Crystalline cellulose was measured

Crystalline cellulose was measured in non-pre-treated and
pre-treated grape marc using a modified Updegraff (1969) method
as described in Harris et al. (2009). For non-pre-treated samples,
the soluble carbohydrates were removed prior to cellulose analysis:
dried homogenized grape marc (50 mg) was incubated three
times with 70% v/v ethanol at 55 C for 1 h. The material was
washed with 1 mL acetone for 2 min at room temperature and
dried overnight under vacuum.
The cellulose content was determined in triplicate using 5 mg of
dry non-pre-treated grape marc or pre-treated grape marc boiled
in 3 mL acetic-nitric reagent for 30 min. Samples were cooled to
room temperature, centrifuged (2500 rpm/10 min) and the supernatant
discarded. The residual material was washed twice with
8 mL water, followed by 4 mL acetone, and dried under vacuum.
To hydrolyze the cellulose, 1 mL H2SO4 (12.5 M) was added, and
incubated for 1 h at room temperature. The liberated glucose content
was quantified using the anthrone method outlined in Harris
et al. (2009) using a spectrophotometer (Thermo Fischer, Waltham,
MA, USA) set at a wavelength of 620 nm. Filter paper (20 mg) was
carried through the experiment as a control and a standard curve
was calculated from known concentrations of glucose (0–50 lg).
The cellulose content was calculated by multiplying the glucose
value for each sample by the total reaction volume and the hydration
factor of 0.9.