Exopolysaccharide Purification from K. pneumoniae

Fresh TSB medium was inoculated with an overnight culture of K. pneumoniae strain 2226 and incubated without agitation at 37°C for 5 days. EPS purification was performed as described previously (Bales et al., 2013 (link)). Briefly, 60 μl of formaldehyde solution (36.5%) was added to every 10 ml of culture, and the culture was incubated at 100 rpm for an hour. Then, 1 M NaOH was added to the culture, followed by agitation at room temperature for 3 h. Cell suspensions were centrifuged at 16,800×g for 1 h (Beckman, JA-25.50, USA), and the supernatant was treated with trichloroacetic acid (TCA, 20% w/v) to remove protein and nucleic acid impurities. The solution was centrifuged at 16,800×g for 1 h (Beckman, JA-25.50, USA); the supernatant was collected, and 1.5 volumes of cold ethanol (96%) was added to precipitate the exopolysaccharides at −20°C for 24 h. The precipitate was pelleted by centrifugation at 16,800×g for 1 h and resuspended in ddH₂O. The EPS mixture was dialyzed against an excess of ddH₂O at 4°C for 24 h using a 12–14 kDa MWCO membrane (Thermo Scientific, USA). The dialyzed EPS was lyophilized.