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The rapid characterization of lactic acid bacteria (LAB) with probiotic and technological properties is crucial for functional food design. In this study, fourteen LAB strains belonging to the species Lactiplantibacillus (L.) plantarum, Lentilactobacillus (L.) parabuchneri, and Leuconostoc (L.) mesenteroides were differentiated using Raman spectroscopy. By integrating Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA), we achieved a clear inter-generic separation while simultaneously enabling the intra-specific grouping of L. plantarum strains. Our results demonstrate that the Raman spectral fingerprint, coupled with supervised chemometric models, successfully categorized the strains into three distinct clusters based on their macromolecular profiles. Specifically, the analysis provided high-resolution differentiation between genera and, more importantly, allowed for the fine-scale clustering of diverse L. plantarum isolates. This highlights Raman spectroscopy as a robust, non-destructive tool for the rapid identification and taxonomic classification of LAB, offering a high-throughput alternative to traditional molecular methods for strain-level discrimination.
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