Safety evaluation of indigenous probiotic Limosilactobacillus fermentum NCDC 400 using whole genome sequences and in vitro approaches

The safety of probiotic bacterial strains has been the subject of considerable discussion due to emerging health risks associated with their use in various health conditions. Herein, we integrate and execute both in silico and in vitro approaches to evaluate the safety of an indigenous probiotic strain, Limosilactobacillus fermentum NCDC 400. Our analysis of the strain's whole genome sequencing using established pipelines and universally accepted databases find no potential antibiotic-resistance or virulence genes of public health concern. Functional annotation of the genome indicates that NCDC 400 does not produce unusual metabolites or harmful enzymes. In vitro testing confirms that the strain does not exhibit any harmful phenotypes, activities of harmful enzymes, or overproduction of harmful metabolites. The strain is sensitive to the antagonistic properties of human serum as well as to all antibiotics enlisted by EFSA guidelines. Targeted PCR assays confirm the absence of genes conferring unusual antibiotic resistance and virulence factors. Cell culture assays reveal that NCDC 400 is non-cytotoxic to Caco-2 cells and does not affect their cellular integrity. Overall, these results demonstrate the safe and non-toxic nature of NCDC 400 and call for further in vivo trials to evaluate and establish its safety in healthy and diseased preclinical models.