利用比色法與層析法測定果寡醣與果聚醣

Abstract

果寡/聚醣依其醣結構骨幹可分為五大類群，包含分別以β-(2,1)、β-(2,6)為主幹的兩種直線型果寡/聚醣，以及包含兩種鍵結的三種分支型果寡/聚醣。不僅如此，同類型的果寡/聚醣有GFn 與Fn 兩種形式，自然界中大部分為GFn 型，以α-glucose 為核心，以fructose 進行延伸的異質多醣，不具有還原端；少部分為Fn 型，僅由fructose 進行延伸的同質多醣，具有還原端。由於果寡/聚醣種類繁多，且其含量分布及組成較不明確，為了確認農作物及果寡醣商品中含有何種果寡/聚醣，需建立判別β-(2,1)、β-(2,6) 鍵結果寡/聚醣的方式。本研究利用市售果寡醣樣品以及農作物，先以 sucrase、maltose、beta-amylase、pullulanase等酵素先水解非果寡/聚醣的醣類分子以減少干擾，接著以 Exo-inulinase、Endo-inulinase 及 Endo-levanase 等專一性酵素將保留了果寡/聚醣的水解液進一步水解出葡萄糖及果糖等單醣，再以高效陰離子交換層析-脈衝安培流檢測法求得單醣含量後，將兩次水解後的單醣含量進行比較進而得到果寡/聚醣含量。同種樣品中，若使用 Endo-levanase 處理後單醣含量明顯上升，代表該樣品具有 β-(2,6) 鍵結的果寡/聚醣，此方式可以推導出樣品中含有不同鍵結類型的果寡/聚醣結構及含量差異。為進一步驗證這些結果，另外進行了比色法測定樣品中果寡/聚醣含量，一樣利用Endo-inulinase 及 Endo-levanase專一性酵素的差異來比較是有含有不同種類之果寡/聚醣，結果顯示部分樣品含有 β-(2,6) 鍵結的果寡/聚醣，且觀察層析圖中的波峰分布，可以推論有些樣品可能含有分支型的果寡/聚醣。本研究之結果可應用於開發更精確的營養配方，以將這種功能性食品成分包含在人類和動物飲食中，也可應用於具有還原以及非還原端的果寡醣結構特徵鑑別。

Fructan/fructooligosaccharide (FOS) can be classified into five subfamilies according to the backbone of their structure, including two linear types fructan/FOS composed with β-(2,1) or β-(2,6) backbone, and three branched types fructan/FOS with both types of linkages. Not only that, the same type fructan/FOS has two forms, GFn and Fn. Most of which are GFn type in nature, the heterooligosaccharide with α-glucose as the core and fructose for β-extension, without reducing end; A few are Fn type, the homooligosaccharides that are β-extended only by fructose, with reducing ends. Due to the wide variety of fructan/FOS, and their content distribution and composition are not clear, in order to confirm what kind of fructan/FOS are contained in crops and FOS products., it is necessary to establish a method to differentiate the bonding between β-(2,1), β-(2,6). In this study, commercially available fructan and FOS samples and crops were used to hydrolyze sugar molecules other than fructan with sucrase, maltose, beta-amylase, pullulanase and other enzymes in order to reduce interference, followed by specific enzymes such as exo-inulinase, endo-inulinase and endo-levanase, they will further hydrolyze the hydrolyzate that remains fructan/FOS to monosaccharides such as glucose and fructose, and then use high-performance anion-exchange chromatography with pulsed amperometric detection method to detect the content of monosaccharides, comparing the monosaccharide content in first and second hydrolyzation to obtain the fructan/FOS content. In the same sample, if the monosaccharide content increases significantly after treatment with endo-levanase, it means that the sample has β-(2,6) linkage fructan or FOS. This method can deduce that the sample contains different types and content of fructan/FOS. In order to further verify these results, a colorimetric method was also carried out to measure the content of fructan/FOS. The difference of specificity between the endo-inulinase and endo-levanase was also used to compare the contents of different types of fructan/FOS. The results indicate that some samples contain β-(2,6)-linked fructan/FOS. Additionally, based on the peak distribution observed in the chromatogram, it can be inferred that certain samples may contain branched fructan/FOS. The results of this study can be applied to the development of more precise nutritional formulas to include this functional food ingredient, It can also be used to identify the structural characteristics of FOS with reducing and non-reducing ends.