A Novel Bacteriocin from Insectivorous Bat (Hipposideros speoris) Intestine Mimicking Mammalian FAM216B: Probiotic, Antioxidant, and Antimicrobial Properties of Enterococcus hirae BNT6.

The alarming rise of antibiotic resistance necessitates the search for novel and safe alternatives to conventional therapies. Probiotics and their metabolites, particularly bacteriocins, are increasingly recognized for maintaining gut health, combating pathogens, and alleviating oxidative stress. Bats, with their diverse and underexplored gut microbiota, represent a unique reservoir of probiotic strains and bioactive peptides. This study aimed to isolate and characterize a novel probiotic strain from the intestine of the insectivorous bat Hipposideros speoris and evaluate its probiotic attributes, antioxidant potential, safety traits, and bacteriocin production. The isolated strain was identified as Enterococcus hirae BNT6 through 16S rRNA sequencing. Probiotic properties including acid and bile tolerance, cell surface hydrophobicity, adhesion to SW480 epithelial cells, and antioxidant activities (DPPH and ABTS scavenging) were assessed. Safety traits were evaluated through hemolysis, DNase activity, and antibiotic susceptibility testing. Bacteriocin purification was carried out using ammonium sulfate precipitation, gel filtration chromatography and RP-HPLC. Antimicrobial activity was tested against pathogenic bacteria, while molecular characterization was performed by SDS-PAGE, MALDI-TOF MS, peptide mass fingerprinting, spectroscopic analysis, and 3D in silico modeling. E. hirae BNT6 survived at pH 3, tolerated 0.3% bile salts, showed high hydrophobicity (51.7%), and adhered efficiently to SW480 cells. It exhibited strong antioxidant activity (65.7% DPPH and 81.6% ABTS scavenging). Safety evaluation confirmed its non-hemolytic, DNase-negative phenotype and susceptibility to most clinically relevant antibiotics. The purified bacteriocin, Enterocin BNT6, displayed broad-spectrum antimicrobial activity against Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, and Bacillus subtilis, and remained stable under varying pH, temperature, and enzymatic conditions. Molecular analyses indicated a size of ~ 27 kDa (SDS-PAGE) and ~ 15 kDa (MALDI-TOF MS), with peptide mass fingerprinting showing 59% similarity to mammalian FAM216B protein. Structural studies revealed a predominantly α-helical conformation. Our findings establish Enterococcus hirae BNT6 as the first bat-derived probiotic strain producing a FAM216B-like bacteriocin with strong probiotic, antioxidant, and antimicrobial activities. In silico structural modeling revealed a predominantly α-helical, stable conformation with striking similarity to mammalian FAM216B, suggesting potential evolutionary mimicry. These unique attributes highlight its promise as a next-generation therapeutic and microbiome-based intervention.