Extension of Shelf-Life of Tomato Using Irradiated Chitosan and its Physical and Biochemical Characteristics

. The effect of irradiated chitosan coating on post-harvest preservation of tomato was observed in this study. Irradiated chitosan (40 kGy) solution of various concentrations (500, 750, 1000, 1500 and 2000 ppm) were applied on post-harvest preservation of tomato. Both chitosan treated and untreated (control) tomato were stored at room temperature in open and zip bag conditions. The effect of coating of various chitosan solutions on tomato was observed during storage period. The percentage of weight loss and spoilage rate of the preserved and control tomato samples were investigated. Several parameters (such as total bacteria count, total mold count, moisture, ash, acidity, vitamin C, sugar, protein and fat) were analysed for irradiated chitosan coated tomato in open condition after 3-weeks storage period. In addition, the same parameters were also analysed for control tomato. Considering all parameters, the results revealed that 1500 ppm chitosan solution performed better in extending the shelf-life of tomato as compared to the control and other treated samples. Thus, this observation recommend that irradiated chitosan coating have the potential to be used as natural preservative to maintain quality and extending shelf-life of tomato.


Introduction
Tomato (Lycopersicon esculentum) is the most common vegetable crop in all areas of tropical countries in the world and is available throughout the year [1]. It is produced mainly for the consumption of fresh vegetable, juice, jams, sauces or salad. As a tropical commodity, the storage suffers from severe problems, which results in their rapid deterioration and high economic losses. It is found that the main culprit is microbiological diseases. A careful management is necessary to control diseases and safely preservation of post-harvest tomato for consumer. However, the shelflife of tomatoes vary depending on storage conditions. It ranges from 4 to 8 days at room temperature [2]. This short period seriously limits the long distance commercial transport of tomato. Researchers are working on several methods in order to minimize the postharvest losses of fruits and vegetables and consequently extend its shelf-life, such as, application of fungicides, heat treatment, irradiation, use of bio-control agents, use of natural coating (essential oil, chitosan) and others [3][4][5][6][7]. In addition, the use of irradiated chitosan coatings on strawberry fruits were reported that greatly extend the shelf life due to antimicrobial properties of irradiated chitosan [8]. These coatings act as barriers to water loss and gas exchange by creating a micro-modified atmosphere around the product [7,9,10]. The use of chitosan coatings has been successfully investigated to extend the shelf life of several food products, such as, mangoes [11], water caltrop [12], Chinese water chestnut [13], fruit based salad [14]. Recently, another review reported that chitosan films and coatings prevent losses of fresh fruit nutritional quality [15].
Chitosan is a linear amino polysaccharide of glucosamine and N-acetyl glucosamine units and is obtained by alkaline deacetylation of chitin extracted from the exoskeleton of crustaceans such as shrimps and crabs, as well from the cell walls of some fungi [16]. Chitosan, as the most abundant naturally occurring amino-polysaccharide, possesses many of these attributes and has attracted attention because of its unique physiochemical characteristics and biological activities [17,18,16 ]. From a biological standpoint, chitosan and its derivatives are very attractive for agriculture applications, which are closely related to human safety and fitness. Owing to its high biodegradability, nontoxicity, and antimicrobial properties, chitosan is widely-used as an antimicrobial agent either alone or blended with other natural polymers. The antimicrobial activity depends on several factors such as molecular weight, degree of deacetylation, solubility, positive charge density, chemical modification, pH, concentration, hydrophilic/hydrophobic characteristic, chelating capacity, and type of microorganism. Early research reported the antimicrobial potential of chitin, chitosan, and their derivatives dated from the 1980-1990s [16,[19][20][21][22][23]. The antimicrobial activity of chitin, chitosan and their derivatives against diverse groups of microorganisms, such as bacteria, yeast and fungi have been receiving great attention over recent years [24][25][26]. In addition, the antimicrobial activities of chitosan are greatly depending on molecular weight and degree of deacetylation (DD). Another report suggested that chitosan having a higher degree of deacetylation (DD) tends to have higher antimicrobial activity [27]. There are some related works reported the preservation of tomato [28][29][30][31][32] but no specific observation has been reported yet for extending shelf life of post-harvest tomato treated with irradiated chitosan (40 kGy) solution in this manner. This research was aimed to investigate the effect of several concentrations of irradiated chitosan coating on post-harvest tomato for extending shelf-life as well as evaluating the quality changed during its storage period at ambient temperature in open condition.

Extraction and Irradiation of chitosan
Chitosan was extracted from prawn shell in Institute of Radiation and Polymer Technology (IRPT) laboratory. High molecular weight chitosan was dissolved in 2% acetic acid solution to make a homogeneous chitosan solution. Chitosan solution (2% w/v in 2% acetic acid) was gamma irradiated at 40 kGy by a 120 kCurie radiation sources at 3.2 kGy per hour dose rate. Other chemicals used were laboratory grade.

Preparation of irradiated chitosan solution
Irradiated chitosan solution varying the concentrations (500, 750, 1000, 1500 and 2000 ppm) were prepared in distilled water to apply for coating over a wide range of tomato samples.

Source of tomatoes and preservation by irradiated chitosan solution
Matured green tomatoes were directly collected from local cultivator and brought to the laboratory. The tomatoes were selected without any infection and damage. The selected tomatoes were coated by immersing them in 500, 750, 1000, 1500 and 2000 ppm irradiated chitosan solution for two minutes respectively. The control consisted of tomatoes without washing or immersing in chitosan solution. After air drying at room temperature, tomatoes were stored at ambient temperature in open and in zip bag. The control samples were also stored at the same conditions. The room temperature variation was 25-28 °C and the relative humidity of the air ranged from 70 to 85 % during the whole storage period.

Percentage of weight loss
At the first day of sampling, the initial weight of all chitosan coated and control tomatoes were recorded. In a definite time, the weight losses of tomatoes were checked and recorded up to fresh International Letters of Natural Sciences Vol. 67 condition. The percentage of weight losses in a given time intervals were calculated as the total weight loss divided by initial weight multiplied by 100.

Spoilage rate
The spoilage of tomatoes due to fungal or any other microorganism infection was counted every 7 days interval and recorded as percentage. 500 g (average 6 pieces) tomatoes were observed for each batch. The percentage of spoilage rate was calculated as the number of spoiled samples divided by the initial number of all tomatoes multiplied by 100.

Microbiological (Total bacteria count and Total mold count) analysis
Bacterial and fungal counts were performed by Plate Count Agar (PCA) and Potato Dextrose Agar (PDA) media respectively. 1 g tomato sample was mixed in 10 mL of saline water and the 10 µL sample was spread in PCA and PDA plates. The plates were incubated at 37 0 C and 25 0 C for 24 hours respectively. Then the colony forming units (cfu) were counted. All cfu counts were performed in triplicate.

Chemical analysis
Various irradiated chitosan solution coated tomato samples in open condition were analyzed for different parameters (such as moisture content, ash content, acidity, vitamin C, sugar, protein, fat) after three-weeks storage periods by standard methods. On the other hand, the control tomatoes were also analyzed for the same parameters within a week after preservation. All experiments were performed in triplicate.

Characterization of irradiated chitosan solution
The average molecular weight and the degree of deacetylation (DD) of irradiated chitosan was determined by viscometric method through Mark Houwink equation and FTIR spectroscopy respectively. The effect of various gamma radiation doses on the average molecular weight and degree of deacetylation of chitosan solution were reported previously by khan et.al [8,33] research group in the institute of radiation and polymer technology (IRPT) laboratory, Bangladesh Atomic Energy Commission. The degree of deacetylation of chitosan at 40 kGy radiation dose was approximately 79 %.

Percentage of weight loss
The weight changes of different chitosan solution coated and uncoated (control) postharvest tomatoes during 6-weeks storage (Fig. 1a) in open and 4-weeks storage (Fig. 1b) in zip bag were observed weekly at ambient temperature respectively. The result showed that 2000 ppm chitosan solution coated tomatoes in open significantly maintained weight compared to control and other treated samples. It was found that the treated and untreated tomatoes exhibited the same rate of weight loss during one-week storage period. One week later, the percent of weight loss of control samples sharply increased with increasing storage period however, it was increased slowly for treated tomatoes. In addition, it was recorded that 29.87 % weight loss for control (shrinkage and dried condition), whereas 10.71 to 18.66 % weight loss for treated tomatoes (almost fresh) depending on chitosan concentration during 3-weeks storage periods. Owing to the high spoilage, the weight loss of control tomatoes was not recorded after 3-weeks storage periods. Although the weight loss of 1500 and 2000 ppm chitosan treated tomatoes were found only 35.68 and 30.48 % at 6-weeks storage periods respectively. In case in zip bag condition, no significant weight loss was found during 4-weeks storage periods but all samples were spoilt (Fig. 1b) due to no evaporation and transpiration. In addition to reducing respiration rate, chitosan coating acts as a hydrophobic barrier and thus prevent evaporation of water from inner cell of tomatoes. Chitosan coating

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improves the other quality such as slower softening, texture changes and color retention. This was similar with the recent study reported that the loss of firmness during storage of tomatoes that could be the result of endogenous enzymes; was linked to the cell wall degradation [28]. Another report showed that chitosan films and coatings prevent losses of fresh fruit [15]. According to the method used in this study, control tomatoes both in open and in zip polybag at ambient temperature lost its shelf life as firmness after 3-weeks and 2-weeks storage periods respectively.

Spoilage rate
One week later, control tomatoes in open condition started suffering from shrinkage, spoilage and dryness but all treated tomatoes were almost fresh up to 4-weeks storage periods. Chitosan treated tomatoes begun to shrinkage, spoilage and dryness after 4-weeks storage period but few control tomatoes were found in compressible and dried condition at 3-weeks storage period. Moreover, chitosan treated tomato samples showed excellent extending shelf life up to 7-weeks (Table 1). International Letters of Natural Sciences Vol. 67 On the other hand, the control tomatoes in zip bag at ambient temperature started spoilage after a week and were completely spoiled after two weeks (Table 2). Whereas the chitosan treated tomatoes in zip bag were found to be edible condition up to 4-weeks storage periods. This finding were attributed due to the antimicrobial activity of irradiated chitosan resulting in increasing the shelf life of tomatoes. This result supported by the previous study of several research groups that reported the antimicrobial activity of chitosan and received great attention [24][25][26][27].

Microbiological analysis (TBC and TMC)
The total bacteria count (TBC) and total mold count (TMC) of control and various chitosan solutions coated tomato in open and in zip bag shown in Fig. 2a and 2b respectively after 3-weeks storage periods. On the other hand, the TMC was 11.0 and 16.5 cfu/g for 1500 ppm chitosan solution coated tomato in the same conditions. Based on the results, it was suggested that irradiated chitosan significantly performed as antimicrobial agent. Hence, this result supported by early reported work that suggested the mechanism of antimicrobial activity of irradiated chitosan is that it prevents the bacteria and mold growth and thus killing them by reducing nutrients and oxygen in the culture medium [29].

Chemical analysis (Nutritional facts)
Moisture content: The moisture content of control and irradiated chitosan (500, 750, 1000, 1500 and 2000ppm) coated postharvest tomato within a week and after 3-weeks storage period shown in Table 3 respectively. The lowest value of moisture content (92.7 %) was observed for control within a week while highest value was 94.2 % for 1500 ppm chitosan coated tomato after 3-weeks storage period. The highest moisture content for treated tomato may be due the fact that irradiated chitosan coating acts as a better protective shield between inner and outer environment of the tomato during preservation period. The similar result was reported that the loss of moisture may be due to high respiration and transpiration rate [2,30] and also reported that chitosan coatings decreased the water vapor permeability [15].  Vitamin C: It was found that vitamin C content remained satisfactory level with the increases of chitosan concentration for a long (after 3-weeks) storage period. The value of ascorbic acid (vitamin C) was 17.6 % for control tomato within a week, whereas it was 14.9 % for 2000 ppm chitosan treated tomato after 3 weeks ( Table 3). The cause for maintaining vitamin C content by chitosan coating may be due to slow ripening rate. In addition, ascorbic levels may be decreased with time due to oxidation of vitamin C. This finding was also supported by a research work on mango that reported the vitamin C was mostly high in mature but unripe mango fruit and it decreased as the ripening time progressed [2]. This finding was also supported by the report that revealed chitosan based edible coatings prevent vitamin C losses in fruits during storage [15]. Acidity: The values of acidity indicate the quality and acceptability of fruits and vegetables, very low and very high values of acidity are not acceptable for good food. This effort revealed that the value of titrable acidity increases with increasing chitosan concentration ( Table 3). The highest value was observed for 2000 ppm chitosan coated tomato due to acidic nature of chitosan solution, and the lowest value was observed for control. In addition, the increasing trend of acidity may have occurred due to an elevation of CO 2 concentration and a reduction of O 2 during its storage period in a modified atmospheric condition caused by the chitosan coating. Sugar: It was observed that the sugar content (Table-3) of chitosan coated tomato slightly decreased with increasing chitosan concentration after 3-weeks storage time. The decreasing trend of sugar in high chitosan concentration coated tomato might be due to slow ripening process. This result agreed with the report that suggested by Moneruzzaman et al. [32]. Ash content: This observation revealed that control sample contained the lowest ash, whereas chitosan coated samples showed ash content in increasing trend with respect to control sample (Table-3). Protein: No significant changes of protein were observed for all tomatoes after 3-weeks storage periods (Table-3). This result might be due to slow metabolism conversion during preservation process.
Fat content: Changing of fat were observed chitosan coated tomato after 3-weeks storage periods (Table-3). The lowering of fat content for chitosan treated tomato with respect to control may be due to the facts that chitosan acted as a fat reducer.

Conclusion
Based on this study, the application of irradiated chitosan appeared to bear a significant potential as a natural preservative for extending the shelf life of postharvest preservation of tomato. It was revealed from the observation that irradiated chitosan coating maintained the quality of preserved tomato during storage period. Moreover, 1500 ppm chitosan solution showed the best result compared to control and others treatment in terms of percentage of weight loss, spoilage rate, total bacterial counts (TBC), total mold counts (TMC) and nutritional facts. Thus, these results recommended that the application of irradiated chitosan solution on postharvest tomato can be a promising natural preservative for extending shelf life as well as maintaining the desired quality during storage in open at room temperature.
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