<p>The most common food and supplement prebiotic fiber is inulin – most commonly extracted from chicory root. Fructo-oligosaccharide (FOS) is a smaller version of inulin, both containing mainly fructose units with β-1,2 linkages. FOS/inulin has been used, and studied, as a prebiotic for decades. The potential of alternative prebiotics intrinsic in whole foods, such as in tubers, grains, vegetables, and pulses – the world’s most common staple crops – are not as commonly recognized as prebiotics, though have this potential if fermentable in the gut. If such alternative sources of prebiotic ingredients could be established it would allow for cheaper, possibly more effective, and more diverse food product development options beyond FOS/inulin. </p> <p>This study demonstrates the potential of tubers, grains, and pulses as prebiotics in relation to their <i>in vitro</i> human fecal fermentation rate, short chain fatty acid (SCFA) production, and microbiota indicators of alpha diversity and impact on key bacterial genera. Fecal samples were obtained from three diverse healthy human donors and used as the initial bacterial inoculum to simulate conditions in the human gut (colon). Substrates (n=35), after undergoing an upper gastrointestinal tract simulated digestion, were fermented by each individual donors’ inoculum separately, and measurements after 6, 12, and 24 h of fermentation were made on gas production, SCFA metabolite production, and microbiome composition. </p> <p>The results of this study establish high fermentability and potential prebiotic effects of dietary fibers from tubers, grains, and pulses. Whole foods, ground and cooked the same way, produced dietary fibers that were largely insoluble, but surprisingly fermentable with high SCFA levels, mostly slow fermentation profiles indicating high tolerability, and mostly promoting diverse microbiota responses compared to FOS. Generally, whole food fibers had higher fermentability than similar isolated fibers. Overall, the processing steps, such as atmospheric or pressure cooking, tested in some pulses did not detract, or add to, the prebiotic abilities of the substrates. Each food fiber substrate had unique effects on the gut microbiota parameters tested. Gut microbiome compositional responses to the same substrate varied significantly among the three donors, but notably SCFA metabolite responses were similar among donors. </p>