Strain-level identification and quantitative detection of Lactiplantibacillus plantarum in fermented milk using MALDI-TOF MS.

The efficacy of probiotics is highly strain-specific; however, accurate strain-level identification remains challenging. In this study, Lactiplantibacillus plantarum, a species widely used in probiotic products, was selected as the target organism to develop a strain-level identification method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). First, comparative analysis of ribosomal protein sequences from 11 strains revealed significant interstrain variation in both number and sequence, theoretically supporting the feasibility of MALDI-TOF MS for strain discrimination. Subsequently, an orthogonal experimental design was employed to optimize the formic acid extraction method. The optimal conditions were harvesting 1000 μL of decline-phase culture, extraction with 75% FA, and a formic acid-to-acetonitrile volume ratio of 80:80 (v/v). By evaluating the number, relative intensity, and positional variation of characteristic peaks within the main spectrum profiles, the optimal number of raw spectra required to construct a single-strain database was determined to be 10. Based on these optimized parameters, a MALDI-TOF MS database for L. plantarum, designated as YLp10-2025, was successfully established. Using this database, individual identification and accurate quantitative detection of different L. plantarum strains were achieved in commercially available fermented milk artificially inoculated with these strains. The strain identification approach established in this study, based on an external MALDI-TOF MS database, provides an effective tool for evaluating the quality of probiotic products and offers technical support for developing strain-level identification methods for other microorganisms.