赤霉病污染大麦作物中结合态脱氧雪腐镰刀菌烯醇检测体系构建研究
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摘要
赤霉病是大麦小麦以及其他小作物中常见的一种真菌病害,其流行不仅导致作物减产,更会产生真菌毒素威胁人和动物的安全。近年来研究表明,在大麦和小麦中存在着结合态真菌毒素,由于这种形式的毒素,尤其是结合脱氧雪腐镰刀菌烯醇(DON)不能被常规溶剂提取并检测其含量,一旦在人和动物体内消化释放出这种毒素,将成为潜在的食品安全威胁。
本研究比较了三种不同粉碎颗粒度对大麦中DON测定的影响,其中细颗粒的测定结果高于粗颗粒,不过并不是所有样品都能够做出均匀响应；在三种不同提取条件的比较中,5g/40mL的比率,较之其他两种能够减少取样误差,因而取得较理想的提取效果。同时比较了三种DON的测定方法：GC-ECD, GC/MS和EZ-TOX试剂盒,试验结果发现GC的优点是结果比较稳定,但日常维护费用较高,对标准校正物的依赖性较强,样品处理以及测定时间也较长；EZ-TOX测定结果迅速,在测定范围内较结果理想,但是测定范围有限,而且每5个样品的测定至少需要3个标准物绘制标准曲线,因而难免会影响结果的稳定性。通过比较木瓜蛋白酶,α-淀粉酶／戊基葡糖苷酶,纤维素酶／木质素酶处理之后对大麦样品中DON测定的影响,在大麦样品中存在结合态DON,而其中木瓜蛋白酶的处理效果较好,DON的值增加了16-28%。
采用三氯乙酸(TCA)溶剂水解污染大麦样品,发现样品中结合态DON普遍存在。但是限于高温条件下,TCA水解的样品测定图谱噪音较大,干扰因素较多,直接影响到测定结果,因而选用具有相似的水解效果(试验过程中不需要加碱中和反应),测定图谱更优的TFA水解处理样品。采用Doehlert Matrix实验设计,对TFA溶剂水解测定大麦中总DON的试验条件进行了优化,通过PCSAS以及专业的Design-Expert软件分析,从不同角度对构建的模型进行了验证和确认,证明该试验模型很理想,因变量总DON测定值在模型中得到了最大响应,即在优化条件为：反应时间54min,温度133℃, TFA浓度1.25M。经过反复重复实验验证,确认该方法对于测定不同大麦麦芽样品的可行性。同时还测定了优化条件下DON的回收率为90-98%,说明了该定量方法的准确性。在该优化实验条件下比较了8种不同净化柱对测定大麦中DON的影响,计算不同净化柱对DON的回收率,并比较了每两种柱子测定样品的图谱与标准工作曲线的叠加图,在综合评价上述两个指标之后,发现在该实验条件下,Alumina:C18和Mycosep227是8种柱子中净化效果最好的。
同时在TFA优化实验条件下还筛查了污染大麦中的结合态DON的情况,其中40%的大麦中存在此结合化合物；在制麦芽的浸泡阶段,有大量的结合态DON流失,而其他过程则变化不明显,表明结合态DON可溶于水。也进一步证明这种测定方法的有效性和可观的应用前景。
Fusarium Head Blight (FHB) is a fungal disease commonly occurred in barley, wheat and some other small grains. It prevelance results in not only the reduction of quality and quantity of crops, but the contamination of mycotoxins. Recently the bound mycotoxins, particlarly bound deoxynivalenol(DON) has raised more interest since it escapes the routine detection, but can be released after digestion in human and animals, which poses potential threat on the food safety.
The impact of particle size and enzymatic treatments were investigated on the determination of deoxynivalenol (DON) in FHB-infected barley samples. The results showed that the fine-grind samples generally yielded higher reults than medium and coarse grinds. Three determination methods(GC-ECD, GC-MS, ELISA) were used to compare the accuracy to detect DON. This trend was most pronounced in samples with higher DON content. Enzyme treatments involved either amylolytic(alpha-amylase/AMG), proteolytic (papain) or cell wall degrading (cellulase/xylanase) enzymes. Papain treatment resulted in significant increases (16-28%) in the amount of DON detected in five of the seven samples tested when compared to the untreatment samples or enzyme controls. The results strongly suggested that FHB infected barley samples can contain bound DON.
Doehlert matrix design was performed to determine the optimal conditions for the time, temperature of heat hydrolysis and the concentration of TFA to determine the total DON content in barley samples with TFA hydrolysis. The optimal conditions were 133℃for temperature,54 min for time and 1.25M for TFA concentration. Statistic analysis shows that variable the total DON had largest response on the optimal point in the model established. Verification of the method was also investigated on different barley samples. Recovery tests of DON,3-ADON and 15-ADON and additional verification tests proved that this procedure could be applied for analysis of other FHB infected barley samples with this matrix.
With the optimal conditions of TFA hydrolysis, the occurrence of bound DONin FHB infected barley and malt samples on GC-ECD. The presence of bound DON was observed in almost 40% of barley samples. The results of bound DON in malting samples showed that after steeping the content of free DON decreased dramatically together with bound DON. The fact that so much bound DON was lost during steeping suggests that bound DON appears likely to be water soluble and DON-glucosides would be expected to be at least partially water soluble.
本研究比较了三种不同粉碎颗粒度对大麦中DON测定的影响,其中细颗粒的测定结果高于粗颗粒,不过并不是所有样品都能够做出均匀响应；在三种不同提取条件的比较中,5g/40mL的比率,较之其他两种能够减少取样误差,因而取得较理想的提取效果。同时比较了三种DON的测定方法：GC-ECD, GC/MS和EZ-TOX试剂盒,试验结果发现GC的优点是结果比较稳定,但日常维护费用较高,对标准校正物的依赖性较强,样品处理以及测定时间也较长；EZ-TOX测定结果迅速,在测定范围内较结果理想,但是测定范围有限,而且每5个样品的测定至少需要3个标准物绘制标准曲线,因而难免会影响结果的稳定性。通过比较木瓜蛋白酶,α-淀粉酶／戊基葡糖苷酶,纤维素酶／木质素酶处理之后对大麦样品中DON测定的影响,在大麦样品中存在结合态DON,而其中木瓜蛋白酶的处理效果较好,DON的值增加了16-28%。
采用三氯乙酸(TCA)溶剂水解污染大麦样品,发现样品中结合态DON普遍存在。但是限于高温条件下,TCA水解的样品测定图谱噪音较大,干扰因素较多,直接影响到测定结果,因而选用具有相似的水解效果(试验过程中不需要加碱中和反应),测定图谱更优的TFA水解处理样品。采用Doehlert Matrix实验设计,对TFA溶剂水解测定大麦中总DON的试验条件进行了优化,通过PCSAS以及专业的Design-Expert软件分析,从不同角度对构建的模型进行了验证和确认,证明该试验模型很理想,因变量总DON测定值在模型中得到了最大响应,即在优化条件为：反应时间54min,温度133℃, TFA浓度1.25M。经过反复重复实验验证,确认该方法对于测定不同大麦麦芽样品的可行性。同时还测定了优化条件下DON的回收率为90-98%,说明了该定量方法的准确性。在该优化实验条件下比较了8种不同净化柱对测定大麦中DON的影响,计算不同净化柱对DON的回收率,并比较了每两种柱子测定样品的图谱与标准工作曲线的叠加图,在综合评价上述两个指标之后,发现在该实验条件下,Alumina:C18和Mycosep227是8种柱子中净化效果最好的。
同时在TFA优化实验条件下还筛查了污染大麦中的结合态DON的情况,其中40%的大麦中存在此结合化合物；在制麦芽的浸泡阶段,有大量的结合态DON流失,而其他过程则变化不明显,表明结合态DON可溶于水。也进一步证明这种测定方法的有效性和可观的应用前景。
Fusarium Head Blight (FHB) is a fungal disease commonly occurred in barley, wheat and some other small grains. It prevelance results in not only the reduction of quality and quantity of crops, but the contamination of mycotoxins. Recently the bound mycotoxins, particlarly bound deoxynivalenol(DON) has raised more interest since it escapes the routine detection, but can be released after digestion in human and animals, which poses potential threat on the food safety.
The impact of particle size and enzymatic treatments were investigated on the determination of deoxynivalenol (DON) in FHB-infected barley samples. The results showed that the fine-grind samples generally yielded higher reults than medium and coarse grinds. Three determination methods(GC-ECD, GC-MS, ELISA) were used to compare the accuracy to detect DON. This trend was most pronounced in samples with higher DON content. Enzyme treatments involved either amylolytic(alpha-amylase/AMG), proteolytic (papain) or cell wall degrading (cellulase/xylanase) enzymes. Papain treatment resulted in significant increases (16-28%) in the amount of DON detected in five of the seven samples tested when compared to the untreatment samples or enzyme controls. The results strongly suggested that FHB infected barley samples can contain bound DON.
Doehlert matrix design was performed to determine the optimal conditions for the time, temperature of heat hydrolysis and the concentration of TFA to determine the total DON content in barley samples with TFA hydrolysis. The optimal conditions were 133℃for temperature,54 min for time and 1.25M for TFA concentration. Statistic analysis shows that variable the total DON had largest response on the optimal point in the model established. Verification of the method was also investigated on different barley samples. Recovery tests of DON,3-ADON and 15-ADON and additional verification tests proved that this procedure could be applied for analysis of other FHB infected barley samples with this matrix.
With the optimal conditions of TFA hydrolysis, the occurrence of bound DONin FHB infected barley and malt samples on GC-ECD. The presence of bound DON was observed in almost 40% of barley samples. The results of bound DON in malting samples showed that after steeping the content of free DON decreased dramatically together with bound DON. The fact that so much bound DON was lost during steeping suggests that bound DON appears likely to be water soluble and DON-glucosides would be expected to be at least partially water soluble.
引文
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