以石墨化碳液相層析串聯軌道阱質譜法探討非消化性寡醣之多樣性結構組成

Abstract

非消化性寡醣是低分子量的膳食纖維，其益生質效能取決於結構組成，然而非消化性寡醣的結構複雜性造成分析上的困難，本研究設計了多元的分析策略藉此克服此挑戰，以多孔性石墨化碳液相層析有效地分離了多數量的異構物，以還原法區別還原性與非還原性結構，並以四級桿串聯軌道阱式質譜儀的工作模式有效地鑑別高聚合度結構，已成功開發了非消化性寡醣的高通量分析平台，平台可鑑別38種異麥芽寡醣雙醣至六醣、29種清酒中雙醣至八醣、24種β-葡萄糖苷酶轉醣產物、84種半乳寡醣雙醣至六醣、47種蜂蜜中的二三醣、30種母乳寡醣三醣至九醣，相較過去研究能鑑別更多的寡醣結構。分析平台可應用於建立食品中的寡醣指標性成分，多元樣品分析結果顯示，異麥芽寡醣原料具有5組主要的雙醣至四醣成分群，以1→4與1→6鍵結構成，佔總寡醣量的55%以上; 清酒中的主要寡醣包含isomaltose、isomaltotriose、panose、63 glucosyl panose、isomaltotetraose、66 glucosyl maltohexaose，於相同製造商不同等級清酒中含量差異大; 半乳寡醣原料具有15組主要的雙醣至五醣成分群，大多為多元鍵結型態的還原性直線型結構，佔總寡醣量65%以上，相同製造商不同生產批次的原料發現了寡醣含量變異的情形; 洋槐、荔枝、龍眼、麥盧卡蜂蜜中寡醣譜型差異大，其差異來自於turanose、maltulose、isomaltose、maltose、nigerose、erlose。分析平台也可應用於探討寡醣的酵素合成特異性，β-葡萄糖苷酶Td2F2以cellobiose為受質之轉醣反應分析結果顯示，產物sophorose、laminaribiose、gentiobiose、α,β-trehalose先後抵達最大產率，且非還原端帶有β-1→6鍵結的結構不易被水解，造成產物累積。本研究所開發的PGC-LC-Orbitrap-MS/MS分析平台未來更可應用於探討寡醣結構與益生質效能的關聯性。

Non-digestible oligosaccharides (NDO) are low-molecular-weight dietary fibers. The prebiotic efficacy is dependent on their chemical profile. However, profiling analysis of non-digestible oligosaccharides is still challenging due to structural complexity. We adopted multiple analytical strategies to overcome this challenge. Porous graphitic carbon liquid chromatography (PGC-LC) effectively separates numerous isomers. The reduction method differentiates non-reducing structures from reducing structures. The working mode of Q-Orbitrap facilitates the structural characterization of structures with a high degree of polymerization (DP). The high-throughput analytical platform for NDO was developed. The platform can characterize 38 isomalto-oligosaccharides (IMO) with DP2-DP6, 29 oligosaccharides in Sake with DP2-DP8, 24 transglycosylation products of β-glucosidase, 84 galacto‐oligosaccharides (GOS) with DP2-DP6, 47 oligosaccharides in honey with DP2-DP3, and 30 human milk oligosaccharides (HMO) with DP3-DP9. The platform is able to characterize more structures than previous studies. The platform can be applied to investigate marker oligosaccharides in foods. In IMO materials, we investigated five major DP2-DP4 with 1→4 and 1→6 linkages accounting for over 55% of total oligosaccharides content. In Sake samples, the main oligosaccharides were isomaltose and isomaltotriose, panose, 63 glucosyl panose, isomaltotetraose, and 66 glucosyl maltohexaose. Oligosaccharide profiles varied in Sake samples of different grades from the same manufacturer. In GOS materials, we investigated fifteen major group components up to DP5, accounting for at least 65% of total oligosaccharides content: most were linear structures with versatile linkages. Substantial variations in components occurred in GOS materials from different batches. In Acacia, Lychee, Longan, and Manuka honey samples, oligosaccharide profiles significantly varied. The difference contributed from turanose, maltulose, isomaltose, maltose, nigerose, and erlose. The platform can be also applied to study enzyme specificity of oligosaccharides synthesis. We studied the transglycosylation reaction using cellobiose catalyzed by β-glucosidase Td2F2. As a result, sophorose, laminaribiose, gentiobiose, and α,β-trehalose achieved maximum yield time successively. The structures with β-1→6 linkages at the non-reducing end were not easily hydrolyzed, leading to the accumulation of products. The developed platform can be further applied to investigate the structural and prebiotic-efficacy relationships in the future.