Effect of active lipid‐based coating incorporated with nanoclay and orange peel essential oil on physicochemical properties of Citrus sinensis

Abstract The aim of this study was to evaluate the different lipid‐based coating on the physicochemical properties and shelf life of blood orange. In this study, four different carnauba wax coatings formula were used: carnauba wax, carnauba wax incorporated with orange peel essential oil (OPEO) (1%), carnauba wax with montmorillonite nanoclay (MMT) (2%), and carnauba wax combination by OPEO (0.5%) and MMT (1%). Physicochemical properties (total phenol content, antioxidant activity, °Brix, titratable acidity, vitamin C, color, firmness, and pH) of fruits were determined throughout the storage. According to the results, carnauba wax with MMT was better than the other treatments. The highest antioxidant activity was observed in carnauba wax coating containing MMT and total phenol and DDPH gained 733.00 ± 1.204 (mg gallic acid/100 g) and 78.327 ± 0/364%, respectively, at 100th day. Blood orange coated by carnauba wax with MMT had the least of deformation and dissolved solid and the highest acidity rather than other treatments. Moreover, time storage and coating had significant effect on vitamin C content in which maximum and minimum amount was observed in wax coating incorporated by MMT and combination with MMT and OPEO treatments, respectively. Fruits coating with MMT showed better brightness.

However, potential functions and applications of the coatings warrant increased considerations. Extensive research is still needed on the methods of coating formation and improvement of their properties and applications.
The aim of this study was to preserve the physicochemical traits and prolonging the shelf life of blood orange with coating. In this study, we evaluated the effect of carnauba wax alone and carnauba wax incorporated with orange peel essential oil (OPEO), carnauba wax with MMT and carnauba wax combination by orange peel essential oil and MMT and then investigated their physicochemical properties.

| Fruits coating
In this study, different wax coatings, including carnauba wax +2% MMT, carnauba wax +1% OPEO, carnauba wax +1% MMT + 0.5% OPEO, and carnauba wax alone, were evaluated. Each formulation was sprayed on Citrus sinensis fruits and then stored at 7°C and 85% RH for 100 days. The quality properties of fruits were measured every 20 days.

| Total phenol content
Total phenols were determined by the method of McDonald, Prenzler, Antolovich, and Robards (2001) using the Folin-Ciocalteu reagent. An aliquot (20 ml) of orange juice was added to 1.16 ml of distilled water, followed by 100 ml of Folin-Ciocalteu solution.
After 5 min, 300 ml of 20% sodium carbonate was added and the contents of the tube were thoroughly mixed before being incubated in a water bath (40°C) for 30 min. The tube was allowed to cool in the dark. The absorbance of samples was read at 760 nm using gallic acid as standard. Results were expressed in mg gallic acid per 100 g dry weight.

| DPPH radical assay
An ethanol solution of 0.1 mmol/L DPPH was added to 1 ml orange juice and after mixing for 10 s, the solution was stored in dark room for 30 min. Afterward, the absorbance of resulting solution was measured at 515 nm by spectrometer (T80 + UV/VIS, PG Instrument Ltd, America). DPPH solution was used as control which had maximum absorbance (Koleva, van Beek, Linssen, Groot, & Evstatieva, 2002). The activity was measured according to the following equation:

| Titratable acidity
Titratable acidity was determined by titration of orange juice with 0.1 N NaOH solution and expressed as lactic acid percentage (AOAC, 2005). Phenolphthalein was used as detergent.

| Vitamin C
A modified method of Egan, Kirk, and Sawyer (1981) was used for the measurement of vitamin C from orange juice. 5 ml orange juice was added to 60 ml distilled water and then was homogenized with 4% potassium iodide, 0.4 ml acetic acid (10%), and 0.3 ml starch (1% concentration). The mixture was then titrated by NBS solution.

| Color
To evaluate the color of oranges, samples were placed in a dark box white D65 lamp. The photography was carried out by a digital camera (Canon, 16 Mega Pixels) with perpendicular distance of 30 cm.
The color components (L*, a*, and b*) were calculated according to the method described by Yaman and Bayoιndιrlι (2002), using image j software.

| Statistical analysis
The data obtained from experiments were analyzed using completely randomized factorial design by SAS software (SAS Institute, Cary, NC, USA), and the differences between means were evaluated using Duncan's multiple range test. All experiments were done in triplicate.  (Table 1).

| DPPH radical assay
The antioxidant activity of samples was determined by the DPPH radical scavenging assay. DPPH scavenging activity assay is widely used to evaluate the ability of compounds to scavenge free radicals or donate hydrogen, and determine the antioxidant activity in foods (Bidchol, Wilfred, Abhijna, & Harish, 2011). According to Table 2, time and coating had significant effect on inhibition of DPPH radicals (p > 0.5). The antioxidant activity of samples increased during storage (p < 0.05), then remained relatively constant at the end of the storage. On the other hand, the initial DPPH from 59.39 reached to 72.72%. The best antioxidant activity of juice was observed for wax + MMT-coated fruits, whereas the lowest was attributed to control, however, had no significant difference with wax + OPEO treatment (p < 0.05). Also there were no differences between 80th and 100th day (Table 2).
F I G U R E 1 Effect of storage and coating on total phenol

| Total soluble solids (°Brix)
According to analysis of variance (Figure 2), the effects of time and coating had significant effect on soluble solids; however, there was no significant interaction between time and coating (p < 0.05).
Moreover, the results showed that total soluble solids gradually increased during storage, that is reached (12.4) after 100-day storage.
Also the highest one was observed in control coating which had no significant difference with Wax treatment (p < 0.05) and the lowest one was related to MMT + wax coating. The interaction effect of coating and time indicates maximum and minimum, respectively, were relevance to wax coating and MMT + wax at 100th day which had no difference with MMT + OPEO and OPEO (p < 0.05).  (Table 3). Moreover, the analysis of time and coating interaction effect showed high amount of vitamin C at 100th day in MMT + OPEO-coated fruits which was the same as MMT treatment and the lowest one was related to control which had no difference with wax coating alone (p < 0.05).

| Titratable acidity
According to Figure 3, coating beneficially influenced titratable acidity of blood orang after 100 days of storage (p < 0.05).
However, acidity increased gradually in all samples and reached to 1.06% by coating. The MMT + wax-treated fruits had higher acidity rather than others. Thereafter, wax + OPEO + MMT, wax + OPEO, and wax, respectively, showed higher acidity compared with control.

| Firmness
Firmness is one of the most critical quality attributes influencing consumer appeal and marketing of fresh fruit. Figure 4

| Color
The color parameters of fruits coated are illustrated in Figure 5.
The results showed that the color of samples changed during the storage period (p < 0.05). According to Figure 5a, it can be noticed that component "L" decreased in all treatments during the storage time (p < 0.05) and changed approximately from 83.31 at the beginning to 59.43 at 100th day. Moreover, oranges coated with carnauba wax+MMT had higher "L" parameter and lower one was related to those treated with OPEO. However, no significant differences were observed between control, MMT + OPEO, and wax treatment.

| pH
The pH values showed significant changes during the storage although there were no differences among the samples. According to

| D ISCUSS I ON
Citrus fruits are non-climacteric; hence, their respiration rate and ethylene production do not increase remarkably during the ripening as in climacteric fruits. However, they suffer from some physiologic postharvest disorders such as water loss, peel pitting, and chilling injury at unappropriated storing conditions (Lester & Hodges, 2008;Wardowski, Hall, & Grierson, 2006). Fruit coating is a common approach to reduce the weight loss and improve appearance which is typically based on various waxes such as beeswax or carnauba

F I G U R E 4 Effect of storage time and coating on firmness
In addition, the amount of anthocyanin, flavanols, and hydroxy cinnamic acid in the blood orange usually increases during the storage (Rapisarda et al., 2008). So high antioxidant activity of blood oranges during storage could be due to the synthesis of phenolic compounds as mentioned above although it can be attributed to the species. However, the temperature and storage time should be considered (Klimczak et al., 2007).
The results showed that total soluble solids gradually increased during storage which might be due to sugar synthesis from organic acids (Rapisarda et al., 2008). Degradation of cell wall may also lead to increase in total dissolved solids (Burns, 1990).
Nonetheless, total soluble solids during storage have the uptrend as a result of hydrolytic enzymes activity or waste water under storage conditions (Dris, Niskanen, & Jain, 2003). The results of this study were conformable with the study of Shahid and Abbasi (2011) which reported that the increase in soluble solids of orange over storage and lower amount was observed in fruits beeswax and cellulose coated.
In citrus, the predominant form of vitamin C is ascorbic acid, whereas dehydroascorbic acid (DHA) is less than 10% of total vitamin C content. Moreover, the changes in DHA values during the storage are negligible; thus, the amount of ascorbic acid can be assumed as vitamin C content in juice (Rapisarda et al., 2008).
The results of vitamin C indicated the reduction trend in all samples; however, those coated with wax reinforced with MMT and MMT + OPEO showed higher vitamin C content. The reduction of ascorbic acid during the storage might be related to the increasing in respiration rate because vitamin C is sensitive to oxidation F I G U R E 5 Effect of storage time and coating on (a) color component "L," (b) color component "a," and (c) color component "b" deterioration (Hassan, Lesmayati, Qomariah, & Hasbianto, 2014). It seems control had higher respiration and higher vitamin C loss. In contrast, fruits treated with wax containing MMT had higher vitamin C content. This results are attributed to excellent oxygen barrier properties of reinforced coatings with MMT which reduce the rate of vitamin C oxidation (Azeredo et al., 2012;Bendahou, Kaddami, Espuche, Gouanvé, & Dufresne, 2011;Sanchez-Garcia & Lagaron, 2010). However, the amount of vitamin C in the fruit is influenced by storage time and temperature (Klimczak et al., 2007). The same observation was gained from Azeredo et al. (2012) who claimed that nanocomposite coating (MMT and nano-cellulose) enhanced further maintenance of vitamin C.
In addition, Arena, Fallico, and Maccarone (2001) observed the reduction of vitamin C during the long time storage in commercial oranges.
The results of titratable acidity showed a reduction trend in all fruits coated during the storage; however, the wax coatings incorporated with MMT had higher acidity rather than others. It seems that MMTs play an important role in maintenance of acidity. Titratable acidity is an important quality indicator for citrus fruit ripening and directly related to the concentration of organic acids such as citric acid and malic acid. This reduction of acidity might be related to the consumption of the malic and citric acid during ripening (Shahid & Abbasi, 2011) or used as source of carbon for alcoholic fermentation and even synthesis of polyphenols such as anthocyanins and non anthocyanins phenols (Rapisarda et al., 2008). In addition, coating acts as barrier to gas permeation which causes the accumulation of CO 2 and motivate of anaerobic respiration (Arnon, Zaitsev, Porat, & Poverenov, 2014). Furthermore, MMT clays disperse in polymer coating matrix and create a maze path that causes slowly gases permeation rate (Kim & Cha, 2014). The similar results were gained by Hassan et al. (2014) who examined the effect of bees wax coating on quality of tangerine citrus. They obtained the decline in titratable acidity; however, there were no significant difference. Also, Zeng, Zhang, Chen, and Fu (2012) found that the amount of titratable acidity decreases over time and oranges coated by clove bud extracts had higher acidity than control. Thus, it seems low temperature storage and the use of appropriate coating is necessary for maintaining and increasing biologic properties citrus postharvest (Rapisarda et al., 2008). There are numerous researches that verified the reduction of titratable acidity for other fruits (Dris et al., 2003;Obenland, Collin, Mackey, Sievert, & Arpaia, 2011;Robards, Li, Antolovich, & Boyd, 1997;Tietel et al., 2010).
The results of firmness revealed that the wax + MMT coating maintained the appropriate firmness of fruits while wax-coatingtreated fruits and control had the lowest stiffness. Water loss during the storage causes rapid deterioration by shriveling. Maintaining the quality of fruit could be achieved by reducing water loss from fruit during storage (Chien et al., 2007). In this manner, edible coatings can be stated as a barrier against water loss, thereby preventing the reduction of cell pressure and of the cell membrane break which prevent the reduction of the firmness (Bartolozzo, Borneo, & Aguirre, 2016). In addition, coating supplemented with MMT clay nanomaterials got reinforced and as nanofillers improved the water vapor barrier properties (Azeredo et al., 2012). The results of this study are in agreement with Hashemi and Taghinezhad (2012) who evaluated the effect of nanocomposites (nanoclay-chitosan) on lemon. They claimed coating had significant effect on firmness which verified our results. Moreover, according to the results of the Njombolwana et al. (2013), carnauba coating showed the low firmness loss ratio (0.74) on sweet orange fruit as our experimental data exhibited. In addition, there are various studies about coating effects on quality and firmness. For instance, the same results were observed about of chitosan for strawberry fruit (Del-Valle, Hernández-Muñoz, Guarda, & Galotto, 2005). Also, the study on the Persian lime wax coating indicated that the use of wax causes greater uniformity, increased stiffness, and reduced defects treated fruits (Chien et al., 2007). The results of Valencia-Chamorro et al. Berry based on alginate-acerola including the MMT and nanocellulose indicated that MMT-coated samples were preferred rather than others because they had the color properties as same as fresh Acerola (Azeredo et al., 2012).
The results of pH were conformed with titratable acidity results and as predicted an increase in pH was observed. The change in pH during whole storage period might be due to number of reasons; first, the alteration of biochemical condition of fruit due to treatments and second, due to lower rate of respiration and metabolic activity. pH value increases but at a slower rate particularly at the end of storage period, as there might be the saturation of atmosphere inside the pack with water vapors (Biasi & Zanette, 2000;Shahid & Abbasi, 2011). In addition, reduction of pH during storage could be related to oxidation of food components such as aldehydes and ketones as well as high CO 2 concentration because of the oxygen and water vapor barrier properties of coatings, especially MMT impact on accumulation of CO 2 and increasing the pH (Manzano & Diaz, 2001;Pesis & Ben-Arie, 1986).
Our results are in agreement with finding of Hashemi and Taghinezhad (2012) which evaluated the chitosan nanocomposite coating (chitosan-clay) on the lemon quality.

| CON CLUS ION
In this study, the physicochemical properties, maintenance of quality and the extension of shelf life of blood orange by coating are demonstrated.
Fruits were coating based on carnauba wax with MMT (2%), carnauba with OPEO (1%), carnauba combination with MMT (1%) and OPEO (0.5%), and coating based on carnauba wax alone. The results showed that coating had significant effect on decay and no trace of decay was observed. Also wax coating had maximum deformation which was same as control and minimum amount was related to MMT that had no significant difference with orange peel essential oil and MMT + OPEO. Furthermore, the carnauba + MMT coating, respectively, had the maximum and minimum amount of total acidity and dissolved solid. In addition, time and coating had significant effect on vitamin C in which maximum and minimum amount was observed in MMT and combination of MMT + OPEO treatments, respectively. Total phenol analysis of coated samples had a difference with control. Moreover, DPPH evaluation showed the highest antioxidant activity of MMT coating. In addition, component "L" and "a" had the maximum changes in OPEO treatment and MMT coating, respectively.
To conclude, the incorporation of MMT into the carnauba wax coating is an alternative approach for elongation of fruit shelf life like blood range while preserving their freshness.

CO N FLI C T O F I NTE R E S T
None declared.

ACK N OWLED G M ENTS
The authors are grateful to chemical department of Gorgan University of Agricultural Sciences & Natural Resources. We thank Dr. Alireza Sadeghi Mahoonak for comments that greatly improved the manuscript.

E TH I C A L R E V I E W
This study involved no human or animal testing.