Early Probiotic Supplementation of Healthy Term Infants with Bifidobacterium longum subsp. infantis M-63 Is Safe and Leads to the Development of Bifidobacterium-Predominant Gut Microbiota: A Double-Blind, Placebo-Controlled Trial

Abstract

Bifidobacteria are important intestinal bacteria that provide a variety of health benefits in infants. We investigated the efficacy and safety of Bifidobacterium longum subsp. infantis (B. infantis) M-63 in healthy infants in a double-blind, randomized, placebo-controlled trial. Healthy term infants were given B. infantis M-63 (n = 56; 1 × 10⁹ CFU/day) or placebo (n = 54) from postnatal age ≤ 7 days to 3 months. Fecal samples were collected, and fecal microbiota, stool pH, short-chain fatty acids, and immune substances were analyzed. Supplementation with B. infantis M-63 significantly increased the relative abundance of Bifidobacterium compared with the placebo group, with a positive correlation with the frequency of breastfeeding. Supplementation with B. infantis M-63 led to decreased stool pH and increased levels of acetic acid and IgA in the stool at 1 month of age compared with the placebo group. There was a decreased frequency of defecation and watery stools in the probiotic group. No adverse events related to test foods were observed. These results indicate that early supplementation with B. infantis M-63 is well tolerated and contributes to the development of Bifidobacterium-predominant gut microbiota during a critical developmental phase in term infants.

Keywords: Bifidobacterium longum subsp. Infantis; GI motility; gut microbiota; probiotics; secretory IgA; short-chain fatty acids; term infant.

Figure 1

CONSORT flow diagram showing participant flow through the trial.

Figure 2

Enterotype of subjects during the intervention. (a) Principal coordinate analysis (PCoA) as seen through the first two principal coordinates (PCo1 and PCo2) based on Jensen–Shannon distance (JSD) calculated by the fecal microbiota composition at the ASV level; (b) PAMs clustering with JSD. The estimated suitability of the cluster number Calinski–Harabasz index; (c) top 20 ASVs in the fecal microbiota composition in each enterotype; (d) relative abundance of ASVs annotated as the B. longum group, including B. longum subsp. infantis and B. longum subsp. longum; (e) the relative abundance of ASVs annotated as Enterobacteriaceae; (f) the relative abundance of ASV annotated as Lactococcus. Asterisks (*) indicate q < 0.05 calculated by ALDEx2.

Figure 3

Dynamics of enterotype in each group during the intervention. PCoA of fecal microbiota in each group based on JSD calculated by the fecal microbiota composition at the ASV level. (a) Before ingestion; (b) 1 week after ingestion; (c) 1 month; (d) 3 months of age. Blue and red symbols indicate subjects in the placebo and M-63 groups, respectively.

Figure 4

Correlation between the breastfeeding rate and the relative abundance of Bifidobacterium at 1 month of age. Red squares (◆) indicate the M-63 group, and blue circles (●) indicate the placebo group. Spearman’s correlation coefficients and the corresponding p values are shown.

Figure 5

Number of infant stools and consistency. (a) PPS population; (b) exclusively breastfed infants; (c) mixed-fed infants and exclusively formula-fed infants. ** p < 0.01 vs. placebo group (Wilcoxon rank sum test).