Purification of Pythium insidiosum Biomass

The Pythium insidiosum Pi-S strain was subcultured on Sabouraud dextrose (SD) agar for 1 week. Several small pieces of agar-attaching hyphae were transferred to a 250-ml flask containing 100 ml of SD broth for shaking incubation (∼100 rpm) at 37°C for 7 days. The hyphal material was removed by filtration through a 0.2-μm-pore-size membrane (Merck Millipore, USA). The harvested hyphae were extracted to obtain SABH, as previously described (24 (link), 54 (link)). The cell-free culture broth was concentrated using an Amicon centrifugation tube (10,000 molecular weight cutoff [MWCO]; Merck Millipore) to obtain CFA, according to the reported protocol (24 (link), 54 (link)). Both SABH and CFA were stored at –30°C until use.