Production of Exopolysaccharide

Physico-chemical characterization of EPS

1% (w/v) FeCl ₃ was added. Upon 30 min incubation, the

In each experiment, one factor was varied by holding

absorbance is measured at 700 nm. Higher absorbance

other factors constant. Different carbon sources, nitrogen

of the reaction mixture indicated greater reducing power.

sources and pH were initially studied by employing

2. Hydrogen peroxide scavenging assay

single factor experiment. The FTIR spectra were

characterized using GX-FTIR model, Perkin Elmer, USA

The free radical scavenging ability of EPS was determined

spectrophotometer. The dried EPS was mixed with pure

by hydrogen peroxide assay (Malik et al., 2011).

KBr powder (1:99) and pressed into pellet for FTIR

Hydrogen peroxide (10 mM) solution was prepared in

spectroscopy at a frequency between 4000-450 cm -1 (El-

phosphate buffered saline (0.1M, pH 7.4). 1 ml of EPS

dem, 2004). Protein content was determined by the Lowry

sample at various concentrations was rapidly mixed with

method using Bovine Serum Albumin as standard. Total

2 ml of hydrogen peroxide solution and incubated for 10-

carbohydrate was determined by phenol sulfuric method

60 min. After incubation of 10, 20, 30, 40 and 60 min, the

(Dubois et al., 1956) using glucose as standard.

absorbance of the reaction mixture was measured at 230

nm against blank using UV-Visible spectrophotometer

Antimicrobial activity of EPS

(Shimadzu).

To determine the antimicrobial activity of EPS, EPS

3. ABTS Inhibition assay

samples were solubilized in D/W at concentrations of

50 and 100 mg/ml. The antimicrobial activity of EPS

ThefreeradicalscavengingactivityofEPSwasdetermined

was tested against various pathogenic gram-positive

by ABTS (2, 2 azinobis (3-ethylbenzothiazoline-

( Staphylococcus aureus) and gram-negative ( Salmonella

6-sulphonicacid)

diamonium

salt)

radical

cation

typhi, Pseudomonas aeruginosa , Escherichia coli )

decolourization assay (Li et al., 2009). The reaction

bacterial sp. by agar well diffusion assay (AWDA)

mixture was prepared with 2.45 mM potassium

(Nagarajan et al., 2009).

persulphate. The mixture was incubated in the dark at

room temperature for 12-16 h before use. The radical

Evaluation of hydrophilic activity of EPS

cations scavenging activity was assessed by mixing 3.9

Hydrophilic activity of EPS was determined in terms of

ml ABTS solution with 0.1 ml crude exopolysaccharide.

D/W absorption capacity by tea bag method. The EPS

The final absorbance was measured at 743 nm with

sample is placed in a pre-weighed tea bag and the bag

spectrophotometer.

is dipped in an excess amount of water at different time

Determination of flocculating activity

intervals. The bag is removed from the D/W, and excess

water is drained by hanging the bag until no water drops

In 2 ml of EPS samples having concentration of 2.5 mg/

(Jamil and Ahmed, 2008).

ml, 1 ml of 6.8 mM CaCl ₂ and 10 ml of 5g/l activated

charcoal were added. The whole mixture was mixed

Analysis of Antioxidant properties of EPS

thoroughly and vortex for 30 sec. The mixture was

incubated at room temperature. Control was prepared

1. Ferric Reducing Antioxidant Power (FRAP)

without adding EPS solution. The upper layer absorbance

TheferricreducingpowerofEPSsamplesweredetermined

was measured at 550 nm. Flocculation activity was

by using the potassium ferricyanide-ferric chloride

calculated according to following formula (Abdel-Aziz

method (Liu et al., 2011). Different concentrations of

et al., 2012).

EPS were added to 2.5 ml phosphate buffer (0.2 M,

Flocculating activity= [(B-A)/ B]*100*Dilution Factor

pH 6.6) and 2.5 ml potassium ferricyanide (1%). The

mixtures were incubated at 50ºC for 20 min, and reaction

Where B = Turbidity of control, A=Turbidity of

was stopped with addition of 2.5 ml trichloroacetic acid

experimental

(10%). An aliquot of the mixture was taken and 0.5 ml

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