Using in vitro gastrointestinal (GI) tract digestive products to investigate fermentation dynamics of Lactobacillus rhamnosus GG, Bifidobacterium animalis subsp. lactis BB-12, and Escherichia coli

Gastrointestinal digestion shapes nutrient availability and microbial substrates in early life, yet how milk digestion products regulate probiotic–pathogen dynamics remains unclear. Using in vitro digestion products of human milk (HM), infant formula (IF), and control milk (CM), we evaluated the fermentation responses of Lactobacillus rhamnosus GG (LGG), Bifidobacterium animalis subsp. lactis BB-12 (BB-12), and Escherichia coli (E. coli) in static reactors (ST) and peristalsis-mimicking soft elastic tubular reactors (SETR). HM-derived residues selectively enhanced probiotic activity by increasing reducing-sugar utilization, acidification (ΔpH), and overall metabolic output by 110% (LGG) and 130% (BB-12), while suppressing E. coli growth by 28%. In the absence of digestive products, LGG and BB-12 declined by 0.99 and 3.19 log units, whereas E. coli increased by 0.33 log. Mechanical compression in SETR further boosted BB-12 metabolic output by 23% compared with ST. These results demonstrate that infant milk digestion products differentially regulate microbial strategies. We acknowledge that this simplified three-strain model does not capture full gut community complexity, and broader consortia studies are needed for future validation.