Philippa Kaur

Quality controls for the validation of MICODE protocolTo validate the MICODE experimental approach in the version of fecal batch of the human proximal colon, we chose to monitor and check some parameters as quality controls (QC) related to metabolites and microbes at the end of fermentations, and in comparison, to the baseline. QCs adopted were; (i) the Firmicutes/Bacteroidetes ratio (F/B), which is related to health and disease11, was maintained at a low level, confirming the capacity to simulate a healthy in vivo condition for 24 h. (ii) The presence of Archea (e.g., Methanobrevibacter smithii and Methanosphaera stadtmanae), which are pretty sensible to oxygen content12, was retained from the baseline to the end point in each vessel and repetition, indicating that the environmental conditions were strictly maintained. (iii) Good’s rarity index of alpha biodiversity remained similar during time of fermentation (p  > 0.05), indicating enough support to the growth of rare species. (iv) Observed OTUs richness index scored approximately 400 OTUs at the end point. (v) The paradigm of prebiotics was confirmed when the positive control (FOS) was applied on MICODE; high probiotic and SCFAs increases and limitation of enteropathogens. (vi) Each GC/MS analysis had quantified some stool-related compounds (urea, 1-propanol, and butylated hydroxy toluene), that ranged across the complete chromatogram and were adsorbed at the same retention times.Changes in bacterial alpha and beta diversitiesThe microbiota diversity indices were analyzed to study the impact of HPBA on microbial population, to assess population’s stability during fermentation, and to compare its microbiota to that of other bioreactors (Figure S1). The baseline of value was compared to the endpoints of fermentation of different treatments. It is undisputable that a part of the effect of reduction in richness (Observed OTUs) was derived by the passage from in vivo to in vitro condition, but the focus must be set on the different trend that other alpha diversity indices had. For example, abundance (Chao 1) for HBPA was significantly higher at the end of fermentation (p  0.05) and HPBA (p  0.05), while oppositely, FOS decreased in evenness (p  > 0.05) and raised in dominance (p  0.05). Among these, 31 variables were significant and their Log2 fold changes in respect to the baseline were compared by post-hoc test (Table 1). The 41 OTUs selected were those that recorded shifts after fermentation and that from literature are susceptible to the effect of prebiotic or fiber substrates. We have included even three OTUs of Archea relative to QC of the experiments (previously discussed).Table 1 Abundances (% ± S.D.) and changes in phylum taxa (Log2 F/C) after 24 h in vitro fecal batch culture fermentations from healthy donors and administrated with HBPA, HB, and FOS as the substrates, and also including a blank control.Full size tableThe first group of OTUs included beneficial or commensal bacteria that usually respond to prebiotics. In this group, three Bifidobacterium were picked showing increases on the substrates and reduction on the blank control. HB and HBPA fostered Bif. bifidum, but just the latter did it significantly, making this taxon grew up to the 3.30% of relative abundance (p  More

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