L. Hao et al. / Carbohydrate Polymers 141 (2016) 54–59
55
Liberation Army. The strain was inoculated at petri plate contain-
2.5. Determination of molecular weight
ing potato dextrose agar for 7 days at 30 ◦C. F. pinicola fruiting body
was obtained from Changbaishan in Jilin province, China. Buty-
lated hydroxytoluene (BHT) was purchased from Solarbio Co. Ltd
(Beijing, China). 1-phenyl-3-methyl-4-benzene formyl pyrazolone
(PMP) was from Ziyi Bio. Co. Ltd (Shanghai, China). 1-Diphenyl-2-
picrylhydrazyl (DPPH) and reference monosaccharides (d-glucose,
d-xylose, l-arabinose, d-mannose, l-rhamnose, d-galactose) were
purchased from Sigma-Aldrich (St. Louis, MO, USA).
The molecular weights of EPS and IPS were determined on an
Agilent 1200 HPLC system equipped with an evaporative light scat-
tering detector (ELSD) and TSK-gel G5000 PWXL (7.8 mm × 30 cm,
TOSOH Corp., Japan). The column was eluted with double-distilled
water at a flow rate of 0.8 mL/min. Standard dextrans (T10, T40,
T70, T100, T380, T500, Sigma, USA) were used for molecular weight
measurement.
2.6. Infrared spectroscopy
2.2. Preparation of crude polysaccharide extract
The Fourier-transform infrared (FTIR) spectrum of the polysac-
IPS was extracted twice from F. pinicola fruiting body with hot
water at 70 ◦C in a 1:30 (w/v) ratio for 2 h, and then four volumes
of ethanol were added and precipitated overnight at 4 ◦C. The pre-
cipitate was collected by centrifugation at 10,000 × g for 15 min
and lyophilized, thus yielded a crude polysaccharide extract. In
the preparation of EPS, fermentation medium (sucrose 5%, yeast
extract 1%, KH2PO4 0.2%, MgSO4·7H2O 2%, w/v) was inoculated,
and then incubated for 5 d. Similar to IPS, the fermentation broth
was processed by concentration, precipitation, centrifugation and
lyophilization to obtain crude polysaccharide extract.
charides was detected on
a Bruker-Vector 22 spectrometer
(German). The polysaccharides were mixed with KBr powder,
ground and pressed into 1 mm pellets in the frequency range of
4000–500 cm−1
.
2.7.1. DPPH radical scavenging activity
The methods of Shimada et al. was employed with some modifi-
cations (Shimada, Fujikawa, Yahara, & Nakamura, 1992). Different
concentrations of EPS and IPS (2.0–10.0 mg/mL) were prepared
with distilled water. A total of 0.5 mL each sample with differ-
ent concentrations was mixed with 1.5 mL of 0.4 mmol/L methanol
solution of DPPH, and the mixed solution was reacted for 30 min in
darkness. Then absorbance was measured at 517 nm with BHT as
control. The activity of scavenging the DPPH radical was calculated
using the following equation: Y% = [1 − (A1 − A2)/A0] × 100, where
A0 was the absorbance of the blank group (distilled water + DPPH),
A1 was the absorbance of the sample reaction (sample + DPPH), and
A2 was the background absorbance of the sample (distilled water
replaced DPPH).
2.3. Isolation and purification of the polysaccharides
Both the crude EPS and IPS (100 mg each) were dissolved in
cellulose. Eluting process was performed with a linear gradient
from 0 to 0.5 mol/L NaCl at a flow rate of 1 mL/min. Total sugar con-
tent of the eluent was determined by phenol-sulfuric acid method
(DuBois, Gilles, Hamilton, Rebers, & Smith, 1956). The fractions
eluted with linear gradient of NaCl solution were pooled, desalted
and further purified on a Sephadex G-200 column eluting with dis-
tilled water at 0.5 mL/min. The major polysaccharide fractions were
pooled and lyophilized. From this process, the purified polysac-
charides were obtained for further identification of structure and
monosaccharide compositions.
2.7.2. Hydroxyl radical scavenging activity
Hydroxyl radical scavenging activity was measured according to
Fenton method described before (Zhong, Jin, Lai, Lin, & Jiang, 2010).
Polysaccharides of different concentrations (2.0–10.0 mg/mL) were
prepared. Then added 5 mmol/L of FeSO4 (2 mL), 5 mmol/L of sal-
icylic acid-ethanol solution (2 mL), 2 mL of polysaccharide sample
solution sequentially. Then the reaction started with 5 mmol/L
H2O2 (2 mL) in water bath at 37 ◦C. Absorbance was measured
at 510 nm with distilled water as reference and BHT as positive
control. The hydroxyl radical scavenging effect was calculated as
follows: Y% = [1 − (A1 − A2)/A0] × 100, where A0 was the absorbance
of blank, A1 was the absorbance of the sample, and A2 was the
background absorbance of the sample.
Monosaccharide compositions were determined by high
performance liquid chromatography (HPLC) after pre-column
derivatization (Sun et al., 2009). In brief, 50 mg of purified polysac-
charide powder was hydrolyzed with 2 mol/L of trifluoroacetic
acid aqueous solution at 120 ◦C for 6 h in a sealed tube. After the
hydrolysis was completed, the excess acid was removed by co-
distillation with methanol to yield dry hydrolysate, which was
dissolved in 0.3 mol/L of NaOH (100 L), then 0.5 mol/L of methanol
solution (200 L) of PMP was added. The resulted solution was
incubated at 70 ◦C for 1 h. After derivatization, the solution was
cooled to room temperature followed by adjustment of pH to
neutral with 0.3 mol/L HCl, then 1.5 mL of distilled water was
added by vigorously shaking. Finally, the mixture was extracted
with chloroform three times, and the aqueous phase was filtered
through a 0.22 m nylon membrane (Westborough, MA, USA).
The resulted solution (10 L) was injected onto a C18 column
(4.6 mm × 250 mm) connected with a DAD-UV detector (Agilent
Technologies, USA). The mobile phase was a mixture of 0.1 mol/L
KH2PO4 (pH 10)–acetonitrile (83:17) at a flow rate of 1.0 mL/min.
The column temperature was 30 ◦C. Sugar identification was
done by comparison with reference monosaccharides (d-glucose,
d-xylose, l-arabinose, d-mannose, l-rhamnose, d-galactose). Cal-
culation of the molar ratio of monosaccharides was based on the
peak area of the monosaccharides.
2.7.3. Protective effect on yeast cells from UV
Firstly, the yeast cells were cultured to the early stage of the log-
arithm in yeast extract peptone dextrose medium. Then 20 mL of
fermentation broth was centrifuged for 5 min at 4000 × g, and the
supernatant was discarded for yeast paste collection. Then the cells
were washed with phosphate buffer solution twice, and the yeast
paste was mixed with 20 mL of saline. A total of 1 mL yeast suspen-
sion was mixed with 4 mL of different concentrations of EPS and
IPS (5–25 mg/mL) in petri dish. Then the cells were disposed with
UV radiation for lethal time (1 min treatment could kill all yeast
cells in the control group) respectively, then the blank group was
mixed with distilled water. Finally, the mixture after UV radiation
were diluted to appropriate concentration for spread plate viable
count method at 28 ◦C for 2 d with BHT as control. The survival rate
was calculated as follows: Y% = [(A0 − A1)/A0 × 100], where A0 was
the amount of cells which had not been disposed by UV radiation,