The aim of this study was to investigate five lactic acid bacteria (LAB) strains, isolated from breast milk, selected as producers of potential therapeutic biomolecules – S-proteins and exopolysaccharides (EPSs). Proteomic analysis of strains Levilactobacillus brevis MB1, MB2, MB13 and MB20 confirmed the presence of surface S-proteins, whose tertiary structure was determined using the I-TASSER server. The functional role of S-proteins was demonstrated in terms of survival under simulated gastrointestinal tract conditions, adhesion to Caco-2 cells and competitive exclusion of pathogens. Genes involved in the Wzx/Wzy-dependent pathway of EPSs biosynthesis were detected in the genome of Limosilactobacillus fermentum MC1, and nuclear magnetic resonance (NMR) structural analysis revealed that EPSs consist of two polysaccharides. The role of EPSs in adhesion to Caco-2 cells and competitive exclusion of Escherichia coli 3014 was demonstrated. The antimicrobial activity of the supernatants of the tested LAB strains against pathogenic bacteria and the antioxidant activity of the tested strains were confirmed. The increase in the expression of JAM-A, occludin, ZO-1, MUC2 and ZG16, and the decrease in the expression of the proinflammatory cytokines IL-6, IL-8 and IL-1β indicate the regenerative and immunomodulatory effects of the LAB strains and their biomolecules. Positive effects of L. fermentum MC1 and its EPSs on inflammatory processes in the intestine of mice, the regeneration of the intestinal mucosa and the microbiota indicate potential application of the strain L. fermentum MC1 as probiotic and its EPSs as prebiotics. The LAB strains were protected by microencapsulation in alginate, with and without the addition of galacto- or fructo-oligosaccharides, and by nanoencapsulation, as evidenced by a high survival rate under unfavourable conditions (> 10⁸ CFU/mL).