Journal of Applied Phycology 16: 333–340, 2004.

C 2004 Kluwer Academic Publishers. Printed in the Netherlands.

333

Anti-oxidation of agar oligosaccharides produced by agarase from a marine

bacterium

Jingxue Wang∗ , Xiaolu Jiang, Haijin Mou & Huashi Guan

Department of Food Science and Technology, Ocean University of China, No. 5 Yushan Road, Qingdao, 266003,
Shandong Province, People’s Republic of China
∗
Author for correspondence (e-mail: jxwang2001@public.qd.sd.cn; phone: +86-532-2032290;
fax: +86-532-2894024)
Received 26 January 2004; revised and accepted 10 May 2004

Key words: agar oligosaccharide, agarase, antioxidation, MALDI-TOF-MS, marine bacterium

Abstract

In order to prepare the active agar oligosaccharide, agarase extracted from a strain of unidentified marine bacterium
from the South China Sea coast was selected for the agar depolymerization. The optimum decomposing conditions
were determined to be pH 7.0, 35 ◦ C and halophilic properties 2%. Three main degraded products, AOS-1, AOS-2
and AOS-3, were separated by ethanol fractionation and anion exchange chromatography. The molecular mass was
analyzed by MALDI-TOF-MS. The agar oligosaccharides exhibited antioxidative activities in scavenging hydroxyl
free radical, scavenging superoxide anion radical and inhibiting lipid peroxidation.The fragment with the sulfate
group showed stronger antioxidative activities than that without the sulfate group. Higher antioxidative activities
were found when the molecular mass was increased. The results indicated that the antioxidative activities were closely
related to the molecular mass of the agar oligosaccharides and the substitute groups binding the carbohydrate ring.

Abbreviations: MALDI-TOF-MS, Matrix-assisted laser desorption ionization time-of-flight mass spectrometry;

DNS, 3,5-dinitrosalicylic acid; PBS, phosphate buffer solution; MDA, malonic dialdehyde; MM, molecular mass;
DS, degree of substitute; DHB, 2,5-dihydroxybenzoic acid

Introduction fractionation, gave a highly sulfated, agar-type po-

lysaccharide that inhibited the transplantation of
Agar, a polysaccharide present in the cell walls of Ehrlich ascites carcinoma in mice (Fern·ndez et al.,
some red algae, is commonly used in the fields of bio- 1989).
chemistry, cosmetic, medicine and aquaculture. Bioas- Neoagarooligosaccharides, prepared by partial en-
says have shown that sulfated polysaccharides from zyme hydrolysis, showed biological activity towards
agar have antiviral properties that are expressed by Gracilaria conferta, demonstrated by increase of
interfering with the efficient attachment of certain oxygen consumption, release of hydrogen perox-
viruses to host cells (Takemoto, 1966). Mazumder ide, elimination of epiphytic bacteria and induction
et al. (2002) found that a high-molecular-mass galac- of thallus tip bleaching (Weinberger et al., 2001).
tan sulfate, agar-type polysaccharide, exhibited se- Water extracted polysaccahrides from Porphyra hai-
lective antiviral activity against herpes simplex virus tanesis (Zhang et al., 2003) exhibited antioxidative
types 1 and 2, likely due to an inhibition of the activities.
initial virus attachment to the host cell. Cold water Thus it can be seen that the agar polysaccharide or
extraction of the red alga Gracilaria dominguen- agar oligosaccharide can be attained by many meth-
sis, followed by cetyltrimethylammonium bromide ods. Different methods including the water extraction,
334

ethanol extraction, acid hydrolysis and enzyme hydrol- 2% NaCl, 1.5% agar, 0.1% K2 HPO4 , 0.5% peptone,
ysis, etc., lead to different products with the different and 0.1% yeast extract. The plates were incubated at
activities. A special enzyme hydrolyzing the agar, 37 ◦ C for 24 h. Five millilitres of overnight culture of
agarase (agarose 4-glycanohydrolase, E.C.3.2.1.81), isolated colonies was transferred to 125 mL of fresh
has been found in certain marine mollusks (Usov & medium containing 0.2% agar. Incubation was carried
Miroshnikova 1975). However, the most was reported out in a shaking incubator at 150 rpm and 30 ◦ C for
from several bacterial genera, including Cytophaga 24 h, and then followed straight away by the enzyme
(Van der Meulen et al., 1974), Vibrio (Aoki et al., 1990; assays.
Sugano et al., 1993), Streptomyces (Stanier, 1942), All the agar used was commercial agar powder ex-
Alteromonas (Leon et al., 1992; Potin et al., 1993), tracted from Gelidum amausii (Beijing Shuangxuan
Pseudoalteromonas (Romanenko et al., 2002; Vera Co.).
et al., 1998) and Pseudomonas (Groleau & Yaphe,
1977; Ha et al., 1997), etc. Most of these bacte-
ria were isolated from marine environments, while Preparation of crude agarase
a few species isolated from rivers (Agbo & Moss,
The cells were grown in a Lab-line orbital shaker at 150
1979), hot spring (Shieh & Jean, 1998), soil (Sampietro
rpm and 30 ◦ C until the stationary phase, then were cen-
& Vattuone de Sampletro, 1971) and sewage
trifuged at 7000 × g for 15 min. The supernatant was
(van Hofsten & Malmqvist, 1975) have also been
collected and fractionated at 80% ammonium sulfate
described.
saturation. The raw agarase was prepared by lyophiliz-
By now, the antioxidative study on the products
ing the pellet.
prepared by agarase hydrolysis from the red algae was
Agarase activity was determined by measuring the
comparatively deficient. Furthermore, superoxide an-
increase in the concentration of reducing sugar as de-
ion (O− 2 ·), hydroxyl radical (OH·) and lipid peroxide scribed by von Borel et al. (1952). After centrifuging to
radical (ROO− ·) are the most representative free rad-
remove bacterial cells and gel residues, a 1-mL culture
icals. In oxidation reactions, superoxide anion is the
supernatant was added to 20 mL 0.3% agar substrate
first free radical species normally formed in the cells,
and incubated at 35 ◦ C for 30 min. Then the 1-mL reac-
and its oxidative effects can be magnified because it
tion solution was mixed with 1.5 mL DNS. The mixture
produces other kinds of cell-damaging free radicals.
was heated at 100 ◦ C for 5 min, cooled, and diluted
Among them, the damaging action of the hydroxyl
to 25 mL with deionized water. Optical density was
radical is the strongest among free radicals. Lipid per-
read at 520 nm and values for reducing sugars were ex-
oxidation, oxidative damage of polyunsaturated fatty
pressed as D-galactose equivalents. One unit of agarase
acids in membrane phospholipids is one of the mul-
activity was defined as the amount of enzyme that re-
tiple toxic effects of oxidative stress that is related to
leased 1 µg of D-galactose per min under the above
several pathological conditions (Zimniak et al.,1977).
conditions.
Therefore, the antioxidative study was planed from
these three aspects. In the experiment the enzymol-
ysis products were selected as the experimental ob- Preparation of agar oligosaccharides
jects. The aim is to evaluate their in vitro antioxidant
activity and characterize the relationship between an- Ten gram of agar powder was scattered in 500 mL
tioxidant activity and chemical characteristic, includ- deionized water. A 50-mL agarase solution (agarase
ing the molecular mass and content of the sulfate activity was 15,000 U) extracted from strain QJH-12
group. was added to the agar solution. The reaction was carried
out at 35 ◦ C for 12 h, and then was stopped by heat-
ing the solution in boiling water for 10 min. Twofold
Materials and methods ethanol was added to the reaction mixture to remove
the high-molecular-mass polysaccharides. After cen-
Isolation of strains and cell growth trifugation and concentration, the water-soluble frac-
tions were produced by ethanol gradient fractionation.
Strain QJH-12 was isolated from the South China Sea The F1 and F2 samples were the degraded products
coast in Sanya, Hainan Province. The screening was by 10 times and 9 times ethanol fractionation, respec-
carried out on agar plates in a medium containing tively. For further separation F1 and F2 were applied
 335

to a column (28 × 1.8 cm) of AG MP-1 resin (Bio- Hydroxyl radical was generated by an innovated
rad, USA) pre-equilibrated with 0.5 M acetic acid so- method of Smirnoff and Cumbes (1989) in sodium
lutions. The column was eluted with a linear gradient phosphate buffer (150 mM, pH 7.4) containing 0.15
of acetic acid (0.5–2.0 M) at a rate of 0.6 mL min−1 . mM FeSO4 -EDTA, 2 mM sodium salicylate, 6 mM
The amount of total sugar in the column fractions H2 O2 , and varying concentrations of oligosaccharides
was determined by the method of phenol-sulfuric acid (25 mg L−1 , 50 mg L−1 and 100 mg L−1 ). In the
(Dubois et al., 1965). Optical density was read at 490 essential control, sodium phosphate buffer replaced
nm and the values for total sugar were expressed as D- H2 O2 . The solution was incubated at 37 ◦ C for 1 h,
galactose equivalents. The separated oligosaccharide and was detected by monitoring the absorbance at
peaks were collected and lyophilized. The oligosac- 510 nm.
charide samples were stored under −18 ◦ C before The antioxidant activities of the samples were eval-
determination. uated according to the inhibition percentage of free rad-
icals production: Inhibition rate (%) = (A0 − A)/A0 ×
100%, where A0 is the absorbance of control group in
MALDI-TOF-MS of agar oligosaccharides the free radicals generation system, A is the absorbance
of the test group.
The molecular mass distribution of the agar
oligosaccharides was determined by MALDI-TOF-MS
(Matrix-assisted laser desorption ionization time-of- Results
flight mass spectrometry) using LDI-1700 instrument
(Linear Scientific, Inc., USA). The instrument was fit- Strain properties
ted with a pulsed nitrogen laser at 337 nm with 3ns pulse
duration. 2,5-Dihydroxybenzoic acid (DHB) was used Strain QJH-12 was gram negative, polar flagellated.
as the matrix. Electron micrograph of the strain showed an arced
cell, 0.5 × 1.2–1.5µ m (Figure 1). The strain was ox-
Antioxidative activities of agar oligosaccharides idase positive, catalase positive and O/129 test posi-
tive. The preliminary identification result showed that
The system of the peroxidation of polyunsaturated the morphologic and physiological characteristics of
fatty acid from yolk lipoprotein induced by iron was this strain were in accordance with Vibrio, according
constructed according to the method of Zhang et al. to Bergey’s Manual of Systematic Bacteriology (Holt
(1996). The lipid peroxidation reaction system con- et al., 1994). The isolated strains produced soft pits
tained 0.2 mL 1/25–1/32 diluted yolk suspension, 0.1 on the agar surface with clear haloes around the bac-
mL varying concentrations of oligosaccharides (25 teria colonies, which were obvious marks indicating
mg L−1 , 50 mg L−1 and 100 mg L−1 ), 0.2 mL 25 mM
FeSO4 ·7H2 O and 1.5 mL pH 7.45 PBS. In the con-
trol group, 0.1 mL PBS replaced the oligosaccharide.
All tubes were treated in 37 ◦ C water bath for 15 min.
Determination of lipid peroxidation involved measur-
ing the amount of MDA by the method of thiobarbi-
turic acid reaction according to Stewart and Bewley
(1980).
To assay superoxide anion radical scavenging
properties, the method of pyrogallol autooxidation
(Zou et al., 1986) was used. The reaction sys-
tem contained 2.4 mL pH 8.2 Tris-HCl buffer, 0.1
mL varying concentrations of agar oligosaccharides
(25 mg L−1 , 50 mg L−1 and 100 mg L−1 ) and 0.3
mL 7 mM pyrogallol solution. After reaction for 4
min, one drop of 10 M HCl was added to terminate
the reaction. Then the optical density at 325 nm was
measured. Figure 1. Electron micrograph of strain QJH-12.
336

the high ability of degrading agar. When the agar

was the sole carbon source, the maximum agarase
activity of fermentation medium of QJH-12 reached
310 U mL−1 after cultivation in conventional batch
culture for 24 h at 30 ◦ C. However, the agarase was
not detected in the medium without agar. Two percent
NaCl concentration was helpful on the production of
agarase. Patent application on this strain is ongoing
now.

Enzyme properties

The effect of pH on agarase activity was determined

in K2 HPO4 –NaOH buffer at pH 6–8. The effect of Figure 2. Elution profile of agar oligosaccharids F1 and F2 on AM
GP-1 anion exchange chromatography. The colunm was eluted with
temperature on the agarase activity was measured over
a linear gradient of acetic acid (0.5–2.0 M) at a rate of 0.6 mL min−1 .
the range of 25–40 ◦ C. The optimum pH was 7.0 and
optimum temperature was 35 ◦ C.
Since the strain QJH-12 came from the ocean,
high NaCl concentration was necessary for its growth agar oligosaccharide containing the sulfate group with
and enzyme production. When enzyme activity was the polymerization degree 16 was 2546 Da. When
measured in the presence of salt, a significant el- the polymerization degree was over 10, the oligosac-
evation could be observed. The enzyme activity charides with even polymerization degree were
reached the maximum in the presence of 2% NaCl, dominant.
and then declined when the NaCl concentration was
increased.
Antioxidative activities of agar oligosaccharides

MALDI-TOF-MS of the agaroligosaccharides In the experiment, the antioxidant activities of

enzymatic-depolymerized agar oligosaccharides (MM
After fractionation by ethanol precipitation, two frac- < 3800 Da) were analyzed. Agar oligosaccharides
tions, F1 and F2, were produced. The fractions were showed antioxidative activities in inhibiting lipid per-
applied to anion-exchange chromatography column oxidation, scavenging superoxide anion and hydroxyl
for further purification and the result was shown in free radical.
Figure 2. One significant peak was showed in F1 curve In the test of inhibiting lipid peroxidation, the
and marked as AOS-1. Two main peaks, AOS-2 and three samples, AOS-1, AOS-2 and AOS-3, exhib-
AOS-3, were separated from sample F2, which were ited a similar effect and had no obvious effect on
showed in F2 curve. inhibiting lipid peroxidation (Figure 4). Figure 5
The MALDI-TOF mass spectra of the three sam- showed the results of superoxide anion radical scav-
ples were showed in Figure 3. According to the mass enging activity of agar oligosaccharides. No evident
spectra, AOS-1 was an oligosaccharides mixture with effect could be found in the test of AOS-1. How-
polymerization degree from 3 to 14. No sulfate group ever, AOS-2 and AOS-3 showed effective antioxida-
was linked in the sugar ring according to the molecular tive activity, which illustrated the positive relation to
mass. The main composition was trisaccharide (MM- the treating dose. Figure 6 showed the results of hy-
486 Da), pentasaccharide (MM-792 Da), hexasaccha- droxyl radical scavenging activity of agar oligosac-
ride (MM-936 Da) and octosaccharide (MM-1242 charides. All three samples showed a dose-response
Da). AOS-2 was mainly composed of oligosaccha- relation and presented the highest antioxidative ac-
rides with polymerization degree from 6 to 22 without tivity at the highest test dose. AOS-3 exhibited the
the sulfate group. AOS-3 was composed of oligosac- highest hydroxyl radical scavenging activity in the
charides with even polymerization degree from 16 experimental samples, with the inhibition percentage
to 24, with one sulfate group on each oligosaccha- of 27.5, 51.4 and 76.1% at 25, 50 and 100 mg L−1 ,
ride molecule. For example, the molecular mass of respectively.
 337

Figure 3. MALDI-TOF-MS spectra of agar oligosaccharides AOS-1, AOS-2 and AOS-3.

338

Discussion

Among various naturally occurring substances,

polysaccharides extracted from marine algae were
proved to be useful candidates in the search for effec-
tive, nontoxic substances with free radicals scaveng-
ing activities. Polysaccahrides from Fucus vesiculosus
(Ruperez et al., 2002), Laminaria japonica (Xue et al.,
2001) and Porphyra haitanesis all exhibited antiox-
idative activities. However, the study on the oligosac-
charides, especially the oligosaccharides from the red
algae was comparatively by deficient.
In the experiment, agar oligosaccharides prepared
by agarase showed antioxidative activities in inhibiting
Figure 4. Inhibitory effects of agar oligosaccharide fractions on lipid lipid peroxidation, scavenging superoxide anion and
peroxidation. hydroxyl free radical. Higher activity could be found
in the test of scavenging hydroxyl radical than that
of scavenging superoxide anion and inhibiting lipid
peroxidation. AOS-3 exhibited the highest inhibition
percentage of 76.1% at 100 mg L−1 . By now, several
explanations of in vitro antioxidation mechanism of
polysaccharide have been given (Zhou et al., 2002). The
key role of polysaccharide in scavenging hydroxyl rad-
ical is to participate in the oxidation so as to scavenge
directly the reactive oxygen species. The chelation be-
tween polysaccharide and metal ions that are necessary
for the production of reactive oxygen species is also an
important factor in its antioxidation effects. The reac-
tion mechanism of agar oligosaccharide will be studied
further by the method of dynamic calculation.
The fragments with different molecular mass distri-
bution and different substitute degree of sulfate group
Figure 5. Inhibitory effects of agar oligosaccharide fractions on showed different antioxidative activities. This indi-
superoxide radicals.
cated that the structure of oligosaccharide plays an
important role in antioxidation. Higher antioxidative
activities were found when the molecular mass in-
creased. The agar oligosaccharide with high molecular
mass (MM 2000–3800 Da) had higher antioxidation ac-
tivity than that with low molecular mass (MM < 2000
Da). Xue et al. (2001) reported that low-molecular-
mass polysaccharide from Laminaria japonica had
higher antioxidative activity than did high-molecular-
mass polysaccharide. Chondroitin sulfate fraction with
a molecular mass of 14,430 had the capacity to inhibit
the lipid oxidation, while the fraction with a molec-
ular mass of 8570 did not have this activity (Volpi &
Tarugi, 1999). The data showed that the molecular mass
of oligosaccharide is necessary to the formation of ef-
fective structure in antioxidative activity.
Figure 6. Inhibitory effects of agar oligosaccharide fractions on The antioxidation tests (Figures 4, 5 and 6)
hydroxyl free radicals. showed that higher activity occurred in the agar
 339

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