2.1. Sample collection Ashwina variety of mango at full mature and ripen stages was collected from Fruit Research Institute, Rajshahi. After discarding the disease associated and injured ones, the mangoes were divided into two groups, unripe (mature green) and full ripe. They were finally stored at -20°C for analysis. Unless stated otherwise, all the reagents, synthetic and other substrates used for performing the experiments were from Sigma-Aldrich and CarlRoth GmbH, Germany.
2.2. Extraction of fruit juice from mango About 50 g of fruit tissue was homogenized with a blender and filtered through two layers of muslin cloth. The filtrate was then centrifuged for 20 min at 8,000 g at 40C and the clear supernatant was collected.
2.3. Total soluble solids (TSS) and titratable acidity (TA) TSS was determined according to the method as described by Mazumdar et al. using Digital-Bench-Refractometer (Range 0-32%) [6]. An appropriate quantity of each sample was placed on the prism-plate of the refractometer and the reading appearing on the screen was directly recorded as total soluble solids (°Brix). Titratable acidity (TA) was determined according to the method as described by Hortwitz. 10 mL of crude extract was taken in a beaker and titrated with 0.1N NaOH after adding 2-3 drops of phenolphthalein as an indicator.
2.4. Vitamin C Approximately 2-3 g of mango flesh was cut into small pieces and homogenized well with 20 mL of 3% meta-phosphoric acid and filtered through double layer of muslin cloth. The filtrate was centrifuged at 3,000 g for 10 min and the clear supernatant was titrated with 2, 6-dichlorophenol indophenol solution as described elsewhere. The amount of vitamin C in the extract was determined by comparing with the titration curve of standard vitamin C solution. Result was expressed as mg/100 g of fresh fruit.
2.5. Starch content The starch content of the mango flesh was determined by the Anthrone method. 2 g of mango was cut into small pieces and homogenized well in 20 mL water. It was then filtered through double layer of muslin cloth. Excess ethanol was added to the filtrate to precipitate the polysaccharide, mainly starch. After keeping overnight at 40C, the precipitate was collected by centrifugation at 3,000 g for 15 min. The precipitate was heated to dryness and dissolved in 40 mL of 1M HCl and then heated at 70°C for few min. It was then transferred to a volumetric flask and diluted to 100 mL with 1M HCl. Aliquot of 1 mL of the extract of each sample was pipetted into test tubes and 4 mL of the anthrone reagent was added to each test tube and mixed well. The tubes were placed in a boiling water bath for 10 min and cooled. A blank reagent was prepared by using 1 mL of water and 4 mL of anthrone reagent in a test tube and treated similarly. The absorbance of the blue-green solution was measured at 680 nm in UV-VIS spectrophotometer (U-1800, Hitachi, Japan). The amount of starch present in mango flesh was calculated from the standard curve of different concentrations of glucose and expressed as g/100 g of fresh fruits.
2.6. Total sugar (TS), reducing sugar (RS), non-reducing sugar (NRS) and phenol content (PC) 2 g of mango flesh was cut into small pieces and plunged into 20 mL of boiling ethanol for 5-10 min. After cooling, it was crushed thoroughly in a mortar with a pestle. The homogenate was filtered through two layers of muslin cloth and re-filtered through a Whatman no-41 filter paper. The extract was evaporated to dryness over a steam bath and subsequently cooled. The residues were dissolved in 100 mL of distilled water and the resulting solution was used as flesh extract (sample stock) for the estimation of total sugar (TS), reducing sugar (RS) and phenol content (PC).
2.7. Enzymatic analysis The activity of amylase, invertase, polyphenol oxidase, β -glucanase, β-galactosidase, α- mannosidase, and β-hexosaminidase was assayed.