Phytonutrient-enriched prebiotic mixtures (PEPs), composed of phytonutrients and prebiotics that serve as substrates for beneficial gut microbes, are widely recognized for their potential to promote gut health. However, despite this established role, direct evidence demonstrating their synergistic effects when co-administered with probiotics remains limited. To address this gap, we employed a three-phase design integrating ex vivo evaluation and clinical validation to assess how individual components influence microbial responses, how PEP interacts with probiotics, and whether these effects translate to humans. In ex vivo analyses, PEP components were associated with increased levels of acetate, butyrate, total short-chain fatty acids (SCFAs), and lactate, with fiber-rich components showing the strongest effects. Co-treatment with probiotics further enhanced these metabolite levels compared to single treatments, particularly for butyrate and total SCFAs, along with elevated lactate levels. Notably, these effects became more pronounced with higher doses of co-administered probiotics, suggesting a dose-dependent enhancement of microbial metabolic activity and cross-feeding–mediated metabolic flux. In a randomized clinical study including PEP-only, probiotic-only, and combined PEP plus probiotic groups, all groups showed increases in fecal metabolites. However, the combined PEP and probiotic group exhibited the greatest increases in butyrate (~6 µmol/g) and total SCFAs (22.9 µmol/g). Across all groups, participants with constipation-type stool patterns shifted toward normal stool types, as assessed by the Bristol stool scale. These findings demonstrate that PEP modulates microbiome-derived metabolic activity, and that its combination with probiotics further enhances the production of butyrate and total SCFAs. Overall, this study provides both mechanistic and clinical evidence supporting synbiotic strategies and demonstrates the potential of PEP as an effective approach to improve gut microbial metabolism.

Phytonutrient-enriched prebiotic mixtures (PEPs), composed of phytonutrients and prebiotics serving as substrates for gut microbes, are recognized for their potential to modulate gut microbial metabolic activity. However, direct evidence of enhanced effects following co-administration with probiotics remains limited. Using a three-phase design integrating ex vivo evaluation and clinical validation, we assessed how PEP components influence microbial responses and whether co-administration with probiotics enhances these effects. PEP components increased acetate, butyrate, total short-chain fatty acids (SCFAs), and lactate, with fiber-rich components showing the strongest effects (all q < 0.0001 relative to negative control). Co-treatment with probiotics further enhanced butyrate and total SCFAs in a dose-dependent manner. In a randomized clinical study, all groups showed increases in fecal metabolites, with the combined group exhibiting the greatest increases in butyrate (+6.0 µmol/g, ~1.5-fold, p < 0.05) and total SCFAs (+22.9 µmol/g, ~1.3-fold, p < 0.05). Participants with constipation-type stool patterns shifted toward normal stool types across all groups. These findings support the utility of combined PEP and probiotic interventions for enhancing microbiome-derived metabolic activity.