Engineering, 2012, 5, 184-188
doi:10.4236/eng.2012.410B048 Published Online October 2012 (
Copyright © 2012 SciRes. ENG
Preparation of Water Soluble Yeast Glucan by Four Kinds of
Solubili zing Processes
Liping Du, Xuekua ng Zhan g, Chao Wang, Dongguang Xiao
Key Laboratory of Industrial Microbiology, Ministry of Education, Tianjin Industrial Microbiology Key Laboratory,
College of Bi otechnology Tianjin University of Science and Technology, Tianjin University of Science and Technology,
Tianjin 300457, P. R. China
Received 2012
(1→3)-β-D-glucan from the inner cell wall of Saccharomyces cerevisiae is considered a member of a class of drugs known as bio-
logical response modifiers (BRM). However the glucan was an insoluble polysaccharide, which could be the major barrier to the
utilization of glucan. In this case, the insoluble glucan was convent into a soluble form by four kind of solubilizing processes. The
yield, solubility, chemistry structure and immunoprophylaxis efficacy of the soluble products were compared. Our date suggest that
urea has a significant e ffect on yield, and D MSO has a sign ificant effect o n solubilit y. FT-IR s pectra, 13C NMR spectra an d helix-coil
transition analysis demonstrate that the chemistry structure of native and solubilizing glucans have no significant difference. They
still have the triple helical structure. The solubility and immunoprophylaxis efficacy assay indicate that the introduction of phosphate
group not only enhanced the solubility of glucan, but also improved the survival rate of mice challenged with E. coli.
Keywords: Glucan; Solubility; FT-IR Spectra; 13C NMR Spectra; Immunoprophylaxis Efficac y
1. Introduction
Glucan is a β-linked polyglucose immune stimulant that can be
isolated from the cell wall of Saccha rom yces cerevisi ae [1,2]. It
can be used as fat replacer, gell ing agent, thickening agent [3-6].
It is also considered a member of a class of drugs known as
biological response modifiers (BRM) [7]. It has been reported
that (1→3)-β-glucan enhances the innate host defense response
by binding to a specific receptor on the plasma membrane of
macrophages, resulting in their activation, and this activity is
associated with anti-tumor, anti-bacterial and wound-healing
activities [8-12]. However, the native glucan was an insoluble
polysaccharide, whi ch became a major b arrier to the utilization
of (1→3)-β-glucan as BRMs. If (1→3 )-β-glucan is to become
clinically applicable, it has to be converted into biologically
effective, water-soluble form that can be safely administered
via the systemic route. Numerous studies have therefore fo-
cused on converting these glucans into a water-soluble form
through chemical modification such as amination [13], sulfation
[14] and phosphorylation [15]. It has also been demonstrated
that such modified polysaccharides exhibit anti-inflammatory,
anti-tumor, anti-viral and immunomodulatory activities [16-18].
In this study, we have attempted to convert insoluble yeast
glucans into a soluble form through phosphorylation process.
However, in this process we found that only DMSO or DMSO
plus urea (without H3PO4) could conver t i nso lub l e glu can i nt o a
soluble form. Therefore, four kind of preparation of soluble
glucan process were investigated. And four different soluble
products were compared by solubility, FT-IR spectra, 13C NMR
spectra, and helix-coil transition analysis and immunoprophy-
laxis ef ficacy assay.
2. Materials and Methods
2.1. Materials
Isolated cell walls fro m Baker’s yeast were sup plied as “spray-
dried” powder by Angel Yeast LLC, China. Male ICR/HSD
mice (18-20 g) were supplied by the Experimental Animal
Center of the Academy of Military Medical Science (AMMS),
Beijing, China. Mice were housed in a specific pathogen-free
(SPF) environment. All other major reagents were purchased
from Sigma (St. Louis, MO, USA).
2.2. Preparation of Particulate Glucans
Particulate glucans were isolated from the cell wall of S. cere-
visiae by a modification of the method of Williams et al [15].
Briefly, the cell wall of S. cerevisiae was dispersed in 0.3 L of
0.75 M (3 %) sodium hydroxide (NaOH), and extracted in wa-
ter bath at 75 ºC for 2 hours. The suspension was cooled to
room temperature and centrifuged (4000 rpm, 10 min), the
resulting pellet collected and this material then washed three
times using 0.5 L distilled water. The residue was then mixed
with 0 .2 L of 0.75 M NaOH, and ext racted in bo iling water for
3 hours. Distilled water was then added to the cooled suspen-
sion, the pH adjusted to 7 with HCl, and the supernatant after
centrifugation discarded. This water wash was repeated until
the residue became white and flocculent, and finally the pellet
was washed with abso lute ethanol u ntil the sup ernatant became
colorless. Particulate glucan was obtained by drying the washed
2.3. Preparation of Soluble Glucans
Different soluble glucans were prepared by using the improved
Copyright © 2012 SciRes. E NG
method of Williams et al [1 5]. Briefly, particulate glucan (1 g)
was suspended in 60 ml dimethyl sulfoxide (DMSO) containing
15 g u rea. H3PO4 (85%, 15 ml) was added dropwise to the solu-
tion at room temperature (total volume 75 ml, volume ratio
between H3PO4 and DMSO 1:4). The solution was then heated
to 100 ºC for 5 hours, while the solution was constantly stirred,
to bring about the reaction. A crystalline precipitate (presuma-
bly urea phosphate) formed after about 1.5 hours. The reaction
mixture was then cooled to ambient temperature, and 500 ml
distilled water added to dilute the solution. The solution was
then dialyzed against deionizer water for 5 days to remove
Me2SO, H3PO4, urea and any other small compounds, and then
filtrated , concentrated, and freeze-dried to obtain the pure sam-
ples. Different soluble glucans were generated by varying the
dosage of urea and H3PO4, which were tabulated in Table 1.
2.4. Determination of Water Solubility
The water solubility of glucans was measured using a modifica-
tion of the method of Chang et al [19]. Briefly, each sample (1
g) was suspended in distilled water (5 ml) and the resulting
suspension agitated at 25 ºC for 24 hours. The sample was cen-
trifuged at 1600 × g for 15 min. The supernatant (2 ml) was
mixed with three volumes of ethanol to precipitate the glucan.
The precipitates were recovered by centrifugation at 1600 × g
for 15 min, vacuum-dried at 60 ºC, and weighed. The solubility
was calculated using the formula below:
Solubility(%)100(2.5Dry precipitate weight)(Dry sample weight)=××
2.5. Infrared Spectroscopy
IR spectra were recorded using the KBr-disk method with a
Bruker VECTOR 22 Fourier transform infrared (FTIR) spec-
trometer (Bruker, Germany) in the range 400 to 4000 cm-1.
Sixteen scans at a reso lution of 4 cm-1 were averaged an d refe-
renced agai nst air.
2.6. Nuclear Magnetic Resonance Spectroscopy
13C NMR spectra were recorded on Bruker AVANCE 600 MHz
spectrometers (Bruker BioSpin, Swiss) at room temperature.
All samples were dissolved in Me2SO-d6 (heat at 80 ºC for 30
min) to a final concentration of 80 mg/ml. Tetramethylsilane
(TMS) was used as internal standard.
2.7. Analysis of Helix-co il Transition
The conformational structure of the glucans in solution was
determined by characterizing Congo red-polysaccharide com-
plexes. The transition from a triple-helical arrangement to the
single-stranded conformation was examined by measuring the
Table 1. Dosage of ure a and H3PO4.
Group Gl u c an name Urea (g) H3PO4 (ml)
A Glucan A 0 0
B Glucan B 18 0
C Glucan C 0 10
D Glucan D 18 10
λmax of Congo red-polysaccharide solutions at NaOH concen-
trations ranging from 0.0025 to 0.5 M. Polysaccharide aqueous
solutions (0.5 mg/ml) containing 2 ml of 312 μM Congo red
were treated with different concentrations of NaOH (2 ml).
Visible absorption spectra were recorded with a UV/visible
spectrophoto meter (type 2401PC) at each alkali concentrati on.
2.8. Immunoprophylaxis Efficacy Assay
Fifty ICR/HSD mice were divided into five groups. Groups
A to D were injected intraperitoneally with Glucan A, Glucan B,
Glucan C and Glucan D respectively at a dose of 200 mg/kg (4
mg per mouse) 24 hours prior to intraperitoneal challenge with
1×109 viable E. coli. While group O was injected with isovo-
lumetric dextrose 24 hours prior to challenge with E. coli
served as contr ol. The same clini cal isolate of E. coli was used
in all cases, and was maintained in our laboratory. E. coli was
cultured in trypticase soy broth for 18 hours at 37 ºC in an air
bath shaker at 180 rpm. The culture was then centrifuged (3500
rp m, 8 min ), the cel l pell et washe d three ti mes, and this mixtu re
diluted to provide 1 × 109 E. coli/ml. Survival of each group of
mice was monitored hourly during the 24 hour period.
3. Results and Discussion
3.1. The Yield and Solubility of Glucan Samples
The yield and solubility of glucans were shown in Figure 1.
The yield of glucan A and C was very low (~18%). However,
the yield of glucan B and D was higher. This could be con-
clud ed that DMSO and urea had effects o n the yield of glucan s.
DMSO and urea was used as denaturant could facilitate the
breakdown of the intermolecular hydrogen bonds between the
glucan chains. As a result, more hydroxyl group exposed,
which was beneficial to the solubility of glucan. Meanwhile the
influence of urea on the yield was more powerful than DMSO.
The solubility of glucan C and D was significantly higher than
glucan A and B, which could be attribute to the introduction of
phosphate group.
3.2. FT-IR Spectra of Di fferent Glucan Samples
The FT-IR spectra of the glucans are shown in Figure 2. The
Glucan sample
Figure 1. The yield an d solu bil it y o f glucan sam p l es .
Copyright © 2012 SciRes. ENG
peak assignment of native glucan is as follows (cm-1): 3390
(O-H stretch), 2919, 1383, and 1305 (C-H stretch), 1645 (in-
tra-molecular hydrogen bonds), 1372 (CHOH stretch), 1254
(CH2OH stretch), 1159 (bridge O stretch), 1076 and 1041 (C-O
stretch), and 890 (characteristic of β-linked polymer). Com-
pared with native glucan, the absorption peaks of glucan A and
glucan B have no change. While the new absorption peaks of
glucan C and glucan D appeared at 930 cm-1 and 857 cm-1
(C-O-P and O-P-O stretch). These data confirmed that the
phosphate groups had been successfully introduced into the
glucan molecule by esterification. And the new absorption peak
of glucan D appeared at 17 19 cm-1 originating from C=O vibra-
tion due to the carboxyl group, which indicated that there were
COOH moieties pres ent in the glucan D.
3.3. 13C NMR Spectra of Different Glucan Samples
13C NMR spectra from glucans are shown in Figure 3. Ch emi-
cal shifts from all the glucans were observed at 103.5, 86.7,
76.8, 73.3, 68.9 and 61.4 ppm, which correspond to C-1, C-3,
C-5, C-2, C-4, and C-6, respectively. In addition, another signal
was observed at 70.3 ppm (C6’), which is a characteristic peak
due to β-(1→6) branching. Therefore, all the glucan appeared
to be composed of (1→3) -β-glucans with (1→6) -linked
branches. This indicated that the placement of all the glucans
did not cha ng e.
Wavenumber cm
Native Glucan
Glucan A
Glucan B
Glucan C
Glucan D
2919 1645 1372
1076 1014
1383 1305
Figure 2. FT-IR spectra of different glucan samples.
Glucan A
Glucan B
Glucan C
Glucan D
C1 C3 C5 C2 C6
C1 C3 C5 C2 C4 C6
C2 C4
Figure 3. 13C NMR spectra of different glucan samples.
Compared with glucan A and B, three major new signals at
76.0 (C2*), 72.4 (C4*) and 70.2 (C6*) ppm were observed
from glucan C and D, showing that the substitution positions of
glucan C and glucan D were C2, C4 and C6. In addition, the
signal at 72.4 ppm (C4*) was weaker than the signal at 76.0
(C2*) and 70.2 (C6*) ppm, indicating that the phosphate group
concentration at the C4 position was significantly lower than
those at C2 and C6. It can therefore be concluded that phos-
phorylation at C4 is hindered, possibly due to steric hindrance
by the neighboring oxygen in the glucose ring. A new peak
appeared at 160.3 ppm for glucan D, which could be attributed
to COOH, suggesting that COOH moieties were present in
glucan D.
3.4. Helix-Coil Transition Analysis of Different
Glucan Sa mple s
Figure 4 shows the change of maximum absorbance (λmax ) of
Congo red-polysaccharide complex solution with alkaline con-
centration. Laminarin served as the β-1,3-linked triple helical
control. Dextran T-500 served as the random coil control.
Congo red in NaOH served as the negative co ntrol. In the pres-
ence of NaOH (0.025-0.05 M), the Congo red-laminarin or
Congo red-glucan (glucan A, B, C and D) solution exhibited a
red shift from low to high. And the λmax keep stable when the
concentration of NaOH from 0.05-0.25 M. The red shift of λmax
arising from the laminarin or glucan with Congo red is sugges-
tive of the existence of triple helical in all the glucan sample
solution. It is well established that triple helical existing in glu-
can solutions tend to form a complex with Congo red, resulting
in a red shift of the maximum absorbance λmax of Congo red.
3.5. Effect of Different Glucan Samples on the
Survival o f Mice Cha lle nged with E. c oli
Experimental peritonitis induced by Escherichia coli could be
modified by the addition of glucan and its derivatives, which
have immunostimulatory activities. The bioactivity of glucans
was therefor e stu di ed usin g the survival rat e of mice with E. col i
induced peritonitis as an assay. ICR/HSD mice were injected
intraperitoneally with glucans (200 mg/kg) 24 hours prior to
intraperitoneal challenge with 1 × 109 viable E. coli. Compared
0.0 0.1 0.2 0.3 0.4 0.5
Congo Red
Dextran T-500
Glucan A
Glucan B
Glucan C
Glucan D
Figure 4. Helix-coil transition analysis of glucans according to the
absorption maximum of the Congo red-polysaccharide complex at
various con cen t r at ions of NaOH
Copyright © 2012 SciRes. E NG
05 10 15 20
Glucan A
Glucan B
Glucan C
Glucan D
Figure 5. Effects of different glucan samples on survival rate of
mice cha llenged wit h E. co li .
to the control group (dextrose), all glucans had a potent immu-
noprophylaxis efficacy that significantly (p < 0.05) enhanced
the survival rate and the survival time of ICR/HSD mice chal-
lenged with E. coli (Figure 5). The survival rate of mice was
60 %, 40 %, 70 % and 80 % respectively, for glucan A, glucan
B, glucan C and glucan D. In contrast, the control group mice
showed 0 % survival after 9 hours. This indicated that introduc-
tion of phosphate group not only enhanced the solubility, but
also improved the immunoprophylaxis efficacy.
4. Conclusion
Four kinds of soluble glucans were successfully prepared by
four processes. DMSO and urea was used as denaturant could
facilitate the breakdown of the intermolecular hydrogen bonds
between the glucan chains. As a result, more hydroxyl group
exposed, which was beneficial to the solubility and substitution
reaction of glucan. Meanwhile, urea has a significant effect on
yield, and DMSO has a significant effect on solubility. From
FT-IR and 13C NMR spectra we can see that there was no dif-
ference between native and glucan A, B. Glucan C and D was
successfully phosphorylated. And the substitution positions
were C2, C4 and C6. However the hydroxyl group of glucan D
at C6 was partially oxidized into a carboxyl group, which was
caused by urea and H3PO4 together. All the glucans had the
triple helical structure and the immunoprophylaxis efficacy.
However the immunoprophylaxis efficacy of phosphorylated
glucan was better. The introduction of phosphate group not
only enhanced the solubility of glucan, but also improved the
survival rate of mice challenged with E. coli.
5. Acknowledgements
This study was financially supported by the 863 (Hi-tech re-
search and development program of China) program under
contract NO. 2012AA021505. We also would like to express
our deepest appreciation to Tianjin University of Science &
Technology for the financial support (Grant number:20100208)
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