Plant Material

The selected strawberry, during this work, was of the variety “Oso Grande”, coming from the canton Chambo of the province of Chimborazo-Ecuador, whose location has a latitude of 1°44’00’’ and a longitude of 78°35’00’. The selection was made according to ^{Barquero (2007)}, considering the degree of maturation, size, uniformity and health of the fruit, without containing any dirt or foreign matter, in addition to having a minimum of 75% red coloration.

Preparation of the Coatings

The brands of the different substances used were: Gel’hada gelatin, Home Chef pectin and Apicare beeswax, the clove essential oil brand Isabrú Botanik with a 100% purity; distilled water with 98% of Purity Mark Prodont; Glycerin with a 99% purity Mark Ofmagnet and the CMC brand of magnets with a 99.5% purity. The formulation of edible coatings is shown in Table 1.

Table 1 Formulation of edible coatings. 

The edible coatings were prepared by heating the distilled water at 65 - 75 °C, with constant stirring. The ingredients were added in the following order: The main component (gelatin, pectin or beeswax), glycerin, carboxymethylcellulose until a homogeneous mixture was obtained; finally, the temperature was reduced to 20 °C, and the clove essential oil was added. In the case of beeswax, it was first diluted at 62 - 65 °C, which was subsequently incorporated into the mixture with the other components.

The fruit was washed and disinfected by immersion in ozonated drinking water for 15 min. Then, left to dry; after that, the coating was applied by dipping the fruit and leaving it to drain for 1.5 min. Finally, it dried at temperature of 14 - 24 °C until its compaction. The storage was carried out on plastic trays, at an ambient temperature of 13 °C, with a relative humidity of 66.30%, and at refrigerated temperature of 4 °C, with a relative humidity of 95% (^{FRN - ESPOCH, 2016}).

Physical and chemical properties assessment

Weight loss (%): performed by the gravimetric method, using a pioneer precision analytical balance. (^{Restrepo and Aristizabal, 2010}).

Texture: the measurement of this property was done according to the ^{INEN 1909 (2015)}; using a penetrometer mark QA. supplies, fruit pressure tester, FT 327 model and 3.5 mm diameter plunger.

Soluble solids (%): A refractometer: ATAGO brand, Model PAL1, was used as described in ^{INEN 380 (1985)}.

pH: in this property measurement a potentiometer Testr 30 waterproof, Mark Oakton was used as described in the ^{INEN 389 (1986)}. Titratable acidity (%) was performed according to the ^{INEN 381 (1986)}, using an acid titrator: burette Dornic Marca BRIXCO (^{Camacho et al., 2009}).

Lifetime Valuation

This was carried out by establishing Alvarado´s method, through the count of molds and yeasts ^{(Alvarado, 1996)}.

Economic valuation

Production costs were determined based on projection for one year, according to the established ^{FAO (2016a)} method.

Experimental design

The experimental design was based on the evaluation of physical-chemical properties (VD: effects): weight loss (1); texture (2); soluble solids (%) (3); pH (4) and titratable acidity (% citric acid) (5) of the “Oso Grande” strawberry variety, in the post-harvest conservation through the addition of edible coatings, in front of a control. The process was carried out using a completely randomized design, with factorial arrangement, involving the following independent variables (VI): three types of edible coatings based on gelatin, pectin and beeswax; temperature: ambient and cooling; time: 11 days, with three replicates each. This same procedure was repeated, but with 12 levels for VI Time. The results were subjected to analysis of variance and the means separation according to Tukey test.

The experimental treatments number for each VD measurement: physical and chemical properties: weight loss (1), texture (2), soluble solids (%) (3), pH (4) and titratable acidity (5) were calculated through the product independent variables levels (VI), according to the following mathematical model:

Where:

N _T: Treatments number nT: Levels number of the independent variable (VI): Time nt: Levels number VI temperature: ambient (A) and refrigerated (R) nTR: Levels number the VI type coating: gelatin, pectin and beeswax

Then, the total experimental runs number (NCET) was obtained by multiplying the number of treatments by the number of repetitions (coefficient k) (Equation 2).

By explicitly replacing expressions 1 and 2, the total treatments number and the experimental runs were obtained, respectively.

The total treatments number, according the modified Equation 1, in the measurement the physicochemical properties studied, is given according to the following mathematical models 3, 4 and 5.

The experimental runs were performed with three replicates (coefficient k) for each VD. The total experimental runs value was given by the sum of the experiments, considering absence of control (Equation 6) and adding the total sum of control used; the value obtained is given after Equations 7 and 8.

Where,

N _{T VD1}: Treatments measurement number the VD₁ N _{T VD2,3,4 and 5}: Treatments measurement number the VD_{2,3,4 and 5} TEx: Type of Experiment, according to VD type N _{TT VD1,2,3,4 and 5}: Total Treatments number VD_{1,2,3 and 5} N _{CTwithoutVD1,2,3,4 and 5}: Total runs number the VD_{1,2,3,4 and 5} measurement; no control treatment N _{Tsamples VD1,2,3,4 and 5}: Total Samples number used in all trials

Assessment of physico - chemical characteristics

Weight loss (%). In the analysis of the weight loss variable, referential studies, such as those performed by ^{Robinson et al. (1975)} report that the maximum percentage of weight loss, for the marketing of the strawberry is 6%. In this research, these values are within the established parameter up to the third and fourth day (which correspond to the treatments in beeswax and gelatin refrigeration respectively). Other treatments are outside the set parameter (from the third day). Weight loss values (from day 2 to day 12) are shown in Figure 1. The results were re-obtained from the second day, since the initial measurements (first day) constitute the starting point for this research.

Figure 1 Weight loss depending on the experimentation days at room temperature and refrigeration. 

The treatment with gelatin was the one that presented the best characteristics in this parameter, since its weight loss percentage only reached a value of 5.26. This is because gelatin is a hydrocolloid substance (gelling agent), which gives the coating thickening properties that do not allow the fruit to lose the water it has in its composition because it forms a good barrier to the transfer of gases and water vapor (^{Trejo et al., 2007}).

Figure 1 shows a considerable weight loss of the treatments at room temperature in relation to those of refrigeration. This is because, after fruits are harvested, physiological changes occur, such as perspiration (^{Fernández et al., 2015}); and because the fruit is constituted mainly of water (72 - 95%). There is a great dehydration or wilting, which controlled by refrigeration avoids the loss of water that is produced by evaporation (^{Pelayo and Castillo, 2002}).

Texture. The loss of firmness constitutes one of the most significant physiochemical changes; it is associated with the loss of water, generated through perspiration and respiration of the fruit. As a result, wilting and loss of consistency occur. It is one of the main factors used to determine fruit quality and post-harvest shelf life (^{Acuña, 2009}).

With regard to the variable texture, there are investigations, such as ^{Alcántara (2009)}, where they indicate that, for its commercialization, the strawberry must have a firmness oscillating between 9.8 - 11.5 N. In the present investigation, at refrigeration temperature, it is within the parameter until the fifth day in the treatments coated with beeswax, pectin and control and until the sixth day with the treatment of gelatin.

Gelatin coatings are those that delayed the softening of the fruit longer, presenting a value (per day six) of 9.92 N. This was because the gelatin develops excellent mechanical properties that are very favorable to cover food (^{Acosta, 2014}); in this order, treatments with pectin and beeswax were followed which (until day five) presented a value of 10.02 N; because polysaccharides, such as pectin, are capable of constituting a structural matrix, allowing to obtain edible coatings with moderate mechanical properties (^{Eum et al., 2009}). In the case of beeswax, these results were due to the addition of polysaccharide (CMC) in the formulation of the treatment, which provided good mechanical properties since lipids, on their own, present drawbacks in this aspect (^{Campos et al., 2011}; ^{Pavón and Valencia, 2016}).

Analyzing temperature factor, it can be observed that the effect of refrigeration presented better results with respect to the ambient temperature treatments, obtaining values similar to those reported by ^{Trejo et al. (2007)} in his research. For this reason, the application of 1% of gelatin for edible coatings in strawberries influenced the softening of tissues; since the uncoated fruits showed a 62% loss of firmness; meanwhile, the coated strawberries showed 5% values from the third day of storage. In Figure 2, it can be seen that the texture decreases with storage time, until finally softening is achieved.

Figure 2 Texture Behavior (N) as a function of elapsed time at room temperature and refrigeration. 

Soluble solids (%). For the soluble solids variable, researches such as that of ^{Chicaiza (2015)}, assert that for the “Oso Grande” variety, this parameter must have a minimum value of 6.91 ° Bx; while ^{Reyes and Zschau (2012)} confer a maximum value of 8.2 °Bx, so that the fruit is acceptable. In this research, these values are within the established data until the twelfth day (in the treatments in gelatin refrigeration, beeswax and without coating), and the pectin until the eleventh day. The percentages of soluble solids obtained in the research are shown in Figure 3.

Figure 3 Soluble solids (%) depending on the experimentation days at room temperature and refrigeration. 

The treatments with gelatin kept stable the amount of soluble solids present in the strawberry, reporting a percentage of 7.49 because the gelatin, when presenting excellent barriers to oxygen, slows down the process of degradation of sucrose that cause fungi, thus avoiding an increase in soluble solids. Refrigeration (considering the temperature factor) maintains the activity of the microorganisms in the latent state, which allows a lesser variation of soluble solids present in the fruit (^{Ruiz, 2015}). The soluble solids variation can be seen in Figure 3.

pH and titrable acidity. In regard to the pH variable, reference studies such as those of ^{Nunes (2007)} report values between 3.4 - 3.75. In this research these values remained until the eighth day, which correspond to the treatments that are at room temperature, but until day 12 for those that are in refrigerated conditions (this was because bacteria and fungi use, as a source of energy and nutrients, the components present in food, such as sugars, amino acids, and phenolic compounds). In this case, organic acids or glucose may be produced. The production of these compounds causes an increase in the concentration of OH-ions, which results in an increase in pH. This type of process is what causes food to deteriorate (^{Fennema, 1996}).

The gelatin-based coatings maintain the pH of the strawberry stable, reporting a value of 3.69, since the gelatin forms a protective layer, avoiding the entry of oxygen and stopping the activity of the fungi and bacteria, which keeps the pH in the fruit stable. In Figure 4 it is possible to observe the increase of the pH of the treatments at room temperature with respect to those of refrigeration.

Figure 4 pH behavior as a function of elapsed time at room temperature and refrigeration 

As for the titratable acidity variable, researches such as that of ^{Chicaiza (2015)}, indicate that the strawberry should have a minimum percentage of 0.69 g and a maximum of 0.89 g of citric acid per 100 g of product. In this research these values are within the established data until the ninth day; which corresponds to the treatments in gelatin refrigeration, pectin, and beeswax with percentages of 0.73, 0.71 and 0.69, respectively. This was due to the conservation effect provided by the low temperatures; the coatings create a protective layer on the fruit that retains volatile compounds and slows its senescence process, stabilizing the organic acids longer (^{ELIKA, 2010}; ^{Villegas and Albarracín, 2016}).

The acidity reduction coincided with the pH increase. The cause was the use of organic acids as an energy source to support the process of senescence of the fruit (^{Chicaiza, 2015}). In addition, it must be considered that the organic acids present in the food influence the taste, color and stability of the same. Acidity values can be very variable, as in fruit. Citric acid can constitute up to 60% of the total soluble solids in the edible portion (Figure 5).

Figure 5 Citric acid (%) as a function of elapsed time at room temperature and refrigeration. 

Molds and yeasts.In regard to the parameter molds and yeasts, reference studies such as those carried out by ^{Frazier and Westhoff (2003)}; report that in order for a fresh fruit to be consumed, without causing any health risk, it must possess a range acceptable to oscillate between 3 log CFU mL^-1 and 4 log CFU mL^-1. In this research, these values remain until the ninth day in treatments with refrigerated coatings; other treatments are outside the parameter set from the third day.

The incorporation of lipids, such as essential oils in an edible coating, is an effective method to solve some of these problems, as well as in the control of fungal diseases of the fruit (^{Pontigo-Suárez et al., 2015}) by reducing the diffusion processes and the maintenance of high concentrations of active molecules on the surface of the fruit (^{Shao et al., 2015}). Clove essential oil has been shown to have antimicrobial, antifungal, and antiviral activity due to its main chemical component, which is eugenol (^{Hall and Fernandez, 2004}; ^{Anjum and Akhtar, 2012}; ^{Vesaltalab et al, 2012}; ^{Chaieb et al., 2007}; Shao et al., 2015).

The gelatin coatings presented the lowest microbial load, with a value of 45 CFU mL^-1 because when combined with a protein, a polysaccharide and a lipid, this helps to minimize the disadvantages of the individual components, making synergy of their functional and physical properties (^{Pascall and Lin, 2013}).

Beeswax, as a coating, presented good characteristics within this parameter, with a value of 51 CFU mL^-1 b ecause, when using lipids such as beeswax and essential oils, combined with polysaccharides such as CMC, coatings are obtained with excellent barriers against oxygen, carbon dioxide and moisture (^{Parzanese, 2009}).

Treatments with pectin followed, in numerical value, to beeswax with a value of 52 CFU mL^-1; since, when using polysaccharides in combination with essential oils, a good combination is produced, obtaining edible coatings with good barriers against humidity and moderate mechanical properties (^{Parzanese, 2009}; ^{Ruiz et al., 2016}).

The conservation in low temperatures is essential to avoid the development of molds and yeasts, especially the development of the mold Botrytis cinerea, since according to ^{Shao et al. (2015)}; it is this mold that most affects the strawberry (grows at temperatures of 15 - 20 ºC and in conditions of high relative humidity with values between 85 - 90%). The temperature directly affects the conservation of the food, it already keeps the microorganisms in latent life (inactivity), thus increasing the useful life of the product (ISETA, sf.). This is demonstrated in the results of this research, since refrigeration achieved up to 50% fungal growth, compared with treatments at room temperature (Figure 6).

Figure 6 Growth of molds and yeasts as a function of elapsed time at room temperature and refrigeration. 

Value of shelf life

Edible coatings at room temperature reported values between 2 to 3 days of shelf life. These data are consistent with those of the ^{FAO (2016b)}; which states that the strawberry is very perishable and deteriorates within 2 or 3 days of harvesting in natural environmental conditions.

In the treatment of refrigerate fruit, values of 4 to 5 days were obtained. These values exceed that indicated by ^{Acuña and Llerena (2001)}; which shows that the strawberry lasts up to four days in refrigeration; this verifies the effectiveness of the use of coatings in the post-harvest handling of this valuable fruit (^{Villegas and Albarracín, 2016})..

The useful life of the strawberry can improve by performing a good post-harvest management, avoiding in this way the activation of metabolic processes that cause the deterioration of the fruit. Temperature is a very important factor for storage conservation. At high temperatures, senescence processes develop at high speed, and at low temperatures, metabolic activity and moisture loss is reduced, slowing microbial proliferation. For this reason, the preparation of the fruit at refrigerated temperatures after collection is recommended (^{Ariel, 2004}). The values used for the calculation of useful life can be seen in Table 2.

Table 2 Values used for the calculation of useful life of the strawberry. 

Economic valuation

All production costs were projected for one year, with a production capacity of 300 kg of strawberry daily, using 75% capacity. In Table 3, production costs can be seen for obtaining the three types of coatings.

Table 3 Production costs for one year (USD).