2.3.2. Lipid Oxidation Markers

Primary lipid oxidation products: The amount of hydroperoxides was measured using the method of Shantha and Decker [27]. Briefly, two stock solutions were prepared: for solution A, 0.8 g barium chloride was dissolved in 25 mL 0.4 M HCl, and for solution B, 7.5 g ammonium thiocyanate was dissolved in 25 mL ultrapure water: both solutions were stored cold and in the dark. To prepare the reagent, 0.14 M Fe₂SO₄ 7H₂O was mixed with solution A in 1:1 (v/v) ratio and to obtain clear Fe (II) solution, barium chloride precipitate was centrifuged. Then, supernatant was mixed with solution B in 1:1 (v/v) ratio and the reagent was ready for use.

To determine the hydroperoxide concentration, lipids were extracted from 0.3 g emulsion aliquots by mixing with 1.5 mL n-hexane:n-propanol (3:1, v/v). Next, 0.2 mL hexane phase, 2.8 mL methanol:1-buthanol (2:1, v/v) and 30 µL reagent were mixed. After 20 min incubation at room temperature, absorbance was determined at 510 nm against a blank containing all reagents except the sample using a spectrophotometer (DU 720 Beckman Coulter, Brea, CA, USA). The concentration expressed as mmol hydroperoxides per kg of oil was determined using a calibration curve obtained with a cumene hydroperoxide standard solution.

For stripped rapeseed oil and fresh PC, values of 0.05 and 11.23 mmol hydroperoxides per kg oil were found, respectively.

Secondary lipid oxidation products: The para-anisidine value (pAV), which is a global measurement of the formation of aldehydes, was determined by first mixing 0.3 g emulsion with 1.5 mL n-hexane:n-propanol mixture (3:1 v/v). The absorbance of the top hexane phase (Ab) was immediately measured at 350 nm, using pure hexane as blank. Then, 1 mL of this top hexane phase was added to 0.2 mL para-anisidine solution (2.5 M in acetic acid) and mixed. After 10 min the absorbance of this mixture (As) was measured using as blank pure hexane similarly mixed with the para-anisidine solution. The pAV was calculated as shown in Equation (2):

where m is the mass (g) of oil per mL hexane. For stripped rapeseed oil and PC, values of 0.02 and 0.51 (AU) were found, respectively.

Thiobarbituric acid reactive substances (TBARS) were determined according to a previously described procedure [28]. A trichloroacetic acid-thiobarbituric acid (TCA-TBA) solution (15 g TCA, 0.375 g TBA, 1.76 mL 12 N HCl and 82.9 mL of water) was mixed with butylated hydroxytoluene (2 wt.% solution in ethanol), in 100:3 (v/v) ratio. One milliliter emulsion was combined with 2 mL TBA solution, and the mixture was placed in a water bath at 75 °C for 30 min. The tubes were then cooled to room temperature for 10 min and then centrifuged (4000× g) for 15 min. The absorbance was measured at 532 nm. Concentrations of TBARS were calculated from a standard curve prepared with 1,1,3,3-tetraethoxypropane.