**2.8 Functional groups of EPS analysis by Fourier-transform infrared (FT-IR) spectroscopy**

Sample were prepared by grinding dried EPS (1 mg) with KBr (20 mg) in 1:20 w/w ratio and pressed into 1 mm thick pellets for measurement. FT-IR spectra were recorded on an FT-IR spectrometer (Shimadzu, Japan) in the frequency range of 4000–400 cm−1 [37].

#### **2.9 In vitro antioxidant activities of EPS**

#### *2.9.1 Ferric reducing antioxidant power measurement of EPS*

Various concentrations of exopolysaccharide (0, 50, 75, 100, and 125 ppm) was blended with 1 ml of 0.2 M phosphate buffer, pH = 6.6 and 1 ml of 1% potassium ferricyanide solution. The mixture incubated at 50 °C for 20 minutes. Then 1 ml of trichloroacetic acid (10%) was added to the reaction mixture and centrifuged at 3000 rpm for 10 minutes. Then 2.5 ml of the supernatant was mixed with distilled water (2.5 ml) and 0.5 ml of 0.1% ferric chloride solution. The absorbance of the solution

was measured at 700 nm using a UV spectrophotometer. Increasing the absorption of the samples means increasing the reducing power of the samples. The blank sample was distilled water, and ascorbic acid was used as positive reference standard. The ferric ion reducing power were expressed as milligrams of ascorbic acid equivalent (AAE) per ml of EPS sample [38, 39].

#### *2.9.2 Assay of ABTS+ radical scavenging capacity*

The antioxidant activity of EPS was assay based on the radical scavenging capacity of 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS+) radical with slight modification. The maximum absorption wavelength of ABTS radical ions is 734 nm, and hence the absorbance at this particular wavelength is used to detect the concentration of ABTS radical ions. Briefly, ABTS solution (7.4 mM) was mixed with potassium persulphate solution (2.6 mM) (1:1 v/v) and was left at 20–22°C in the dark for 24 hours. The ABTS stock solution was diluted with absolute ethanol to an absorbance of 0.7 ± 0.02 at 734 nm. Then, 0.2 and 0.8 ml of ABTS working solution were mixed thoroughly. VC was used as the positive control. The absorbance of the mixture solution was determined at 734 nm after 5 minutes of incubation in the dark, using a microplate reader. The ABTS radical scavenging activity (%) was calculated using the following formula:

$$\text{Scavenging rate} \left( \% \right) = \left( \text{Ac} - \text{As} \right) / \text{Ac} \times \text{zoo} \tag{1}$$

Whereas is the absorbance of the test sample and Ac is the absorbance of the control at 734 nm [40–42].

### **2.10 Data analysis**

Data were analyzed by one way ANOVA with three replications using SPSS ver 22.0 with *P* = 0.05. This analysis was then followed by Duncan multiple range test.
