The fruit sample used in this experimental study was Musa Spp. (locally known as Sagor kola), a hybrid developed by Bangladesh Agriculture Research Institute (Azam et al., 2010). Both green and ripe banana samples were collected from the local wholesale market (Sadarghat). Ripe banana samples were also collected from banana orchards. In this experimental study, reagent grade sodium hydroxide, vitamin C, hydrochloric acid and sulfuric acid (MERCK KGaA, Germany) were used. Calcium carbide ethephon and ethylene glycol were collected from local markets in Dhaka, Bangladesh. Spectrophotometry (HACH DR-6000 UV-Vis spectrophotometer), multi-gas analyzer (NOVA model600), XRF (Shimadzu, XRF-1800) and Scanning Electron Microscopy (acceleration voltage: 5 kV; JSM-7600F SEM) were used for elemental analysis of different ripening agents. Experiments were carried out at room temperature (26°C).
3.1. Sample treatment Different treatment methods to artificially ripen green banana samples are discussed below. 3.1.1. Ethephon treatment Unripe green banana samples were treated with 50 ml of ethephon solution (300 ppm). Treated banana samples were kept in a confined space for 24 h. Afterwards, they were exposed to open air. 3.1.2. Kerosene treatment Banana samples were placed in an airtight container (50 L). An open-lid kerosene lantern was placed at the center of the container and was allowed to burn until the oxygen inside was exhausted. Banana samples were kept in the container for 24 h and then exposed to open air.
3.1.3. Calcium carbide treatment Fresh unripe banana samples were placed in a container (25 L). Then 10 g of calcium carbide powder was placed at the middle of the container. It took around 30 h for the samples to start ripening. After 48 h, the samples were fully ripened. 3.1.4. Ethylene glycol treatment Ethylene glycol solution (20 weight percent, wt%) was applied on the outer surface of the unripe green banana samples using a brush. Then the samples were kept in an open-lid container (25 L). It took around 48 h for the samples to be completely ripened.
3.2. Experimental techniques: Experimental techniques to determine ripening condition, moisture content, TTA, vitamin C, sugar contents of banana samples, and toxicity and diffusivity of ripening agents are discussed in the following sections. 3.2.1. Determination of ripening condition The degree of ripening was determined using starch-iodine test (Blanpied & Silsby, 1992; Ministry of Justice, Canada, 2009, UC Davis, 2010; Dwivany et al., 2012; Hollo and Szejtli 1958). The Starch-iodine test is widely used to determine the maturity of fruits in which amylase present as starch in the unripe fruits produces violet color in the presence of iodine (Hollo & Szejtli, 1958). After dipping the banana samples into iodine solution; the starch content of the unripe samples gave violet color, whereas the ripe samples showed no color change. This is because starch is converted to sugar with the progression of ripening process (Bouzayen et al., 2010). So a ripe fruit will show no color when strained with iodine but an unripe fruit will.
3.2.2. Determination of moisture content The moisture content was determined by oven-dry method (Abano and Amoah 2011; Canet., 1988; Nielsen, 2010; Senthilkumar et al., 2005). Freshly cut banana samples were dried in an oven (National, NB-7500E) at 110°C until a constant weight was reached. Weight readings of the samples were taken before and after drying; the weight difference denoted the amount of moisture in the samples. The moisture content is expressed in gram of moisture available per 100 g of fruit sample (g of moisture/ 100 g of fruit stem) (Shahnawaz, Sheikh, & Nizamani, 1980).
3.2.3. Determination of TTA TTA was measured using the method (method 22.060 of 13th edition) described by the Association of Official Analytical Chemists (AOAC), USA (AOAC 1980). Juice from banana samples was titrated with standardized 0.1M NaOH solution using phenolphthalein indicator. The resultant acid content from the titration is multiplied by the citric acid factor and the result is expressed in gram citric acid per 100 g of fruit stem (John Bean Technologies Corporation 2011). 3.2.4. Determination of vitamin C Vitamin C (ascorbic acid) concentration was determined by redox titration using standardized iodine solution (Majidi and Y-AlQubury 2016; Munir et al., 2013; Mussa & Sharaa, 2014; Patel, 2017; Said et al., 2016). In this method, ascorbic acid in fruit reacts with iodine to produce dehydroascorbic acid and iodide ions (Equation (1)) (Mussa & Sharaa, 2014; Patel, 2017). Excess iodine then reacts with the starch indicator to produce violet color and indicate the end point of titration (Mussa & Sharaa, 2014; Patel, 2017). The results obtained are expressed in ppm (mg/L).
3.2.5. Determination of sugar content The sugar content of the fruit samples was determined by measuring the refractive index of the samples (Cejpek, 2012). A KRUSS (Germany) refractometer was used to measure the refractive index of the banana solutions (10 wt%). Afterward, sugar content (wt%) was determined from this refractive index.
3.2.6. Analysis of ripening agents X-ray fluorescence spectrometer (Shimadzu, XRF-1800) was used for the elemental analysis of calcium carbide. During elemental analysis, high amount of sulfur was found in calcium carbide samples. Energy-dispersive X-ray spectroscopy (EDS, JSM-7600F SEM) was used to cross-check the value of elemental sulfur in calcium carbide samples. EDS was done using a field emission secondary electron microscope (FESEM; JSM-7600F SEM). Kerosene fume was analyzed using a NOVA model 600–8 gas analyzer. Kerosene was burned in a confined space (25 L), fitted with a small stack. The fume was collected from the stack and analyzed using the gas analyzer. Sulfur content in ethephon and ethylene glycol was determined using a HACH DR-4000 UV-Vis spectrophotometer (Company 2018) (USEPA). 3.2.7. Analysis of diffusion of sulfur from ripening agent to fruit To understand the diffusion of sulfur from calcium carbide to the flesh and peel of fruits, sulfate and sulfide content of fresh and carbide-treated banana samples (flesh and peel) were measured using HACH DR-6000 UV-Vis spectrophotometer (Company 2018, Company 2018) (USEPA).