2.3. Preparation of chitin nanofibers

The preparation of the chitin nanofibers was adapted from previous work (Lee et al., Food Chem., submitted). Briefly, 20 g of chitin powder was incubated in 200 mL of 1 M NaOH and 1 M HCl to remove the residual protein and calcium carbonate. The pellets were washed with water until a constant pH (∼4.2) is reached. Purified chitin was stored at −20 °C overnight and freeze-dried for subsequent storage. Three g of purified chitin were wetted with water and mixed with 150 mL of phosphoric acid in a shaking bath for 12 h to form a clear solution. Afterward, 750 mL of water was added to dilute the chitin suspensions to obtain a milky dispersion, followed by centrifugation at 17 000×g (Beckman Coulter J-20 XPI, Indianapolis IN, USA) for 15 min. After the supernatant was removed, the pellets were dialyzed with water until a constant pH value (∼4.2). The concentration of chitin in the final dispersion was gravimetrically determined to be 1.0% w/v.