Sulfated exopolysaccharide produced by Labrenzia sp. PRIM-30, characterization and prospective applications

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Sulfated exopolysaccharide produced by Labrenzia sp. PRIM-30, characterization and prospective applications

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Priyanka P, Arun Ab, Rekha Pd ∗ Yenepoya Research Centre, Yenepoya University, University Road, Deralakatte, Mangalore 575018, India

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a r t i c l e

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a b s t r a c t

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Article history: Received 18 January 2014 Received in revised form 14 May 2014 Accepted 15 May 2014 Available online xxx

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Keywords: Sulfated-exopolysaccharide Labrenzia Marine Alphaproteobacteria Antioxidant activity

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1. Introduction

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Exopolysaccharides (EPS) are biopolymers produced by bacteria, having potential industrial applications. An EPS produced by a bacterium designated as PRIM-30 isolated from the deep seawater collected from offshore region of Cochin, India was studied. The isolate was identified as Labrenzia sp. based on 16S rRNA gene sequencing. Under optimum conditions for EPS production, the EPS yield was 840 mg L−1 culture medium. The average molecular weight of the EPS was 269 kDa and composed of glucose, arabinose, galacturonic acid and mannose in the ratio 14.4:1.2:1:0.6. Importantly, the purified EPS contained 4.76% (w/w) sulfate groups. Viscosity of the (1% w/v) EPS was 3.92 cP (shear rate 300 s−1 , 25 ◦ C). Anodic peak potential (Epa ) of the EPS by cyclic voltametric measurement was −0.7 V. The EPS showed antioxidant activities with IC50 values of 640 and 190 ␮g mL−1 , respectively, for the inhibition of DPPH and superoxide radicals. The EPS displayed a linear dose dependent increase in total antioxidant capacity and ferric reducing power activities. To date, only a very few marine alphaproteobacterial representatives have been reported for EPS production and this study for the first time, shows the production of a sulfated EPS by a member of the genus Labrenzia. © 2014 Published by Elsevier B.V.

Exopolysaccharides (EPS) produced by bacteria exhibiting significant structural diversity with novel material and biological properties are considered as invaluable source of natural polymers with multiple biotechnological applications [1]. These high molecular weight carbohydrate polymers synthesized by bacteria have different physiological role and aid in biofilm formation which is an important trait for adherence and community behavior [2]. It is understood that environmental factors influence the diversity of bacteria and in turn the chemical composition of the EPS produced by them [3]. Marine habitats challenged by osmotic stress harbor bacteria producing EPS with unique composition, and hence been studied for prospective application in various sectors [4]. Structural diversity of marine bacterial EPS in terms of the monosaccharide composition, its spatial orientation and presence of attached bioactive functional groups has attracted research interest [5]. In particular, EPS having sulfate groups resemble vital

∗ Corresponding author. Tel.: +91 824 2204668/+91 9741501821; fax: +91 824 2204673. E-mail addresses: [email protected], [email protected], [email protected] (R. Pd).

eukaryotic polymers such as chondroitin sulfate and heparin sulfate and hence find potential biomedical applications. Presence of sulfate groups in bacterial EPS is comparatively rare. However, it has been a characteristic of EPS produced by bacteria such as Halomonas stenophila and Alteromonas infernus isolated from hypersaline habitats [6–8]. These sulfated EPS are associated with potential biological activities such as antiproliferation of cancer cells, anticoagulation and wound healing in vitro [5,9]. EPS having antioxidant activities are used as food supplements or as prophylactic agents in pharmaceutics [10]. Bacterial EPS have also been established for various other applications, in cosmeceutical industry as an active principle in the formulations [11], in food industry as texture modifiers and emulsifiers [12] and also in bioremediation sector for enhanced oil recovery and removal of toxic contaminants [13,14]. Due to the versatility of the EPS molecule, chemical modifications to deliver a tailored product to suit the pharmaceutical demands is also found possible [1]. Considering the developments in the pharmaceutical industry toward the development of non toxic, biocompatible therapeutics from natural sources, exploitation of marine bacteria for novel biomolecules such as EPS is viewed as an attractive alternative to plant or synthetic polymers. Microbial sources of EPS are advantageous over these polymers due to the ease of culturing thus enabling continuous production of industrially important compounds.

http://dx.doi.org/10.1016/j.ijbiomac.2014.05.054 0141-8130/© 2014 Published by Elsevier B.V.

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In the present study, detailed investigation on a sulfated EPS produced by a marine bacterium isolated from deep seawater collected from offshore of Cochin (India) was made.

were used for calibration and the Mw of the sample was determined by plotting against standard graph. 2.6. Biochemical characterization of the EPS

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2. Materials and methods 2.1. Chemicals and reagents Chemicals 2, 2,-diphenyl-1-picryl hydrazyl (DPPH), dextran molecular weight standards, DEAE-cellulose, sephacryl S-500 mhydroxy biphenol, dimethyl acetamide, dichloromethane and BSTFA-TMCS were purchased from Sigma Aldrich (USA). Potassium ferricyanide, phenol, ethanol, Hydrochloric acid, sulphuric acid, perchloric acid, Tween-80, xylene, toluene and hexane were purchased from Merck (India). Dialysis membrane 12 kDa cutoff, MY media components, tris-base, d-glucose, bovine serum albumin, glucose penta acetate, glucuronic acid, pyrogallol were purchased from HiMedia (India). All the reagents were of analytical grade. 2.2. Isolation and culture conditions

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Seawater sample was collected from offshore of Cochin, India (9◦ 55 0.95 N, 73◦ 45 0.75 E, depth > 1500 m) using a Niskin water sampler. The sample was plated was on a complex media (MY) [15], supplemented with 7.5% (w/v) sea salts [16]. Based on the mucoid phenotype of colonies, an EPS producing bacterial isolate was selected and designated as PRIM-30. The EPS production was monitored for 7 days in MY media at 32 ◦ C in an incubator shaker. To establish the optimum conditions required for EPS production by the isolate following variables were separately studied: salinity (1, 2.5, 5, 7.5, 10 and 15% w/v sea salts to MY media) [16], temperature (22, 32 and 42 ◦ C) and incubation either in rotator (100 rpm or 200 rpm) or static conditions. The cultures were monitored for growth (CFU) and EPS yield by incubating for 72 h (log phase). All the experiments were conducted in 100 mL broth media taken in quadruples.

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2.3. Taxonomic identification of the isolate

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The EPS produced by Labrenzia sp. PRIM-30 was characterized by estimating the total sugar [20], protein [21], uronic acid [22], acetyl [23], sulphate [24] and phosphate [25] content spectrophotometrically (Shimadzu UV-1800, Japan). Sulfate content was further confirmed using CHNS analysis using an Elementar Vario EL III, CHNS analyzer (Germany). 2.7. Monosaccharide analysis Monosaccharide analysis was carried out using a GC–MS. For this, 0.1 mg of the purified EPS sample was subjected to methanolysis using methanolic HCl (1 M) for 16 h at 80 ◦ C. The mixture was dried under nitrogen and dissolved in dichoromethane (DCM) and dimethyl acetamide. The contents were evaporated under nitrogen and re-dissolved in N,O-bis(trimethylsilyl)trifluoroacetamidetrimethyl chlorosilane (BSTFA–TMCS) in DCM and kept at 60 ◦ C for 1 h [26]. The derivatised sample was analysed on a GC–MS system (Agilent 7890 GC with 5975 C MS, USA) with DB 5 ms 30 × 0.25 mm column (Agilent Technologies). For MS analysis 1 ␮L sample was injected using a splitless injector. Oven programming included an initial column temperature of 50 ◦ C for 2 min followed by a ramp at 20 ◦ C min−1 to 310 ◦ C and held at 310 ◦ C for 7 min. Mass spectral analysis of the peaks obtained were carried out for the identification of the monosaccharides. 2.8. Fourier transform infrared (FTIR) spectroscopy For FTIR analysis, EPS (10 mg) was ground and pelleted with anhydrous KBr, the spectrum was recorded in the frequency range of 4000–500 cm−1 using a Shimatzu Prestige 21 IR spectrometer (Japan). 2.9. Rheological property of the EPS

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Taxonomic identification of the isolate was carried out using 16S rRNA gene sequencing as described earlier [17]. Sequence data was aligned and compared with available standard sequences of bacterial lineage in the web based EzTaxon-e server [18]. The sequences were submitted to NCBI Genbank. 2.4. Extraction and purification of the EPS

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For EPS extraction, bacteria was grown in 1 L MY broth supplemented with 7.5% w/v sea salts, pH 7.2 at 32 ◦ C, for 120 h in an incubator shaker (100 rpm). Extraction of the EPS from the cell free culture supernatants was carried out by precipitating with cold ethanol according to Quesada et al. [19]. The precipitated EPS was re-dissolved, dialysed against deionised water (Direct Q5, Millipore, India) using a 12 kDa cutoff dialysis membrane and lyophilized. Subsequently, the EPS was purified by gel permeation chromatography (GPC) on a Sephacryl S-500 column followed by anion exchange chromatography (AEC) on a diethylaminoethylcellulose column.

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2.5. Molecular weight determination

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Molecular weight (Mw) of the EPS was determined by GPC through a 20 × 1.4 cm Sephacryl S-500 column eluted with 0.05 M tris–HCl buffer, pH 7.2. Dextran standards (5, 50, 150 and 670 kDa)

For rheological studies, the purified EPS (1% w/v in water) was subjected to varying shear rate and the resultant viscosity was measured using a rheometer (Brookfield DV-III Ultra Programmable Rheometer, USA) equipped with spindle CPE-40. Viscosity of different concentrations (1, 0.5, 0.25, 0.12% w/v) of EPS solution was determined at 25 ◦ C and the thixotropic behavior of the EPS was evaluated. The effect of acidic pH on the viscosity of the EPS was studied in1 mM HCl (pH 3). 2.10. Emulsifying activity of the EPS The EPS solution (0.5% w/v in distilled water) was mixed with an equal volume of different hydrophobic substrates (olive oil/sunflower oil/petrol/kerosene/hexane/xylene/toluene). The mixture was vortexed vigorously for 2 min and kept for 24 h at 4 ◦ C. The emulsification index (EI) in percentage was calculated as the ratio between the volume of emulsified layer and total volume [27]. Tween 80 at a concentration of 0.5% (v/v) was taken as a positive control. 2.11. Cyclic voltammetric analysis of EPS Cyclic voltammetric measurements were made to assess the redox state of the EPS. Experiments were done using an Autolab PGSTAT 30 voltammeter (Netherlands) with three electrode system where, two platinum electrodes were used as working and counter electrodes and a saturated calomel electrode was used as

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reference electrode. The electrodes were immersed in EPS solution (1 mg mL−1 in deionised water). Microelectrode potential was recorded at 10 mV s−1 at 25 ◦ C, and the voltammogram obtained was analysed.

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2.12. Analysis of antioxidant property of EPS

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Antioxidant activity of the EPS in terms of 1,1-diphenyl2-picrylhydrazyl (DPPH) radical scavenging was assayed at concentrations of 0.25, 0.50 and 1.0 mg mL−1 according to previously described method [28]. Superoxide radical scavenging activity of the EPS was measured according to Li [29] at concentrations 40, 80, 160 and 330 ␮g mL−1 . The IC50 (inhibitory concentration for 50% reduction of the free radicals DPPH and superoxide) was calculated by plotting the values obtained for each tested concentration and then estimate the concentration required for 50% inhibition through regression analysis. Ferric (Fe3+ ) reducing power (FRP) of EPS was determined according to the method of Oyaizu [30] and total antioxidant capacity (TAC) was measured by assaying the ability of the EPS to covert Mo(VI) to Mo(V) [31] at concentrations 0.25, 0.5 and 1.0 mg mL−1 .

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2.13. Statistical analysis

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All values represent the mean from quadruplets (n = 4) and differences between treatments were analysed by one way analysis of variance (ANOVA) using the STATISTICA (Stat Soft, Inc.).

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3. Results and discussion

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3.1. Taxonomic identification and EPS production by the isolate

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The isolate produced light pink colored mucoid colonies with entire margins on MY media supplemented with 7.5% (w/v) sea salts. It was identified as Labrenzia sp. belonging to the class Alphaproteobacteria with 100% similarity to Labrenzia aggregata IAM 12614T . The partial 16S rRNA gene sequence (740 bp) was submitted to GenBank under the accession number KF015426. Production of EPS in relation to growth was studied in the media initially used for isolation (MY media supplemented with 7.5% (w/v) sea salts). It was observed that optimum incubation time for growth was between 72 and 96 h and after that a significant decrease (p < 0.05) in the growth was observed. However, maximum EPS yield was recorded at 120 h (Fig. 1A). This may be due to the increased dispersion of the EPS from the cells at later stages of incubation following optimum growth as reported in some species of Halomonas [32]. The isolate is a moderately halotolerant bacteria showing growth between 1 and 10% (w/v) sea salts in accordance to other members of the genus Labrenzia that show tolerance to a wide range of salinity [33]. Maximum EPS production was obtained at a higher salinity (7.5% w/v) than that required for optimum growth (5% w/v sea salts) indicating the possible production of EPS in response to salinity stress (Fig. 1B). In many marine bacterial isolates, EPS plays a protective role against osmotic stress and overproduction under increased salinity is also reported [3]. Under static conditions, the growth and EPS production was significantly lower (p < 0.001) as compared to cultures grown under 100 rpm agitation. A slight decrease in growth and EPS production was also observed under increased agitation (200 rpm). The isolate was able to grow at 22 ◦ C, however, the EPS yield and growth were significantly lower (p < 0.001) compared to that at 32 ◦ C. At 42 ◦ C, the isolate could not grow. Under optimum conditions for EPS production (MY media supplemented with 7.5% sea salts, pH 7, at 32 ◦ C, 100 rpm, 120 h) the isolate was able to produce 840 mg EPS L−1 culture. Purification of

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Fig. 1. (A) Growth and EPS production kinetics (B) Salinity tolerance and relative EPS production by the isolate Labrenzia sp. PRIM-30. Growth expressed as log CFU mL−1 and EPS yield expressed as mg L−1 culture, n = 4.

EPS by GPC and AEC yielded a single major peak of the carbohydrate fraction which confirms the homogeneity of EPS. 3.2. Characterization of the EPS 3.2.1. Biochemical characterization The average molecular weight of the EPS as determined by GPC was 269 kDa (Fig. 2). It is reported that the EPS produced by bacteria have a molecular weight range of 10–1000 kDa [34]. Hence, it can be confirmed that the EPS produced by Labrenzia sp. PRIM-30 is a high molecular weight biopolymer. Monosaccharide analysis by GC–MS revealed that EPS was composed of glucose, arabinose,

Fig. 2. Standard graph using dextran for molecular weight determination by GPC. Dotted lines indicate the elution time and the corresponding log molecular weight of the EPS produced by Labrenzia sp. PRIM-30.

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Fig. 3. FTIR spectra of the EPS produced by Labrenzia sp. PRIM-30.

galacturonic acid and mannose in the ratio of 14.4:1.2:1:0.6. Biochemical composition of the EPS indicated the presence of uronic acid sugars and functional groups such as sulfate, phosphate and 241 acetyl (Table 1). CHNS analysis showed 1.48% sulphur content in 242 the EPS and conversion of sulphur into sulfate (sulfate = S% × 3.22) 243 revealed a similar quantity of sulfate as obtained by biochemical 244 analysis (4.76% w/w). Functional groups present in the EPS are 245 ionisable at the sea water pH 8, thereby giving a negative charge 246 to the polymer. This negative charge plays an important physio247 logical role in bacteria by buffering the cells against fluctuating 248 temperature, salinity and pressure experienced in the deep sea 249 environment [35]. Conversely, the presence of these functional 250 groups imparts important biological activities to the EPS. The per251 centage substitution of uronic acid influences the ion exchange 252 and gel strength properties of alginate [36]. Presence of functional 253 groups like sulfate and phosphate enhance the therapeutic poten254 tial of EPS produced by A. infernus [37] and Lactococcus lactis [38], 255 Q2 respectively (Table 2). 256 239 240

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3.2.2. FTIR analysis FTIR spectrum of the EPS showed a peak at 3417 cm−1 representing stretching of H-bonded OH groups, 2926 cm−1 for C H stretching vibrations and spectral peaks in the region 1000–1200 cm−1 characteristic for EPS (Fig. 3). The other characteristic absorption peaks were at 1736 cm−1 , 1040 cm−1 corresponding to the absorbance of uronic acids and phosphate group, respectively, and at 1250 and 825 cm−1 characteristic of sulfate group [37].

Table 2 Emulsifying activities of the EPS produced by Labrenzia sp. PRIM-30 at a concentration of 0.5% (w/v) against the selected hydrophobic substrates. Tween 80 is taken as a positive control. Values are mean ± SD, n = 4. Hydrophobic substrate

Labrenzia sp. PRIM-30 EPS

Sunflower oil Olive oil Kerosene Petrol Xylene Toluene Hexane

41 44 3 49 9 8 49

± ± ± ± ± ± ±

1.8 0.1 0.4 2.3 1.7 1.6 1.9

Tween 80 62 60 56 23 10 52 38

± ± ± ± ± ± ±

1.6 0.7 0.6 1.3 2.3 1.8 2.6

3.3. Rheological behavior of the EPS The EPS produced by Labrenzia sp. PRIM-30 (1% w/v) showed a low viscosity of 3.92 cP at a shear rate of 300 s−1 and shear stress of 1.17 nm−2 at 25 ◦ C. Apparent viscosities of the EPS solution were 3.8, 2.7, 1.8 and 1.2 cP at concentrations of 1, 0.5, 0.25 and 0.12% w/v, respectively. Between shear rates of 300 to 410 s−1 the EPS showed a slight pseudoplastic behavior. However, above a shear rate of 410 s−1 , it showed Newtonian behavior (Fig. 4). The EPS also

Table 1 Biochemical composition of the EPS produced by Labrenzia sp. PRIM-30, expressed as % (w/w). Values are mean ± SD, n = 4. Biochemical composition

% w/w of EPS

Total sugar Protein Uronic acid Sulfate Acetyl Phosphate

73.39 10.52 2.26 4.36 1.05 0.17

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Fig. 4. Viscosity of the EPS produced by Labrenzia sp. PRIM-30 at a concentration of 1% (w/v) subjected to different shear stress.

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Fig. 5. Cyclic voltammogram of the 1 mg mL−1 solution of EPS produced by Labrenzia sp. PRIM-30, recorded at a microelectrode potential of 10 mV s−1 at 25 ◦ C.

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exhibited thixotropic behavior wherein, the viscosity lost during the increased shear rate was regained upon removal of stress inducing forces. The EPS was soluble in 1 mM HCl (pH 3) and the viscosity was not affected by the lowering of pH. Low viscosity and slight pseudoplastic nature of the EPS adds an advantage for commercial production as efficient agitation and aeration of the cultures can be attained during fermentation with low energy requirements as compared to highly viscous polysaccharides [39].

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3.4. Emulsification activity of the EPS

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The EPS produced by Labrenzia sp. PRIM-30 could emulsify olive oil, sunflower oil, hexane and petrol. The activities against hexane and petrol were higher and the activity against olive oil and sunflower oil was lesser compared to Tween 80 at the same concentration. Hence, for biological applications, EPS can be used as a safe alternative to chemical emulsifiers. The emulsifying activity of some bacterial EPS e.g., Halomonas sp. strain TG39 is positively influenced by the presence of proteins and anionic sulfate and phosphate groups [40]. Presence of these functional groups in the EPS produced by Labrenzia sp. PRIM-30 might have attributed to the emulsifying activity of the EPS. Property of the EPS to emulsify edible oils such as sunflower oil or olive oil is generally exploited in the food and cosmeceutical industry.

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3.5. Cyclic voltammetry

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Cyclic voltammogram of the EPS solution (1 mg mL−1 ) showed a reversible wave function. The anodic peak potential (Epa ) −0.7 V, indicates the oxidation potential of EPS. The reduction potential measured at cathode (Epc ) corresponded to a peak value of −0.3 V (Fig. 5). Oxidation potentials obtained from a cyclic voltammogram are used to assess the antioxidant properties of a compound. Generally, compounds with the low oxidation potential (Epa < 0.45) demonstrate significant antioxidant activity [41]. Hence, the EPS isolated from Labrenzia sp. PRIM-30 with an Epa value of −0.7 V can work as a potential antioxidant molecule. Moreover, this EPS can also take part in mediated electron transfer (MET) a property that often finds application in electro-catalysis or in the development of small scale microbial fuel cells [42].

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3.6. Antioxidant activity

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The EPS showed a concentration dependent increase in the DPPH and superoxide radical scavenging activities with IC50 values being 0.64 mg mL−1 and 0.19 mg mL−1 , respectively. Antioxidant activity obtained for this EPS is higher compared to the DPPH and superoxide radical scavenging activities reported for some non

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Fig. 6. Antioxidant capacity of EPS produced by Labrenzia sp. PRIM-30 in terms of ferric reducing power and total antioxidant capacity expressed as ␮g eq. of ascorbic acid, n = 3.

sulfated EPS produced by Bacillus licheniformis and ␤-glucan [43]. Earlier reports on superoxide scavenging capacity of the polysaccharides have shown the possible role of sulfate content in the EPS for the activity [44]. The FRP and TAC of the polymer exhibited a dose dependent linear increase in the activity as presented in Fig. 6. These properties indicate the ability of the EPS to change the oxidation state or to chelate transition metal ions. This property may render the metal ions less active to fenton’s reaction, thereby inhibiting the generation of harmful hydroxyl radicals [45]. Marine bacteria producing EPS are mainly reported from the genus Alteromonas, Pseudoalteromonas, Vibrio and Halomonas belonging to the class Gammaproteobacteria. Halophilic Alphaproteobacteria reported for EPS production is limited to two type strains Palleronia marisminoris B33T [46] and Salipiger mucosus A3T [8]. Genetic evidence on the presence of some extra-chromosomal elements responsible for EPS production in Labrenzia alexandrii is recently reported [47]. However; detailed studies on the EPS has not yet been reported for the members of this genus. It can be concluded from the present study that the EPS produced by Labrenzia sp. PRIM-30 has unique chemical composition, with biologically active functional groups such as sulfate and phosphate. The low viscosity, emulsifying and antioxidant activities project the prospective applications of the EPS in food and pharmaceutics. Acknowledgments We acknowledge Prof CC Young, National Chung-Hsung University, Taiwan; Dr AC Hegde, and research scholars at the Dept. of Chemical Engineering, NITK, Surathkal for the technical support extended for the analysis and BRNS for the financial support. Q3 Q4 The author Priyanka P acknowledges, Yenepoya University for the Research fellowship. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]

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