The composition of human breast milk is highly variable, and it can be influenced by genetics, diet, lifestyle, and other environmental factors. This study aimed to investigate the impact of geographical location and mode of delivery on the nuclear magnetic resonance spectroscopy (NMR) metabolic profile of breast milk and its relationship with the milk microbiome. Human milk metabolic and microbiota profiles were determined using NMR and 16S rRNA gene sequencing, respectively, in 79 healthy women from Finland, Spain, South Africa, and China. Up to 68 metabolites, including amino acids, oligosaccharides, and fatty acid-associated metabolites, were identified in the milk NMR spectra. The metabolite profiles showed significant differences between geographical locations, with significant differences (p < 0.05) in the levels of galactose, lacto-N-fucopentaose III, lacto-N-fucopentaose I and 2-fucosyllactose, 3-fucosyllactose, lacto-N-difucohexaose II, lacto-N-fucopentaose III, 2-hydroxybutyrate, 3-hydroxybutyrate, proline, N-acetyl lysine, methyl-histidine, dimethylamine, kynurenine, urea, creatine and creatine phosphate, formate, lactate, acetate, phosphocholine, acetylcholine, LDL, VLDL, ethanolamine, riboflavin, hippurate, spermidine, spermine and uridine. Additionally, the effect of caesarean section on milk metabolome was dependent on the geographical region. Specific interrelations between human milk metabolites and microbiota were also identified. Proteobacteria, Actinobacteria, and Bacilli were most significantly associated with the milk metabolites, being either positively or negatively correlated depending on the metabolite. Our results reveal specific milk metabolomic profiles across geographical locations and also highlight the potential interactions between human milk’s metabolites and microbes.
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