2.3.3 Emulsifying properties
The emulsifying activity index (EAI, m2/g) and emulsion stability index (ESI, min) were determined according to Pearce and Kinsella (1978) and Cheunget al. (2014). In brief, a 0.25% (w/v) protein solution (corrected by protein content) was prepared at pH 3, 5, and 7 and stirred at room temperature (21-23℃) overnight. To prepare the emulsion, 5.0 g of the protein solution was added to 5.0 g of canola oil, followed by homogenization using an Omni Macro Homogenizer (Omni International, Marietta, GA, USA) with a 20 mm saw tooth generating probe positioned at the oil/water interface at speed 4 (~7,200 rpm) for 5 min. Immediately after homogenization, 50 μL of the emulsion from the bottom of the tube was transferred to 7.5 mL of 0.1 % (w/v) sodium dodecyl sulphate (SDS), followed by vortexing for 10 s. The absorbance of the diluted emulsion was determined using a Genesys 20 UV-visible spectrophotometer (Thermo Scientific, Madison, WI, USA) at 500 nm using plastic cuvettes. A second absorbance reading was taken from the dilution after 10 min. The EAI and ESI were calculated using the following equations:
\begin{equation} EAI\ (\frac{m^{2}}{g}\mathrm{)}=\frac{2\times 2.203\times A_{0}\times N\ \times l}{c\times\varphi\times 100}\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ (1)\nonumber \\ \end{equation}\begin{equation} \text{ESI\ }\left(\min\right)=\frac{A_{0}}{A}\times t\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ (2)\nonumber \\ \end{equation}
where A 0 is the absorbance of the diluted emulsion immediately after homogenization, N is the dilution factor, l is the 0.01 m path length of the plastic cuvette,c is the weight of protein per volume (g/mL), φ is the oil volume fraction of the emulsion, ΔA is the difference in absorbance between 0 and 10 min (A 0A 10), and t is the time interval (10 min). All measurements were reported as the mean ± one standard deviation (n=3).