Structural characterization and antioxidant activity of an extracellular polysaccharide isolated from Brevibacterium otitidis BTS 44

Food Chemistry 123 (2010) 315–320

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Structural characterization and antioxidant activity of an extracellular polysaccharide isolated from Brevibacterium otitidis BTS 44 Mohsen Mohamed Selim Asker a,*, Bahaa Talaat Shawky b a b

Department of Microbial Biotechnology, National Research Center, Dokki, Cairo, Egypt Department of Microbial Chemistry, National Research Center, Dokki, Cairo, Egypt

a r t i c l e

i n f o

Article history: Received 8 September 2009 Received in revised form 15 February 2010 Accepted 20 April 2010

Keywords: Brevibacterium otitidis BTS 44 Polysaccharide Structural Radical-scavenging activity

a b s t r a c t A water-soluble acidic extracellular polysaccharide reaching a maximum concentration of 23.4 g/l growth medium, coded as BSMA, was isolated from the non-pathogenic soil bacteria Brevibacterium otitidis BTS44, by precipitating with two volumes of ethanol. BSMA consisted of arabinose, mannose, glucose and mannouronic acid in ratios of 2.7:3.6:2.1:1.0. No protein was detected in the BSMA fraction, and its molecular weight was about 127 kDa. It has a backbone composed of (1 ? 5)-linked arabinose, (1 ? 6)-linked mannose with three branches attached to O-3 of (1 ? 6)-linked mannose and terminated with either mannose, or mannose and glucose; all the glucose and most of the mannouronic acid are distributed in branches. Partial acid hydrolysis of BSMA gave four sub-fractions termed BSMA-1, BSMA-2, BSMA-3 and BSMA-4. BSMA-1 was composed of arabinose, mannose and trace amounts of mannouronic acid; BSMA-2 was only composed of arabinose and mannose; BSMA-3 was composed of mannose, mannouronic acid and glucose, and BSMA-4 was only composed of mannose and glucose. In the in vitro antioxidant assay, BSMA was found to possess DPPH radical-scavenging activity, with an IC50 value of 120 lg/ml. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Oxidation is essential to many microorganisms for the production of energy to fuel biological processes. However, oxidative stress as well as the uncontrolled production of oxygen-derived free radicals induced cell damage, which triggers both the physiological process of ageing (Harman, 1993) and pathological progressions, which are involved in the onset of many diseases (Mau, Lin, & Song, 2002; Raouf, Patrice, Andre, Jean-Michel, & Yvan, 2000; Uchida, 2000). Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), superoxide anion radical (O2 ), and hydroxyl radical (OH) are inevitably generated during normal and/or aberrant consumption of molecular oxygen. These free radicals are able to attack numerous biological substances, including DNA, and exert some detrimental effects, which may even result in cell death (Dean, Gieseg, & Davies, 1993). Living cells protect themselves from oxidative damage through several defence mechanisms, such as the enzymatic conversion of ROS into less toxic substances (Cotgreave, Molde´us, & Orrenius, 1988) and through detoxification by reaction with antioxidants (Aruoma, 1996). Antioxidants that have an important role in the prevention of these diseases must be obtained. However, the synthetic antioxidants most commonly used in industrial processing are suspected

* Corresponding author. Tel.: +20 2 33335982; fax: +20 2 33370931. E-mail address: [email protected] (M.M.S. Asker). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.04.037

to have cytotoxicity (Qi et al., 2005; Valentao et al., 2002). Thus, it is essential to develop effective, non-toxic, and natural antioxidants that can protect the human body from free radicals and retard the progress of many chronic diseases (Kinsella, Frankel, German, & Kanner, 1993; Nandita & Rajini, 2004). In recent years, increasing evidence highlights that some polysaccharides isolated from plants have antioxidant activities (Hu, Xu, & Hu, 2003; Jiang, Jiang, Wang, & Hu, 2005; Liu, Ooi, & Chang, 1997). Among various naturally substances, polysaccharides from some microorganisms may harbour antioxidant activity. Polysaccharides represent a class of high-value polymers with many industrial applications in food, cosmetic, textile and pharmaceutical industries, due to their rheological properties. Because of these properties, they have been used as emulsifiers, stabilizers, and texture enhances in food industry. Traditionally, these important polysaccharides have been obtained from plant or algal sources. However, in the last 50 years, polysaccharides from microorganisms have received increased attention. The genus Brevibacterium was proposed by Breed (1953) for some gram-positive, non-spore-forming, non-branching rods. A number of species with diverse morphological, physiological and biochemical properties were subsequently included in the genus. B. otitidis was recognized for the first time by Pascual, Collns, Funke, and Pitcher (1996). Antioxidant activity of B. otitidis exopolysaccharide has not been described yet. In this study, we report on the isolation, purification, chemical characterization and the antioxidant potential of the main polysaccharide (BSMA) from B. otitidis BTS 44.

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2. Materials and methods 2.1. Bacterial strain A mucous bacterial colony was isolated from calcareous soil sample obtained from Ras El-Hekma, Egypt, and was identified as B. otitidis BTS 44. The bacteria was grown and maintained on agar-modified Ashby’s medium consisting of (g/l) sucrose, 10; mannitol, 10; K2HPO4, 0.5; MgSO47H2O, 0.2; NaCl, 0.2; CaSO42H2O, 0.5; MnSO4H2O, 0.0002; FeCl3, 0.0002; NaMoO42H2O, 0.0002 and agar, 15 (Abd El-Malek & Ishac, 1968), fortified with 0.2 g/l yeast extract. The bacteria was cultured at 30 °C for 3 days and stored at 4 °C for one month. 2.2. Production, isolation and purification of polysaccharide B. otitidis BTS 44 was grown in modified liquid Ashby’s medium, fortified with 0.2 g/l yeast extract. This medium (150 ml) was distributed in 250-ml Erlenmeyer flasks and sterilized at 121 °C for 20 min. The flasks were inoculated aseptically by a standardized volume (1 ml) of B. otitidis suspension, to give an optical density of 0.2 at 550 nm. Incubation was carried out at 30 °C for 5 days on a 100 rpm rotary shaker. The culture broth was diluted with water and centrifuged at 3500  g for 30 min (Sigma-Laborzentrifugen, 2K 215, Sigma Co., D-37520 Osterode-am-Harz, Germany) to remove bacterial cells. Trichloroacetic acid (5 g/100 ml) was added to the culture broth, which was left overnight at 4 °C and centrifuged at 3500  g for 20 min. The pH of the clear solution was adjusted to 7.0 with 0.1 M NaOH and dialyzed three times (1000 ml). The supernatant was concentrated under reduced pressure to 200 ml, and precipitated with 4 volumes of ethanol at 4 °C overnight. The crude polysaccharide was recovered by centrifugation (3500  g, 20 min), and dried at 45 °C under reduced pressure after washing successively with ethanol and ether. The polysaccharide was dissolved in 100 ml distilled water, vortexed and centrifuged at 3500  g for 20 min to remove insoluble materials. Crude polysaccharide was precipitated with 1, 2, 3, 4 volumes ethanol, and supernatant was recovered by centrifugation. Polysaccharide fraction named BSM was obtained from the supernatant mentioned above by precipitating with 2 volumes ethanol. BSM was further purified by gel permeation chromatography (GPC) on a Sephadex G-150 column (2.6 cm  70 cm) eluted with 0.1 M NaCl at a flow rate of 0.5 ml/min. An aliquot of the fractions (5 ml) was tested for total carbohydrate by the phenol–H2SO4 method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956), and the main polysaccharide fraction (BSMA) was collected, dialyzed and lyophilized. BSMA was used for structural analysis and antioxidant activity. 2.3. Molecular weight determination The molecular weight of the BSMA polysaccharide was determined by gel permeation chromatography (GPC) on a Sephadex G-150 column (2.6 cm  70 cm). Standard dextrans (40, 500 and 2000 kDa, Fluka Chemical Co., Buchs, Switzerland) and glucose were used, and the elution volumes were plotted against the logarithm of their respective molecular weights. The elution volume of the purified polysaccharide was plotted in the same graph, and the molecular weight was determined (Luo, 2008).

Tharanathan, & Prasad, 2004). The monosaccharides contents were quantified by HPLC on a Shimadzu Shim-Pack SCR-101N column (7.9 mm  30 cm), using deionized water as the mobile phase (flow rate 0.5 ml/min), as described by El-Sayed, Ismail, Ahmed, Abd El-Samei, and Asker (2005). Uronic acid content was determined according to m-hydroxydiphenyl colorimetric method, by using glucouronic acid as standard (Filisetti-Cozzi & Corpita, 1991). 2.5. Infra-red spectroscopy Infra-red spectrum BSMA fraction was obtained by grinding a mixture of sample with dry KBr and pressing in a mould. An IR spectrum was recorded on a Fourier-transform infra-red spectrophotometer (Brucker Scientific 500-IR) (Ray, 2006). 2.6. Periodate oxidation BSMA (50 mg) was dissolved in 12.5 ml of distilled water and 12.5 ml of 30 mM NaIO4 were added. The solution was kept in the dark at room temperature; 0.1-ml aliquots were withdrawn at 24-h intervals, diluted to 25 ml with distilled water and read using a spectrophotometer at 223 nm (Linker, Evans, & Impollomeni, 2001). Consumption of periodate was measured as described by Aspinall and Ferrier (1957), and formic acid production was determined by titration with 5 mM NaOH. Ethylene glycol (2 ml) was added, to terminate the experiment. The solution of periodate product was extensively dialyzed against tap water and distilled water for 48 h. The solution was concentrated using a rotary evaporator at 50 °C and reduced with NaBH4 (100 mg); the mixture was left for 24 h at room temperature, neutralized to pH 6.0 with acetic acid (50% v:v), dialyzed as described above, and concentrated to 10 ml. One-third of the solution mentioned above was freeze dried and fully hydrolyzed for HPLC on a Shimadzu Shim-Pack SCR-101N column (7.9 mm  30 cm), using deionized water as the mobile phase at a flow rate of 0.5 ml/min (El-Sayed et al., 2005); two-thirds were added to the same volume of 1 M sulfuric acid, kept for 40 h at 25 °C, neutralized to pH 6.0 with BaSO4, and filtered for analysis by Smith degradation. The filtrate was dialyzed (molecular weight cut off of 3 kDa); the contents outside of the dialysis bag were analyzed by HPLC as described above, whereas the contents inside the dialysis bag were mixed with 4 volumes of ethanol and centrifuged. The supernatant and precipitate were also analyzed by HPLC (Zhang, 1987). 2.7. Partial acid hydrolysis BSMA (100 mg) was hydrolyzed with 0.05 M TFA (3 ml) for 16 h at 80 °C, the hydrolysate was mixed with 4 volumes of ethanol and kept at 4 °C overnight. The precipitate was removed by centrifugation at 3500  g for 20 min (BSMA-1), and the supernatant was dialyzed against distilled water for 48 h in a dialysis bag (molecular weight 3 kDa cut off). Each solution inside and outside of the dialysis bag was collected for further analysis. Ethanol was added to the solution in the bag after dialysis, and the precipitate and supernatant, designated as BSMA-2 and BSMA-3, respectively, were recovered after centrifugation at 3500  g for 20 min (Tong, Liang, & Wang, 2008). The fraction outside of the dialysis bag (BSMA-4) and all other fractions were analyzed by HPLC, to determine monosaccharides.

2.4. Analysis of monosaccharide composition 2.8. Methylation analysis The polysaccharide BSMA (30 mg) was hydrolyzed with 2 M trifluoroacetic acid (TFA) at 100 °C in a sealed tube for 8 h. Excess acid was removed by flash evaporation on a water bath at a temperature of 40 °C and co-distilled with water (1 ml  3) (Sudhamani,

Prior to methylation, BSMA was reduced to the corresponding neutral sugars (York, Darvill, O’Neill, Stevenson, & Albersheim, 1985). BSMA and reduced BSMA were methylated separately using

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2.9. Radical-scavenging activity (RSA) of BSMA fraction toward DPPH radical The free radical-scavenging activity of BSMA was measured by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals using the method of Shimada, Fujikawa, Yahara, and Nakamura (1992). Five millilitres of DPPH ethanol solution (freshly prepared at a concentration of 0.1 mM) were added to 1 ml of BSMA solution of different concentrations (40–240 lg) in water. After 30 min, absorbance was measured at 517 nm using a UV–visible spectrophotometer (2401PC; Shimadzu, Kyoto, Japan). Lower absorbance of the reaction mixture indicated higher free radical-scavenging activity, which was analyzed from the graph (inhibition percentage plotted against concentration of compound). Ascorbic acid was used as a positive control. The experiment was carried out in triplicate and averaged. The ability to scavenge the DPPH radical was calculated using the following equation:

Scavenging ability ð%Þ ¼



DA517 of control  DA517 =DA517 of control   100:

of sample




The EC50 value is the effective concentration of BSMA at which the DPPH radicals were scavenged by 50%. 2.10. Statistical analysis The obtained data were subjected to one-way ANOVA and the differences between means were measured at the 5% probability level using Duncan’s new multiple range tests. SPSS Version 10 (SPSS, Chicago, IL) was used as described by Dytham (1999).

0.8

BSMA

O.D at 490 nm

the method of Ciucanu and Kerek (1984). The methylated products were extracted with CHCl3, washed with distilled water three times and evaporated to dryness. The product was then hydrolyzed with TFA (2 M) at 100 °C for 6 h. The methylated products were converted into their corresponding alditols by reduction with NaBH4 and acetylation (Guilherme, Marcello, & Philip, 2005). The resulting product was subjected to linkage analysis by GC–MS on a DB-5 capillary column (30 m  0.25 mm) with a film thickness of 0.25 lm (He et al., 2007). The GC temperature was isothermal at 140 °C for 2 min, followed by a 4 °C/min gradient up to 280 °C. The components were identified by a combination of the main fragments in their mass spectra and relative GC retention times; the molar ratios for each sugar were calibrated using the peak areas and response factors.

0.6

0.4

0.2

0 5

25

45

65

85

105

125

Tube number Fig. 1. The gel permeation chromatography of polysaccharide fraction (BSMA) on Sephadex G-150.

was a homogeneous polysaccharide. BSMA contained 14% uronic acid as evaluated by m-hydroxydiphenyl colorimetric method and HPLC analysis. Analysis by HPLC indicated that BSMA was composed of arabinose, mannose, glucose, and mannouronic acid, with a relative molar ratio of 2.7:3.6:2.1:1.0. 3.2. Structural characterization of BSMA The infra-red spectrum of BSMA (Fig. 2) revealed a typical major broad stretching peak around 3423 cm1 for the hydroxyl group, and a weak band at 2925 cm1 showing the C–H stretching vibration. The absorbance at 1739.5 cm1 indicated the presence of uronic acid. The broad band at 1618 cm1 was due to the bound water. The band at 841 cm1 was ascribed to a-pyranoses in the polysaccharide (Bao, Fang, & Fang, 2001; Park, 1971). By partial acid hydrolysis BSMA was fractionated, giving four sub-fractions termed BSMA-1, BSMA-2, BSMA-3 and BSMA-4. The monosaccharide components of them are shown in Table 1. It is evident that BSMA-1 was composed of arabinose, mannose and trace of mannouronic acid; BSMA-2 was only composed of the first two monosaccharides and may be the backbone structure of BSMA; BSMA-3 was composed of mannose, mannouronic acid and glucose, and BSMA-4 was only composed of mannose, glucose and may be the branched structure of BSMA. The polysaccharide of BSMA showed abundant periodate uptake, while it was oxidized. The consumption of periodate (0.89 mol) was four times higher than the amount of formic acid (0.238 mol), that was produced after periodate treatment, indicating the existing of little amounts

3. Results and discussion 3.1. Isolation, purification and composition of BSMA Polysaccharide production reached a maximum of 23.4 g of crude product per litre of growth medium after 5 days and the relationship between product formation and cell growth of B. otitidis BTS 44 was 3.8 g exopolysaccharide per gram cell dry weight. The main fraction (BSMA) was purified on Sephadex G-150 at a yield of 63% after fractionation with ethanol precipitation from the crude exopolysaccharide. BSMA was collected for further analysis of structure and activity. It appeared as a white powder, with a negative response to the ninhydrin test. The fact that no absorption was detected by the UV spectra at both 260 and 280 nm indicated the absence of nucleic acids and protein. The average molecular weight of BSMA was determined as 127 kDa by gel permeation chromatography (GPC). The GPC profile (Fig. 1) also demonstrated that BSMA gave a single, symmetrical peak, revealing that BSMA

Fig. 2. Infra-red spectrum of polysaccharide fraction (BSMA) from B. otitidis BTS 44.

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Table 1 HPLC analysis results of fractions from partial acid hydrolysis. Fractions

BSMA-1 BSMA-2 BSMA-3 BSMA-4

Molar ratios Arabinose

Mannose

Glucose

Mannouronic acid

1.00 1.92 0.00 0.00

1.00 1.00 1.00 1.00

0.00 0.00 1.52 0.95

0.1 0.00 1.21 0.00

of monosaccharides, which are (1 ? 4)-linked or (1 ? 6)-linked. The fact that the amount of periodate consumption was more than the amount of formic acid produced demonstrated that there were other linkages oxidized by periodate, such as (1 ? 4) or (1 ? 2). The periodate-oxidized products were fully hydrolyzed and analyzed by HPLC (Table 2).

Table 2 HPLC results of Smith degradation of polysaccharide fraction BSMA. Fractions

a b c d e f g

Molar ratios Glya

Eryb

Ery Ac

Mand

Arae

Man Af

Glcg

Full acid hydrolysis

4.0

2.6

0.0

1.0

0.0

0.7

0.0

Smith degradation Outside of bag Supernatant in bag Precipitate in bag

3.7 0.0 0.0

2.9 0.0 0.0

0.0 0.0 0.0

1.0 0.0 0.0

0.0 0.0 0.0

0.6 0.0 0.0

0.0 0.0 0.0

Glycerol. Erythritol. Erythric acid. Mannose. Arabinose. Mannouronic acid. Glucose.

Table 3 GC–MS results of methylation analysis of BSMA and reduced BSMA. Methylation product

2,3,4,6-Tetra-O-methyl-glucose 2,3-Di-O-Methyl-arabinose 2,3,4,6-Tetra-O-methylmannose 2,4,6-Tri-O-methyl-mannose 2,3,6-Tri-O-methyl-glucose 2,3,6-Tri-O-methyl-mannose 2,3,4-Tri-O-methyl-mannose 2,3-Di-O-methyl-glucose 2,4-Di-O-methyl-mannose

Type of linkage

Molar ratios BSMA

Reduced BSMA

(1 ? )Glc (1 ? 5) Ara (1 ? ) Man

1.2 4.0 1.2

1.1 4.0 1.0

(1 ? 3) Man (1 ? 4) Glc (1 ? 4) Man (1 ? 6) Man (1 ? 4,6) Glc (1 ? 3,6) Man

0.6 0.8 0.7 1.5 1.0 1.4

1.7 0.8 0.7 1.6 1.0 1.3

The presence of mannose and mannouronic acid revealed some residues of mannose and mannouronic acid were (1 ? 3)-linked, (1 ? 2,3)-linked, (1 ? 2,4)-linked, (1 ? 3,4)-linked, (1 ? 3,6)linked or (1 ? 2,3,4)-linked that cannot be oxidized. No rhamnose and glucose were observed and large amounts of glycerol and erythritol were obtained, demonstrating that rhamnose and glucose were all linkages which can be oxidized by periodate. HPLC analysis for Smith degradation (Table 2) indicated that there was no precipitation in the dialysis bag and this demonstrated that the backbone of BSMA should be oxidized completely by HIO4. Hence, it may be concluded that the linkages of backbone are (1 ? ), (1 ? 2), (1 ? 6), (1 ? 2,6), (1 ? 4) and (1 ? 4,6) that may be oxidized, producing glycerol and erythritol detected out of dialysis bag. The fact that no erythric acid was detected suggested there were (1 ? 3) and (1 ? 2,3) linkages in mannouronic acid (Abd-El-Akher, Hamilton, Montgomeny, & Smith, 1952; Danishefeky, Whistler, & Bettelheim, 1970; Inoue, Korenaga, & Kadoya, 1982; Wang, Luo, & Liang, 2004). The fully methylated BSMA and reduced BSMA were hydrolyzed with acid, converted into alditol acetates, and analyzed by GC–MS. As summarized in Table 3 BSMA and reduced BSMA showed the presence of nine derivatives, namely 2,3,4,6-tetra-O-methyl-glucose; 2,3-di-O-methyl-arabinose; 2,3,4,6-tetra-O-methyl-mannose; 2,4,6-tri-O-methyl-mannose; 2,3,6-tri-O-methyl-glucose; 2,3,6-tri-O-methyl-mannose; 2,3,4-tri-O-methyl-mannose; 2,3-diO-methyl-glucose and 2,4-di-O-methyl-mannose, in molar ratios of 1.2:4.0:1.2:0.6:0.8:0.7:1.5:1.0:1.4 and 1.1:4.0:1.0:1.7:0.8:0.7: 0.6:1.0:1.3, respectively. According to the difference in molar ratio of 2,3,4,6-tetra-O-methyl-mannose, 2,4,6-tri-O-methyl-mannose, 2,3,6-tri-O-methyl-mannose, 2,3,4-tri-O-methyl-mannose and 2,4-di-O-methyl-mannose in BSMA and reduced BSMA (Table 3), it can be deduced the linkage of mannouronic acid is a (1 ? 3)linkage (De S-F-Tischer et al., 2006; Ray, 2006; Sudhamani et al., 2004; Urai et al., 2006). The results from GC–MS analysis, which were consistent with the results from partial acid hydrolysis, periodate oxidation and Smith degradation, indicated that 2,3-di-O-methyl-rhamnose (1 ? 5)-linked arabinose and 2,3,4-tri-O-methyl-mannose (1 ? 6)-linked mannose were major components of the backbone structure, with 3 branches attached to O-3 of (1 ? 3)-linked mannose; all glucose and the majority of mannouronic acid were distributed in branches, and residues of branches terminated with either mannose, or mannose and glucose were composed of (1 ? 3)-linked mannouronic acid (1 ? 4)-linked mannose, and (1 ? 4)-linked glucose or (1 ? 4,6)-linked glucose (Wang et al., 2004). In addition, small amount of (1 ? 3)-linked mannose and trace of (1 ? 3)-linked mannouronic acid was found in one of the branches and backbone, respectively (Fig. 3). In short, the monomer of BSMA was evaluated as below according to analysis of GC–MS, partial acid hydrolysis, periodate oxidation and Smith degradation.

−4)-Glc-(1−3)-Man A-(1−4)-Man-(1 Glc-(1− 6) Man-(1 3) −5)-Ara-(1−6)-Man-(1−5)-Ara-(1−6)-Man-(1−5)-Ara-(1−6) - Man-(1−5)-Ara-(1−6)-Man-(1−5)-Ara-(1−6) - Man-(1−5)-Ara-(1−6)-Man-(1−5) - Ara-(1− 3) 3)

(1 Man-(1−3)-Man A-(1−4)-Glc-(1−3)-Man

G lc-(1−4)-Glc-(1−3)-Man A-(1−4)-Man-(1 6)

Man-(1 Fig. 3. The structure of BSMA isolated from Brevibacterium otitidis.

% Remaining DPPH radicals

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319

saccharide BSMA possesses strong scavenging effect, which may be comparable to vitamin C and so it should be explored as a novel potential antioxidant. Further studies are necessary to relate such activities with the pharmacological effect of B. otitidis BTS44 exopolysaccharide.

100 80 60

References 40 20 0 0

40

80

120

160

200

240

Amount (ug/mL) Fig. 4. Scavenging effect of polysaccharide fraction BSMA (–j–), vitamin C (––) during DPPH test by changes in absorbance at 517 nm. Each value is expressed as mean ± standard deviation (n = 3).

3.3. Radical-scavenging activity (RSA) of BSMA fraction The model of scavenging the stable DPPH radical is a widely used method to evaluate the free radical-scavenging ability of natural compounds (Lee, Hwang, Ha, Jeong, & Kim, 2003; Nagai, Inoue, Inoue, & Suzuki, 2003). In the DPPH test, the antioxidants were able to reduce the stable DPPH radical to the yellow-coloured diphenylpicryl hydrazine. The effect of antioxidants on DPPH radical-scavenging was conceived to be due to their hydrogen-donating ability. The DPPH radical-scavenging activities of BSMA and vitamin C used as a positive control were determined (Shimada et al., 1992), and the results are plotted in Fig. 4. As illustrated, BSMA exhibited scavenging activity towards DPPH radicals in a concentration-dependent manner, with an IC50 value of 120 lg/ ml. Under the same conditions, vitamin C, a free radical scavenger, showed a slightly weaker effect on the hydroxyl radicals, with an IC50 value of 160 lg/ml. The scavenging effect of the purified polysaccharide BSMA and standard on the DPPH radical increased dosedependently and were 91.5% and 80.4% at a dose of 240 lg/ml, respectively. Both of them possessed similar trends in antioxidant activity. These results indicated that BSMA had a noticeable effect on the scavenging free radical, especially at high concentration. It is well-known that reactive oxygen species (ROS), such as hydroxyl radicals, superoxide anion and hydrogen peroxide, are related to the pathogenesis of various diseases (Abe & Berk, 1998; Busciglio & Yankner, 1995). Hydroxyl radical is the most reactive among the oxygen radicals and induces severe damage on the adjacent bio-molecules (Chance, Sies, & Boveris, 1979). It has been found that the exopolysaccharide isolated and purified from B. otitidis BTS 44 may prove to be a useful candidate in the search for natural, effective, non-toxic substances with antioxidant activity. A similar phenomenon was observed by Zhou, Chen, Ouyang, Liu, and Pang (2000), and Chen, Zhang, Qu, and Xie (2008), who demonstrated that the polysaccharides are not only energy resources but play key biological roles in many life processes as well. Further elucidation of possible mechanisms and evaluation of the bioactivities of the polysaccharides from B. otitidis shaken culture (BOSC) will be important for their application in the food and medicinal fields.

4. Conclusion The results obtained in the present study clearly demonstrate that one water-soluble acidic exopolysaccharide coded as BSMA isolated from B. otitidis BTS 44 contained predominantly four monosaccharides: arabinose, mannose, glucose and mannouronic acid. Antioxidation test in vitro showed that the natural exopoly-

Abd El-Malek, Y., & Ishac, Y. Z. (1968). Evaluation of methods used in counting Azotobacter. Journal Applied Bacteriology, 31, 267–275. Abd-El-Akher, M., Hamilton, J. K., Montgomeny, R., & Smith, F. (1952). A new procedure for the determination of the fine structure of the polysaccharides. Journal of the American Chemistry Society, 74, 4970. Abe, J., & Berk, B. C. (1998). Reactive oxygen species as mediators of signal transduction in cardiovascular diseases. Trends in Cardiovascular Medicine, 8, 59–64. Aruoma, O. I. (1996). Characterization of drugs as antioxidant prophylactics. Free Radical Biology & Medicine, 20, 675–705. Aspinall, G. O., & Ferrier, R. J. (1957). A spectrophotometer method for the determination of periodate consumed during the oxidation of carbohydrates. Chemistry and Industry (London), 7, 1216–1221. Bao, J., Fang, X., & Fang, J. (2001). Chemical modification of the (1–3)-a-glucan from spores of Ganoderma lucidum and investigation of their physicochemical properties and immunological activity. Carbohydrate Research, 336, 127–140. Breed, R. S. (1953). The Brevibacterium from nov. of order Eubacterials, Riassunti delle comnuicazione VI. Congresso Internaziole di Microbilogia, Roma, 1, 13–14. Busciglio, J., & Yankner, B. A. (1995). Apoptosis and increased generation of reactive oxygen species in Down’s syndrome neurons. Nature, 378, 776–779. Chance, B., Sies, H., & Boveris, A. (1979). Hydroperoxide metabolism in mammalian organs. Physiological Reviews, 59, 527–605. Chen, H., Zhang, M., Qu, Z., & Xie, B. (2008). Antioxidant activities of different fractions of polysaccharide conjugates from green tea (Camellia Sinensis). Food Chemistry, 106, 559–563. Ciucanu, I., & Kerek, F. (1984). A simple and rapid method for the per-methylation of carbohydrates. Carbohydrate Research, 131, 209–217. Cotgreave, I., Molde´us, P., & Orrenius, S. (1988). Host biochemical defense mechanisms against prooxidants. Annual Review of Pharmacology and Toxicology, 28, 189–212. Danishefeky, J., Whistler, L. R., & Bettelheim, F. A. (1970). Introduction to polysaccharide chemistry. In P. Waid, H. Dprek, & H. Anthory (Eds.). Carbohydrate Chemistry (Vol. IIA, pp. 375–411). New York and London: Academic Press. De S-F-Tischer, C. P., Talarico, B. L., Noseda, D. M., Guimaraes, B. P. M. S., Damonte, B. E., & Duarte, R. E. M. (2006). Chemical structure and antiviral activity of carrageenans from meristiella gelidium against herpes simplex and dengue virus. Carbohydrate Polymers, 63, 459–465. Dean, R. T., Gieseg, R., & Davies, M. J. (1993). Reactive species and their accumulation on radical damaged proteins. Trends in Biochemical Science, 18, 437–441. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28, 350–356. Dytham, C. (1999). Choosing and using statistics: A biologist’s guide. London UK: Blackwell Science Ltd. El-Sayed, O. H., Ismail, S. A., Ahmed, Y. M., Abd El-Samei, M., & Asker, M. M. S. (2005). Studies on the production of sulfated polysaccharide by locally isolated bacteria. Egyptian Pharmaceutical Journal, 4, 439–452. Filisetti-Cozzi, T. M. C. C., & Corpita, N. C. (1991). Measurement of uronic acid without interference from neutral sugars. Analytical Biochemistry, 197, 157–162. Guilherme, L. S., Marcello, I., & Philip, A. J. G. (2005). Methylation GC–MS analysis of arabinofuranose and galactofuranose containing structures: rapid synthesis of partially O-methylated alditol acetate structures. Anais. Da Academia Brasileira deCiencias, 77, 223–234. Harman, D. (1993). Free radical involvement in ageing path physiology and therapeutic implications. Drug and Ageing, 3, 60–80. He, Y., Liu, C., Chen, Y., Ji, A., Shen, Z., Xi, T., et al. (2007). Isolation and structural characterization of a novel polysaccharide prepared from Arca subcrenata Lischke. Journal of Bioscience and Bioengineering, 104, 111–116. Hu, Y., Xu, J., & Hu, Q. H. (2003). Evaluation of antioxidant potential of aloe vera (Aloe barbadensis Miller) extracts. Journal of Agricultural and Food Chemistry, 51, 7788–7791. Inoue, K., Korenaga, H., & Kadoya, S. (1982). A sulfated polysaccharide produced by an Arthobacter species. Journal of Biochemistry, 92, 175–1784. Jiang, Y. H., Jiang, X. L., Wang, P., & Hu, X. K. (2005). In vitro antioxidant activities of water-soluble polysaccharides extracted from Isaria farinosa B05. Journal of Food Biochemistry, 29, 323–335. Kinsella, J. E., Frankel, E. N., German, J. B., & Kanner, J. (1993). Possible mechanisms for the protective role of antioxidants in wine and plant foods. Food Technology, 4, 85–89. Lee, S. E., Hwang, H. J., Ha, J. S., Jeong, H. S., & Kim, J. H. (2003). Screening of medicinal plant extracts for antioxidant activity. Life Sciences, 73, 167–179. Linker, A., Evans, L. R., & Impollomeni, G. (2001). The structure of a polysaccharide from infectious strains of Burkholdria cepacia. Carbohydrate Research, 335, 45–54.

320

M.M.S. Asker, B.T. Shawky / Food Chemistry 123 (2010) 315–320

Liu, F., Ooi, V. E. C., & Chang, S. T. (1997). Free radical scavenging activities of mushroom polysaccharide extracts. Life Science, 60, 763–771. Luo, D. (2008). Identification of structure and antioxidant activity of a fraction of polysaccharide purified from Dioscorea nipponica Makino. Carbohydrate Polymers, 74, 544–549. Mau, J. L., Lin, H. C., & Song, S. F. (2002). Antioxidant properties of several specialty mushroom. Food Research International, 5, 519–526. Nagai, T., Inoue, R., Inoue, H., & Suzuki, N. (2003). Preparation and antioxidant properties of water extract of propolis. Food Chemistry, 80, 29–33. Nandita, S., & Rajini, P. S. (2004). Free radical scavenging activity of an aqueous extract of potato peel. Food Chemistry, 85, 611–616. Park, F. S. (1971). Application of I.R. spectroscopy in biochemistry, biology, and medicine. New York: Plenum (p. 601). Pascual, C., Collns, M. D., Funke, G., & Pitcher, D. C. (1996). Phenotypic and genotypic characterization of two Brevibacterium strains from the human ears description of Brevibacterium otitidis sp. Nov. Med. Microbiology Letter, 5, 113–123. Qi, H. M., Zhang, Q. B., Zhao, T. T., Chenc, R., Zhang, H., Niu, X. Z., et al. (2005). Antioxidant activity of different sulfate content derivatives of polysaccharide extracted from Ulva pertusa (Chlorophyta) in-vitro. International Journal of Biological Macromolecules, 37, 195–199. Raouf, O., Patrice, A. R., Andre, B., Jean-Michel, W., & Yvan, T. (2000). Evidence of prooxidant and antioxidant action of melatonin on human liver cell line HepG2. Life Science, 68, 387–399. Ray, B. (2006). Polysaccharides from Enteromorpha campressa: Isolation, Purification and structural features. Carbohydrate Polymers, 66, 408–416. Shimada, K., Fujikawa, K., Yahara, K., & Nakamura, T. (1992). Antioxidative properties of xanthin on auto oxidation of soybean oil in cyclodextrin emulsion. Journal of Agricultural and Food Chemistry, 40, 945–948.

Sudhamani, S. R., Tharanathan, R. N., & Prasad, M. S. (2004). Isolation and characterization of an extracellular polysaccharide from Pesudomonase ceryophlli CFR1705. Carbohydrate Polymers, 56, 423–427. Tong, H., Liang, Z., & Wang, G. (2008). Structural characterization and hypoglycemic activity of a polysaccharide isolated from the fruit of Physalis alkekengi L. Carbohydrate Polymers, 71, 316–322. Uchida, K. (2000). Role of reactive aldehyde in cardiovascular diseases. Free Radical Biology & Medicine, 28, 1685–1696. Urai, M., Anzai, H., Ogihara, J., Iwabuchi, N., Harayama, S., Sunairi, M., et al. (2006). Structural analysis of an extracellular polysaccharide produced by Rhodococcus rhodchrous strain S-2. Carbohydrate Research, 341, 766–775. Valentao, P., Fernandes, E., Carvalho, F., Andrade, P. B., Scabra, R. M., & Bastos, M. L. (2002). Antioxidative properties of cardoon (Cynara cardunculus L.) infusion against superoxide radical, hydroxyl radical, and hypochlorous acid. Journal of Agricultural and Food Chemistry, 50, 4989–4993. Wang, Z. J., Luo, D. H., & Liang, Z. Y. (2004). Structure of polysaccharides from the fruiting body of Hericium erinaceus Pers. Carbohydrate Polymers, 57, 241–247. York, W. S., Darvill, A., O’Neill, M., Stevenson, T., & Albersheim, P. (1985). Isolation and characterization of plant cell wall and cell wall components. Methods in Enzymology, 118, 3–40. Zhang, W. J. (1987). Technology of biochemical research on compound polysaccharide. Hangzhou: Zhejiang University Press (pp. 108–111). Zhou, M., Chen, Y., Ouyang, Q., Liu, S. X., & Pang, Z. J. (2000). Reduction of oxidative injury to the rabbits with established atherosclerosis by protein bound polysaccharide from Coriolus vesicolor. American Journal of Chinese Medicine, 2, 239–249.