**Background:** The World Health Organization recommends exclusive breastfeeding for the first six months of life. During weaning, the intestinal microbiota evolves toward an adult-like profile, and disruption can increase the risk of acute infectious diseases. Infant formulas are constantly being modified to better mimic human milk, including adjustments to protein content, fatty acid profiles, and the addition of bioactive compounds such as postbiotics. This study aimed to determine whether a novel starting formula (INN) provides gut microbiota compositions more similar to those of breastfed infants from 6 to 12 months of age compared to a standard formula (STD).
**Methods:** This was a multicenter, randomized, double-blind, parallel, comparative clinical trial conducted in 21 centers in Spain. A total of 210 infants (70 per group) completed the intervention until 12 months of age. Group 1 received the INN formula (lower protein content, casein-to-whey protein ratio approximately 70/30, twice as much DHA as STD, twice as much ARA as STD, and thermally inactivated postbiotic Bifidobacterium animalis subsp. lactis BPL1™ HT). Group 2 received the STD formula. Group 3 was exclusively breastfed for exploratory purposes. Visits were conducted at 6 months and 12 months of age. Fecal samples were analyzed using 16S rRNA gene sequencing (V3-V4 regions) on the Illumina NovaSeq 6000 platform. Biochemical analyses included calprotectin, immunoglobulin A, and short-chain fatty acids (SCFAs) measured by ELISA and HPLC. Functional profiles were predicted using PICRUSt2, and microbial signatures were identified using the Rivera-Pinto method.
**Key Results:** At 6 months, Bacillota phylum levels were significantly lower in the INN group compared to the BF and STD groups. Alpha diversity indices (Shannon, Simpson, species richness, Pielou's evenness) in the BF and INN groups differed significantly from the STD group at 6 months. At 12 months, Verrucomicrobiota levels in the STD group were significantly lower than in the BF and INN groups. From 6 to 12 months, Bacteroidota levels increased significantly only in the BF group, while Bacillota increased in both formula groups. Pseudomonadota decreased significantly in all groups. Bifidobacterium was the dominant genus at 6 months in all infants, with significantly lower levels in the STD group compared to BF and INN groups. Clostridium sensu stricto 1 was significantly higher in the INN group compared to both BF and STD groups. The STD group had significantly higher levels of Ruminococcus gnavus group, Clostridioides, Flavonifractor, and Lachnoclostridium compared to BF and INN groups. Calprotectin levels were significantly higher in the STD group (median not reported as exact number) than in the INN and BF groups at 6 months, decreasing significantly in all groups by 12 months. Immunoglobulin A levels were significantly lower in the STD group than in the INN and BF groups at 6 months, with no differences at 12 months. Both formula groups had significantly higher propionic acid levels than the BF group at 6 months. At 6 months, the STD group showed significantly higher abundance of 10 metabolic pathways (glycolysis, superpathway of hexuronide and hexuronate degradation, D-galactarate degradation I, D-galacturonate degradation I, D-glucarate degradation I, superpathway of D-glucarate and D-galactarate degradation, superpathway of β-D-glucuronosides degradation, NAD salvage pathway III, superpathway of phospholipid biosynthesis [E. coli], and L-tryptophan biosynthesis) compared to the BF group. The INN group showed only D-glucarate degradation as significantly higher than BF. At 12 months, no significant differences in metabolic pathways were observed between groups. The Rivera-Pinto analysis showed moderate discrimination between INN and BF groups (AUC 0.709), higher discrimination between STD and BF groups (AUC 0.733), and the highest discrimination between the two formulas (AUC 0.781).
**Clinical Implications:** The INN formula promoted a gut microbiota composition more similar to that of breastfed infants than the STD formula, with higher Bifidobacterium levels, lower calprotectin (suggesting less intestinal inflammation), and higher immunoglobulin A levels at 6 months. The metabolic pathway profiles of the INN group more closely resembled the BF group than the STD group. These findings suggest that reformulating infant formulas with reduced protein content, adjusted fatty acid profiles, and added postbiotics may support gut health and immune development during the weaning period. The study's strengths include its randomized, double-blind, multicenter design. Limitations include that the sample size was calculated for growth rate differences rather than microbiota outcomes, the use of 16S rRNA amplicon sequencing rather than whole-genome sequencing, and the inability to attribute effects to specific formula components since the INN formula differs from STD in multiple respects. Future longitudinal studies with larger sample sizes and single-ingredient modifications are recommended.