2.3. Extraction and determination of the main cell wall components
Sodium dodecyl sulfate solution (15 g/L, containing 5 mmol/L pyrosulfite) was added to the dried precipitate obtained as described in 2.2 and centrifuged twice at 4000 × g for 15 min. The new precipitate was treated with 90% dimethyl sulfoxide to remove starch and then cleaned by several cycles of washing with distilled water and centrifugation to obtain cell wall material (CWM) (Li, Han, & Jin, 2014). The CWM was treated with 50 mL 2-cyclohexanediamine-N,N,N’,N ’-acetic acid (CDTA; 50 mmoL/L, pH 6.5) for 6 h at 25 °C. CDTA-soluble pectin in the supernatant was obtained by filtration through a nylon membrane with 10 μm pore diameter. The remaining solid was incubated in 50 mL Na2CO3 solution (50 mmoL/L, containing 20 mmol/L NaBH4) at 25 °C for 6 h. Na2CO3-soluble pectin in the supernatant was obtained by filtration through a nylon membrane with 10 μm pore diameter. Cellulose in the supernatant and hemicellulose in the precipitation were obtained after adding 50 mL 4 M potassium hydroxide to the remaining solid and reacting for 12 h at 25 °C (He, 2015). The amount of pectin was determined by the carbazole colorimetric method (Hou, 2004), and the amount of hemicellulose and cellulose was determined by the anthrone colorimetric method (Han,1992; Gao, 2006).