Identification and Quantification of Nonviable Lactobacillus pentosus Cells in a Health Food Product

Background: The lack of formal protocol to verify the nonviable cell probiotic product authenticity blocks its registration and supervision process. Objective: To develop a protocol for identification, enumeration, and purity determination of a Lactobacillus pentosus nonviable cell product. Methods: The 16S ribosomal RNA (rRNA) sequencing, 16S rRNA metagenomic analysis, whole-genome sequencing, matrix-assisted laser desorption ionization time of flight MS (MALDI-TOF-MS), and FACSMicroCount (TM) system were applied to establish a protocol of identification, enumeration and purity determination of a Lactobacillus pentosus nonviable cell product. Results: The 1530 bp of 16S rRNA sequence could only identify the bacteria at genus level, but the MALDI-TOF-MS could identify both the nonviable cell and fresh culture to species level with high confidence. Metagenomic analysis of the 16S rRNA amplicon could recognize Lactobacillus as the dominant genus in the nonviable cell product. The total number of matching k-mers between the nonviable cell product and the L. pentosus BGM48 in the GenBank was the highest. The 95% confidence interval of the nonviable cell concentration in the product was determined as 4.31-4.68x10(10) cells/g through the BD FACSMicroCount system. Conclusions: This validation protocol offers an executable approach that can verify microbial contents in nonviable cell products and ensure the compliance with label claims. Highlights: The established validation protocol could determine the nonviable cell species through MALDI-TOF-MS, the concentration through FACSMicroCount system, and the purity and strain level identification through metagenomic analysis of 16S rRNA and the genomic deoxyribonucleic acid.