Using Response Surface Methodology to Optimize Edible Coating Formulations to Delay Ripening and Preserve Postharvest Quality of Tomatoes

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Introduction
Tomato (Solanum lycopersicum L.) is one of the most consumed fruits in the world [1]. It is important in human nutrition and health due to high nutrient content and signifcant amount of bioactive substances such as lycopene, ascorbic acid, tocopherols, folic acid, and favonoids [2][3][4]. Due to its high nutritive value and water content, postharvest tomatoes are susceptible to diseases. Tese fruits are also sensitive to lowtemperature storage [5,6]. Tis leads to the loss of the quality parameters of the fruits such as color, texture, aroma, and appearance responsible for their commercial interest [4,7]. Previous studies reported an increase in tomato's shelf life through modifed atmosphere storage (relatively high CO 2 and low O 2 ) and controlled atmosphere storage [8], active packaging of cardboard [9], and genetic engineering [10]. In order to meet the increasing demand and consumption of minimally processed and additive-free foods, diferent means have been used to extend the shelf life of tomatoes such as edible coatings. Many edible coatings are made from waste agricultural resources through bio-production [11]. Edible coatings are thin layers of the edible component such as hydrocolloids (polysaccharides and proteins), lipids (waxes and resins), and synthetic polymers, applied to the fruit's surface in addition to or as a replacement for natural protective waxy coatings [12]. Tey act as a physical barrier towards carbon dioxide, oxygen, and moisture movement for the fruits [13]. Te uses of edible flms and coatings containing synthetic antimicrobial agents, organic, and vegetable material have been shown to be useful in preserving the quality of tomatoes [13,14]. Moreover, these materials have been used to incorporate functional ingredients such as antioxidants, antimicrobial agent, plants extract, byproduct extract, and nutraceuticals in fruits [15][16][17][18][19]. Pineapple (Ananas comosus L. Merr.) is a fruit rich in several nutrients and bioactive compounds including vitamins C, calcium, and nonvolatile organic acids such as malate and citrate which has been used to preserve the postharvest quality of tomato and strawberry [20][21][22][23]. Luo et al. [24] reported that the polysaccharides contained in pineapple peels have some degree of antioxidant activity. Arabic gum (AG) has been used as barrier to CO 2 and O 2 in some edible coating formulations to extend the shelf life of certain fruits such as guava, mango, and tomato [17,25,26]. Response surface methodology (RSM) was used to study properties of edible flms and the main formulation that have efects on the preservation shelf-life and quality of some fruits [27,28]. Terefore, the objective of this study was to use the RSM to determine the optimum concentrations of aqueous extract of pineapple peel and Arabic gum as well as the optimal time to coat tomato in order to increase shelf life and reduce postharvest losses.

Biological Material.
Healthy tomatoes at the mature green stage ( Figure 1) were collected from the local farm in Dschang (Cameroon) and kept in the laboratory. Te pineapples used as coating material were harvested from farmers feld in Melong (Cameroon) at ripening stage four (with low sugar content) [29]. Arabic gum was harvested on Acacia Senegal plants in Garoua, northern Cameroon [30].

Coating Preparation.
Pineapples were peeled after washing with water. Te peels were dried in the shade, scrambled in the mill, and then grounded to obtain a homogeneous paste. Diferent quantities of paste were weighted (Table 1) and macerated in a water/ethanol mix (1/1, v/v). 230 μl/l of bleach was added to disinfect the medium. Te macerate was transferred on a sieve for fltration. In order to thicken the extract and form an adhesive and transparent flm on the surface of the tomatoes, Arabic gum was added to of fltrate as coating matrix according to the quantities shown in Table 1. Te mixtures were macerated for 15 hours, allowing pineapple peel extract to adhere to the Arabic gum.

Coating.
Tomatoes were washed and soaked in the different coatings for 10, 20, or 30 minutes depending on the experimental design for coatings T1 to T20 (Table 1). Ten fruits were left without coating as the control. All the fruits were left on the bench at room temperature (24 ± 0.5°C) and 82 ± 1.5% RH.

Analytical Methods.
Two physical parameters and three physiological parameters including ripening rate (RR), frmness, total favonoid content, chlorophyll a content, and titratable acidity (TA) of fruits were evaluated after 8 days of storage.

Ripening Rate.
Te ripening rate was evaluated by counting the number of red ripped fruits ( Figure 2) at the 8 th day after treatment according to ripening rate defned by Te United Fresh Fruit and Vegetable Association, in cooperation with USDA [31].

Determination of Chlorophyll a Content.
Quantitative analysis for chlorophyll a content in tomato pulp was performed using a Biochrom Libra S22 spectrophotometer. Chlorophyll a content was determined using the method described by Nagata and Yamashita [32]. Six (6) grams of the tomato pulp was crushed and introduced in a test tube, then 10 ml of acetone/hexane (4/6, v/v) was added. Te mixture was stored at 4°C for 48 hours. Subsequently, chlorophyll a in the hexanolic extracts was detected by spectrophotometry at 663 and 645 nm. Te chlorophyll a content was calculated using the following equation: Chlorophyll a(μg/100 ml) � 0.0999A663 − 0.0989A645.

Firmness.
Firmness is the force (N) required to press the fruit against the tip of a penetrometer. Epicarp was removed at equatorial and top region of tomato fruits. Te cylindrical tip of the penetrometer was pressed down gradually on tomato notches, and the measurements were read on the board of the penetrometer [23].

Total Flavonoid Content.
Te concentration of total favonoids was measured using the aluminum chloride colorimetric method [33] with some modifcations. One milliliter (1 ml) of fltered tomato juice was added to a 10 ml Erlenmeyer fask containing 4 ml of distilled water. Ten, 0.3 ml of 5% NaNO 2 was added. After 5 min, 0.3 ml of 10% AlCl 3 was added. Finally, 2 ml of 1 M NaOH was added after 6 minutes; the volume was completed to 10 ml with distilled water. Te solution was mixed thoroughly, and the absorbance was measured at 510 nm using a spectrophotometer. Flavonoid compounds were determined according to a catechin standard curve in μg/ml.

Titratable
Acidity. Forty milliliters of distilled water were added to 20 ml of tomato juice. Te volume was made up to 40 ml with distilled water. After adjusting the pH to 8.1, the titrate value was measured and was used to calculate the titrable acidity following the method of Gharezi et al. [34].

Experimental Design and Statistical
Analysis. RSM was used to generate the experimental design statistical analysis and regression model with the help of Minitab software. Te central composite rotatable design (CCRD) with a quadratic model [35] was employed as Nandane et al. [28]. Each independent variable had three (3) levels: −1.809, 0 and +1.809 (Table 2). Six replicates of the center points were chosen in random order according to a CCRD confguration for three factors divided in two blocks. Te p values in the design outside the ranges were selected for rotatability of the design [36]. Te center points for these designs were selected with ingredients at levels expected to yield satisfactory experimental results. Twenty (20) edible coating formulations with diferent concentrations of pineapple peel extracts Te coefcients of the polynomial equation were represented by a 0 (constant term), a 1 , a 2, and a 3 (linear efects), a 12 , a 13, and a 23 (interaction efects), and a 11 , a 22, and a 33 (quadratic efects).
Te analysis of regression was made and regression tables were generated; the efect and regression coefcients of individual linear, quadratic, and interaction terms were determined. Te signifcance of all terms in the polynomial equation was appreciated statistically by computing the Fvalue and comparing response variables at standard signifcance levels of 0.1, 0.05, 0.01, and 0.001. Because tomato fruits are perishables, agricultural products with big variations in the quality attribute between one another [27]. Te adequacy of the model was determined using regression coefcient (R 2 ) analysis. Using Minitab Software, numerical and graphical optimization procedures were applied to determine the optimum level of the independent variables.

Efect of Edible Coating on Ripening Rate of Tomato Fruits.
As shown in Figure 3, the ripening rate (RR) of the coated tomato decreased with the pineapple peel extract concentration, while the optimum predicted time of treatment was 20 min. Te CGA was fxed at 10%. Ali et al. [17] observed that fruit coated with 10% arabic gum delayed the ripening process by slowing down the rate of respiration and ethylene production. Te regression coefcient table of RSM analysis with ripening rate as response variable is shown in Table 3. Te model F-value of 69.25 obtained for the efect on ripening rate (%) of treated tomato fruit implies that this model was signifcant. Values of "Prob ˃ F" less than 0.1 indicates signifcance of the model terms (Table 3). In this case, a 1 a 3 , a 13 , a 11 , a 22 , and a 33 are signifcant model terms. Tus, the ripening rate is afected by linear efect of pineapple peel extract concentrations and time, interactions efects of time of treatment, and concentration of pineapple peel extract and by the quadratic efect of tree factors. Te "Lack of Fit p -value" of ˂0.001 implies that the Lack of Fit is signifcantly relative to the pure error. Tus, independent variables had a signifcant efect on the ripening rate. Observations from RSM analysis suggested that the ripening rate was negatively related to the concentration of the pineapple peel extract used ( Figure 3). As the concentration of the pineapple peel extract in the solution increased, there was a relative decrease in ripening rate of the fruit. Tis shows that the calcium and antioxidants compounds in the pineapple extract may have induced the delay of ripening [37][38][39][40]. Te fnal equation in terms of actual factors for ripening rate is as follows:

Efect of Edible Coating on Chlorophyll a in Tomatoes.
Chlorophyll a is a more appropriate biomarker for evaluation of the ripening-retarding efects of edible coatings, because it is part of ripening process through the conversion of chlorophyll a and chlorophyll b into chlorophyllide a and then pheophorbide a before its complete degradation in nongreen products [11,37]. As shown in Figure 4, concentration of chlorophyll a gradually increased with Arabic gum and with the increase of time of treatment. Te regression coefcient table for RSM analysis for chlorophyll a as response variable is shown in Table 3.

Efect of Edible Coating on the Firmness of Tomatoes.
As shown in Figure 5, the response surface frmness of fruits increased with Arabic gum concentration in the coating solution. Firmness was afected by pineapple peel extract concentration and time of treatment. Te regression coefcient table for RSM analysis with frmness as response variable is shown in Table 3. Te F-value (9.41) obtained on frmness of treated tomato fruit implies that this model is signifcant. Ali et al. [41] showed that tomato fruit coated with Arabic gum at 10% resulted in a signifcant delay in change of frmness. Low levels respiration gas (O 2 , CO 2 ) exchanges limit pectin esterase and polygalacturonase activities and allow retention of the frmness. Calcium ion, as a frming agent, in edible coatings could improve the rigidity of the cell wall of coated fruits [37,39]. Te fnal equation in terms of actual factors for frmness is given as follows:

Efect of Edible Coating on Total Flavonoid Content of
Tomatoes. Figure 6 shows that the total favonoid content (TFC) value is increased with pineapple peel extract concentration in the coating solution. Tis parameter decreased with the time of treatment. Te favonoid content value was afected by interaction efect of pineapple peel extract and Arabic gum, quadratic efect of pineapple peel extract, and quadratic efect of time of treatment in the coating formulation. Te regression coefcient table for RSM analysis of favonoid content as response variable is as shown in Table 3. Te F-value of 866.44 obtained implies that the model is signifcant. Flavonoid compounds are secondary

Efect of Edible Coating on Titrable Acidity of Tomatoes.
Te titratable acidity (TA) values of coated fruit during storage were maintained with Arabic gum concentration and decreased with concentration peel extract (Figure 7), and the value of linear term was signifcant (p < 0.1). Te TA value was positively related to Arabic gum concentration. Regression coefcient table for RSM analysis for titrable acidity as response variable is shown in Table 3. Te F-value of 3.06 obtained implies that the model is not signifcant. Te same was observed by Ali et al. [41] who reported that the arabic gum coating delayed ripening of tomato by providing a semipermeable flm around the fruit. Since organic acids, such as malic or citric acid, are primary substrates for respiration, a reduction in acidity is expected in highly respiring fruit as reported by El-Anany et al. [42]. Te fnal equation in terms of actual factors for TA is as follows:

Conclusion
Increasing concentration of pineapple peel extract and Arabic gum improved the thickness of edible coating and had important efects on their quality. Te ripening rate was correlated with the alterations level of chlorophyll a which decreased simultaneously with the ripening of tomato fruits. Te thickness of edible coating was confrmed by the correlation between the production of secondary metabolites as favonoid compounds and the increasing of concentration of pineapple peel extract and Arabic gum. Te optimum concentration of CPE, CGA, and time of treatment were predicted to be 0.70 kg/l, 17.04%, and 18.72 min, respectively, with predicted values of response variables denoted as RR 40.75%, chlorophyll a 8.106 μg/g, frmness 4.00 N, TFC 43.51 μg/ml, and TA 0.302%. Edible coating formulation with pineapple peel extract and Arabic gum can be used in extending the shelf life and delaying the ripening process of tomatoes at ambient conditions. Te RSM method can be efective to study the efect of edible coatings on the ripening of tomato fruits postharvest.

Data Availability
Te data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te authors declare that they have no conficts of interests.