Lactobacilli represent a wide range of bacterial species with several 
implications for the human host. They play a crucial role in maintaining the 
ecological equilibrium of different biological niches and are essential for 
fermented food production and probiotic formulation. Despite the consensus about 
the 'health-promoting' significance of Lactobacillus genus, its genotypic and 
phenotypic characterization still poses several difficulties. The aim of this 
study was to assess the integration of different approaches, genotypic (16S rRNA 
gene sequencing), proteomic (MALDI-TOF MS) and metabolomic (1H-NMR), for the 
taxonomic and metabolic characterization of Lactobacillus species. For this 
purpose we analyzed 40 strains of various origin (intestinal, vaginal, food, 
probiotics), belonging to different species. The high discriminatory power of 
MALDI-TOF for species identification was underlined by the excellent agreement 
with the genotypic analysis. Indeed, MALDI-TOF allowed to correctly identify 39 
out of 40 Lactobacillus strains at the species level, with an overall 
concordance of 97.5%. In the perspective to simplify the MALDI TOF sample 
preparation, especially for routine practice, we demonstrated the perfect 
agreement of the colony-picking from agar plates with the protein extraction 
protocol. 1H-NMR analysis, applied to both culture supernatants and bacterial 
lysates, identified a panel of metabolites whose variations in concentration 
were associated with the taxonomy, but also revealed a high intra-species 
variability that did not allow a species-level identification. Therefore, 
despite not suitable for mere taxonomic purposes, metabolomics can be useful to 
correlate particular biological activities with taxonomy and to understand the 
mechanisms related to the antimicrobial effect shown by some Lactobacillus 
species.