2.4. Accelerated oxidation test
For the purpose of the accelerated oxidation test, 10 g of oleogel or
emulgel were placed into Reacti-vials™ within a
Reacti-Therm™ heating and stirring module (Thermo
Fisher Scientific, Waltham, MA, USA) fitted with aluminum heating
blocks. The Reacti-Therm™ module was set at 60 °C, and
samples were removed for testing on days 4, 7, 10, and 14 with an
initial test on day 0. The peroxide values (PV) of oils and emulsions
were assessed according to the AOCS Official Method Cd 8b-90 (American
Oil Chemists’ Society, 2011). p- Anisidine values (p AV)
were assessed on the same days as PV using the AOCS Official Method Cd
18–90 (American Oil Chemists’ Society, 2011). The TOTOX value on each
given day was calculated using PV and p AV. PV and p AV
tests were conducted in triplicate, and all reagents were prepared fresh
on the day of analysis. TOTOX values were reported as mean ± standard
deviation (SD).
Fatty acid composition was also measured for algal oil in oleogel and
emulgel samples on days 4, 7, 10, and 14 with an initial test on day 0.
This was done in order to track the change in fatty acid profile over
the accelerated oxidation study. The change in fatty acid composition
was determined by following AOAC Official Method 996.01
(Satchithanandam, Fritshce, & Rader, 2001) to prepare fatty acid methyl
esters (FAMEs) and analyzed using an Agilent 6890 N GC system with an
FID detector (Agilent, Santa Clara, CA, USA) and a Supelco SP-2560
capillary column (100 m x 0.25 mm ID, 0.20 µm film) (Sigma-Aldrich Co.,
St. Louis, MO, USA). GC analysis followed procedure set forth previously
(Hyatt, Zhang, & Akoh, 2021). Briefly, 1 µL of sample was injected at a
split ratio of 5:1, the carrier gas (He) flow was 1.1 mL
min-1 and the detector temperature was 250 °C. The
oven was held at 140 °C for 5 min, then increased to 240 °C at a rate of
4 °C min-1, and held for 15 min. In order to analyze
FAMEs of emulgels with the same methods, a 1.0 mL aliquot which would
contain the necessary amount of oil, 50 mg, was pulled and centrifuged
at 2,000 rpm for 10 min, in order to extract required oil. The required
amount of oil was extracted from the resulting supernatant to test the
oxidation of oil in the emulgel matrix. FAMEs analysis was conducted in
triplicate for each sample, and results were expressed as average mg/g
concentrations and normalized percentages for notable fatty acids and
calculated using standard curve with heptadecanoic acid (C17:0).