Additional file 1: Figure S1. Separation of different FOS types using HPLC analysis. I-FOS InuGB-V3: Process solution of the I-FOS production from 800 g L-1 sucrose using crude inulosucrase. GFn I-FOS standards: 1-kestose (GF2), 1,1-kestotetraose (GF3) and 1,1,1-kestopentaose (GF4) (Megazyme). Fn L-FOS standards: levanbiose (F2) and levantriose (F3) (Megazyme). Fn + GFn L-FOS: L-FOS produced as described by Hövels et al. 2021. Sugar and FOS concentrations were determined via isocratic HPLC using column Shodex Ashiapak NH2P-50 4E with 58 % [v/v] acetonitrile at a flow rate of 0.7 mL min−1. Figure S2. Influence of Ca2+, Mg2+, Mn2+, and EDTA on sucrose transfructosylation and hydrolysis by crude inulosucrase. Crude enzyme (4000 U L−1) was added to prewarmed solutions of 570 g L−1 sucrose in 25 mM sodium acetate buffer (pH 4.6) supplemented with 1 mM CaCl2, MgCl2, MnSO4, or EDTA. Activity assays were perfomed in biological triplicates at 37 °C in a 1 mL scale. Sucrose and fructose concentrations before and after 24 h of reaction time were determined via isocratic HPLC using 65 % [v/v] acetonitrile at a flow rate of 1 mL min−1. Figure S3. Conversion and hydrolysis of 570 (a) and 800 g L-1 (b) sucrose by crude InuGB-V3 during I-FOS production. The bioconversion reactions were started by adding 4000 or 6000 U L-1 crude inulosucrase to prewarmed solutions of 25 mM sodium acetate buffer (pH 5.5) and 1 mM CaCl2 with 570 or 800 g L-1 sucrose, respectively. The reactions were perfomed in biological (570 g L−1 sucrose) or technical (800 g L−1 sucrose) triplicates at 40 °C at a 1 mL scale. Sugar and I-FOS concentrations were determined via isocratic HPLC using 58 % [v/v] acetonitrile at a flow rate of 0.7 mL min−1. Table S1. Product amounts of the I-FOS production reaction starting with 570 g L−1 sucrose after 18 hours and calculation of the recovery of the substrate. Table S2. Product amounts of the I-FOS production reaction starting with 800 g L−1 sucrose after 20 hours and calculation of the recovery of the substrate. Figure S4. Determination of a suitable ratio of activated charcoal to I-FOS-rich syrup. Bars show the percentage of the applied fructose, glucose, sucrose, and I-FOS adsorbed to the activated charcoal. 100 mg activated charcoal were incubated with varied volumes of process solution from the 10 L scale I-FOS production diluted in 1 mL water for 45 min at 40 °C and 250 rpm in a temperature-controlled shaking incubator. Sugar and I-FOS concentrations in the solution were determined via isocratic HPLC using 58 % [v/v] acetonitrile at a flow rate of 0.7 mL min−1. The experiment was perfomed in triplicates. Figure S5. Different elution protocols to purify I-FOS from I-FOS-rich syrup with activated charcoal. Diplayed are the amounts of the products fructose, glucose, sucrose, and I-FOS that eluted in the consecutive elution steps using various elution protocols. 100 mg activated charcoal was incubated with 21 μL process solution from the 10 L scale I-FOS production diluted in 1 mL water for 45 min at 40 °C and 250 rpm in a temperature-controlled shaking incubator (elution fraction 1). Elution steps were carried out at 40 °C, 250 rpm and the duration indicated in the graphs with 1 mL solvent (0 – 100 % ethanol [v/v]) each. Between the elution steps, the activated charcoal was separated from the elution fractions by centrifugation. Sugar and I-FOS concentrations were determined via isocratic HPLC using 58 % [v/v] acetonitrile at a flow rate of 0.7 mL min−1. The purifications depicted in (a) and (b) were perfomed in triplicates, experiments (c) and (d) in duplicates.