*4.3. Respiration Rate and Ethylene Emission*

The carbon dioxide (CO2) and ethylene production were measured by placing each tomato in a 1 L glass jar hermetically sealed with a rubber stopper for 1 h at ambient room temperature. Fruits were weighed and volume was measured. Additionally, CO2 and ethylene of room air were tested and subtracted from the measurements, by equipment zeroing, prior to and during experimentation. For respiration rate determination, the holder atmosphere was sucked by a dual gas analyzer (International Control Analyser Ltd., UK) for 30 s. Results were the mean of two determinations for each jar (eight jars per treatment and storage period; *n* = 8) and expressed as milliliter of CO2 per kilogram per hour. Ethylene was quantified by using an ethylene analyzer (ICA 56 Analyser, International Control Analyser Ltd., UK) whereas container air sample was sucked for 30 s. Results were the mean of two determinations for each jar and expressed as microliter of ethylene per kilogram per hour (eight jars per treatment and storage period; *n* = 8). CO2 and ethylene evolution were calculated according to the following Equation: rate of evolution = M × (V1 − V2) × (1/w) × (1/t); where, M represents the measurement; V1, V2 represent jar and

fruit volume (mL), respectively; w represents fruit weight (g); and t represents incubation time (h).

#### *4.4. Weight Loss, Colour and Fruit Firmness*

Individual tomato weights were measured on the day of harvesting (day 0) and after the different sampling dates. Weight loss was calculated for each fruit (*n* = 8) per treatment and storage time as follows: weight loss % = 100 (*W*<sup>o</sup> − *W*f)/*W*o, with *W*<sup>o</sup> being the initial weight and *W*<sup>f</sup> the final weight of the fruit.

Color was determined using the Hunter Lab System and a Minolta colorimeter model CR400 (Konica Minolta, Osaka, Japan). Following the recording of individual *L*\*, *a*\*, and *b*\* parameters, and chroma value (C) was calculated by the following equations C=(*a\**<sup>2</sup> + *b\**2) 1/2 as described previously [24]. Results were the mean of determinations made on four points for each fruit (*n* = 8) along the equatorial axis, for each treatment and storage time.

Fruit firmness was measured at two points on the shoulder of each tomato fruit (1 cm<sup>2</sup> of skin removed), respectively for each treatment by applying a plunger of 8 mm in diameter, using a texturometer FT 011 (TR Scientific Instruments, Forli, Italy). The amount of force (in Newtons; N) required to break the radial pericarp (i.e., surface) of each tomato (*n* = 8) was recorded at ambient (21–23 ◦C) temperature for each treatment and storage time.

#### *4.5. Soluble Solids, Titratable Acidity, Ripening Index, Ascorbic Acid and Carotenoids*

Total soluble solids concentration was determined in triplicate from the juice obtained from two pooled tomatoes for each replication (*n* = 8) with a temperature-compensated digital refractometer (model Atago PR-101, Atago Co. Ltd., Tokyo, Japan) at 20 ◦C, and results were expressed in percentage (%). The titratable acidity was measured via potentiometric titration (Mettler Toledo DL22, Columbus, OH, USA) of 5 mL juice diluted to 50 mL with distilled water using 0.1 N NaOH up to pH 8.1. The results were expressed as percentage of citric acid. The ratio of TSS/TA was used to evaluate the sweetness/ripening index of the fruit.

Ascorbic acid (being the major part in Vitamin C) in eight independent pools of tomato juice was determined by the 2,6-Dichloroindophenol titrimetric method [67]. An aliquot of 5 mL of pooled tomato juice was diluted with 5 mL of water and was titrated by the dye solution until the color changed. Data were expressed as mg of ascorbic acid per gram of fresh weight.

Carotenoids (lycopene and *β*-carotene) were determined according to the Nagata and Yamashita [68] method following modification [69]. Eight individual samples (each sample pooled of two fruits) were examined per treatment and storage period. Thus, 1 g of blended tomatoes were placed in 50 mL falcons and stored in −20 ◦C till analysis (within 48 h). A volume of 16 mL of acetone:hexane 4:6 (*v:v*) were added to each sample, the samples were shaken vigorously and the two phases were separated automatically. An aliquot was taken from the upper solution for measurement of optical density at 663, 645, 505, and 453 nm in a spectrophotometer, using a reference acetone:hexane (4:6) ratio. Lycopene and *β*-carotene contents were calculated according to the Nagata and Yamashita [68] equations:

Lycopene (mg 100 mL−<sup>1</sup> of extract) = <sup>−</sup>0.0458 <sup>×</sup> A663 + 0.204 <sup>×</sup> A645 + 0.372 <sup>×</sup> A505 <sup>−</sup> 0.0806 <sup>×</sup> A453.

<sup>β</sup>-carotene (mg 100 mL−<sup>1</sup> of extract) = 0.216 <sup>×</sup> A663 <sup>−</sup> 1.22 <sup>×</sup> A645 <sup>−</sup> 0.304 <sup>×</sup> A505 + 0.452 <sup>×</sup> A453.

Results were expressed as nmol per gram of fresh weight.

#### *4.6. Total Phenols and Antioxidant Activity*

Eight individual samples (each sample pooled of two fruits) were examined per treatment and storage period. Samples of 5 g were milled in an Ultraturrax (T25 digital ultra-turrax, IKA, Germany) with 10 mL methanol (50% *v/v*) for 30 s, and polyphenol extraction was assisted with ultrasound (Ultrasonic cleaning baths-150, Raypa, Spain) for 5 min. The slurry was centrifuged for 30 min on 5000× *g* at 4 ◦C (Sigma 3–18 K, Sigma Laboratory Centrifuge, Germany). The supernatant was transferred to a 15 mL falcon tube, and was stored at 4 ◦C until analysis (within 48 h) for evaluation of total phenolic content and total antioxidant activity.

The total phenols content of the methanolic extracts was determined by using Folin– Ciocalteu reagent (Merck), according to the procedure described by Tzortzakis et al. [70]. Briefly, 125 μL of plant extract was mixed with 125 μL of Folin reagent. The mixture was shaken, before addition of 1.25 mL of 7% Na2CO3, adjusting with distilled water to a final volume of 3 mL, and thorough mixing. After incubation in the dark for 90 min, the absorbance at 755 nm was measured versus the prepared blank. Total phenolic content was expressed as μmol of gallic acid equivalents (GAE) per gram of fresh weight, through a calibration curve with gallic acid. All samples were analyzed in triplicate.

A sample of 3 mL of freshly prepared ferric-reducing antioxidant power solution (0.3 mol L−<sup>1</sup> acetate buffer, pH 3.6), containing 10 mmol L−<sup>1</sup> TPTZ (Tripyridil-s-triazine) and 40 mmol L−<sup>1</sup> FeCl3·10H2O and 20 <sup>μ</sup>L of extract (50 mg mL<sup>−</sup>1) were incubated at 37 ◦<sup>C</sup> for 4 min and the absorbance was measured at 593 nm. The absorbance change was converted into a FRAP value, by relating the change of absorbance at 593 nm of the test sample to that of the standard solution of trolox ((±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2 carboxylic acid). Standard curve was prepared using different concentrations of trolox, and the results were expressed as mg trolox per gram of fresh weight [69]. All samples were analysed in triplicate.

Radical-scavenging activity was determined according to Wojdylo et al. [71] with some modifications. The 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity of the plant extracts was measured from the bleaching of the purple-colored 0.3 mM solution of DPPH. One milliliter of the DPPH solution in ethanol, 1.98 mL (50% *v/v*) methanol and 0.02 mL of plant extract were mixed. After shaking, the mixture was incubated at room temperature in the dark for 30 min, and then the absorbance was measured at 517 nm. The results were expressed in mg trolox per gram of fresh weight. All samples were analyzed in triplicate.
