A. Rehman et al.
The Journal of Nutrition xxx (xxxx) xxx
Microbial composition. When assessing between-group differences in species-level OTUs, we found that changes in relative abun- dances of 17 OTUs signi fi cantly differed between groups, of which 9 OTUs related to Ruminococcus albus _OTU#1468 ( P ¼ 0.002), Alistipes ihumii_O TU#528 ( P ¼ 0.002), Anaeromassilibacillus sene- galensis _OTU#1422 ( P ¼ 0.017), Saccharofermentans acetigene- s _OTU#1479 ( P ¼ 0.017), Oribacterium sinus _OTU#1283 ( P ¼ 0.027), Clostridium carnis _OTU#994( P ¼ 0.035), Ruthenibacterium lactatiformans _OTU#1478 ( P ¼ 0.039), Alistipes obesi _OTU#532( P ¼ 0.046), and Faecalitalea cylindroides _OTU#1564 ( P ¼ 0.046) were elevated signi fi cantly in the Fruit fl ow group when compared with placebo. In addition, 4 OTUs related to Parabacteroides gold- steinii _OTU#448( P ¼ 0.025), Bacteroides acidifaciens _OTU#364( P ¼ 0.027), Veillonella parvula _OTU#1638 ( P ¼ 0.030), and Rumi- nococcus faecis _OTU#1473 ( P ¼ 0.039) were lower in the Fruit fl ow group when compared to changes in the placebo group (Figure 3A). There were also signi fi cant between-group differ- ences at genus level with decreases in the relative abundance of Prevotella ( P ¼ 0.007)and Veillonella ( P ¼ 0.023) and increases in 5 taxa related to Saccharofermentans ( P ¼ 0.01), Alistipes ( P ¼ 0.023), Anaeromassilibacillus ( P ¼ 0.033), Ruthenibacterium ( P ¼ 0.039), and Oribacterium ( P ¼ 0.042) in the Fruit fl ow group when compared with the changes evident in the placebo group ( Figure 3B). Within-group changes from baseline to end of the intervention were assessed using ALDEx2. In the Fruit fl ow group, 4 species-level OTUs related to Ruminococcus faecis _OTU#1473 ( P ¼ 0.0005), Bacteroides uniformis _OTU#406 ( P ¼ 0.018), Bacteroides ovatu- s _OTU#392 ( P ¼ 0.025), and Hungatella hathewayi _OTU#1083 ( P ¼ 0.036) had lower relative abundance at the end of intervention than at baseline ( Figure 3C ) . In contrast, in the placebo group, 3 species-level OTUs related to Coprococcus catus _OTU#1222 ( P ¼ 0.023), Anaeromassilibacillus senegalensis _OTU#1422 ( P ¼ 0.028), and Barnesiella intestinihominis _OTU#428( P ¼ 0.043) were signif- icantly depleted at the end of intervention when compared with baseline (Figure 3D ). At the genus level, 3 taxa related to Strepto- coccus ( P ¼ 0.005), Rumniococcus ( P ¼ 0.021), and Hungatella ( P ¼ 0.039) had signi fi cantly lower relative abundance at the end of intervention than that at baseline in the Fruit fl ow group. In contrast, in the placebo group, Anaeromassilibacillus ( P ¼ 0.028), Alistipes ( P ¼ 0.038), and Eubacterium ( P ¼ 0.038) were signi fi - cantly depleted at the end of intervention when compared with baseline ( data not shown ). We did not observe any signi fi cant between or within-group changes at the phylum level ( data not shown ).
between individuals in postprandial TMAO responses (for indi- vidual pro fi les, see Supplemental Figure 3). When calculating mean area under the concentration-time pro fi le (total and in- cremental Area Under Curve) and maximum peak plasma con- centrations (C max ), we did not observe a signi fi cant difference between the groups. In addition, we failed to observe any dif- ference between groups for urine TMAO that was collected over 8 hours postprandially ( data not shown ).
Fecal microbiota
Alpha and beta diversity. No signi fi cant between-group differ- ences or within-group changes were observed for species di- versity and richness using observed species, Chao1, Shannon, and Simpson diversity indices (Supplemental Figure 4). To gain insights into the temporal dynamics of microbial communities, fecal samples were also subjected to a multivariate analysis using Bray – Curtis and Jaccard distance methods. However, no signif- icant shift in beta diversity was observed in global or pairwise PERMANOVA analysis between and within groups. In contrast, a signi fi cant shift in Jaccard Principal Component (PC1) was observed when comparing the end of intervention time points of Fruit fl ow and the placebo group ( P ¼ 0.019, Figure 2).
Plasma metabolites
Plasma untargeted metabolomics. Principal component analysis (PCA) of the NMR data shows a clear distinction between plasma samples collected after Fruit fl ow intervention and control sam- ples (Figure 4A). By means of VIP, the top 15 ranking features driving this distinction included TMAO, formic acid, valine, glucose, and lactate, with TMAO being the most discriminant metabolite (low in Fruit fl ow samples, high in control samples, Figure 4B). Plasma LPS. There was no signi fi cant between-group difference in plasma LPS concentrations. However, we observed a signi fi - cant within-group change with reduced plasma LPS
FIGURE1. Effects of 4-wk of supplementation of Fruit fl ow or placebo (maltodextrin) on trimethylamine-n-oxide (TMAO) in overweight and obese adults. Boxes represent median and interquartile range (IQR), and the diamond shape, symbol mean values of absolute changes in (A) plasma and (B) urine TMAO from baseline to end of the inter- vention in each group. There were within-group changes, i.e. different from baseline at P 0.05 and between-group differences, i.e. differ- ences in changes between Fruit fl ow and placebo group at P 0.05. N ¼ 22.
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