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

Homoexopolysaccharides (HoEPS) such as alpha-glucans and beta-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 alpha-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 alpha-glucans requires 2 M trifluoroacetic acid (TFA) for 60/90 min at 121 degrees C while levan is hydrolyzed in 1 M TFA for 30 min at 70 degrees C. Nevertheless, levan was also detectable after hydrolysis in 2 M TFA at 121 degrees 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.

Automated summary

Homoexopolysaccharides (HoEPS) like alpha-glucans and beta-fructans are synthesized by certain types of bacteria. Methylation analysis is an important tool for studying the structure of these polysaccharides, but the conditions for analysis need to be adjusted for different types of HoEPS. Ultrasonication is crucial for swelling and deprotonation of water insoluble alpha-glucans, while water soluble HoEPS do not require ultrasonication. Acid hydrolysis conditions also differ for different HoEPS. Overall, our results demonstrate the need for customized conditions in methylation analysis of bacterial HoEPS.