抗黄曲霉毒素B_1单抗独特型抗体的制备

目的研制黄曲霉毒素B1标准品的替代品并应用于酶联免疫吸附试验(ELISA)。方法在研制了抗黄曲霉毒素B1单抗的基础上,将抗体用无花果蛋白酶进行酶切,制备出抗黄曲霉毒素B1单抗F(ab’)2片段,并以F(ab’)2片段作为免疫原免疫日本大耳白兔,制备出抗黄曲霉毒素B1单抗独特型抗体,并对该独特型抗体进行鉴定。结果该独特型抗体是Ab2β型,与黄曲霉毒素B1存在内影像关系。结论该抗体可以替代黄曲霉毒素B1标准品应用于ELISA检测。     

绿色无毒黄曲霉毒素B_1免疫检测方法

建立了一种基于抗独特型抗体的绿色无毒的ELISA方法,并用于检测面粉中黄曲霉毒素B1。通过酶解2种抗黄曲霉毒素小鼠单克隆抗体(11A9和1G3)制备F(ab’)2片段,并将其免疫新西兰大白兔制备抗独特型抗体。最终得到两种抗独特型抗体,并对抗独特型抗体和AFB1进行相关性分析。通过实际样品添加回收,其批内回收率为115.60%~121.88%,变异系数在5%以内;而批间回收率为111.89%~126.98%,变异系数低于8%。分析结果准确可靠,表明此无毒绿色ELISA是一种安全可靠的AFB1分析方法。     

量子点标记荧光免疫法检测花生中黄曲霉毒素B_1

建立了基于量子点标记二抗的间接竞争荧光免疫吸附测定方法（indirect competitive fluorescence-linked immunosorbent assay,cFLISA）,并研究检测花生中黄曲霉毒素B1的可行性。采用谷胱甘肽为稳定剂,在水相中直接合成碲化镉（CdTe）量子点,并利用1-（3-二甲氨基丙基）-3-乙基碳二亚胺盐酸盐（EDC）与兔抗鼠二抗进行共价偶联,以黄曲霉毒素B1的单克隆抗体建立cFLISA方法。结果表明,该方法的灵敏度和最低检测限值分别为0.023ng/mL和0.001ng/mL,与传统的有机染料FITC-二抗法比较,灵敏度提高了30倍,花生样品加标0.1、0.05和0.025ng/g,回收率范围在88%～116%之间,变异系数均小于10%。建立的cFLISA方法可以较好的检测花生中黄曲霉毒素B1,并为其它真菌毒素的检测提供参考。     

黄曲霉毒素B_1脱毒菌株的分离鉴定及在花生粕中的应用

用以香豆素为唯一碳源的培养基进行初筛,结合固态发酵降解黄曲霉毒素(AFB1)实验和抑制黄曲霉生长实验进行复筛,获得一株能够高效降解AFB1的菌株。经形态观察与16S r DNA序列比对,确定该菌株为解淀粉芽孢杆菌。该菌不仅对低AFB1含量(12.32μg/kg)的花生粕样品有88.23%的AFB1降解率,而且对高AFB1含量(145.17μg/kg)的花生粕样品也有81.39%的AFB1降解率,降解后其AFB1含量低于30μg/kg,符合国家对饲料中AFB1含量(≤50μg/kg)的限量要求。     

酶联免疫吸附法（ELISA）测定黄曲霉毒素B_1的样品基质效应研究

为了探寻与免疫分析相配套的简易前处理方法,以黄曲霉毒素B1（AFB1）为靶标,花生样品为对象,研究了可能影响ELISA测定结果的样品前处理方法;采用实际样品加标回收的方法,研究比较了4种不同浓度（5%、10%、20%和30%）甲醇稀释的样品基质同源ELISA灵敏度。结果表明,甲醇稀释度越小基质效应越明显;样品前处理中除油处理可显著提高回收率;样品前处理中免疫亲和柱净化后可消除相当一部分基质效应的影响,提高了回收率。研究结果为进一步开发简单快速的前处理方法提供了借鉴。     

植物油黄曲霉毒素B_1含量的检测及能力验证

用酶联免疫法测定食用植物油中黄曲霉毒素B1,对酶联免疫法前处理条件进行了优化,结果表明,选择一步萃取与甲醇水溶液(7+3)提取相结合的方法,回收率较理想,在0.1~2.0μg/kg浓度范围内,黄曲霉毒素B1具有良好的线性关系,方法检出限为1.0μg/kg,加标回收率为101.3%~104.3%,精密度试验RSD为1.04%~3.16%,并将建立的方法应用于2012年和2013年度植物油中黄曲霉毒素B1的能力验证,结果|Z|比分数均远远小于2,取得了满意的结果。酶联免疫法操作简单,分析速度快,因此可广泛应用于食用植物油中黄曲霉毒素B1检测。     

抗黄曲霉毒素B1独特型纳米抗体的表达及复性

目的 快速制备大量具有生物活性的独特型纳米抗体F7。方法 构建pET22b-F7原核表达载体, 转化至大肠杆菌E. coli BL21(DE3)中进行诱导表达, 对诱导温度、诱导剂浓度和诱导时间进行优化。结果 独特型纳米抗体F7表达量有所增加但主要以包涵体形式存在。经过包涵体的溶解、复性获得了具有生物活性的抗AFB1独特型纳米抗体, ELISA证实复性后的蛋白依然存在对鼠源AFB1抗体的结合特性。结论 为后续抗体的性质研究和改造奠定了基础。     

间接竞争酶联免疫分析方法检测花生中黄曲霉毒素B1

目的:针对花生中强毒性污染物黄曲霉毒素B_1(AFB_1)建立了一种准确、灵敏的间接竞争酶联免疫分析方法(ic-ELISA)。方法:制备了AFB_1包被抗原,包被量为0.1μg/孔;抗体稀释64 000倍;选用0.1%脱脂乳粉为方法封闭液,0.01 mol/L磷酸盐缓冲液(PBS,pH 7.4)为样品稀释液;结果:在最优的试验条件下,建立的ic-ELISA对目标物AFB1的检测线性范围为0.0097～0.088μg/L(R2=0.999);灵敏度(IC50)和检出限(IC15)分别达到0.027μg/L和0.0066μg/L。板内变异系数(n=6)为0.29%～16.9%,板间变异系数(n=6)为0.22%～21.19%。在花生样品中AFB_1的检测灵敏度(IC50)为1.04μg/kg,回收率为96.67%～106.51%。结论 :本研究建立的ic-ELISA方法操作相对简便,检测灵敏度高、准确性好,可稳定地用于花生样品中AFB_1污染物的定量分析检测。     

2011~2015年花生和小麦制品中黄曲霉毒素B1的检测

目的 对2011~2015年花生和小麦制品中黄曲霉毒素B1进行检测分析和安全评价。方法 采用酶联免疫法对2011~2015年的1140个花生类和6475个小麦类制品的黄曲霉毒素B1的含量进行检测。结果 2011~2015年检测的花生类制品合格率分别为95.5%、98%、97.4%、98.3%和98.7%; 检测的各种小麦类制品合格率均为100%。结论 花生类制品合格率逐年上升, 但仍存在一定的质量隐患; 小麦类制品质量均符合国家规定限量。     

基于激光诱导荧光光谱技术检测花生中黄曲霉毒素B1

目的 探究激光诱导荧光（laser induced fluorescence,LIF）技术检测花生中黄曲霉毒素B1（aflatoxin B1,AFB1）的可行性,定性和定量分析花生中的AFB1。方法 制备不同浓度梯度的污染花生,经LIF系统采集荧光光谱,平滑后分析光谱数据结构。基于全波长光谱使用5种不同建模方法对污染花生定性判别,采用偏最小二乘法回归（partial least squares regression,PLSR）和BP神经网络（BP neural networks,BPNN）进行定量预测。通过竞争性自适应重加权采样（competitive adaptive reweighted sampling,CARS）提取特征波长,研究其对定性和定量预测的影响。结果 对于全波长光谱数据,线性核函数的支持向量机（support vector machine with linear kernel function,SVM（Linear） ）建立的判别模型效果最优,预测正确率100%。PLSR和BPNN均获得较好的定量预测效果,剩余预测偏差（residual predictive deviation,RPD）＞3.0,检出限（limit of detection,LOD）＜20 μg/kg;对于特征光谱数据,SVM（Linear）定性判别预测正确率93.94%,F1值为0.94,ROC曲线下面积（area under the curve,AUC）为0.989。建立的PLSR模型性能优于未提取特征波长的两种定量模型,RPD为3.36,LOD为14.76 μg/kg。结论 LIF技术检测花生中的AFB1简单快速,定性定量预测模型准确性好,具有一定可行性。     

高特异性黄曲霉毒素B_1单克隆抗体的制备及特性研究

本研究将黄曲霉毒素B1转化为半缩醛B2a,在硼氢化钠（NaBH4）还原作用下与载体蛋白偶联制备完全抗原。将制备的完全抗原免疫Balb/c小鼠,经4次免疫后取其脾脏与小鼠骨髓瘤细胞Sp2/0细胞融合,采用半固体培养基筛选后鉴定,获得杂交瘤细胞株3A12,抗体的灵敏度可达6.1±0.025ng/mL,抗体与其它黄曲霉毒素B2、G1及G2的交叉反应率依次为7.8%、20.2%及0.6%,与黄曲霉毒素M1交叉反应率小于0.1%。本研究为研发花生等农产品黄曲霉毒素B1特异性免疫分析技术及产品奠定了重要基础。     

QuEChERS-表面增强拉曼光谱法测定花生中黄曲霉毒素B1

目的 建立基于QuEChERS净化和表面增强拉曼光谱法(surface-enhanced Raman spectroscopy,SERS)测定花生中黄曲霉毒素B1(aflatoxin B1,AFB1)的分析方法.方法 以纳米银(nano silver,AgNPs)作为SERS活性基底,结合QuEChERS样品预处理技术...     

Degradation of aflatoxin B1 in peanut meal by electron beam irradiation

The effects and safety of electron beam irradiation (EBI) treatment on the detoxification of aflatoxin B₁ (AFB₁) in the peanut meal were evaluated in this article. The AFB₁ degradation was predominantly affected by both initial AFB₁ and water concentrations. The degradation of AFB₁ in the selected concentrations (0.5–5 ppm) was proven to follow pseudo first-order reaction kinetics (R² > 0.95). The AFB₁ degradation was faster when the initial concentration was 5 ppm and the moisture content was 21.47%, in comparison with the initial concentration of 1 ppm and 0.5 ppm and the moisture content of 14.32% and 8.74%, respectively. The Ames and cytotoxicity tests were employed to evaluate the residual toxicity of EBI-treated peanut meal. The mutagenic activity of EB-treated samples was completely lost compared with that of untreated samples and the degradation products in peanut meal has almost no cell toxicity.     

环糊精对花生黄曲霉毒素B_1荧光增强作用与应用研究

本文探讨了一种新型无毒的黄曲霉毒素荧光增强剂—β-环糊精及其衍生物。结果表明,在2mL黄曲霉毒素B1溶液中加入1mL0.01mol/Lβ-环糊精或其衍生物时荧光增强倍数最大,其中β-环糊精可使荧光信号增强5倍,2,6-二甲基-β-环糊精荧光增强倍数可达到8倍,表明β-环糊精及其衍生物对黄曲霉毒素荧光增强作用极为显著;利用该增强剂在黄曲霉毒素荧光增强前和荧光增强后的信号变化量和黄曲霉毒素浓度之间的线性关系建立了新型荧光增强剂免疫亲和柱荧光光度检测方法,最低检出限可达0.3μg/kg,相关系数都达到0.99以上;对花生样品的添加回收率在90%～120%之间;与基于高效液相色谱国家检测标准方法比对分析,两种检测方法无显著差异,相对误差小于4%,为黄曲霉毒素高灵敏快速检测技术开辟了新的途径。     

黄曲霉毒素限量标准对我国居民消费安全和花生产业的影响

2009-2010年对全国产后花生黄曲霉毒素污染进行了普查监测,根据检测结果,利用蒙特卡罗方法,开展了我国与国际食品法典委员会（CAC）、欧盟、日本、澳大利亚和新西兰等国际组织和主要贸易国不同花生黄曲霉毒素限量对我国人群直接食用产后花生的致癌风险和对产业影响的研究。结果表明,不同花生黄曲霉毒素限量标准对我国人群摄入产后花生导致的原发性肝细胞癌年发生率影响差异不显著,但对经济和产业造成的影响差异显著。研究结果为制定我国花生黄曲霉毒素限量标准以及促进花生生产、贸易提供技术参考。     

酶联免疫法测定食品和饲料中的黄曲霉毒素

本实验应用酶联免疫方法对食品和饲料中的黄曲霉毒素进行测定，奶粉中黄曲霉毒素M1的多次重复测定的结果的变异系数小于12％，回收率接近90％。说明本试剂盒测定结果比较精确，重现性较好。食品辅料及饲料中的黄曲霉毒素B1的测定结果的变异系数小于15％。说明实验结果的重现性较好。     

酶联免疫法测定食品和饲料中的黄曲霉毒素

本实验应用酶联免疫方法对食品和饲料中的黄曲霉毒素进行测定,奶粉中黄曲霉毒素M1的多次重复测定的结果的变异系数小于12%,回收率接近90%。说明本试剂盒测定结果比较精确,重现性较好。食品辅料及饲料中的黄曲霉毒素B1的测定结果的变异系数小于15%,说明实验结果的重现性较好。     

Comparison of nixtamalization and extrusion processes for a reduction in aflatoxin content

Traditional nixtamalization and an extrusion method for making the dough ( masa ) for corn tortillas that requires using lime and hydrogen peroxide were evaluated for the detoxification of aflatoxins. The traditional nixtamalization process reduced levels of aflatoxin B 1 (AFB 1 ) by 94%, aflatoxin M 1 (AFM 1 ) by 90% and aflatoxin B 1 -8,9-dihydrodiol (AFB 1 -dihydrodiol) by 93%. The extrusion process reduced levels of AFB 1 by 46%, AFM 1 by 20% and AFB 1 -dihydrodiol by 53%. Extrusion treatments with 0, 0.3 and 0.5% lime reduced AFB 1 levels by 46, 74 and 85%, respectively. The inactivation of AFB 1 , AFM 1 and AFB 1 -dihydrodiol in the extrusion process using lime together with hydrogen peroxide showed higher elimination of AFB 1 than treatments with lime or hydrogen peroxide alone. The extrusion process with 0.3% lime and 1.5% hydrogen peroxide was the most effective process to detoxify aflatoxins in corn tortillas, but a high level of those reagents negatively affected the taste and aroma of the corn tortilla as compared with tortillas elaborated by the traditional nixtamalization process.     

Potential ACE-inhibitory activity and nanoLC-MS/MS sequencing of peptides derived from aflatoxin contaminated peanut meal

Our lab has developed a process for sequestering aflatoxin from contaminated peanut meal (PM) using commercial bentonite clays while protein is simultaneously extracted and hydrolyzed by a commercial protease. The objectives of this study were to sequence generated peptides and evaluate their potential ACE-inhibitory properties. Aflatoxin in the unprocessed PM was 610 μg kg⁻¹ compared to 9.7 μg kg⁻¹ on a dry weight basis in the 120 min hydrolysate. This hydrolysate displayed significant ACE-inhibitory activity with an IC₅₀ of 295.1 μg mL⁻¹. Ultrafiltration and size exclusion chromatography (SEC) improved the ACE-inhibitory properties, with the SEC fraction containing the smallest peptides having an IC₅₀ = 44.4 μg mL⁻¹. Additionally, 271 unique peptides were identified by nanoLC-MS/MS, of which 147 belonged to major seed storage proteins. This advanced characterization data will ultimately allow for more efficient production of hydrolysates with ACE-inhibitory activity or other bioactivities of interest from PM.     

Safety evaluation of aflatoxin B1 in peanut oil after ultraviolet irradiation detoxification in a photodegradation reactor

The high incidence of aflatoxin B₁ (AFB₁) in peanut oil has caused wide public concern in the world. Many studies have verified that ultraviolet (UV) irradiation can degrade AFB₁ in foods. A new photodegradation reactor has been developed to study the photodegradation efficiency of AFB₁ in peanut oil, and the safety of peanut oil was evaluated after UV irradiation detoxification based on the mutagenicity of Salmonella typhimurium tester strains and cytotoxicity of HepG2 cells. The results showed that AFB₁ in peanut oil could be decomposed efficiently using the photodegradation reactor. AFB₁ was decreased from 51.96 ± 4.24 to 7.23 ± 0.59 μg kg^?1 in 10 min and reduced by 86.08% compared with that of the negative control. The residual AFB₁ in peanut oil was far less than the limit level set by Chinese government (20 μg kg^?1). The Ames test and cell viability assay revealed that 10 min of UV irradiation reduced significantly the toxicity of AFB₁ in peanut oil. All the results suggest that the deleterious effects of AFB₁ can be highly reduced by UV irradiation in the photodegradation reactor, and the reactor can be applied in a large scale in detoxification of AFB₁ in peanut oil in the oil industry.