以高效能液相層析串聯質譜法分析半乳寡醣樣品之組成結構

Abstract

食品工業上利用乳糖為基質以β-galactosidase進行轉醣反應生產半乳寡醣 (galacto-oligosaccharide, GOS) 來補足嬰兒配方奶粉中相對於母乳中寡醣成份之不足，以維持嬰兒腸道益生菌相的增生，而GOS中的半乳糖與葡萄糖單體主要以β構型鍵結且其組成結構複雜。為針對半乳寡醣結構產物進行解析，本研究建立高效能液相層析串聯質譜法分析並檢測市售GOS產品之異構物組成。先以分子篩層析法初步檢視樣品聚合度組成分布，而後將原態及經對胺基苯甲酸乙酯 (p-aminobenzoic acid ethyl ester, ABEE) 衍生之衍生態分別以GCC-ESI-MSn和Polyamine II-PAD-ESI-MSn兩種方式分析。分析結果顯示，市售六種半乳寡醣產品聚合度均小於六，且以二、三醣結構為主，更有單一樣品三醣組成超過五成；其樣品異構物可被鑑定共含有九種二糖、十八種三醣、二十一種四醣及複數之五、六醣結構，且各產品間組成差異性大。由鍵結型態觀察結果得知樣品結構組成多含有β-1→4、β-1→6鍵結，其次為β-1→3，含有β-1→2者種類最少，部分樣品存在β-1→1，可知半乳寡醣轉醣反應中β-galactosidase確實存在鍵結合成之傾向性。總體來說，本研究所發展之兩種分析方式在半乳寡醣組成輪廓之解析上確實能達到同步解析其聚合度、異構物組成和結構判定之目標，可期應用於其他樣品之研究分析甚至作為相關產業生產應用上之品管用途。

As a supplement for infant formulae, Galacto-oligosaccharides (GOS) are considered as prebiotic compounds to maintain the proliferation of colonic microflora. GOS are producted by transgalactosylation from lactose with β-galactosidase in food industry, and result in several linkage isomers. In this study, we developed a fast and effective analytical method to solve the complicated isomers of GOS products. First, six commercial GOS products were subjected to degree of polymerization (DP) analysis using High-performance size exclusion chromatography. Then, native (without derivatization) sample and p-aminobenzoic acid ethyl ester (ABEE)-derived sample were analyzed with two HPLC-tandem MS methods: GCC-ESI-MSn and Polyamine II-PAD-ESI-MSn respectively. Experimental results showed the DP of all structures was less then six, while DP2 and DP3 were the major components. In total, 9 different kinds of DP2, 18 of DP3 and 21 of DP4 isomers of GOS have been identified, and the composition between each product were varied. For linkage isomers determination, isomers contained β-1→4 and β-1→6 linkages were the major, ones followed by β-1→3 and then β-1→2 linkages in quantity; β-1→1 was also detected in some of the samples. These results demonstrated the tendency to linkage formation of β-galactosidase in transgalactosylation. Overall, the methods developed in this study are capable of analyzing the isomer components and structure characterization simultaneously. The proposed method of determining GOS products can be used for other relevant samples or for a qulity control measurement in related industries.