Structural properties of polysaccharides from cultivated fruit bodies and mycelium of Cordyceps militaris

Article abstract of DOI:10.1016/j.carbpol.2016.01.040

The structural properties of polysaccharides, respectively, obtained from the fermented mycelium and cultivated fruiting bodies of the Cordyceps militaris were investigated and compared in this paper. First, the crude polysaccharides were extracted from the mycelium and the fruiting bodies, respectively. The polysaccharides were successively purified by Sevag and chromatography on Sephadex G-100 column to produce two polysaccharides fractions termed CMPS-II and CBPS-II, respectively. The average molecular weights of CMPS-II and CBPS-II were 1.402 × 10³ kDa and 1.273 × 10³ kDa, respectively, and they were mainly composed of mannose, glucose and galactose in the mole ratios of 1:28.63:1.41 and 1:12.41:0.74, respectively, for CMPS-II and CBPS-II. Afterward, the structural features of CMPS-II and CBPS-II were investigated by a combination of chemical and instrumental analysis, such as FT-IR, periodate oxidation-Smith degradation, GC-MS, NMR and methylation analysis. The results indicated that structurally, both CMPS-II and CBPS-II were 1,3-branched-galactomannoglucan that had a linear backbone of (1 → 4)-linked α-d-glucopyranose (Glcp). Congo-red test revealed that CMPS-II and CBPS-II existed as triple-helical chains in 0.05-0.15 M NaOH solution.

Full text of DOI:10.1016/j.carbpol.2016.01.040

Accepted Manuscript
Title: Structural properties of polysaccharides from cultivated
fruit bodies and mycelium of Cordyceps militaris
Author: Xiao-Cui Liu Zhen-Yuan Zhu Ya-Li Tang Ming-fei
Wang Zheng Wang An-Jun Liu Yong-Min Zhang
PII:
S0144-8617(16)30004-2
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.carbpol.2016.01.040
CARP 10719
To appear in:
Received date:
Revised date:
Accepted date:
12-11-2015
14-1-2016
18-1-2016
Please cite this article as: Liu, X.-C., Zhu, Z.-Y., Tang, Y.-L., Wang, M.-f., Wang, Z.,
Liu, A.-J., and Zhang, Y.-M.,Structural properties of polysaccharides from cultivated
fruit bodies and mycelium of Cordyceps militaris, Carbohydrate Polymers (2016),
http://dx.doi.org/10.1016/j.carbpol.2016.01.040
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Highlights (for review)
Highlights
• Two polysaccharides were obtained from the mycelium and fruiting
bodies.
• The structural features of polysaccharides were investigated and
compared.
• They had a backbone of (1→4)-linked α-D-glucopyranosyl residues.
Page 1 of 30

Structural properties of polysaccharides from cultivated fruit bodies and
mycelium of Cordyceps militaris
Xiao-Cui Liua, Zhen-Yuan Zhua,b,*, Ya-Li Tanga, Ming-fei Wanga, Zheng Wanga, An-
Jun Liua, Yong-Min Zhangb
aKey Laboratory of Food Nutrition and Safety, Ministry of Education, College of
Food Science and Biotechnology, Tianjin University of Science and Technology,
Tianjin 300457, P.R. China
bTianjin Food Safety & Low Carbon Manufacturing Collaborative Innovation Center ,
300457, Tianjin, P.R. China
cUniversité Pierre et Marie Curie-Paris 6, Institut Parisien de Chimie Moléculaire,
UMR CNRS 8232, 4 place Jussieu, 75005, Paris, France
*Corresponding author.
(Z.-Y. Zhu): Tel.: +86 22 60912390, Fax: +86 22 60601437; E-mail:
zhyuanzhu@tust.edu.cn;
1
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ABSTRACT
The structural properties of polysaccharides respectively obtained from the
fermented mycelium and cultivated fruiting bodies of the Cordyceps militaris were
investigated and compared in this paper. Firstly, the crude polysaccharides were
extracted from the mycelium and the fruiting bodies, respectively. The
polysaccharides were successively purified by Sevag and chromatography on
Sephadex G-100 column to produce two polysaccharides fractions termed CMPS-II
and CBPS-II, respectively. The average molecular weights of CMPS-II and CBPS-II
were 1.402×103 kDa and 1.273×103 kDa respectively and they were mainly composed
of mannose, glucose and galactose in the mole ratios of 1:28.63:1.41 and 1:12.41:0.74
respectively for CMPS-II and CBPS-II. Afterward, the structural features of CMPS-II
and CBPS-II were investigated by a combination of chemical and instrumental
analysis, such as FT-IR, periodate oxidation-Smith degradation, GC-MS, NMR and
methylation analysis. The results indicated that structurally, both CMPS-II and CBPS-
II were 1,3-branched-galactomannoglucan that had a linear backbone of (1→4)-linked
α-D-glucopyranose (Glcp). Congo-red test revealed that CMPS-II and CBPS-II
existed as triple-helical chains in 0.05 M-0.15 M NaOH solution.
Keywords: Cordyceps militaris; Polysaccharide; Structure
1. Introduction
Cordyceps, a rare edible and medicinal herb, has long been used in the human
traditional therapies. Cordyceps militaris is known as the Chinese rare caterpillar
fungus, and has similar pharmacological activities to the famous Chinese traditional
medicine Cordyceps sinensis (Gai, Jin, Wang, Li, & Li, 2004; Zheng & Cai, 2004), a
special form of mushroom with the fungal fruit body formed on an insect larva (Ji,
1999). As new food materials, Cordyceps militaris have multiple pharmacological
actions such as anti-inflammatory (Won & Park, 2005), anti-hyperlipidemia (Yu et al,
2004), enhancing insulin resistance and insulin secretion (Chio et al., 2004). Also, it
was indicated that the antioxidant activity of Cordyceps militaris was even stronger
than that of Crodyceps sinensis and Cordyceps kyushuensis (Chen et al., 2004).
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Polysaccharides are major bioactive constituents of Cordyceps, contributing to the
health-promoting effects and the antitumor activity (Chen, Wu, & Huang, 2013; Li &
Tsim, 2004). Polysaccharides are well-known for their roles as energy storage
molecules in cells and structural elements of the cell walls of living organisms. More
interestingly, they also function as important multi-cytokine inducers in cell-cell
communication, for example, in inducing gene expression of various
immunomodulatory cytokines and cytokine receptors (Ooi & Liu, 2000). Recently,
several studies have demonstrated that many natural polysaccharides and
polysaccharide-protein complexes have been applied or are under clinical trials as
immune modifiers and drugs for cancer therapies (Yang & Zhang, 2009; Zhang et al.,
2007). In addition, it had shown that the activities of polysaccharides were highly
dependent on the chemical composition and structural characteristics (Methacanon et
al., 2005).
The limited natural resource and difficult artificial cultivation of Cordyceps
militaris to obtain fruit body make it impossible to obtain large quantity of
polysaccharides. In facing of such a quagmire, submerged cultures offer a promising
alternative, which is fast, cost-effective, easy to control and free from heavy metal
contamination. Many medicinal fungi polysaccharides are being commercially
produced by submerged culture (Chandra & Ohga, 2006; Fan et al., 2007; Gao & Gu,
2007) because of the higher productivity compared to production from fruit bodies.
Although previous studies have demonstrated the immune modulatory properties of
polysaccharides isolated from intra- (Akaki et al., 2009) and extra-cellular extracts
(Kuo et al., 2007), there is no study investigate and compare the structural
characteristics of the polysaccharides extracted from the fermented mycelium and
cultivated fruiting bodies of Cordyceps militaris, respectively. In the present study,
the polysaccharides extracted from the fermented mycelium and cultivated fruiting
bodies were characterized by chemical methods. They were isolated and purified by
Sevag and gel-filtration chromatography to analyze the similarity and difference of
monosaccharide compositions, molecular weight, glycosidic linkage, morphological
characteristics and appearance shape.
2. Materials and methods
3
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2.1. Material and chemical
The strain used in this study was Cordyceps militaris CICC 14015, which was
obtained from China Center of Industrial Culture Collection (CICC), and stored in the
Key Laboratory of Food Nutrition and Safety (Ministry of Education, China), College
of Food Science and Biotechnology (Tianjin University of Science and Technology,
Tianjin, China). D-glucose, D-galactose, L-rhamnose, D-xylose, D-mannose, D-
arabinose and Sephadex G-100 were purchased from Sigma Chemical Co. (St. Louis,
MO, USA). All other chemicals used in this paper were of analytical grade.
2.2. Cultivated fruit bodies and fermented mycelium of C. militaris
The fruit bodies grown on solid rice medium were obtained. The mycelium was
obtained by liquid submerged fermentation. The strain was cultivated in a 250-mL
Erlenmeyer flask containing 100 mL of seed culture medium contained (% w/v)
glucose 2, peptone 1.0, KH2PO4 0.1, and MgSO4·7H2O 0.1 for 3 days at 25 ºC, 250
rpm, uncontrolled pH. The harvested seeds were added into 250-mL Erlenmeyer
flasks containing 100 mL liquid fermentation mediums composed of (% w/v) glucose
4, peptone 1.0, KH2PO4 0.05, and MgSO4·7H2O 0.05. The inoculate rate was 8%
(v/v). The mycelium was harvested after incubated at 25 ºC on a rotary shaker
maintained at 150 rpm for 5 days (Chen et al., 2011).
2.3. Extraction, fractionation and purification of polysaccharides
2.3.1. Extraction from cultivated fruit bodies
The fresh fruit bodies were dried and grinded into fine powder. The mixed powder
(10 g) was extracted with 200 mL of distilled water at 80 ºC for 2 h, and then repeated
for three times. After vacuum filtration, the filtrate was combined and condensed to
150 mL with a rotary evaporator under reduced air pressure at 60 ºC. The resultant
extract was disposed by precipitation with 4 times volume of 95% (v/v) ethanol,
deproteinization with the Sevag method and lyophilization to produce the substance
termed CBPS-I. The total sugar content of CBPS-I was measured by a phenol sulfuric
acid method (Chaplin et al., 1986).
2.3.2. Extraction from fermented mycelium
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For the intra-cellular product isolation, the mycelium was obtained by filtration of
the culture broths. They were washed three times with sufficient distilled water then
dried and grinded into fine powder. Then the polysaccharides in mycelium were
extracted as above, to produce the CMPS-I fraction.
2.3.3. Fractionation and purification of polysaccharides
The polysaccharide (CMPS-I and CBPS-I) was subjected to gel filtration on a
column (30 cm × 3 cm) of Sephadex G-100, eluted with deionized water at a flow rate
of 0.3 mL/min and monitored using the phenol-sulfuric acid method.
2.4. Determination of molecular weight distribution
The molecular weight (Mw) distribution of polysaccharides was determined by
using a HPGPC (Agilent-1200) equipped with a TSK gel G4000 PWxl column (7.8
mm × 300 mm, column temperature 30 ºC) and Refractive Index Detector (RID,
detecting temperature 35 ºC). A sample solution (20 µL) was injected and run with
deionized water at 0.6 mL/min as mobile phase. The standard curve was established
using T-series Dextran as the standards (T-10, T-40, T-70, T-500 and T-2000) (Zhu et
al., 2011).
2.5. FT-IR and NMR analyses
1 mg of sample was mixed with 150 mg of dried KBr, and pressed into a 1 mm
thick disk for the analysis. The IR spectrum was recorded at the absorbance mode in
the range of 4000-400 cm-1 on a Fourier transformed IR spectrophotometer
(VECTOR-22) (Yan et al., 2010).
1H NMR and 13C NMR spectra were recorded on a Bruker spectrometer (600 MHz)
at a probe temperature of 298 K. The freeze-dried samples were exchanged twice with
D2O and measured, respectively (Leeuwen et al., 2008).
2.6. Monosaccharide composition analysis
The polysaccharides were hydrolyzed with 2 M TFA at 110 ºC in a sealed-tube for
3 h. After TFA was removed, one part of the hydrolysate was analyzed by TLC. The
other part was acetylated and analyzed by GC (Zhao et al., 2005). D-glucose, D-
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galactose, L-rhamnose, D-xylose, D-mannose, and D-arabinose were also derivatized
as standards.
2.7. Periodate oxidation and Smith degradation analysis
The periodate oxidation and Smith degradation analysis were performed based on
the literature methods (Linker, Evans, & Impallomeni, 2001; Dixon & Lipkin, 1954).
2.8. Congo-red test
The interaction of Congo red with polysaccharide was evaluated from the shift in
the visible absorption maximum of Congo red. The solution of samples (0.5 mg/mL)
were mixed with 2 mL, 50 μmol/L Congo red and 1 M NaOH to make the finally
concentration of NaOH to 0, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35 and 0.40 M. The
UV-Vis absorption spectrum was recorded on spectrophotometer (400-600 nM) after
keeping the samples at room temperature for 10 min.
2.9. Scanning electron microscopy (SEM)
The samples were fixed onto a copper stub, respectively. After sputtering with a
layer of gold, the samples were examined on a SU1510 scanning electron microscope
(Lai & Yang, 2007).
3. Results and discussion
3.1. Isolation and purification
In this study, polysaccharides CMPS-I and CBPS-I, derived respectively from
fermented mycelium and cultivated fruit bodies of C. militaris, were obtained by hot
water extraction, along with ethanol precipitation and deproteination. The weight-
average molecular weight (Mw) distribution of CMPS-I and CBPS-I were investigated
and showed in Fig. 1. The chromatographic result of HPGPC showed that CMPS-I
had one peak while CBPS-I had three peaks (peak a, b and c), which indicating that
CMPS-I main contained one kind of polysaccharide and CBPS-I was composed by
three kinds of polysaccharides with different molecular weights. The relative content
of the three fractions of CBPS-I was 24.75%, 46.82 % and 28.43%. It indicated that
the second fraction was the main component part of CBPS-I.
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The CMPS-I and CBPS-I were purified by gel-filtration chromatography on a
Sephadex G-100 column, eluted with deionized water and the elution profiles were
showed in Fig. 2. CMPS-I generated one single elution peak, collected designated as
CMPS-II. It was not similar to the results from Lee et al. (Lee et al., 2010), who
obtained a water soluble crude polysaccharide from C. militaris mycelia, which
contained four polysaccharide fractions. It perhaps caused by the different strain
source and cultivation method of C. militaris mycelia. However, three elution peaks
(peak a, b and c) appeared for CBPS-I in Sephadex G-100 column, and the relative
content of the second fraction was higher than others, which was coincided with the
molecular weight distribution result of CBPS-I. It indicated that the main component
of CBPS-I was medium molecular weight polysaccharide which was collected named
as CBPS-II.
3.2. Chemical contents and monosaccharide compositions
Table 1 showed the major chemical contents of the extracts isolated from the
fermented mycelium and cultivated fruit bodies. The protein contents in
polysaccharides were very few. It indicated that the deproteination process was
effective. The total sugar content of CMPS-I and CBPS-I was 56.6% and 47.5%
respectively. It indicated that CMPS-I had higher polysaccharide content than CBPS-I.
Polysaccharides purified by gel-filtration chromatography on a Sephadex G-100
column (yielding CMPS-II and CBPS-II) decreased the impurities by 29.2% and
42.1% for CMPS-I and CBPS-I, respectively. The freeze-dried powders of CMPS-II
and CBPS-II were a mixture of polysaccharides (between 85 and 90 wt.%) and other
components (between 15% and 10%), which might be attributed to crystallization
water and other unknown components in the extracts. The difference of total sugar
content between CMPS-II and CBPS-II was existent, but non-significant.
The monosaccharide compositions of CMPS-II and CBPS-II were measured by
TLC technique and GC analysis. The results of TLC showed that both of them had no
uronic acid generated (data not shown). It indicated that CMPS-II and CBPS-II were
neutral polysaccharides. As shown in Fig. 3, 6 standard monosaccharides were
separated rapidly within 30 min. The peaks of standard monosaccharides were
identified in the order of L-Rhamnose, D-Arabinose, D-Xylose, D-Mannose, D-
Glucose, and D-Galactose. The samples were identified by matching their retention
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times with those of standard monosaccharides under the same analytical conditions.
The monosaccharide compositions of CMPS-II and CBPS-II were showed in Fig. 3
and the mole ratios were listed in Table 1. The results showed that CMPS-II and
CBPS-II were both primarily composed of D-Mannose, D-Glucose, and D-Galactose
in the mole ratios of 1:28.63:1.41 and 1:12.41:0.74 respectively. The results were
in accordance with the experiment result of the polysaccharides from Yu et al. (Yu et
al., 2007). And Lee et al. (Lee et al., 2010) reported that the major sugar constituents
of a C. militaris mycelia polysaccharide were Man, Gal and Glc. In addition, Wu et al.
(Wu et al., 2011) reported that CM-hs-CPS2, a kind of sulfating acidic polysaccharide
isolated from fruiting bodies of cultured Cordyceps militaris grown on solid rice
medium, was composed mainly of mannose, glucose and galactose in a molar ration
of 1.35:8.34:1.00. Comparing the results in Table 1 and Fig. 3, it indicated that
glucose may be the backbone of the structure of CMPS-II and CBPS-II, and mannose
and galactose may be close this backbone. The results indicated that the
monosaccharide compositions of CMPS-II and CBPS-II were approximately similar,
however, the contents of each component had some differences.
3.3. Molecular weight and purity of CMPS-II and CBPS-II
The molecular weight of polysaccharide was determined by universal calibration
curve by using dextran as standards (Dou et al., 2015). The calibration curve of
Dextran standards was plotted as the molecular weights on a log scale versus the
retention time. The regression equation was y = -0.3226 x + 8.865, R2= 0.9866 (y = lg
Mw, x = Rt). As shown in Fig. 4, the HPGPC profiles of CMPS-II and CBPS-II had a
single peak, which indicated that both of them were homogeneous polysaccharides.
Based on calibration with standard dextrans, the weight-average molecular weight
(Mw) of CMPS-II and CBPS-II were estimated to 1.402×103 kDa and 1.273×103 kDa,
respectively. Lower molecular weights (36 kDa) were reported for water-solube
polysaccharide from the fermentation broth of C. militaris (Lee, Kwon, et al., 2010).
In other studies, Lee et al. (Lee et al., 2010) reported that the molecular weight of an
immunostimulatory polysaccharide was 210 kDa from C. militaris mycelium, and
Chen et al. (Chen, Wu, & Huang, 2013) showed the molecular weight of the main
component of C. militaris fruiting bodies polysaccharide was 8.97 kDa. The
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differences might be due to the formulation of strain origins, extraction methods and
purification procedures of polysaccharide of C. militaris.
3.4. Structural characterization
3.4.1. Structural composition of CMPS-II and CBPS-II
The UV absorption spectra of CMPS-II and CBPS-II showed no absorption at 280
and 260 nm indicated that these two polysaccharides contained on protein and nucleic
acid.
To characterize polysaccharide fractions, the characteristic absorption of
polysaccharides was performed in the range of 4000-400 cm−1 by FT-IR (Dou et al.,
2015). As shown in Fig. 5, the broad stretching peaks at 3416.56 cm−1 (CMPS-II) and
3385.75 cm−1 (CBPS-II) were ascribed to the hydroxyl groups with stretching
vibration. The weak absorption peaks at 2926.91 cm−1 (CMPS-II) and 2926.66 cm−1
(CMFB-II) were characteristic of C-H stretching vibration. In addition, at 1632.68
cm−1 (CMPS-II) and 1647.83 cm−1 (CBPS-II) had a strong absorption peak which
were C=O stretching vibration. The band at 1402.02 cm−1 (CMPS-II) and 1405.95
cm−1 (CBPS-II) were assigned to C-H bending vibration. Moreover, The three
stretching peaks at 1154.24 cm−1, 1079.98 cm−1, 1023.50 cm−1 (CMPS-II) and
1153.25 cm−1, 1079.76 cm−1, 1023.66 cm−1 (CBPS-II) indicated the presence of
pyranoside, and the absorption band at 856.11 cm−1 (CMPS-II) and 852.26 cm−1
(CBPS-II) were attributed to the presence of the α-type glycosidic linkages (Barker et
al., 1954). It was coincided with the results of Chen et al. (Chen, Wu, & Huang, 2013),
who reported that W-CBP50 II, polysaccharide isolated from C. militaris fruiting
body, was contained mainly mannose, glucose, galactose and arabinose with α-type
glycosidic linkage. Comparison and analysis of the IR spectra, CMPS-II and CBPS-II
were basically indistinguishable only with slight differences in the intensity of bonds,
and both of them were mainly composed of α glycosidic bond-linked glucopyranose
(Glcp).
3.4.2. Linkage analysis of CMPS-II and CBPS-II polymer chain
The periodate oxidation of CMPS-II and CBPS-II produced 0.020 mol and 0.017
mol formic acid respectively was indicative of 1→6 linkage. As shown in Table 2, the
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periodate oxidation of CMPS-II and CBPS-II consumed 0.74 mol and 0.71 mol of
periodate respectively. It indicated the existence of 1→3 linkage in CMPS-II and
CBPS-II, when less than 1 mol of periodate consumption per mole of glucose residue.
Because 1→3 linkage does not consume periodate during the oxidation (Wang et al.,
2011).
After Smith degradation of the oxidized product of CMPS-II and CBPS-II,
glycerol, erythritol and glucose were found in the degradation products (Table 2). The
molar ratios of glycerol, erythritol and glucose were 1:9.88:2.83 and 1:35.95:11.95
respectively in CMPS-II and CBPS-II. The presence of glucoses were indicative of
CMPS-II and CBPS-II exist 1→3, (1→2,3), (1→3,4), (1 →3,6) or (1→2,3,4) linkage
which cannot be oxidized (Zhang et al., 2010). The result was coincided with the
finding from periodate oxidation analysis. The relatively high content of erythritol
was indicative of 1→4 linkage might be consisted in the main chain or branch. The
relatively lower content of glycerol suggested the small amount of 1→6 or 1→2
linkage. The results from periodate oxidation-Smith degradation showed that 1→3,
1→4, 1→2, 1→6 and 1→3,6 linkages or non-reducing terminal might exist in CMPS-
II and CBPS-II.
The fully methylated products of CMPS-II and CBPS-II were hydrolyzed with acid,
converted into alditol acetates and analyzed by GC-MS. As summarized in Table 3,
CMPS-II and CBPS-II showed five same derivatives, namely 2,3,6-tri-O-Me-D-Gal,
2,3,4,6-tetra-O-Me-D-Glc, 2,3,6-tri-O-Me-D-Glc, 2,4,-di-O-Me-D-Glc, and 2,4,-di-O-
Me-D-Man. The results from GC-MS analysis indicated that (1→4)-linked-glucose
was major component of the backbone of the polysaccharide structure. Residues of
branch structure might were (1→4)-linked-galactose, (1→3,6)-linked-glucose,
(1→3,6)-linked-mannose and (1→)-linked-glucose terminal, which was
in accordance with those data of partial acid hydrolysis and Smith degradation. The
above results indicated that CMPS-II and CBPS-II had highly similar glycosidic
linkage types and a trace difference in the number of glycosidic linkages.
3.4.3. Structural characteristics of CMPS-II and CBPS-II derived from
NMR
1
In the H-NMR spectrum of CMPS-II and CBPS-II (Fig. 6), showed three main
anomeric H at δ 4.3-δ 5.9 ppm (around 5.32, 5.16 and 4.95 ppm) which indicated that
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both of them were mainly composed of three types of sugars. The peaks of CMPS-II
and CBPS-II at around 4.700 ppm (4.706 and 4.707 ppm) were attributed to the
chemical shifts of HOD. In addition, the 13C NMR spectrum of CMPS-II and CBPS-II
(Fig. 6) had no signal at low field from 160~180 ppm, which illustrated both of them
do not contain uronic acid. The characteristics of the carbon signal suggested the α-
anomeric configuration of Glcp moieties (Zhang et al., 2010), which was
in accordance with the analysis of IR spectrum. It also showed three anomeric C at the
91.98 ppm, 95.86 ppm and 99.97 ppm (99.86 ppm) of CMPS-II (CBPS-II), which was
attributed to the anomeric carbon atoms of mannopyranose (Manp), galactopyranose
(Galp) and glucopyranose (Glcp), respectively. Comparing the 1H NMR spectrum and
13C NMR spectrum of CMPS-II and CBPS-II, the results supported the conclusion
that the main monosaccharides for both CMPS-II and CBPS-II were α-D-Mannose, α-
D-Glucose, and α-D-Galactose. Therefore,it was concluded that CMPS-II and CBPS-
II had the similar characteristic group of polysaccharide.
3.5. Morphological characteristics of CMPS-II and CBPS-II in sodium
hydroxide solution
Some linear and branched α-D-glucans had been reported to have a triple-helical
conformation in aqueous or weakly alkaline solution (< 0.15 M NaOH), and is
transformed into single chains in highly alkaline solutions (> 0.25 M NaOH) (Zhang
et al., 2010). The order-disorder transitions in a helical polymer are usually
accompanied with changes in the visible absorption spectra of the complexes formed
with Congo red.
The changes of maximum absorption wavelength of Congo red, Congo red+
CMPS-II and Congo red+CBPS-II at various concentrations of sodium hydroxide
solution were showed in Fig. 7. Compared with Congo red, the maximum absorption
wavelengths of Congo red+CMPS-II and Congo red+CBPS-II were changed when the
concentration of NaOH was 0.05 M. It indicated that both CMPS-II and CBPS-II
formed Congo red complex which were showed large bathochromic shift (from 492
nm to 505 nm). When the concentration of NaOH was greater than 0.05 M, the
maximum absorption wavelength of Congo red+CMPS-II and Congo red+CBPS-II
were decreased gradually and slowly close to the wavelength of Congo red at 0.15 M
NaOH. It indicated that Congo red+CMPS-II and Congo red+CBPS-II existed triple-
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helical structures in 0.05 M NaOH solution. In addition, the spiral began to uncoiling
when NaOH solution concentration was greater than 0.05 M and the spiral was
unlocking completely at 0.15 M NaOH solution. On the whole, CMPS-II and CBPS-II
both had triple helical structures in weakly alkaline solution.
3.6. Appearance shape of CMPS-II and CBPS-II
It is a qualitative tool to analyze the surface morphology of polysaccharides by
Scanning Electron Microscopy (SEM) (Wang et al., 2015). The SEM images of
CMPS-II and CBPS-II were showed in Fig. 8, the surface of CMPS-II (× 200) (Fig.
8A) showed crumbs accumulation, intertwined, and the regularity of structure was not
strong. It indicated that CMPS-II was easily curled to form a variety of irregular
shapes. CMPS-II SEM images enlarge to × 1000 times (Fig. 8B) showed that the
surface topography was smooth with balls on the edge of the chain structure. It
indicated that polysaccharide molecules existed crosslinking to form mesh structure.
The microstructure of CBPS-II (× 200) was showed in Fig. 8C. The surface was
rough, more closely and gathered. CBPS-II SEM images enlarge to × 950 times (Fig.
8D) showed that the surface present a small lamellar or granular structure. It indicated
that CBPS-II was also mainly of amorphous structure, and a part of the
polysaccharide was scattered.
4. Conclusions
It was clearly demonstrated from all the results obtained in the present studies that
both the water-soluble polysaccharide CMPS-II and CBPS-II of C. militaris are
neutral heteropolysaccharides with few branches. The total polysaccharide (wt %)
contents of CMPS-II and CBPS-II were 85.8 and 89.6 respectively. The average
molecular weights of CMPS-II and CBPS-II were different around to 1.402×103 kDa
and 1.273×103 kDa respectively. The monosaccharide composition results indicated
that CMPS-II and CBPS-II were mainly composed of mannose, glucose and galactose
in the molar ratios of 1:28.63:1.41 and 1:12.41:0.74 respectively. By contrast, both
polysaccharides were consisted primarily of glucose, which were similar to
monosaccharide compositions of several well-known fungi polysaccharides, such as
Poria cocos (Huang et al., 2007), Ganoderma lucidum (Zhao et al., 2010) and
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Lentinus edodes (Wang et al., 2011) et al. It indicated that CMPS-II and CBPS-II had
similar monosaccharide composition with differences contents.
Although several studies had reported the characterization of polysaccharides
isolated from C. militaris, no reports had ever been recorded on the structural
properties comparison for polysaccharides of C. militaris with different cultivation
status. The results from the series of chemical and spectroscopic analysis showed that

both CMPS-II and CBPS-II might be composed of (1→4)-linked α-D-glucopyranosyl,
(1→4)-linked α-D-galactopyranosyl, (1→3,6)-linked α-D- glucopyranosyl, (1→3,6)-
linked α-D-mannopyranosyl residues and (1→)-linked-glucose at the terminal. It
indicated that CMPS-II and CBPS-II had similar glycosidic linkage and link manner.
In addition, CMPS-II and CBPS-II existed triple helix chains in NaOH solution, and
certain similarities were showed in their appearance shapes.
In conclusion, CMPS-II and CBPS-II had difference in the total polysaccharide
content, molecular weight size and monosaccharide mole ratio, however, they had the
same structure characteristics: glycosidic linkage types and link types, triple-helical
conformation and appearance shape. The difference and similarity of the
polysaccharide obtained from fermented mycelium and cultivated fruit bodies of C.
militaris were studied for the first time. It accelerated substantially the pace of using
cultured C. militaris mycelium in traditional Chinese medicine. It also provided the
experimental evidence and scientific explanation for the folkloric uses of mycelium as
a substitute for fruiting body of C. militaris. To understand the activity relationships
between CMPS-II and CBPS-II, our laboratories had researched the antioxidant
activity, antitumor activity and inhibitory activity to α-glucosidase of CMPS-II and
CBPS-II. The research results will be showed in the next article. In addition, to
improve the yield of mycelium polysaccharides of C. militaris in submerged
fermentation, biosynthesis mechanism of them is being currently researched in our
laboratories.
Acknowledgment
This work was financially supported by the National Spark Key
Program of China (2015GA610001), the Foundation of Tianjin
University of Science and Technology (Nos. 20120106), the International
13
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Science and Technology Cooperation Program of China
(2013DFA31160), and the Foundation of Tianjin Educational Committee
(20090604).
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Figure captions:
Fig. 1. The weight-average molecular weight (Mw) distribution of (a) CMPS-I and (b)
CBPS-I.
Fig. 2. Elution profiles of (a) CMPS-I and (b) CBPS-I in Sephadex G-100 column
chromatography.
Fig. 3. The GC chromatograms of derivatives of (a) 6 standard monosaccharides, (b)
CMPS-II and (c) CBPS-II. Peaks: (1) L-Rhamnose, (2) D-Arabinose, (3) D-Xylose, (4)
D-Mannose, (5) D-Glucose, (6) D-Galactose, (7) Internal standard (Inositol).
Fig. 4. The weight-average molecular weight (Mw) of (a) CMPS-II and (b) CBPS-II.
Fig. 5. FT-IR spectrum of (a) CMPS-II and (b) CBPS-II.
Fig. 6. 1H-NMR spectrum of (a) CMPS-II and (b) CBPS-II; 13C-NMR spectrum of (c)
CMPS-II and (d) CBPS-II.
Fig. 7. The maximum absorption wavelength of Congo red, Congo red+CMPS-II and
Congo red+CBPS-II at various concentrations of sodium hydroxide solution.
Fig. 8. SEM images of CMPS-II (A:×200, B:×1000) and CBPS-II (C:×200, D:×950).
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Table 1 Polysaccharide properties of CMPS-I, CBPS-I, CMPS-II, and CBPS-II from C. militaris
Total sugar
(wt.%)
Monosaccharide component (mol)
Samples
D-Mannose
D-Glucose
D-Galactose
CMPS-I
CBPS-I
56.6
47.5
85.8
89.6
-
-
-
-
-
-
CMPS-II
CBPS-II
1
1
28.63
12.41
1.41
0.74
-: Not detected in the sample.
19
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Table 2 Analytical results from periodate oxidation and Smith degradation of CMPS-II and
CBPS-II
Periodate consumed
Linkage
Smith degradation products
(mol/mol Glc)
1→2
1→3
1
-
Glycerol
Glucose, Mannose or Galactose
Erythritol
1→4
1
1→6
2
Glycerol
CMPS-II
CBPS-II
0.74
0.71
Glycerol, Erythritol, Glucose
Glycerol, Erythritol, Glucose
20
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Tabie 3 Results of the methylation analysis of CMPS-II and CBPS-II
Relative
molar
ratio
TR/mi
n
Methylation sugar
residues
Major mass fragments (m/z) (Relative
Linkages types
abundance, %)
CMPS-II
16.415
2,3,6-tri-O-Me-D-Gal
→4) Gal (1→
T-
0.13
0.11
1.21
0.82
0.42
43.0, 88.0 (34.9, 99.9)
43.0, 45.0,101.0 (99.9, 31.4, 54.2)
43.0, 117.0 (99.9, 45.7)
16.949 2,3,4,6-tetra-O-Me-D-Glc
19.035
20.261
22.329
CBPS-II
16.410
2,3,6-tri-O-Me-D-Glc
2,4,-di-O-Me-D- Glc
2,4,-di-O-Me-D-Man
→4) Glc (1→
→3, 6) Glc (1→
→3, 6) Man (1→
43.0, 87.0, 189.0 (99.9, 48.7, 27.8)
43.0, 129.0 (99.9, 43.6)
2,3,6-tri-O-Me-D-Gal
→4) Gal (1→
T-
0.14
0.12
0.35
0.70
0.52
43.0, 88.0 (34.9, 99.9)
43.0, 45.0, 101.0 (99.9, 31.4, 54.2)
43.0, 117.0 (99.9, 45.7)
16.933 2,3,4,6-tetra-O-Me-D-Glc
19.029
20.260
22.330
2,3,6-tri-O-Me-D-Glc
2,4,-di-O-Me-D- Glc
2,4,-di-O-Me-D-Man
→4) Glc (1→
→3, 6) Glc (1→
→3, 6) Man (1→
43.0, 87.0, 189.0 (99.9, 48.7, 27.8)
43.0, 129.0 (99.9, 43.6)
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(a)
(b)
Fig. 1. The weight-average molecular weight (Mw) distribution of (a) CMPS-I and (b) CBPS-I.
1
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(a)
(b)
Fig. 2. Elution profiles of (a) CMPS-I and (b) CBPS-I in Sephadex G-100 column
chromatography.
2
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(a)
(b)
(c)
Fig. 3. The GC chromatograms of derivatives of (a) 6 standard monosaccharides, (b) CMPS-II and
(c) CBPS-II. Peaks: (1) L-Rhamnose, (2) D-Arabinose, (3) D-Xylose, (4) D-Mannose, (5)
D-Glucose, (6) D-Galactose, (7) Internal standard (Inositol).
3
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(a)
(b)
Fig. 4. The weight-average molecular weight (Mw) of (a) CMPS-II and (b) CBPS-II.
4
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(a)
(b)
Fig. 5. FT-IR spectrum of (a) CMPS-II and (b) CBPS-II.
5
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(a)
(b)
(c)
(d)
Fig. 6. 1H-NMR spectrum of (a) CMPS-II and (b) CBPS-II; 13C-NMR spectrum of (c) CMPS-II
and (d) CBPS-II.
6
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515
510
505
500
495
490
485
Congo red
Congo red+CMPS-II
Congo red+CBPS-II
0
0.1
0.2
0.3
0.4
0.5
C (NaOH)/(mol/L)
Fig. 7. The maximum absorption wavelength of Congo red, Congo red+CMPS-II and Congo
red+CBPS-II at various concentrations of sodium hydroxide solution.
7
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