探討木聚醣之呋喃醣苷修飾對人體腸道菌相之影響

Abstract

發展精準營養之基礎在於明瞭食品原料在人體中之消化過程與生理反應。越來越多研究指出，飲食型態與代謝性疾病及發炎反應間存在高度關聯性。阿拉伯木聚醣（arabinoxylan）是一種來自穀物的膳食纖維，可作為人類結腸微生物生長之碳源。其結構由木聚醣主鏈構成，並在木醣基單元的 C(O)-2 或 C(O)-3 位置上接有阿拉伯醣取代基。阿拉伯木聚醣的結構特徵會因原料來源而異，進而影響其物理化學性質與腸道發酵性。然而，目前系統性探討阿拉伯木聚醣結構與腸道菌相反應的研究仍相當有限。因此本研究旨在釐清小麥阿拉伯木聚醣的呋喃醣苷構型與分子大小對其在結腸中降解之影響。我們以 α-L-阿拉伯呋喃醣苷酶（α-L-arabinofuranosidase）對三種小麥阿拉伯木聚醣（LV、MV及HV）進行結構修飾，選擇性切除單取代的阿拉伯醣取代基，產生去支化形式之處理組（LVDB、MVDB及HVDB）。透過酸水解與分子篩高效液相層析法分析樣品的單醣組成與分子大小分布，另進一步進行甲基化分析以鑑定醣苷鍵結型態。隨後將原始與去支化的木聚醣樣品接種於含有人類糞便菌相的體外發酵系統中，並在厭氧條件下培養 24 小時。研究結果顯示，經酵素處理的樣品其阿拉伯醣與木醣的比例明顯下降，且隨著樣品分子大小減少，C(O)-3 單取代呋喃醣苷也被有效去除。多樣性分析結果顯示，去支化樣品的 Shannon 指數高於原始樣品，反映其微生物群落的多樣性相對較高。阿拉伯木聚醣結構的改變影響了菌相的組成，尤其在 Bacteroidota 與 Actinomycetota 兩菌門之間出現相對消長的趨勢。值得注意的是，Bacteroides plebeius 在去支化處理組中的相對豐富度普遍高於原始樣品；相對地，Bifidobacterium 則在原始樣品組中有較高的豐富度。例如，B. plebeius 的相對豐度從 LV 組的 8.3% 上升至 LVDB 組的 18.3%，而 Bifidobacterium 則從 25.3% 降至 9.9%。以線性判別分析效應值（Linear Discriminant Analysis Effect Size, LEfSe）進行的差異豐富度分析進一步指出，菌群豐度的變化與木聚醣的呋喃醣苷構型有密切關聯。而主座標分析（PCoA）圖也證實了原始與去支化樣品在菌相結構上呈現明顯分群。微生物組成的變化亦調控了短鏈脂肪酸（SCFA）的生成。在所有去支化處理組中皆觀察到丙酸上升、乙酸與丁酸下降的趨勢。 Spearman相關性分析顯示，丙酸的生成與 Bacteroides 和 Phascolarctobacterium 的豐富度相關；而乙酸、丁酸、乳酸與甲酸的生成則與 Bifidobacterium、Blautia 與 Megasphaera 有關。本研究驗證了我們的假設：調整阿拉伯木聚醣的呋喃醣苷構型可改變其發酵產物組成，並進而調控腸道微生物群落。此成果為膳食纖維結構導向的設計提供了新的思路，有助於發展針對腸道菌相精準調控的營養策略。

Understanding the connections among food materials, digestive processes, and physiological responses is the foundation of precision diets. An increasing number of studies have indicated that dietary patterns are highly associated with health outcomes such as metabolic disorders and inflammatory responses. Arabinoxylan is a cereal-derived dietary fiber that serves as a substrate for human colonic microorganisms, comprising a xylan backbone substituted with arabinose residues at the C(O)-2 or C(O)-3 positions of xylosyl units. The structural features of arabinoxylan vary depending on the raw material source, governing its physicochemical and fermentative properties. Presently, systematic research linking arabinoxylan structures with gut microbiota responses remains limited. In this thesis work, we aimed to elucidate the impact of the furanoside patterns and molecular size of wheat arabinoxylan (WAX) on colonic degradation. Three types of wheat arabinoxylan—LV, MV, and HV—were enzymatically modified using α-L-arabinofuranosidase to selectively cleave monosubstituted arabinose residues, yielding the debranched forms LVDB, MVDB, and HVDB. The acid hydrolysis and size exclusion chromatography were applied to reveal the monosaccharide composition and molecular size distribution of these substrates; methylation analysis was further conducted to identify the glycosidic linkage types. Both native and debranched xylan substrates were subjected to an in vitro fermentation system inoculated with human fecal microbiota under anaerobic conditions for 24 hours. Our results demonstrated that enzymatically treated samples exhibited reduced arabinose/xylose ratios; as the substrate molecular size decreased, monosubstituted C(O)-3 furanosides were effectively removed. Diversity analyses indicated that the Shannon index of debranched substrates was higher for the debranched substrates compared to the native ones, indicating increased microbial diversity in the debranched WAX. The structural alterations of arabinoxylan led to distinct shifts in microbial abundances, presented as a trade-off between the phyla Bacteroidota and Actinomycetota. Notably, Bacteroides plebeius consistently exhibited higher relative abundance in the debranched WAX treatments compared to their native counterparts, while Bifidobacterium was more enriched in the native substrates. For instance, the relative abundance of B. plebeius increased from 8.3% (LV) to 18.3% (LVDB), whereas Bifidobacterium decreased from 25.3% to 9.9%. Differential abundance analysis via Linear Discriminant Analysis Effect Size (LEfSe) revealed that microbial taxa were selectively enriched based on the distinct furanoside patterns of xylan substrates. Principal coordinates analysis (PCoA) plots confirmed that native and debranched substrates formed clearly separated clusters. The altered microbiota composition modulated the short-chain fatty acid profiles, with elevated propionate and reduced acetate and butyrate levels observed in all debranched substrate treatments. Spearman’s correlation analysis indicated that propionate production was associated with genera Bacteroides and Phascolarctobacterium, whereas acetate, butyrate, lactate, and formate production were associated with genera Bifidobacterium, Blautia, and Megasphaera. These findings validate our hypothesis that reordering furanoside patterns diverts the fermentation outcomes of wheat arabinoxylan, offering insights into modulating intestinal microbial communities through the structural design of dietary fibers. This work contributes to the development of precision fiber strategies for gut microbiota-targeted nutrition.