Gassericins from Lactobacillus Paragasseri K7: Preparative Isolation and Discovery of Dose-Dependent...

Gassericins K7 are heterodimeric bacteriocins produced by Lactobacillus paragasseri K7. Their biological characterisation has been limited by challenges in obtaining these compounds in sufficient purity and quantity. In this study, we used a preparative isolation and purification workflow for gassericins K7, combining ammonium sulphate precipitation, Amberlite XAD-16 extraction, and reversed-phase chromatography. Mass spectrometry confirmed the presence of all four peptide components GasK7A α, GasK7A β, GasK7B α, and GasK7B β in the pooled preparation, including GasK7A β, identified here for the first time in natural isolates of this strain. The purified bacteriocin preparation exhibited strong antibacterial activity against indicator strain Latilactobacillus sakei NCDO 2714. Cytotoxicity testing in RAW 264.7 macrophages revealed minimal effects at concentrations up to 50 µg/ml, with only a modest reduction in cell viability observed at 100 µg/ml. Subsequent evaluation of immunomodulatory properties demonstrated a clear dose-dependent anti-inflammatory effect in LPS-stimulated macrophages. Treatment with gassericins K7 reduced nitric oxide (NO) production, downregulated inducible nitric oxide synthase (iNOS) expression, and suppressed secretion of the pro-inflammatory cytokines TNF-α and IL-6. Importantly, exposure of macrophages to gassericins alone did not elicit any inflammatory response. These findings provide the first evidence of anti-inflammatory activity of purified gassericins K7 in eukaryotic immune cells and highlight their potential as postbiotic components or biotherapeutic candidates for inflammatory disorders. The preparative method used in this study enables the production of gassericins K7 in quantities sufficient for advanced in vitro and in vivo investigations, facilitating future development of bacteriocin-based therapeutics.