Prebiotic-Driven Dynamics of Lactobacillus Co-Cultures

Probiotics, which include specific species of Lactobacillus, are microorganisms that support human health and utilize available nutrients in the gut to grow. Galacto-oligosaccharides (GOS), prebiotics composed of galactose units with a terminal glucose unit, serve as fermentable substrates that promote the growth of beneficial microbes, including Lactobacillus. Our group previously developed humanized GOS (hGOS), a LacNAc-enriched β(1-4)-linked GOS. Gut microorganisms do not exist in isolation within the intestinal environment. To understand their behavior in communities, we studied interactions between co-cultures of Lactobacillus strains. To enable strain-specific tracking, we transformed strains of Lactobacillus rhamnosus GG (LGG), L. rhamnosus AMC143 (an infant fecal isolate), and Lactiplantibacillus plantarum BAA-793 (Lp) with plasmids that encode fluorescent marker genes. These strains were cultured with GOS or hGOS as the sole carbon source, with lactose and N-acetylglucosamine as controls. In monocultures, Lp showed the highest growth rates (μmax = 0.42-0.63 h⁻¹), compared to LGG and AMC143 (0.27-0.42 h⁻¹). In co-cultures, Lp also showed the highest growth rates, especially in hGOS (0.96 h⁻¹) and GOS (0.90 h⁻¹), compared to LGG and AMC143 (0.06–0.39 h⁻¹). The co-culture conditions enhanced Lp growth in all carbohydrates relative to monoculture conditions, indicating potential cross-feeding interactions. Because growth was estimated from fluorescence intensity, changes in pH may influence fluorescence intensity and could partially explain the relatively high growth rates observed, representing a limitation of this approach. Together, these results highlight the complex interspecies interactions of Lactobacillus, particularly in hGOS, providing a framework for designing synbiotics to support gut health.