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

High-yield production and purification of prebiotic inulin-type fructooligosaccharides

“…The recently introduced recombinant variant of inulosucrase InuGB from L. gasseri DSM 20604 designated as InuGB-V3 (GenBank accession: ACZ6728.1 (Wienberg et al 2021 )) was heterologously produced in Escherichia coli BL21 pASK3_InuGB-V3 to obtain crude enzyme solutions. Liquid cell cultures were grown in Terrific-Broth (TB) medium (12 g L −1 tryptone/peptone, 24 g L −1 yeast extract, 5 g L −1 glycerol, 2.31 g L −1 KH 2 PO 4 , 12.54 g L −1 K 2 HPO 4 ; phosphate buffer autoclaved separately) supplemented with 100 µg mL −1 carbenicillin at 37 °C and 180 rpm unless otherwise specified.…”

“…Samples were taken immediately after the start of the reaction and after 24 h of incubation. Sampling and subsequent quantification of sucrose by HPLC was performed as previously described (Wienberg et al 2021 ). These experiments were performed in biological triplicates.…”

“…The reactions were started by adding 4000 U L −1 crude inulosucrase to prewarmed solutions. Samples were taken immediately after the reaction had been started and after 24 h. Sucrose and fructose concentrations were measured by HPLC as described previously (Wienberg et al 2021 ). Free fructose represented the proportion of sucrose that was hydrolyzed.…”

“…4000 U L −1 crude enzyme was added to prewarmed solutions of 570 g L −1 sucrose, 1 mM CaCl 2 , and 25 mM sodium acetate buffer (pH 5.5). Samples were incubated at 40 °C and sampled periodically for 24 h. Samples were mixed with four volumes of 96% [v/v] ethanol and vortexed vigorously to stop the reaction and to precipitate inulin (Wienberg et al 2021 ). I-FOS production from 800 g L −1 sucrose was performed in four replicates at an upscaled volume of 10 L for 20 h. 6000 U L −1 crude enzyme was added to solutions of 800 g L −1 household sugar (sucrose), 1 mM CaCl 2 , and 25 mM sodium acetate buffer (pH 5.5) prewarmed to 40 °C.…”

“…In our previous work, we presented a truncated version of the inulosucrase from Lactobacillus gasseri DSM 20604 named InuGB-V3 (Wienberg et al 2021 ). The enzyme was used in purified form to synthesize I-FOS from sucrose as an analyte to establish an HPLC method suitable for the analysis and quantification of I-FOS.…”