Effect of the Biodegradable Coatings the Base on Microalgae and Oil of the Seed of the Pomegranate in the Conservation Powder-Crop of the Papaya ‘ Golden ’

It is very challenging to the quality of the papaya culture’s fruits (Carica papaya L.) after the crop, especially due to their significance to the international market, that is, their elevated exportation demands. The purpose of this work was the application of biodegradable coatings composed of Scenedesmus sp. and Chlorella sp. associated or not with pomegranate seed oil in ‘Golden’ papaya and to evaluate their effect in the conservation powder-crop. The installation of the experiment was carried out in a completely randomized design, with a 6 x 6 factorial outline, that is, six concentrations (C: control; SO: 0.5% of Scenedesmus sp. + 0.3% of pomegranate seed oil; S: 0.5% of Scenedesmus sp.; CO: 0.5% of Chlorella sp. + 0.3% of pomegranate seed oil; CH: 0.5% of Chlorella sp.; O: 0.3% of pomegranate seed oil) and six evaluation periods (0, 3, 6, 9, 12, and 15 days), stored at a temperature of 18±2 °C with 60±5% RH with three repetitions of two fruits per portion. The use of coverings composed of Scenedesmus sp. and Chlorella sp. in association or not with pomegranate seed oil was proven efficient in the reduction of the breathing tax of ‘Golden’ papaya, delaying the ripening process, and therefore representing a promising alternative for these fruits’ powder-crop conservation. The coating composed of 0.5% of Chlorella sp. + 0.3% of pomegranate seed oil (CO) provided a better powder-crop conservation of ‘Golden’ papaya during 15 days of storage.


Introduction
Papaya (Carica papaya L.) is a fruit of great economic importance all over the world.It is consumed in natura in several countries (Lorenzi et al., 2015).Its main producing countries are India, Brazil, Indonesia, Nigeria, and Mexico.Brazil, the second greatest producer, represents 12.6% of the world production (FAOSTAT, 2015).The papaya tree's fruit is produced in most of Brazil's territory especially in the Northeastern region from which Bahia and Espírito Santo are the best producing states, representing 70% of the Brazilian production (IBGE, 2016).They are cultivated almost for the whole year and their commercialization perspectives for consumption in natura in the internal market or for exportation are quite favorable, establishing the culture as one of the most promising fruits for exploration.However, Brazil is considered as one of the top ten food-wasting countries in the world, with virtually 30% of the production thrown away during the powder-crop phase (Fernandes et al., 2016).In spite of this culture's importance and of the all necessary care during the cultivation phases performed in the production chain, the fruits' commercialization has some problems related to damages caused during the powder-crop (Catanho et al., 2017).The main fruit loss causes are mechanical, or caused by cold, powder-crop diseases, and advanced ripeness (Bautista-Baños et al., 2013).If those wastes were minimized, the papaya fruit farmers and exporters could achieve maximum profits and seize a larger portion of the world market (Ali et al., 2011).Therefore, we can state that one of the Brazilian horticulture's current great challenges is the postharvest fruit quality preservation (Botelho et al., 2016).Consequently, alternative methods are necessary to extend the powder-crop storage of papaya during chain logistics.
A technique that is becoming more popular as a powder-crop treatment for fruits, including the papaya, is the application of biodegradable coatings (Hamzah et al., 2013).Biodegradable coatings are used as an alternative for fruit conservation due to their capacity of changing the atmosphere, reducing the breathing, perspiration and humidity indexes, maintaining pulp firmness, and delaying fruit senescence (Mannozzi et al., 2017).Edible and biodegradable coatings have a great potential to act as transporters of several active ingredients, including antimicrobial agents (Fai et al., 2016;Yousuf & Srivastava, 2017).
Among the several new compounds studied, the pomegranate seed oil, due to its nutritional and medicinal properties, was highlighted as a potential ingredient in the food industry with beneficial attributes to health (Verardo et al., 2014).Its biological potential also makes it an attractive nutraceutical ingredient as a consequence of innovation.It is available at a low cost and is widely accepted by the consumers (Caligiani et al., 2010).
Researches for the development of new products, starting from microalgae, have been proven increasingly promising, since they are sources of natural products that were recently studied for biotechnological applications (Dantas, 2013;Dantas et al., 2015).Scenedesmus is a green alga with great potential for industrial applications (Koller et al., 2017;Abdulsamad & Varghese, 2017).It contains all of the essential amino acids and good amounts of proteins, lipids, and macro and microelements (Cheban et al., 2015).Chlorella is a unicellular green alga that contains several valuable proteins (40-60%) and that has been thoroughly used in aquaculture, food, and biotechnology industries (Dantas et al., 2015).Microalgae have excellent coating properties or biofilms.Therefore, they are a promising coating or film-forming material due to their rheological and functional characteristics.In the last years, the efficiency of elaborated coatings was evaluated, starting with microalgae in the fruit application (Onias et al., 2016;Queiroga et al., 2017;Oliveira et al., 2018aOliveira et al., , 2018b)).In this context, this work's objective was to evaluate the application of biodegradable coatings of the Scenedesmus sp. and Chlorella sp.algae in association or not with pomegranate seed oil for the conservation of powder-crop 'Golden' papaya.

Material and Methods
The experiment was developed in the Federal University of Campina Grande (UFCG), Agri-food Science and Technology Center (CCTA), in the Fruit and Vegetable Powder-crop Laboratory.The fruits employed in the experiment were acquired from an orchard located in the meadows of Mamanguape, Paraíba (PB), 385 km from the municipal district of Pombal-PB, in which only the 'Golden' variety was cultivated.
The crop was performed at the morning.The fruits were pre-selected in the field, avoiding the ones with disease symptoms, pathogen presence, or mechanical damage.The fruits' maturation was visually determined.To standardize the experimental sample, we selected the fruits with the following characteristics: fruits with color change, whose yellow spots do not cover more than 15% of their peel (Teodosio, 2014).Afterwards, they were conditioned in a single layer, in cardboard boxes (with the dimensions of 640 × 480 cm), previously covered with bubble plastic to minimize the impact and friction between them, and taken into the laboratory, where the fruits were selected according to their size uniformity and color.Those with defects or apparent damages caused by the transport were discarded.Later, they were washed with a 1% detergent solution and sanitized with a sodium hypochlorite solution to 200 ppm of active chlorine for 20 minutes.They were then rinsed with water and air dried.The experiment was set up in a completely randomized design, with a 6 × 6 factorial outline, which comprised six coating techniques (C = control; SO = 0.5% of Scenedesmus sp.+ 0.3% of pomegranate seed oil; S = 0.5% of Scenedesmus sp.; CO = 0.5% of Chlorella sp.+ 0.3% of pomegranate seed oil; CH = 0.5% of Chlorella sp.; O = 0.3% of pomegranate seed oil) and six evaluation periods (0, 3, 6, 9, 12, and 15 days), with three repetitions of two fruits per portion.
The microalgae employed in this study were produced according to Lima (2016), in organic production tanks, at Tamanduá Farm, located in the city of Patos-Paraiba.Once the biomasses were obtained, they were diluted into 2 L of water at constant stirring until the complete homogenization of the solution.Afterwards, the fruits were plunged in the solutions for 20 minutes, after which they were dried at room temperature, conditioned at a bench, and stored at the temperature of 18±2 °C and 60±5% RH for subsequent evaluation.For each evaluation period, the fruits were processed in centrifuges and their following features were evaluated: Loss of Fresh Mass (PMF): determined by a gravimeter at a semi-scale analytical precision of±0.01g.The results were expressed in percentage losses, employing the ratio before the mass loss and after each storage period.
Firmness of the Pulp: evaluated by a texturometer (Fruit Hardness Tester), with a penetration depth of 2.0 mm, speed of 2.0 mm s -1 and a TA 8/1000 ferrule.The readings were made with the whole fruits, and each fruit was measured four times: on their opposite faces and after the removal of portions of their peel.The results were expressed in Newtons (N).
Color of the Peel and Pulp: checked through the digital colorimeter Konica Minolta CR-400 and the CIELAB System, which sets a three-dimensional chromatic space with 3 axes in rectangular coordinates (L*a*b*), which respectively indicate the brightness (L*), the red (*a positive) to green (-a* negative) tones, and the yellow (b* positive) to blue (-b* negative) tones.It also sets the cylindrical coordinates (L*, C*, H°).The values of a* and b* are converted into Hue angle (H°), which represents the color intensity, and their chromes (C*), that is, the color purity, according to the equations of (Pinheiro, 2009).
Potential of Hydrogen (pH): measured by a digital peg for the direct reading of the homogenized pulp, according to IAL (2008).
Total Titratable Acidity (ATT): measured according to the methodology recommended by the Adolfo Lutz Institute (2008), using 10 grams of homogenized pulp diluted into 100 mL of distilled water, followed by titration with standardized solution of NaOH 0.1N, using the phenolphthalein turning point as indicator.The results were expressed in citric acid g by 100 g-1 of the sample.
SS/ATT Ratio: calculation of the ratio between soluble solids and total titratable acidity (SS/ATT).The results were expressed in decimals.
Vitamin C: it was measured through Tillman's methods.We weighed 1 g of the sample, which was transferred into Erlenmeyer vials, in which the volume was completed for 50 mL with 0.5% of oxalic acid, whose titration was performed with Tillman's solution until the turning point (AOAC, 2006).
Total Soluble Sugars: The total soluble sugars were measured using Antrona's method (Yemn & Willis, 1954), through a spectrophotometer analysis at 620 nm, and the results were expressed in g 100 g -1 of the pulp.
Total of Carotenoids: measured according to Linchtentháler's methodology (1987).We employed the wavelengths of 470, 646, and 663 nm in a spectrophotometer to perform the reading to determine the carotenoid and chlorophyll concentrations used in a and b of the sample.The results were expressed in g 100 g -1 of the pulp.

Statistical Analysis:
The data were submitted for analysis of variance and regression through the Sisvar Program (Ferreira, 2011).

Results and Discussion
Starting with the analysis of variance presented in Table 1, we can notice that there was significant interaction between the factors under study (coating and storage time) for the parameters of mass loss, firmness, and peel color (brightness, chromaticity, and hue angle), while the colorimetric parameters of the pulp (brightness, chromaticity, and hue angle) directly proportional to the storage time.Note.ns: not significant; ** significant at 1%; * significant at 5%.
The loss of fresh mass was directly proportional to the storage in all coatings types (Figure 1A).Fruits without coating (C) presented a greater mass loss (10.59%), while the coated ones (CO) had a smallest loss of fresh mass (7.84%) in comparison with the other coatings: O: 9.24%, CH: 9.52%, SO: 8.76%, and S: 9.63%.Studies related to the use of microalga-based (Scenedesmus sp. and Chlorella sp.) biodegradable coatings associated with pomegranate seed oil with the objective of reducing the breathing process of papaya fruits were not found in the literature.Pego et al. (2015), while evaluating the effect of different concentrations of cassava starch used as eatable covering of 'Sunrise soil' papaya, also obtained a directly proportional behavior, presenting an average   Note.ns: not significant; ** significant at 1%; * significant at 5%.
As for the pH, there was not a significant interaction between the evaluated factors (coating × storage period).However, there was significance for the isolated factor (time of storage), in which there was oscillation of the mean pH values of all samples, increasing during the first days of storage, decreasing from the 3rd day (5.74) to the 12th day (5.04), and increasing again (5.43), as seen in Figure 3A.According to Barragán-Iglesias et al. ( 2018), the pH level increase happens due to a decrease in the amount of hydrogen ions supplied by organic acids during the ripening process.We can observe that the CO treatment had its lower pH value at its 15th day of storage; there was a tiny decrease of acidity, reflecting its delayed maturation.This pH decrease happened due to the formation of organic acids and sugar in association to the fruits' breathing.Those pH variations over the storage can be attributed to the initial degradation and the subsequent synthesis of organic acids with different potentials of ionic dissociation (Almeida et al., 2006).A similar behavior was reported by Soares et al. (2015), who obtained a pH of 5.85 in 'Golden' fruits using eatable coatings.
The values of the titratable total acidity (ATT) variable are displayed in Figure 3B, which present significant differences in the interaction between the coatings and the storage period.Analyzing the ATT's behavior, we notice that the C treatment kept its ATT values until the 9th day of storage.Afterwards, there was a significant increase of these values to 0.81%.For the CO, SO, and S treatments, there was happened a decrease until the 3rd day, presenting significant increases until the 15th day of 0.71%, 0.73%, and 0.72%.In the O and CH treatments, there was a reduction of acidity up to the 3rd day and, consequently, a significant increase until the 12th day, with reductions until the 15th of 0.39% and 0.57%.We can infer that the treatments under a temperature of 18 °C slowed the papayas' normal ripening process down because the decrease of the acidity is associated to the consumption of acids in the breathing process caused by maturation (Pego et al., 2015), while the increase in the acidity in their coatings is probably caused by to the galacturonic acids liberated during the hydrolysis of the cell wall's components, responsible for the tissue firmness (Soares et al., 2015).
Soluble Solids (SS) are considered a quality index in papaya.Their concentration and the composition play an important role in the fruits' flavor, which reflect the maturation phase (Corrêa et al., 2008).The increase of the SS levels contributes to increase the sugar level of the fruit (Nunes et al., 2017).SS have a quadratic behavior and a significant difference during the storage period.At the end of the conservation period, there was an increase of this parameter, for all cases with mean values of 12.15°Brix (Figure 3C).Souza et al. (2014), Teodosio (2014), Mendes et al. (2015), Soares et al. (2015) and Nunes et al. (2017) reported similar behaviors.
The data related to soluble solids and titratable acidity ratio showed that there was significant interaction between the treatments and the time of storage, decreasing from the 3rd day onwards, which corresponds to a quick inhibition of the ripening process.After comparing the data, the final value of the control treatment (15.21) was lower in comparison with the other treatments , which indicates that the coating grants them a sweeter flavor (Figure 3D).According to Dias et al. (2011), the SS/ATT ratio is an important quality variable in the powder-crop because it expresses the balance between the sweetness and the acidity of the fruit, partly playing an important role in the pleasant sensation in the consumer's palate.Therefore, the greater this value is, the better the level of sweetness will be (Pego et al., 2015).Similar results were reported by Nunes et al. (2017) for 'Formosa' papaya using cassava-starch-based coatings.

Figure papaya
As Figure 4

Table 1 .
Summary of the analysis of variance for mass loss, firmness, pulp and peel color of 'Golden' papaya with and without the application of biodegradable coatings stored at 18±2 °C with 60±5% RH

Table 2 .
Summary of the analysis of variance for pH, total titratable acidity, soluble solids, SS/ATT ratio, vitamin C, carotenoids, and total sugar of 'Golden' papaya with and without the application of biodegradable coatings stored at 18±2 °C with 60±5%RH