Comprehensive Evaluation of Probiotic Properties and Biosafety of Lactiplantibacillus plantarum D25: Insights from Genomic and Phenotypic Analyses

Lactiplantibacillus plantarum, a probiotic widely used in the food industry and healthcare, requires rigorous safety assessment for industrial development. This study aimed to systematically evaluate the biosafety and probiotic properties of strain D25 through whole-genome sequencing and phenotypic analyses.We performed whole-genome sequencing on the Illumina HiSeq platform and carried out phenotypic assays to assess antibacterial activity, environmental tolerance, and toxicological properties.The genome of D25 measured 3,290,333 bp, had a GC content of 44.44%, and was predicted to encode 3,077 coding sequences. The cell-free supernatant of this strain exhibited broad-spectrum inhibitory activity against 19 pathogenic bacteria; inhibition of Vibrio cholerae and Vibrio alginolyticus was particularly strong, with inhibition-zone diameters of 22.54 mm and 21.44 mm, respectively, and inhibition rates exceeding 70%.Safety assessment indicated that the strain lacked hemolytic and gelatinase activities. Although eight antibiotic-resistance genes were detected, they were all chromosomal and showed no association with mobile genetic elements, which reduced the risk of horizontal gene transfer. Environmental tolerance tests showed that D25 grew at pH 3.5-6.0, tolerated bile salts up to 20 mM, and retained good thermal stability at 50-55 oC.