming predominantly sorghum-based food. Nevertheless, further toxicity data on TA are required with high priority to assess potential health hazards.     

栗树类芽孢杆菌S3胞外粘多糖毒理安全性评价

对栗树类芽孢杆菌（Paenibacillus castaneae）S3胞外粘多糖进行毒理学安全性评价,为其食用安全性提供依据。通过小鼠急性经口毒性试验、骨髓细胞染色体畸变、精子畸形及30 d喂养试验分别考察低（1.0 g/kg）、中（2.0 g/kg）、高剂量（4.0 g/kg）粘多糖的急性、致突变及亚急性等毒理性质。结果表明,小鼠的最大粘多糖耐受剂量＞12 g/kg,该粘多糖属于无毒物质;各剂量粘多糖对小鼠骨髓细胞染色体及精子无明显畸变作用;30 d喂养各剂量粘多糖组,小鼠生长发育良好,体质量增加、脏器系数、血常规及血生化等各项指标与对照组均无显著差异（P＞0.05）,未见明显的病理改变。因此,在该试验研究剂量和条件下,栗树类芽孢杆菌S3胞外粘多糖具有良好的食用安全性。     

Simple and high-throughput method for the multimycotoxin analysis in cereals and related foods by ultra-high performance liquid chromatography/tandem mass spectrometry

A rapid, reliable and sensitive method was developed to determine 12 mycotoxins (deoxynivalenol, aflatoxins B1, B2, G1, G2 and M1, fumonisins B1 and B2, ochratoxin A, HT-2 and T-2 toxin and zearalenone) simultaneously in maize, walnuts, biscuits and breakfast cereals. The method is based on a single extraction step using acetonitrile/water mixture (80/20 v/v) followed by ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC–MS/MS). The selectivity of the MS/MS detection allowed the elimination of further clean up steps. Extraction, chromatographic and detection conditions were optimised in order to increase sample throughput and sensitivity. Matrix-matched calibration was used for quantification and recoveries of the extraction process ranged from 70.0% and 108.4%, with relative standard deviations lower than 25% in all the cases, when samples were fortified at 5 and 50 μg/kg. Limits of detection ranged from 0.01 to 2.1 μg/kg and limits of quantification ranged from 0.03 to 6.30 μg/kg, which were always below the tolerance levels of mycotoxins set by European Union in the matrices evaluated. Several samples were analysed and aflatoxins B1, B2, G1, G2 and T-2 toxin were detected in one maize sample, with concentrations lower than 6.0 μg/kg and deoxynivalenol was detected in a breakfast cereal at 42.1 μg/kg.     

新食品原料食叶草的安全性评估

摘要:目的 本研究旨在系统评估食叶草的食用安全性。方法 依据现行《新食品原料安全性审查管理办法》的规定,采用国标或其他相关方法对食叶草开展成分分析（营养成分、可能存在的天然有毒物质）、卫生学检验（重金属、农药残留）、毒理学试验（包括急性经口毒性试验、三项遗传毒性试验（Ames试验、哺乳动物红细胞微核试验和小鼠精母细胞染色体畸变试验）、90 d经口毒性试验和致畸试验）,结合食用人群、食用历史和不良反应调查结果,综合评估食叶草作为新食品原料,在成人（孕妇、乳母除外）每日推荐摄入量50 g情况下的食用安全性。结果 食叶草富含蛋白质和钾元素（均值分别为3.42 g/100 g和0.489 g/100 g）,其所含生物活性物质粗多糖和总黄酮含量分别为348.9 mg/100 g和40.67 mg/100 g。蒽醌含量为1.29 mg/100 g,按成人体重60 kg、每日食叶草推荐摄入量50.0 g计算,经食叶草摄入的蒽醌量为0.011 mg/kg BW,远小于其未观察到有害作用剂量（31.3 mg/kg BW）;而草酸含量与菠菜等蔬菜相比含量较低（0.32 g/100 g）。植物中重金属和农药残留均符合我国食品安全国家标准的规定。食叶草（粉）急性经口毒性试验表明,该物质属实际无毒级;三项遗传毒性试验结果均为阴性;90 d经口毒性试验未观察到有害作用剂量为8.0 g/kg BW,致畸实验结果显示试验剂量下食叶草（粉）对SD大鼠无致畸作用。对食用食叶草及其制品的人群调查结果中未见不良反应的报告。结论 评估认为,食叶草作为新食品原料,按照我国成年人（除孕妇、乳母外）每日摄入不超过50.0 g,对人体健康造成的潜在风险较低     

HPLC法测定食用动物油脂中丙二醛残留量

建立柱前衍生高效液相色谱法测定动物油脂中丙二醛残留量的方法。试样经三氯乙酸混合液提取,与硫代巴比妥酸(TBA)进行衍生化反应后,采用Thermo Syncronis C₁₈色谱柱(250 mm×4.6 mm,5μm),以0.01 mol/L乙酸铵-甲醇(体积比65︰35)为流动相,等度洗脱,二极管阵列检测器检测。结果表明,丙二醛在0.01~2.0μg/mL范围均有良好线性关系,相关系数(r)为0.9999;方法检出限为0.0064 mg/kg;3个不同水平(0.1,0.2和0.6 mg/kg)的加标平均回收率为97.41%~102.25%,RSD值为1.08%~2.15%。方法具有操作简便、灵敏度高、重现性佳等特色,适用于动物油脂中丙二醛残留量检测。     

磺胺甲噁唑和磺胺嘧啶在青石斑鱼组织中的分布及代谢规律

通过一次性投喂各含有200 mg/kg磺胺甲噁唑（sulfamethoxazole,SMZ）和磺胺嘧啶（sulfadiazine,SDZ）的饲料,研究两种药物在青石斑鱼中各组织分布与消除规律。采用超高效液相色谱-串联质谱检测青石斑鱼各组织中SMZ和SDZ的含量,并用内标法定量。结果表明,SMZ在青石斑鱼各组织中的最大含量：肝脏、背肌、血浆、肾脏和鳃依次为827.97μg/kg、776.70μg/kg、610.29μg/L、432.14μg/kg和345.18μg/kg。SDZ在青石斑鱼各组织中的最大含量：肝脏、背肌、鳃、血浆和肾脏依次为895.30μg/kg、660.55μg/kg、431.88μg/kg、419.56μg/L和310.67μg/kg。SMZ在青石斑鱼各组织中的半衰期：肾脏、鳃、背肌、血浆和肝脏半衰期依次为26.65、21.00、20.38、18.73h和16.90h。SDZ在青石斑鱼各组织中的半衰期：肾脏、血浆、鳃、背肌和肝脏依次为31.50、27.72、24.75、21.66h和18.24h。SMZ和SDZ在青石斑鱼肝脏中半衰期最短,代谢速度最快;在肾脏中半衰期最长,代谢速度最慢。在水温（25±2）℃条件下,SMZ和SDZ各200mg/kg的剂量同时单次投喂青石斑鱼,建议休药期不低于3d。SMZ和SDZ代谢规律研究为磺胺类药物在水产品中的合理使用提供了参考。     

固相萃取-液质联用测定辣椒粉中苏丹红

该试验建立了分子印迹固相萃取-超高效液相色谱-串联质谱法检测辣椒粉中苏丹红Ⅰ、Ⅱ、Ⅲ、Ⅳ的方法。辣椒粉样品经过正己烷提取,氮吹浓缩,苏丹红专用分子印迹固相萃取柱净化和富集,以体积分数为0.2%的甲酸水溶液和乙腈为流动相梯度洗脱,经Agilent ZORBAX Eclipse Plus C₁₈(2.1 mm×50 mm,1.8μm)柱分离,ESI+电喷雾模式扫描,MRM多反应模式监测,外标法定量。结果显示,该方法下4种苏丹红的检出限均为5μg/kg,4种化合物在0~50μg/L质量浓度范围内线性关系良好,相关系数均在0.9990以上;当加标浓度为10,50和100μg/kg时,回收率在86.5%~102.2%之间,相对标准偏差均小于6.0%。该方法可为辣椒粉中4种苏丹红的定性定量分析提供参考。