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).