2.6.3. Gas chromatography analysis of squalene
The content of squalene was determined by the gas chromatography. 0.2-2 g oil samples and 300 μL squalane internal standard solution were saponified with 50 mL ethanolic potassium hydroxide solution at 80 °C for 50 min, then were transferred to a separatory funnel containing deionized water. The saponification solution was extracted three times with n-hexane, and the extract was combined. The organic phase was washed to neutral by adding 25 mL ethanol solution. Then the residual water was removed by anhydrous sodium sulfate and the solvent was evaporated in a vacuum evaporator at 40 °C. The analysis was carried out using the GC-2010 PLUS (Shimadzu, Kyoto, Japan) and squalene was separated on a DB-5 capillary column (25 m × 0.32 mm × 0.17 μm; Agilent, Santa Clara, CA, USA). The operating conditions were as follows: split ratio 1:10; injector 250 °C; column 160 °C increased to 220 °C at 15 °C/min, increased to 280 °C at 5 °C/min, maintained for 20 min, and finally to 300 °C at 5 °C/min for 2 min. Squalane was identified by standard substances and the contents were determined by the concentration of internal standard.
2.6.4. Determination of the contents of oryzanol
According to the method of China Grain Sector Standard LS/T 6121.1-2017, the oryzanol content in RBO was calculated and analyzed by UV-VIS spectrophotometer (UV2800-A, UNICO instruments Co. Ltd., Shanghai, China). 0.02 g oils were weighed and diluted to 25 mL with n-heptane. The absorbance of the solution was measured at 315 nm with n-heptane as blank. The measured absorbance should be between 0.2-0.8, beyond which further dilution was required. The content of oryzanol was calculated according to the formula given below, and the specific extinction coefficient of oryzanol was known (359 g / 100 mL).
Oryzanol (%) =\(\frac{\mathrm{A\times N\times 25}}{\mathrm{m(g)\times 359}}\)\(\ \frac{\mathrm{\text{\ A\ }}\mathrm{\ }\mathrm{\text{N\ }}\mathrm{\ }\mathrm{25}}{\mathrm{m\ (g)\ }\mathrm{\ 359}}\)
where A, N, m, were the absorbance of samples, dilution factor after samples volume determination (N=1 if not diluted) and weight of samples, respectively. \(\ \)
2.7.Thermal behavior analysis
Thermal behavior of RBO was analyzed following the method given by Samaram, Mirhosseini, Tan & Ghazali (2014). The thermal properties of the RBO were studied by differential scanning calorimetry (DSC) using a DSC-60A (Shimadzu, Kyoto, Japan). The purge gas used was 99.99% nitrogen with a pressure of 0.5 MPa and a flow rate of 100 mL/min. 5-7 mg of the RBO was weighed in a aluminum pan, heated to 60 ℃ and held for 5 min, then cooled from 60 to -50 ℃ by a rate of 10 ℃/min with 5 min holding time, finally heated from -50 ℃ to 60 ℃ at a rate of 10 ℃/min, and maintained for 5 min.