*3.7. Particle Size Distribution Analysis*

Particle size determination was performed using a zetasizer, Nano-ZS device (Malvern Instruments, Malvern, Worcestershire, UK). The purified protein solutions prepared in Tris–HCl buffers (50 mmol/L) were subjected to scattered light wavelength (532 nm) and 15◦ laser reflection angle at a measuring temperature of 25 ◦C. The size measurements were taken as the mean of five readings.

#### *3.8. CD Spectral Measurement*

The purified protein solutions were subjected to CD spectra measurement with a spectropolarimeter (JASCO J-1500, Tokyo, Japan). The samples of treated protein solutions were prepared in Tris–HCl buffer (50 mmol/L) using this buffer as a blank. The secondary structure of protein solutions was determined in the range of far-ultraviolet (196–260 nm) with the scanning speed of 120 nm/min and the bandwidth of 1 nm. Data for CD spectra were presented as changes in the molar extinction coefficient (Δε, M−<sup>1</sup> cm−1). The contents of secondary structure were calculated from the CD spectra by the estimation software of Spectra Manager (JASCO, Japan) [5].
