Natural incidence of zearalenone in Croatian pig feed,urine and meat in 2014

The aim of this study was to determine the levels of zearalenone (ZEN) in different feed materials and feedstuffs for pigs, as well as in pig urine and pig meat following contaminated feed consumption. In total, 253 feed material and feedstuff samples were collected from Croatian pig farms. The results revealed the presence of ZEN in significant concentrations, the maximal being found in maize (5522 µg/kg), wheat (3366 µg/kg) and pig fattening feed (1949 µg/kg). In farms in which high feed contamination and pig hyperestrogenism were observed, samples of pig urine (n = 30) and meat (n = 30) were retrieved as well. The mean ZEN concentrations in pig urine and pig meat were 206 ± 20.6 µg/L and 0.62 ± 0.14 µg/kg, respectively. Despite high contamination of feedstuffs responsible for farmed pigs’ intoxication, ZEN levels determined in pig meat were shown to be of little significance for human safety.     

Impact of post-anthesis rainfall,fungicide and harvesting time on the concentration of deoxynivalenol and zearalenone in wheat

Field experiments were conducted to identify the impact of post-anthesis rainfall on the concentration of deoxynivalenol (DON) and zearalenone (ZON) in harvested wheat grain. Winter wheat plots were inoculated with Fusarium graminearum at stem extension (GS31) and prothioconazole was applied at mid-anthesis (GS65) to split plots and plots were subsequently mist irrigated for 5 days. Plots were either covered by polytunnels, irrigated by sprinklers or left as non-irrigated uncovered control plots after medium-milk (GS75). Plots were harvested either when ripe (GS92; early harvest) or three weeks later (late harvest). Fusarium head blight (FHB) was assessed each week from inoculation. At harvest, yield and grain quality was measured and grains were analysed for DON and ZON. Differences in rainfall resulted in contrasting disease pressure in the two experiments, with low FHB in the first experiment and high FHB in the second. Difference in FHB resulted in large differences in grain yield, quality and mycotoxin content. DON concentration was significantly (P < 0.05) higher in irrigated compared to covered and control plots in the first experiment, whereas in the second experiment, DON was significantly (P < 0.05) higher in the covered plots compared to the control and irrigated plots. ZON concentration was significantly (P < 0.05) higher in irrigated plots in both experiments. Later harvesting resulted in an approximate fivefold increase in ZON in the first experiment, but was not significantly different in the second experiment. Prothioconazole significantly (P < 0.05) reduced DON in both experiments, but gave inconsistent reductions to ZON. This is the first report to show that the post-anthesis rainfall can significantly increase ZON in wheat, which can increase further with a delayed harvest but may be significantly reduced with the application of prothioconazole. Importantly, in the absence of moisture late season, ZON remains at very low concentrations even when wheat is severely affected by FHB.     

Multi-walled carbon nanotubes-based magnetic solid-phase extraction for the determination of zearalenone and its derivatives in maize by ultra-high performance liquid chromatography-tandem mass spectrometry

A simple and rapid magnetic solid-phase extraction (M-SPE) procedure using multi-walled carbon nanotube-magnetic nanoparticles (MWCNT-MNPs) as sorbents was established for purification of zearalenone (ZEA), α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), zearalanone (ZAN), α-zearalanol (α-ZAL) and β-zearalanol (β-ZAL) in maize. The main parameters affecting the clean-up efficiency were thoroughly investigated, and high purification efficiencies for all analytes were obtained. The resulting MWCNT-MNP-ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was validated for maize samples. The matrix effects were greatly minimized using the M-SPE approach, with signal suppression/enhancement values decreased from 69.9–127.6% to 92.1–103.8%. Consequently, complex matrix-matched calibration curves were not necessary and the calibrations constructed in acetonitrile could be applied for accurate quantification of the targeted mycotoxins in real samples. The average recoveries ranged from 75.8 to 104.1% and the inter- and intra-day precision values expressed as RSDs, were all lower than 14%. Limits of detection and quantification were in the range of 0.03–0.04 and 0.07–0.10 μg/kg, respectively. The analytical performance of the developed method was also successfully evaluated with maize samples, and this method was proved to be a powerful tool for monitoring ZEA and its derivatives in maize.     

玉米赤霉烯酮多克隆抗体的制备及特性分析

合成玉米赤霉烯酮半抗原,应用液相色谱-质谱联用法进行鉴定,并采用活泼酯法将半抗原与载体蛋白OVA或BSA偶联,分别作为免疫原或包被原,并通过SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)和紫外扫描法鉴定偶联效果,免疫3只BALB/c小鼠,制备玉米赤霉烯酮多克隆抗体。结果表明:抗血清效价最高达1∶32 000,以此多抗建立的玉米赤霉烯酮间接竞争标准曲线IC50为39.8ng/mL,IC10为0.71ng/mL,多抗与玉米赤霉烯酮类似物β-zearalenol、zear-alanone、α-zearalanol、β-zearalanol交叉反应率分别为4.80%,3.07%,0.96%,0.09%。说明试验成功制备了玉米赤霉烯酮人工抗原及高特异性多克隆抗体。     

重组过氧化物酶A4-Prx酶学特性及其降解DDGS中ZEN的研究

初步研究了一种重组过氧化物酶Acinetobacter sp.SM04(A4-Prx)的酶学特性,并先对pH、温度和H2O2浓度对过氧化物酶A4-Prx活性的影响进行单因素优化,然后采用正交实验设计对A4-Prx降解玉米酒糟中玉米赤霉烯酮的反应条件进行了优化。结果表明,重组过氧化物酶A4-Prx不含有血红素,其过氧化物酶活性的最适pH、温度和H2O2浓度分别为pH8.5、75℃、25mmol/L;A4-Prx降解DDGS中ZEN的最佳反应条件为:H2O2浓度40mmol/L,料液比1:2(g/mL),温度70℃,时间9h,且在该条件下ZEN降解率为99.2%±0.2%。     

Mycotoxins in breakfast cereals from the Canadian retail market: A 3-year survey

One hundred and fifty-six samples of breakfast cereals were collected from the Canadian retail marketplace over a 3-year period. The samples were analysed for the mycotoxins deoxynivalenol, nivalenol, HT-2 toxin, zearalenone, ochratoxin A, and fumonisins B₁ and B₂ to contribute to dietary exposure estimates in support of the development of Canadian guidelines for selected mycotoxins in foods. The samples included corn-, oat-, wheat- and rice-based cereals, as well as mixed-grain cereals, and were primarily from North American processors. Overall, deoxynivalenol was the most frequently detected mycotoxin  — it was detected in over 40% of all samples analysed. Fumonisins and ochratoxin A were each detected in over 30% of all samples. Zearalenone was detected in over 20% of all samples. Nivalenol and HT-2 toxin were each detected in only one sample. The survey clearly demonstrated regular occurrence of low levels of multiple mycotoxins in breakfast cereals on the Canadian market.     

Fate of the fusarium mycotoxins, deoxynivalenol, nivalenol and zearalenone, during extrusion of wholemeal wheat grain

In the European Union, deoxynivalenol in cereals and cereal products is controlled by recent legislation with the objective of minimizing consumer exposure to this mycotoxin. Relatively few studies have examined the loss of Fusarium mycotoxins during processing and whether this is accurately reflected by the processing factors. The behaviour of deoxynivalenol, nivalenol and zearalenone during extrusion of naturally contaminated wholemeal wheat flour has been examined using pilot-scale equipment. Factors examined were temperature and moisture content. Concentrations of the three mycotoxins were little changed by extrusion although the amount of deoxynivalenol decreased at the lowest moisture content. However, this effect did not appear to be temperature-dependent, suggesting that the apparent loss is either due to binding or inability to extract the residue. Under some conditions, concentrations of the mycotoxins, particularly nivalenol, were higher after extrusion.     

过氧化物酶A4-Prx在毕赤酵母中的优化表达

以来源于不动杆菌Acinetobacter sp.SM04的过氧化物酶A4-Prx的毕赤酵母工程表达菌株GS115/pPIC9K-A4-Prx为研究对象,优化其表达培养条件以提高该菌株对于目的蛋白A4-Prx的表达量。本论文首先研究了培养基成分与诱导条件对表达量的影响,结果表明培养基的pH、甘氨酸浓度和诱导温度对外源蛋白的产量均有显著影响。采用Box-Behnken设计,利用Design Expert软件进行二次回归分析得到了目的蛋白的最优表达条件为:诱导培养基pH 7.0、甘氨酸浓度为0.11%及诱导温度30℃。在此优化条件下重组蛋白的理论表达量达129.87 mg/L,约为未优化下的2倍,实验验证实际表达量达128.94 mg/L,且重组表达的过氧化物酶A4-Prx对酒糟蛋白饲料(DDGS)和食品中的玉米赤霉烯酮毒素(Zearalenone,ZEA)具有高效降解能力。本研究为过氧化物酶的工业化高密度发酵奠定了基础,推动生物降解ZEA研究的进展。     

酰肼介导的抗体定向偶联策略提高免疫层析试纸条检测性能

以酰肼功能团修饰的胶体金作为试纸条的标记材料,利用酰肼基团可特异性地与抗体Fc片段醛基发生亲核加成反应的特性,实现抗体在胶体金纳米粒子（gold nanoparticles,AuNPs）表面的定向偶联。对比于碳二亚胺介导的共价偶联法,酰肼功能团介导的定向偶联需要更少的抗体标记量,更短的偶联时间,酰肼化胶体金免疫层析试纸条（hydrazided gold nanoparticles-based immunochromatographic assay,hAuNPs-ICA）检测玉米赤霉烯酮（zearalenone,ZEN）展示出更好的检测灵敏度;将hAuNPs-ICA检测ZEN加标的玉米样品,结果显示加标回收率介于83.1%～118.0%,批内、批间变异系数为3.9%～13.1%,表明hAuNPs-ICA具有较好的精密度和准确性。     

玉米赤霉烯酮样品前处理与快速检测技术 研究进展

玉米赤霉烯酮是一种毒性极强的小分子,具有弱雌激素活性,其性质相对稳定,缺乏特效解毒剂。其检测方法主要有生物检测法、理化检测法和免疫学检测法,其中免疫检测法因快速、简便、灵敏而得到较为广泛的应用。本文综述了玉米赤霉烯酮的样品前处理技术、免疫学快速检测技术及相关专利的研究进展情况,并对玉米赤霉烯酮的样品前处理技术及其检测产品的开发进行了展望。