Probiotics are widely recognized for their health benefits, and as the global demand for probiotic products continues to rise, ensuring their quality becomes increasingly important. Rigorous quality control is crucial for ensuring the safety, stability, and efficacy of probiotics. Recently, genome analysis has been utilized as a tool for probiotic quality control. Genome analysis has revealed that some probiotic products lack the essential genes that are responsible for their intended functions. However, this method can't detect minor sequence mutations within heterogeneous subpopulations. To overcome this limitation, we proposed a single-cell comparative genomic analysis pipeline, a comparative genomic analysis method using single-cell genome sequence data. This novel approach successfully elucidated the population structure within a single strain culture medium. Our change-point quality filter reduced raw SNP calls by (1172 to 113), indicating substantial removal of amplification-induced artifacts while retaining bona-fide variants. Our pipeline identified twelve clusters within the lyophilized powder of Bifidobacterium longum subsp. longum BB536. The most abundant cluster accounted for approximately 70% of the total sequences, with no SNPs in its representative sequence compared to the reference. Moreover, all these clusters showed a high Average Nucleotide Identity (ANI) value of over 99.99% with the reference genome, suggesting the stability of the manufactured probiotic powder. These results suggest that this innovative methodology could monitor the dynamics of minor difference clusters in stable probiotic products. Therefore, we propose that a novel single-cell comparative genomic analysis could be a viable method for more rigorous probiotic quality control, ensuring the safety, stability, and efficacy of probiotic products.
Keywords: Bifidobacterium; Comparative genomics; Probiotics; Safety.
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