Fig. 1: Overview of experiments and analyses performed.

n = 6 biologically independent samples per age group. A basic research model consisting of ex vivo colonic incubation bioreactors that mimic the early life colon, seeded with cryopreserved fecal inocula, and supplemented with different pre- and probiotic combinations. Fecal inocula were harvested from the same six donors at infant (approx. 3 months old) and toddler age (approx. 12 months old). The microbiota contained in this fecal material provided a background for the system. Five different combinations of pre- and probiotics were supplemented to the bioreactors: (1) blank condition in which no prebiotic and no probiotic is added, (2) probiotic only condition in which Bifidobacterium longum subsp. infantis LMG 11588 is added, (3) prebiotic only condition in which an age-adapted human milk oligosaccharide (6-HMO) mix is added, (4) synbiotic condition in which the combination of B. infantis LMG 11588 and 6-HMO is added, and (5) additional probiotic supplementation condition in which B. infantis LMG 11588, 6-HMO, and Bifidobacterium animalis subsp. lactis CNCM I-3446 are added. Conditions 1–4 allow the methodical quantification of any potential synergy between 6-HMO and B. infantis LMG 11588. Condition 5 enables to assess if this synergy is influenced by additional probiotic supplementation. Triphasic analysis is performed to quantify key microbial metabolites through HPAEC and GC, to track the strains added to the system through strain-specific qPCR, and to characterize the background microbiota dynamics through 16 S rDNA sequencing.