Characterization of EPS from Ligilactobacillus salivarius CRL 2217 by transmission and scanning electron microscopy techniques

Abstract

Bacterial exopolysaccharides (EPS) are macromolecules usually synthesized during growth under certain incubation conditions. Depending on their location, they may be of a capsular type (tightly bound to the cell surface) and/or secreted to the extracellular environment (with weak bonds or completely secreted). These compounds can exhibit diverse functional and technological properties based on their structure; e.g.: they may possess antioxidant, prebiotic, anti-inflammatory, immunostimulant, or antibacterial properties. The objective of this study was to characterize the EPS produced by Ligilactobacillus salivarius (Lg. salivarius) CRL 2217 in a modified MRS broth employing scanning electron microscopy (SEM) and transmission electron microscopy (TEM) techniques. Growth curves of Lg. salivarius CRL 2217 were performed in MRS broth (control) and MRS modified by replacing glucose with 5% sucrose as the energy source. Samples were taken at different times to determine O.D.560nm, CFU/mL, EPS concentration was determined using the DuBois et al. (1956) method with D-glucose as a standard, and residual glucose using the glucose oxidase method (Wiener Lab., Rosario, Argentina). The results obtained indicated that EPS synthesis was stimulated in the medium with sucrose. It occurred simultaneously with cell growth and reached a maximum peak concentration of 1.40 ± 0.04 mg/mL at 16 h. In MRS with glucose, a basal EPS production of approximately 0.2 mg/mL was determined along the curve. A higher electron density was observed around the bacteria in the medium with sucrose when the cells were evaluated in the late exponential phase in both media using TEM. In addition, morphological changes on the cells were observed in the different culture media. The extracted EPS was purified, lyophilized, and analyzed by SEM at different magnifications. A structure of meshes and superimposed layers, which could confer a good water retention capacity, was observed. Thus, the water retention capacity and the solubility in water of the EPS were evaluated according to the method of Du et al. (2018), obtaining values of 445.35 ± 12.23 and 98.24 ± 0.61 %, respectively. The results of this study suggest that the EPS of Lg. salivarius CRL 2217, synthesized in an MRS medium with sucrose as the energy source, was secreted around the bacterium in a discontinuous manner and may be weakly bound to the cell surface. It exhibits a lattice structure that confers excellent solubility and water-holding capacity. This EPS could be a potential additive for use as a thickener, stabilizer, and emulsifier in farm animal feed products. Also, its potential in drug delivery could be explored.