Determination of antioxidant activity with DPPH, ABTS, radical scavenging assay

Se-enriched Lb and St were obtained from optimum conditions and inoculated in no-Se fresh MRS broth, cultivated to their post-log phase at 35 °C. After 6000 r/min for 10 min, the supernatant and cell pellets were collected, respectively. The pellets were washed six times by sterile saline to ensure that there was no sodium selenite residue. Meanwhile, all the cell pellets were further diluted to 10⁹ cfu/mL suspensions with sterile saline. The strains (Lb and St) grown in selenite-free MRS were enrolled as the control.

The scavenging of DPPH by Se-enriched strains was analyzed by a modified method reported by Lin and Chang (2000). 2 mL of cell suspension (or supernatant) and 2 mL of freshly prepared DPPH solution (0.2 mmol/L in ethyl alcohol) were mixed and reacted for 30 min in the dark. Blank samples contained sterile distilled water. The scavenged DPPH rate was monitored by measuring the decrease in absorbance at 517 nm. The DPPH free radical scavenging rate was defined as follows:

With a modification of the method reported by Wootton-Beard et al. (2011), the scavenging of ABTS by Se-enriched strains was analyzed. Stable, dark blue-green ABTS radical solution was generated after ABTS (7 in 20 mmol/L sodium acetate buffer, pH 4.5) reacted with an oxidant (2.45 mmol/L potassium persulfate) in the dark for 12–16 h at 4 °C, which was then diluted to the practical reagent with an absorbance of 0.7 ± 0.02 at 734 nm. In our assay on scavenging of ABTS, 2 mL of sample and 2 mL of reagent were mixed for 10 min at room temperature. The sterile distilled water took place of bacteria solution as blank samples. The strains grown in selenite-free MRS were used as the control. The ABTS scavenging was monitored by measuring the decrease of absorbance at 734 nm. The ABTS free radical scavenging rate was defined as follows: