Biofilm formation is one of the most important adaptation processes in Bacillus subtilis. The exopolymeric substances (EPS) are crucial element in the process of biofilm formation. The essential EPS component is polysaccharide EpsA-O, which is necessary for the formation of mature biofilm. With the cultivation of highly productive mutant strain B. Subtilis (ΔsinR) on the solid MSgg medium for 24h with increased glycerol concentration, we obtained extracellular material from which the polysaccharide EpsA-O was isolated. For negative control we used mutant strain B. subtilis ΔepsA-O. We have focused on purity, rheology and HPSEC analysis of isolated material. At the microscopic, macroscopic and biochemical levels, we observed differences between the highly productive strain and the negative control. The differences between the isolated materials of strains ΔsinR and ΔepsA-O are also evident on the chromatograms obtained by HPSEC method. The refractive index chromatogram from the strain ΔsinR contains an additional peak, which is assumed to belong to an EpsA-O polysaccharide. We estimated that it belongs to the size class > 3000 kDa. By monitoring the purity of isolates, we found that the most impurities represent biological molecules that absorb light in the UV part of the spectrum, the most important step of purification process represents a change in the pH of the solution. With the rheological methods, we confirmed that the viscosity of isolates solutions was much higher if the cells were cultivated on a solid and not on the liquid medium. We also found that the behaviour of isolates solutions from strain ΔsinR is pseudoplastic even at very low concentrations, while isolates from the strain ΔepsA-O behave like Newtonian fluids. High intrinsic viscosity supports the estimated HPSEC of the EpsA-O polysaccharide. By oscillatory assay we found that the purified isolate at a concentration of 1% has a gel-like structure, all samples below this concentration have liquid-like structure.
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