2.2. Extraction of proteins and oil bodies
Peanut protein and oil bodies were extracted using the graded extraction method (Sukhotu, 2014) and aqueous enzymatic extraction of maize germ (Li, Peng, & Li, 2015), respectively, with some modifications. The skinless peanut seeds were ground in a high-speed universal grinder (FW-100; Beijing Ever Bright Medical Treatment Inc, Beijing, China). Twenty grams of peanut powder was dispersed in deionized water (the solid-to-liquid ratio of 1:7, 1:6, 1:5, 1:4, 1:3, and 1:2 w/v). Viscozyme® L (0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, and 2.0%, calculated by the weight of total substrate) was added to the mixture and enzymolysis was conducted in a digital water-bathing constant temperature vibrator (THZ-82; Jinhua Huafeng Instrument Inc, Jinhua, China) for different times (20, 40, 60, 80, 100, 120, and 140 min) and at different temperatures (35, 40, 45, 50, 55, 60, 65, and 70 °C). Thereafter, the enzyme was deactivated by placing the samples in a boiling water bath for 5 min. The cooled solution was centrifuged at 5000 × g for 20 min (DZ267-32C6; Anting Scientific Instrument Factory, Shanghai, China). The supernatant (oil bodies) was decanted and dried at 50 ℃ for 10 h in a vacuum drying oven (DZF-2B; Beijing Ever Bright Medical Treatment Inc, Beijing, China), whereas the precipitation was dried in a freezer dryer (LGJ-25; Beijing Sihuan Scientific Instrument Inc, Beijing, China) for 24 h and weighed. The amount of protein was measured by an Automatic Kjeldahl Apparatus (K1100; Jinan Haineng Instrument Inc, Shandong, China), and the yields of protein and oil bodies were calculated using the formula1 and 2, respectively:
\begin{equation} \mathrm{Yield\ of\ peanut\ protein=}\frac{\mathrm{\text{protein\ content\ of\ precipitate}}}{\mathrm{\text{protein\ content\ of\ peanut}}}\mathrm{\times 100\ }\left(\mathrm{\%}\right)\mathrm{\text{\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ }}\mathrm{\ (1}\mathrm{)}\nonumber \\ \end{equation}\begin{equation} \mathrm{Yield\ of\ oil\ bodies\ =}\frac{\mathrm{\text{dry\ weight\ of\ oil\ bodies}}}{\mathrm{\text{weight\ of\ peanut\ sample}}}\mathrm{\times 100\ }\left(\mathrm{\%}\right)\mathrm{\text{\ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ }}\mathrm{\text{\ \ \ \ \ \ \ \ \ \ \ \ }}\mathrm{\text{\ \ }}\mathrm{(2)\ }\nonumber \\ \end{equation}