Polysaccharide extraction

As polysaccharides have different extractability and solubility we extracted them with three different solvents. All the HMWDOM freeze dried samples and the POM AIR samples were individually well-homogenised with a spatula. Samples were weighed out in 8-strip tubes (approximately 10 mg of each sample) and a 1.6-mm stainless steel bead was added in each tube to aid sample mixing. Polysaccharides were sequentially extracted with: autoclaved MilliQ water, 50 mM EDTA pH 7.5 and 4 M NaOH with 0.1% w/v NaBH₄. For each of the extracting solvents the following was performed: for every 10 mg of sample 300 µL of solvent were added to the tube (volume of solvent was adjusted to the weight i.e. sample extractions were normalised by weight). The tubes - set in an appropriate holder - were placed on a TissueLyser II (Qiagen) with which samples were shaken at 30/s for 2 min and then at 6/s for 2 h. After the 2 h extraction, samples were spun down at 6000 × g for 15 min at 15 °C. Extracts (supernatants) were collected in 1.5 mL tubes and stored at 4 °C. The pellets were resuspended in the next extracting solvent using the same extraction procedure (with the TissueLyser II) as depicted above. The three sequential extractions of all the samples were performed on the same day.