Isolation and in vitro characterization of Liquorilactobacillus ghanensis D15 from water kefir and its anti-Vibrio bacteriocin

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Bacteriocins of natural origin are recognized for their safety, high efficacy, and broad-spectrum antibacterial activity, and have been widely used in controlling pathogenic bacteria such as Staphylococcus aureus and Escherichia coli . However, their application against Vibrio parahaemolyticus , a major pathogen in aquaculture, remains underexplored. In this study, we isolated and screened bacteriocin-producing lactic acid bacteria from water kefir and its starter cultures using Vibrio parahaemolyticus as the indicator strain. The selected isolate underwent evaluation for its probiotic properties and was identified via 16S rDNA sequencing as Liquorilactobacillus ghanensis D15 (deposited as KD9 at CCTCC under accession No. M2024604), representing its first reported isolation from water kefir. The strain exhibited strong acid tolerance, bile salt resistance, auto-aggregation, co-aggregation, a survival rate of 29.67% in simulated gastrointestinal fluid, and a 55.90% adhesion rate to Caco-2 cells. Fermentation conditions for bacteriocin production were optimized via single-factor experiments, revealing inoculum size as a critical factor. The fermented supernatant of KD9 was further subjected to ethyl acetate extraction, ultrafiltration, and preparative chromatography, resulting in the isolation and purification of bacteriocin B-KD9, which exhibited a 23.43-fold enhancement in its anti- Vibrio activity. LC-MS/MS analysis of the active B-KD9 fraction revealed the presence of five candidate peptide sequences, namely FTGPDVKFP, PVGLADPDGPA, PDSPLFDKT, FPSLVGRPR, and TDDLLEDLK. The bacteriocin demonstrated exceptional stability across a wide pH range (2.0–8.0) and under high-temperature conditions (78.18% activity retention after 120°C for 20 min). It exhibited broad-spectrum antibacterial activity against key aquaculture pathogens including Vibrio parahaemolyticus and Vibrio alginolyticus , as well as against Staphylococcus aureus and Escherichia coli . These findings indicate that KD9 and its active peptide fraction represent promising candidates for further investigation as natural anti- Vibrio agents.