小麦中脱氧雪腐镰刀菌烯醇的测定及方法研究

利用免疫亲和柱对脱氧雪腐镰刀菌烯醇(DON)的特异吸附性,使用高效液相色谱法对样品进行测定.研究了提取方式、样品粒度、样品溶液稳定性、振荡提取时间等因素对检测结果的影响.当标准曲线在0～2.0 μg/mL之间,线性关系良好,相关系数达到0.999 1.检测结果RSD小于8.0％,不同水平的加标回收率处于92.0％～107.4％之间,通过回收率试验和标准物质试验验证了方法的可靠性和准确性.     

Influence of inoculum and climatic factors on the severity of Fusarium head blight in German spring and winter barley

Fusarium head blight (FHB) of small cereals is a disease of global importance with regard to economic losses and mycotoxin contamination harmful to human and animal health. In Germany, FHB is predominantly associated with wheat and F. graminearum is recognised as the major causal agent of the disease, but little is known about FHB of barley. Monitoring of the natural occurrence of FHB on Bavarian barley revealed differences for individual Fusarium spp. in incidence and severity of grain infection between years and between spring and winter barley. Parallel measurement of fungal DNA content in grain and mycotoxin content suggested the importance of F. graminearum in winter barley and of F. langsethiae in spring barley for FHB. The infection success of these two species was associated with certain weather conditions and barley flowering time. Inoculation experiments in the field revealed different effects of five Fusarium spp. on symptom formation, grain yield and mycotoxin production. A significant association between fungal infection of grain and mycotoxin content was observed following natural or artificial infection with the type B trichothecene producer F. culmorum, but not with the type A trichothecene-producing species F. langsethiae and F. sporotrichioides. Trichothecene type A toxin contamination also occurred in the absence of significant damage to grain and did not necessarily promote fungal colonisation.     

高灵敏度磁荧光免疫层析试纸模式构建及在呕吐毒素检测中的应用

构建基于磁荧光纳米材料的免疫层析试纸模式,弥补现在免疫层析技术的不足,为更灵敏的免疫学快速检测提供技术支撑。以呕吐毒素（deoxynivalenol,DON）为靶标,采用溶剂热法制备羧基修饰的超顺磁颗粒,碳二亚胺法将磁颗粒、绿色荧光蛋白及DON单克隆抗体进行偶联,一步法制备磁荧光抗体探针,以DON人工抗原（DON-BSA）为检测线建立磁荧光免疫层析试纸。同时用胶体金标记DON单克隆抗体,以DON-BSA为检测线建立胶体金免疫层析试纸;制备的磁荧光抗体探针具有很好的磁性、荧光特性及抗体反应性,基于该探针成功制备了DON磁荧光免疫层析试纸,该试纸回归方程为y=－0.562x＋0.921,R2=0.990,IC50为5.611 ng/mL,检出限为1.089 ng/mL;制备了DON胶体金免疫层析试纸,该试纸裸眼检测灵敏度为500 ng/mL;定量检测回归方程为y=－0.543x＋1.485,R2=0.991,IC50为65.16 ng/mL,检出限为11.94 ng/mL。DON磁荧光免疫层析试纸的灵敏度是胶体金免疫层析试纸的10.96 倍。本实验建立的磁荧光免疫层析试纸模式可以同时实现样品的富集及荧光信号检测,提高检测灵敏度,并成功用于DON的检测,为磁荧光纳米颗粒广泛应用于免疫层析领域提供参考。     

粮油食品中呕吐毒素危害及风险分析

该文分析呕吐毒素性质,在自然界和粮油食品中存在情况,及对人类和动物危害,并进行风险评估,同时提出预防呕吐毒素危害建议。     

Survey of ochratoxin A and deoxynivalenol in stored grains from the 1999 harvest in the UK

Three hundred and twenty samples from the 1999 UK harvest comprising wheat (201 samples), barley (106) and oats (13) were analysed for ochratoxin A and deoxynivalenol. A small number of organic samples was also obtained. Samples were collected from farms, central stores, mills, maltings and ports from across the UK from February to April 2000. Ochratoxin A and deoxynivalenol analysis was by affinity column clean up and high-performance liquid chromatography with fluorescence and ultraviolet light detection, respectively, with limits of detection of 0.2 and 20 μg kg^-1. The survey found ochratoxin A at below 5 μg kg^-1 in 97% of the samples indicating satisfactory storage conditions. The remaining 3% of the samples contained ochratoxin A at levels between 5.2 and 231 μg kg^-1, but none of these samples was intended for human consumption. Deoxynivalenol was detected in 88% of all samples, with 83% below 100 μg kg^-1; the maximum level was 600 μg kg^-1. Twenty samples containing deoxynivalenol at or above 150 μg kg^-1 by high-performance liquid chromatography were all confirmed by gas chromatography/mass spectrometry. Nivalenol was also detected by gas chromatography/mass spectrometry at levels of 50 μg kg^-1 or higher in 18 of 20 samples where deoxynivalenol was confirmed.     

Influence of temperature and pH on the degradation of deoxynivalenol (DON) in aqueous medium: comparative cytotoxicity of DON and degraded product

Deoxynivalenol (DON), a toxic fungal metabolite, is stable under different processing conditions; however, its stability in aqueous medium at different temperatures and low pH (1–2) (present in the gastrointestinal tract) has not been investigated. In the present study, DON standard was used to study the influence of temperature and pH on DON stability in aqueous medium, the characterisation of the degraded product, and the comparative toxicity profile of the degraded and the parent compound. The results suggest that standard DON was unstable at 125–250°C showing 16–100% degradation whereas DON at pH 1–3 had 30–66% degradation, with a concomitant increase in the formation of a degraded product. Further ESI-MS characterisation of the dominant precursor ion of the HPLC eluate of the DON-degraded product was found to be m/z 279, resembling the known metabolite DOM-1. The degraded product of DON was reconfirmed as DOM-1 by comparison with standard DOM-1 and both gave a similar λ_max at 208 nm. Comparative studies of both standard DOM-1 and the degraded product of DON showed no cytotoxicity up to 6400 ng ml^–1 while significant cytotoxicity was observed for DON (400 ng ml^–1). The results suggest that a highly acidic environment (pH 1–2) could be responsible for the de-epoxydation of DON leading to the formation of DOM-1.     

Impact of durum wheat milling on deoxynivalenol distribution in the outcoming fractions

The milling behaviour of two naturally infected samples of durum wheat grain with contrasting levels of mycotoxins was studied. Although the two samples showed a similar milling behaviour, an increase of ～20% in deoxynivalenol (DON) levels was found in semolina from the sample containing the higher level of mycotoxin. However, even if the highest concentration of DON was found in fractions originating from the grain outer layers, the mycotoxin contamination in semolina and flours were not related to the amount of two compounds (ash or phytic acid) used to monitor these external tissues. The presence of the trichothecene-producing fungi in the inner-most semolina fraction was also shown using specific DNA primers and PCR amplification. Comparison of DON concentrations in the feed stock and corresponding output at each milling step or grinding of semolina fractions followed by sizing showed that concentration of mycotoxin occurs in the finest particles at the first processing steps. Therefore, DON contamination of milling fractions is not simply due to the presence of peripheral grain tissues.     

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.     

Assessment of trichothecene chemotypes of Fusarium culmorum occurring in Europe

Fusarium trichothecenes are a group of fungal toxic metabolites whose synthesis requires the action of gene products from three different genetic loci. We evaluated, both chemically and by PCR assays, 55 isolates of Fusarium culmorum from eight European countries and different host plants for their ability to produce trichothecenes. Specific sequences in the Tri6-Tri5 intergenic region were associated with deoxynivalenol production. Sequences in the Tri3 gene were also associated with deoxynivalenol production and specific primer sets were selected from these sequences to identify 3-acetyl-deoxynivalenol or 15-acetyl-deoxynivalenol chemotypes. Specific sequences in the Tri5 and Tri7 genes were associated with the nivalenol chemotype but not with the deoxynivalenol chemotype. Two chemotypes were identified by chemical analysis and confirmed by PCR. Strains of the nivalenol chemotype produced nivalenol (up to 260 µg g^-1) and 4-acetyl-nivalenol (up to 60 µg g^-1), strains with the 3-acetyl-deoxynivalenol chemotype produced deoxynivalenol (up to 1700 µg g^-1) and 3-acetyl-deoxynivalenol (up to 600 µg g^-1). Three strains of F. culmorum from France, previously reported as 15-acetyl-deoxynivalenol producers, had the 3-acetyl-deoxynivalenol chemotype. The results are consistent with data from other European countries on the occurrence of the nivalenol and 3-acetyl-deoxynivalenol chemotypes and provide support for the hypothesis that European isolates of F. culmorum producing deoxynivalenol belong only to the 3-acetyl-deoxynivalenol chemotype. The production of trichothecenes from F. culmorum isolates from walnut (3-acetyl-deoxynivalenol chemotype) and leek (nivalenol chemotype) is reported for the first time.     

Nonaccumulation of Residues in Swine Tissue Following Extended Consumption of Deoxynivalenol-Contaminated Diets

Several tissues from 62 pigs fed deoxynivalertol (DON)-contamirtated diets (6.0–7.6 mg DON/kg feed) for periods of 3–7 wk were analyzed to determine if residues accumulated following extended ingestion of the toxin. In the liver, kidney and backfat samples analyzed, trace levels were found, measuring on average 3.0 ± 3.5, 5.0 ± 9.2, and 6.7 ± 9.6 ng DON/g wet tissue, respectively. Only 13% (24/186) of samples had more than 10 ng/g, and of these only two contained more than 50 ng/g. These results indicate that when pigs are fed DON-contaminated grains, residues do not appear to accumulate in tissues to any appreciable extent.