荧光标记-酶识别-高效液相色谱法测定婴儿配方乳粉中反式低聚半乳糖

Determination of trans-galactooligosaccharides in infant formula by combined analytical techniques of fluorescence labeling, enzymatic identification and high performance liquid chromatography

中文关键词:  婴儿配方乳粉  反式低聚半乳糖  荧光标记  酶识别  β-半乳糖苷酶

英文关键词:infant formula  trans-galactooligosaccharides  fluorescence labeling  enzymatic identification  beta-galactosidase

中文摘要:

      目的 建立荧光标记-酶识别-液相色谱法的婴儿配方乳粉中反式低聚半乳糖(trans- galactooligosaccharides, TGOS)的测定方法。方法 利用还原氨化反应将奶粉中的TGOS进行荧光标记, 利用β-半乳糖苷酶专一性水解TGOS的特性识别样品中的荧光标记TGOS, 采用高效液相色谱-荧光检测器进行分离检测, 通过分析β半乳糖苷酶处理与非酶解处理组样品色谱图中各相同聚合度TGOS保留时间区间的峰面积差异, 计算TGOS含量, 并用液相质谱联用法进行了验证。结果 在本研究色谱条件下, 乳糖和另外3种低聚半乳二糖实现了分离, 通过质谱验证表明, 低聚麦芽糖(DP2~DP6)与各聚合度TGOS呈现规律性出峰顺序。昆布三糖和实验中各聚合度TGOS的摩尔灵敏度无显著差异(P>0.05)。在0.032~0.321 μmol添加范围内, 荧光标记昆布三糖摩尔量和色谱峰面积呈现出良好的线性关系(r=0.9992)。以已知含量的TGOS糖浆做加标实验, 回收率为90.0%~99.1%, 满足实验要求。结论 该方法能实现高乳糖含量样品中乳糖和TGOS的色谱分离, 适用于高乳糖含量样品中TGOS的定性定量检测。

英文摘要:

      Objective To establish a method for the determination of trans- galactooligosaccharides (TGOS) in infant formula milk powder by fluorescent labeling-enzyme recognition-liquid chromatography. Methods The TGOS in the milk powder was fluorescently labeled by reductive amination reaction, and the fluorescent labeling TGOS in the sample was identified by the specific hydrolysis of TGOS by β-galactosidase. The separation was detected by high performance liquid chromatography-fluorescence detector. The TGOS content was calculated by analyzing the difference of peak area in the retention time interval of the same degree of polymerization TGOS in the chromatographic map of the samples in the treatment group of β-galactosidase and non-enzymatic hydrolysis, and was verified by liquid chromatography-mass spectrometry. Results In this study, lactose and three other galactooligosaccharides were separated under the chromatographic conditions. The results of mass spectrometry showed that the galactooligosaccharides (DP2~DP6) and TGOS presented regular peak order. There was no significant difference in the molar sensitivity of laminaritriose and TGOS (P>0.05). In the range of 0.032~ 0.321 μmol, the molar amount of fluorescently labeled laminaritriose and the peak area of the chromatogram showed a good linear relationship (r=0.9992). Standard test was conducted with TGOS syrup with known content, and the recovery rate was 90.0%-99.1%, meeting the experimental requirements. Conclusion This method can achieve chromatographic separation of lactose and TGOS in high lactose content samples and is applicable to qualitative and quantitative detection of TGOS in high lactose content samples.