*2.4. Determination of Weight Loss, General Appearance, Firmness, and Total Soluble Solids*

Green bean samples were weighed immediately after (drying in a laminar airflow hood for 2 h) and at every sampling time to measure weight loss by using a digital laboratory scale. Another set of the green bean samples of 250 g each in triplicates) were used for further chemical analysis. To determine general appearance, the following scale was used: 9 = excellent, 7 = very good, 5 = good, 3 = poor, and 1 = unacceptable. The appearance score was assessed by a group of three trained laboratory panelists.

The firmness values of each pod were determined at three different points by using the digital penetrometer (PCE-PTR 200, PCE Americas Inc., Jupiter, FL, USA) with 6 mm diameter probe (range 0 to 1 kg\*) [26]. The firmness values were expressed in Newton (N). To determine total soluble solids (TSS), a digital refractometer (model PR101, Atago [0–45%] Co. Ltd., Tokyo, Japan) was used at room temperature [27].

#### *2.5. Determination of Total Sugar, Vitamin C, Total Phenolic Compounds, and Chlorophyll Content*

The total sugar content was determined by the anthrone method at 630 nm as described in [28]. Briefly, 200 mg fruit were extracted three times with ethanol (80%) at 80 ◦C. Then, the extracts were evaporated to dryness and redissolved in 2 mL distilled water. One millilitre of sample extracts was added to 1.5 mL of anthrone reagent (0.2% in H2SO4) and mixed thoroughly. The sample was brought to boil using a boiling water bath. The solution was cooled to room temperature and absorbance was measured. The formation of the blue-green complex indicates the presence of total sugars. Glucose was used as a standard. Vitamin C content was determined using the titrimetric method with 2,6-dichlorophenolindophenol described by the Association of Official Analytical Chemistry [29].

TPC was calculated by using the Folin–Ciocalteu reagent with some alteration by using gallic acid as a standard curve [17]. Five grams of the sample was diluted using 5 mL of methanol (80%). The solution was blended with 2.5 mL of Folin–Ciocalteu (10-fold with distilled water) and added to 2.5 mL of distilled water. Afterwards, 2 mL of aqueous sodium carbonate solution (7.5%, *w*/*v*) was added after incubation for 5 min. The final solution was mixed and incubated in the dark at room temperature for 1 h. The absorption was assessed at 765 nm using the spectrophotometer, and the results were expressed as milligrams of gallic acid equivalent (GAE) per 100 mg of fresh fruit weight.

Chlorophyll content was determined as described in [30]. In brief, 0.5 g of fresh sample were homogenized with 5 mL dimethyl formamide and kept in the dark in the refrigerator for 48 h. The absorbance was then measured at 470, 647 and 663 nm with a spectrophotometer (model UV-2401 PC, International Equipment Trading LTD. (IET), Milano, Italia).

#### *2.6. Determination of Mould, Yeast and Total Microbial Count*

Ten grams of each treatment were homogenized with 90 mL sterile saline for 2 min by a stomacher (Stomacher BW-400P, Turelab, Shanghai, China). Total count and mould and yeast count (MY) were enumerated on plate count agar and potato dextrose agar after incubation at 37 ◦C for 48 h and 28 ◦C for 5 d, respectively [31]. The results were expressed as log10 of colony-forming units per gram sample (CFU g<sup>−</sup>1).
