酶法合成芥酸海藻糖酯的分离纯化与表征.docVIP

酶法合成芥酸海藻糖酯的分离纯化与表征 王海霞1,2，李舒生1,2，贾承胜*1,2，张晓鸣1,2，冯骉1,2 (1. 食品科学与技术国家重点实验室, 江南大学, 江苏 无锡 214122; 2. 江南大学食品学院, 江苏 无锡 214122) 摘 要：脂肪酶催化合成芥酸海藻糖酯产物中可能存在单酯和二酯。采用薄层层析(TLC)和硅胶柱层析分离纯化反应完成液，然后用高效液相色谱(HPLC)、液质谱(HPLC-MS)和核磁共振(NMR)法鉴定了纯化组分的分子结构。TLC分离芥酸海藻糖酯的最佳条件：点样量2 μl，以氯仿/甲醇(4:1, V/V)展开约30 min，然后用5%硫酸乙醇溶液喷雾、120℃显色20 min。硅胶柱层析分离芥酸海藻糖酯的适宜条件：5 ml反应液上12 mm × 600 mm 硅胶(100～200目)层析柱，流动相为氯仿/甲醇(4:1, V/V)，流速80 ml/h，按1管/200s收集洗出液。两种分离组分分别为6-O-芥酸海藻糖酯和6,6- 关键词：芥酸海藻糖酯；芥酸海藻糖二酯；薄层层析；硅胶柱层析 Enzymatic Synthesis, Separation and Characterization of Erucoyl Trehaloses WANG Hai-Xia1,2, LI Shu-sheng1,2, JIA Cheng-sheng*1,2, ZHANG Xiao-ming1,2, FENG Biao1,2 (1. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China; 2. School of Food Science and Technology, Jiangnan University, Wuxi 214122, China) Abstract: Erucoyl trehaloses which were synthesized using immobilized lipase may contain monoester and diester. The reaction mixture were separated and purified by the thin layer chromatography (TLC) and silica gel column chromatography, then the purified products were analyzed by HPLC, HPLC-MS, and NMR. The optimum conditions for TLC were confirmed: 2 μl of the reaction mixture were applied to silica gel G plates, developed by chloroformmethanol (4:1, V/V), and then detected by spraying with 5% of sulfuric acid in ethanol and heated at 120 oC for 20 min. The optimum conditions for silica gel column chromatography were: 5 ml of the reaction mixture was applied to 12 mm×600 mm silica gel column (100～200 mesh) and chloroform-methanol (4:1, V/V) was used as mobile phase, whose flow rate was 80 ml/h, the eluent of 1 tube/200s was collected. The purified products were 6-O-erucoyl trehaloses and 6, Key words：erucoyl trehaloses, dierucoyl trehaloses, TLC, silica gel chromatography 基金项目: 江苏省自然科学基金项目 (BK2008100)；国家863 计划项目 (2006AA10Z312)。 * 通讯作者: 贾承胜(1968-)，男，安徽太湖人，理学博士，副教授，主要从事食品化学研究。 Email: chshjia@jiangnan.edu.cn 海藻糖是由两个葡萄糖分子以α,α,1,1-糖苷键构成的非还原性糖，广泛存在于自然界中许多动植物和微生物体内[1,2]，随着工业化海