Colonic In Vitro Model Assessment of the Prebiotic Potential of Bread Fortified with Polyphenols Rich Olive Fiber

Abstract

The use of olive pomace could represent an innovative and low-cost strategy to formulate healthier and value-added foods, and bakery products are good candidates for enrichment. In this work, we explored the prebiotic potential of bread enriched with Polyphenol Rich Fiber (PRF), a defatted olive pomace byproduct previously studied in the European Project H2020 EcoProlive. To this aim, after in vitro digestion, the PRF-enriched bread, its standard control, and fructo-oligosaccharides (FOS) underwent distal colonic fermentation using the in vitro colon model MICODE (multi-unit colon gut model). Sampling was done prior, over and after 24 h of fermentation, then metabolomic analysis by Solid Phase Micro Extraction Gas Chromatography Mass Spectrometry (SPME GCMS), 16S-rDNA genomic sequencing of colonic microbiota by MiSeq, and absolute quantification of main bacterial species by qPCR were performed. The results indicated that PRF-enriched bread generated positive effects on the host gut model: (i) surge in eubiosis; (ii) increased abundance of beneficial bacterial groups, such as Bifidobacteriaceae and Lactobacillales; (iii) production of certain bioactive metabolites, such as low organic fatty acids; (iv) reduction in detrimental compounds, such as skatole. Our study not only evidenced the prebiotic role of PRF-enriched bread, thereby paving the road for further use of olive by-products, but also highlighted the potential of the in vitro gut model MICODE in the critical evaluation of functionality of food prototypes as modulators of the gut microbiota.

Keywords: 16S-rDNA MiSeq; FOS; MICODE; VOCs; microbiota; olive byproduct; prebiotic; qPCR.

Figure 1

Principal component’s analysis (PCA) of relative abundances (%) of significative (ANOVA p < 0.05) assigned Operational Taxonomic Units (OTUs) at the species level, after 24 h (EP = Endpoint; EP_2 = duplicate sample) of in vitro batch culture fermentations inoculated with human feces (n = 3 healthy donors) and administrated with FOS, Eco0%, and Eco4% as the substrates and a blank control (X). Variables in red font are the main descriptors of Eco4% or FOS cases.

Figure 2

PCAs of the volatilome sorted by chemical classes of significative (ANOVA p < 0.05) VOCs, including the baseline and three different timepoints (t1 = 5 h; t2 = 10 h; t3 = 24 h). (A) = Aldehydes; (B) = Ketones; (C) = Alcohols. Left-side diagrams are for PCAs of cases, while right-side diagrams are for PCAs of variables.

Figure 3

Endpoint changes in main microbial metabolites related to prebiotic activity, expressed as normalized scale from relative abundances with respect to the baseline (red line). The baseline absolute quantifications in mg/Kg are found in the Supplementary Material (Table S2). Changes were recorded after 24 h of in vitro batch colonic fermentations inoculated with human feces (n = 3 healthy donors) and administrated with FOS, Eco 0%, and Eco 4%. Samples were analyzed in duplicate from two independent experiments (n = 4). Box = mean; Rectangles = mean * S.D.; Whiskers = min and max values. Cases with different letters or numbers or symbols among a single independent variable are significantly different according to Tukey’s HSD test (p < 0.05). (A) Short-Chain Fatty Acids: gray plot = Acetic acid; white plot = Propanoic acid; black plot = Butanoic acid. (B) Medium-Chain fatty Acids: Fuchsia plot = Hexanoic acid; green plot = Heptanoic acid; blue stripes plot = Octanoic acid; black plot = Nonanoic; pale blue plot = n-Decanoic acid. (C) Branched-Chain Fatty Acids: red plot = Propanoic, 3-metyl acid; green plot = Butanoic, 3-methyl acid; blue plot = Pentanoic, 2-methyl acid. (D) Indoles: gray plot = Indole; white plot = Skatole.

Figure 4

Interomics, Spearman Rank Correlations among main microbial metabolites from the volatilome and ANOVA significant (p < 0.05) species OTUs from the microbiota. Prop2M = Propanoic acid, 2-metyl; Buty3M = Butanoic acid, 3-methyl; Penta3M = Pentanoic acid, 3-methyl. Left-side dendrogram identifies by Pearson analysis three major different clusters among bacterial species. Significance of correlations are provided as supplementary material (Figure S6).