大肠菌群特异性检测荧光底物MUGal的合成及应用

Synthesis and Application of MUGal which is the Specific Fluorescent Substrate for Detection of Coliform

摘要:

4-甲基伞形酮基-β-D-吡喃半乳糖苷（MUGal）是食品安全中重要指示菌大肠菌群的特异性快速检测荧光底物。本文采用相转移催化方法使4-甲基伞形酮（4-MU）和四乙酰基-α-D-吡喃溴代半乳糖缩合生成保护的糖苷，再在碳酸钠的水/甲醇溶液中去保护生成4-甲基伞形酮基-β-D-吡喃半乳糖苷。通过对温度、时间和催化剂用量对糖苷化一步产率的研究发现，当温度为30 ℃，反应时间为16 h时，产率最高可达73.12%。醇解反应一步以碳酸钠为碱产率可达91.01%。在对产物进行IR和1HNMR结构表征后，进一步将产物应用于大肠菌群显色培养基，并和进口底物比较，其产荧光管数经X2检验无显著性差异（P>0.05），在实际样品检测中，采用GB4789.3-2010（48 h）和MUGal方法（24 h）的阳性检出率均为80%，MUGal方法的检出时间明显缩短。因此，本研究制备的荧光底物能和进口产品相媲美，可以作为进口底物的替代品。

Abstract:

4-Methylumbelliferyl-β-D-galactopyranoside (MUGal) is specific fluorescent substrate for rapid detection of coliform bacteria which are the important indicator bacteria for food safety. Protected glycoside was produced from condensation of 4-methylumbelliferone (4-MU) and tetraacetyl-α-D-galactopyranosyl bromide under the conditions of phase transfer catalysis, and then deprotection in H2O/CH3OH solution of sodium carbonate gave 4-methylumbelliferyl-β-D-galactopyranoside. Through the study of temperature, time and amount of catalyst on the yield of glycosylation，we found that when the temperature was 30 ℃ and the reaction time was 16 h, the yield could be up to 73.12%. Yield of alcoholysis reaction with sodium carbonate as base was 91.01%. Structural characterization of target compound was conducted by IR and 1H NMR. The product was further applied to coliform chromogenic medium. Compared with imported products, they were no significant differences in the number of fluorescent tubes by X2 test (P>0.05). In sample test, both positive rate of GB4789.3-2010 (48 h) and MUGal(24 h) were 80%. The detection time was significantly shorter than GB 4789.3-2010. Therefore, the product could be comparable with imported products and could be used as a substitute for imported products.