Safety Assessment of GABA-Producing Levilactobacillus brevis LAB6 MTCC 25662 and Its Anti-inflammatory Effects in Murine Macrophages

Abstract

To ascertain safety, the assessment of bacterial strains to be used as probiotics needs rigorous and well-designed in vitro and in vivo studies. Here, the safety of a GABA-producing Levilactobacillus brevis LAB6 MTCC 25662 was assessed using in (silico, vitro and vivo) approaches. Firstly, the genomic analysis suggested that LAB6 is non-pathogenic to humans, as it does not harbour the genes for virulence, pathogenesis-related and horizontally transferable antimicrobial resistance. LAB6 neither produces biogenic amines nor degrades mucin, has no haemolytic activity and does not exert cytotoxicity on HEK-293 cells. In vivo safety of LAB6 was assessed in acute, subacute and sub-chronic oral feeding experiments following revised OECD guidelines 425, 407 and 408, respectively. Histopathological, blood biochemical, haematological parameters, gut permeability and oxidative stress levels were assessed. In vivo studies indicated that LAB6 did not negatively impact haematological markers or cause deleterious histological alterations in the vital organs. The anti-inflammatory potential of LAB6 in alleviating lipopolysaccharide (LPS)-induced inflammation in murine macrophages was assessed in the presence of GABA A and GABA B receptor antagonists. LAB6, owing to its GABA-producing ability, prevented LPS-induced inflammation by reducing TNF-α, IL-6 and IL-1β levels by 62.3%, 27.2% and 74.8%, respectively. Antagonism of the GABA A receptor with bicuculline methiodide (BMI) partially blunted the protective effects of LAB6, while GABA B antagonism has no significant impact in curtailing its protective effects. Overall results indicated that oral consumption of anti-inflammatory and GABA-producing LAB6 is safe to test in human studies further.