In vitro and in vivo evaluation of the safety of Levilactobacillus brevis CGMCC1.5954 with probiotic potential based on tri-generation whole genome sequencing and animal studies

Abstract

In this study, the safety of Levilactobacillus brevis CGMCC1.5954 was evaluated, which has probiotic properties, through both in vitro methods such as antibiotic susceptibility testing, biogenic amine testing, and virulence gene analysis, and in vivo methods including biochemical testing, routine blood index testing, and bacterial displacement capacity analysis. The results of HPLC analysis revealed that during incubation, L. brevis CGMCC1.5954 did not secrete cadaverine, histamine, putrescine, or tyramine. It exhibited sensitivity to tetracyclines, macrolides, cephalosporins, β-lactamase inhibitor complex, and other antibiotics, but not to aminoglycosides and quinolones, which is consistent with the behavior of other lactic acid bacteria. Its genome encodes a total of 2314 genes, including 1743 proteins and 109 signal peptides. Notably, no virulence genes were found in this genome. Additionally, the gene encoding the production of γ-aminobutyric acid was found in it. In vivo experiments indicated that gavage feeding of L. brevis CGMCC1.5954 for 28 d had no significant effect on normal growth, blood hemoglobin level, erythrocytes, white blood cells, and other routine blood parameters in mice. Furthermore, there was no migration of it observed in the liver, kidney, spleen, or blood during the feeding trial, which indicated that it did not cause any infections. Moreover, H&E staining showed no evidence of lesion damage to the liver, kidney, or other organs. To sum up, L. brevis CGMCC1.5954 is considered safe for producing fermented foods, especially those that contain high levels of γ-aminobutyric acid.