Dietary Supplementation with Popped Amaranth Modulates the Gut Microbiota in Low Height-for-Age Children: A Nonrandomized Pilot Trial

Abstract

Amaranth has been recognized as a nutraceutical food because it contains high-quality proteins due to its adequate amino acid composition that covers the recommended requirements for children and adults. Since pre-Hispanic times, amaranth has been consumed as popped grain; the popping process improves its nutritive quality and improves its digestibility. Popped amaranth consumption has been associated with the recovery of malnourished children. However, there is no information on the impact that popped amaranth consumption has on gut microbiota composition. A non-randomized pilot trial was conducted to evaluate the changes in composition, structure, and function of the gut microbiota of stunted children who received four grams of popped amaranth daily for three months. Stool and serum were collected at the beginning and at the end of the trial. Short-chain fatty acids (SCFA) were quantified, and gut bacterial composition was analyzed by 16S rRNA gene sequencing. Biometry and hematology results showed that children had no pathology other than low height-for-age. A decrease in the relative abundance of Alistipes putredinis, Bacteroides coprocola, and Bacteroides stercoris bacteria related to inflammation and colitis, and an increase in the relative abundance of Akkermansia muciniphila and Streptococcus thermophiles bacteria associated with health and longevity, was observed. The results demonstrate that popped amaranth is a nutritious food that helps to combat childhood malnutrition through gut microbiota modulation.

Keywords: 16S rRNA sequencing; diet; gut microbiota; popped amaranth; short-chain fatty acids.

Figure 1

Study design to analyze the effect of supplementing the normal diet with amaranth on gut microbiota of children between 6 to 7 years old with moderate acute undernutrition living in rural areas.

Figure 2

The effect of amaranth consumption on the gut microbiota composition of children living in rural areas. Alpha-diversity expressed as (A) observed richness, (B) Shannon index, (C) Inv-Simpson, and (D) Chao 1 indexes. Boxes express the IQR (interquartile range), bars indicate the minimum and maximum values. A Kruskall–Wallis test and Dunn’s post hoc analysis (p < 0.05) were performed. No significant differences were found amongst the groups and different indexes. Ctrl = control group of children with normal height-for-age; UN = undernutrition group with low height-for-age; UNA = UN group after three months of amaranth consumption.

Figure 3

The effect of amaranth consumption on the gut microbiota composition of children living in rural areas. Beta-diversity measured using principal coordinate analysis (PCoA) based on the Bray Curtis dissimilarity (p < 0.05). Ctrl = control group of children with normal height-for-age; UN = undernutrition group with low height-for-age; UNA = UN group after three months of amaranth consumption.

Figure 4

(A) Relative abundance in gut microbiota at phylum level; (B) Ratio of Firmicutes to Bacteroidetes (B/F). Significant differences were calculated using the non-parametric Wilcoxon test and non-significant differences were detected. (C) Relative abundance in gut microbiota at family level. Ctrl = control group of children with normal height-for-age; UN = undernutrition group with low height-for-age; UNA = undernutrition group after three months of amaranth consumption.

Figure 5

Heatmap of differential ASVs observed between: UN/Ctrl, undernutrition group (U/N) vs. control group (Ctrl); UNA/UN, undernutrition group after amaranth consumption (UNA) vs. undernutrition group at the beginning of assay (UN); UNA/Ctrl, undernutrition group after amaranth consumption (UNA) vs. control group (Ctrl). Values were determined using LogFC, at p < 0.05 and FDR < 0.1.

Figure 6

Short-chain fatty acid (SCFA) levels in children’s feces samples. One-way ANOVA was carried out followed by Tukey’s post hoc test. (A) acetic acid; (B) butyric acid; (C) propionic acid; (D) Total SCFA. Data is presented as mean of mg/mL ± standard deviation. Asterisk shows significant differences at p < 0.05. Ctrl = control group of children normal weight-for-age; UN = undernutrition children group with low height-for-age; UNA = undernutrition children group after three months of amaranth consumption.

Figure 7

Heatmap of predicted functions of the gut microbiome by Tax4fun2 evaluated according to the differences in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Differences in the KEGG pathway: undernutrition group (UN) vs. control children (Ctrl); undernutrition group after amaranth consumption (UNA) vs. undernutrition group at the beginning of assay (UN); undernutrition group before amaranth consumption (UN) vs. control group (Ctrl).

Figure 8

Association between taxa and pathways. Taxa are represented by yellow circles and pathways by green squares. The network’s nodes are taxa and pathways. The association between the nodes and edges was calculated using the Spearman correlation methodology. Only correlations of absolute co-ordinates > 0.5 were considered. The nodes correlations are represented by the vertex, where red are positive and blue negative correlations.