Red palm olein biscuit supplementation modulates gut microbiota in vitamin A deficient rural Malaysian schoolchildren: a randomised controlled trial

Emerging evidence suggests a bidirectional relationship between vitamin A deficiency (VAD) and gut microbiota, with implications to host health. This double-blind randomised controlled trial (NCT03256123) aims to investigate the effects of 6-month red palm olein (RPO)-enriched biscuit supplementation on gut microbiota of 328 vitamin A-deficient primary schoolchildren (aged 8-12 years) in rural Malaysia, randomised to receive either RPO-enriched biscuits (experimental group, n = 145) or palm olein (PO)-enriched biscuits (control group, n = 183). Stool samples are collected at baseline, 3-month and 6-month of supplementation, and analysed using 16S rRNA gene sequencing. The primary outcome is the changes in gut microbiota composition, while secondary outcomes include alteration in gut microbiota diversity, correlations with blood biomarkers, and microbial network structure. Compared to the control group, the experimental group shows significant reductions in alpha diversity indices (e.g., observed ASVs and chao1, P < 0.001), and increased abundance of potentially beneficial gut bacteria, such as Anaerostipes, UCG-010 and Lachnospiraceae_NK4A136_group (P_adj < 0.05), which also positively correlate with improvements in alpha-carotene, beta-carotene, alpha-tocopherol, packed cell volume, and mean corpuscular volume (P_adj < 0.05). Microbial network analysis reveals that the experimental group exhibits a more cohesive and stable network, evidenced by higher average degree and clustering coefficient values, and lower average path lengths (P < 0.001). Our findings shed light on the potential of RPO-enriched biscuit supplementation in modulating gut microbiota in vitamin A-deficient children by enhancing microbial network cohesiveness and enriching potentially beneficial bacterial taxa which are linked to enhanced micronutrient and haematological outcomes. These findings lay a valuable foundation and expand the existing evidence base for understanding the relationship between provitamin A carotenoids and gut microbiota.