ROS destroying the bacterial membrane generally accompanies with the production of lipid peroxide radical, thereby forming the malondialdehyde (MDA). To verify the role of ROS in breaking the bacterial membrane (Ayaz Ahmed and Anbazhagan, 2017 (link)), MDA expression was detected by TBA assay. Briefly, 10% TCA was introduced into the bacterial liquid that was co-cultured with EG for 24 h, followed by the addition of 0.67% TBA to incubate for 1 h at 95°C. After being cooled to room temperature, the reaction mixture was centrifuged at 6000 rpm for 15 min, and the absorbance of the supernatant was measured at 532 nm using a microplate reader. Bacteria treated with 10 μM hydrogen peroxide and untreated bacteria were used as positive and negative controls, respectively.
In addition, excessive ROS is inclined to reduce the intracellular concentration of GSH and then weaken the anti-oxidation ability of bacteria, thus leading to lipid peroxidation of the membrane (Rahman et al., 2007 (link)). Hence, GSH expression induced by EG was detected by DTNB assay. Briefly, the bacterial liquid, co-cultured with EG for 24 h, was collected and cracked on ice with 10% TCA solution for 15 min. Then, 200 μl of the bacterial lysate was mixed with 1,800 μl Tris buffer (30 mM, pH 8.3) and 100 μl 0.1% DTNB for the incubation of 90 min in the dark at room temperature. After that, the absorbance of solutions was monitored at 412 nm using a microplate reader. Bacteria treated with 10 μM hydrogen peroxide and untreated bacteria were used as positive and negative controls, respectively. The relative production of MDA or GSH was estimated as the following formula:
Where ODs, ODn, and ODp represent the detected absorbance of solutions for the sample group, negative and positive control, respectively.
In addition, excessive ROS is inclined to reduce the intracellular concentration of GSH and then weaken the anti-oxidation ability of bacteria, thus leading to lipid peroxidation of the membrane (Rahman et al., 2007 (link)). Hence, GSH expression induced by EG was detected by DTNB assay. Briefly, the bacterial liquid, co-cultured with EG for 24 h, was collected and cracked on ice with 10% TCA solution for 15 min. Then, 200 μl of the bacterial lysate was mixed with 1,800 μl Tris buffer (30 mM, pH 8.3) and 100 μl 0.1% DTNB for the incubation of 90 min in the dark at room temperature. After that, the absorbance of solutions was monitored at 412 nm using a microplate reader. Bacteria treated with 10 μM hydrogen peroxide and untreated bacteria were used as positive and negative controls, respectively. The relative production of MDA or GSH was estimated as the following formula:
Where ODs, ODn, and ODp represent the detected absorbance of solutions for the sample group, negative and positive control, respectively.
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