## 2 Materials and Methods
### Analytical Methods  
#### 2.3.1 Total Phenolic Content and Antioxidant Capacity  
The extract preparation involved making a ratio of 1:10 (m:v) of sample and methanol (Sigma-Aldrich, Germany), followed by Ultra-Turrax homogenizer (IKA LABORTECHNIK T25 basic, Janke & Kunkel  
Figure 1: Schematic representation of a longitudinal section of a pineapple fruit and flow diagram of the abiotic stress treatments (wounding and thermal treatment). GmbH &Co., Germany) at 8000 rpm for 2 min and incubation overnight at 4 \({}^{\circ}\)C. The extracts were obtained by centrifugation (HERMLE Z383K LABORTECHNIK, Germany) at 8000 rpm for 20 min (4 \({}^{\circ}\)C), and the supernatants were stored at 4 \({}^{\circ}\)C protected from light until needed for analysis. The total phenolic content (TPC) was determined according to the method of [PERSON] (1959) [24] and [PERSON] (2009) [25], with some alterations. Aliquots of the extract supernatant after centrifugation (150 \(\mu\)L) were diluted with 2400 \(\mu\)L nanopure water, followed by the addition of 150 \(\mu\)L of 0.25 M Folin-Ciocalteu (Panreac AppliChem, Germany), and the sample was incubated for 3 min at room temperature. The reaction was interrupted by adding 300 \(\mu\)L of 1M Na\({}_{2}\)CO\({}_{3}\) (Panreac AppliChem, Germany) and the mixture was incubated for a further 2 h, protected from light. Afterwards, the absorbance of the solution was measured at 725 nm. The total phenolic content was defined using a standard curve developed with equivalent gallic acid (GAE) and expressed as mg GAE.g\({}^{-1}\) dry weight. The average of three replicates was used for each condition. The antioxidant capacity was evaluated by DPPH (2,2-diphenyl-1-picrylhydrazyl) method following the procedure of [PERSON] et al. (1995) [26], with some modifications. In this case, the DPPH solution was formulated with methanol until it achieved 1.1 units of absorbance at 515 nm. The sample extracts were prepared as described above. Sample aliquots of 100 \(\mu\)L were taken from the supernatants and then added to 3900 \(\mu\)L DPPH solution. This mixture was homogenized, and the reaction took place for 40 min in the dark. The sample absorption was read at 515 nm. The blank was prepared with methanol and used as control and to calibrate the spectrophotometer for readings. The antioxidant capacity was determined using a standard curve developed with Trolox, and the results are expressed as Trolox equivalent antioxidant capacity (TEAC; umol Trolox.g\({}^{-1}\) dry weight). The average of three replicate samples was used for each condition. Ferric-reducing antioxidant power (FRAP) was performed according to [PERSON] and [PERSON] (1996) [27], with some modifications. Solutions of 300 mM acetate buffer (3.1 g sodium acetate (C\({}_{2}\)H\({}_{3}\)NaO\({}_{2}\)\(\cdot\)3H\({}_{2}\)O; Panreac AppliChem, Germany) and 16 mL acetic acid glacial (C\({}_{2}\)H\({}_{4}\)O\({}_{2}\); FisherChemical (United Kingdom)), pH 3.6, 10 mM TPTZ (2,4,6-tripyridyl-s-triazine; Sigma-Aldrich, Germany) in 40 mM hydrochloric acid (HCI; Sigma-Aldrich, Germany), and 20 mM iron (III) chloride (FeCl\({}_{3}\); Sigma-Aldrich, Germany) were prepared. The working solution was made by combining 35 mL acetate buffer 300 mM, 3.5 mL TPTZ solution, and 3.5 mL FeCl\({}_{3}\) solution. The procedure involved mixing 2.7 mL of the FRAP solution with 270 \(\mu\)L H\({}_{2}\)O and 90 \(\mu\)L of the extract samples, which had been warmed in a water bath at 37 \({}^{\circ}\)C for 30 min. The coloured result (ferrous tripyridyltriazine complex) was then read at 595 nm using water as the blank. The antioxidant capacity was determined using a standard curve established with Trolox, and the results are expressed as Trolox equivalent antioxidant capacity (TEAC; umol Trolox.g\({}^{-1}\) dry weight). The average of three replicates was used for each condition. The antioxidant capacity was also measured using the ABTS (2,2\({}^{\prime}\)-azino-bis(3-ethylbenzothazoline-6-sulphonic acid)) method as described by [PERSON] et al. (1999) and [PERSON] et al.