Observation of exopolysaccharides (EPS) from Lactobacillus helveticus SBT2171 using the Tokuyasu method

Some species of lactic acid bacteria used for the production of natural cheese produce exopolysaccharides (EPS). Electron microscopy is useful for analyzing the microstructure of EPS produced by lactic acid bacteria. However, pretreatments used to observe the microstructure of EPS by electron microscopy, such as dehydration and resin embedding, can result in EPS flowing out easily from the cell. Therefore, in this study, the Tokuyasu method was conducted on cryosection to reduce EPS outflow. Two types of observation method, namely, using lectin and ruthenium red, were conducted in an attempt to observe EPS produced by Lactobacillus helveticus SBT2171. Observation using the lectin method confirmed that colloidal gold particles conjugated with a lectin recognizing β-galactoside were present in the capsule. Structures that appeared to be β-galactoside-containing slime polysaccharides that were released from the cell wall were also observed. Observation using ruthenium red showed that capsular polysaccharides (CPS) in the capsule were present as a net-like structure. Colloidal gold conjugation with an anti-β-lactoglobulin antibody, in addition to ruthenium red staining, allowed the identification of slime polysaccharides released from the cell wall in the milk protein network derived from the culture medium. Based on these results, the Tokuyasu method was considered to be a useful pretreatment method to clarify and observe the presence of EPS. In particular, both CPS in the capsule and slime exopolysaccharides released from the cell wall were visualized.