Biofilm-inspired encapsulation enhances the viability of probiotic Lacticaseibacillus paracasei NN4–1 under simulated gastrointestinal conditions

The survival of probiotics during transit through the gastrointestinal tract (GIT) remains a significant challenge, limiting their in vivo functional efficacy. Microorganisms often resist adverse conditions by forming biofilms. Leveraging this property, the current study introduces a novel biofilm-inspired encapsulation approach using single- and multilayer-coated sodium alginate gel beads (SAGBs) to promote in situ biofilm formation by Lacticaseibacillus paracasei NN4–1. Comparative analyses were conducted to assess bacterial viability in SAGBs, planktonic cells, and biofilm cells under simulated GIT conditions. In vitro studies showed enhanced resistance in SAGBs, with a survival rate of 81.48% compared to unencapsulated cells. Additionally, biofilm encapsulation increased biochemical production, yielding average protein and polysaccharide concentrations of 0.633 mg/mL and 1.056 mg/mL, respectively. The scanning electron microscope revealed clusters of bacterial colonization inside the SAGBs. Whole-genome sequencing revealed multiple genes associated with biofilm formation, stress tolerance, adhesion, acid, and bile salt resistance. Multilayer of SAGBs reduced bacterial leakage by 52.52%, slowed small-molecule diffusion, and slightly improved textural properties without compromising bacterial metabolic activity or growth. Furthermore, SAGBs exhibited markedly higher survival (99.43%) than planktonic (76.3%) and biofilm cells (77.5%) after 21 days of refrigerated storage in milk. This approach offers promising applications in designing next-generation functional foods and targeted probiotic delivery systems, warranting higher viability of probiotics under adverse conditions of the GIT.