Isolation and characterization of Bifidobacterium spp. from breast milk with different human milk oligosaccharides utilization and anti-inflammatory capacity

Breast milk is the best source of nutrition for infants. Human milk oligosaccharides (HMOs) and the corresponding HMOs-consuming Bifidobacterium positively influence infant health. This study aims to isolate and characterize Bifidobacterium from breast milk of healthy Chinese mothers, identifying the most efficacious strains for inclusion in simulated maternal milk formulas. Nine Bifidobacterium strains (two of B. breve and seven of B. infantis) were isolated, exhibiting a broad spectrum of probiotic potential. This included tolerance to simulated infant gastrointestinal conditions, notable adhesion, antibacterial, antioxidant activities, and HMOs utilization ability. Lacto-N-Tetraose (LNT) is preferred in early growth among Bifidobacterium isolates. B. breve showed a preference for LNT, whereas B. infantis showed a preference for fucosylated HMOs, and displayed reduced utilization of sialylated HMOs. They also exhibited robust safety profiles, including no hemolytic activity, an appropriate D/L lactate-producing ratio, and non-toxicity in an acute oral toxicity assay on mice. It is noteworthy that B. breve N-90, O-147, B. infantis O-161 and R-1 exhibited anti-inflammatory effects in LPS-induced RAW 264.7 cells. Specifically, a notable reduction in TNF-α levels was observed in pre-treatment, while a decrease in IL-1β and IL-6 levels in co-treatment. B. breve N-90 and B. infantis R-1 were identified finally as promising probiotic candidates. Their whole-genome sequencing analysis confirmed presence of functional genes associated with gastrointestinal colonization, antioxidation, and glycoside hydrolase activity on HMOs. The annotation for antibiotic resistance and virulence genes concurred with phenotypes, further validating the safety. Breast milk is a good source for Bifidobacteria isolation, while Bifidobacteria utilize HMOs in a strain-dependent manner. The two selected strains, B. breve N-90 and B. infantis R-1, are potential candidates for inclusion in simulated maternal milk formulas and deserved further in vivo investigation for their health-promoting effects.