Carbohydrate Polymers 99 (2014) 553–555

Contents lists available at ScienceDirect

Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Preparation of polysaccharides from cyanobacteria Nostoc commune

and their antioxidant activities
Hong-Bin Wang, Sheng-Jun Wu ∗ , Dou Liu
School of Food Engineering, Huaihai Institute of Technology, 59 Cangwu Road, Xinpu 222005, China

a r t i c l e i n f o a b s t r a c t

Article history: In this study, water soluble polysaccharides were prepared from cyanobacteria Nostoc commune by water
Received 9 July 2013 extraction. Factors affecting the polysaccharide yields were investigated, and the optimum extraction
Received in revised form 20 August 2013 conditions were determined as follows: time, 4 h; temperature, 90 ◦ C; the ratio of liquid to solid, 60:1
Accepted 23 August 2013
(v/w); and extraction times, 4. The extract was filtered, concentrated to ∼10% (w/v), precipitated with 3
Available online 2 September 2013
volumes of ethanol, freeze-dried, and ground to yield a water soluble power. The polysaccharide content
of the product was 96.7%, and the yield was 9.18% (w/w). Fourier transform infrared spectra demon-
Keywords:
strated that the product samples were mainly composed of polysaccharides. The polysaccharides showed
Polysaccharides
Extraction
high hydroxyl radical scavenging activity (92.71%) and reducing capacity (0.445) at the concentration of
Antioxidant activities 10 mg/mL.
© 2013 Elsevier Ltd. All rights reserved.

1. Introduction ability (Tang, Hu, & Chen, 2007). However, the antioxidant activ-
ity of the EPSs of N. commune has not been frequently reported (Li
Nostoc is a genus of the nitrogen-fixing cyanobacteria family et al., 2011).
of Nostocaceae and has the ability to use atmospheric nitrogen In the present study, the extraction conditions of the EPSs of N.
when combined nitrogen is not available (Morsy, Kuzuha, Takani, commune were optimized, the EPSs of N. commune were partially
& Sakamoto, 2008). The species Nostoc commune is a prominent characterized, and the antioxidant activities, including reducing
component of microbial populations worldwide, distributed from capacity and high hydroxyl radical scavenging activity (HRSA),
the tropics to the polar regions of the earth (Jensena et al., 2013). were determined.
Traditionally, some species of Nostoc, including N. commune, have
been used in China as a food source or as medicine to treat illness 2. Materials and methods
(Li et al., 2011).
N. commune forms macroscopic colonies in natural habitats 2.1. Materials
with filaments embedded in extracellular polysaccharides (EPSs)
(Pereira et al., 2009). It is believed that the EPSs of N. commune play N. commune was purchased from a local supermarket (Xinpu,
a role in the stabilization of cells in the air-dried state, inhibiting China) and stored at 4 ◦ C until use. All chemicals were reagent-
the fusion of membrane vesicles during desiccation and acting as grade.
an immobilization matrix for secreted enzymes, which stay fully
active after long-term air-dried storage (Jensena et al., 2013). Thus, 2.2. Extraction of N. commune EPSs
N. commune can resist extreme desiccation and readily restore
metabolic activity upon rehydration (Potts, 1994). The dried colonies were powdered and extracted with organic
However, the main function attributed to the EPSs of N. com- solvents in a Soxhlet apparatus (light petroleum, acetone and
mune is to play important roles in protecting the organism itself methanol). The materials were soaked in distilled to yield a sus-
from a range of physical as well as biological stresses. The EPSs of pension with varying ratios of liquid to solid (30, 40, 50, 60, 70, and
N. commune have been studied considerably in recent years for their 80, respectively, v/w). The reactor was maintained in a thermostatic
viscosities (Huang, Liu, Paulsen, & Klaveness, 1998), strong effect on water bath at different temperatures (75, 80, 85, 90, 95, and 100 ◦ C,
the complement system, and reactive oxygen species scavenging respectively) for varying times (1, 2, 3, 4, 5, and 6 h, respectively).
After the first extraction, the suspensions were filtered through a
Whatman GF/A filter paper. The filter residues were suspended in
∗ Corresponding author. Tel.: +86 0518 85895427; fax: +86 0518 85895428. distilled water for the second extraction as described above. The
E-mail address: [email protected] (S.-J. Wu). extraction processes were performed for 6 times.

0144-8617/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carbpol.2013.08.066
554 H.-B. Wang et al. / Carbohydrate Polymers 99 (2014) 553–555

2.3. Recovery of N. commune EPSs 12

The suspensions were filtered through a Whatman GF/A filter 10

paper, concentrated to ∼15% (w/v), proteins removed by Sevag
method, precipitated with 3 volumes of absolute ethanol, filtered 8
through Whatman GF/A filter paper again, and freeze-dried. The %

Yield (%)
yield of N. commune EPSs was calculated using Eq. (1). 6

W2
Yield = 100 × (1) 4
W1
where W1 and W2 represent the weights of the recovered N. com- 2
mune EPSs and the original N. commune, respectively.
0
2.4. Analytical methods 1 2 3 4 5 6
T ime (h)
Ash, moisture, and total sugar contents of the samples
were determined according to standard methods (Hou, 2004). Fig. 1. Effect of time on extraction of N. commune EPSs. Data are shown as mean ± SD
(n = 3).
The Fourier transform infrared (FTIR) spectra of representative
hydrolysate samples were obtained in KBr pellets by using a Nico-
let Nexus FTIR 470 spectrophotometer over a wavelength range of 3.2. Effect of temperature, the ratio of liquid to solid, and
400–4000 cm−1 . extraction times on extraction of N. commune EPSs

2.5. Antioxidant activity assays The reaction temperature, the ratio of liquid to solid, and
extraction times affected extraction of N. commune EPSs. The tem-
Hydroxyl radical scavenging activity (HRSA) of the hydrolysates peratures studied were from 75 ◦ C to 100 ◦ C. The maximum yield
was measured according to the method of Andrews (1986). N. com- (9.18%) was obtained at 90 ◦ C (Fig. 2), ratio of liquid to solid 60:1
mune EPSs hydroxyl scavenging activity was calculated as follows: (v/w) (Fig. 3), and extraction 4 times (Fig. 4). Higher temperature
increased the yields of N. commune EPSs due to the greater diffu-
sion rate. On the other hand, the decrease in yields at 100 ◦ C can
A1 − A2
HRSA (%) = × 100 (2) be attributed to the evaporation of H2 O. Similarly, higher ratio of
A1 − A0
liquid to solid and more extraction times favored the yield of N.
where: A0 is the absorbance of the reagent blank absorbance, A1 is commune EPSs. In contrast to our findings, Sheng, Zhai, and Chen
the positive control absorbance, A2 is the absorbance of the sample. (2001) extracted N. commune EPSs at 98 ◦ C for 3.8 h and 3 times.
Reducing capacity was measured according to method
described by Qiao et al. (2009) with slight modifications. Briefly, 3.3. Product characterization
One milliliter of N. commune EPSs solution, 1.0 mL phosphate buffer
(0.2 M, pH 6.6) and 1.0 mL potassium ferricyanide (1%, w/v) were The FTIR spectra of the product samples peaked at ∼3396 cm−1
mixed and incubated at 50 ◦ C for 20 min. After cooling down, 1.0 mL (O H) (symmetrical deformation of CH3 and CH2 ), ∼1072 cm−1
trichloroacetic acid (10%, w/v) and 0.2 mL fresh ferric trichloride (stretching vibration of the C O C in glucose circle), and
(FeCl3 , 0.1%, w/v) were added to the reaction mixture. Then the ∼1619 cm−1 (special absorbance peaks of aldehyde in peach gum
reaction mixture was shaken and its absorbance was detected at polysaccharides) (Fig. 5). These data also demonstrated that the
700 nm against a blank (water instead of N. commune EPSs solu- product samples were mainly composed of polysaccharides. Ash,
tion) 10 min later. Absorbance of the reaction mixture indicates the moisture, and total sugar contents were 0.8%, 1.1% and 96.7% (w/w),
reduction capability of sample. respectively. All product samples consisted of water-soluble white
powders.
Reducing capacity = A1 − A2 (3)

where A1 is the absorbance of the sample and A2 is the absorbance

of the sample under identical conditions as A1 with water instead 12
of FeCl3 solution.
10
2.6. Statistical analysis
8
Yield ( %)

All data are presented as mean ± S.D. Statistical analysis was

performed using Statgraphics Centurion XV Version 15.1.02. A mul- 6
tifactor ANOVA with posterior multiple range test was used to find
significant differences of two groups. 4

3. Results and discussion 2

3.1. Effect of time on extraction of N. commune EPSs 0

75 80 85 90 95 100
The effect of time on the extraction of N. commune EPSs was Temperature (°C)
determined over a period of 6 h (Fig. 1). The yields increased with
time up to 6 h and then leveled off. Therefore, the optimal reaction Fig. 2. Effect of temperature on extraction of N. commune EPSs. Data are shown as
time was 6 h. mean ± SD (n = 3).
 H.-B. Wang et al. / Carbohydrate Polymers 99 (2014) 553–555 555

12 0.5 120
0.45
100
10 0.4

Absorbance (700 nm)

0.35
80

HRSA (%)
8 0.3
0.25 60
Yield (%)

0.2 HRSA
6 40
0.15 Absorbance
0.1
4 20
0.05
0 0
2 2 4 6 8 10
Concentration (mg/mL)
0
30 40 50 60 70 80 Fig. 6. Hydroxyl radical scavenging activity (HRSA) and reducing capacity of N.
Ratio of liquid to solid commune EPSs. Data are shown as mean ± SD (n = 3).

Fig. 3. Effect of the ratio of liquid to solid on extraction of N. commune EPSs. Data
are shown as mean ± SD (n = 3).
Reducing capacity is also essential to assess the antioxidant
activity (Duh, Du, & Yen, 1999). Fig. 6 shows the reducing capacity
of N. commune EPSs using the K3 Fe(CN)6 reduction method. The
12
reducing capacity of N. commune EPSs increased with the sam-
ple concentration. At the concentration of 10 mg/mL, the reducing
10
capacity (absorbance at 700 nm) of N. commune EPSs was 0.445.
It has been reported that there was a direct correlation between
8
antioxidant activity and reducing capacity (Qiao et al., 2009). The
Yield (%)

results indicate that there could be a direct correlation between

6 antioxidant activity and reducing capacity in N. commune EPSs.

4 4. Conclusions

2 In this study, water soluble N. commune EPSs were prepared

by water extraction. The extraction process was monitored by
0 the yield of the polysaccharides. Higher temperature and ration
1 2 3 4 5 6
of liquid to solid, more extraction times, and prolonged time
Extraction times favored polysaccharides extraction. The polysaccharides were par-
tially characterized and their antioxidant activity was estimated.
Fig. 4. Effect of extraction times on extraction of N. commune EPSs. Data are shown The polysaccharides had high antioxidant activity and reducing
as mean ± SD (n = 3).
capacity.

3.4. HRSA and reducing capacity of N. commune EPSs References

Hydroxyl radicals (HO• ) have the highest activity among reac- Andrews, A. T. (1986). Electrophoresis: Theory, techniques and biochemical and clinical
applications (2nd ed., pp. 53–75). Oxford: Clarendon.
tive oxygen species and induce severe damage to biomolecules. HO• Duh, P. D., Du, P. C., & Yen, G. C. (1999). Action of methanolic extract of mung bean
has been widely accepted as a tool for estimating the free-radical hulls as inhibitors of lipid peroxidation and non-lipid oxidative damage. Food
scavenging activities of antioxidants (Qiao et al., 2009). The HRSA and Chemical Toxicology, 37, 1055–1061.
Hou, M. L. (2004). Food analysis. Beijing, China: Chemical Industry Press., in chinese.
of N. commune EPSs was presented in Fig. 6. The scavenging effect Huang, Z., Liu, Y. D., Paulsen, B. S., & Klaveness, D. (1998). Studies on polysaccha-
increased with concentration up to 10 mg/mL. At a concentration rides from three edible species of Nostoc (cyanobacteria) with different colony
of 10 mg/mL, the HRSA was 92.71%. Apparently, N. commune EPSs morphologies: Comparison of monosaccharide compositions and viscosities of
polysaccharides from field colonies and suspension cultures. Journal of Phycol-
showed high HO• scavenging activity. ogy, 34, 962–968.
Jensena, S., Petersen, B. O., Omarsdottira, S., Paulsen, B. S., Duus, J. Ø., & Olafsdot-
tir, E. S. (2013). Structural characterisation of a complex heteroglycan from the
cyanobacterium Nostoc commune. Carbohydrate Polymers, 91, 370–376.
Li, H. F., Xu, J., Liu, Y. M., Ai, S. B., Qin, F., Li, Z. W., et al. (2011). Antioxidant and
moisture-retention activities of the polysaccharide from Nostoc commune. Car-
bohydrate Polymers, 83, 1821–1827.
Morsy, F. M., Kuzuha, S., Takani, Y., & Sakamoto, T. (2008). Novel thermostable gly-
cosidases in the extracellular matrix of the terrestrial cyanobacterium Nostoc
commune. Journal of General and Applied Microbiology, 54, 243–252.
Pereira, S., Zille, A., Micheletti, E., Moradas-Ferreira, P., De Philippis, R., & Tamagnini,
P. (2009). Complexity of cyanobacterial exopolysaccharides: Composition,
structures, inducing factors and putative genes involved in their biosynthesis
and assembly. FEMS Microbiology Reviews, 33, 917–941.
Potts, M. (1994). Desiccation tolerance of prokaryotes. Microbiology and Molecular
Biology Reviews, 58, 755–805.
Qiao, D. L., Ke, C. L., Hu, B., Luo, J. G., Ye, H., Sun, Y., et al. (2009). Antioxidant activities
of polysaccharides from Hyriopsis cumingii. Carbohydrate Polymers, 78, 199–204.
Sheng, J., Zhai, C., & Chen, C. (2001). Study of polysaccharide of Nostoc Commune.
Jingxi Huagong/Fine Chemicals, 18, 617–619.
Tang, J., Hu, Z. Y., & Chen, X. W. (2007). Free radical scavenging and antioxidant
enzymes activation of polysaccharide extract from Nostoc sphaeroides. American
Fig. 5. FT-IR spectra of peach gum polysaccharides. Journal of Chinese Medicine, 35, 887–896.