Polyphasic characterization of exopolysaccharides produced by Lactobacillus plantarum Lp90 strain

ABSTRACT Introduction Lactic acid bacteria (LAB) occur in a variety range of fermented foods. Lactobacillus plantarum is a widespread LAB species which is encountered in diverse niches and some L. plantarum strains have been recognized as probiotics. Several LAB are able to secrete exopolysaccharides (EPS), which can be either highly adherent or loosely bound to the microbial cell surface, thus distinguished into capsular and secreted forms, respectively; they are thought to provide protection against adverse environment. The ability to produce EPS by LAB has been reported to be strictly correlated to the presence of specific eps/cps gene clusters. EPS and EPS-producing LAB have been investigated in relation to their application in food industry and in bacteria-host interaction. Moreover, the prebiotic and pro-technologicals functions of exopolysaccharides produced by LAB are topics of growing interest. Aims of the research In this study, a polyphasic characterization of exopolysaccharides (EPS) produced by a Lactobacillus plantarum strain, named Lp90, was performed. The strain was previously isolated from wine and selected for a typical ropy phenotype. Materials and methods EPS produced by L. plantarum Lp90 were purified and quantified by phenol-sulfuric acid method. Furthermore, chemical characterization was performed by gas-liquid chromatography (GLC). The genome of L. plantarum Lp90 was sequenced using the Illumina GAIIx platform and annotated by RAST (Rapid Annotation using Subsystem Technology) server, allowing a comparative genome analysis with L. plantarum strains already sequenced. 7 Knockout of genes responsible for the ropy phenotype was performed and L. plantarum Lp90 non-ropy mutant strains obtained. Host-lactobacilli (EPS producing) interaction was performed in order to understand the probiotic potential of L. plantarum Lp90 and the possible prebiotic actions of exopolysaccharides produced by this strain. Bacterial survival during the simulation of the gastro-intestinal tract was assayed. The ability of L. plantarum strain Lp90 to adhere and compete for adhesion sites with E. coli O157: H7 on Caco-2 cells, and the colonization of L. plantarum strain Lp90 fluorescently labeled on enterocytic cells of zebrafish larvae, was performed. The potential immune-modulation effects of Lp90 on Caco-2 cells as well as on macrophage-differentiated THP-1 cells with digested yogurt containing this bacterial strain were also evaluated. Moreover, the affinity with abiotic surfaces was observed by the biofilms formation on glass tubes. The potential role of exopolysaccharides produced by L. plantarum Lp90 in relation to its original habitat (wine) was analysed during microvinification assays and in presence of typical wine stresses, such as ethanol, pH and sulfur dioxide. Results Transmission Electron Microscopy (TEM) images clearly showed the presence of exopolysaccharides around the cell wall of Lactobacillus plantarum Lp90. Moreover, the chemical analysis suggested that they are hetero-polysaccharides, composed by rhamnose, glucose, galactose, glucosamine and galactosamine. L. plantarum Lp90 genome is about 3,324,076 bps long with a total of 3,273 predicted genes. Four different cps/eps gene clusters involved in exopolysaccharides biosynthesis were identified; in particular the cps2 gene cluster presented three glycosyltransferase genes apparently unique in Lp90 but homologous to Lactobacillus fabifermentans T30PCM01. Following the entire or partial cps2 cluster deletion, we obtained two non-ropy mutant strains, (Lp90Δcps2 and Lp90Δcps2.5 respectively), thus suggesting that ropy phenotype of L. plantarum Lp90 is inherent to the cluster cps2. 8 EPS produced by L. plantarum Lp90 do not seem to promote in vitro and in vivo bacterial adhesion on intestinal epithelium, as well as the immune-modulation after the interaction of Caco-2 cells, while their inhibitory effect on E. coli adhesion on Caco-2 was observed. Furthemore, L. plantarum Lp90 showed a moderate survival during in vitro models of the gastro-intestinal tract, which is an added value for this strain considering its origin habitat. Exopolysaccharides produced by L. plantarum strain Lp90 mask the ability of this strain to form biofilm on glass surface. Exopolysaccharides produced by L. plantarum strain Lp90 confer increased tolerance to certain stressful conditions (ethanol, low pH, sulfur dioxide, lysozyme) usually encountered during winemaking. Finally, preliminary analysis of yogurt produced with L. plantarum strain Lp90, showed a positive technological features and immune-modulation of cytokine-mediating genes.