2.5. Acid Value, Peroxide Value, and p-Anisidine Value (p-AV)

Acid value of tree-borne seed oils was measured using the American Oil Chemist's Society (AOCS) method [14]. After adding 1 g oil to a 150 mL conical flask, 30 mL of the solution was mixed with ethanol-ether in 1 : 1 (v/v) ratio to dissolve oils. To this solution, we added (and mixed well) 100 μL of 1% phenolphthalein as an indicator and then the mix was titrated with 0.1 M potassium hydroxide (KOH)/ethanol solution until it became light red. Acid value was finally identified through a mock experiment without sample.

Peroxide value of seed oils were measured using the AOCS method [15]. After collecting 1 g oil in a 250 mL conical flask, oil samples were dissolved with 25 mL of solution mixed with acetic acid-chloroform in 3 : 2 (v/v) ratio, to which 1 mL saturated potassium iodide (KI) solution was added. The mix was then shaken for 1 min, incubated in a dark condition for 10 min, and mixed well with 75 mL distilled water. Then, 1 mL of 1% starch solution was added, and the solution was titrated with 0.01 N sodium thiosulfate (Na₂S₂O₃) solution until it became transparent. Peroxide value was finally identified through a mock experiment without sample.

To identify amounts of aldehydes and ketones that are secondary oxidation products generated during acidification of seed oils, p-AV was utilized. After mixing 100 mg oil with 25 mL isooctane, optical density (OD) was measured at 350 nm. Following reaction of 2.5 mL of this solution with 0.5 mL of 0.25% (w/v) p-anisidine/acetic acid solution for 15 min, OD was measured at the same wavelength. In this way, OD values before and after reaction with anisidine solution were measured to find p-AV value [16].