Genome mining and proteomics approach confirms the production of antifungal peptides in Lactobacillus plantarum UC8491

Antifungal compounds from LAB able to inhibit food-spoilage fungi are of greatest interest for the food industry. Until today, the antifungal activity of LAB remains poorly understood and need to be continuously study to development of new commercial formulations applicable to the control of food-borne yeasts and molds. A bacteriocin producing strain Lactobacillus plantarum UC8491 has been demonstrated largely its ability to inhibit the development of foodborne pathogenic bacteria and spoilage fungi in fermented food such as cheese and yogurt. In this study, the analysis of the whole genome sequence of L. plantarum UC8491 and de novo proteomic approach was applied to understand the nature of the antifungal activity. The genomic DNA of L. plantarum UC8491 was sequenced using a whole-genome shotgun strategy with an Illumina Genome analyzer Hiseq1000. The draft genome has a size of 3.30 Mb and contain 334 subsystems (sets of related functional roles), respectively, according to the RAST Server. L. plantarum UC8491, genome analysis confirm the presence of the plantaricin biosynthesis cluster containing the pln genes organized in five operons: plnABCD; plnEFI; plnJKLR, plnMNOP, and plnGHSTUV. A bottom-up proteomic approach was carried out through QTOF mass spectrometry from a cell free supernatant precipitated with ammonium sulfate and from a band excised of the inhibition zone observed in the direct detection of anti- microbial activity on Tricine-SDS-PAGE gel. Peptides were filtered through a 10 kDa membrane, reduced and alkylated, then separated via nanoscale CHIP liquid chromatography in reverse mode, and finally analyzed in data-dependent tandem mass spectrometry. Spectra were processed in SpectrumMill versus Bactibase and Bagel database, in similarity mode, using a false discovery rate of 1%. The analysis of the fraction 6410 kDa resulted in the identification of peptides belonging to bacteriocins or related proteins, such as Bioactive peptide 1; acidocin, pyiocin/carocin, lantibiotic, ABC transporter, ATP-binding protein. Peptides belonging to many types of colicins were also identified. Peptides sequences from tricine gel shows homology with Enterocin AS-48 and divergicin 750 both from class IIc of bacteriocins, this results are in accordance with the Bagel database analysis, showing the presence in the genoma of L. plantarum UC 8491 of more tham 30 small ORF from putative bacteriocin from Class IIb and IIc diffretent than plantaricin. Results obtained in this study reveled that this strain produces additional antimicrobial peptides, probably responsible of its antifungal activity