Effect of molecular weight and M/G of alginate on the gastrointestinal tolerance of biofilm-state probiotic encapsulated by calcium alginate

Biofilm-state probiotics can effectively improve their tolerance in harsh environments. Previous studies have shown that calcium alginate is an effective carrier for promoting biofilm formation, but its mechanism is unclear. Here, we show that tuning alginate molecular weight (Mw) and mannuronic/guluronic (M/G) directs Lactobacillus rhamnosus GG (LGG) biofilm formation and gastrointestinal tolerance. Our key finding is that calcium alginate beads with low Mw and low M/G alginates produce denser biofilms and superior gastrointestinal tolerance. These formulations markedly improved LGG survival in simulated challenges (gastric acid: 0.02–0.28 log CFU/g loss; bile: 1.54–2.31 log CFU/g), outperforming free cells. Mechanistic analysis revealed that low Mw and low M/G alginate matrices, with larger specific surface area, pore size, and volume, enhanced quorum-sensing autoinducer-2 signaling and subsequent protective extracellular substance synthesis, thereby improving the gastrointestinal tolerance of biofilm-state LGG. Overall, the findings emphasized that controlling alginate structure provides a rational, material-based strategy for designing advanced probiotic or bioactive delivery systems.