2.11. Surface hydrophobicity

The hydrophobicity of the MP samples was determined using 8-anilino-1-naphthalenesulphonic acid (ANS) as a hydrophobic probe, following the method reported by Liu et al. [2] with minor modifications. Each sample was diluted to concentrations ranging from 0.2 to 1.0 mg/mL in 50 mM phosphate buffer. Subsequently, 4.0 mL of each diluted sample was mixed with 20 μL of 8.0 mM ANS solution and incubated in the dark for 20 min. The fluorescence intensity of the mixed solution was measured using an F-4500 spectrophotometer (Tokyo, Japan) at wavelengths of 380 nm (excitation) and 410–570 nm (emission), with excitation and emission slit widths set at 5.0 nm. The hydrophobicity of the MP samples was determined as the initial slope of the linear regression equation of a fluorescence intensity vs protein concentration plot.