2.7. Peroxide Value Determination

Peroxide value (PV) was used to analyze the oxidative stability of the algae oil. It was determined using the colorimetric ferric-thiocyanate method described by Shantha and Decker [31]. This was based on the principle that lipid peroxides are able to oxidize Fe²⁺ to Fe³⁺ and oxidation can be spectrophotometrically quantified by means of ferric ion complexation with thiocyanate. Peroxide value was determined following the International Dairy Federation standard method ISO3976:1977 [32] with slight modification. Briefly, 0.4 g BaCl₂·2H₂O was dissolved in 50 mL of distilled water. Separately, a ferrous solution was prepared by dissolving 0.5 g of FeSO₄·7H₂O in 50 mL of distilled water. The barium solution was slowly added to the ferrous one under magnetic stirring, then 2 mL HCl 10 N were added. The BaSO₄ precipitate was filtered to obtain a clear FeCl₂ solution, which was stored in an opaque flask. Freshly prepared FeCl₂ solution was used in each procedure. To prepare the complexing agent, 30 g of NH₄SCN were dissolved in 100 mL of distilled water.

To determine the peroxide value of the neat algae oil, 8 mg of algae oil were dissolved in 1mL of ethanol 85%. In case of particles, the oil was extracted according to the Bligh and Dyer method [33]. For this, 0.5 or 1 g of sample (in case of 2:1 or 9:1, respectively) were dissolved in 1 mL of deionized water. 0.5 mL of the previous solution were mixed with 1.5 mL of isooctane/isopropanol (2:1 v/v) mixed in the vortex and centrifuged at 1000 rpm for 4 min. The organic phase containing the oil was removed for further analysis.

After that, an aliquot of 200 µL of the oil solutions were mixed with 9.6 mL chloroform-methanol (7:3 v/v). Then, 50 µL FeCl₂ and 50 µL of NH₄SCN were added and mixed in the vortex. After 5 min of reaction protected from light, the absorbance was measured at 500 nm against a blank containing all reagents excepting the sample.

To construct the standard curve of absorbance versus Fe³⁺ concentration, a standard solution of iron (III) chloride was prepared. 0.121 g of FeCl₃·6H₂O was dissolved in water and make up to 25 mL. 0.5 mL of the previous solution were made up to 50 mL with chloroform/methanol (7:3 v/v). Standard Fe³⁺ samples containing 0–40 µg Fe³⁺ were analyzed following the previous method by UV-Vis spectrophotometry at 500 nm.

Peroxide value expressed as milliequivalents of peroxides per kilogram of oil, was calculated using the following equation:

where As and Ab are the absorbance of the test sample and blank, respectively, m is the slope of the calibration curve, m₀ is the weight sample of oil, 55.84 g/mol is the atomic weight of iron, S is the volume of the aliquot of the oil solution, V is the volume used to dissolve the oil. The samples were measured by triplicate.