The aim of this study was to demonstrate the effect of single or mixed fibers (arabinoxylan, β-glucan, xyloglucan, and inulin) on the metabolome of cecum content in mice with obesity caused by a high-fat diet. Twenty-eight six-week-old male mice were divided randomly into seven groups (n = 4/group), including a normal-diet group (CON), a high-fat-diet group (HFD), and groups with the same high-fat diet but supplemented with arabinoxylan (HFAX), arabinoxylan + β-glucan (HFAβ), arabinoxylan + xyloglucan (HFAG), xyloglucan (HFXG), and xyloglucan + inulin (HFXI). A total of 66 molecules were identified and quantified in cecum content by proton nuclear magnetic resonance (1 H-NMR). The metabolomic profiles combined with statistical analysis revealed compounds distinguishing the control group from those supplemented with fibers. In detail, a high-fat diet could significantly elevate the concentrations of acetone and methionine (p < 0.05) while decreasing the levels of methanol, arabinose, acetate, and 3-hydroxyphenylacetate (p < 0.05) in the cecum contents of mice. Compared to HFD, the supplementation caused higher levels of fumarate and hypoxanthine (p < 0.05) and lower levels of phenylacetate, acetate, fucose, formate, proline, betaine, and trimethylamine N-oxide (TMAO) (p < 0.05). An enrichment analysis highlighted that the pathways mainly altered were amino sugar metabolism, aspartate metabolism, and arginine and proline metabolism. In conclusion, non-starch polysaccharide (NSP) supplementation could change the metabolomic profiles of cecum contents in obese mice as a result of a high-fat diet. Moreover, mixed NSPs exhibited more beneficial effects than singular form on gut metabolism.
