*3.10. Antioxidant Capacity*

### 3.10.1. ABTS Assay Scavenging of Free Radical in Exopolysaccharides and Biomass

The ability of the polysaccharides to scavenge the free radicals was evaluated using an ABTS assay according to Re et al. [92], with few modifications. ABTS radical cation was produced through the reaction with the 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) 7-mM solution with 2.45-mM potassium persulfate for 16h in the dark at 4 ◦C. After incubation, the well-mixed solution was diluted to an absorbance of 0.7 at 727 nm with the deionized water. For biomass and EPS, 10 mg were weighted, and 1 mL of phosphate buffer was added, mechanically disrupted and centrifuged at 4 ◦C for 5 min at 3500 rpm. A total of 50 μL of these samples (supernatant in case of biomass) were mixed with 940 μL of phosphate buffer and 10 μL of ABTS solution. The resulting mixture was measured with a spectrophotometer at 727 nm. ABTS radical scavenging capacity was calculated according to Equation (6) [93]:

$$\mathbf{A}\mathbf{A}^{\diamondsuit} \mathbf{\color{red}{=}} (\mathbf{A}\mathbf{b}\mathbf{s}\_0 - \mathbf{A}\mathbf{b}\mathbf{s}\_1 / \mathbf{A}\mathbf{b}\mathbf{s}\_0) \times 100\tag{6}$$

where Abs0 is the absorbance of the ABTS radical in phosphate buffer at time 0, and Abs1 is the absorbance of the ABTS radical solution mixed with the sample after 8 min. A calibration curve was performed with different concentrations of Trolox® (0 to 5 μg mL−1) from a stock of Trolox® 2.5 mM. The % inhibition was determined by interpolation of the absorbance values in the Trolox standard curve fitted to the equation of a linear regression line (y = 13.593x + 0.8717; R<sup>2</sup> = 0.99). All determinations were performed in triplicate (*n* = 3).

### 3.10.2. DPPH Free-Radical Method in Biomass from *T. suecica*

Radical scavenging and antioxidant activities of the extracts of biomass were assessed by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical method by Brand-Williams et al. [94]. To obtain the extracts from the biomass, 10 mg of the sample was weighted, and 1 mL of 80% methanol was added, homogenized by mechanical disruption, and left for 16 h in the dark at 4 ◦C. After incubation, samples were centrifuged at 4 ◦C for 5 min at 3500 rpm. Aliquots of 200 μL of the supernatant samples were added to 90 μL of instantly prepared DPPH solution (0.358 mM) and 910 μL 80% methanol. The samples were incubated in the dark for 30 min at room temperature, and the absorbance (abs) was read at 517 nm against 80% methanol as blank. The absorbance was then transformed into a percentage of inhibition versus 80% methanol. The percentage of the antioxidant activity was calculated according to Equation (7) [93].

$$\mathbf{A}\mathbf{A}\% = \left[ \left( \mathbf{A}\text{bs}\_0 - \mathbf{A}\text{bs}\_1 \right) \mathbf{A}\mathbf{s}\_0 \right] \times 100 \tag{7}$$

where Abs0 is absorbance at time zero, and Abs1 is absorbance at the end of the reaction (30 min) at 517 nm. A calibration curve was performed with di fferent concentrations of Trolox ® (0 to 7.5 μg mL−1) from a stock of Trolox ® 2.5 mM. The % inhibition was determined by interpolation of the absorbance values in the Trolox standard curve fitted to the equation of a linear regression line (y = 13.593x + 0.8717; R<sup>2</sup> = 0.99). All determinations were performed in triplicate (*n* = 3).

### *3.11. Fourier-Transform Infrared Spectroscopy (FTIR)*

Fourier-transform infrared (FTIR) spectra of the polysaccharides from *T. suecica* were obtained by using self-supporting pressed discs of 13 mm in diameter of a mixture of polysaccharides and KBr (1% *w*/*w*) with a hydrostatic press at a force of 15.0 tcm−<sup>2</sup> for 2 min. The FTIR spectra were obtained with a Thermo Nicolet Avatar 360 IR spectrophotometer (Thermo Electron Inc., Franklin, MA, USA) having a resolution of 4 cm<sup>−</sup><sup>1</sup> with a deuterated triglycine sulfate (DTGS) detector and using OmnicTM 7.2 software (bandwidth 50 cm<sup>−</sup><sup>1</sup> and enhancement factor 2.6) in the 400–4000 cm<sup>−</sup><sup>1</sup> region. Baseline adjustment was performed using the Thermo Nicolet OMNIC software to flatten the baseline of each spectrum. The OMNIC correlation algorithm was used to compare sample spectra with those of the spectral library (Thermo Fisher Scientific, San Jose, CA, USA).
