Probiotic lactic acid bacteria from stingless bee products: isolation, characterization, and enhanced viability using prebiotic-fortified microencapsulation

Abstract Purpose Stingless bee products, such as honey and bee bread, are rich sources of beneficial microbes and bioactive compounds. This study aimed to develop a novel multi-strain probiotic formulation by isolating and characterizing lactic acid bacteria (LAB) from stingless bee honey (SBH) and stingless bee bread (SBB), and then enhancing their viability through prebiotic-fortified microencapsulation. Methods A total of 34 LAB isolates were obtained from SBH and SBB collected from five stingless bee species. Among them, three LAB strains - Lactiplantibacillus sp. SBB-HI4 (closest to L. plantarum ATCC 14917 T , 99.7% similarity), Lactiplantibacillus sp. SBB-HI8, (closest to L. pentosus DSM 20314 T , 99.8% similarity), and Lacticaseibacillus sp. SBB-GT4 (closest to L. paracasei JCM 1171 T , 99.8% similarity) - were selected based on their probiotic potential. Their acid and bile salt tolerance, cell surface properties (hydrophobicity, auto-aggregation, and co-aggregation), and antimicrobial activity against common enteropathogens were evaluated. Safety was assessed by hemolytic activity and antibiotic susceptibility. A double-layered alginate-chitosan microencapsulation system supplemented with galacto-oligosaccharides (GOS) was applied to enhance probiotic stability. Results The selected LAB isolates exhibited high acid (pH 3.0) and bile (pH 8.0) tolerance, with survival rates exceeding 80%. Notably, SBB-HI4 showed the highest hydrophobicity (34.03%), auto-aggregation (20.70%), and co-aggregation with E. coli (23.92%) and S. Typhimurium (19.46%). All three strains exhibited broad-spectrum antibacterial activity and passed safety assessments. Encapsulated probiotics demonstrated superior viability, maintaining counts of about 2.6 × 10⁶ CFU/g for four months at 4 °C. The addition of 1.0% (w/v) GOS significantly improved probiotic survival under simulated gastrointestinal conditions. Conclusion Stingless bee-derived LAB, particularly when co-encapsulated with GOS in alginate–chitosan beads, offer promise for applications in the development of stable, functional probiotic supplements aimed at improving gut health across diverse hosts, including bees, animals, and humans.