4.8. Determination of Peroxidase (POD), Superoxide Dismutase (SOD), Phenylalanine Ammonia-Lyase (PAL), and Polyphenol Oxidase (PPO) Activities

The extractions of the PPO and POD were performed by homogenizing the sample (2.0 g) with ice-cold acetic acid–sodium acetate buffer (100 mmolL⁻¹, pH 5.5) in a test tube. The mixture was centrifuged at 10,000× g at 4 °C for 20 min. According to the methods of Adhikari et al. and Lo’ay et al. [72,73], the supernatant was taken to determine the enzyme activity. For the PPO assay, the reaction system consisted of 4 mL of acetic acid–sodium acetate buffer (50 mmolL⁻¹, pH 5.5), 1 mL of catechol solution (50 mmolL⁻¹), and 0.1 mL of supernatant. One unit of PPO activity was represented as an increase in the OD₄₂₀ of 1 per kilogram (fresh weight) per minute. For the POD assay, the reaction system consisted of 0.2 mL of H₂O₂ solution (50 mmolL⁻¹), 3 mL of guaiacol solution (25 mmolL⁻¹), and 0.5 mL supernatant. One unit of POD activity was represented as an increase of 1 in the OD₄₇₀ per kilogram (fresh weight) per minute. The enzyme activities of the PPO and POD were expressed as U/g FW.

For the extraction of the enzymes, the sample was ground to powder in liquid nitrogen and homogenized in a prechilled mortar and pestle in 1.5 mL ice-cold extraction buffer containing 50 mM Na-phosphate buffer (pH 7.8), 1 mM ethylene diaminete traacetic acid (EDTA), and 1.0 percent (w/v) polyvinyl-pyrrolidone (PVP). The supernatant was used to determine the SOD activity. The superoxide dismutase (SOD) activity was assayed by measuring its capacity to reduce nitro-blue tetrazolium (NBT). The absorbance of the reaction solution was measured at 560 nm. One unit of SOD was defined as the enzyme activity that inhibited the reduction of nitroblue tetrazolium to blue formazan by 50 percent. The total SOD activity was expressed as U/g FW [70].

For the phenylalanine ammonia-lyase (PAL)-activity assay, 1.0 g of fresh yellow-peach fruit sample was homogenized with 5.0 mL of ice-cold sodium borate buffer (100 mM, pH 8.8) containing 5 mM β-mercaptoethanol, 2 mM ethylene diaminetetraacetic acid, and 4% (w/v) polyvinyl pyrrolidine. The homogenized sample was then thoroughly ground at 4 °C. The homogenate was centrifuged at 12,000× g for 30 min at 4 °C. The supernate was then collected for the enzymatic assay. The PAL activity was determined according to the method described by Shi et al. [74]. The specific enzyme activity was expressed as units (U) per gram of FW. One unit of PAL activity was defined as the amount of enzyme that caused an increase of 0.01 in the absorbance at 290 nm in 1 h under specified conditions.