Mechanism analysis of enhanced gastrointestinal tolerance of biofilm-state probiotics within calcium alginate by initial medium pH

The poor gastrointestinal tolerance of probiotics limits their efficacy. Probiotics encapsulated by calcium alginate can be cultured to form biofilm to improve their gastrointestinal tolerance. In this work, the tolerance of Lacticaseibacillus rhamnosus GG (LGG) was further enhanced by the adjustment of the initial medium pH, and its mechanism was analyzed. The results showed that the encapsulated LGG cultured at pH 4.0 and 8.0 yielded denser biofilms with superior protective effects, reducing viability loss to 1.04 and 0.34 log CFU/g, respectively, during digestion—significantly outperforming free cells and extreme pH conditions. Mechanism analysis showed that the optimized pH environments strengthened the alginate network, increasing the biomechanics of gel on LGG, exhibited mainly by storage modulus and hardness. The biomechanics promoted AI-2 accumulation, triggering quorum sensing mediated extracellular matrix synthesis to enhance tolerance. These findings highlight initial pH regulation as a simple, process-based strategy for designing robust biofilm-state probiotic delivery systems.