Impact of a fermented soy beverage supplemented with acerola by-product on the gut microbiota from lean and obese subjects using an in vitro model of the human colon

Abstract The aim of this study was to evaluate the effects of soy-based beverages manufactured with water-soluble soy extract, containing probiotic strains (Lactobacillus acidophilus LA-5 and Bifidobacterium longum BB-46) and/or acerola by-product (ABP) on pooled faecal microbiota obtained from lean and obese donors. Four fermented soy beverages (FSs) (“placebo” (FS-Pla), probiotic (FS-Pro), prebiotic (FS-Pre), and synbiotic (FS-Syn)) were subjected to in vitro digestion, followed by inoculation in the TIM-2 system, a dynamic in vitro model that mimics the conditions of the human colon. Short- and branched-chain fatty acids (SCFA and BCFA) and microbiota composition were determined. Upon colonic fermentation in the presence of the different FSs formulations, acetic and lactic acid production was higher than the control treatment for faecal microbiota from lean individuals (FMLI). Additionally, SCFA production by the FMLI was higher than for the faecal microbiota from obese individuals (FMOI). Bifidobacterium spp. and Lactobacillus spp. populations increased during simulated colonic fermentation in the presence of FS-Syn in the FMLI and FMOI. FS formulations also changed the composition of the FMOI, resulting in a profile more similar to the FMLI. The changes in the composition and the increase in SCFA production observed for the FMLI and FMOI during these in vitro fermentations suggest a potential modulation effect of these microbiotas by the consumption of functional FSs. Graphical abstract Key points • Soy beverages increased Bifidobacterium abundance in microbiota from obese individuals. • The synbiotic beverage increased Bifidobacterium abundance in microbiota from lean individuals. • The synbiotic beverage changed the microbiota from obese individuals, approaching the lean profiles. Supplementary Information The online version contains supplementary material available at 10.1007/s00253-021-11252-8.

using the alkaline solution described above, containing bile and pancreatin maintained at concentrations of, respectively, 10 g/L and 1 g/L. A new incubation took place for another 2 h at 37 ºC for enteric II phase simulation.

Supplementary Information Text S2 (Microbiota analysis description)
Barcoded amplicons from the V3-V4 region of 16S rRNA genes were generated using a 2step PCR. An amount of 10-25 ng genomic DNA was used as template for the first PCR with a total volume of 50 μl using the 341F (5'-CCTACGGGNGGCWGCAG-3') and the 785R (5'-GACTACHVGGGTATCTAATCC-3') primers appended with Illumina adaptor sequences. PCR products were purified, and the size of the PCR products were checked on a Fragment analyser (Advanced Analytical) and quantified by fluorometric analysis. Purified PCR products were used for the 2nd PCR in combination with sample-specific barcoded primers (Nextera XT index kit, Illumina).
Subsequently, PCR products were purified, checked on a Fragment analyser (Advanced Analytical) and quantified, followed by multiplexing, clustering, and sequencing on an Illumina MiSeq with the paired-end (2x) 300 bp protocol and indexing. The sequencing run was analysed with the Illumina CASAVA pipeline (v1.8.3) with demultiplexing based on sample-specific barcodes. The raw sequencing data produced was processed removing the sequence reads of too low quality (only "passing filter" reads were selected) and discarding reads containing adaptor sequences or PhiX control with an in-house filtering protocol. A quality assessment on the remaining reads was performed using the FASTQC quality control tool version 0.10.0. Quality trimming was applied based on Phred quality scores.

Supplementary information Text S3 (Bioinformatics description)
In order to ensure that comparable regions of the 16S rRNA gene were analysed across all reads, sequences that started before the 2.5-percentile or ended after the 97.5-percentile in the alignment were filtered. Potentially chimeric sequences were removed. Sequences were aligned and clustered into operational taxonomic units (OTUs; defined by 97% similarity). A biom table file was generated which was subsequently employed for diversity analysis. Rarefaction curves were computed with the "alpha_rarefaction.py", using Simpson metric and a rarefaction depth value of 1225 sequences. Principal Coordinate Analysis (PCoA) plots were obtained using the "beta_diversity_through_plots.py" command, selecting the weighted and unweighted UniFrac as desired metric to generate the distance matrix (Caporaso et al. 2010;Pruesse et al. 2007).    Figure S4. Short-chain fatty acids and secondary organic acids as ratios (%) from the different test compounds at 48 h for the faecal microbiota from lean individual (FMLI) and faecal microbiota from obese individuals (FMOI) tested. The simulated lumen were fed the following media: Control = SIEM + dialysate solution; FS-Pla-= SIEM + fermented soy beverage without the probiotic strains or the acerola by-product (ABP); FS-Pro = SIEM + fermented soy beverage with the probiotic strains but without the ABP; FS-Pre = SIEM + fermented soy beverage with the ABP but without the probiotic strains; FS-Syn = SIEM + fermented beverage soy with the probiotic strains and the ABP.

Table S1
Composition of dialysate solution (Dial)

Concentration (g.L -1 or mL.L -1 )
Pectin from citrus (Sigma-Aldrich) 12 Xylan (Sigma-Aldrich), 12 Arabinogalactan (   Average (standard error) of α-diversity measures (Shanonn Index, PD_whole_tree, Chao 1, and Observed_OTUs) observed for faecal microbiota from lean individuals (FMLI) and faecal microbiota form obese individuals (FMOI), obtained from TIM-2 trials with the different test compounds tested. Greek letters in a column indicate significant differences (Mann-Whitney U, P <0.05) between different simulated lumen for the same test compound and the same time. *Test compounds fed to the simulated lumen were: Control = SIEM + dialysate solution; FS-Pla-= SIEM + fermented soy beverage without the probiotic strains or the ABP; FS-Pro = SIEM + fermented soy beverage with the probiotic strains but without the ABP; FS-Pre = SIEM + fermented soy beverage with the ABP but without the probiotic strains; FS-Syn = SIEM + fermented beverage soy with the probiotic strains and the ABP. Value showed as mean (standard error). FMLI -Faecal microbiota from lean individuals. FMOI -Faecal microbiota from obese individuals. # Significant difference (P<0.05) between 0 h and 48 h obtains from non-parametric Mann-Whitney U test. *Test compounds fed to the simulated lumen were: Control = SIEM + dialysate solution; FS-Pla-= SIEM + fermented soy beverage without the probiotic strains or the ABP; FS-Pro = SIEM + fermented soy beverage with the probiotic strains but without the ABP; FS-Pre = SIEM + fermented soy beverage with the ABP but without the probiotic strains; FS-Syn = SIEM + fermented beverage soy with the probiotic strains and the ABP.