Extending the shelf life of mango (Mangifera indica L.) fruits by using edible coating based on xanthan gum and pomegranate peel extract

The effect of various concentration of xanthan gum (0.5%, 1%, and 2%) based edible coating supplemented with pomegranate peel extract (0.5 mL) on functional and physico-chemical properties of mango (Mangifera indica L.) fruits were studied during the storage period of 15 days at 22 °C. The application of xanthan gum (XG) based edible formulations with pomegranate peel extract (PPE) was found to be effective to maintain the quality attributes and characteristics like reducing weight loss, respiration rate, ethylene production, maintained total soluble solids (TSS), acidity, pH, texture property, ascorbic acid, phenols, and antioxidant activity as compared to control samples. In general, all tested formulations are effective; but edible coatings based on 2% of XG were found the most potential to prevent the postharvest characteristics of mango fruits while maintaining the quality attributes.


Introduction
Among the tropical fruits, the mango (Mangifera indica) is the most important fruit in the Anacardiaceae family. Owing to its sensory characteristics and complexity for immediate consumption, has tremendous consumer appeal in fresh form. Fresh mangoes are highly appreciated for their texture, color, good fragrance and nutritional value [1]. Mango fruits and its products such as puree, pickles, nectar, canned slices and chutneys are popular globally and increasing in value on the western market [2,3]. Mangoes are species of a climacteric type; after harvesting, they cannot be well-preserved at room temperature for a long period of time because they mature within 2-10 days [4]. A sequence of metabolic activities occur during maturation resulting in chemical changes, increasing respiration, changes in structural polysaccharides leading to fruit softening, deterioration of chlorophyll and biosynthesis of carotenoids, starch hydrolysis to sugars resulting in the ripening of fruit with texture softening to acceptable quality [5]. Low shelf-life and lack of post-harvest maintenance are limiting factors to export mango fruits to distant markets. Different treatments i.e. low-temperature storage, drying and use of inorganic chemicals such as potassium metabisulphite have been investigated to sustain quality to enhance the shelf life of fresh mango fruits [6]. Shelf life of other fruits after harvest can be successfully enhanced by keeping them at lowtemperatures [7,8]. In general, low-temperature storage is the ultimate technique for extending the storage-life of fruits & vegetables; however, its complete benefit cannot be realized for mango due to its chilling susceptibility, and due to its residual effect, inorganic chemicals contribute to adverse health conditions for consumers. Thus, different techniques such as the use of a plant growth regulator for ionizing radiation, plant extracts, modified atmosphere (MA), controlled environment, and edible coatings (ECs) can be used to prolong mango fruit's post-harvest life [9][10][11]. The need for high-quality foods and the minimal food processing and storage technology have highlighted the idea of using ECs to prolong the storage-life of fresh and minimally processed foods as well as to preserve them from adverse environmental effects. ECs have been advanced for this purpose; they are biodegradable packaging material [12,13] that are formed by applying a layer of edible film on an intact or minimally processed fruit surface, forming a protective layer [14][15][16][17]. This protects the consistency of these fruits by developing a film that acts as partial barriers to gases such as O 2 and CO 2 , avoiding water vapor and aroma compounds, creating a modified atmosphere outer the fruit, limiting the rate of respiration and lack of water, and retaining texture & taste [18]. However, work remains to be conducted on antimicrobial, antioxidant or combined effects of edible coatings. Ultimately, the function of these coatings may significantly improve the storage-life of fruits [19]. The usage of ECs like Xanthan Gum (XG), synthesized by Xanthomonas campestris as an exo-polysaccharide under unfavorable circumstances, is a compound commonly recognized as a safe (GRAS) compound (FDA, 21CFR172.695, 2020) [20] for use as a stabilizer, emulsifier or thickener. It forms an extremely viscous solution in cold/hot water with outstanding consistency at low concentration over a broad range of pH and temperatures; therefore it is resistant to enzymatic degradation. It enables the storage of particulate matter for a long time, also in complicated formulations. XG known as an additive, has been generally used in the food processing industries such as bakery, beverages, milk, and pet food [21][22][23]. It is worth noting that xanthan gum is used as artificial saliva in people with Sjogren's syndrome, which manifests itself as dry mouth [24]. In addition, xanthan gum is found in some protective and anti-reflux medications. Many researchers have been examined the effects of XG based edible packaging on pears, guavas [18], fresh-cut melons [19], baby carrots [25], grapes [26] and fresh cut pears/fuji apples [22,27,28] and extended their storage life with maintaining physicochemical, sensorial, textural properties, color and slowing down the respiratory rate and the output of ethylene during storage [29,30]. Therefore, this study aimed to access whether XG (at different concentrations) has the potential to be used as an EC for delaying ripening, enhancing shelf-life and protecting the quality of stored mango during storage for 15 days at room temperature. Physiological loss in weight, TSS, TA, pH, the content of ascorbic acid, respiration rate, ethylene production, firmness, TPC, and antioxidant activity were analyzed and reported hereby.

Pomegranate peel extracts (PPE) preparation
The pomegranate peel of Bhagwa cultivar was freeze drying for 32 h at − 45 °C using a freeze dryer (Benchtop, Vir Tis, USA) to obtained peel powder. The method of preparation of extraction from the peel powder was prepared according to the method followed by Kumar and Neeraj [14] 0.2 g of pomegranate peel powder was dissolved in water (10 mL) as solvent and sonicated at 45 °C for 30 min using an ultrasonic bath (CUB-5, Citizen, 40 kHz, 220-240 V, India). The sonicated mixture was centrifuged (Sigma, 3-18, KS, Germany) for 10 min at 5 °C using 8654 rpm. The prepared mixtures were filtered using Whatman No. 11 and the 0.02 g/mL extract was stored for further incorporation in edible formulation.

Coating preparation
The solutions for XG were prepared by dissolving 0.5%, 1%, and 2% XG in deionized water at room temperature, continuous stirring until full dissolution, followed by heating at 60 °C and eventual cooling at 20 °C; glycerol (0.5 g) was added as plasticizer and 0.5 mL of PPE (0.02 g/mL); again stirred using magnetic stirrer for 30 min. The purified solution of XG was then retained at 20 °C before further usage.

Treatment application and storage conditions
Mangoes were divided into four random lots and each lot was allocated to be dipped in one of four treatments: (A) 0.5% XG (w/v), (B) 1% XG solution (w/v), (C) 2% XG (w/v), and (D) control (distilled water). The treated mango fruit was drained and stored at 22 °C, 60-65% relative humidity for 15 days. Sample analyzes were made every 3rd day, with three replicates of each treatment. However, the sampling date was assessed for the weight loss, TSS, TA, pH, ascorbic acid content, respiration rate, ethylene production, firmness, total phenolic content (TPC) and antioxidant activity. To ensure reproducibility of the results, the whole procedure was replicated, with the results from the first procedure included in this study.

Determination of physiological loss in weight (PLW)
Mass loss of mango fruits during storage period was investigated and calculated using the methods followed by Kumar et al. [31]. At each sampling interval date, these fruits were subsequently weighed to track their changes in weight for the entire experiment until the mangoes were spoiled. The weight samples were taken and their percentage calculated to quantify the overall differences in weight. The percentage change (loss) of weight was determined using Eq. 1.

Total soluble solids (TSS)
The TSS of mango fruits was determined by digital refractometer at room temperature. Before using refractometer, it was clean and calibrated by distilled water. The percentage of TSS was revealed as the results in terms of degree Brix (°Bx) [32].

Titratable acidity (TA) and pH
TA of mango fruits was determined using titration method followed by Kumar et al. [33] with minor modification. 10 g of mango pulp homogenized with distilled water (90 mL) and phenolphthalein indicator (3-4 drops) was also added. The mixture was titrated against 0.1 M NaOH solution.
pH was determined according to the standard methodology using digital pH meter. 20 g of sample suspension was prepared in 100 mL of purified water and the pH was measured by pH meter. Before the use, the pH meter was calibrated using buffer solution of pH 4 and pH 7. The average value was reported as result of pH.

Determination of respiration rate and ethylene production
Using a closed system approach and a CO 2 /O 2 analyzer, the respiration rate of samples was measured. Two mangoes were placed in a 1 L container hermetically sealed with a silicone rubber septum for 3 h to determine the respiration rate. The head-space gas was sucked through a hypodermic hollow needle after a predetermined amount of time, and the respiration rate was measured using an autogas analyzer (PBI Dansensor, Denmark). The results were revealed in milliliters of CO 2 per kilogram of fruit released per hour (mL CO 2 /kg h). Ethylene production was assessed by sealing two mangoes in a 1 L jar with a sub seal septum for 3 h. A gas chromatograph fitted with a flame ionization detector and Porapak-N 80/100 mesh packed stainless steel column was used to extract 1 mL of the head-space atmosphere, and the ethylene was quantified. The results are given in microliters of ethylene released per kilogram of fruit per hour (µL/ kg h).

Firmness
Firmness of the fruit samples during the storage period was determined using texture analyzer (Stable, Micro system, UK). 5 kg load cell with 2 mm aluminum needle probe at 10 mm/min of texture analyzer speed used to puncture the fruits samples at five equatorial surfaces. For each sample, firmness was measured as mean of three measurements and reported in Newton (N) force needed for penetration of fruit surface.

Ascorbic acid
For the determination of ascorbic acid, 50 mL of mango juice was immersed with 2 g of potassium iodide in solid form. 10 mL of both 0.5 M H 2 SO 4 and 0.01 M solutions of potassium iodate were then concurrently applied to the flask. The mixture was titrating against 0.07 M sodium thiosulphate until it lost almost all of its color. 2 mL of starch as indicator was then applied, completing the titration cycle [34]. Titrated values were obtained and their means calculated to obtain the values for ascorbic acid. Results were revealed in terms of mg/100 g.

Total phenolic content (TPCs)
TPCs of control and treated mango fruits were determined using standard Folin Ciocalteu (FC) reagent method with some modification [35]. 1 mL of sample extract immersed with distilled water (70 mL) and 5 mL of FC reagent (ten time fold) was also added. Mixture was mixed vigorously, added 15 mL of 20% sodium carbonate solution and make up volume of solution up to 10 mL using distilled water.
The solution was mixed and incubated for 2 h at dark place. The absorbance of the samples was recorded using UV spectrophotometer at 765 nm wave length. Gallic acid was used as standard references and results were revealed as mg/g on dry basis equivalent.

Antioxidant activity
The samples antioxidant activity was determined using 2,2-diphenyl-1-picryl hydrazyl (DPPH) assay method [36]. Sample extract (0.1 mL) was mixed with 3.9 mL of DPPH solution. The solution was incubated at dark place for 2 h. The absorbance of the samples was recorded at 517 nm wave length using UV spectrophotometer. Methanol was used as blank samples to calibrate DPPH free reaction mixture was used as control sample. The results of antioxidant activity were revealed in terms of percentage (%).

Statistical analyses
All the experiment were performed in replicates of three (n = 3) using completely randomized design and results were indicated in terms of mean ± standard deviation. The statistical analysis of data were performed by analysis of variance using SPSS (24.0) statistical software. Sigma-Plot 14 software was used for the graphical representation of data. Duncan multiple post-hoc comparison test was appoint for evaluate the statistical differences between the means (p < 0.05).

Physiological loss in weight (PLW)
XG coating treatments and storage period has performed a significant impact on mango fruit weight loss (Fig. 1a). With the progression of the storage period, the weight loss increased significantly and reached the maximum for the control on the 9th day and 15th day for the treatments after which the mangoes were spoiled, respectively. Moreover, all coating treatments showed less fruit loss than control fruits. 2% XG coated mango fruit showed 10.41%, while control PLW of mango fruit showed 21.90% at the end of the storage time (15 days). Water loss is responsible for the decline weight of fruits and shriveling during post-harvest storage [37]. However, the fact that coating restricts water loss and maintains high fruit weight during storage is well documented [38]. This is supported by Adetunji et al. [39], who also observed weight retention in XG-treated papaya fruit. The results of this study show that increased concentration of XG reduced mango fruit weight loss as compared to control. Daisy et al. [40], Minh et al. [41], and Kumar et al. [31] are reducing the PLW of mango, rambutan and tomato fruits at different storage conditions by using gum arabic, XG and chitosan-pullulan composite ECs supplemented with PPE by controlling water loss and respiration rate, respectively.

Total soluble solids (TSS)
TSS of mango fruits was significantly (p ≤ 0.05) affected by treatments for XG coating, storage-period, and their interactions. The control mangoes showed a significant rise (p < 0.05) in TSS, reached a peak at the end of storage (14.02 °Bx); however, XG coating treatments (p ≤ 0.05) significantly delayed mango TSS rise during storage time (Fig. 1b). Among the XG treatments, 2% surface coating treatment with XG showed insignificant change in TSS up to the 15th day (10.48 °Bx). Such discoveries are in a good agreement with Adetunji et al. [39], they have concluded that the XG coating maintains the papaya fruit TSS significantly. The results are in a good agreement with the previous study by Kumar et al. [42] and Kumar et al. [16] have noted the application of edible coatings functionalized with PPE on litchi and mango fruits as effective packaging to maintain the TSS of fruits by reducing hydrolytic conversion of sugar and leakage of juice that could be due to the minimizing oxidation, respiration rate and moisture barrier property of the EC.

Titratable acidity (TA)
Similarly, during the storage period, TA of mangoes was significantly affected by XG coating (p ≤ 0.05). There was a decline in TA for control and treatment with an increased storage period (Fig. 1c). In the 2% XG coating treatments, TA was higher than in control and other storage treatments. The values ranged from 0.31 to 0.40% at the end of storage on the 15th day. With little increments in TSS and low TA decreases in all XG treatments, this means that the XG treatment was slower to ripen. Increased or reduced acidity during maturation may be due to their conversion into sugars and further utilization in the fruit's metabolic processes. Gowda and Huddar [43] have recorded a similar pattern on various mango fruit varieties stored at 18-34 °C. The results of our experiment are in a good agreement with the findings by Adetunji et al. [39] that is due to XG coating, pH and acidity shift in papaya fruits.
pH Figure 1d presents the pH values in mango samples using various concentrations of XG coatings. The results indicate that mango fruit treated with 2% of XG coating showed lower pH values. It may be due to the composition of the solution XG, which had a pH of 4.13. By comparison, at the end of the storage period, pH of control mango fruit was 5.06. Result is similar to the study on guava fruit with XG coating Gad and Zagzog [44], which reported 1% XG mix with 0.2% chitosan nano-particles coating has improved overall quality of guava fruits during extended cold storage and shelf-life periods.

Respiration rate
During the starting 3 days of storage, respiration rates of both samples (control and coated mango fruits) were reduced. Both subsequently experienced a significant rise, with control fruit showing peak respiration after 9 days while the coated fruit after 12 days of storage. All treatments for the coating delayed respiratory peak periods during 15 days of storage. However, 2% XG treatment (37.51 mg CO 2 / kg h) significantly (p ≤ 0.05) suppressed the fruit oxygen consumption during 15 days of storage (Fig. 2a) compared to the control sample (48.31 mg CO 2 /kg h). Respiration rates are directly related to metabolic activity and therefore considered an excellent fruit shelf-life indicator [45]. The shortened storage life of climacteric fruits is responsible for an increase in respiration during maturation [46]. Besides, mango cultivars have also reported a reduction in the respiration rate as a result of polysaccharide-based coating [37]. Likewise, with the increasing thickness of chitosan coating, Zhu et al. [47] have observed a decreasing trend in mango fruit respiration rate. Similarly, after increasing decline occurrence, Cosme Silva et al. [48] have demonstrated an improvement in mango respiration rate. This indicates that XG coating keeps the mango fruit respiration rate low during storage. According to Dang et al. [49] application of carnauba based EC on mango delayed the climacteric peak in the coated fruit as compared to the control.

Ethylene production
The major fruit ripening hormone is ethylene and its production increases during maturation. Control mango fruit obtained an ethylene production rate after 6 days, while the coated treatments reached peak values after 12 days of storage. However, 2% XG coating maintained a steady production of ethylene (p ≤ 0.05) compared to control and other storage treatment (Fig. 2b). The production of fruit ethylene is controlled by the enzymes ACC-synthase, ACC-oxidase, and the decrease in the production of ethylene following exposure to simulated condition is likely due to decreased activity of these enzymes [50]. However, as the production of ethylene also responds to fruit respiration levels and the respiration rate was significantly inhibited by 2% XG coating, i.e. 0.41 µL/kg h at the end of the storage period, significantly less ethylene could be produced in 2% coated mango fruit. Similarly, Shah et al. [51] have observed a decrease in mango ethylene production by application of a chitosanaloe vera EC. This study also shows that the increase in the concentration of XG inhibits the production of ethylene in mango fruit.

Firmness
Entire mango fruits were softened during 15 days of storage (Fig. 2c). The coated fruit, however, softened to a lesser degree than controlled mango fruit. Also, 2% XG coated fruit (1945.53 N) was found to be slightly firmer (p < 0.05) than control mango fruit (1141.21 N) and other treated fruits. Firmness is an essential sensory feature, indicative of fruit quality and storage life [52]. However, XG slows the maturation by reducing the rate of respiration with increased XG concentration, fruit yields greater firmness. Softening is the catabolic action of polygalacturonases and pectin methylesterase enzymes during maturation, resulting in degradation of middle lamella between parenchyma cells, destruction of the cell wall and loss of cellular turgidity [53]. By increasing the thickness of the chitosan coating, Cosme Silva et al. [48] have recorded greater firmness in mango fruits. Likewise, by using a highly impermeable carnauba wax EC, Baldwin et al. [29] have maintained the firmness of mango fruit during the storage time.

Ascorbic acid
The ascorbic acid is the strongest antioxidant that protects the fruit from the harmful effects of reactive oxygen species [54]. During storage, ascorbic acid degraded in entire mango fruits. Control samples (20.81 mg/100 g) showed more ascorbic acid degradation (p ≤ 0.05) from 6 to 15 days than 2% ascorbic acid degradation (38.66 mg/100 g) during storage (Fig. 2d). Ascorbic acid is also an antioxidant and degrades after oxygen reaction. Hence, avoiding oxygen contact with food could delay ascorbic acid's oxidative breakdown [55]. Additionally, treatment with XG by 2% resulted in higher phenolic content. Baldwin et al. [29] have noticed a strong association between the ascorbic acid content and fruit phenolic concentration. Our findings are in-line with the study of Robles-Sánchez et al. [56], they have monitored mango fresh cut the ascorbic acid degradation by coating them with antioxidant-incorporated alginates. This study presents that efficiency of EC was increasing during storage with increasing the concentration of XG coating on mango fruit.

Total phenolic content (TPC)
Phenolic content of all treated samples decreased in mango flesh significantly until the end of the storage period. However, there was a substantial retention of phenolic content (p ≤ 0.05) in 2% XG-coated samples (31.40 mg/g) relative to control (21.82 mg/g) and 0.5% (26.40 mg/g) and 1% (28.02 mg/g) coated mango fruits. However, during the entire storage period, 2% of XG treatment maintained significantly (p ≤ 0.05) higher phenolic content (Fig. 3). Coatings produce mild fruit stress, and composite coating can increase stress [57]. The decline in control phenolic content at the end of storage could be attributable to the fact that the phenolic content of fruits decreases during senescence [58]. These results are consistent with successful retention in the phenolic content of mango treated with chitosan and spermidine composite coating observed by Jongsri et al. [59]. This study revealed that the application of XG reduced the degradation of phenolic content of mango fruit synergistically.

Antioxidant activity
During the 15 days of storage, the antioxidant content decreases significantly. XG coating also retained high antioxidant activity for up to 15 days, and slowly decreased afterwards. The 2% XG-coated fruit antioxidant activity was significantly higher (p ≤ 0.05) than control and other coated fruits ( Table 1). The values ranged from 63.52 to 51.03% at the end of storage on the 15th day. This recommends that the rise in respiration rate during maturation stimulates antioxidant activity in the fruit. The control samples after 6 days resulted in a peak respiration rate, while the coated treatments did after 12 days of storage. Therefore, declines  in antioxidant activity could be observed on these days for control and coated treatments, respectively. The phenolic content of 2% XG coated samples was higher, so 2% XG coating possesses an antioxidant activity higher than control and other types of coated mango fruits during the storage. Additionally, Palafox-Carlos et al. [58] have found that antioxidant activity decreases during mango fruit over maturation. This study showed that during post-harvest storage 2% of XG maintains high antioxidant activity in mango fruit. It was a positive relationship between antioxidant activity (%), TPC, and XG coating treatment concentration that suggest the effect of polyphenol content and XG concentration on antioxidant activity.

Conclusion
Xanthan gum (0.5%, 1%, and 2%) as EC supplemented with PPE was used for extending the postharvest shelf life of mango (Mangifera indica L.) fruits during storage period of 15 days at 22 °C. The results of present study proved that the application of this composite EC to be effective to extending storage-life of mango fruits while maintaining postharvest quality attributes i.e. reducing weight loss, respiration rate, ethylene production, maintained total soluble solids (TSS), acidity, pH, texture property, ascorbic acid, total phenolic content and antioxidant activity as compared to control samples. The increasing concentration of XG solution was found the most effective to maintain quality attributes of mango fruits and extending the shelf life. The results established that the XG coating supplemented with PPE can have potential application to reduce the postharvest losses of variety of fruits and vegetables.