Ema Damayanti, Tsania Taskia Nabila, Nur Fitrianto, Yeyen Prestyaning Wanita, Fenny Amilia Mahara, Vika Tresnadiana Herlina, Vita Taufika Rosyida, Teguh Wahyono, Muslih Anwar, Rofiq Sunaryanto, Puspita Lisdiyanti
Stingless bee honey (SBH) is recognized as a functional food with antibacterial, antioxidant, and anticancer properties. This study aimed to isolate and characterize lactic acid bacteria (LAB) with potential probiotic, antibacterial, and antioxidant activities from SBH. LAB were isolated from honey samples of seven Indonesian stingless bee species and screened for antibacterial activity. Five isolates with the highest antibacterial activity were selected for further characterization, including whole genome sequencing (WGS), probiotic property assessment, antioxidant activity, α-amylase inhibitory potential of their cell-free supernatants (CFS), and metabolomic profiling. The isolates were identified as Lacticaseibacillus rhamnosus (TB-3, HI-1, and TS-4) and Pediococcus acidilactici (HI-5 and LT-3) based on WGS analysis. Genome-mining revealed biosynthetic gene clusters (BGCs), including T3PKS, RiPP-like and bacteriocin. All isolates exhibited good probiotic properties with high survival rates under simulated gastrointestinal conditions. CFS of the isolates exhibited strong antibacterial activity against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, as well as significant antioxidant capacity (DPPH, ABTS, and FRAP), high polyphenol and flavonoid contents, and α-amylase inhibition. Untargeted metabolomic analysis using UHPLC-HRMS revealed distinct metabolite profiles with several potential bioactive compounds. This study highlights the potential of SBH-derived LAB as probiotic candidates with antibacterial and antioxidant properties. The integration of genomic and metabolomic approaches offers valuable insights into their application in functional foods and nutraceuticals.