The main purpose was to determine the abundance of dominant phyla, Bifidobacterium spp., and Lactobacillus in breast milk of obese mothers versus normal-weights in fourth month of lactation in Iranian population. Sixty health women at the fourth month of breastfeeding, aged 18–40 years, were included and categorized based on body mass index (BMI) to the obese (BMI ≥ 30 kg/m2) and normal-weights (18.5 ≤ BMI ≤ 24.9). Bacterial DNA was extracted and qPCR of the 16S region was performed after human milk donation in a sterile condition. A multiple linear mixed model was used to determine the effective factors on the phyla population. Bifidobacterium spp. was significantly higher in milk of normal-weight group than the obese. The current weight showed a significant effect on the Actinobacteria abundance in milk. The Bacteroidetes and Firmicutes were significantly lower in mother’s milk with cesarean section (p = 0.04). Pre-pregnancy obesity decreased the Firmicutes and Lactobacillus abundance in maternal milk (p = 0.04 and p = 0.01). The Actinobacteria and Bifidobacterium spp. showed a significant effect on infant’s height (p = 0.008 and p = 0.04). The maternal current and pre-pregnancy weight showed an important effect on abundance of Actinobacteria and Bifidobacterium spp., as the good phyla and genus in milk which are associated with the infant’s height.
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