The Effect of Quintuply-Fortified Salt on the Gut Microbiome of Young Children 1–5 y of Age in Punjab, India; A Substudy of a Randomized, Community-Based Trial

Abstract Background Young children in India often face multiple micronutrient deficiencies, yet interventions such as micronutrient powders (MNPs) have raised concerns about potential adverse effects on the gut microbiome. Large‐scale food fortification (LSFF) is an effective strategy to improve micronutrient intake, however, its impact on the gut microbiome of children remains unclear. Objective To determine whether intake of quintuply fortified salt (QFS) for 12 months adversely affects gut microbiome composition in children aged 1-5 years. Methods In a double‐blind, randomized, controlled trial in Punjab, India, children received: 1) QFS with iron as encapsulated ferrous fumarate, zinc, vitamin B12, folic acid, and iodine (eFF‐QFS); 2) QFS with the same micronutrients, but iron as encapsulated ferric pyrophosphate plus EDTA (eFePP‐QFS); or 3) standard iodized salt for 12 months. Stool samples were collected from 125 children (eFF-QFS, n=43; eFePP-QFS, n=45; iodized salt, n=37) at baseline and 12 months and analyzed via 16S rRNA gene sequencing. Changes in alpha diversity (Shannon, ACE index) between groups were assessed with linear mixed models, beta diversity (Bray-Curtis dissimilarity) with linear regression and PERMANOVA, and relative abundance of Enterobacteriaceae, Lactobacillus, Bifidobacterium, Bacteroides, Prevotella or Escherichia-Shigella with zero‐inflated negative binomial mixed models. Results Average discretionary salt utilization was estimated to be 3.5 g/child equivalent/day across groups. ACE index was higher in the iodized salt arm versus eFePP-QFS, but similar to eFF-QFS. PERMANOVA revealed no overall group differences; however, pairwise Bray-Curtis distances from baseline were modestly greater in eFF-QFS versus the other groups. No significant changes in relative abundance were identified. Conclusions After 12 months, QFS resulted no major changes in abundance of key taxa and minimal, inconsistent shifts in certain diversity metrics and relative to the iodized salt control, suggesting no adverse effects on microbiome composition among young children in this setting. Additional studies in settings with improved iron status are needed. Clinical Trial Registration ClinicalTrials.