Preparation and biological activities of an exopolysaccharide produced by Enterobacter cloacae Z0206

Article abstract of DOI:10.1016/j.carbpol.2010.03.020

A water-soluble extracellular polysaccharide (EPS-1) was isolated from the submerged culture broth of Enterobacter cloacae Z0206 through fermentation, ethanol precipitation, anion-exchange and gel-permeation chromatography. Its structural analysis showed that the average molecular weight of EPS-1 was 23,928 Da, and it was composed of glucose, mannose and galactose with a molar ratio of 6.860:1.180:0.455. It was hypothesized that EPS-1 belongs to the α-type heteropolysaccharide with pyran group. Bioactivity tests in vivo showed that the administration of EPS-1 to cyclophosphamide (CP)-exposed animals resulted in significant increase and recovery of B lymphocyte proliferation, tumor necrosis factor α (TNF-α) production and activities of antioxidant enzymes (superoxide dismutase (SOD) and glutathione peroxidase (GPx)). It is suggested that EPS-1 could provide protection against CP-induced immunosuppression and oxidative damage in mice model, and it may act as a potent immunomodulatory and antioxidant agent.

Full text of DOI:10.1016/j.carbpol.2010.03.020

Carbohydrate Polymers 81 (2010) 607–611
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Preparation and biological activities of an exopolysaccharide produced by
Enterobacter cloacae Z0206
Mingliang Jina, Youming Wanga, Chunlan Xua,b, Zeqing Lua, Ming Huanga, Yizhen Wanga,∗
a The Key Laboratory of Molecular Animal Nutrition of Ministry of Education, College of Animal Sciences, Zhejiang University,
No. 164 Qiutao North Road, Hangzhou 310029, PR China
b College of Life Sciences, Northwestern Polytechnical University, Xi’an 710072, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A water-soluble extracellular polysaccharide (EPS-1) was isolated from the submerged culture broth
of Enterobacter cloacae Z0206 through fermentation, ethanol precipitation, anion-exchange and gel-
permeation chromatography. Its structural analysis showed that the average molecular weight of
EPS-1 was 23,928 Da, and it was composed of glucose, mannose and galactose with a molar ratio of
6.860:1.180:0.455. It was hypothesized that EPS-1 belongs to the ␣-type heteropolysaccharide with
pyran group. Bioactivity tests in vivo showed that the administration of EPS-1 to cyclophosphamide
(CP)-exposed animals resulted in significant increase and recovery of B lymphocyte proliferation, tumor
necrosis factor ␣ (TNF-␣) production and activities of antioxidant enzymes (superoxide dismutase (SOD)
and glutathione peroxidase (GPx)). It is suggested that EPS-1 could provide protection against CP-induced
immunosuppression and oxidative damage in mice model, and it may act as a potent immunomodulatory
and antioxidant agent.
Received 28 December 2009
Received in revised form 7 March 2010
Accepted 9 March 2010
Available online 16 March 2010
Keywords:
Exopolysaccharide
Enterobacter cloacae
Immunomodulation
Antioxidant
© 2010 Elsevier Ltd. All rights reserved.
1. Introduction
this Se-enriched exopolysaccharide to cyclophosphamide (CP)-
exposed mice resulted in improvement of cellular and humoral
In recent years, many polysaccharides have been isolated from
mushrooms, fungi, yeasts, algae, lichens and plants (Liu et al., 2007).
They have emerged as an important class of bioactive natural prod-
ucts in the biochemical and medical areas due to their specific
biological activities such as immunostimulating (Xu, Wang, Jin, &
Yang, 2009), antioxidant (Luo & Fang, 2008), antiviral (Wang, Ooi,
& Ang, 2007), antitumor effects (Tong, Xia, Feng, Sun, & Gao, 2009).
In many Asian countries, several immunoceuticals composed of
polysaccharides have been accepted such as lentinan, krestin and
schizophyllan (Zhang, Cui, Cheung, & Wang, 2007).
Enterobacter cloacae Z0206, a bacterial strain, can produce large
amounts of exopolysaccharides. It has been reported that glycopro-
teins from E. cloacae showed antitumor effects on mice with S180
tumors (real), and F3, one of the glycoprotein components, could
distinctly inhibit QGY7703 (liver cancer), A549 (glandular cancer of
the lungs), Kato III (gastric carcinoma) and Sw1116 (intestinal can-
cer) cell strains (Zhang, Ren, & Chen, 2002). In our previous studies
(Xu et al., 2009), we found that E. cloacae Z0206 could accumu-
late selenium (Se) in the form of Se-enriched exopolysaccharide
efficiently during cultivation with selenium. Administration of
immune responses.
However, little is known about the exact pharmacological
effects and structure–function relationship of exopolysaccharide
without Se derived from E. cloacae, which would allow a better
understanding of the functional effects about this macromolecule.
The objective of the current study was to extract and purify
the major exopolysaccharide produced by E. cloacae Z0206 using
DEAE-52 cellulose and Sephadex G-100 column chromatogra-
phy. Based on the experiments above, the possible effects of this
exopolysaccharide on the immune functions and antioxidant sta-
tus of immunocompromised hosts were further investigated by
establishment of CP-induced immunosuppressive mice.
2. Materials and methods
2.1. Materials
The exopolysaccharide-producing bacterial strain identified
as E. cloacae Z0206 was identified and kept in our laboratory,
and it has been collected by China General Microbiologi-
cal Culture Collection Center (CGMCC). CP, lipopolysaccharide
(LPS), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bro-
mide (MTT), dimethyl sulfoxide (DMSO) and RPMI-1640 medium
were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The
∗
Corresponding author. Tel.: +86 571 86951729; fax: +86 571 86994963.
E-mail address: yzwang@zju.edu.cn (Y. Wang).
0144-8617/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2010.03.020

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M. Jin et al. / Carbohydrate Polymers 81 (2010) 607–611
sheep red blood cells (SRBCs), used as an antigenic material, were
delivered by the Institute of Feed Science, Zhejiang University, kept
in Alsever’s medium, and washed three times with PBS before use.
2.6. Monosaccharide components of polysaccharide
The polysaccharide (50 mg) was hydrolyzed into monosaccha-
rides with 2 mL of 1 M H2SO4 at 100 ◦C for 10 h, and neutralized with
BaCO3 to pH 7.0. After filtration through a 0.45 ␮m membrane, the
mixture was analyzed by HPLC (Waters-2690, USA) equipped with
RID and a Sugar-pak-1 column (Waters, USA). Ultrapure water was
used as the solvent at a flow rate of 0.7 mL/min and 25 ␮L of sample
or glucose, mannose and galactose each as a standard were injected.
2.2. Microorganism cultivation
E. cloacae Z0206 was initially grown on PDA medium (fresh
potato, 20% (w/v); dextrose, 2.0%; agar, 2.0%) at 30 ◦C for 1 day, and
then transferred to 250 mL flasks containing 80 mL of seed culture
medium (fresh potato, 20% (w/v); dextrose, 2.0%; peptone, 0.2%;
yeast extract, 0.3%) and incubated on a rotary shaker at 250 rpm for
18 h at 30 ◦C.
2.7. Infrared spectrophotometer (IR)
The IR spectrum of the polysaccharide was determined using
a Fourier transform infrared (FTIR) spectrophotometer (Nicolet
Nexus 670, Thermo Electron, USA) equipped for detecting func-
tional groups. The purified polysaccharide was ground with KBr
powder and pressed into pellets for FTIR measurement in the fre-
Cultivation medium contained: dextrose, 2.5%; peptone, 0.5%;
yeast extract, 0.5%, K2HPO4, 0.2%; KH2PO4, 0.1% and MgSO4·7H2O,
0.05%. The exopolysaccharides production was performed in a
10 dm3 bioreactor (Shanghai Biotech Ltd., China) with 7 dm3 cul-
tivation medium at 30 ◦C for 2 days. The initial pH was 7.5 and the
inoculation volume was 5.0% (v/v). Aeration rate (1.0 vvm), growth
temperature, foam level, pH and dissolved oxygen tension were
controlled and/or measured by the bioreactor control unit.
quency range of 4000–400 cm−1
.
2.8. Animals and treatment
40 ICR (Institute of Cancer Research) male mice (18 2 g) were
randomly divided into four groups of ten each. Two immunosup-
pressed groups (CP and CP + EPS-1) were administered with EPS-1
(0 and 400 mg/kg body weight (B.W.)) by gavage once daily for 14
days, and CP was given intraperitoneally at 50 mg/kg B.W. on the
12th day. Control mice received the same volume of 0.9% normal
saline. The EPS-1 group was given EPS-1 at 400 mg/kg B.W. by gav-
age once daily for 14 days. Animals of all groups were challenged
with 0.2 mL of 0.1% SRBCs (i.p.) on the 10th day. The animals were
sacrificed by cervical dislocation 12 h after the last dose. Spleen,
liver and blood samples were collected for further analysis.
2.3. Preparation of crude exopolysaccharides
After cultivation, the broth was centrifuged at 4500 × g for
20 min to remove the mycelia. The supernatant was evaporated
under reduced pressure at 50 ◦C, then precipitated upon addition
of 4 volumes of cold 95% EtOH and kept at −20 ◦C overnight. The
resulting precipitate was collected by centrifugation at 7600 × g for
15 min at 4 ◦C. The precipitates were dissolved in distilled water
and deproteinized by a combination of papain and trypsin enzy-
molysis and the Sevag method (Staub, 1965), then precipitated
with 4 volumes of cold 95% EtOH at −20 ◦C overnight. After cen-
trifugation at 7600 × g for 15 min at 4 ◦C, successive washes with
anhydrous ethanol, acetone and ether, and drying under vacuum
at 40 ◦C, the crude exopolysaccharides were obtained.
2.9. Splenic lymphocyte proliferation assay
Single-cell suspensions of splenic lymphocytes were prepared
(Kaspers, Lillehoj, Jenkins, & Pharr, 1994) and placed into 96-well
flat-bottomed microplates at a concentration of 5 × 106 cells/mL
with RPMI-1640 media, adding 100 ␮L of LPS (10 ␮g/mL) or com-
plete medium (controls). After incubation for 44 h at 37 ◦C in a
humidified 5% CO2 atmosphere, 20 ␮L MTT (5 mg/mL) was added
into each well, followed by incubation for another 4 h, and then
100 ␮L of DMSO was added into each well to dissolve the precip-
itation completely. The light absorbance was measured at 570 nm
with Enzyme-linked Immunosorbent Assay Reader (Model BIO-
RAD-550, USA).
2.4. Purification of exopolysaccharides
The crude exopolysaccharides were redissolved in distilled
water and subsequently applied to a DEAE-52 anion-exchange
chromatography column (2.6 cm × 50 cm), eluting successively
with distilled water and a linear gradient of 0–0.2 M NaCl with
a flow rate of 0.5 mL/min. The yielded fractions were combined
according to the total carbohydrate content quantified by the
phenol-sulfuric acid method (Dubois, Gilles, Harmilton, Rebers, &
Smith, 1956). The main peak obtained was concentrated and fur-
ther fractionated on a Sephadex G-100 column (1.6 cm × 50 cm)
eluted with 0.05 M NaCl at a flow rate of 0.4 mL/min in the same
manner to yield completely separated fractions. The main fraction
was collected, dialyzed and lyophilized to obtain a white purified
exopolysaccharide, which was subjected to subsequent analyses.
2.10. Cytokine determination in serum
Blood was obtained from the heart of treated and control ani-
mals and allowed to stand for 1 h at 37 ◦C. After centrifugation
at 6000 × g for 10 min at 4 ◦C, the blood serum was collected and
kept frozen until use. The presence of TNF-␣ in blood serum was
determined using the mouse ELISA Set (BD OptEIA, BD Biosciences
PharMingen, San Diego, CA, USA).
2.5. Assay for molecular weight
The average molecular weight of the polysaccharide was
measured by gel-permeation chromatography (GPC) with an Ultra-
hydrogel Linear Column (7.8 mm × 300 mm) on a Waters high
performance liquid chromatography (HPLC, Waters-2690, Mas-
sachusetts, USA). The column was operated at 34 ◦C and eluted
with 0.1 M phosphate buffer (pH 7.0) at a flow rate of 0.8 mL/min.
The eluent was monitored with a refractive index detector (RID,
Waters-2410, USA). The column was calibrated by various stan-
dard dextrans. All data provided by the GPC system were collected
and analyzed using the Millennium software package.
2.11. Antioxidant status assay in liver
Livers of mice were excised, cleaned and homogenized in 1:5
volumes of normal saline solution. The homogenate was cen-
trifuged at 1700 × g for 10 min at 4 ◦C. The supernatant fractions
were collected and activities of SOD and GPx were determined by
spectrophotometric methods using corresponding diagnostic kits
(Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

M. Jin et al. / Carbohydrate Polymers 81 (2010) 607–611
609
2.12. Statistical analysis
Data were analyzed by one-way analysis of variance
(ANOVA), followed by Dunnett’s t-test. Results were presented
as means S.D. Differences between groups were considered
statistically significant at the 5% (p < 0.05) level.
3. Results
3.1. Isolation and purification of exopolysaccharides
Crude exopolysaccharides were precipitated with ethanol from
the centrifuged and deproteinated culture broth of E. cloacae Z0206.
The precipitate was fractionated on a DEAE-52 cellulose column
with distilled water and a gradient elution of NaCl in the range
of 0–0.2 M, leading to the isolation of two polysaccharide sub-
fractions, NPS and APS (Fig. 1). Sub-fraction NPS was a neutral
polysaccharide as it was eluted with water, and sub-fraction APS
should be acidic as it was eluted with NaCl solution. NPS was the
major component, shown by its peak areas in anion-exchange chro-
matography. For convenient preparation and high content, NPS
fractions were collected, and further fractionated on a Sephadex G-
100 column eluted with 0.05 M NaCl, resulting in one sub-fraction,
EPS-1 (Fig. 2).
Fig. 3. HPLC analysis of monosaccharide components of EPS-1.
monosaccharide composition of EPS-1 was determined by the sul-
furic acid hydrolysis and HPLC analysis methods (Fig. 3). The results
indicated that EPS-1 was composed of glucose, mannose and galac-
tose with a molar ratio of 6.860:1.180:0.455.
3.2. IR
The FTIR spectrum of EPS-1 (Fig. 4) showed a strong band at
3444.87 cm−1, which was attributed to the stretching vibration of
O–H in the constituent sugar residues. The band at 2927.49 cm−1
was associated with the stretching vibration of C–H in the sugar
ring. The absorptions around 1699.00 cm−1 were due to the stretch-
ing vibration of C O, and the band at 1384 cm−1 represented CH2
and OH bending (Sun, Fang, Goodwin, Lawther, & Bolton, 1998). The
band at 850.60 cm−1 was attributed to ␣-type glycosidic linkages,
while the band at 763.97 cm−1 was associated with ␣-isomeric
pyranose symmetrical stretching vibration (Cheng, Wan, Jin, Wang,
& Xu, 2008). The prominent absorptions at 1144.89, 1081.47, and
1023.10 cm−1 also indicated the ␣-pyranose form of the glucosyl
residue. Therefore, it can be concluded that EPS-1 belongs to the
␣-type heteropolysaccharide with pyran group.
The GPC profile showed that EPS-1 was a homogeneous polysac-
charide with an average molecular weight of 23,928 Da. The
3.3. Immune and antioxidant activities of EPS-1
The proliferative responses of lymphocytes to LPS and the
serum TNF-␣ concentration in CP-treated mice reduced markedly
(p < 0.05) compared with control animals (Table 1). Animals receiv-
ing EPS-1 along with CP showed significant recovery in splenic
lymphocyte proliferation and the level of TNF-␣ (p < 0.05) com-
pared with CP treated alone. Furthermore, treatments with EPS-1
increased the LPS-induced lymphocyte proliferation significantly
(p < 0.05) compared with the control animals.
Fig. 1. Anion-exchange chromatography of crude exopolysaccharides produced by
Enterobacter cloacae Z0206. Column: DEAE-52 cellulose (2.6 cm × 50 cm); flow rate:
0.5 mL/min; fraction volume: 5 mL. NPS: neutral polysaccharide (eluted with dis-
tilled water); APS: acidic polysaccharide (eluted with a linear gradient of 0–0.2 M
NaCl).
CP treatment caused a significant reduction in activities of
antioxidant enzymes SOD and GPx in liver compared with control
animals (p < 0.05) (Table 2). EPS-1 treatment significantly blocked
the decrease in these antioxidant enzyme activities of mice treated
with CP (p < 0.05).
Table 1
Effect of EPS-1 on immune functions.
Group
Lymphocyte proliferation (OD570 nm
)
TNF-␣ (ng/mL)
Control
CP
CP + EPS-1
EPS-1
0.475 0.020a
0.269 0.013b
0.429 0.004a
0.583 0.046c
5.140 0.000a
2.703 0.249b
4.385 0.841a
4.210 0.156a
Values are expressed as means S.D. of six animals. Means within a column with dif-
ferent letters (a–c) differ significantly (p < 0.05). Abbreviation: TNF-␣, tumor necrosis
factor ␣.
Fig. 2. Gel-permeation chromatography of NPS eluting with 0.05 M NaCl. Column:
Sephadex G-100 (1.6 cm × 50 cm); flow rate: 0.4 mL/min; fraction volume: 5 mL.

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M. Jin et al. / Carbohydrate Polymers 81 (2010) 607–611
Fig. 4. FTIR spectra of the polysaccharide EPS-1.
Table 2
its use also introduces the possibility of immunotoxicity, terato-
Effect of EPS-1 on antioxidant status in liver.
genicity and genotoxicity (Bin-Hafeez, Ahmad, Haque, & Raisuddin,
2001; Franke, Pra, Erdtmann, Henriques, & da Silva, 2005). CP was
shown to inhibit the humoral and cellular immune responses in
experimental animal models (Masnaya & Ratner, 2000; Rondinone,
Giovanniello, Barrios, & Nota, 1983). In addition, studies have indi-
cated that CP has a prooxidant character, and its administration
may lead to the generation of oxidative stress in liver, lungs and
serum in mice and rats with a resulting decrease in the activi-
ties of antioxidant enzymes and an increase in lipid peroxidation
in these tissues (Mathew & Kuttan, 1997). Therefore, CP has been
used to establish an experimental model applicable to assay the
immunomodulation and antioxidant properties by antibiotics or
other samples in normal or immunocompromised mice (Bujalance,
Moreno, Jimenez-Valera, & Ruiz-Bravo, 2007).
Lymphocyte proliferation is a crucial event in the activation cas-
cade of both the cellular and humoral immune responses (Xu et
al., 2009). Splenic lymphocyte proliferation induced by LPS can be
used to examine B lymphocyte activity (Han et al., 1998). Some
cytokines such as TNF-␣, interleukin-2 (IL-2) and interferon-␥ (INF-
␥) can induce a cell-mediated immune response and directly or
indirectly regulate immune reactions (Mosmann & Coffman, 1989).
TNF-␣ can induce the production of cytokines that could participate
in the host response to infecting pathogens (Jablons et al., 1989).
The present study showed that combined treatment with EPS-1
and CP significantly promoted the recovery of the lymphocyte pro-
liferation response and the level of TNF-␣ in serum compared to
treatment with CP alone, indicating that EPS-1 had a potent effect
on the cellular immune response.
Oxidative stress is a crucial mediator of cell injury and pathol-
ogy. Reactive oxygen species (ROS) can lead to the autooxidation
of glycosylated protein, induction of membrane damage and oxi-
dation of cellular lipids and proteins (Fang, Yang, & Wu, 2002).
Several antioxidant enzymes, including SOD and GPx, are important
buffers in the interception and degradation of superoxide anion and
hydrogen peroxide. SOD is the only enzyme that disrupts superox-
ide radicals, and it can convert superoxide to hydrogen peroxide
and maintain low superoxide concentrations (Jiang et al., 2008).
GPx is an equally important antioxidant, able to react with hydro-
gen peroxide, thus preventing intracellular damage (Li, Zhou, & Li,
2007). According to this study, EPS-1 can promote the activities of
SOD and GPx in hepatocytes, indicating it has antioxidant activity
Group
SOD (U/mg prot)
GPx (U/mg prot)
Control
CP
CP + EPS-1
EPS-1
300.586 8.021a
285.186 8.143b
301.349 1.887a
327.190 6.010c
871.570 35.572a
709.086 100.588b
867.760 10.431a
864.025 27.672a
Values are expressed as means S.D. of six animals. Means within a column with
different letters (a–c) differ significantly (p < 0.05). Abbreviations: SOD, superoxide
dismutase; GPx, glutathione peroxidase.
4. Discussion
The water-soluble extracellular polysaccharide (EPS-1) was iso-
lated from the fermentation broth of E. cloacae Z0206. EPS-1 was
composed of glucose, mannose and galactose with a molar ratio of
6.860:1.180:0.455 and its average molecular weight was approx-
imately 24 kDa. It was reported that the bioactive glycoprotein
from E. cloacae, with a molecular weight of 33 kDa, was composed
of xylose (Zhang et al., 2002). The partially purified polysaccha-
ride produced by E. cloacae WD7 was characterized as an acidic
heteropolysaccharide, composed of neutral sugars (29.4%), uronic
acids (14.2%) and amino sugars (0.93%); function group analysis
by FTIR spectroscopy showed the presence of hydroxyl, carboxyl,
carbonyl and methoxyl groups (Prasertsan, Dermlim, Doelle, &
Kennedy, 2006). These results indicate that EPS-1 produced by E.
cloacae Z0206 has a different structure. It is noted that the char-
acteristics of polysaccharides, i.e. sugar components, molecular
weight, and the structure of the main chain and branches strongly
affect the activities of polysaccharides (Jiang et al., 2008). These
characteristics may endow EPS-1 with special activities.
The high molecular weight polysaccharides are often called
biological response modifiers (BRM) or immunopotentiators, and
it is generally accepted that they can enhance various immune
responses in vivo and in vitro (Liu et al., 2007). However, immunos-
timulatory effects of a drug or nutritional supplement are difficult
to evaluate in healthy animals (Huang, Wu, Chen, Yang, & Wang,
2007). Therefore, we made an immunosuppressive animal model
to evaluate the biological activities of EPS-1.
CP is a cytotoxic alkylating agent with a high therapeutic index
and broad spectrum of activities against a variety of cancers, as
well as B cell malignant diseases (Sun & Peng, 2008). However,

M. Jin et al. / Carbohydrate Polymers 81 (2010) 607–611
611
in vivo. It can be concluded that EPS-1 treatment might ameliorate
the synthesis of essential antioxidant enzymes, which have a role
in preventing the pathological concentrations of oxygen radicals or
improving biochemical changes induced by CP.
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5. Conclusions
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mediated induction of major histocompatibility complex class II antigens on
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induced by ␥-irradiation in rat liver mitochondria. Carbohydrate Polymers, 69,
172–178.
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activity of a polysaccharide from Strongylocentrotus nudus eggs. Carbohydrate
Polymers, 67, 313–318.
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testing of their antioxidant activities. Carbohydrate Polymers, 72, 376–381.
Masnaya, N. V., & Ratner, G. M. (2000). Peculiarities of primary humoral immune
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Medicine, 129, 370–373.
In summary, the present study is the first to demonstrate the
isolation and biological activities of a novel exopolysaccharide,
EPS-1, from the submerged culture broth of E. cloacae Z0206. Pre-
liminary tests suggested that treatment with EPS-1 resulted in
accelerated recovery of immuno- and antioxidant-suppression in
CP-treated mice. EPS-1 significantly exhibited immunomodulatory
and antioxidant activity in vivo. The structural and pharmacological
results obtained might help increase knowledge of the relationship
between the structure and biological activities of polysaccharides.
However, the exact chemical structure of EPS-1 is not clear yet,
and little is known regarding the essential structure responsible
for its biological activity. This research will be carried out in our
laboratory.
Mathew, S., & Kuttan, G. (1997). Antioxidant activity of Tinospora cordifolia and its
usefulness in the amelioration of cyclophosphamide induced toxicity. Journal of
Experimental and Clinical Cancer Research, 16, 407–411.
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lymphokine secretion lead to different functional properties. Annual Review of
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Acknowledgements
Prasertsan, P., Dermlim, W., Doelle, H., & Kennedy, J. F. (2006). Screening, character-
ization and flocculating property of carbohydrate polymer from newly isolated
Enterobacter cloacae WD7. Carbohydrate Polymers, 66, 289–297.
Rondinone, S. N., Giovanniello, O. A., Barrios, H. A., & Nota, N. R. (1983). Effect
of fractional cyclophosphamide dosage on sheep red blood cell-delayed-type
hypersensitivity response in mice. Journal of Immunology, 130, 1600–1603.
Staub, A. M. (1965). Removal of protein—Sevag method. Methods in Carbohydrate
Chemistry, 5, 5–6.
This study was supported by the National Key Technology R&D
Program, China (2006BAD14B06-4) and the Science and Technol-
ogy Key Program of Zhejiang Province, China (2007C12041).
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