Biofilm-based probiotics have emerged as a compelling approach for administering probiotics.Selecting appropriate wall materials for enclosing probiotics in biofilm structure is viewed as an advanced method of microencapsulation.In this study, Lactiplantibacillus paraplanturm LR-1 was enclosed in three different types of wall materials to create calcium pectin beads (CPB), chitosan-calcium pectin beads (C-CPB), and sodium alginate-pectin-whey beads (SPW).The biofilm cells of LR-1 were cultured externally and then encapsulated to create CPB-ex-LR-1, C-CPB-ex-LR-1 and SPW-ex-LR-1.In contrast, LR-1 was cultured within the microcapsules to form biofilms in situ, resulting in CPB-situ-LR-1, C-CPB-situ-LR-1 and SPW-situ-LR-1.The microcapsules displayed a relatively uniform morphol. with the water contents below 10%.Nevertheless, there were noticeable edge effects in the distribution of strains within the in situ-type microcapsules.The detection of distinctive peaks unique to Lactiplantibacillus within the microcapsules confirmed the successful encapsulation.Addnl., a decrease in the intensity of the peaks linked to the wall materials indicated the formation of an amorphous structure within the microcapsules.The bacterial concentrations of situ-type microcapsules were found to surpass 10 lg CFU/g.The SPW-situ-LR-1 demonstrated superior tolerance performance, optimal release properties in simulated gastrointestinal fluids, and exceptional stability during the storage period.Addnl., SPW-situ-LR-1 showed similar effectiveness in alleviating colitis compared to fresh Biofilm-LR-1.Both Biofilm-LR-1 and SPW-situ-LR-1 pre-treatments exhibited pos. effects on regulation, with SPW-situ-LR-1 administration significantly increasing the presence of Lactobacillus.This study provides valuable insights into the development of the next-generation probiotics.