This study investigated the non-inferiority of feeding term healthy infants with enriched formula milk powder containing 1,3-dioleoyl-2-palmitoylglycerol (OPO) and milk fat globular membrane (MFGM), compared to breast milk, in terms of the formation of gut microbiota, neurodevelopment and growth. Infants were divided into three groups: breast milk group (BMG, N = 50), fortified formula group (FFG, N = 17), and regular formula group (RFG, N = 12), based on the feeding pattern. Growth and development information was collected from the infants at one month, four months, and six months after the intervention. Fecal samples were collected from infants and analyzed for gut microbiota using 16S ribosomal DNA identification. The study found that at the three time points, the predominant bacterial phyla in FFG and BMG were Proteobacteria, Firmicutes, and Bacteroidetes, which differed from RFG. The abundance of Bifidobacterium in the RFG was lower than the FFG (one month, p = 0.019) and BMG (four months, p = 0.007). The abundance of Methanoprebacteria and so on (genus level) are positively correlated with bone mineral density (BMD) of term infants, and have the potential to be biomarkers for predicting BMD. The abundance of beta-galactosidase, a protein that regulates lactose metabolism and sphingoid metabolism, was higher in FFG (six months, p = 0.0033) and BMG (one month, p = 0.0089; four months, p = 0.0005; six months, p = 0.0005) than in the RFG group, which may be related to the superior bone mineral density and neurodevelopment of infants in the FFG and BMG groups than in the RFG group. Our findings suggest that formula milk powder supplemented with OPO and MFGM is a viable alternative to breastfeeding, providing a practical alternative for infants who cannot be breastfed for various reasons.

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