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Physicochemical and biosorption properties of novel exopolysaccharide produced by Enterococcus faecalis
LWT - Food Science and Technology 68 (2016) 606e614
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LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Physicochemical and biosorption properties of novel exopolysaccharide produced by Enterococcus faecalis Perumal Venkatesh, Meleppat Balraj, Repally Ayyanna, Dasari Ankaiah, Venkatesan Arul* Department of Biotechnology, School of LifeSciences, Pondicherry University, Puducherry 605014, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 July 2015 Received in revised form 25 November 2015 Accepted 4 January 2016 Available online 7 January 2016
Duck intestinal homogenate isolate Enterococcus faecalis DU10 produced exopolysaccharide (EPS) (840 mg/L) in MRS medium (35 C, pH 6.5), was characterized for its physicochemical, biosorption, antioxidant, antibiofilm and rheological properties. HPTLC analysis revealed fructose and arabinose are as major, xylose and mannose are as minor constituents of EPS. And further, this EPS DU10 was characterized through FTIR, 1H and 13C NMR. EPS DU10 efficiently adsorbed heavy metals Zn(II) (515.78 ± 0.7 mg/g), Pb(II) (424.42 ± 0.9 mg/g), Cd(II) (334.4 ± 14 mg/g), and Cu(II) (232.9 ± 0.3 mg/g). EPS DU10 exhibits broad spectrum of biofilm inhibition for bacterial pathogens and good in vitro antioxidant properties by scavenging free radicals at very low concentration (0.1 mg/mL). Along with these properties, high viscosity of EPS makes it suitable for pharmaceutical and industrial applications. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Enterococcus faecalis DU10 Exopolysaccharides Biosorption Antioxidant Antibiofilm
1. Introduction The majority of bacterial species could able to synthesize exopolysaccharide (EPS) which mainly consist of polysaccharides, proteins, extracellular DNA and lipids (Flemming & Wingender, 2010) and they are widely diverse in structure, physicochemical, rheological and other unique properties. Since in the last two decades, bacterial polysaccharides have become an alternative of interest for use as antioxidant, antibiofilm, immunostimulatory, immunomodulatory, antitumor, antiviral and anti-inflammatory agents in various medical and pharmaceutical industries (Liu et al., 2010; Pan & Mei, 2010) and also used in the food industry as viscosifying, stabilizing, gelling, or emulsifying agents (De Vuyst & Degeest, 1999). In recent times, there has been increasing interest in active application of EPS in new area of research include use as bioflocculants, bioabsorbents, drug delivery, and heavy metal removal agents (biosorption) (Wang, Ahmed, Feng, Li, & Song, 2008). Due to application of heavy metals in various industrial processes such as electroplating, manufacturing, mining, and automotive results in contamination of environmental sources. The contamination of aquatic systems by these toxic heavy metals and
* Corresponding author. E-mail address: [email protected] (V. Arul). http://dx.doi.org/10.1016/j.lwt.2016.01.005 0023-6438/© 2016 Elsevier Ltd. All rights reserved.
their subsequent accumulation in the ecosystem has become a worldwide concern (Axtell, Sternberg, & Claussen, 2003). Since they are non-biodegradable and tend to accumulate in the food chain, causing harm to the human being. For instance, intoxication by high levels of Zn(II), Cd(II), Pb(II) and Cu(II) tend to cause respiratory disorders, artherosclerosis, damage of the pancreas, kidney failure, cancer, hypertension, breakdown of central nervous system, affect red blood cell and others (Davis, Ramirez, Mucci, & Larsen, 2004; Davis, Volesky, & Vieira, 2000; Nriagu, 2007). For removal of heavy metal contaminant effluent, number of conventional methods is in practice such as ion exchange, adsorption, chemical precipitation, electrochemical treatment, membrane separation and flotation. Among them adsorption is highly effective and economical. Adsorption using microbial biomass can retain relatively high quantities of metals by means of a passive process known as biosorption-a novel method developed in the 1990s (Pümpel & Schinner, 1993). Many natural materials have been studied in biosorption including yeast, fungi, bacteria and seaweed for recovery of metals from aqueous solution with persuasive results (Arica, Kacar, & Genc, 2001). The microbial biomass produced by microorganisms is mostly biopolymers and is often EPS. The biosorption of heavy metals by EPS is consider as non-metabolic, and energy independent and performed by interaction between metal cations and negative charge of acidic functional groups of EPS (Kim, Kim, Kim, & Oh, 1996). Strain Enterococcus faecalis included in lactic acid bacteria (LAB)
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group and preferably using in many commercial probiotic feed additives to poultry, cattles, etc., due to Generally Recognized As Safe (GRAS) status with many beneficial attributes (Fernandez, Martinez-Bueno, Martin, Valdivia, & Maqueda, 2007; Javed, Masud, Ain, Imran, & Maqsood, 2011). And bacteriocins producing E. faecalis strains have been isolated from different sources and reported by many researchers for its antagoninstic activity against important pathogenic bacteria. In this aspect, in our previous study, probiotic LAB strain E. faecalis DU10 have been isolated from duck intestine and studied its beneficial probiotic attributes and antagonistic activity against important poultry pathogen Salmonella (Venkatesh, Ayyanna, Ankaiah, & Arul, in press). This LAB E. faecalis could also be able to synthesize EPS. However, an isolation and characterization study of EPS from E. faecalis remains scarce or limited. Therefore in this present study, EPS produced by probiotic LAB E. faecalis DU10 was isolated and studied with respect to its physicochemical characterization using multivariate experimental design methodologies. Some important properties such as biosorption capacity to remove heavy metal ions Cu(II), Zn(II), Pb(II) and Cd(II), antioxidant properties, antibiofilm activity, thermostability and rheological properties were investigated. In addition, shifting in peak positions of potential functional groups of EPS involved in biosorption were unraveled by FTIR. Hence, this work will afford some fundamental information, to act as a practical guide to use of this EPS for various biological tasks. Within the limits of our knowledge, this is the first report on detailed characterization of EPS from probiont E. faecalis isolated from the intestinal homogenate of duck. 2. Materials and methods 2.1. Microorganism and chemicals The bacterial strain E. faecalis DU10 (GenBank: KJ622043) was isolated from duck intestinal homogenate in our laboratory and maintained in MRS (deMan, Rogosa and Sharpe) agar medium and glycerol stock. MRS, TSB (tryptone soya broth), BHI (brain heart infusion broth), and xanthan were procured from Himedia (Mumbai). AKTA prime plus and Phenyl Sepharose column were purchased from GE Healthcare (Sweden). Nitroblue tetrazolium (NBT), Phenazine methosulfate (PMS), NADH (b-nicotinamide adenine dinucleotide), butylated hydroxyl toluene (BHT), D2O (Deuterium oxide), and trimethylsilyl (TMS) were purchased from SigmaeAldrich (USA). Synthetic metal solution of Cu(II), Zn(II), Pb(II), and Cd(II) was prepared by dissolving calculated quantity of copper sulphate, zinc sulphate, lead nitrate, and cadmium chloride respectively in double distilled water. All reagents were used analytical grade. 2.2. Extraction, purification, quantification and estimation of molecular weight of EPS Bacterial strain was inoculated (6.0 log cfu/mL) in 1000 mL MRS medium and incubated (35 C, 48 h) then heated (100 C, 15 min) in order to deactivate EPS degrading enzymes. Cell free supernatant (CFS) obtained by centrifugation (8000g, 15 min, 4 C) was added with two fold ice cold 99% ethanol was kept at 4 C for overnight. The EPS pellet was collected by centrifugation (10,000g, 20 min, 4 C) and then was dissolved in minimal volume of phosphate buffer (50 mM, pH-7) and dialyzed against the same buffer to overnight in the dialysis membrane (10 kDa). Dialyzed EPS was lyophilized (Virtis) and dissolved in phosphate buffer containing 0.5 M NaCl and gel filtered through phenyl sepharose column (AKTA prime plus) using the same phosphate buffer with flow rate of 1 mL/min. The eluted fractions were collected and lyophilized for
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further characterization. The EPS was quantified by phenol-sulfuric acid method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956), which was expressed relative to a glucose standard. Approximate molecular weight of EPS was estimated by AKTA prime plus with size exclusion column filled with Sephadex G75. EPS was eluted with 0.5 M NaCl solution at a flow rate of 0.5 mL/ min. Different molecular weight of dextrans 10, 15e20, 30e40, and 500 kDa (Himedia, Mumbai) were used as standards to establish a calibration curve and from this the molecular mass of EPS was calculated (Pan & Mei, 2010). 2.3. Characterization of EPS The monosaccharide analysis of the EPS was carried out in HPTLC. The EPS was hydrolyzed with 3 M trifluoroacetic acid (TFA) (120 C, 4 h) and then syringe filtered (0.45 mm). HPTLC analysis was performed using winCATS Planar Chromatography Manager (Switzerland) with 10.0 10.0 cm TLC silica plate (Sigma, USA). Ten mL of samples sprayed on TLC plates (8 mm band length) using N2 gas with 17.5 mm distance between tracks. Acetonitrle and HPLC grade water (90:10) were used as mobile phase. After 30 min of run, the plate was dried (60 C, 5 min) and then the bands were scanned at 254 nm. Monosaccharides were identified by comparison of their Rf (retardation factor) values with pure standards analyzed under the same conditions. Functional groups of EPS was determined using Fourier transformed infrared (FTIR) spectroscopy analysis. A pressed pellet of 1 mg EPS with 180 mg potassium bromide (KBr) was analyzed using IR spectrometer (Thermo Nicolet Model: 6700) in transmission mode with a resolution of 4 cm1 in the 4000e400 cm1 range. The hydrolyzed EPS was dissolved in 0.5 mL D2O and the proton and carbon number of EPS was identified and confirmed by 1H and 13 C NMR experiments using a Bruker Avance-II 400 NMR spec13 trometer (1H frequency ¼ 400.13 MHz, C frequency ¼ 100.62 MHz) at 298 K using 5-mm broad band inverse probe head equipped with shielded z-gradient and XWIN-NMR software version 3.5 using TMS as an internal reference and the chemical shifts are expressed in ppm. 2.4. Thermal properties of EPS by TGA and DSC To develop the polymer nature, the pyrolysis and combustion were carried out in thermal analyzer instrument (Model: Q600 SDT) operating at atmospheric pressure. Briefly, 10 mg EPS was placed in a platinum crucible and heated at a linear heating rate (10 C/min) from 25 to 300 C. The experiments were performed separately in air and nitrogen atmosphere at a flow rate of 50 mL/ min. Prior to the experiment, TGA/DTA unit was calibrated for temperature reading using indium as melting standard. For differential scanning calorimeter (TA e Q20 DSC), 5 mg EPS was placed in an aluminum pan and analyzed. Empty pan was used as a reference, for determining the melting point and enthalpy change. The heating rate was 10 C/min from 20 to 300 C. Xanthan gum was used as reference material for both TGA and DSC analysis. 2.5. Applications of EPS 2.5.1. Quantification of metal adsorption The metal adsorbed by EPS was quantified according to Shuhong et al., (2014). Briefly, 50 mL (10 mg/L) metal solution was mixed with 2.0 mL (10 mg/L) EPS solution. Then the reaction mixture was incubated in dark (25 C, 5 h) and then centrifuged (10,000g, 10 min, 4 C). The concentration of metal ions in the supernatant was determined by AAS (GBC-SavantAA).
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2.5.2. In vitro antioxidant assays The antioxidant activities of EPS at different concentrations (0.005e0.25 mg/mL) on scavenging of DPPH, OH and superoxide radical were performed and BHT was used as positive control. Scavenging of DPPH radical on EPS was determined according to the method described by Shimada, Fujikawa, Yahara, and Nakamura (1992). Briefly, 4 mL reaction mixture contains 2 mL DPPH (0.1 mM in absolute ethanol) and 2 mL EPS. The mixture was incubated (25 C, 15 min) and then the absorbance was measured at 517 nm. The scavenging activity was determined by the following formula (%) ¼ [1 Asample/Ablank] 100. Scavenging of OH radical was evaluated by the method described by Winterbourn and Sutton (1984). The reaction mixture of 4.5 mL which contains 1 mL PBS (pH 7.4, 0.15 M), 1 mL safranin (40 mg/mL), 1 mL EDTA-Fe(II) (0.945 mM), 1 mL H2O2 (3% (V/V)) and 0.5 mL EPS was prepared. The reaction mixture was incubated (37 C, 30 min) then the absorbance was measured at 560 nm. The hydroxyl scavenging activity was evaluated by using the following formula (%) ¼ [(Ablank e Asample)/Ablank] 100. The superoxide radical scavenging activity was measured according to the method described by Liu, Ooi, and Chang (1997). Briefly, 3 mL reaction mixture which prepared with 0.1 M sodium phosphate buffer (pH 7.4), contains 156 mM NADH, 52 mM NBT, 20 mM PMS and1 mL EPS. The reaction mixture was incubated (25 C, 5 min) then the absorbance was red at 560 nm. The superoxide scavenging activity was measured using the following formula (%) ¼ (1 Asample/Ablank) 100.
2.5.3. In vitro antibiofilm of EPS The antibiofilm activity was investigated by a modified microtiter plate method. The reaction mixture (200 mL) was prepared by adding 180 mL TSB/BHI broth, 10 mL biofilm forming pathogenic bacteria culture (Listeria monocytogenes, Vibrio vulnificus, Vibrio fischeri and Salmonella typhi), 10 mL EPS at different concentrations (0.025, 0.050, 0.1 mg/mL). After incubation (37 C, 18 h) the reaction mixture was discarded and washed with 200 mL PBS. The biofilm fixed by adding 50 mL methanol and incubated for 10 min. Then the methanol was discarded and washed with PBS. The 96 well microtiter plate was stained with 0.2% crystal violet solution. After washing with dis. H2O, 100 mL acetic acid (0.5 M) was added and the absorbance was measured at 570 nm. Control was prepared with broth (without pathogens). Antibiofilm activity was calculated by the following formula (%) ¼ Control Test/Control 100.
2.5.4. Rheological properties The viscosity measurements were carried out using Brookfield LVDV-3 ultra programmable rheometer (USA) at room temperature with 10 rpm. Two percentage EPS sample was made in dis. H2O to measure the viscosity at different temperature (25 and 45 C) and pH (3, 6 and 9). To determine the effect of ionic solutions, EPS sample was made by dissolving with 0.1 M NaCl, and KCl solutions. 2.6. Statistics study All the experiments were carried out in triplicate and the results
Fig. 2. (A) Sugar composition of EPS DU10 by HPTLC. T1-hydrolyzed EPS DU10, T2xylose, T3-mannose, T4-arabinose, T5-fructose. (B) FT-IR spectra analysis of EPS DU10.
Fig. 1. Purification of EPS DU10 through phenyl sepharose column at 280 nm using Akta Prime plus protein purification system.
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expressed as mean ± standard deviation (SD). Comparisons of data on antioxidant and antibiofilm activity were performed using oneway analysis of variance (ANOVA) followed by Tukey's test by using OriginPro 8.5. Results were considered statistically different at P < 0.05.
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exclusion column (Fig. A). The correlation coefficient R2 value 0.9376 of this calibration curve indicates a highly significant accuracy and from this calibration curve the average molecular weight of EPS DU10 was calculated to be 2.26 105 Da. 3.2. Characterization of EPS
3. Results and discussion 3.1. Purification, quantification and molecular mass of EPS The EPS produced by E. faecalis DU10 was purified and the purity was determined through phenyl sepharose column using AKTA prime plus (Fig. 1). The EPS production was found to be 840 mg/L, which is significantly greater than that reported for other LAB strains such as Lactobacillus plantarum KF5 (75.57 mg/L) (Yanping et al., 2010), L. plantarum (140 mg/L) and Lactobacillus paraplantarum (297 mg/L) (Zotta, Piraino, Parente, Salzano, & Ricciardi, 2008). However, it is lower than Enterococcus faecium MC13 (1 g/L) (Kanmani et al., 2013). A calibration curve of known molecular weight dextrans was established by SEC (AKTA prime plus) with Sephadax G75 size
HPTLC analysis showed that EPS DU10 is composed of fructose and arabinose are as major constituents and xylose and mannose are as minor constituents in the approximate proportion of 45.37%, 28.65%, 15.56%, 10.52% respectively (Fig. 2A). The IR spectrum of EPS DU10 (Fig. 2B) showed an intense broad peak around 3400 cm1, is common to all polysaccharides, represents rounded trait typical of hydroxyl groups (OeH) and bound water, which overlaps in part with the weak CeH stretching peak of CH2 groups appearing at 2940 cm1. The well-defined envelope found between 1200 and 900 cm1 represents skeletal CeO and CeC vibration bands of glycosidic bonds and pyranoid ring, which also represents the presence of carbohydrates. Observed peaks between 850 and 970 cm1 found in EPS DU10 correspond to the linkages taken place between monosaccharides. A unique peak at
Fig. 3. NMR analysis of EPS DU10. (A) 1H NMR of EPS DU10, (B)
13
C NMR of EPS DU10.
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1240 cm1 of EPS DU10 will appear only in bacterial EPS including algae (Wang et al., 2008). Further, 1H and 13C NMR (Fig. 3) exhibits the typical bands and peak characteristics of EPS DU10. The 13C absorption at ppm 66.55 (C-1), 114.44 (C-2), 70.63 (C-3), 72.32 (C-4), 72.99 (C-5), and 68.41 (C-6) correlated with 1H absorption at ppm 3.73 (C-1), 4.45 (C-2), 3.97 (C-3), 4.56 (C-4), 3.75 (C-5), and 3.77 (C-6) are suggest the presence of a-D-fructose. The 13C absorption at ppm 114.44 (C-1), 93.97 (C-2), 76.08 (C-3), 93.60 (C-4), 66.55 (C-5), 60.9 (C-6) correlated with 1H absorption at ppm 5.11 (C-1), 4.45 (C-2), 3.97 (C-3), 4.43 (C-4), 3.93 (C-5), and 3.68 (C-6) are corresponding to the presence of a-D-arabinose. The 13C absorption at ppm 96.39 (C-1), 72.99 (C-2), 68.41 (C-3), 76.08 (C-4), 60.90 (C-5), and 66.90 (C-6) correlated with 1H absorption at ppm 4.52 (C-1), 3.33 (C-2), 3.48 (C3), 3.73 (C-4), 3.97 (C-5), and 3.42 (C-6) are suggest the presence of b-D-xylose. The 13C absorption at ppm 96.36 (C-1), 72.30 (C-2), 71.16 (C-3), 68.41 (C-4), 66.79 (C-5), and 60.90 (C-6) correlated with 1H absorption at ppm 5.11 (C-1), 3.93 (C-2), 3.53 (C-3), 3.62 (C-4), 3.82 (C-5), and 3.73 (C-6) are corresponding to the presence of a-Dmannose. 3.3. Thermal analysis by TGA and DSC The thermostable property of EPS plays an essential role for its industrial utilization (Marinho-Soriano & Bourret, 2005). The TGA analysis (weight loss versus temperature) showed that EPS DU10 degrades in two steps (Fig. 4A). In the first step, due to moisture content the weight loss (12.5%) was observed up to 90 C. Thereafter, in the second step, depolymerization occurred up to 275 C and weight loss of 46.85% was observed. This depolymerization temperature of EPS DU10 is modest similar to reference material xanthan gum (278 C with 30% of weight loss) (Fig. 4A), and also comparable with early reported polymers such as KF5 EPS (279.59 C) isolated from L. plantarum KF5, locust gum (278.46 C) (Yanping et al., 2010), at the same time, lower than EPS isolated from Oceanobacillus iheyensis (380 C) (Kumari, Vijay, Avinash, & Bhavanath, 2014). Beside chemical properties, the energy level of exopolysaccharides by thermal characterization determines its commercial value in industrial purposes. So, the thermal transition of EPS DU10 was studied by differential scanning calorimetric analysis. EPS DU10 has exhibited amorphous to crystalline transition (Tc) temperature at 167 C (Fig. 4B), thereafter the melting transition started from 270 C. Whereas, the reference material xanthan gum analog showed significantly low Tc at 92 C and slightly high melting transition at 290 C. The melting transition of EPS DU10 was significantly higher than some early reported EPS such as EPS of O. iheyensis (176 C) (Kumari et al., 2014), L. plantarum KF5 (86.35 C) (Yanping et al., 2010), mutant Bacillus polymyxa (183.25 C) (Kwon, Joo, & Oh, 1992). These thermal properties of EPS DU10 exhibits its potential stability and proving to be useful in model food matrices or hydrocolloids.
Fig. 4. A. Thermogravimetric analysis (TGA), B. Differential scanning calorimetric (DSC) analysis of EPS DU10 isolated from Enterococcus faecalis DU10.
Hou, and Zhang (2009) also reported a novel EPS from deep sea mesophilic bacterium able to adsorb Cu(II) and Pb(II) comparable to EPS DU10. This is the first report of EPS produced by a bacterium
3.4. Potential applications of EPS 3.4.1. Metal adsorption The metal adsorption capacity by EPS DU10 was varied against different metal ions (Fig. 5). EPS DU10 showed excellent biosorption for Zn(II) (515.78 ± 0.7 mg/g) than Pb(II) (424.42 ± 0.9 mg/ g), Cd(II) (334.4 ± 1.4 mg/g), and Cu(II) (232.9 ± 0.3 mg/g). These biosorption are higher than EPS from Arthrobacter ps-5 for Pb(II) (331.8 mg/g) and Cu(II) (257.9 mg/g) (Shuhong et al., 2014). Similarly a biofloculant isolated from Achromobacter sp. isolated from oil refinery waste shows less Zn(II) (430 mg/L) and Pb(II) (30 mg/L) adsorption than EPS DU10 (Subudhi et al.,2014). Zhou, Wang, Shen,
Fig. 5. The metal biosorption capacity of EPS DU10 for Cu(II), Zn(II), Pb(II), Cd(II). The results are represented as mean ± SD of three independent experiments.
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Fig. 6. IR Spectra of EPS after metal absorption (A) Cu(II), (B) Zn(II), (C) Pb(II), (D) Cd(II). The continuous line denotes the IR spectra of dialyzed EPS DU10. The discontinuous dot line denotes the IR spectra of metal bearing EPS after biosorption.
isolated from duck intestine showing four different metal adsorption activities. When comparing IR spectrum of purified EPS with metal bearing EPSs, the peak positions noted that clearly shifted (Fig. 6 A,B,C,D). The intense broad peak of hydroxyl groups (OeH) in EPS found at 3358 cm1 shifted to 3422, 3390, 3397, 3402 cm1 in the metal bearing EPSs of Cu(II), Zn(II), Pb(II) and Cd(II) respectively. Similarly, weak CeH stretching peak of CH2 groups of EPS DU10 appeared at 2950 cm1 shifted to 2917, 2910, 2930 and 2936 cm1 respectively, likewise C]O peak of EPS DU10 shifted from 1645 cm1 to 1632, 1645.7, 1651 and 1623 cm1 respectively. Similarly, peak found at 1242 cm1 of EPS DU10 represents skeletal CeO and CeC vibration bands of glycosidic bonds and pyranoid ring shifted to 1236.7, 1236, 1236 and 1229 cm1 respectively. The C O C shifted from 1044.6 cm1 to 1019, 1050.9, 1057 and 1057 cm1 respectively. Moreover, decrease in intensity of peaks was observed in all metal bearing IR spectrums.
3.4.2. Antioxidant properties The antioxidant property of EPS DU10 was evaluated by DPPH, OH radical and superoxide radical scavenging activity at different concentrations (0.005e0.25 mg/mL) (Fig. 7A,B,C). The DPPH is a stable free radical that can readily undergo scavenging by antioxidant. Hence, DPPH free radical has been widely accepted as a tool for evaluating the free radical scavenging activities of natural compounds (Leong & Shui, 2002). EPS DU10 showed maximum
scavenging (26.6 ± 0.47%) of DPPH at 0.1 mg/mL and it was observed that the scavenging was increased on dose dependent manner and showed similar or slightly lower activity when compared with BHT (Fig. 7A). Among the oxygen radicals, hydroxyl radical is the most reactive and induces severe damage to adjacent biomolecules. Since hydroxyl radicals can easily cross cell membranes, they react with most biomolecules causing tissue damage and cell death. Therefore, removing of hydroxyl radicals is important for the protection of living systems (Cheng, Ren, Li, Chang, & Chen, 2002) and (Sakanaka, Tachibana, & Okada, 2005). As shown in Fig. 7B, the hydroxyl radical scavenging activity by EPS DU10 was dose dependent and attained maximum activity (30.49 ± 0.38%) at 0.25 mg/mL. BHT was showed to have higher activity in all tested concentrations than EPS. On the contrary, mushroom Gomphidius rutilus produced EPS (GREP) reported to be slightly higher than that of BHT in hydroxyl radical scavenging activity at the tested concentrations (Chanjuan, Zhanyong, Tingting, Jing, & Xianghua, 2012). The occurrence of superoxide anion could cause cellular damage due to it produces other free radicals and oxidizing agents, therefore indirectly commences lipid peroxidation (Athukorala, Kim, & Jeon, 2006). The rate of superoxide scavenging of EPS DU10 and BHT were directly proportional to their concentrations and the inhibition by EPS DU10 is significantly similar to BHT in all used concentrations (Fig. 7C). This result is highly supported by Chanjuan et al., (2012). These results remarkably enlighten the
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Fig. 7. Antioxidant activities of EPS DU10. (A) DPPH scavenging activity of EPS DU10. (B) OH radical scavenging activity of EPS DU10. (C) Superoxide scavenging radical activity of EPS DU10. White and black dots bars denoted control BHT and EPS DU10 respectively. Each value is the mean ± S.D of three replicate analysis; bars with different superscript letters are statistically significant (Oneway Anova test; p < 0.05 and subsequently multiple comparison with Tukey's method).
potential antioxidant properties of EPS DU10.
3.4.3. Antibiofilm activity EPS DU10 displayed significant inhibition of biofilms formation by pathogenic strains at all tested concentrations and the reduction was significantly (p < 0.05) increased in dose dependent manner (Fig. 8). EPS DU10 showed maximum activity of 46.31 ± 0.68% against S. typhi than V. fischeri (44.52 ± 0.39%), L. monocytogenes
(43.36 ± 0.47%) and V. vulnificus (40.63 ± 0.39%) at 0.1 mg/mL concentration. The inhibition for S. typhi (46.31 ± 0.68%) by EPS DU10 is greater than that previously reported EPS of Streptococcus phocae PI80 (25%) with 1 mg/mL (Kanmani et al., 2011). EPS of E. faecium MC13 and S. phocae PI80 reported that 60% and 67% of biofilm inhibition respectively for L. monocytogenes at 1 mg/mL EPS concentration (Kanmani et al., 2011, 2013). Hence it is worth to notice that EPS DU10 attained 43.36 ± 047% inhibition for
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4. Conclusion EPS DU10 produced by duck intestinal isolate E. faecalis showed heterogeneity in sugar composition, and thermally stable with good intrinsic viscosity at different conditions. This EPS DU10 exhibited excellent metal biosorption for Zn(II), Pb(II), Cd(II) and Cu(II). The antioxidant properties and excellent antibiofilm activity against pathogenic bacteria of EPS DU10 promising its pharmacological applications. Thus, this EPS DU10 may find use as a biosorbant agent in heavy metal removal as well as desirable properties with reference to be use in biofilm making and food industry. Acknowledgments Council of Scientific and Industrial Research (CSIR), New Delhi is gratefully acknowledged by Venkatesh Perumal for his research fellowship. Appendix A. Supplementary data Fig. 8. Antibiofilm activity of EPS DU10. Each value is the mean ± S.D of three replicate analysis; bars with different superscript letters are statistically significant (Oneway Anova test; p < 0.05 and subsequently multiple comparison with Tukey's method).
Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.lwt.2016.01.005. References
L. monocytogenes at much lower concentration (0.1 mg/mL). The mechanism of inhibition of biofilm formation by the EPS is by inhibiting the initial attachment and autoaggregation of E. coli cells by partially affecting the bacterial surface properties (Kim, Oh, Kim., .2009) (see Fig. 8).
3.4.4. Rheological properties The effect of temperature (25 and 45 C), pH (3, 6, 9) and ionic conditions (0.1 M CaCl2, NaCl, KCl) in the viscosity of EPS were done. The result showed that EPS DU10 (Table 1) has high viscosity (263 ± 5 mPa) at 25 C than 45 C (208 ± 4 mPa), it is due to decreased interaction between molecules at higher temperature loosened the polymer structure, resulting in lower viscosity agreeing with the previous report by Freitas et al., (2009). Among three different pH conditions EPS DU10 showed high viscosity (271 ± 1 mPa) at pH 3 and decreased viscosity at pH 9 (231 ± 2 mPa); this is probably because the intermolecular arrangement of charged polymers extended by electrostatic repulsion or contracted by electrostatic attraction between the polymer chains of ionic solution (Kanmani et al., 2011). With the presence of 0.1 M NaCl solution EPS DU10 showed significantly increased viscosity (277 ± 6 mPa) than 0.1 M KCl (271 ± 2 mPa) solution. These results suggest that EPS DU10 has good stable intrinsic viscosity.
Table 1 Viscosity of EPS DU10. The results are represented as mean ± SD of three independent experiments. Rheological factors Temperature pH
Solution
EPS DU10 (mPa) 25 C 45 C 3 6 9 NaCl KCl
263 208 271 269 231 277 271
± ± ± ± ± ± ±
5 4 1 1 2 6 2
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Shuhong, Y., Zhiyang, M., Zhaofang, L., Yan, L., Meiping, Z., & Jihui, W. (2014). Effects of carbohydrate sources on biosorption properties of the novel exopolysaccharides produced by Arthrobacter ps-5. Carbohydrate Polymers, 112, 615e621. Subudhi, S., Batta, N., Pathak, M., Bisht, V., Devi, A., Lal, B., et al. (2014). Bioflocculant production and biosorption of zinc and lead by a novel bacterial species, Achromobacter sp. TERI-IASST N, isolated from oil refinery waste. Chemosphere, 113, 116e124. Venkatesh, P., Ayyanna, R., Ankaiah, D., & Arul, V. (2015). Partial purification and characterization of bacteriocin produced by Enterococcus faecalis DU10 and its probiotic attributes. Preparative Biochemistry and Biotechnology. in press. Wang, Y., Ahmed, Z., Feng, W., Li, C., & Song, S. (2008). Physicochemical properties of exopolysaccharide produced by Lactobacillus kefiranofaciens ZW3 isolated from Tibet kefir. International Journal of Biological Macromolecules, 43, 283e288. Winterbourn, C. C., & Sutton, H. C. (1984). Hydroxyl radical production from hydrogen peroxide and enzymatically generated paraquat radicals: catalytic requirements and oxygen dependence. Archives of Biochemistry and Biophysics, 235, 116e126. Yanping, W., Chao, L., Peng, L., Zaheer, A., Ping, X., & Xiaojia, B. (2010). Physical characterization of exopolysaccharide produced by Lactobacillus plantarum KF5 isolated from Tibet Kefir. Carbohydrate Polymers, 82, 895e903. Zhou, W., Wang, J., Shen, B., Hou, W., & Zhang, Y. (2009). Biosorption of copper(II) and cadmium(II) by a novel exopolysaccharide secreted from deep-sea mesophilic bacterium. Colloids and surfaces. B, Biointerfaces, 72(2), 295e302. Zotta, T., Piraino, P., Parente, E., Salzano, G., & Ricciardi, A. (2008). Characterization of lactic acid bacteria isolated from sourdoughs for Cornetto, a traditional bread produced in Basilicata (Southern Italy). World Journal of Microbiology and Biotechnology, 24, 1785e1795.
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LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Physicochemical and biosorption properties of novel exopolysaccharide produced by Enterococcus faecalis Perumal Venkatesh, Meleppat Balraj, Repally Ayyanna, Dasari Ankaiah, Venkatesan Arul* Department of Biotechnology, School of LifeSciences, Pondicherry University, Puducherry 605014, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 July 2015 Received in revised form 25 November 2015 Accepted 4 January 2016 Available online 7 January 2016
Duck intestinal homogenate isolate Enterococcus faecalis DU10 produced exopolysaccharide (EPS) (840 mg/L) in MRS medium (35 C, pH 6.5), was characterized for its physicochemical, biosorption, antioxidant, antibiofilm and rheological properties. HPTLC analysis revealed fructose and arabinose are as major, xylose and mannose are as minor constituents of EPS. And further, this EPS DU10 was characterized through FTIR, 1H and 13C NMR. EPS DU10 efficiently adsorbed heavy metals Zn(II) (515.78 ± 0.7 mg/g), Pb(II) (424.42 ± 0.9 mg/g), Cd(II) (334.4 ± 14 mg/g), and Cu(II) (232.9 ± 0.3 mg/g). EPS DU10 exhibits broad spectrum of biofilm inhibition for bacterial pathogens and good in vitro antioxidant properties by scavenging free radicals at very low concentration (0.1 mg/mL). Along with these properties, high viscosity of EPS makes it suitable for pharmaceutical and industrial applications. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Enterococcus faecalis DU10 Exopolysaccharides Biosorption Antioxidant Antibiofilm
1. Introduction The majority of bacterial species could able to synthesize exopolysaccharide (EPS) which mainly consist of polysaccharides, proteins, extracellular DNA and lipids (Flemming & Wingender, 2010) and they are widely diverse in structure, physicochemical, rheological and other unique properties. Since in the last two decades, bacterial polysaccharides have become an alternative of interest for use as antioxidant, antibiofilm, immunostimulatory, immunomodulatory, antitumor, antiviral and anti-inflammatory agents in various medical and pharmaceutical industries (Liu et al., 2010; Pan & Mei, 2010) and also used in the food industry as viscosifying, stabilizing, gelling, or emulsifying agents (De Vuyst & Degeest, 1999). In recent times, there has been increasing interest in active application of EPS in new area of research include use as bioflocculants, bioabsorbents, drug delivery, and heavy metal removal agents (biosorption) (Wang, Ahmed, Feng, Li, & Song, 2008). Due to application of heavy metals in various industrial processes such as electroplating, manufacturing, mining, and automotive results in contamination of environmental sources. The contamination of aquatic systems by these toxic heavy metals and
* Corresponding author. E-mail address: [email protected] (V. Arul). http://dx.doi.org/10.1016/j.lwt.2016.01.005 0023-6438/© 2016 Elsevier Ltd. All rights reserved.
their subsequent accumulation in the ecosystem has become a worldwide concern (Axtell, Sternberg, & Claussen, 2003). Since they are non-biodegradable and tend to accumulate in the food chain, causing harm to the human being. For instance, intoxication by high levels of Zn(II), Cd(II), Pb(II) and Cu(II) tend to cause respiratory disorders, artherosclerosis, damage of the pancreas, kidney failure, cancer, hypertension, breakdown of central nervous system, affect red blood cell and others (Davis, Ramirez, Mucci, & Larsen, 2004; Davis, Volesky, & Vieira, 2000; Nriagu, 2007). For removal of heavy metal contaminant effluent, number of conventional methods is in practice such as ion exchange, adsorption, chemical precipitation, electrochemical treatment, membrane separation and flotation. Among them adsorption is highly effective and economical. Adsorption using microbial biomass can retain relatively high quantities of metals by means of a passive process known as biosorption-a novel method developed in the 1990s (Pümpel & Schinner, 1993). Many natural materials have been studied in biosorption including yeast, fungi, bacteria and seaweed for recovery of metals from aqueous solution with persuasive results (Arica, Kacar, & Genc, 2001). The microbial biomass produced by microorganisms is mostly biopolymers and is often EPS. The biosorption of heavy metals by EPS is consider as non-metabolic, and energy independent and performed by interaction between metal cations and negative charge of acidic functional groups of EPS (Kim, Kim, Kim, & Oh, 1996). Strain Enterococcus faecalis included in lactic acid bacteria (LAB)
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group and preferably using in many commercial probiotic feed additives to poultry, cattles, etc., due to Generally Recognized As Safe (GRAS) status with many beneficial attributes (Fernandez, Martinez-Bueno, Martin, Valdivia, & Maqueda, 2007; Javed, Masud, Ain, Imran, & Maqsood, 2011). And bacteriocins producing E. faecalis strains have been isolated from different sources and reported by many researchers for its antagoninstic activity against important pathogenic bacteria. In this aspect, in our previous study, probiotic LAB strain E. faecalis DU10 have been isolated from duck intestine and studied its beneficial probiotic attributes and antagonistic activity against important poultry pathogen Salmonella (Venkatesh, Ayyanna, Ankaiah, & Arul, in press). This LAB E. faecalis could also be able to synthesize EPS. However, an isolation and characterization study of EPS from E. faecalis remains scarce or limited. Therefore in this present study, EPS produced by probiotic LAB E. faecalis DU10 was isolated and studied with respect to its physicochemical characterization using multivariate experimental design methodologies. Some important properties such as biosorption capacity to remove heavy metal ions Cu(II), Zn(II), Pb(II) and Cd(II), antioxidant properties, antibiofilm activity, thermostability and rheological properties were investigated. In addition, shifting in peak positions of potential functional groups of EPS involved in biosorption were unraveled by FTIR. Hence, this work will afford some fundamental information, to act as a practical guide to use of this EPS for various biological tasks. Within the limits of our knowledge, this is the first report on detailed characterization of EPS from probiont E. faecalis isolated from the intestinal homogenate of duck. 2. Materials and methods 2.1. Microorganism and chemicals The bacterial strain E. faecalis DU10 (GenBank: KJ622043) was isolated from duck intestinal homogenate in our laboratory and maintained in MRS (deMan, Rogosa and Sharpe) agar medium and glycerol stock. MRS, TSB (tryptone soya broth), BHI (brain heart infusion broth), and xanthan were procured from Himedia (Mumbai). AKTA prime plus and Phenyl Sepharose column were purchased from GE Healthcare (Sweden). Nitroblue tetrazolium (NBT), Phenazine methosulfate (PMS), NADH (b-nicotinamide adenine dinucleotide), butylated hydroxyl toluene (BHT), D2O (Deuterium oxide), and trimethylsilyl (TMS) were purchased from SigmaeAldrich (USA). Synthetic metal solution of Cu(II), Zn(II), Pb(II), and Cd(II) was prepared by dissolving calculated quantity of copper sulphate, zinc sulphate, lead nitrate, and cadmium chloride respectively in double distilled water. All reagents were used analytical grade. 2.2. Extraction, purification, quantification and estimation of molecular weight of EPS Bacterial strain was inoculated (6.0 log cfu/mL) in 1000 mL MRS medium and incubated (35 C, 48 h) then heated (100 C, 15 min) in order to deactivate EPS degrading enzymes. Cell free supernatant (CFS) obtained by centrifugation (8000g, 15 min, 4 C) was added with two fold ice cold 99% ethanol was kept at 4 C for overnight. The EPS pellet was collected by centrifugation (10,000g, 20 min, 4 C) and then was dissolved in minimal volume of phosphate buffer (50 mM, pH-7) and dialyzed against the same buffer to overnight in the dialysis membrane (10 kDa). Dialyzed EPS was lyophilized (Virtis) and dissolved in phosphate buffer containing 0.5 M NaCl and gel filtered through phenyl sepharose column (AKTA prime plus) using the same phosphate buffer with flow rate of 1 mL/min. The eluted fractions were collected and lyophilized for
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further characterization. The EPS was quantified by phenol-sulfuric acid method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956), which was expressed relative to a glucose standard. Approximate molecular weight of EPS was estimated by AKTA prime plus with size exclusion column filled with Sephadex G75. EPS was eluted with 0.5 M NaCl solution at a flow rate of 0.5 mL/ min. Different molecular weight of dextrans 10, 15e20, 30e40, and 500 kDa (Himedia, Mumbai) were used as standards to establish a calibration curve and from this the molecular mass of EPS was calculated (Pan & Mei, 2010). 2.3. Characterization of EPS The monosaccharide analysis of the EPS was carried out in HPTLC. The EPS was hydrolyzed with 3 M trifluoroacetic acid (TFA) (120 C, 4 h) and then syringe filtered (0.45 mm). HPTLC analysis was performed using winCATS Planar Chromatography Manager (Switzerland) with 10.0 10.0 cm TLC silica plate (Sigma, USA). Ten mL of samples sprayed on TLC plates (8 mm band length) using N2 gas with 17.5 mm distance between tracks. Acetonitrle and HPLC grade water (90:10) were used as mobile phase. After 30 min of run, the plate was dried (60 C, 5 min) and then the bands were scanned at 254 nm. Monosaccharides were identified by comparison of their Rf (retardation factor) values with pure standards analyzed under the same conditions. Functional groups of EPS was determined using Fourier transformed infrared (FTIR) spectroscopy analysis. A pressed pellet of 1 mg EPS with 180 mg potassium bromide (KBr) was analyzed using IR spectrometer (Thermo Nicolet Model: 6700) in transmission mode with a resolution of 4 cm1 in the 4000e400 cm1 range. The hydrolyzed EPS was dissolved in 0.5 mL D2O and the proton and carbon number of EPS was identified and confirmed by 1H and 13 C NMR experiments using a Bruker Avance-II 400 NMR spec13 trometer (1H frequency ¼ 400.13 MHz, C frequency ¼ 100.62 MHz) at 298 K using 5-mm broad band inverse probe head equipped with shielded z-gradient and XWIN-NMR software version 3.5 using TMS as an internal reference and the chemical shifts are expressed in ppm. 2.4. Thermal properties of EPS by TGA and DSC To develop the polymer nature, the pyrolysis and combustion were carried out in thermal analyzer instrument (Model: Q600 SDT) operating at atmospheric pressure. Briefly, 10 mg EPS was placed in a platinum crucible and heated at a linear heating rate (10 C/min) from 25 to 300 C. The experiments were performed separately in air and nitrogen atmosphere at a flow rate of 50 mL/ min. Prior to the experiment, TGA/DTA unit was calibrated for temperature reading using indium as melting standard. For differential scanning calorimeter (TA e Q20 DSC), 5 mg EPS was placed in an aluminum pan and analyzed. Empty pan was used as a reference, for determining the melting point and enthalpy change. The heating rate was 10 C/min from 20 to 300 C. Xanthan gum was used as reference material for both TGA and DSC analysis. 2.5. Applications of EPS 2.5.1. Quantification of metal adsorption The metal adsorbed by EPS was quantified according to Shuhong et al., (2014). Briefly, 50 mL (10 mg/L) metal solution was mixed with 2.0 mL (10 mg/L) EPS solution. Then the reaction mixture was incubated in dark (25 C, 5 h) and then centrifuged (10,000g, 10 min, 4 C). The concentration of metal ions in the supernatant was determined by AAS (GBC-SavantAA).
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2.5.2. In vitro antioxidant assays The antioxidant activities of EPS at different concentrations (0.005e0.25 mg/mL) on scavenging of DPPH, OH and superoxide radical were performed and BHT was used as positive control. Scavenging of DPPH radical on EPS was determined according to the method described by Shimada, Fujikawa, Yahara, and Nakamura (1992). Briefly, 4 mL reaction mixture contains 2 mL DPPH (0.1 mM in absolute ethanol) and 2 mL EPS. The mixture was incubated (25 C, 15 min) and then the absorbance was measured at 517 nm. The scavenging activity was determined by the following formula (%) ¼ [1 Asample/Ablank] 100. Scavenging of OH radical was evaluated by the method described by Winterbourn and Sutton (1984). The reaction mixture of 4.5 mL which contains 1 mL PBS (pH 7.4, 0.15 M), 1 mL safranin (40 mg/mL), 1 mL EDTA-Fe(II) (0.945 mM), 1 mL H2O2 (3% (V/V)) and 0.5 mL EPS was prepared. The reaction mixture was incubated (37 C, 30 min) then the absorbance was measured at 560 nm. The hydroxyl scavenging activity was evaluated by using the following formula (%) ¼ [(Ablank e Asample)/Ablank] 100. The superoxide radical scavenging activity was measured according to the method described by Liu, Ooi, and Chang (1997). Briefly, 3 mL reaction mixture which prepared with 0.1 M sodium phosphate buffer (pH 7.4), contains 156 mM NADH, 52 mM NBT, 20 mM PMS and1 mL EPS. The reaction mixture was incubated (25 C, 5 min) then the absorbance was red at 560 nm. The superoxide scavenging activity was measured using the following formula (%) ¼ (1 Asample/Ablank) 100.
2.5.3. In vitro antibiofilm of EPS The antibiofilm activity was investigated by a modified microtiter plate method. The reaction mixture (200 mL) was prepared by adding 180 mL TSB/BHI broth, 10 mL biofilm forming pathogenic bacteria culture (Listeria monocytogenes, Vibrio vulnificus, Vibrio fischeri and Salmonella typhi), 10 mL EPS at different concentrations (0.025, 0.050, 0.1 mg/mL). After incubation (37 C, 18 h) the reaction mixture was discarded and washed with 200 mL PBS. The biofilm fixed by adding 50 mL methanol and incubated for 10 min. Then the methanol was discarded and washed with PBS. The 96 well microtiter plate was stained with 0.2% crystal violet solution. After washing with dis. H2O, 100 mL acetic acid (0.5 M) was added and the absorbance was measured at 570 nm. Control was prepared with broth (without pathogens). Antibiofilm activity was calculated by the following formula (%) ¼ Control Test/Control 100.
2.5.4. Rheological properties The viscosity measurements were carried out using Brookfield LVDV-3 ultra programmable rheometer (USA) at room temperature with 10 rpm. Two percentage EPS sample was made in dis. H2O to measure the viscosity at different temperature (25 and 45 C) and pH (3, 6 and 9). To determine the effect of ionic solutions, EPS sample was made by dissolving with 0.1 M NaCl, and KCl solutions. 2.6. Statistics study All the experiments were carried out in triplicate and the results
Fig. 2. (A) Sugar composition of EPS DU10 by HPTLC. T1-hydrolyzed EPS DU10, T2xylose, T3-mannose, T4-arabinose, T5-fructose. (B) FT-IR spectra analysis of EPS DU10.
Fig. 1. Purification of EPS DU10 through phenyl sepharose column at 280 nm using Akta Prime plus protein purification system.
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expressed as mean ± standard deviation (SD). Comparisons of data on antioxidant and antibiofilm activity were performed using oneway analysis of variance (ANOVA) followed by Tukey's test by using OriginPro 8.5. Results were considered statistically different at P < 0.05.
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exclusion column (Fig. A). The correlation coefficient R2 value 0.9376 of this calibration curve indicates a highly significant accuracy and from this calibration curve the average molecular weight of EPS DU10 was calculated to be 2.26 105 Da. 3.2. Characterization of EPS
3. Results and discussion 3.1. Purification, quantification and molecular mass of EPS The EPS produced by E. faecalis DU10 was purified and the purity was determined through phenyl sepharose column using AKTA prime plus (Fig. 1). The EPS production was found to be 840 mg/L, which is significantly greater than that reported for other LAB strains such as Lactobacillus plantarum KF5 (75.57 mg/L) (Yanping et al., 2010), L. plantarum (140 mg/L) and Lactobacillus paraplantarum (297 mg/L) (Zotta, Piraino, Parente, Salzano, & Ricciardi, 2008). However, it is lower than Enterococcus faecium MC13 (1 g/L) (Kanmani et al., 2013). A calibration curve of known molecular weight dextrans was established by SEC (AKTA prime plus) with Sephadax G75 size
HPTLC analysis showed that EPS DU10 is composed of fructose and arabinose are as major constituents and xylose and mannose are as minor constituents in the approximate proportion of 45.37%, 28.65%, 15.56%, 10.52% respectively (Fig. 2A). The IR spectrum of EPS DU10 (Fig. 2B) showed an intense broad peak around 3400 cm1, is common to all polysaccharides, represents rounded trait typical of hydroxyl groups (OeH) and bound water, which overlaps in part with the weak CeH stretching peak of CH2 groups appearing at 2940 cm1. The well-defined envelope found between 1200 and 900 cm1 represents skeletal CeO and CeC vibration bands of glycosidic bonds and pyranoid ring, which also represents the presence of carbohydrates. Observed peaks between 850 and 970 cm1 found in EPS DU10 correspond to the linkages taken place between monosaccharides. A unique peak at
Fig. 3. NMR analysis of EPS DU10. (A) 1H NMR of EPS DU10, (B)
13
C NMR of EPS DU10.
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1240 cm1 of EPS DU10 will appear only in bacterial EPS including algae (Wang et al., 2008). Further, 1H and 13C NMR (Fig. 3) exhibits the typical bands and peak characteristics of EPS DU10. The 13C absorption at ppm 66.55 (C-1), 114.44 (C-2), 70.63 (C-3), 72.32 (C-4), 72.99 (C-5), and 68.41 (C-6) correlated with 1H absorption at ppm 3.73 (C-1), 4.45 (C-2), 3.97 (C-3), 4.56 (C-4), 3.75 (C-5), and 3.77 (C-6) are suggest the presence of a-D-fructose. The 13C absorption at ppm 114.44 (C-1), 93.97 (C-2), 76.08 (C-3), 93.60 (C-4), 66.55 (C-5), 60.9 (C-6) correlated with 1H absorption at ppm 5.11 (C-1), 4.45 (C-2), 3.97 (C-3), 4.43 (C-4), 3.93 (C-5), and 3.68 (C-6) are corresponding to the presence of a-D-arabinose. The 13C absorption at ppm 96.39 (C-1), 72.99 (C-2), 68.41 (C-3), 76.08 (C-4), 60.90 (C-5), and 66.90 (C-6) correlated with 1H absorption at ppm 4.52 (C-1), 3.33 (C-2), 3.48 (C3), 3.73 (C-4), 3.97 (C-5), and 3.42 (C-6) are suggest the presence of b-D-xylose. The 13C absorption at ppm 96.36 (C-1), 72.30 (C-2), 71.16 (C-3), 68.41 (C-4), 66.79 (C-5), and 60.90 (C-6) correlated with 1H absorption at ppm 5.11 (C-1), 3.93 (C-2), 3.53 (C-3), 3.62 (C-4), 3.82 (C-5), and 3.73 (C-6) are corresponding to the presence of a-Dmannose. 3.3. Thermal analysis by TGA and DSC The thermostable property of EPS plays an essential role for its industrial utilization (Marinho-Soriano & Bourret, 2005). The TGA analysis (weight loss versus temperature) showed that EPS DU10 degrades in two steps (Fig. 4A). In the first step, due to moisture content the weight loss (12.5%) was observed up to 90 C. Thereafter, in the second step, depolymerization occurred up to 275 C and weight loss of 46.85% was observed. This depolymerization temperature of EPS DU10 is modest similar to reference material xanthan gum (278 C with 30% of weight loss) (Fig. 4A), and also comparable with early reported polymers such as KF5 EPS (279.59 C) isolated from L. plantarum KF5, locust gum (278.46 C) (Yanping et al., 2010), at the same time, lower than EPS isolated from Oceanobacillus iheyensis (380 C) (Kumari, Vijay, Avinash, & Bhavanath, 2014). Beside chemical properties, the energy level of exopolysaccharides by thermal characterization determines its commercial value in industrial purposes. So, the thermal transition of EPS DU10 was studied by differential scanning calorimetric analysis. EPS DU10 has exhibited amorphous to crystalline transition (Tc) temperature at 167 C (Fig. 4B), thereafter the melting transition started from 270 C. Whereas, the reference material xanthan gum analog showed significantly low Tc at 92 C and slightly high melting transition at 290 C. The melting transition of EPS DU10 was significantly higher than some early reported EPS such as EPS of O. iheyensis (176 C) (Kumari et al., 2014), L. plantarum KF5 (86.35 C) (Yanping et al., 2010), mutant Bacillus polymyxa (183.25 C) (Kwon, Joo, & Oh, 1992). These thermal properties of EPS DU10 exhibits its potential stability and proving to be useful in model food matrices or hydrocolloids.
Fig. 4. A. Thermogravimetric analysis (TGA), B. Differential scanning calorimetric (DSC) analysis of EPS DU10 isolated from Enterococcus faecalis DU10.
Hou, and Zhang (2009) also reported a novel EPS from deep sea mesophilic bacterium able to adsorb Cu(II) and Pb(II) comparable to EPS DU10. This is the first report of EPS produced by a bacterium
3.4. Potential applications of EPS 3.4.1. Metal adsorption The metal adsorption capacity by EPS DU10 was varied against different metal ions (Fig. 5). EPS DU10 showed excellent biosorption for Zn(II) (515.78 ± 0.7 mg/g) than Pb(II) (424.42 ± 0.9 mg/ g), Cd(II) (334.4 ± 1.4 mg/g), and Cu(II) (232.9 ± 0.3 mg/g). These biosorption are higher than EPS from Arthrobacter ps-5 for Pb(II) (331.8 mg/g) and Cu(II) (257.9 mg/g) (Shuhong et al., 2014). Similarly a biofloculant isolated from Achromobacter sp. isolated from oil refinery waste shows less Zn(II) (430 mg/L) and Pb(II) (30 mg/L) adsorption than EPS DU10 (Subudhi et al.,2014). Zhou, Wang, Shen,
Fig. 5. The metal biosorption capacity of EPS DU10 for Cu(II), Zn(II), Pb(II), Cd(II). The results are represented as mean ± SD of three independent experiments.
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Fig. 6. IR Spectra of EPS after metal absorption (A) Cu(II), (B) Zn(II), (C) Pb(II), (D) Cd(II). The continuous line denotes the IR spectra of dialyzed EPS DU10. The discontinuous dot line denotes the IR spectra of metal bearing EPS after biosorption.
isolated from duck intestine showing four different metal adsorption activities. When comparing IR spectrum of purified EPS with metal bearing EPSs, the peak positions noted that clearly shifted (Fig. 6 A,B,C,D). The intense broad peak of hydroxyl groups (OeH) in EPS found at 3358 cm1 shifted to 3422, 3390, 3397, 3402 cm1 in the metal bearing EPSs of Cu(II), Zn(II), Pb(II) and Cd(II) respectively. Similarly, weak CeH stretching peak of CH2 groups of EPS DU10 appeared at 2950 cm1 shifted to 2917, 2910, 2930 and 2936 cm1 respectively, likewise C]O peak of EPS DU10 shifted from 1645 cm1 to 1632, 1645.7, 1651 and 1623 cm1 respectively. Similarly, peak found at 1242 cm1 of EPS DU10 represents skeletal CeO and CeC vibration bands of glycosidic bonds and pyranoid ring shifted to 1236.7, 1236, 1236 and 1229 cm1 respectively. The C O C shifted from 1044.6 cm1 to 1019, 1050.9, 1057 and 1057 cm1 respectively. Moreover, decrease in intensity of peaks was observed in all metal bearing IR spectrums.
3.4.2. Antioxidant properties The antioxidant property of EPS DU10 was evaluated by DPPH, OH radical and superoxide radical scavenging activity at different concentrations (0.005e0.25 mg/mL) (Fig. 7A,B,C). The DPPH is a stable free radical that can readily undergo scavenging by antioxidant. Hence, DPPH free radical has been widely accepted as a tool for evaluating the free radical scavenging activities of natural compounds (Leong & Shui, 2002). EPS DU10 showed maximum
scavenging (26.6 ± 0.47%) of DPPH at 0.1 mg/mL and it was observed that the scavenging was increased on dose dependent manner and showed similar or slightly lower activity when compared with BHT (Fig. 7A). Among the oxygen radicals, hydroxyl radical is the most reactive and induces severe damage to adjacent biomolecules. Since hydroxyl radicals can easily cross cell membranes, they react with most biomolecules causing tissue damage and cell death. Therefore, removing of hydroxyl radicals is important for the protection of living systems (Cheng, Ren, Li, Chang, & Chen, 2002) and (Sakanaka, Tachibana, & Okada, 2005). As shown in Fig. 7B, the hydroxyl radical scavenging activity by EPS DU10 was dose dependent and attained maximum activity (30.49 ± 0.38%) at 0.25 mg/mL. BHT was showed to have higher activity in all tested concentrations than EPS. On the contrary, mushroom Gomphidius rutilus produced EPS (GREP) reported to be slightly higher than that of BHT in hydroxyl radical scavenging activity at the tested concentrations (Chanjuan, Zhanyong, Tingting, Jing, & Xianghua, 2012). The occurrence of superoxide anion could cause cellular damage due to it produces other free radicals and oxidizing agents, therefore indirectly commences lipid peroxidation (Athukorala, Kim, & Jeon, 2006). The rate of superoxide scavenging of EPS DU10 and BHT were directly proportional to their concentrations and the inhibition by EPS DU10 is significantly similar to BHT in all used concentrations (Fig. 7C). This result is highly supported by Chanjuan et al., (2012). These results remarkably enlighten the
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Fig. 7. Antioxidant activities of EPS DU10. (A) DPPH scavenging activity of EPS DU10. (B) OH radical scavenging activity of EPS DU10. (C) Superoxide scavenging radical activity of EPS DU10. White and black dots bars denoted control BHT and EPS DU10 respectively. Each value is the mean ± S.D of three replicate analysis; bars with different superscript letters are statistically significant (Oneway Anova test; p < 0.05 and subsequently multiple comparison with Tukey's method).
potential antioxidant properties of EPS DU10.
3.4.3. Antibiofilm activity EPS DU10 displayed significant inhibition of biofilms formation by pathogenic strains at all tested concentrations and the reduction was significantly (p < 0.05) increased in dose dependent manner (Fig. 8). EPS DU10 showed maximum activity of 46.31 ± 0.68% against S. typhi than V. fischeri (44.52 ± 0.39%), L. monocytogenes
(43.36 ± 0.47%) and V. vulnificus (40.63 ± 0.39%) at 0.1 mg/mL concentration. The inhibition for S. typhi (46.31 ± 0.68%) by EPS DU10 is greater than that previously reported EPS of Streptococcus phocae PI80 (25%) with 1 mg/mL (Kanmani et al., 2011). EPS of E. faecium MC13 and S. phocae PI80 reported that 60% and 67% of biofilm inhibition respectively for L. monocytogenes at 1 mg/mL EPS concentration (Kanmani et al., 2011, 2013). Hence it is worth to notice that EPS DU10 attained 43.36 ± 047% inhibition for
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4. Conclusion EPS DU10 produced by duck intestinal isolate E. faecalis showed heterogeneity in sugar composition, and thermally stable with good intrinsic viscosity at different conditions. This EPS DU10 exhibited excellent metal biosorption for Zn(II), Pb(II), Cd(II) and Cu(II). The antioxidant properties and excellent antibiofilm activity against pathogenic bacteria of EPS DU10 promising its pharmacological applications. Thus, this EPS DU10 may find use as a biosorbant agent in heavy metal removal as well as desirable properties with reference to be use in biofilm making and food industry. Acknowledgments Council of Scientific and Industrial Research (CSIR), New Delhi is gratefully acknowledged by Venkatesh Perumal for his research fellowship. Appendix A. Supplementary data Fig. 8. Antibiofilm activity of EPS DU10. Each value is the mean ± S.D of three replicate analysis; bars with different superscript letters are statistically significant (Oneway Anova test; p < 0.05 and subsequently multiple comparison with Tukey's method).
Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.lwt.2016.01.005. References
L. monocytogenes at much lower concentration (0.1 mg/mL). The mechanism of inhibition of biofilm formation by the EPS is by inhibiting the initial attachment and autoaggregation of E. coli cells by partially affecting the bacterial surface properties (Kim, Oh, Kim., .2009) (see Fig. 8).
3.4.4. Rheological properties The effect of temperature (25 and 45 C), pH (3, 6, 9) and ionic conditions (0.1 M CaCl2, NaCl, KCl) in the viscosity of EPS were done. The result showed that EPS DU10 (Table 1) has high viscosity (263 ± 5 mPa) at 25 C than 45 C (208 ± 4 mPa), it is due to decreased interaction between molecules at higher temperature loosened the polymer structure, resulting in lower viscosity agreeing with the previous report by Freitas et al., (2009). Among three different pH conditions EPS DU10 showed high viscosity (271 ± 1 mPa) at pH 3 and decreased viscosity at pH 9 (231 ± 2 mPa); this is probably because the intermolecular arrangement of charged polymers extended by electrostatic repulsion or contracted by electrostatic attraction between the polymer chains of ionic solution (Kanmani et al., 2011). With the presence of 0.1 M NaCl solution EPS DU10 showed significantly increased viscosity (277 ± 6 mPa) than 0.1 M KCl (271 ± 2 mPa) solution. These results suggest that EPS DU10 has good stable intrinsic viscosity.
Table 1 Viscosity of EPS DU10. The results are represented as mean ± SD of three independent experiments. Rheological factors Temperature pH
Solution
EPS DU10 (mPa) 25 C 45 C 3 6 9 NaCl KCl
263 208 271 269 231 277 271
± ± ± ± ± ± ±
5 4 1 1 2 6 2
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