A Fermented Food-Derived Putative Anti-Helicobacter pylori Peptide from Lactiplantibacillus pentosus Isolated from Fermented Mushroom Sausage: Activity-Guided Enrichment and Activity in Simulated Gastric Fluid

Functional fermented foods are increasingly recognized as sources of microbial metabolites with potential biological activity. In this study, MRK2-3, a protease-sensitive anti-Helicobacter pylori component produced by Lactiplantibacillus pentosus MRK2-3 isolated from fermented mushroom sausage, was enriched and preliminarily characterized for in vitro anti-H. pylori activity. Activity appeared during the stationary phase. Sequential ethyl acetate extraction, Sep-Pak C18 chromatography, and reverse-phase HPLC increased specific activity, although the final activity recovery was 0.72%. MALDI-TOF analysis of the active fraction showed a dominant ion at approximately m/z 1848 together with several lower-intensity ions; therefore, chemical homogeneity and sequence identity were not established. The MRK2-3 fraction retained activity after incubation at pH 2–6 and after heating at 80 °C and 100 °C for 30 min, whereas activity decreased under alkaline conditions and after treatment at 121 °C for 15 min. Trypsin, α-chymotrypsin, pepsin and proteinase K reduced activity. The fraction inhibited all tested H. pylori strains (agar-dilution MIC ranged from 12.5 to 100 μg/mL). In a simulated gastric fluid, higher concentrations produced a rapid reduction in viable H. pylori 3949 counts. These findings support MRK2-3 as a fermentation-derived putative antimicrobial peptide candidate with anti-H. pylori activity under gastric-like conditions. However, its amino acid sequence, structural identity, and novelty require confirmation. Antimicrobial mechanism, cytotoxicity, and in vivo efficacy also remain to be established.