粮油产品黄曲霉毒素B1检测技术研究进展

对黄曲霉毒素检测技术进行了简要介绍和评述,并对近年来粮油及制品黄曲霉毒素B1快速检测方法研究进展和发展趋势进行了综述和讨论.     

抗黄曲霉毒素B1单链抗体(scFv)库的建立和筛选

从抗黄曲霉毒素B1单克隆抗体细胞系中扩增到重链可变区(VH)和轻链可变区(VL),经PCR将VH、Linker和VL连接成单链抗体(scFv),并克隆到载体pCANTAB-5E,经电转化大肠杆菌TG1,构建了库容量为1×108的单链抗体库.通过辅助噬菌体M13K07感染后,使单链抗体展示在噬菌体衣壳蛋白pⅢ的N端,得到客容量为1×108的噬菌体展示的抗体库.将抗体库用于"淘洗-富集-扩增"4轮以后,得到针对AFB1特异性的噬菌体抗体.进一步以噬菌体感染大肠杆菌HB2151,在细菌细胞周质中可溶性大量表达,最终经竞争EUSA分析表明获得了3株可以稳定分泌抗黄曲霉毒素B1 scFv菌株,分别命名为3C3、383和4A2.     

黄曲霉毒素B1标准替代物的制备及其在花生样品检测中的应用

以抗黄曲霉毒素B1（AFB1）单克隆抗体的F(ab’)2 片断为抗原免疫兔子,得到AFB1抗独特型抗体。经过酶联免疫吸附法（ELISA）条件的优化,建立AFB1抗独特型抗体最佳竞争抑制曲线。该曲线与AFB1竞争抑制曲线相比较可知,AFB1抗独特型抗体与AFB1之间存在指数增长关系,且相关性系数R为0.999 8。以AFB1抗独特型抗体浓度与其相应的抑制率作标准曲线,ELISA法测定花生中AFB1添加回收率,范围在90.4%~100.2%之间。综上,AFB1抗独特型抗体可以作为AFB1无毒替代标准品,为黄曲霉毒素检测无毒化提供了广阔的应用前景。     

高特异性黄曲霉毒素B1单克隆抗体的 制备及特性研究

本研究将黄曲霉毒素B1转化为其半缩醛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%。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。     

粮油黄曲霉毒素B1 高效快速检测微柱的研制

研制并比较了7种不同类型的黄曲霉毒素B1 纯化微柱,优选出AFTB1 - Ⅰ亲和微柱,具有分离高效、操 作简便适于快速检测的特点。设计出与微柱净化配套使用的半自动化负压装置,可简化净化操作步骤、缩短操作 时间。对亲和微柱的进样条件、淋洗条件、洗脱条件、流速、结合容量、加标回收率、稳定性等进行了研究,结果表 明:微柱的结合容量> 25μg/kg,不同水平加标回收率范围为80. 00% ～91. 50%。不同批次微柱的加标回收率相对 标准偏差为1. 76% ,无显著差异。同时,采用现有国标方法,利用本实验研制的亲和微柱对实际样品净化,不同水 平加标回收率范围为80. 00%～92. 00% ,能满足国标检测限量范围内粮油中黄曲霉毒素B1 的快速检测要求。     

黄曲霉毒素B1检测中的应用

 采用黄曲霉毒素时间分辨荧光免疫层析试纸条及配套的时间分辨荧光速测仪,对油料饼粕中黄曲霉毒素B1的快速检测进行了应用研究。该时间分辨荧光免疫分析技术是基于时间分辨荧光免疫层析试纸条和载有Eu(Ⅲ)标记特异性单克隆抗体的样品瓶建立的检测技术。时间分辨荧光速测仪可内置标准曲线,直接输出检测结果。对6种油料饼粕做黄曲霉毒素B1添加回收率实验,回收率在70%～120%之间,批间、批内变异系数＜15%。在实际样品的检测中,时间分辨荧光免疫层析试纸条检测技术与液相色谱-串联质谱法相比,检测结果相对误差＜15%。时间分辨荧光免疫层析试纸条检测技术测定快速、准确,技术稳定、可靠,设备经济、小型,适用于大批量油料饼粕样品的快速检测和风险评估,具有广阔的应用前景。     

免疫亲和净化-高效液相质谱法检测茯砖茶中黄曲霉毒素B1

为建立检测茯砖茶中黄曲霉毒素B1的免疫亲和净化-高效液相色谱质谱分析方法,并对20份茯砖茶样品中黄曲霉毒素B1含量进行测试.样品经过70％甲醇溶液匀浆提取,免疫亲和柱富集净化,经C18色谱柱分离后采用三重四级杆质谱检测.结果表明,HPLC-MS检测线性关系良好,方法检出限为0.03μg/kg,加标浓度2～20μg/kg...     

黄曲霉毒素B_1对小鼠肝脏蛋白质组影响的初步研究

采用双向电泳及质谱分析等方法分析了黄曲霉毒素B1对小鼠肝脏蛋白表达的影响。结果表明,黄曲霉毒素B1不仅能使小鼠逐渐消瘦,肝脏组织出现病变,且能诱导或抑制肝脏部分蛋白的表达。经双向电泳发现,有35个蛋白质点发生变化。对其中新出现或明显上调的5个蛋白质点以及缺失的或明显下调的7个蛋白质点进行质谱分析,结果发现,有5个蛋白属于引发小鼠肝脏发生病变甚至是癌变的蛋白,7个蛋白属于参与小鼠肝脏能量与物质代谢的蛋白,这些蛋白对小鼠肝脏癌变过程都有一定的影响。黄曲霉毒素B1可能通过作用于这些蛋白质使小鼠肝脏发生病变甚至是癌变。本研究鉴定出了12个蛋白,为后续研究黄曲霉毒素B1诱发肝脏癌变的机理及其对人类肝脏蛋白的影响打下基础。     

量子点标记荧光免疫法检测花生中黄曲霉毒素B1

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。     

酶联免疫吸附法（ELISA）测定黄曲霉毒素B1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思路。     

甘蓝型油菜分枝籽粒油酸含量的光谱估测

为寻找最佳光谱检测部位,创新一种高油酸油菜种质资源筛选方法,以44个高油酸含量的甘蓝型油菜为材料,按照从主茎、一次分枝到二次分枝的顺序,采集籽粒反射光谱及油酸含量数据,分析不同部位的原始及一阶微分光谱与对应籽粒油酸含量的相关关系,建立了基于全波长、特征波长的逐步多元线性回归(stepwise multiple line...     

转基因高油酸油菜株系W-4种子脂肪酸组成

为研究fad2基因下调表达对油菜种子脂肪酸组成的影响,分析比较了转基因高油酸油菜品系W-4的T5、T6和T7种子以及非转基因对照Westar种子中的脂肪酸组成。数据显示W-4种子中油酸平均含量为84.61%±1.41%,较对照增加了25.91%,达到极显著水平;亚油酸和亚麻酸含量分别为3.22%±0.56%和3.45%±0.51%,较对照分别下降了80.89%和47.00%,降幅均达极显著水平。此外,W-4种子中棕榈酸平均含量为3.42%±0.18%,较对照Westar下降18.10%,达到极显著水平;而硬脂酸平均含量为1.87%±0.19%,较对照下降了8.33%,亦达到显著水平;W-4种子中廿碳烯酸的平均含量为1.54%±0.06%,较对照平均增幅为18.46%,达到极显著水平;W-4平均芥酸含量较对照略有增加,但不显著。结果表明油菜种子中fad2基因下调表达对种子的脂肪酸合成与积累影响较大,其不仅显著降低种子中多不饱和脂肪酸含量,增加油酸的含量;而且也显著降低饱和脂肪酸含量,并促进长链单烯酸的合成。     

高油酸花生遗传改良研究进展

油酸和亚油酸是花生种仁中的主要脂肪酸,其含量和比例是花生和花生油的重要品质指标,花生中的油酸含量存在丰富的遗传变异,与普通花生相比,高油酸花生因含有较高的不饱和脂肪酸更受到消费者的青睐。提高花生油酸含量、降低亚油酸含量(即提高油酸、亚油酸比值,O/L值) 是花生品质改良的重点,也是国内外研究的热点之一。通过育种途径改良花生脂肪酸成分及其含量,对于提高人民生活水平和增进营养健康具有重要意义。本文围绕着调控油酸含量的脂肪酸脱氢酶FAD2 基因的特点,高油酸花生分子标记的研究进展,油酸含量的检测技术和高油酸花生育种的方法进行了综述,总结了中美两国高油酸花生育成的新品种以及对高油酸花生育种的现状,并进行了展望。     

基于RNA-Seq技术的向日葵油酸形成的转录组学分析

本研究以高油酸材料J9和低油酸材料NK244为研究对象,在授粉后20d取材,采用RNA-Seq技术对其进行转录组学研究。经比较分析,2个材料共鉴定到差异表达基因（DETs）5 447个,其中上调表达基因2 709个,下调表达基因2 738个。对DETs进行生物信息学分析,GO信息分析表明,有2 218个DETs富集到57个功能条目,其中上调表达基因主要涉及催化活性、异构酶活性、碳水化合物结合、DNA结合、脂结合、碳水化合物过程、脂代谢过程和脂肪酸生物合成等;下调表达基因主要涉及蛋白激酶活性、ATP结合、蛋白磷酸化、tRNA氨酰化的蛋白质翻译和纤维素生物合成过程。KEGG分析中799个差异表达片段被显著富集在28个通路,主要为碳水化合物代谢通路和脂代谢通路。本研究为向日葵油酸调控机理研究提供了一定理论依据,为向日葵品质育种与种质创新提供理论参考。     

The physicochemical and tribological properties of oleic acid based triester biolubricants

Currently, increasing pollutants in the environment lead to an increase in the use of plant oil based biolubricants. These products could lead to a significant reduction in environmental pollution and thus contribute to the discovery of a replacement for petroleum-based lubricants. Plant oils are advantageous because they are rapidly biodegradable, renewable, excellent lubricants and inexpensive to produce. However, these oils show poor stability to oxidation and unfavorable properties at low temperatures. Thus, this work outlines modifications in the epoxidation, oxirane ring opening, esterification and acylation reactions used to produce oleic acid based triester derivatives. These products have improved physicochemical and tribological properties that make them good biolubricant base stock candidates. Octyl 9-(lauroyloxy)-10-(behenoxy)octadecanoate 22 has the lowest pour point (−45 °C) while octyl 9-(lauroyloxy)-10-(octyloxy)octadecanoate 16 has the highest onset temperature. Based on the results, an increase in mid chain substituent length improves the pour point and anti-wear properties but conversely lowers the onset temperature.     

基于主成分和聚类分析的冀花高油酸花生品种综合评价

利用3年表型和重测序数据分别对11个冀花高油酸花生品种进行主成分分析和聚类分析。结果发现,15个性状中脂肪含量变异系数最小为1.91%,除亚油酸外产量性状变异均大于品质变异系数,百果重与百仁重呈极显著正相关,油酸和亚油酸呈极显著负相关。主成分分析提取的前2个主成分累计贡献率达78.99%,利用表型性状对品种进行分类,结果与系谱关系部分一致。质控后获得320 000个分布均匀的高质量SNP位点,利用基因型数据将11个品种聚为4类,能够将同一组合衍生品种划分为同一类,结果与品种系谱关系一致。因此,利用基因型数据更能准确反映品种内在遗传基础,为种质资源的分类和利用提供参考。     

The Inhibitory Effect of Phycocyanin Peptide on Pulmonary Fibrosis In Vitro

Phycocyanin is an excellent antioxidant with anti-inflammatory effects on which recent studies are growing; however, its specific target remains unclear. Linear tetrapyrrole compounds such as bilirubin have been shown to lead to the induction of heme oxygenase 1 expression in vivo, thus achieving antioxidant and anti-inflammatory effects. Phycocyanin is bound internally with linear tetrapyrrole phycocyanobilin in a similar structure to bilirubin. We speculate that there is probably a way of inducing the expression of heme oxygenase 1, with which tissue oxidative stress and inflammation can be inhibited, thus inhibiting pulmonary fibrosis caused by oxidative damage and inflammation of lung. By optimizing the enzymatic hydrolysis process, phycocyanobilin-bound phycocyanin peptide were obtained, and its in vitro antioxidant, anti-inflammatory, and anti-pulmonary fibrosis activities were investigated. The results show that the phycocyanobilin peptide was able to alleviate oxidative and inflammatory damage in cells through the Keap1-Nrf2-HO-1 pathway, which in turn relieved pulmonary fibrosis symptoms.     

The Potential Protective Effect and Possible Mechanism of Peptides from Oyster (Crassostrea hongkongensis) Hydrolysate on Triptolide-Induced Testis Injury in Male Mice

Peptides from oyster hydrolysate (OPs) have a variety of biological activities. However, its protective effect and exact mechanism on testicular injury remain poorly understood. This study aimed to evaluate the protective effect of OPs on triptolide (TP)-induced testis damage and spermatogenesis dysfunction and investigate its underlying mechanism. In this work, the TP-induced testis injury model was created while OPs were gavaged in mice for 4 weeks. The results showed that OPs significantly improved the sperm count and motility of mice, and alleviated the seminiferous tubule injury. Further study showed that OPs decreased malonaldehyde (MDA) level and increased antioxidant enzyme (SOD and GPH-Px) activities, attenuating oxidative stress and thereby reducing the number of apoptotic cells in the testis. In addition, OPs improved the activities of enzymes (LDH, ALP and ACP) related to energy metabolism in the testis and restored the serum hormone level of mice to normal. Furthermore, OPs promoted the expression of Nrf2 protein, and then increased the expression of antioxidant enzyme regulatory protein (HO-1 and NQO1) in the testis. OPs inhibited JNK phosphorylation and Bcl-2/Bax-mediated apoptosis. In conclusion, OPs have a protective effect on testicular injury and spermatogenesis disorders caused by TP, suggesting the potential protection of OPs on male reproduction.     

PTP1B Inhibitory and Anti-Inflammatory Effects of Secondary Metabolites Isolated from the Marine-Derived Fungus Penicillium sp. JF-55

Protein tyrosine phosphatase 1B (PTP1B) plays a major role in the negative regulation of insulin signaling, and is thus considered as an attractive therapeutic target for the treatment of diabetes. Bioassay-guided investigation of the methylethylketone extract of marine-derived fungus Penicillium sp. JF-55 cultures afforded a new PTP1B inhibitory styrylpyrone-type metabolite named penstyrylpyrone (1), and two known metabolites, anhydrofulvic acid (2) and citromycetin (3). Compounds 1 and 2 inhibited PTP1B activity in a dose-dependent manner, and kinetic analyses of PTP1B inhibition suggested that these compounds inhibited PTP1B activity in a competitive manner. In an effort to gain more biological potential of the isolated compounds, the anti-inflammatory effects of compounds 1–3 were also evaluated. Among the tested compounds, only compound 1 inhibited the production of NO and PGE₂, due to the inhibition of the expression of iNOS and COX-2. Penstyrylpyrone (1) also reduced TNF-α and IL-1β production, and these anti-inflammatory effects were shown to be correlated with the suppression of the phosphorylation and degradation of IκB-α, NF-κB nuclear translocation, and NF-κB DNA binding activity. In addition, using inhibitor tin protoporphyrin (SnPP), an inhibitor of HO-1, it was verified that the inhibitory effects of penstyrylpyrone (1) on the pro-inflammatory mediators and NF-κB DNA binding activity were associated with the HO-1 expression. Therefore, these results suggest that penstyrylpyrone (1) suppresses PTP1B activity, as well as the production of pro-inflammatory mediators via NF-κB pathway, through expression of anti-inflammatory HO-1.     

脱落酸处理对油菜幼苗镉积累和毒害作用的影响

为缓解镉毒害,研究脱落酸(ABA)对油菜幼苗镉(Cd)毒害的调节作用机理,以南方主栽油菜品种沣油737为材料,通过水培试验,采用不同浓度的Cd和ABA处理,分析了ABA对Cd胁迫下油菜幼苗的干鲜重、叶片黄化程度和根系形态等农艺指标,根系活力、丙二醛(MDA)和活性氧含量等氧化损伤指标,Cd积累量及其转运系数和抗氧化酶活性等的影响。结果显示,Cd处理显著抑制了油菜幼苗的生长,导致油菜幼苗的生物量、叶片SPAD值、根系活力随着Cd处理浓度的增加而显著下降,而根系MDA和活性氧等损伤指标则显著增加。外源ABA处理可缓解Cd对油菜幼苗的毒害作用,以15μmol/L处理浓度效果最佳,与单独Cd处理相比油菜根系形态显著改善,幼苗地上部和根系中Cd的积累分别显著下降了31.17%和34.89%,MDA和O₂^·–含量分别下降了20.87%和38.89%,而超氧化物歧化酶、过氧化氢酶和过氧化物酶的活性则分别增加了158.05%、28.59%和34.62%,但是ABA应用对Cd的转运系数无显著影响。表明外源ABA可通过抑制Cd吸收,增强抗氧化防御系统以维持油菜体...