Protein solubility

The samples were placed on a multi-stirrer (MS-MP8, Wisd Laboratory Instruments, Wertheim, Germany) for 1 h at 350 rpm and were subjected to centrifuge (Beckman Coulter, Fullerton, CA, USA) at 6,000×g for 20 min. The protein solubility of the samples (unheated and heated samples) were calculated by quantifying proteins before and after centrifugation. The protein content of the samples was determined using the bicinchoninic acid assay (Cortés-Ríos et al., 2020). Briefly, sample (25 μL) was mixed with bicinchoninic acid (BCA) solution (200 μL) in a 96-well plate and placed in a plate reader (Biotek Instruments, Winooski, VT, USA) for 30 min at 37°C. The absorbance was taken at 562 nm and protein content was calculated from the standard curve prepared using bovine serum albumin (BSA).