Isolation of Starch Granules

Starch granules were isolated according to the method of Wei et al.¹⁵ and Lu³⁵, with minor modifications. The maize grains were steeped in 0.2% NaOH for 48 h at room temperature. The samples were then rinsed in ultrapure water and then ground using a blender for 3 min. The suspensions were filtered through a 100-mesh sieve. The materials collected on the screen were again homogenized for 2 min, and then passed through the same sieve. The filtrates were adjusted to a pH level of 9.5 with 0.2% NaOH and a pH controller, mixed with excess alkaline protease (Enzyme Commission (EC) Number 3.4.21.62, Beijing Solarbio Science & Technology Co., Ltd., China), and stirred at 42 °C the pH was adjusted when it dropped below pH 8.5) . Approximately 18 h later, the starch was washed with 0.2% NaOH and centrifuged (4,000 g for 10 min) until no biuret reaction occurred (or protein content was less than 0.5%), and finally washed with water. The starch was further treated with anhydrous ethanol and a mixture of methanol and chloroform (v/v = 1:1) three times. Finally, the starch was collected and dried under atmosphere, ground into powder, and then passed through a 200-mesh sieve.