Chromeleon software version 7

Preliminary compositional analysis of each obtained GOS mixture was determined by glyco-profile analysis employing a High Performance Anion Exchange Chromatography and pulsed amperometric detection (HPAEC-PAD; Dionex IC-3000 system; Thermo Scientific¹). Separations were performed using a CarboPac PA1 (Thermo Scientific) analytical-anion exchange column (dimensions, 250 mm by 4 mm) with a CarboPac PA1 (Thermo Scientific) guard column (dimensions, 50 mm by 4 mm) and a detector (ED40) in the pulsed amperometric detection PAD mode (Dionex, Thermo Scientific). Qualitative analysis of the GOS Dionex profile was performed with an elution gradient according to a previously published method (Van Leeuwen et al., 2016 (link)) and the qualitative determination of the carbohydrate composition was performed by the use an elution gradient summarized in supplemental Supplementary Table 3 at a constant flow rate of 1.0 ml min^–1 at 30°C using the following eluents with programmed gradient for the analysis: (A) 100 mmol NaOH, (B) 100 mmol NaOH, 500 mmol sodium acetate (NaAC), (C) 50 mmol NaAC and (D) Milli-Q water. The obtained chromatography profiles were analyzed employing CHROMELEON software Version 7 (Dionex, Thermo Scientific).

Oligo- and monosaccharides were analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) on a Dionex ICS-3000 system operated by Chromeleon software version 7 (Dionex). Sugars were loaded onto a CarboPac PA1 2 × 250-mm analytical column (Dionex, Thermo Scientific) coupled to a CarboPac PA1 2 × 50-mm guard column kept at 30°C. Depending on the analytes, the following gradients were used. The system was run at a flow rate of 0.25 ml/min. For manno-oligosaccharides, the elution conditions were as follows: for 0 to 9 min, 0.1 M NaOH; for 9 to 35 min, 0.1 M NaOH with a 0.1 to 0.3 M sodium acetate (NaOAc) gradient; for 35 to 40 min, 0.1 M NaOH with 0.3 M NaOAc, and for 40 to 50 min 0.1 M NaOH. Commercial mannose and manno-oligosaccharides (DP, 2 to 6) were used as external standards.

High Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) was performed on a Dionex ICS-5000 DC HPLC system operated by the Chromeleon software version 7 (Dionex) using a Dionex Carbopac PA200 column. Solvent A was double-distilled water, solvent B was 1 M sodium hydroxide (NaOH), and solvent C was 1 M sodium acetate (NaOAc). The gradient used was: 0–4 min, 10% solvent B and 2.5% solvent C; 4–24 min, 10% B and a linear gradient from 2.5 to 25% C; 24–24.1 min, 50% B and 50% C; 24.1–25 min, an exponential gradient of NaOH and NaOAc back to initial conditions; and 25–31 min, initial conditions.
Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) was performed on a Bruker Daltonics Autoflex System (Billerica, USA). The matrix, 2,5-dihydroxy benzoic acid, was dissolved in 50% methanol in water to a final concentration of 10 mg mL⁻¹. Oligosaccharide samples were mixed 1:1 (v/v) with the matrix solution. One microliter of this solution was placed on a Bruker MTP 384 ground steel MALDI plate and left to air dry for 2 h prior to analysis.