METHODOLOGY
Raw Material Preparation (Sample Extraction )
Without adding water, fresh banana peel is homogenize with a Politron PT 6000 (Kinematica AG, Lucerne, Switzerland) high speed blender at 12,000g for 1 min
Drying Experiment
Drying was done at three temperatures (50 oC, 70 oC, and 90 oC) with a constant air-flow rate of 2.0 ± 0.2 m/s (in triplicate for each temperature). A convective tray dryer was used, with which the temperature and air flow can be controlled. The load density was 30 ± 0.5 kg/m2. The samples were dried until they reached a constant weight (equilibrium condition). The dehydrated samples were then packed and sealed in polyethylene bags. (M. Miranda et al., 2009)
Solvent Extraction
Twelve sets of dry banana peel samples, approximately 2 g each are prepare. Each of extraction process is done at least in triplicate (3-9 times). Firstly, the samples are extracted with 25mL of 70% acetone each using a shaker at 400 rpm for 1 h. The extracts then centrifuged at 5000g for 20 min in a Jouan CR-312 centrifuge (Thermo Electron Corporation, Madrid, Spain). The supernatant then dry and use for the analysis of antioxidant activities, phenolic compounds and minerals. The extractions are carried out in sealed tubes in a water bath for 0min, 60min or 120 min (extraction temperature of 25 or 55 oC). All the sealed tubes are cover with foil paper to perform all the operations under reduced light. The extracts obtained were stored at 80oC for less than three days, at which time the antioxidant potential, extraction yield and bioactive compounds were estimated. The type and polarity of the solvent used to extract antioxidants from banana peel can affect single electron transfer and hydrogen atom transfer, which are key aspects in the measurements of antioxidant capacity (R. González-Montelongo et al., 2010)
Antioxidant Activities
The FRAP assay was done according to (Benzie and Strain, 1996) with some modifications. The stock solutions included 300mM acetate buffer ( 3.1 g C2H3NaO2.3H2O and 16mL C2H4O2 ), pH 3.6, 10mM TPTZ (2, 4, 6- tripyridyl-s-triazine) solution in 40mM HCl, and 20Mm FeCl3.6H2O solution. The fresh working solution was prepared by mixing 25mL acetate buffer, 2.5mL TPTZ solution, and 2.5mL FeCl3.6H2O solution and then warmed at 37 oC before using. Fruit extracts (150 µL) were allowed to react with 2850 µL of the FRAP solution for 30 min in the dark condition. Readings of the colored product [ferrous tripyridyltriazine complex] were then taken at 593 nm. Ascorbic acid were used as standards to calibrate the methods. Additional dilution was needed if the FRAP value measured was over the linear range of the standard curve. All measurements to determine antioxidant activity were made on a Shimadzu UV-visible 160A double-beam spectrophotometer (Kyoto, Japan) equipped with a Hellma (Jamaica, NY) cell (pathlength 10_2 m).
Ascorbic Acid Content
Ascorbic acid in the extracts was determined using LC with UV-visible detection on a Spherisorb ODS-2 RP-C18 (Alltech) column (5 lm particle size, 250 _ 4.6 mm i.d.). A 0.2 M potassium phosphate monobasic solution in Milli-Q water was used as mobile phase (pH adjusted to 2.4 with orthophosphoric acid) at a flow rate of 0.5 ml/min. Detection wavelength for the detector was set at 254 nm.
Phenolic content
The content of soluble phenols was measured using a modified (Folin and Ciocalteu, 1927) method. Briefly, an aliquot (0.5 ml) of the extract, blank or standard was placed in a 25 ml flask, where the Folin-Ciocalteu reagent (0.5 ml) was added and the mixture was allowed to react for 3 min under continuous stirring before a solution of sodium carbonate (75 g/l, 10 ml) was added and mixed well. The volume was then made up to 25 ml with distilled water and left standing at room temperature for 1 h. The absorbance was then measured at 750 nm using a Shimadzu UV-160A spectrophotometer (Kyoto, Japan). The results were expressed as gallic acid equivalents (GAE), using a calibration curve over the range of 50-200 ppm.
Minerals
An atomic absorption spectrophotometer by PerkinElmer, model 3100, equipped with hollow cathode lamp was used. From the homogenized sample, 2g were taken for the study of mineral content. Calcium, iron, magnesium and potassium were calibrated against known standards using atomic absorption.
