酶解法测定多种糖混合体系中海藻糖的含量

Determination of Trehalose in Saccharide Mixtures Based on Trehalase-catalyzed Hydrolysis

摘要:

基于海藻糖水解酶(trehalase)可专一性水解海藻糖为葡萄糖的基本原理，建立了一种快速测定多种糖混合体系中海藻糖含量的分析方法：在trehalase过量的情况下，保证样品中海藻糖短时间内完全水解为葡萄糖，再采用DNS法测定酶解前后样品中还原糖含量并计算其差值，即可折算出样品中海藻糖含量。对含已知浓度海藻糖标准品和麦芽糖、果糖、葡萄糖、蔗糖的多种糖混合溶液中海藻糖含量的测定结果分析表明，酶解法测定海藻糖含量的样品回收率在91.12%~105.92%范围内，相对标准偏差为2.22%~3.43%，测定结果不受样品中其它糖类的干扰。该方法的检出限为70 μg/mL样品。T检验结果表明，酶解法和HPLC对发酵法生产海藻糖中间产物中海藻糖含量的检测结果无显著差异。酶解法测定海藻糖含量具有快速、准确、灵敏等特点，不仅适合于糖成分复杂的样品中海藻糖含量的测定，也具有广谱的应用价值。

Abstract:

Based on the specificity of trehalase in hydrolyzing trehalose to glucose, a simple and rapid method for the determination of trehalose in saccharide mixtures was established. Excess trehalase ensured that trehalose would be hydrolyzed completely into glucose in a short time period, and then, the dinnitrosalicylic acid (DNS) method was used to determine the reducing sugar content in both samples before and after hydrolysis. The trehalose content could be calculated using the difference in reducing sugar content. The data of trehalose content in mixed solutions containing a trehalose standard, maltose, fructose, glucose, and sucrose were analyzed, and the results showed that the recovery rates of trehalose were in the range of 91.12%~105.92% and relative standard deviation was 2.22%~3.43%. Other saccharides in the mixture did not interfere with the content data with a detection limit of 70 μg/mL. The trehalose content in the intermediate product of fermentation was determined via the trehalase hydrolysis method and high performance liquid chromatography (HPLC). A t test showed that there was no difference between the two groups. The trehalase hydrolysis method was rapid, accurate, and sensitive. It can be used for samples with complex sugar compositions, and has a wide range of applications.