Abstract
Probiotics have been applied to a wide range of bacteria, causing gastrointestinal and vaginal infections. However, probiotics generally possess limited antimicrobial spectra and are primarily utilized as dietary supplements. Recognizing the need for more versatile probiotics, this study focuses on isolating and characterizing strains suitable for antibiotic replacement. Among these strains, Weissella sp. SNUL2, derived from a traditional fermented food in Korea (i.e., Sikhae), emerged as a promising candidate. The correlation between optical density at 600 nm and colony-forming units was verified and applied in subsequent experiments. To assess the therapeutic potential of probiotics, antibacterial tests were conducted using a microplate reader to evaluate the inhibition of 60 bacterial strains (including common foodborne pathogens) induced by Weissella sp. SNUL2 cell-free supernatant (CFS). The results confirmed its broad-spectrum antibacterial properties compared to previously known probiotics. Furthermore, enzymatic treatment with proteinases (trypsin and pepsin) and a time-kill assay were conducted to elucidate the nature of the antibacterial substance in Weissella sp. SNUL2 CFS. Through sequential chromatography involving gel filtration and ion-exchange chromatography, specific fractions with enhanced antibacterial properties were identified. LC-MS/MS analysis of the secretome fraction revealed the presence of various proteins from the C39 family, peptidoglycan endopeptidases, and N-acetylmuramoyl-l-alanine amidase domain-containing protein precursors. Hence, the combined action of these proteins may contribute to Weissella sp. SNUL2's broad antimicrobial activity.