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

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. 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 ±â€¯7.4 g inulin-FOSL-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 min. 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.

Biosynthesis of inulin from sucrose using inulosucrase from Lactobacillus gasseri DSM 20604.

Inulin is composed of fructose residues connected by ß-(2, 1) glycosidic linkages with many promising physiochemical and physiological properties. In this study, an inulin-producing inulosucrase gene from Lactobacillus gasseri DSM 20604 was cloned, expressed and purified. SDS-PAGE and gel filtration found that the recombinant inulosucrase is a monomeric protein with a molecular weight of 63KDa. The optimal pH for its sucrose hydrolysis and transfructosylation activities was pH 5.5. The optimal temperatures were measured to be 45, 25, and 35°C for sucrose hydrolysis, transfructosylation, and total activity, respectively. Biosynthesis studies showed that the optimal enzyme dosage was 4.5U/g sucrose. Higher sucrose concentrations immensely contributed to inulin biosynthesis; the inulin yield reached its maximum after 1.5h of reaction. Structural analyses of the polysaccharide produced by the recombinant enzyme from sucrose revealed that it is an inulin-type fructan with a molecular weight of 5.858×106Da.

Synthesis and structural characterization of raffinosyl-oligofructosides upon transfructosylation by Lactobacillus gasseri DSM 20604 inulosucrase.

A new process based on enzymatic synthesis of a series of raffinose-derived oligosaccharides or raffinosyl-oligofructosides (RFOS) with degree of polymerization (DP) from 4 to 8 was developed in the presence of raffinose. This process involves a transfructosylation reaction catalyzed by an inulosucrase from Lactobacillus gasseri DSM 20604 (IS). The main synthesized RFOS were structurally characterized by nuclear magnetic resonance (NMR). According to the elucidated structures, RFOS consist of ß-2,1-linked fructose unit(s) to raffinose: α-D-galactopyranosyl-(1 → 6)-α-D-glucopyranosyl-(1↔2)-ß-D-fructofuranosyl-((1 ← 2)-ß-D-fructofuranoside)n (where n refers to the number of transferred fructose moieties). The maximum yield of RFOS was 33.4 % (in weight respect to the initial amount of raffinose) and was obtained at the time interval of 8-24 h of transfructosylation reaction initiated with 50 % (w/v) of raffinose. Results revealed the high acceptor and donor affinity of IS towards raffinose, being fairly comparable with that of sucrose for the production of fructooligosaccharides (FOS), including when both carbohydrates coexisted (sucrose/raffinose mixture, 250 g L(-1) each). The production of RFOS was also attempted in the presence of sucrose/melibiose mixtures; in this case, the predominant acceptor-product formed was raffinose followed by a minor production of a series of oligosaccharides with varying DP. The easiness of RFOS synthesis and the structural similarities with both raffinose and fructan series of oligosaccharides warrant the further study of the potential bioactive properties of these unexplored oligosaccharides.