Carbopac pa1 analytical column

FPase, pectinase, α-amylase, and α-glucosidase activities were measured as described elsewhere [50 (link)–52 (link)]. The protein concentration was assayed using the Lowry method [53 (link)]. For composition analysis of SPRs, glucose, xylose and galactose were produced according to the National Renewable Energy Laboratory protocols [54 ]. The content of glucose was determined as described elsewhere [6 (link)]. The contents of xylose and galactose were determined using a Dionex ICS 2500 system (Thermo Scientific, Waltham, MA, USA) with a CarboPac™ PA1 analytical column (2 × 250 mm). The elution solution was a mixture of water and 400 mM NaOH at a volume ratio of 95:5. The production and measurement of galacturonic acid followed the protocols as previously described [55 (link)]. The pectin content was based on the released galacturonic acid. Ash and lignin were measured as described elsewhere [56 (link)]. Starch content was determined with the method described previously [48 (link)]. The cellulose content was determined by subtracting the starch content from the glucan content. Three independent replicates were performed.

The oligosaccharides were also analyzed by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using an ICS-3000 system (Dionex, Sunnyvale, CA, USA) and a CarboPac-PA1 analytical column (4 × 250 mm, Thermo Scientific, USA) fitted with a CarboPac PA1 guard column (4 × 50 mm, Thermo Scientific, USA). Before samples were injected, the column was washed with 200 mM NaOH for 10 min at a flow rate of 1 mL min⁻¹ and equilibrated for 5 min with 100 mM NaOH and 5% (v/v) 1 M NaOAc. The samples were eluted with a constant 100 mM NaOH solution and a gradient of 1 M NaOAc from 5% to 25% over 25 min. Pure DP3 (G4G3G, M3) and DP4 (G4G4G3G, M4) (1,3;1,4)-β-d-glucan oligosaccharide standards, and pure oligosaccharide standards derived from laminarin (laminaribiose L2, laminaritriose L3, and laminaritetraose L4) and cellulose (cellotriose C3, cellotetraose C4, cellopentaose C5, and cellohexaose C6) were obtained from Megazyme.

Bacterial cultures in mMRS medium were collected at 6 and 30 h of growth, representing the early-log phase and the late stationary phase. Bacterial cells were removed by centrifugation at 10,000× g for 2 min. Supernatant was collected and diluted 10,000-fold. Dilutions were filtered through a 0.22 μm cellulose acetate membrane (VWR International, Radnor, PA, USA) and 25 µL of these supernatants were injected into a High-Performance Anion-Exchange Chromatograph Coupled with Pulsed Amperometric Detection instrument (HPAE-PAD ICS-5000, Thermo Scientific, Sunnyvale, CA, USA) according to methods from [43 (link)] with some modifications. Briefly, chromatographic separation was carried out on a CarboPac PA1 analytical column (4 × 250 mm, DionexTM, ThermoFisher Scientific, Waltham, MA, USA) and CarboPac PA1 guard column (4 × 50 mm, Dionex) with an isocratic gradient, 0–30 min 73.5% A, 25% B, 1.5% C, at a 1.0 mL/min flow rate, where solvent A was deionized water, solvent B 100 mM NaOH and solvent C was 500 mM NaOAc in 100 mM NaOH. HMOs were quantified using calibration curves generated using reference standards of LNT, LNnT (Dextra, Reading, UK) and 2′-FL (Carbosynth, St. Gallen Switzerland), ranging in concentration from 0.00025 to 0.005 mg/mL. All samples were analyzed in triplicate.