2.1. Collection and Preparation of Sample The study was conducted on five commonly consumed fruits in Bangladesh: Apple (Malus domestica), Grape (Vitis vinifera), Pear (Pyrus xbretschneideri), Plum (Ziziphus mauritiana), Guava (Psidium guajava) were collected from new market area, Agargaon, Dhaka, Bangladesh. The samples were washed with running tap water, as usually practiced in domestic kitchens. After washing, the sample were peeled with a sterile peeler then uniformly sliced with a sterile knife on a clean sterile chopping board and cut into uniform slices.
2.2. Irradiation Treatment The samples were packed into sterilized (with 25 kGy radiation dose by 60Co gamma irradiator) food grade transparent low-density polyethylene (LDPE) and then sealed with a heat sealer (Impulse Sealer, TEW Electronic Heating Equipment CO. Ltd., Taiwan). Four packed of samples each containing 50g were irradiated with three different irradiation doses 0.5, 1.0 and 1.5 kGy and four sets (day-0, day-2, day-4 and day-6) of samples were irradiated at the same doses for evaluation of microbiological, biochemical and organoleptic analysis, respectively. The samples were reserved at 4±1°C in the refrigerator for further evaluation. Non-irradiated sample of each type of fruit was kept as a control sample. All the procedures were done inside laminar airflow cabinet. Doses were applied to the samples at room temperature from the Co-60 gamma irradiator source (Located at Atomic Energy Research Establishment, Institute of Food and Radiation Biology, Dhaka, Bangladesh) by calibrating with dose and time basis on central distance from source to sample where these were placed.
2.3. Microbiological Analysis Determination of microbial population in the sample decimal dilution technique followed by pour plating method was used on the day of irradiation. Stock solution were prepared by taking 5 g (25 g for Listeria spp) of homogenized and filtered sample in 50 ml of sterile saline (0.9% NaCl wate) by an autoclaved mortar and pestle and filtered through a sterile muslin cloth to a conical flask with 50 ml sterilized saline (0.9% NaCl) water to prepare the stock sample under a laminar air flow cabinet. For total aerobic spore count these suspensions were heated at 80°C for 10 min in a water bath. 1 ml sample from conical flask was taken in a test tube containing 9 ml of previously sterilized saline water. Thus 10-1 dilution was got. This procedure was repeated where further dilution was required. With the help of micropipette, 1 ml of the sample from the test tube was poured into Petri dishes then sterilized specific media was poured into Petri dishes and shaken horizontally to spread out the sample uniformly over the media. After solidification of the media the Petri dishes were covered with lids. Then the Petri dishes were placed in upturned position in incubator at 37°C (30°C for yeast and mold) for 24-48 hr. The analyses were enumeration of total aerobic flora, total anaerobic bacteria, total aerobic spore, total yeast and mold, total coliform, Listeria spp. and Staphylococcus aureus. For microbiological purposes Nutrient Agar, Thioglycollate media, Potato Dextrose Agar, MacConkey Agar, Mannitol Salt Agar were purchased from Scharlau Chemie S.A. (Spain). Listeria Selective Agar Base (Oxford formulation) was purchased from Oxoid LTD (England). For anaerobic bacteria Thioglycollate media was used. After spreading, plates were kept into an anaerobic jar. The lid of the jar was closed. After that a vacuum pump was attached to one port of the jar, and a nitrogen source was attached to another port of the jar. Then the air inside the jar was sucked out with vacuum pump and the jar was filled with nitrogen gas to maintain anaerobic condition inside the jar. Then the jar was put inside the incubator.
2.4. Biochemical and Nutritional Analysis 2.4.1. Determination of Ascorbic Acid and Total Carotenoid The estimation of ascorbic acid content was carried out by the titration result of the sample extract with 2, 6- Dichlorophenol-Indophonol dye (Oxford formulation, Oxoid LTD, England) which is reduced by ascorbic acid to colorless form in pH range 1-3.5 [18,19]. For total carotenoid estimation 4 g of homogenized sample was transferred to a mortar and mixed 0.3 g of MgCO3 and 25 ml of cold acetone (Refrigerated about 2 Hours) to prepare acetone extract by filtration. Twenty (20 ml) of petroleum ether was taken in separating funnel and 15 ml of acetone extract were added to allowed 15 min. After that 150 ml of distilled water was added and allowed to separate two phase. After discarding aqueous phase the ether phase was collected in a 25 ml of volumetric flask containing 7.5 g of anhydrous sodium sulfate to remove residual water. The flask was filled up to volume with petroleum ether and total carotenoid were determined from the molar absorptivity β-carotene E1% = 2590 at λmax450 nm and lycopene E1% = 3450 at λmax 472 nm derived from the standard plots [21,21]. 2.4.2. Determination of Moisture Content The moisture content was determined by drying the sample at same elevated temperature and reporting the loss in weight as moisture.
2.5. Organoleptic Analysis Organoleptic analysis was carried out followed by the method described by Miyauchi et al., 1964. Following nine points of hedonic scale was used for sensory evaluation by twenty trained judge’s age between 20 – 39. 9= Like extremely 8= Like very much 7= Like 6= Like slightly 5= Neither like nor dislike 4= Dislike slightly 3= Dislike 2= Dislike very much 1= Dislike extremely Average sensory score 5 (neither like nor dislike) is usually acceptable in organoleptic evaluation. So, the acceptability threshold we considered was around 7, which means “like” in hedonic scale. Sensory quality attributes including colour, flavour, taste and texture of samples were evaluated immediately after irradiation and during refrigeration (4 ± 1°C) storage.
2.6. Statistical Analysis Results were expressed as mean ± SD (Standard deviation of mean). One way ANOVA was performed for data analysis. ANOVA was followed by Fisher’s Least Square Differences (LSD) for post hoc comparisons. The statistical program used was Microsoft Office Excel 2010 and its add-in DSAASTAT (Andera Onofri, Dipartimento di scienze Agrarie Ambientali, Borgo xx Giugno, 7406121 Perugia, Italy). P<0.05 was considered statistically significant.