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

Exopolysaccharides produced by lactic acid bacteria : from health-promoting benefits to stress tolerance mechanisms

A wide range of lactic acid bacteria (LAB) is able to produce capsular or extracellular polysaccharides, with various chemical compositions and properties. Polysaccharides produced by LAB alter the rheological properties of the matrix in which they are dispersed, leading to typically viscous and “ropy” products. Polysaccharides are involved in several mechanisms such as prebiosis and probiosis, tolerance to stress associated to food process, and technological properties of food. In this paper, we summarize the beneficial properties of exopolysaccharides (EPS) produced by LAB with particular attention to prebiotic properties and to the effect of exopolysaccharides on the LAB-host interaction mechanisms, such as bacterial tolerance to gastrointestinal tract conditions, ability of ESP-producing probiotics to adhere to intestinal epithelium, their immune-modulatory activity, and their role in biofilm formation. The pro-technological aspect of exopolysaccharides is discussed, focusing on advantageous applications of EPS in the food industry, i.e., yogurt and gluten-free bakery products, since it was found that these microbial biopolymers positively affect the texture of foods. Finally, the involvement of EPS in tolerance to stress conditions that are commonly encountered in fermented beverages such as wine is discussed

The lactic acid bacteria of fermented fruits and vegetables

Vegetables fermented foods and beverages are traditional worldwide consumed foodstuffs, since fermentation is one of the most important methods of food preservation still in use for vegetables and fruits. Several species of lactic acid bacteria are involved in the fermentation of vegetable matrices. These microorganisms transform raw materials trough their metabolism conferring specific nutritional and organoleptic properties to the product. The industrial demand of standardization of the product and the process, as well as the need to ensure food safety, require to drive a controlled fermentation. Therefore, selected cultures of lactic acid bacteria are recommended as microbial starter for industrial fermentations. In the last years, an increasing interest has been generated about the beneficial role played by lactic acid bacteria in the gut environment. For this reason, fermented food and beverages from vegetables have been proposed as optimal carriers to deliver probiotic bacteria and for the elaboration of functional foods. In this chapter, we shall provide a background on the key role of lactic acid bacteria in vegetable fermentations, focusing on both traditional and innovative applications for the food industry

Barley β-Glucans-Containing Food Enhances Probiotic Performances of Beneficial Bacteria

Currently, the majority of prebiotics in the market are derived from non-digestible oligosaccharides. Very few studies have focused on non-digestible long chain complex polysaccharides in relation to their potential as novel prebiotics. Cereals β-glucans have been investigated for immune-modulating properties and beneficial effects on obesity, cardiovascular diseases, diabetes, and cholesterol levels. Moreover, β-glucans have been reported to be highly fermentable by the intestinal microbiota in the caecum and colon, and can enhance both growth rate and lactic acid production of microbes isolated from the human intestine. In this work, we report the effects of food matrices containing barley β-glucans on growth and probiotic features of four Lactobacillus strains. Such matrices were able to improve the growth rate of the tested bacteria both in unstressed conditions and, importantly, after exposure to in vitro simulation of the digestive tract. Moreover, the effect of β-glucans-containing food on bacterial adhesion to enterocyte-like cells was analyzed and a positive influence on probiotic-enterocyte interaction was observed

Functional Starters for Functional Yogurt

In this study, we investigated the multifunctionality (microbial starters and probiotics) of Lactobacillus plantarum WCFS1 and Lactobacillus plantarum CECT 8328 strains used as microbial starters for the production of yogurt in combination with Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus. The ability of the probiotic strains to survive to oro-gastrointestinal stresses was monitored by an in vitro assay simulating the human digestive tract. The transcriptional level of several genes involved in the immune response suggested that the probiotic strains may have a favorable influence on immunomodulation. Overall, this study revealed that the tested lactobacilli exhibited suitable technological features for yogurt production and might be used to formulate novel food with immunomodulating effects

Strain-specific features of extracellular polysaccharides and their impact on Lactobacillus plantarum-host interactions

Lactobacilli are found in diverse environments and are widely applied as probiotic, health-promoting food supplements. Polysaccharides are ubiquitously present on the cell surface of lactobacilli and are considered to contribute to the species- and strainspecific probiotic effects that are typically observed. Two Lactobacillus plantarum strains, SF2A35B and Lp90, have an obvious ropy phenotype, implying high extracellular polysaccharide (EPS) production levels. In this work, we set out to identify the genes involved in EPS production in these L. plantarum strains and to demonstrate their role in EPS production by gene deletion analysis. A model L. plantarum strain, WCFS1, and its previously constructed derivative that produced reduced levels of EPS were included as reference strains. The constructed EPS-reduced derivatives were analyzed for the abundance and sugar compositions of their EPS, revealing cps2-like gene clusters in SF2A35B and Lp90 responsible for major EPS production. Moreover, these mutant strains were tested for phenotypic characteristics that are of relevance for their capacity to interact with the host epithelium in the intestinal tract, including bacterial surface properties as well as survival under the stress conditions encountered in the gastrointestinal tract (acid and bile stress). In addition, the Toll-like receptor 2 (TLR2) signaling and immunomodulatory capacities of the EPS-negative derivatives and their respective wild-type strains were compared, revealing strain-specific impacts of EPS on the immunomodulatory properties. Taken together, these experiments illustrate the importance of EPS in L. plantarum strains as a strain-specific determinant in host interaction.</p

Zebrafish gut colonization by mCherry-labelled lactic acid bacteria

39 p.-7 fig.-2 fig. suppl.A critical feature of probiotic microorganisms is their ability to colonize the intestine of the host. Although the microbial potential to adhere to the human gut lumen has been investigated in in vitro models, there is still much to discover about their in vivo behaviour. Zebrafish is a vertebrate model that is being widely used to investigate various biological processes shared with humans. In this work, we report on the use of the zebrafish model to investigate the in vivo colonization ability of previously characterized probiotic lactic acid bacteria. Lactobacillus plantarum Lp90, L. plantarum B2 and Lactobacillus fermentum PBCC11.5 were fluorescently tagged by transfer of the pRCR12 plasmid, which encodes the mCherry protein and which was constructed in this work. The recombinant bacteria were used to infect germ-free zebrafish larvae. After removal of bacteria, the colonization ability of the strains was monitored until 3 days post-infection by using a fluorescence stereomicroscope. The results indicated differential adhesion capabilities among the strains. Interestingly, a displacement of bacteria from the medium to the posterior intestinal tract was observed as a function of time that suggested a transient colonization by probiotics. Based on fluorescence observation, L. plantarum strains exhibited a more robust adhesion capability. In conclusion, the use of pRCR12 plasmid for labelling Lactobacillus strains provides a powerful and very efficient tool to monitor the in vivo colonization in zebrafish larvae and to investigate the adhesion ability of probiotic microorganisms.The work performed in Spain was supported at the Centro de Investigaciones Biológicas by grant AGL2012-40084-C03-01 from the Spanish Ministry of Economics and Competitiveness and at AZTI-Tecnalia by the “Departamento de Desarrollo Económico y Competitividad Viceconsergería de Agricultura, Pesca y Politicas Alimentarias” (Project FOODBASK3). The work performed in Italy was supported by the Italian Ministry for Development in the framework of the “Industria 2015 Bando Nuove Tecnologie per il Made in Italy—Realizzazione di una innovativa pasta alimentare funzionale arricchita di componenti bioattivi e probiotici”, and by MIUR (PON02_00186_2937475) in the framework of the project named “Protocolli innovativi per lo sviluppo di alimenti funzionali” “(Pro.Ali.Fun.)”.Peer reviewe