Effect of Nanochitosan Based Coating on Climacteric Behavior and Postharvest Shelf-Life Extension of Apple Cv

LWT - Food Science and Technology 70 (2016) 33e40

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LWT - Food Science and Technology

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Effect of nanochitosan based coating on climacteric behavior and postharvest shelf-life extension of apple cv. Golab Kohanz

* Ali Sahraei Khosh Gardesh a, Fojan Badii b, , Maryam Hashemi c, Ali Yasini Ardakani a, Neda Maftoonazad d, Ahmad Mousapour Gorji e a Department of Food Science and Technology, Islamic Azad University, Yazd, Iran b Food Engineering Department, Agricultural Engineering Research Institute (AERI), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran c Microbial Biotechnology Department, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran d Agricultural Engineering Research Department, Fars Agricultural and Natural Resources Center, Agricultural Research, Education and Extension Organization (AREEO), Zarghan, Fars, Iran e Vegetable Department, Seed and Plant Improvement Institute (SPII), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran article info abstract

Article history: The effect of nanochitosan-based coating on the quality and storage life of apple cv. Golab Kohanz was Received 6 November 2015 studied. Fresh fruits were coated with nanochitosan emulsion (less than 100 nm) with two chitosan Received in revised form concentrations of 0.2 and 0.5%. The fruits were kept at 1 ± 1 C and relative humidity of 85e90% for nine 14 January 2016 weeks. Respiration rate based on the amount of carbon dioxide (CO ) produced, weight loss, enzyme Accepted 2 February 2016 2 activities, color characteristics, ethylene production, firmness of the coated and uncoated apples were Available online 4 February 2016 measured during nine weeks of storage at specified intervals (once a week). The results showed that coating significantly reduced weight loss, respiration rate, ethylene production and peroxidase activity of Keywords: fi Apple the samples compared with the control. Coating had signi cant effect on polyphenol oxidase activity, fl Climacteric respiration slowed down softening process and improved the esh color after the climacteric peak. Nanochitosan Coating coating with 0.5% chitosan concentration significantly extended the quality and prevented the weight Golab Kohanz loss of the fruits, over the entire storage period. These findings confirmed the potential benefits of Nanochitosan applying nanochitosan coatings to extend the shelf life and maintain the quality of this highly sensitive apple. The results showed that nanochitosan coating is particularly effective after the climacteric period. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Therefore, it has a very limited storage life even in optimum conditions of temperature and relative humidity. Proper packaging Apple is considered as one of the most consumed fruits in the method is one of the effective ways of reducing the rate of loss of this world. It is consumed in different forms as fresh fruit or as a wide horticultural product during transportation and marketing. range of industrially processed products (Wang, Liu, Kerry, & Kerry, Various techniques have been studied in order to extend the shelf 2007). Based on respiration behavior and ethylene production, ap- life of fresh products, for example, low temperature and high relative ple is classified as a climacteric fruit. This means that the metabolic humidity, controlled and modified atmosphere packaging, etc. How- activities and ripening continue after harvesting, thus, making apple ever, each technique has its own advantages and drawbacks. One fruits highly perishable commodities. Apple cv. Golab Kohanz is one method of extending postharvest shelf life is the use of edible coat- of the native apple cultivars in Iran with a unique flavor and aroma. ings. Applying physical barriers such as coating on the surface of the This fruit is highly perishable and does not have enough resistance to fruit provides a partial barrier to moisture, oxygen (O2)andcarbon poor conditions of transport compared to other apple cultivars. dioxide (CO2) and can decline the extent of available oxygen; thereby regulating the rate of respiration and retarding the physiological ripening process of horticultural crops (Elsabee & Abdou, 2013; * Corresponding author. Agricultural Engineering Research Institute, PO Box: Maftoonazad & Ramaswamy, 2005; Maftoonazad, Ramaswamy, & 31585-845, Karaj, Iran. Marcotte, 2008; Salvia-Trujillo, Rojas-Graü, Soliva-Fortuny, & E-mail address: [email protected] (F. Badii). http://dx.doi.org/10.1016/j.lwt.2016.02.002 0023-6438/© 2016 Elsevier Ltd. All rights reserved. 34 A. Sahraei Khosh Gardesh et al. / LWT - Food Science and Technology 70 (2016) 33e40

Martín-Belloso, 2015). Edible coatings based on naturally occurring 2.3. Scanning electron microscopy (SEM) studies polymers have been extensively used on different fresh fruits and vegetables (Elsabee & Abdou, 2013; Eshghi et al., 2014; Maftoonazad The structure of the nanochitosan coating was studied using a & Ramaswamy, 2008; Maftoonazad, Badii, & Shahamirian, 2013; scanning electron microscope (Philips XL30, Netherland) at a Maftoonazad et al., 2008; Salvia-Trujillo et al., 2015; Zahid, Ali, voltage of 30 kV. Prior to the analysis, the samples were prepared Manickam, Siddiqui, & Maqbool, 2012; Zambrano-Zaragoza et al., by drop coating of suspension on an aluminum grid, followed by 2014). Emulsion coatings are directly applied on the surface of fresh drying out the grids at room temperature. This was followed by horticultural products which act as modified atmosphere packaging gold coating to achieve a thickness of 10 nm using a Varian in- by modifying and changing the internal atmosphere of the individual strument (Eshghi et al., 2014). Image processing and particle size fruits (McHugh & Senesi, 2000). analysis were performed on electron micrographs using the image Chitosan, a by-product from crustacean shell wastes, is a high analysis software program, imageJ (http://imagej.nih.gov/ij/docs/ molecular weight cationic polysaccharide, normally obtained by guide)(Martin, Goff, Smith, & Dalgleish, 2006). more than 50% deacetylation of chitin and is referred to as a range of polymers that, unlike chitin, are soluble in aqueous acidic solu- 2.4. Sample preparation tions (Huang, Sheu, & Chao, 2009; Rinaudo, 2006). Chitosan-based coatings have found different applications as effective coatings in Apples (cv. Golab Kohanz) were harvested in a commercial prolonging the shelf life and improving the quality of fresh horti- ripening stage. Fruits with uniform maturity and size were selected. cultural products due to their excellent physico-chemical proper- Apples were then split into three lots and a total of 200 apples were ties, antimicrobial activity and non-toxicity (Bautista-Banos et al., used for each condition. The first two lots were dipped in two 2006; Eshghi et al., 2014; Jiang & Li, 2001; Li & Yu, 2000; Qiu, concentrations of 0.2 and 0.5% (W/V) nanochitosan suspension, Jiang, Ren, Huang, & Wang, 2013). Chitosan is very effective in respectively for 1 min at 25 C. The coated apples were dried at inhibiting the growth of several fungi by inducing chitinases and 25 C and 50%RH for 8 h. The rest was kept without coating as interfering with fungal growth (Bautista-Banos et al., 2006; Li & Yu, control. Fruits were then placed onto apple trays by hand and 2000). The effective amount of chitosan used for coating fruits can packed in cardboard-corrugated boxes. All the fruits were stored at be declined extensively, since the penetration and absorption of 1 ± 1 C and relative humidity of 85e90% for nine weeks and the chitosan increases considerably in the form of nanoemulsion or following experiments were carried out every week. nanoparticles (Zahid et al., 2012). The chitosan nanoparticle has different potential benefits over 2.5. Weight loss conventional chitosan, as they improve barrier properties and functionality of edible coating as a result of their increased surface The weight loss of the fruit during storage was measured using a area (Eshghi et al., 2014; Maftoonazad et al., 2013). The chitosan digital balance (Mettler Toledo, PB602 S-FACT, Switzerland). The nanoparticle is considered to have higher antimicrobial activities fruits were equilibrated at 25 C for 1 h before the measurement and barrier properties. In previous research work, a nanochitosan and the results were reported as percentage of water loss based on based coating was successfully applied on strawberry. This type of the original weight. coating significantly improved the storage life and preserved the bioactive components of the strawberry fruit (Eshghi et al., 2014). It 2.6. Respiration rate would be of great importance to evaluate the effect of this coating on the shelf life of other sensitive and perishable horticultural Respiration rate was measured based on the method described crops. Therefore, the purpose of the present study was to evaluate by Maftoonazad and Ramaswamy, (2008) with slightly modifica- the effect of nanochitosan based coatings on the properties and tion. The respiration measurements were performed at 25 C and storage life of apple cv. Golab Kohanz. the apples equilibrated and reached at this temperature before the measurements. Apples with known weights were placed in an 2. Materials and methods airtight plastic container (10 cm 20 cm 20 cm) and a CO2- sensitive sensor (Testo AG-435-2, Germany) was allocated to the 2.1. Materials inside of the container to measure the concentration of CO2 every 1 min over a 30 min period. Respiration rate was obtained from the Chitosan (deacetylation degree of 75e85%, CAS 9012-76-4) and regression slope of CO2 concentration against time and reported as all chemicals used were obtained from SigmaeAldrich Co., USA. ml CO2/kg.h (Maftoonazad & Ramaswamy, 2008). Chemicals were of analytical grade. Apples (cv. Golab Kohanz)were harvested from a commercial garden in Karaj, Iran. 2.7. Ethylene production

2.2. Preparation of coating solutions Apples with known weights were placed in an airtight glass jar (height of 10 cm and diameter of 20 cm) and an ethylene-sensitive The chitosan nanoparticle coating was prepared based on the sensor (BIOCONSERVACION, UK) was placed inside the jar. The method described by Mohammadi, Hashemi, and Hosseini (2015) volume of ethylene produced by the apple over 1 h was measured with a slight modification. 0.5% (w/v) of chitosan was dispersed and expressed as ppm C2H4/kg.h (Guillen et al., 2013). in 1% (v/v) acetic acid solution at pH ¼ 4.6 overnight at room temperature after which, the solution was sonicated (MISONIX Inc. 2.8. Enzyme activities S-4000, USA) at 60 W for 4 min. A solution of polyphosphate (0.75 mg/ml) was gradually added to the chitosan solution and the Polyphenol Oxidase and Peroxidase were extracted according to final pH of the mixture was adjusted to 5.6 by adding 1N NaOH. the method described by Eshghi et al. (2014). The extraction solu- After centrifuging at 27000 g for 14 min, nanoparticles were tion of 0.2 M sodium phosphate buffer at pH 6.5 containing 4% (w/ resuspended in distilled water and freeze dried. Two nanochitosan v) PVPP and 1% (v/v) Triton X-100 was prepared. 10 g of homoge- coatings with chitosan concentrations of 0.2 and 0.5% were pre- nized apple was mixed with 20 ml of the enzyme extraction solu- pared by dispersing in distilled water. tion. The mixture was homogenized at 4 C for 3 min and then, A. Sahraei Khosh Gardesh et al. / LWT - Food Science and Technology 70 (2016) 33e40 35 centrifuged at 4000 rpm for 10 min. The supernatant was used for diameter flat tipped probe at a constant speed of 12 mm/min. The the enzyme assays (Eshghi et al. 2014; Terefe, Matthies, Simons, & maximum force was obtained from force-deformation curves and Versteeg, 2009). the results were expressed in Newton (Konopacka & Plocharski, 2004). 2.8.1. Polyphenol oxidase activity 75 ml of enzyme extract was mixed with 3 ml of 0.07 M catechol in 0.05 M sodium phosphate buffer (pH 6.5) solution. The blank was 2.12. Statistical analysis prepared in the same way, except that distilled water was used instead of the enzyme extract. The absorbance was measured at All experiments were carried out in three replications for nine 25 C for 10 min, using a spectrophotometer at 420 nm (CECIL 1100, weeks. The experiment was designed according to split plot design. England). The activity of the enzyme was expressed as the alter- Sources of variation were coating treatment and storage time. Data ation of absorbance per minute per gram fresh weight of the was analyzed by a two way analysis of variance using SPSS software sample (Terefe et al., 2009). (SPSS version 20, SPSS Inc., Chicago, USA). Duncan's multiple range test was used to determine the differences between means at a 2.8.2. Peroxidase assay significant level of 0.05 (P < 0.05). First, 500 ml of enzyme extract was mixed with 1 ml of 0.05 M phosphate buffer at pH 6.5. Then, 1 ml of 1% p-phenylenediamine in 0.05 M phosphate buffer at pH 6.5 and 500 ml of 1.5%hydrogen peroxide (H2O2) were added. The absorbance was measured at 3. Results and discussion 485 nm and 25 C for 10 min using a spectrophotometer (CECIL 1100, England). The activity of the enzyme was expressed as the According to our preliminary experiments, nanochitosan alteration of absorbance per minute per gram fresh weight of the coating improved significantly the quality and storage life of apples sample (Terefe et al., 2009). compared to the fruits coated with conventional chitosan. There- fore, the results of the conventional chitosan coating have not been 2.9. Color characteristics presented here.

The color characteristics of apples were obtained using a Konica Minolta (CR-400/410, Japan). Apples were cut into half and the three dimensions of L* (lightness or brightness), a* (redness or greenness) and b* (yellowness or blueness) values of apples were measured. The color measurements were made on three different locations on the flesh of five apples. The total color difference (DE) was measured in order to find out the effect of coating on the time- related color changes, using the following equation: rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi DE ¼ DL*2 þ Da*2 þ Db*2 (1) where: DL*, Da* and Db* were the differences of L*, a* and b* of the stored apples at any given time from those of the fresh sample (Maftoonazad & Ramaswamy, 2008). Also, BI or browning index was measured as follow (Zambrano-Zaragoza et al., 2014):

½100ð 0:31Þ BI ¼ (2) 0:17

À *Á a* þ 1:75L ¼ * * (3) ð5:645L þ a* 3:012b Þ

2.10. Titrable acidity, soluble solids and pH

10 g samples were blended with 40 ml distilled water and titrable acidity was measured by titration method (AOAC, 2005). Soluble solids was measured at 25 C using a refractometer (MC- 20181, Korea). A standard calibrated pH meter (Metrohm-691, Switzerland) was used for pH measurements (Maftoonazad et al., 2008).

2.11. Firmness

Fruit ﬁrmness was measured using an Instron Universal Testing Fig. 1. a. SEM results for nanochitosan coating with 0.5% chitosan concentration. b. Size ﬁ Machine (Houns eld, UK) equipped with 50N load cell. After distribution of 0.5% nanochitosan coating, obtained by image analysis of SEM peeling the skin of fruits, they were penetrated using a 10 mm micrograph. 36 A. Sahraei Khosh Gardesh et al. / LWT - Food Science and Technology 70 (2016) 33e40

3.1. Structure of the nanochitosan coating 42% for the apples coated with 0.5 and 0.2% chitosan, respectively. In strawberry, as a non-climacteric fruit, respiration rate was also The structure of the nanochitosan coatings was studied by SEM reduced by coating with the nanoparticle of chitosan (Eshghi et al., and the acquired micrograph for 0.5% chitosan is shown in Fig. 1a. 2014). It was previously shown that chitosan coating reduced Image analysis was performed on the image to obtain a particle size respiration rate in peaches considerably over the whole period of distribution for the nanoparticles (Fig. 1b). It was reported previ- storage (Li & Yu, 2000). The chitosaneglucose complex coating ously that the concentration of chitosan and tripolyphosphate successfully decreased the respiration rate of table grapes as a (TPP) significantly influenced the size of the nanoparticle (Calvo, result of modifying the internal atmosphere of the fruit (Gao, Zhub, Remunan-Lopez, Vila-Jato, & Alonso, 1997; Janes & Alonso, 2003). & Zhang, 2013). Different edible coatings had the same effect on However, in this work, the concentration of nanochitosan did not climacteric fruits such as peach (Maftoonazad et al., 2008), banana have any significant effect on the size of the particles. Fig. 1b shows (Kittur, Saroja, & Tharanathan, 2001), pear (Meheriuk & Lau, 1988), that the particle size of the 0.5% chitosan coating reduced to less kiwifruit (Xu, Chen, & Sun, 2001) and tomato (Park, Chinnan, & than 100 nm and the particle size diameters were in the range of Shewfelt, 1994). 18e120 nm. The same result was obtained for 0.2% chitosan coating. 3.3. Ethylene production

3.2. Respiration rate Ethylene is a biologically active compound and considered as a naturally occurring hormone in plants. It is biologically effective Fig. 2 represents respiratory patterns of coated and uncoated and responsible for fruit ripening. As ripening begins in climacteric apples. Statistical analysis showed significant differences between fruits such as apple, a dramatic increase in the production of the respiration rate of control and that of the coated apples at forty- ethylene occurs which is coincident with a rapid rise in respiration (Kader, 2002). It is of great importance to decrease the amount of five days of storage (P < 0.05). The maximum CO2 evolution rate of 35 ml/kg.h appeared for control apples after forty-five days, produced ethylene as soon as possible in order to extend the whereas it decreased significantly and reached 26 and 17 ml/kg.h storage life of the fruits. for coated samples with 0.2 and 0.5% chitosan concentrations, Fig. 3 shows that the nanochitosan coating declined ethylene fi respectively. Both coatings led to a decrease in the peak respiration evolution signi cantly over the whole storage period. All of the fi rate of apples. In fact respiratory peak was eliminated in coated coated and control apples reached the peak after fty days. As ex- fi fruits with 0.5% concentration. The same finding was obtained for pected, there was a highly signi cant difference between control the peaches coated with sodium alginate or methyl cellulose and coated apples at the peak point and apples with and without fi (Maftoonazad et al., 2008). However, respiration rate before and edible coatings showed signi cant differences in the volume of after the respiratory peak did not change with coating. Further- ethylene release. Nanochitosan coatings with 0.5 and 0.2% chitosan more, the rate of increase in respiration rate (DR/Dt) for the coated concentrations decreased the maximum ethylene production after fi apples with 0.2 and 0.5% were 0.33 and 0.20 ml/kg.h/day, respec- fty days to approximately 33 and 20%, respectively. In other tively as compared with 0.56 ml/kg.h/day for the control. It means words, the volume of ethylene release of the control was about 1.5 that the average increase of respiration rate declined nearly 64 and times higher than that of the coated ones with 0.5% chitosan concentration. It could be seen that the peak of ethylene release usually emerged close to the peak of respiration rate. In plum fruit, the climacteric peak of ethylene production was reduced significantly by alginate coating (Valero et al., 2013). A significant delay on ethylene production was also observed for plums and peaches

Fig. 2. Effect of nanochitosan coatings on the respiration rate of apple stored at 1 ± 1 C Fig. 3. Effect of nanochitosan coatings on the ethylene production of apple stored at and 85e90% RH. Numbers are the mean ± standard errors of three replications. 1 ± 1 Cand85e90% RH. Numbers are the mean ± standard errors of three replications. A. Sahraei Khosh Gardesh et al. / LWT - Food Science and Technology 70 (2016) 33e40 37 coated with aloe arborescens and aloe vera gels (Guillen et al., 2013). Applying aloe vera gels with rosehip oil reduced markedly ethylene production in climacteric fruits such as peach, plum and nectarine (Paladines et al., 2014).

3.4. Weight loss

During storage period, fresh fruits and vegetables loss weight normally due to respiratory process and transpiration (Maftoonazad et al., 2008). Fig. 4 shows the percentage of weight loss as a function of storage time in coated and uncoated apples. It can be seen that the rate of weight loss until week two does not show significant differences among coated and uncoated samples, but from the third week until the end of storage time the percentage of weight loss in control sample was significantly higher than that of coated samples with 0.5% chitosan (P < 0.05). After nine weeks storage at 1 ± 1 C and relative humidity of 85e90%, the weight loss of uncoated apples was 5.5% as compared to 3.4 and 2.2% of their original weight for treated samples with coatings 0.2 and 0.5% nanochitosan concentrations, respectively, which means that coating with 0.5% nanochitosan emulsion had a Fig. 5. Effect of nanochitosan coatings on the peroxidase activity of apple stored at beneficial effect on reducing the weight loss of apple by 59%. This 1 ± 1 Cand85e90% RH. Numbers are the mean ± standard errors of three replications. also means that the control had lost its commercial value completely after nine weeks of storage. fi These results are in agreement with the research report of and untreated samples reached the peak after fty days of storage Eshghi et al. (2014) with the conclusion that nanochitosan coatings which is coincident with the climacteric respiratory and maximum fi significantly reduced water loss in strawberry up to 57% during ethylene production. The nanochitosan coatings inhibited signi - < storage. Chitosan based coatings have been applied to conserve cantly the enzyme activity of apple at this stage (P 0.05). After water content effectively in different fruits such as strawberry that, the changes of peroxidase activity were in line with the (Riberio, Vicente, Teixeira, & Miranda, 2007), banana and mango changes in volume of released ethylene. It was observed that (Kittur et al., 2001). However, size reduction of chitosan particle to nanochitosan coating can inhibit the activity of peroxidase in nano-scale may improve the functionality and properties of chito- strawberry (Eshghi et al., 2014). However, it was reported that san at lower concentrations (Eshghi et al., 2014). chitosan coating can induce the activity of peroxidase in table grapes (Gao et al., 2013). 3.5. Determination of peroxidase activity Peroxidase is an oxireductase enzyme related to enzymatic browning in fruits (Eshghi et al., 2014). The change of peroxidase Fig. 5 compared the peroxidase activity of the coated fruits with activity of fruits is related to the volume of ethylene release. At the that of the control. After thirty days of storage a significant differ- time of releasing ethylene, the activity of peroxidase increased. It fi ence in the peroxidase activity of 0.5% nanochitosan coated and thus certi ed that peroxidase activity was related to auto-catalytic uncoated samples was observed. The peroxidase activity in treated synthesis of ethylene. This also indicated that peroxidase activity was directly related to the process of fruit aging (Eshghi et al., 2014).

3.6. Polyphenol oxidase (PPO) activity

Polyphenol oxidase activities of the control and coated apples during nine weeks of storage at 1 ± 1 C and relative humidity of 85e90% are shown in Fig. 6. It can be seen that the enzyme activity showed a similar increase in all the samples, although the nanochitosan coating reduced the activity of polyphenol oxidase in strawberry (Eshghi et al., 2014), it seems not to have an impact on the activity of the enzyme in Golab apple, until the end of storage time (after climacteric respiration), when the enzyme activity declined significantly in coated apples (P < 0.05). In the presence of molecular oxygen (O2), polyphenol oxidase is responsible for catalyzing the hydroxylation of monophenols to o-diphenols and the oxidation of o-diphenols to o-quinones (Eshghi et al., 2014; Fernandes, Ribeiro, Raposo, Morais, & Morais, 2011). It was reported that enzyme activities were reduced by Chitosan coating, as a result of a decline in the respiration rate of coated fruits (Eshghi et al., 2014; Fernandes et al., 2011). As can be seen from Fig. 2, nanochitosan coatings decreased respiration rate significantly at Fig. 4. Effect of nanochitosan coatings on the weight loss of apple stored at 1 ± 1 C climacteric peak, therefore, the reduction in the enzyme activity and 85e90% RH. Numbers are the mean ± standard errors of three replications. was observed after fifty days of storage. 38 A. Sahraei Khosh Gardesh et al. / LWT - Food Science and Technology 70 (2016) 33e40

Fig. 6. Effect of nanochitosan coatings on the polyphenol oxidase activity of apple stored at 1 ± 1 C and 85e90% RH. Numbers are the mean ± standard errors of three replications.

3.7. Color characteristics

Fig. 7a shows that the results of L* value in the samples was slightly higher in coated apples over the whole period of storage. However, after climacteric respiration the value of L* for the control dropped significantly to approximately 76 whereas, it remained constant in the coated apples with 0.2 and 0.5% chitosan at nearly 78 and 80, respectively. Since the changes in the value of L* are associated with the activity of polyphenol oxidase, the application of nanochitosan coating as an oxygen barrier may restrict the action of polyphenol oxidase (Zambrano-Zaragoza et al., 2014). The same results were observed for the fresh cut Red Delicious apple coated with xanthan gum (Zambrano-Zaragoza et al., 2014). As can be seen in Fig. 7b, the value of total color difference (DE) for the coated apples did not change significantly during the storage time, while for the control, it increased significantly (P < 0.05) after forty days until the end of storage. In other words, the values of DE showed less variation for the coated samples than those of the control apples. Fig. 7c showed that the browning index (BI) in the coated samples were lower than that of the control apples over the entire period of storage. BI evaluates the purity of the brown color and considered as an essential factor for studying enzymatic and non- enzymatic browning in the sample (Zambrano-Zaragoza et al., 2014). It was observed that BI in the control apples had consider- able variations, and specifically increased after 35 days of storage, while the coated apples did not show any significant change over the entire period of storage. Based on the statistical results, significant differences between coated and control samples were observed after 35 days until the end of the storage period. The time dependent color changes in the flesh of fruits toward lower L* value, higher DE are the results of ripening which is extensively lower in coated apples. The results indicated that all the color characteristics of the uncoated samples increased significantly after climacteric peak as compared with those of the coated Fig. 7. a. Effect of nanochitosan coatings on the value of lightness of apple flesh stored apples which were coincident with increasing the activity of the at 1 ± 1 C and 85e90% RH. Numbers are the mean ± standard errors of three repli- polyphenol oxidase enzyme. cations. b. Effect of nanochitosan coatings on the value of total color difference (DE) of apple flesh stored at 1 ± 1 C and 85e90% RH. Numbers are the mean ± standard errors of three replications. c. Effect of nanochitosan coatings on the value of browning index fl ± e ± 3.8. Titrable acidity, soluble solids and pH (BI) of apple esh stored at 1 1 C and 85 90% RH. Numbers are the mean standard errors of three replications.

The variations of titrable acidity (TA), soluble solids and pH for the control and coated fruits during storage are shown in Table 1. of soluble solids in fruits. It was also reported that applying aloe Nanochitosan coatings showed no signiﬁcant effect on the amount A. Sahraei Khosh Gardesh et al. / LWT - Food Science and Technology 70 (2016) 33e40 39

Table 1 Effect of nanochitosan coatings on the pH, Soluble Solids and TA (Titrable Acidity) of apple stored at 1 ± 1 C and 85e90% RH. Numbers are the mean ± standard errors of three replications. Numbers within a column marked with different letters are signiﬁcantly different (P < 0.05) according to Duncan's multiple range test.

Days pH Soluble solids (%) TA (%)

Uncoated Coated 0.2% Coated 0.5% Uncoated Coated 0.2% Coated 0.5% Uncoated Coated 0.2% Coated 0.5%

0 4.5 ± 0.1c 4.5 ± 0.1c 4.5 ± 0.1c 10.5 ± 0.0a 10.5 ± 0.0a 10.5 ± 0.0a 1.9 ± 0.0a 1.9 ± 0.0a 1.9 ± 0.0a 7 4.6 ± 0.0c 4.6 ± 0.0c 4.5 ± 0.1c 10.5 ± 0.1a 10.8 ± 0.0a 10.1 ± 0.0a 1.9 ± 0.0a 1.9 ± 0.1a 1.9 ± 0.1a 14 4.7 ± 0.1b 4.6 ± 0.0c 4.5 ± 0.0c 10.7 ± 0.2a 10.8 ± 0.2a 10.0 ± 0.3a 1.7 ± 0.1b 1.7 ± 0.0b 1.7 ± 0.1b 21 4.7 ± 0.1b 4.6 ± 0.0c 4.6 ± 0.0c 10.9 ± 0.0a 11.0 ± 0.0a 11.0 ± 0.1a 1.6 ± 0.0b 1.7 ± 0.0b 1.7 ± 0.0b 28 4.7 ± 0.0b 4.6 ± 0.0c 4.6 ± 0.0c 11.0 ± 0.0a 11.0 ± 0.4a 11.0 ± 0.0a 1.5 ± 0.0c 1.6 ± 0.1b 1.6 ± 0.1b 35 4.7 ± 0.1b 4.7 ± 0.0b 4.7 ± 0.1b 11.0 ± 0.0a 11.0 ± 0.0a 11.0 ± 0.0a 1.5 ± 0.0c 1.5 ± 0.1c 1.5 ± 0.0c 42 4.8 ± 0.0a 4.7 ± 0.0b 4.7 ± 0.0b 11.0 ± 0.0a 11.0 ± 0.0a 11.0 ± 0.0a 1.3 ± 0.1d 1.5 ± 0.1c 1.5 ± 0.0c 49 4.8 ± 0.0a 4.7 ± 0.0b 4.7 ± 0.0b 11.0 ± 0.2a 11.0 ± 0.1a 11.0 ± 0.1a 1.3 ± 0.1d 1.4 ± 0.0c 1.5 ± 0.1c 56 4.8 ± 0.0a 4.7 ± 0.0b 4.7 ± 0.0b 11.4 ± 0.1a 11.2 ± 0.0a 11.2 ± 0.0a 1.3 ± 0.0d 1.4 ± 0.1c 1.4 ± 0.0c 63 4.8 ± 0.0a 4.7 ± 0.0b 4.7 ± 0.0b 11.4 ± 0.2a 11.2 ± 0.0a 11.2 ± 0.0a 1.3 ± 0.0d 1.4 ± 0.1c 1.4 ± 0.0c

vera gels did not change soluble solids of peach and plum fruits followed by a steady change until the end of storage time. The during storage (Guillen et al., 2013). nanochitosan coatings reduced the rate of softness in apple After 42 days of storage, pH and TA values changed significantly significantly (P < 0.05) between 42 and 56 days which is coincident in uncoated samples compared to the coated fruits. However, both with the climacteric respiratory and maximum ethylene produc- coatings had similar effect on the pH and TA variations over the tion. Fig. 8 shows that 0.5% nanocoating was significantly more whole period of time. TA decreased slightly slower in the nano- effective in firmness retention than 0.2% nanocoating during this chitosan coated samples than those of control fruits during storage. climacteric period. In climacteric fruits, the main factor in fruit Since organic acids are used as substrates for the respiration pro- softening after harvesting is the activity of the enzymatic hydrolysis cess, it is expected that the values of TA decrease during post- of the cell walls which is controlled by ethylene (Valero et al., 2013). harvest storage of fruits (Valero et al., 2013). The results According to the results represented in Fig. 3, the ethylene pro- represented in Fig. 2 show that coatings decline the peak respira- duction was inhibited significantly by nanochitosan coatings. tion rate of apples significantly, which is a reason why nanochitosan Therefore, the coated apples showed significantly lower softening coatings delayed TA losses significantly in the apples after climac- process compared to the control fruits during climacteric period. It teric respiration. Since the control apples had higher variations in was previously reported that the application of chitosan based pH values, it was expected that their variations in titrable acidity coatings results in reducing softening process for different were higher compared to the coated samples. Thus, at the end of climacteric and non climacteric fruits (Eshghi et al., 2014; Gao et al., storage time, the values of titrable acidity in coated samples were 2013; Han et al., 2014; Qiu et al., 2013). significantly higher than those of the control apples. 4. Conclusions 3.9. Firmness Apple cv. Golab Kohanz is a sensitive and highly perishable apple variety with a very limited storage life. Coating of this fruit using Fig. 8 shows that the values of fruit firmness for all the samples nanochitosan emulsion significantly enhanced the quality and experienced a sharp decline over the first 3 weeks of storage storage life of the apple. These coatings were used as a thin layer on the surface of the fruit to prevent products from transferring moisture, gases, and dissolved substances. On the other hand, they reduced the respiratory rate of the products and acted as modified atmosphere packaging. By reducing the size of chitosan and applying nanochitosan coating, the effective concentration of chitosan decreased considerably to 0.5% as compared to those effective concentrations suggested in previous studies for coating different fresh horticultural products (Gao et al., 2013; Han et al., 2014; Jiang & Li, 2001). Nanochitosan coating with 0.5% chitosan markedly eliminated the climacteric respiration peak by subsequently lowering the maximum ethylene production to approximately 33%. The coating positively controlled the enzymatic activities, improved the color quality of the fruit, slowed down the fruit softening and declined the weight loss up to 2.5 times over a nine week period of storage. The overall results showed that nanochitosan coatings slowed down the natural fruit ripening and improved the quality and postharvest storage-life of the apple, specifically after the climacteric period.

Acknowledgments

This study was supported by the Agricultural Engineering Fig. 8. Effect of nanochitosan coatings on the ﬁrmness of apple stored at 1 ± 1 C and Research Institute (AERI) and the Agricultural Biotechnology 85e90% RH. Numbers are the mean ± standard errors of three replications. Research Institute of Iran (ABRII). 40 A. Sahraei Khosh Gardesh et al. / LWT - Food Science and Technology 70 (2016) 33e40

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