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

Background: Young children in India often face multiple micronutrient deficiencies, yet interventions such as micronutrient powders have raised concerns about potential adverse effects on the gut microbiome. Large-scale food fortification is an effective strategy to improve micronutrient intake; however, its impact on the gut microbiome of children remains unclear. Objectives: To determine whether intake of quintuply-fortified salt (QFS) for 12 mo adversely affects gut microbiome composition in children aged 1–5 y. Methods: In a double-blind, randomized, controlled trial in Punjab, India, children received: 1) QFS with iron as encapsulated ferrous fumarate [eFF], zinc, vitamin B12, folic acid, and iodine (eFF-QFS); 2) QFS with the same micronutrients, but iron as encapsulated ferric pyrophosphate [eFePP] plus ethylenediaminetetraacetic acid (eFePP-QFS); or 3) standard iodized salt for 12 mo. Stool samples were collected from 125 children (eFF-QFS, n = 43; eFePP-QFS, n = 45; iodized salt, n= 37) at baseline and 12 mo and analyzed via 16S rRNA gene sequencing. Changes in alpha diversity (Shannon, abundance-based estimator index) between groups were assessed with linear mixed models, beta diversity (Bray-Curtis dissimilarity) with linear regression and permutational multivariate analysis of variance, 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/d across groups. Abundance-based estimator index was higher in the iodized salt arm compared with eFePP-QFS, but similar to eFF-QFS. Permutational multivariate analysis of variance revealed no overall group differences; however, pairwise Bray-Curtis distances from baseline were modestly greater in eFF-QFS compared with the other groups. No significant changes in relative abundance were identified. Conclusions: After 12 mo, 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.This trial was registered at clinicaltrials.gov as NCT05166980 and at Clinical Trials Registry–India as CTRI/2022/02/040333.