The Effect of Quintuply-Fortified Salt on the Gut Microbiome of Nonpregnant Women of Reproductive Age in Punjab, India: A Substudy of a Randomized, Community-Based Trial.

Background: Women of reproductive age in India are vulnerable to multiple micronutrient deficiencies. Large-scale food fortification of staple foods and condiments offers a cost-effective approach to improving micronutrient intake. However, the impact of large-scale food fortification on the gut microbiome remains poorly understood.

Objectives: This study aims to determine whether quintuply-fortified salt (QFS) alters the gut microbiome of nonpregnant women of reproductive age after 12 mo.

Methods: A double-blind, randomized, controlled community-based trial was conducted among 998 women (18-49 y) in Punjab, India. Eligible participants were randomly assigned to receive 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. Stool samples were collected from a subsample of women at baseline and 12 mo and subjected to 16S ribosomal RNA gene sequencing. Outcomes included intervention effects on alpha diversity (Shannon index and abundance-based estimator (ACE) index) assessed via linear mixed regression models, Bray-Curtis dissimilarity (beta diversity) assessed via permutational multivariate analysis of variance (PERMANOVA), and relative abundance of Enterobacteriaceae, Lactobacillus, and Bifidobacterium, Prevotella or Streptococcus modeled using zero-inflated negative binomial mixed regression.

Results: Among the 129 women who provided both a baseline and 12-mo stool sample, 86 had sufficient read depth following sequencing (eFF-Q5S, n = 33; eFePP-Q5S, n = 26; iodized salt, n= 27). Neither alpha diversity nor beta diversity differed significantly at baseline or after the 12-mo intervention. There was no intervention effect on relative abundance of individual taxa (q-value > 0.05).

Conclusions: QFS did not appear to alter the gut microbiome of nonpregnant women of reproductive age in Punjab, India.