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.