High-resolution method for isocratic HPLC analysis of inulin-type fructooligosaccharides

Abstract In recent decades, strategies to improve human health by modulating the gut microbiota have developed rapidly. One of the most prominent is the use of prebiotics, which can lead to a higher abundance of health-promoting microorganisms in the gut [1]. Currently, oligosaccharides dominate the prebiotic sector due to their ability to promote the growth and activity of probiotic bacteria selectively. Extensive efforts are made to develop effective production strategies for the synthesis of prebiotic oligosaccharides, including the use of microbial enzymes. Within the genus Lactobacillus, several inulosucrases have been identified, which are suitable for the synthesis of prebiotic inulin-type fructooligosaccharides (inulin-FOS). In this study, a truncated version of the inulosucrase from Lactobacillus gasseri DSM 20604 was used for the efficient synthesis of inulin-FOS. Product titers of 146.2 ± 7.4 g inulin-FOS L-1 were achieved by the catalytic activity of the purified recombinant protein InuGB-V3. A time and resource-saving HPLC method for rapid analysis of inulin-FOS in isocratic mode was developed and optimized, allowing baseline separated analysis of inulin-FOS up to a degree of polymerization (DP) of five in less than six minutes. Long-chain inulin-FOS with a DP of 17 can be analyzed in under 45 minutes. The developed method offers the advantages of isocratic HPLC analysis, such as low flow rates, high sensitivity, and the use of a simple, inexpensive chromatographic setup. Furthermore, it provides high-resolution separation of long-chain inulin-FOS, which can usually only be achieved with gradient systems.