Influence of ultrasonication and hydrolysis conditions in methylation analysis of bacterial homoexopolysaccharides

Abstract

Homoexopolysaccharides (HoEPS) such as α-glucans and β-fructans are synthesized by lactic and acetic acid bacteria. Methylation analysis is an important and well-established tool for the structural analysis of these polysaccharides, however, multiple steps are required for polysaccharide derivatization. Because ultrasonication during methylation and the conditions during acid hydrolysis may influence the results, we investigated their role in the analysis of selected bacterial HoEPS. The results reveal that ultrasonication is crucial for water insoluble α-glucan to swell/disperse and deprotonate prior to methylation whereas it is not necessary for water soluble HoEPS (dextran and levan). Complete hydrolysis of permethylated α-glucans requires 2 M trifluoroacetic acid (TFA) for 60/90 min at 121 °C while levan is hydrolyzed in 1 M TFA for 30 min at 70 °C. Nevertheless, levan was also detectable after hydrolysis in 2 M TFA at 121 °C. Thus, these conditions can be used to analyze a levan/dextran mixture. However, size exclusion chromatography of permethylated and hydrolyzed levan showed degradation and condensation reactions at harsher hydrolysis conditions. Application of reductive hydrolysis with 4-methylmorpholine-borane and TFA did not lead to improved results. Overall, our results demonstrate that conditions used for methylation analysis have to be adjusted for the analysis of different bacterial HoEPS.