Themonosaccharide compositionof PCanddPC wasdetermined
as follows. Pectin samples (2 mg) were hydrolyzed with 2 mol L−1
TFA (trifluoroacetic acid) (1 mL) for 8 h at 100 ◦C. The product was
successively reduced with NaBH4 (Wolfrom & Thompson, 1963a),
acetylated with Ac2O–pyridine (1:1, v/v) (Wolfrom & Thompson,
1963b), and the resulting alditol acetates were examined by
GC–MS. This was performed with a Varian model 3800 gas chro-
matograph coupled to a Saturn 2000R mass spectrometer using a
DB-225 capillary column (25 m × 0.25 mm i.d.). Temperature used
was 50 ◦C during injection, then programmed at 40 ◦C min−1 to
220 ◦C (constant), with He as carrier gas. The products were iden-
tified by their typical retention times and electron impact profiles.
Uronic acid contents were determined by the colorimetric method
of Filisetti-Cozzi and Carpita (1991). 1H NMR spectroscopy was employed to confirm the DM
(Grasdalen, Bakoy, & Larsen, 1988). Samples were deuterium-
exchanged three times by freeze-drying with D2O, then finally
dissolved in D2O (at 10 mg mL−1) and transferred into 5 mm NMR
tubes. The 1H NMR spectra were acquired at 70 ◦C with 256 scans
on a Bruker AVANCE III 400 NMR spectrometer observing 1H at
400.13 MHz. Chemical shifts were expressed as ppm. The DM was
obtained using the equation below.
DM (%) = IA − IB
IA + IB 100 (1)
IA refers to the intensity of resonance signals of H-1 of all GalA units
(–COO− and COOCH3 forms) and of H-5 of all esterified GalA units
(∼ı 5.12 to ı 4.76). IB refers to the intensity of resonance signals
from H-5 of non-esterified GalA units (∼ı 4.58 to ı 4.52).
The protein content of the pectins was determined by the col-
orimetric method described by Hartree (1972).