Collection and preparation of materials Freshly harvested mature banana (Musa acuminata), carambola (Averrhoa Carambola) and tomato (Solanum lycopersicum) were collected from the commercial farms around Khulna city, Bangladesh. A total 36 of each type of fruits were collected during the period of October to November, which were spotless. The fruits were almost uniform in shape and color. Average weight of the selected banana, carambola and tomato were 105±0.86, 112±0.56 and 120±0.76 g, respectively.
Extraction of chitosan from chitin The chitin was collected from the Forestry and Wood Technology Lab., Khulna University, Bangladesh. In order to prepare the chitosan, firstly the acetyl groups were removed from the chitin by deacetylation process (Puvvada et al., 2012). The purified chitin was further treated in a hot water bath at 100°C for 2 hrs with 50% NaOH solution where the mixing ratio of chitin and NaOHwere 1:25 (gmL-1). Afterwards, the resulted chitosan washed with water and conversion of chitosan from chitin was completed by dissolving the chitosan into ethanol. To form the slurry, chitosan dispersed in water having a concentration of 1, 1.5 and 2 wt%. Then a high-speed blender (Vita-Mix Blender) was used to blend the suspension for 10 minutes with 37000 rpm rotating speed and thus the chitosan was prepared.
Extraction of extracts from guava leaves Mature green guava leaves were collected from a commercial guava farm around Khulna city, Bangladesh. The leaves were washed with distilled water to remove the impurities. Afterward, 250 g guava leaves were transferred to 2000 ml water and boiled at 80°C for 3 hours. Then, the temperature of the solution was maintained at 70°C to evaporate it until 0.5% concentration of the extracted solution (Seo et al.,2014).
Preparation and application of preservatives Three different concentrations, viz. 1.0, 1.5 and 2.0% of chitosan were prepared by adding water in it to apply on fruits as preservative (Herna´ndezMun˜oz et al.,2008). The extracted guava leaves extract having 0.5% concentration were also prepared earlier. These four solutions were sprayed uniformly and separately on the collected fruits with a hand pump sprayer. The fruits were then allowed to dry at room temperature. Untreated samples were taken as control. All the fruits were stored in an open and safe place which is free from insects. The storage duration was determined by considering the complete perishtime of the control fruits.
Evaluation of the fruit quality Collected each type of fruits were grouped into fresh (6), control (6) and remaining 24 for the treatments. Fresh fruit samples were analyzed immediately after collection while remaining samples analyzed after 12 days of storage to compare the effectiveness of the treatments.
Peel color, flavor and firmness Peel color, flavor, firmness and overall acceptability of the fruits were assessed by a panel consists of 10 members. The members were selected randomly from the faculty members of Life Science, School of Khulna University, Bangladesh. The fruits were assessed by comparing with the original color, flavor and firmness and the panel members filled a questionnaire (Brishti et al.,2013).
Measurement of pH of the fruit juice To determine the pH value of the fruit juice, the sample frutis were peeled and blended separately by a digital blender (Vita-Prep 3®) having 37000 rpm to prepare juice. The pH meter (Benchtop pH meter) was used to measure the pH value of the fruit juice (Brishti et al., 2013).
Determination of total carbohydrate content: Total carbohydrate content of the fruits were measured separately by using the Lane-Eynon method (Okoye et al., 2008). The fruit juice (carbohydrate solution) was gradually poured to a flask containing copper sulfate solution and a methylene blue indicator. The carbohydrate solution reacts with the copper sulfate present in the flask. Once all the copper sulfate solution was reacted, any further addition of reducing sugars causes the indicator to change from blue to white. The volume of sugar solution required to reach the end point was recorded and total carbohydrate was determined.
Determination of protein content The protein content of fruit samples were estimated by Lowry’s method using a standard curve of Bovine Serum Albumin (BSA) solution (20-100 Mg/ml) and absorbance at a wavelength of 660 nm using a double beam UV-Visible spectrophotometer.
Measurement of percentage disease index (PDI) PDI was used to observe the effectiveness of preservatives on fruit samples in retarding fruit disease. Among the three different fruits, only banana was used for PDI because of its noticeable appearance at outer surface and thus, for easy detection of color change. PDI was assessed by a scanner (Canon DR M140) and Adobe Photoshop (Version CS6) software (Hossain et al., 2010). The banana peel was scanned and analyzed the percentage of yellow and black portion, which were the results for PDI.
Statistical analysis Analysis of the data was carried out by using SAS system software (version 8.1) at 95% confidence level. The significance of differences among treatment means was determined by analysis of variance (one-way ANOVA) followed by least significant difference (LSD) test.