<![CDATA[Pado is a traditional fermented food from West Sumatra, made by combining pelagic fish with grated coconut and dried seeds of Pangium edule. Lactic acid bacteria are the predominant microorganisms present and generally produce metabolites that contribute to prolonging the shelf life of the product. The neutralized cell-free supernatant (NCFS) of these isolates may contain bioactive peptides, such as antibacterial peptides. This study aimed to obtain partially purified NCFS from pado isolates with the best inhibitory activity against Salmonella typhimurium and Staphylococcus aureus using agar diffusion methods. Crude NCFS was obtained from seven isolates from pado by centrifuging their growth media, and the supernatant was neutralized. The proteinaceous nature of crude NCFS was confirmed after artificial digestion with proteolytic pepsin. Ultrafiltration was used to partially purify crude NCFS. Antibacterial peptides were further analyzed using liquid chromatography-high resolution mass spectrometry (LC-HRMS). All NCFS showed antibacterial activity against S. typhimurium, but only three isolates showed apparent inhibitory activity against S. aureus. The Lactiplantibacillus plantarum B3Iw4 isolate was selected for further study. The low-molecular-weight NCFS fraction (in the range of 317.20–1,494.70688 Da) after ultrafiltration by a 3 kDa membrane exhibited a threefold increase in specific activity against S. aureus compared to the crude NCFS. LC-HRMS analysis identified 20 peptides in the partially purified NCFS. The peptide, AAERAGAAALAMHGR, was predicted to have antibacterial activity after matching with the BIOPEP-UWM database, with a matched sequence AA. This peptide has relatively high hydrophobicity and is interesting for further study.]]>
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