Sensitive and selective determination of glutathione in probiotic bacteria by capillary electrophoresis-laser induced fluorescence
Sensitive and selective determination of glutathione in probiotic bacteria by capillary electrophoresis-laser induced fluorescence
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DOI：
10.1007/s00216-006-0980-6
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发表时间：
2007-02-01
影响因子：
4.3
通讯作者：
Raggi, Maria Augusta
中科院分区：
文献类型：
--
作者：
Musenga, Alessandro;Mandrioli, Roberto;Raggi, Maria Augusta
谷胱甘肽（GSH）是一种硫醇，在保护组织免受氧化应激方面具有重要作用，氧化应激与结肠癌的发生有关。因此，开发生产谷胱甘肽的益生菌物种可能会引起极大的兴趣。为测定双歧杆菌属和乳球菌属部分益生菌中谷胱甘肽的含量，建立了毛细管电泳-激光诱导荧光检测相结合的高灵敏度、高选择性分析方法。细胞裂解液样品的预处理非常简单——用乙腈以1:2的体积比沉淀蛋白质。选择荧光团5-碘乙酰氨基荧光素（5-IAF）进行谷胱甘肽衍生化；它与pH值为12.5的硫醇反应，形成荧光加合物，在488nm的激光激发下进行检测。优化反应条件为温度、时间、5-IAF/GSH摩尔比。电泳以碳酸盐岩缓冲液（25 mmol L-1, pH 9.8）为背景电解质，电压为30 kV；电泳运行在不到7分钟内完成。浓度与响应在2.5 ~ 500 ng mL（-1）范围内呈良好的线性关系，检出限为0.5 ng mL（-1）。采用标准添加法测定益生菌细胞的谷胱甘肽含量，以减少基质效应。结果表明，该方法具有良好的灵敏度和选择性，可用于益生菌细胞裂解物中谷胱甘肽的测定。
Glutathione (GSH) is a thiol with an important function in protecting tissue against the oxidative stress which has been related to carcinogenesis in the colon. For this reason the development of probiotic species producing glutathione could be of great interest. To determine the glutathione content of some probiotic bacteria of the Bifidobacterium and Lactococcus genera, a very sensitive and selective analytical method based on capillary electrophoresis coupled to laser-induced fluorescence detection has been developed. Pretreatment of cell-lysate samples is very simple-precipitation of protein with acetonitrile in 1:2 volume ratio. The fluorophore 5-iodoacetamidofluorescein (5-IAF) was chosen for glutathione derivatisation; it reacts with thiols at pH 12.5, forming a fluorescent adduct which is excited by a laser at 488 nm for detection. The reaction conditions optimised were temperature, time, and 5-IAF/GSH molar ratio. Electrophoresis was performed with a carbonate buffer (25 mmol L-1, pH 9.8) as background electrolyte and a voltage of 30 kV; an electrophoretic run was complete in less than 7 min. There was a good linear relationship between concentration and response in the range 2.5-500 ng mL(-1) and the LOD was 0.5 ng mL(-1). The glutathione content of probiotic cells was determined by using the standard additions method to reduce matrix effects. The method was fully validated and shown to be of suitable sensitivity and selectivity for determination of GSH in probiotic cell lysates.