*3.5. Determination of Endogenous Formaldehyde Content in L. edodes*

Steam distillation was used to extract formaldehyde from *L. edodes* at each growth stage. Each sample was supernatant of 4 g *L. edodes* homogenized with 100 mL Tris-HCl (0.5 M, pH = 7.6) buffer and 10 mL 10% (*v*/*v*) phosphoric acid aqueous solution in a 250 mL distillation flask. Water vapor was collected into a 150 mL flask, and then immersed in an ice-bath. The distillation process was stopped when 6070 mL of the distillate was collected and made up to 100 mL by deionized water. Formaldehyde in the distillate was derived by adding 1 mL of the distillate, 3.5 mL acetate buffer (0.1 M, pH = 4.0) and 0.5 mL DNPH (3 mg/mL) into a centrifuge tube at 25 ◦C for 15 min. Then the derived sample was filtered through a 0.22 μm filter for HPLC analysis. The formaldehyde derivative (formaldehyde-DNPH) of each group was separated and determined by a reverse-phase HPLC system (Waters, Milford, MA, USA). The mobile phase was composed of 0.05% acetic acid in acetonitrile and 0.05% acetic acid in water. The injection volume was 20 μL. All samples were detected at 355 nm as previously reported [18].
