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Fruit and vegetable byproducts (FVBP) present high content of bioactive compounds and dietary fibers and have demonstrated a positive modulatory effect upon gut microbiota composition. In the present study, the prebiotic potential of a FVBP flour obtained from solid byproducts after fruit and vegetable processing was evaluated after in vitro gastrointestinal digestion. An initial screening with three strains of Lactobacillus (Lactobacillus casei 01, Lactobacillus rhamnosus R11 and Lactobacillus acidophilus LA-5®) and one Bifidobacterium strain (Bifidobacterium animalis spp. lactis BB12®) was carried out and then the prebiotic effect of FVBP flour was performed with fecal samples of five donors. The changes in gut microbiota were evaluated at 0, 12, 24 and 48 h of fermentation by the real-time polymerase chain reaction (qPCR) method with 16S rRNA-based specific primers. The pH and short chain fatty acids (SCFA) production at each fermentation time were assessed. The fructooligosaccharides (FOS) were used as positive control. The impact of FVBP flour upon cell viability was also evaluated. FVBP flour showed higher prebiotic effect than FOS on growth enhancement of Lactobacillus after 48 h of fermentation and similar bifidogenic effect as FOS on Bifidobacterium growth at 12, 24 and 48 h of fermentation. SCFA production was observed when FVBP flour was used as carbon source, including butyrate, which supports the prebiotic potential of this flour. Additionally, it was observed that after in vitro gastrointestinal digestion, the FVBP flour at 3% promoted cell metabolism of Caco-2 cell line up to 67%. Thus, the present study demonstrates the viability of using a fruit and vegetable byproducts flour as a potential sustainable prebiotic source., (Copyright © 2020. Published by Elsevier Ltd.)