Ultimate 3000 chromatograph

Average molecular weights of the HA fractions were determined by HP-SEC using Ultrahydrogel 250 column (300 × 7.8 mm, 6 µm, pore size 250 Å; Waters, Milford, MA, USA). The mobile phase was 0.1 M Tris-HCl, pH 8.9 (1 mL/min). We used an Ultimate 3000 chromatograph (Dionex, Sunnyvale, CA, USA) equipped with a vacuum degasser, LPG-3400SD pump, column thermostat TCC-3000SD, and a spectrophotometric detector DAD-3000 operating at 240 nm. The molecular weights of the fractions were estimated by comparison with the retention times of polystyrene sulfonate standards (PSS Polymer Standards Service GmbH, Mainz, Germany).

The dry powder of the hydrolysate was dissolved in ultrapure water at a concentration of 100 mg/mL, centrifuged at room temperature for 5 min at 20,000× g, and passed through a 0.45 μm filter. Gel filtration was performed in ultrapure water on a HiLoad 16/600 Superdex^® 30 pg column (GE Healthcare, Chicago, IL, USA) at a 0.3 mL/min flow rate. Then, 1 mL of the hydrolysate solution was loaded on the column at a specific time point. Collected fractions (1.5 mL) were subjected individually to measure absorbance at 220 nm and 280 nm on a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and independently, to estimate the antioxidant activity. The most active fraction was lyophilised, dissolved in 0.1% (v/v) trifluoroacetic acid (TFA), and subjected to reverse-phase high pressure liquid chromatography (RP-HPLC) using an UltiMate 3000 chromatograph (Dionex™/Thermo™, Sunnyvale, CA, USA). The separation was performed on a Discovery C18 250 × 4.6 mm column (Sigma-Aldrich, Poznan, Poland) at a 1 mL/min flow rate by means of two buffers: (A) 0.1% TFA (v/v) and (B) 0.07% TFA in 80% acetonitrile (both v/v). The linear gradient from 0% to 60% of buffer B was set up in 15 min, while the spectrophotometric detection was carried out at 220 nm and 280 nm. The collected fractions were evaporated in a vacuum centrifuge and redissolved in ultrapure water.