The hydrolysis parameters defined according to the experimental design were: substrate concentration of 8.0% and addition of 70.0 U of protease per mL of reaction, resulting in 424.32 and 16.39 Trolox EQ μmol g−1 for the ORAC and DPPH assays, respectively.
Ordinarily, the reactor was filled with 150 mL of reaction solution.
The experiments were carried out in 250-mL Erlenmeyer flasks with the working volume of 100 mL of reaction mixture.
At predetermined times, 5 ml of reaction mixture was collected and centrifuged (4000 rpm, 15 min) in a centrifuge.
The total 1 mL of reaction mixture consisted of 0.9 mL of pNPX (0.5 mg mL−1) and 0.1 mL of suitably diluted enzyme.
An aliquot (1 ml) of reaction mixture was added with 1 ml of distilled water and 200 µl of 0.1%% FeCl3.
They are grown (a) without surfactants, (b) with 0.1 ml PEI, and (c) with 2.5 mg of sodium citrate (per 40 ml of reaction solution), at 0.05 M, 80°C for 5 h.
One unit of SOD activity was defined as the amount of oxidative inhibition by SOD up to 50% per gram tissue in 1 mL of reaction solution, and the SOD activity was monitored at 550 nm.
The superoxide radicals were generated in 5 mL of reaction mixture containing 1 mL of NBT (300 µmol/L) solution, 1 mL of NADH (468 µmol/L) solution and 3 mL of sample solution were mixed.
Figure 6a, b, c presents the plan-view SEM images of ZnO nanostructures grown without surfactants, with 0.1 ml PEI, and with 2.5 mg of sodium citrate (per 40 ml of reaction solution), respectively.
Take total 3 mL of reaction mixture containing 0.5 mL of culture filtrate, 1 mL of sodium tartrate buffer (50 mM, pH 4.0) and 1 mL of 2 mM 2,6-DMP.
