Effect of Salicylic Acid Treatments on Quality Characteristics of Apple Fruits During Storage

INTRODUCTION

Apples are the natural source of dietary mineral salts, vitamins, antioxidants, fibre, organic acids and sugars. The highest concentration of bioactive substances, including antioxidants, is found in or near the peel, so it is recommended as a dietary supplement (Wolfe et al., 2003; Wolfe and Liu, 2003). Apple is a climacteric fruit with a long post-harvest life in cool storage. Losses in fruit quality are mostly due to its relatively high metabolic activity during storage (Fattahi et al., 2010). Cool storage is widely used to reduce respiration rate, Ethylene production and extend the shelf-life of fruits (Fattahi et al., 2010). Ethylene is a gaseous plant hormone that at very low concentrations plays a major role in the regulation of the metabolism of harvested horticultural crops (Saltveit, 1999). The responses of harvested fruits, vegetables and ornamental crops to endogenously produced and exogenously applied ethylene are numerous and varied and they can be beneficial or detrimental depending on each case (Saltveit, 1999). In general, ethylene can influence the postharvest life of both climacteric and nonclimacteric fruit by affecting their quality attributes and the development of physiological disorders and postharvest diseases (Palou et al., 2003). Respiration and Ethylene production causes a sharp increase in production of oxygen free radicals which is responsible for stress dependent peroxidation of membrane lipids. The effects of ethylene can be reduced by inhibitors of ethylene biosynthesis and increase enzyme antioxidant activity (Khan et al., 2003; El-Tayeb et al., 2006; Shi and Zhu, 2008; Joseph et al., 2010). Salicylic acid is known as a signal molecule in the induction defense mechanisms in plants. SA is a well known phenol that can prevent ACO activity that is the direct precursor of ethylene and decrease Reactive Oxygene Species (ROS) with increase enzyme antioxidant activity (Ansari and Misra, 2007; Mba et al., 2007; Mahdavian et al., 2007; Canakci, 2008). It delays the ripening of banana fruit (Srivastava and Dwivedi, 2000), inhibit ethylene production in kiwifruit (Fattahi et al., 2010) and carrot cell suspension cultures (Roustan et al., 1990). As Zhang et al. (2003) reported, application of SA on kiwifruit increased superoxide free radical and Lipoxygenase (LOX) activity. In that case, climacteric rise in ethylene production was retarded. So, fruit ripening and senescence were delayed (Zhang et al., 2003). Application of exogenous methyl salicylate (MeSA) vapor on kiwifruits led to prevent the softening process of fruit flesh, kept ascorbic acid content and firmness during 5 months storage (Solaimani et al., 2009). The aims of this study was to determine the effect of salicylic acid application on the quality and storage life of apple fruit during storage.

MATERIALS AND METHODS

The experiment was started in season 2010-2011 and Fruit Weight Losses, Fruit Firmness, total soluble solids, titratable acidity, peroxidase activity (POD), ascorbic acid content (Vitamin C) and superoxide dismutase (SOD) activity were measured at 15, 30 and 60th days of postharvest life. Malus domestica var. Jonagold were harvested at commercial maturity stage from an experiment orchard at the apple Research Institute of Iran (uromieh, Iran). Fruits were subsequently transferred to laboratory and sorted based on size and the absence of physical injuries or infections. Fruits were randomly divided into six groups, each group containing 100 fruits in four replicates and immersed into solution of (0, l.5, 3 mM) SA and in distilled water as control for 5 min. Fruits were then dried for about 24 h and then stored at 5°C and 85-90% relative humidity for two months. After15, 30 and 60th days storage, 30 fruits per treatment were taken from cool storage for fruit quality assessment.

Experimental design and statistical analysis: Experiment was arranged in complete randomized design with four replications. Analysis of variance was performed on the data collected using the General Linear Model (GLM) procedure of the SPSS software) Version 16, IBM Inc.). The mean separation was conducted by tukey analysis in the same software (p = 0.05).

Weight loss: Weight loss was determined by using Tefera et al. (2007) method, by periodical weighing of apple fruits 15, 30 and 60 days after storage.

Fruit firmness: Firmness was determined by measuring compression using a hand-held Effegi penetrometer with a 7.9 mm probe after removal of skin to a vertical depth of 1 mm on two sides of the fruit. The firmness considered as an average peak force of 10 fruits and expressed as kg/7.9 mm².

Total soluble solid: Total Soluble Solids (TSS) were measured by the method described by Dong et al. (2001).

Titratable acidity: Titratable acidity was determined using 5 mL of fruit puree from five fruits mixed with 25 mL of distilled water with two drops of phenolphthalein (1%) as indicator, titrated with 0.1N NaOH to an endpoint pink (pH 8.2). The results were expressed as percent anhydrous citric acid since it is the dominant acid in apple.

Ascorbic acid content (Vitamin C): Ascorbic Acid (AA) content of apple was determined by the 2,6-dichlorophenolindophenol method (Tefera et al., 2007). An aliquot of 10 mL apple fruit juice extract was diluted to 50 mL with 3% metaphosphoric acid in a 50 mL volumetric flask. The aliquot was filtered and titrated with the standard dye to a pink endpoint (persisting for 15 sec).

Browning index: Browning index was assessed by measuring the extent of browning area as described by Wang et al. (2005).

Relative electrical conductivity: Relative electrical conductivity was measured by the method described by Fan and Sokorai (2005).

Determination of Acc-oxidase activity: For the measurement of ACC oxidase activity, flesh slices of 1 mm thickness (approximately 1 g) were put into 40 mL Erlenmeyer flasks containing 2 mL of incubation buffer consisting of 1 mM ACC, 0.4 M mannitol and 0.1 M Tricine (pH 7.5). ACC oxidase activity was determined both in the absence and in the presence of 30 mM sodium ascorbate, 0.1 mM FeSO₄ and 20 mM NaHCO₃ according to the method described by Moya-Leon and John (1994). The flasks were incubated at 30°C for 1 h and the ethylene formed was determined as described above. The activity was expressed as ethylene (in nanomoles) produced per gram fresh weight per hour.

Superoxide dismutase (SOD) activity: A 1.0 g aliquot of frozen powder was added to 10 mL of cold ethanol absolute for 30 min, then centrifuged at 0°C and 10,000xg for 10 min and the supernatant discarded. The ethanol extraction was repeated twice. The pellet was then resuspended in 5.0 mL of cold 100 Mm sodium-potassium phosphate buffer (NaKPi), pH 7.0, 0.1% (w/v) polyvinylpolypyrrolidone (PVPP), prepared and stored at 4°C the day before and, after 30 min, centrifuged at 4°C and 10,000x g for 30 min. The supernatant was recovered and used for the enzyme activity assay. Total SOD activity was measured after Madamanchi et al. (1994). For each sample assayed, six tubes were set up containing 10, 20, 40, 60, 80 and 500 μL of the enzyme extract. The reaction mixture contained 2 μM riboflavine, 10 μM l-methionine, 50 μM Nitro Blue Tetrazolium (NBT), 20 μM KCN, 6.6 M Na2EDTA, 10-500 μL of the enzyme extract and 65 μM NaKPi, pH 7.8, to give a total volume of 3.0 mL. SOD activity was assayed by measuring the capacity of the enzyme extract to inhibit the photochemical reduction of NBT to blue formazan. Glass tubes were thermostated at 25°C for 10 min in absence of direct light. The reaction was started by exposing the mixture to four white fluorescent lamps (Leuci, 15 WTS preheat, daylight 6500°K) in a box (80x50x50 cm) with aluminium-foil-coated walls. Blanks were obtained with non-illuminated duplicates. The blue colour developed in the reaction was spectrophotometrically measured at 560 nm and the corresponding non-exposed samples were used as blank. The volume of sample causing 50% inhibition in colour development was taken as one unit of SOD activity.

Peroxidase activity (POD): A 1.0 g aliquot of frozen powder was added to 10.0 mL of cold 200 mM NaPi, pH 7.0, 5 mM Na2EDTA, 0.1% (w/v) PVPP, 3 mM dithiothreitol, 15 mM-mercaptoethanol, 10 mM sodium metabisulfite, prepared and stored at 4°C the day before and after 30 min, centrifuged at 15,000x g for 30 min. The supernatant was recovered and used for the enzyme activity assay. The reaction mixture (3.0 mL final volume) consisted of 50 μL of 10 mM guaiacol, 2.9 mL of 10 mM NaPi, pH 7.0, 10 μL of 40 mM H₂O₂. A 40 μL aliquot of the crude enzyme extract was then added to start the reaction. The activity of the mixture was determined spectrophotometrically at 470 nm after 10 min at 20°C.

RESULTS AND DISCUSSION

Effect salicylic acid on weight loss: The results indicate that treatment with 1.5 mM SA solution slightly reduced the weight loss (Table 1). maximum weight loss occurred in control while lowest loss was recorded in 3 mM SA(p = 0.05) (Table 1). Weight loss was highest during the eighth weeks. Overall highest weight loss occurred in control during the Fifth week (p = 0.05) (Table 1). It’s thought that SA can decrease respiration through inhibition of ethylene biosynthesis or action (Srivastava and Dwivedi, 2000). Salicylic acid also caused decrease in respiration rate and fruit weight losses by closing stoma (Zheng and Zhang, 2004).

Effect salicylic acid on firmness: The results indicate that maximum firmness was recorded in 3 mM SA as compared to control and 1.5 mM SA. Maximum firmness was recorded in 3 mM SA during 60 day (p = 0.05) (Table 1). This result was in agreement with the report of Solaimani et al. (2009) that suggested postharvest application of kiwifruit by MeSA decreased softening and kept firmness during storage. Zhang et al. (2003) reported that rate of fruit ripening related to internal SA concentration. Our results suggested that firmness caused by SA associated with ACO activity inhibitory. This suppression may mostly due to inhibitory effect of SA on ACC conversion to ethylene (Li et al., 1992).

Effect salicylic acid on total soluble solids, titratable acidity: The results in Table 1 show that the storage period has a significant effect on TSS% and TA of fruits (p = 0.05). The results indicate that minimum TSS was observed in 3 mM salicylic acid, the highest TSS was recorded in control. Total soluble solids content of fruits during storage is considered an index of fruit ripening and an increase in TSS corresponds to a conversion of starch to soluble sugars. Also, The results indicate that maximum TA was observed in 3 mM salicylic acid and lowest TA was recorded in control. Titratable acidity is directly related to the concentration of organic acids present in the fruit which are an important parameter in maintaining the quality of fruits. Titratable acidity increased gradually in all treatments except control (Table 1) and did seem to be influenced by the postharvest SA. Delay in fruit ripening and extended shelf-life after SA treatment also reported in banana fruit by Srivastava and Dwivedi (2000). Similarly, Zhang et al. (2003) found that the rate of softening in kiwifruit treated by SA reduced because had remained relatively high levels of SA concentrations.

Effect salicylic acid on ascorbic acid (Vit C) content: The results indicate that the values of Vitamin C significantly increased with increasing SOD and POD activity and decreased ACO activity in the storage duration. All treatments had significant effect on the values of vitamin C except control (p = 0.05).

Table 1:	Mean comparison of fruit weight loss, Firmness, TA, Ascorbic acid, Browning index, REC, SOD, POD, ACC oxidase activity in different concentration SA solution during 60 days storage at 5°C

Means in each column followed by similar letters are not significantly different at 5% level. ¹Total soluble solids, ²Titratable acidity, ³Relative electrical conductivity, ⁴Peroxidase activity, ⁵Superoxide dismutase activity

The results indicate that maximum SOD and POD activity was observed in 3 mM salicylic acid (p = 0.05) also, the results indicate that Acc-oxidase activity decreased with increasing SOD and POD activity in the storage duration. Lamikanra and Watson (2001, 2002) indicated the ascorbate dependency of peroxidase (POD) enzymes in a number of commonly fresh-cut processed fruits whose activities appear to be related to the level of oxidative stress in cut fruit. Ascorbic acid level decreased gradually during the ten weeks storage period. Ascorbic acid is an important nutrient quality parameter and is very sensitive to degradation due to its oxidation compared to other nutrients during food processing and storage. These results showed that SA treatments had a significant effect on retaining ascorbic acid content in apple fruit. As Zhang et al. (2003) reported, application of SA on kiwifruit increased superoxide free radical and enzyme antioxidant activity. In that case, climacteric rise in ethylene production was retarded. So, fruit ripening and senescence were delayed (Zhang et al., 2003). Our result showed that higher concentrations of SA delayed the rapid oxidation of ascorbic acid with increasing SOD and POD activity and decreased Acc-oxidase activity in the storage duration.

Effect salicylic acid on Browning Index (BI) and Relative Electrical Conductivity (REC): The BI and REC decreased with increasing SOD and POD activity and decreased Acc-oxidase activity in the storage duration. salicylic acid (3 mM SA) had a significantly influence in reducing the BI and REC in fruits compared to control in the storage duration (p = 0.05). The results indicate that maximum BI and REC were recorded in control as compared to other treatment. Oxidative membrane injury allows the mixing of the normally separated enzyme (PPO) and oxidizable substrates (polyphenols) which lead to browning. Respiration and Ethylene production causes a sharp increase in production of oxygen free radicals which is responsible for stress dependent peroxidation of membrane lipids. As Zhang et al. (2003) reported application of SA on kiwifruit increased superoxide free radical and Lipoxygenase (LOX) activity. In that case, climacteric rise in ethylene production was retarded. So, fruit ripening, REC, BI and senescence were delayed (Zhang et al., 2003).

CONCLUSION

From the results of the present study, it can be concluded that Salicylic acid treatments significantly retained maximum firmness, total soluble solids, ascorbic acid content, Peroxidase activity(POD), Superoxide dismutase (SOD) activity and reduced Browning Index (BI), Relative Electrical Conductivity (REC) and weight loss compared to the control.