Comparative evaluation of sugar substitutes reveals greater intestinal perturbation associated with sucralose under inflammatory stress

Sugar substitutes used in reduced-sugar foods differ in chemical structure, digestive fate, and microbial accessibility, yet their intestinal responses have rarely been compared under a common exposure framework. D-allulose, erythritol, and sucralose were evaluated as distinct sugar substitutes, with inulin included as a reference prebiotic carbohydrate. The ingredients were comparatively evaluated under a standardized exposure framework using in vitro fecal fermentation, healthy mice, and mice subjected to dextran sulfate sodium (DSS)-induced inflammatory stress. Gut microbiota profiling was integrated with inflammatory and barrier-related measurements, followed by serum metabolomics and Caco-2 validation of the sucralose-associated response. In vitro, D-allulose and inulin supported greater growth or enrichment of selected potentially beneficial bacteria, whereas sucralose showed the weakest growth support. In healthy mice, sucralose reduced microbial diversity and increased serum lipopolysaccharide by 23.2% relative to Control, accompanied by focal inflammatory cell infiltration and lower ZO-1 expression. Under DSS challenge, sucralose was associated with the greatest intestinal perturbation, including a 20.0% shorter colon than in the Model group, higher disease activity index and colonic myeloperoxidase levels, enhanced pro-inflammatory responses, and reduced tight-junction- and mucus-related markers. These changes coincided with enrichment of Faecalibaculum, depletion of Akkermansia and Dubosiella, and elevation of a serum feature annotated as p-cresyl sulfate. In Caco-2 cells, p-cresyl sulfate at 5 and 50 μM altered inflammation- and barrier-related markers without reducing cell viability. Overall, these findings demonstrate ingredient-specific intestinal response profiles and support considering gut microbial and intestinal effects alongside technological and nutritional attributes when evaluating sugar substitutes for reduced-sugar food applications.