Edible Coatings of Aloe Vera Gel and Carnauba Wax Microparticles to Increase Strawberry (Fragaria ananassa) Shelf Life

ABSTRACT

Extending the shelf life of fruits without altering their nutritional or sensory characteristics has led to the search for different preservation technologies. Strawberry (Fragaria ananassa) is prone to rapid decay after harvest. Aloe Vera Gel (AVG) provides antioxidants that help prevent rapid ripening; however, its hydrophilic nature can reduce the effectiveness of the coating. On the other hand, Carnauba wax (CWP) could decrease water vapor permeability and has not been previously used for strawberries. The objective of this study was to evaluate the effects of the coating formed by different concentrations of AVG with CWP microparticles. The samples were immersed in the coating for different times, lyophilized and analyzed in the scanning electron microscope, with different resolutions, to establish the experimental conditions and confirmed the formation of the layer on the surface. A Completely Random Design was applied, with a 3² factorial arrangement: AVG (0%, 30%, 45% v/v) and CWP (0%, 0.3%, 0.4% w/v). Three repetitions and 27 experimental runs were carried out. The treatments that presented the best physicochemical and microbiological conditions were analyzed sensorially. The treatments with higher concentrations of the two components provided the least change in weight loss, pH, and ripening index (p ≤ .05), and the lowest values of the severity index caused by Botrytis cinerea. The coated samples were well accepted sensorially, with no significant difference with the uncoated strawberries (p > .05). The proposed coatings have the potential to extend the shelf life of strawberries and be applied in the fruit marketing chain.

KEYWORDS:

• Aloe vera gel
• Carnauba wax
• microparticles
• Botrytis cinerea
• edible coating

Introduction

Strawberry (Fragaria ananassa) is a non-climacteric fruit with a high respiration rate (at 20°C produces 50 to 100 mL CO2/Kg) which causes transformations in the internal components of the fruit, such as the increase of degrees Brix and acidity, dehydration and senescence. However, it has a low ethylene production, and its ripening does not continue after being detached from the plant. Only chlorophyll degradation occurs, leading to visual changes in color and alterations of the cell wall, which has a very thin epidermis and high water content that make it prone to mechanical or microbiological damage (Pilar Pinzón et al., 2007). Thus, improper harvest or postharvest techniques cause the fruit to have bumps, bruises, or wounds that will, in turn, accelerate its deterioration and ease the growth of Botrytis cinerea (Chávez, 2019). This fungus can grow at temperatures above 15°C, causing damage to the flowers and fruits. On the other hand, strawberry shelf life (usually around 3–4 days) is related to the conditions of storage, transport, and commercialization to which the fruit is put through (Gol et al., 2013).

The need to extend the shelf life of fruits has led to the application of different preservation technologies that usually cause high cost and energy consumption, or damage to its sensory and nutritional properties. Therefore, cheaper ways, such as the addition of coatings, have been investigated. Edible coatings (EC) made of biodegradable components are an alternative for fruit preservation. When applied, an EC forms a thin permeable layer on the fruit’s surface, helping to control the precursor gases of ripening, minimizing the respiration rate, water loss, and preserving the organoleptic characteristics of the fruit. In addition to being nontoxic and colorless, the EC maintains the fruit’s own texture and increases its shelf life (Fernández Valdés et al., 2015).

ECs are made primarily from polysaccharides, lipids, and proteins, alone or in combination. In addition, plasticizers and/or emulsifiers can be added to these formulations to improve the coating’s final properties. Examples of strawberry’s coating materials include beeswax, potato starch and chitosan (Fernández Valdés et al., 2015). Aloe Vera (AV) is another compound that can be used as an EC component. It contains water, mucilage (made of glucuronic acid, pectin, and sugars), and phenolic compounds with significant antioxidant, anti-fungal, and anti-bacterial activities (Domínguez-Fernández et al., 2012). On the other hand, Carnauba wax (CW) is obtained from the leaves of the palm Copernicia cerifera, found in dry areas of northeast Brazil. The wax contains hydrocarbons, esters, alcohols, and resins. The application of this wax in fruits’ coatings has provided benefits by delaying senescence, reducing weight loss, and improving appearance (Silva de Freitas et al., 2019). However, its application on strawberry has not been explored.

Due to the fact that the strawberry has short periods of shelf life, the present investigation had the objective of developing an edible coating based on Aloe Vera gel and microparticles of Carnauba Wax to extend the fruit’s useful shelf life. Although many combinations have been studied, it was planned to find the effective concentration of the proposed combination or failure to extend the shelf life of this fruit after the evaluation of the physicochemical properties, B. cinerea severity index, and sensory analysis across time.

Materials and Methods

Sample Preparation

Strawberries were harvested in the early hours of the day by a local producer in Patate, Tungurahua, Ecuador. The ripening state was maintained at number 4, according to the scale of eight points (1–2: not harvestable, 3–6: fresh consumption, and 7–8: for agroindustry) proposed by Villagrán et al. (2012). To assure their correct selection, the strawberries were analyzed against a white board. The selected strawberries were washed with tap water and disinfected with 0.2%v/v of commercial disinfectant “Star-bac” (citric acid, benzalkonium chloride and propylene glycol) in water. Strawberries were immersed for 5 minutes, drained, rinsed, and dried by forced air convection for 1 hour at 20°C.

Edible Coating Preparation

Aloe Vera Gel (AVG) Extraction

Aloe Vera leaves were classified according to the presence or absence of dents and washed with tap water. The thorns were removed, and the mucilage was extracted by peeling the cortex of the leaves. The gel was blended with water to formulate 3 different treatments: 0, 30, and 45% v/v (these concentrations were previously studied and established in the laboratory). The resulting gel was filtered, and citric and ascorbic acid (1 and 0.5 w/v%, respectively) were added as preservatives. The pH was adjusted to 5.8, and the solution was heated for 45 minutes at 85°C, and subsequently cooled to 25°C (Sophia et al., 2015).

Carnauba Wax Microparticles (CWP)

Carnauba wax microparticles were generated by an emulsion technique. In a conical tube, 0.25 g of Carnauba wax, 0.5% v/v of Tween 80, and 25 mL of distilled water were added, without mixing. The tubes were placed in a water bath at 85°C for 30 minutes to melt the wax. Samples were homogenized using a vortex mixer for 1 minute to form the emulsion. To obtain the wax microparticles, the emulsion was frozen and lyophilized (BIOBASE).

Edible Coating Elaboration

Prepared solutions of AVG (aqueous phase) and CWP (oleic phase), with Tween 80 (surfactant), glycerol (plasticizer), and distilled water were mixed, with a final volume of 100 mL that was stirred at 250 rpm for 20 minutes. Finally, the strawberries were submerged in the coating for 1 minute for further analysis (see the following sub-sections).

Coating Application

Preliminary Coating Conditions

The objective of a preliminary application was to verify the formation of the coating on the surface of the fruit. Therefore, strawberry samples were immersed for 1 minute in the coating with 45% v/v AVG; 0.3% w/v CWP; 1.5% v/v glycerol and 0.5% v/v Tween 80. Subsequently, they were dried by forced air convection at 20°C for 1 hour; stored at 4°C for 24 hours and packed in bioriented polyethylene boxes with perforations. Cuttings of size 1 × 1 cm x 2 mm were made in the epidermis of strawberries with and without coating. The samples were lyophilized and analyzed under the scanning electron microscope (JOEL JSM-IT399LA) with resolutions of 100×, 250×, 500×, and 1000× (Voltage: 10Kv and Pressure: 50 Pa).

Definition of Coating Application Time and CWP Working Concentrations

Once the application of the coating was verified, the immersion time and concentration of Carnauba wax were analyzed to establish the best conditions for the experiment. The strawberry samples were mixed with 0.3, 0.4; 0.5% w/v of Carnauba wax. The concentrations of AVG (45% v/v), glycerol (1.5% v/v), and Tween 80 (0.5% v/v) were kept constant (based on previous laboratory trials). The immersion time was varied at 30, 60, and 120 seconds. Samples were coated and analyzed by scanning electron microscopy (SEM), as previously explained. According to the results obtained during preliminary tests in the laboratory (data not shown), 30 seconds immersion produced irregular coats, while 120 seconds immersion generated agglomerations; hence, the most suitable immersion time was 60 seconds.

Experimental Design

A completely randomized design with a 3² factorial arrangement was applied (Qamar et al., 2018), corresponding to the combination of two factors with three levels each: AVG (0%, 30%, and 45% v/v) and CWP (0%; 0.3% and 0.4% w/v). Three repetitions of each treatment were performed, and 27 experimental units were obtained. The 9 treatments were: T1 (0%AVG, 0%CWP); T2 (0%AVG, 0.3%CWP); T3 (0%AVG, 0.4%CWP); T4 (30%AVG, 0%CWP); T5 (30%AVG, 0.3%CWP); T6 (30%AVG, 0.4%CWP); T7 (45%AVG, 0%CWP); T8 (45%AVG, 0.3%CWP); T9 (45%AVG, 0.4%CWP).

The storage temperature (4°C), concentration of Tween 80 (0.5% v/v), and glycerol (1.5% v/v) were kept constant. The output variables of the experiment were:

• Physicochemical properties: weight loss, maximum 10% (Pavón-Vargas and Valencia-Chamorro, 2016); pH, 3-4.2 (Cordenunsi et al., 2003); degrees Brix, minimum 6.4 °Brix (ICONTEC, 1997); and titratable acidity, maximum 0.93% (ICONTEC, 1997).

• Microbiological assay: Botrytis cinerea Severity Index.

• Sensory analysis: Appearance, odor, flavor, overall acceptance (in relation to the control samples).

Physicochemical Properties

For each treatment, pH, percentage of weight loss, degrees Brix, and titratable acidity were determined (each parameter was analyzed in triplicate) during days 0, 2, 4, 6, 8, 10, 12 and 14.

Weight Loss Percentage

The percentage of weight loss was measured based on the method proposed by Muñoz and Naranjo (2012).

pH

The pH is associated with the senescence of the fruit. Thus, this parameter was determined according to the method proposed by García-Mera et al. (2017): pH was measured from a solution of shredded strawberry at 10% w/v.

Ripening Index

To obtain the ripening index of the strawberries, a ratio between degrees Brix and the titratable acidity was calculated (Pilar Pinzón et al., 2007).

Microbiological Assay

According to Chávez (2019), the severity index in strawberries allows its visual classification based on its superficial damage at room temperature across time. Strawberries were classified starting from the ones that did not show any wound up to the ones that showed the development of Botrytis cinerea. In triplicate, each strawberry sample was classified based on the number of hyphae on its surface following the scale: 0, 1–10%, 11–25, 26–50, and 51–100% of hyphae. Each treatment was maintained at room temperature (20°C). This analysis was made on days 1, 2, 3, 4, and 7 in treatments 1, 5, 6, 7, 8, and 9, since these samples obtained the best results for the physical-chemical parameters (Results and Discussion Section).

Sensory Analysis

This stage was carried out in the Sensory Analysis Laboratory at Universidad San Francisco de Quito. Participated Sixty-two untrained judges (33 males and 29 females), with an age range between 18 and 23 years old. A 9-point hedonic scale was used to evaluate the attributes of appearance, odor, flavor and overall acceptance (Ruiz-Martínez et al., 2020), following the scale from extremely dislike (1) to extremely like (9). The samples were arranged in a randomized complete block design (RCBD).

Following the methodology proposed by Jouquand et al. (2008), each panelist was presented with 4 samples (1 sample for each treatment), in a polystyrene plastic tray. Strawberries were stored at 4°C, and transferred to room temperature (20°C) 2 hours before the evaluation began. Each fruit was coded with 3 random numbers: the control sample (149) and 3 samples from the treatment that showed the best physicochemical and microbiological conditions (T9: 45% v/v AVG and 0.4% w/v CWP), with different storage days, day 0 (342), day 3 (267) and day 6 (581). Additionally, the judges were given water as a palate cleanser. The rest time between tests was 30 seconds.

Statistical Analysis

All quantitative data were reported as (average ± standard deviation). Statistically significant differences were determined by analysis of variance (ANOVA) and Tukey test at 95% confidence. Minitab 2018 Software was used.

Results and Discussion

Preliminary Coating

The application of the preliminary coating had the objective of creating a thin layer over the surface of the strawberry. To corroborate the EC deposition, coated and uncoated (control) samples were analyzed through SEM, which allowed to obtain images at a high resolution of the strawberry’s surface.

As shown in Figure 1, after 60 seconds of immersion in distilled water, it can be seen that at 250× from the surface of the control sample (Figure 1a), the achenes were clear in sight, showing the rough and uneven surface, common in a natural strawberry. On the contrary, in the coated treatment (Figure 1b), a thin layer of EC uniformly covered the strawberry, producing a smoother and more homogenous surface, with the appearance of microparticles of CW (CWP). In addition, Tween 80 was used as a surfactant; while glycerol was used as a plasticizer to give elasticity to the coat. Mehyar et al. (2012) found similar results when using an EC made of pea starch, whey protein isolate (WPI) and Carnauba wax (1: 1: 1) to coat walnuts. The film was homogenous; however, it showed droplets of CW on the surface of the nut, due to the differences in surface energy between pea starch and CW.

Figure 1. Scanning electron micrographs of strawberry surface: (a) uncoated and (b) with preliminary coating (b). Magnification: 250x; Calibration bar: 100 $\mu \mathrm{m}$.

Definition of Working Range of Concentrations of Carnauba Wax Microparticles

This process was carried out to find the best CWP concentration that allowed the coating to not only adhere correctly to the whole surface of the strawberry, but also conferred the maximum protection and extension of the fruit’s shelf life. Concentrations of 0.3 (Figure 2a) and 0.4% w/v (Figure 2b) of CWP showed a similar formation of a thin layer on top of the strawberry’s surface; however, the concentration of 0.5% w/v (Figure 2c) produced the most irregular surface due to the development of big agglomerations of microparticles that could potentially alter the organoleptic properties. Therefore, 0.3 and 0.4% w/v CWP were determined as adequate concentrations to use for the subsequent experimentation because of the reduction in size of the lipid clusters, allowing a uniform coverage and higher stability. Chiumarelli and Hubinger (2014) found similar results when using an edible coat composed by Cassava starch, glycerol, carnauba wax, and stearic acid. As the wax content increased, the microstructure analysis showed a more non-homogenous surface of the films, larger pores, χlusters, and less flexibility, translating into less protection provided by the coat. Jiménez et al. (2010) concluded that this occurs due to poor cohesion forces and mechanical properties; hence, increasing the water vapor permeability (WVP) and reducing the shelf life of the product.

Figure 2. Scanning electron micrographs of strawberries coated for 60 seconds under different CWP concentrations: 0.3% (a), 0.4% (b), and 0.5% (c). Magnification: 250x; Calibration bar: 100 $\mu \mathrm{m}$.

Physicochemical Properties

Weight Loss (%)

The percentage of weight loss increased during storage time (Figure 3 and Table 2). It was greater in the samples that contained the lowest amount of EC. T1, T2, and T3 maintained at 4°C had a rapid degradation, ending with a lifespan of only 10 days (D10), with significant damage and, in many cases, great microorganism growth (Table 2). Before this time, T1 and T2 had the highest weight loss (3.75 ± 0.14)% and (3.62 ± 0.04)%, respectively, values statistically different (p < .05) from treatments T5, T6, T7, T8 and T9 (See statistical analysis in Table 1). On the other hand, the rest of the coated strawberries had a potential for longer shelf life. This indicates that the AVG coat produced a greater decrease in weight loss than CWP alone. At day 14 (D14), T9 showed the least amount of weight loss (2.04 ± 0.03)% (p < .05) compared to T4, T5, T6, T7 and T8; where, T4 had the highest weight loss (2.85 ± 0.11)%.

Figure 3. Weight loss (%) of strawberry samples coated with different EC compositions of aloe vera gel and carnauba wax microparticles.

Table 1. Summary of analysis of variance (ANOVA) of percent weight loss of treatments.

Source of variation	Degrees of freedom	MS
		Day 2	Day 4	Day 6	Day 8	Day 10	Day 12	Day 14
Total	26
Treatment	8	0,08*	0,3*	1,4*	1,4*	1,35*	4,14*	4,55*
Aloe vera gel (A)	2	0,2*	1,05*	3,2*	3,83*	4,2*	16,4*	18,01*
Carnauba gel (B)	2	0,03*	0,2*	1,07*	0,9*	0,8*	0,11*	0,12*
Interaction (A*B)	4	0,01*	0,003*	0,67*	0,4*	0,21*	0,04*	0,03*
Error	18	0,005	0,003	0,003	0,01	0,004	0,0006	0,003

 * Significant at 5% probability by the F test.

Table 2. Weight loss (%) of the treatments.

Treatment/Days	Day 2	Day 4	Day 6	Day 8	Day 10	Day 12	Day 14
T1	$1,26\pm 0,05$ $AB$c	$1,99\pm 0,06$ $A$b	$3,38\pm 0,12$ $Aa$	$3,69\pm 0,29$ $Aa$	$3,75\pm 0,14$ $Aa$	-	-
T2	$1,21\pm 0,01$ $ABe$	$1,92\pm 0,03$ ABd	$2,36\pm 0,07$ $Bc$	$3,24\pm 0,07$ $Bb$	$3,62\pm 0,04$ $Aa$	-	-
T3	$1,29\pm 0,17$ $Ad$	$1,78\pm 0,09$ $Bc$	$1,61\pm 0,02$ $Cc$	$2,31\pm 0,09$ $Cb$	$2,67\pm 0,04$ $Ba$	-	-
T4	$1,27\pm 0,05$ ABe	$1,62\pm 0,03$ Cd	$1,57\pm 0,05$ $CDd$	$2,16\pm 0,05$ $CDc$	$2,61\pm 0,02$ $Bb$	$2,75\pm 0,04$ $Aab$	$2,85\pm 0,11$ $Aa$
T5	$1,19\pm 0,05$ $ABf$	$1,47\pm 0,04$ CDe	$1,56\pm 0,04$ $CDe$	$2,13\pm 0,05$ $CDd$	$2,31\pm 0,05$ $Cc$	$2,48\pm 0,03$ $Bb$	$2,67\pm 0,07$ $Ba$
T6	$1,17\pm 0,04$ $ABd$	$1,30\pm 0,10$ $Ed$	$1,49\pm 0,02$ $CDc$	$2,08$ $\pm 0,09$ $CDb$	$2,25\pm 0,08$ $Cab$	$2,30\pm 0,03$ $Ca$	$2,43\pm 0,03$ $Ca$
T7	$1,07\pm 0,06$ BCe	$1,37\pm 0,05$ $DEd$	$1,42\pm 0,04$ $DEd$	$1,98\pm 0,03$ $Dc$	$2,17\pm 0,02$ $CDb$	$2,22\pm 0,03$ $Dab$	$2,32\pm 0,09$ $CDa$
T8	$0,96\pm 0,06$ CDf	$1,22\pm 0,04$ EFe	$1,35\pm 0,05$ $EFd$	$1,92\pm 0,04$ $Dc$	$2,04\pm 0,03$ $DEb$	$2,15\pm 0,04$ $Eab$	$2,20\pm 0,01$ $Da$
T9	$0,80\pm 0,06$ Df	$1,08\pm 0,04$ Fe	$1,21\pm 0,02Fd$	$1,60\pm 0,04$ $Ec$	$1,86\pm 0,06$ $Eb$	$1,98\pm 0,02$ $Fa$	$2,04\pm 0,03$ $Ea$

Means followed by at least one capital letter in the columns do not differ from each other, by Tukey’s test at 95% confidence.

The response variable was determined each day and when it exceeded the established specification, the analysis was stopped for the following days, which is why cells with a hyphen appear.

It was observed that, at long term, the maximum concentration of AVG + CWP produced the smallest weight loss due to the protection given by the edible coat. All treatments had a weight loss percentage below 10% and were, thereby, within the acceptable commercial weight loss range for fruits (Pavón-Vargas and Valencia-Chamorro, 2016). This effect may be due to the polysaccharide present in AVG, which allows the formation of a physical barrier for protection. However, polysaccharide coats are highly hygroscopic; thus, the gel acts as a poor barrier to water vapor, since it absorbs moisture from the environment. Hence, the addition of lipids, in the form of CWP, helps reduce water vapor permeability, increasing relative humidity on the surface of the fruit, consequently reducing the moisture gradient from the exterior (Singh et al., 2016), effect that could be proven with T9 (45% AVG, 0.4% CWP).

Sogvar et al. (2016) found that a coating of AVG and 5% ascorbic acid applied to strawberries had the greatest influence in reducing weight loss and ripening, thus increasing the shelf life of the fruit.

pH

As the ripening of a fruit increases over time, pH increases as well, due to the degradation of the cellular wall and organic acids that leads to a release of total solids and glucose, reducing the acidity of the fruit (Mazur et al., 2014). This phenomenon was observed during the present research, where, in every treatment, pH values rose. As shown in Figure 4(See Table 4 for values), at day 0 (D0), treatments T1 through T5 had the highest pH and were statistically different (p < .05) from T6 through T9. However, starting from day 4 (D4) until day 10 (D10) (maximum lifespan of the control sample),

Table 4. pH of the treatments.

Treatment/Days	Day 0	Day 2	Day 4	Day 6	Day 8	Day 10	Day 12	Day 14
T1	3,38 ±0,07 Ad	3,42 ± 0,03 Ad	3,51 ± 0,02 Ac	3,64 ± 0,03 Ab	3,69 ± 0,02 Aab	3,72 ± 0,02 Aa	-	-
T2	3,3 ± 0,01 Ad	3,37 ± 0,02 ABc	3,39 ± 0,03 Bc	3,45 ± 0,04 Bb	3,51 ± 0,01 Bb	3,65 ± 0,04 Ba	-	-
T3	3,34 ± 0,03 Ac	3,31 ± 0,03 BCc	3,36 ± 0,03 Bc	3,36 ± 0,02 CDc	3,44 ± 0,01 BCb	3,58 ± 0,04 Ba	-	-
T4	3,35 ±0,05 Ad	3,33 ± 0,02 ABd	3,34 ± 0,03 Bd	3,38 ± 0,01 Ccd	3,37 ± 0,04 CDcd	3,44 ± 0,02 Cbc	3,48 ± 0,03 Ab	3,69 ± 0,02 Aa
T5	3,35 ± 0,01 Ac	3,36 ± 0,02 ABc	3,36 ± 0,02 Bbc	3,37 ± 0,01 Cbc	3,39 ± 0,02 CDbc	3,39 ± 0,03 CDbc	3,43 ± 0,02 ABb	3,6 ± 0,05 Ba
T6	3,17 ± 0,04 Bd	3,29 ± 0,08 BCc	3,35 ± 0,01 Bbc	3,34 ± 0,01 CDbc	3,37 ± 0,01 Dbc	3,39 ± 0,02 CDbc	3,41 ± 0,02 Bab	3,51 ± 0,05 Ca
T7	3,13 ± 0,01 Be	3,22 ± 0,02 CDd	3,26 ± 0,02 Ccd	3,29 ± 0,03 Dcd	3,34 ± 0,01 Dc	3,39 ± 0,02 CDb	3,43 ± 0,02 ABab	3,44 ± 0,02 CDa
T8	3,1 ± 0,02 Be	3,18 ± 0,01 DEd	3,2 ± 0,05 CDcd	3,1 ± 0,02 Ecd	3,26 ± 0,04 Ec	3,35 ± 0,02 Db	3,38 ± 0,02 Bab	3,43 ± 0,02 Da
T9	3,08 ± 0,01 Bf	3,13 ± 0,02 Eef	3,18 ± 0,01 Dde	3,18 ± 0,03 Ede	3,22 ± 0,03 Ecd	3,28 ± 0,03 Ebc	3,31 ± 0,05 Cb	3,42 ± 0,03 Da

Means followed by at least one capital letter in the columns do not differ from each other, by Tukey’s test at 95% confidence. Means followed by at least one lowercase letter in the rows do not differ from each other, by Tukey’s test at 95% confidence.

The response variable was determined each day and when it exceeded the established specification, the analysis was stopped for the following days, which is why cells with a hyphen appear.

Figure 4. pH changes over time for strawberry samples coated with different EC compositions of aloe vera gel and carnauba wax microparticles.

Table 3 summarizes the results of the statistical analysis. T1 had the highest pH value (3.72 ± 0.02), while T9 had the lowest pH (3.28 ± 0.03) (p < .05).

Comparing treatments T2 (3.65 ± 0.04) and T7 (3.39 ± 0.02), it can be seen that CWP had a significantly greater effect than AVG in preventing a pH increase. Until day 12 (D12) of storage, the treatment that had the maximum combination of both EC components (T9), was the one who had the best effect in controlling pH changes, since it produced the significantly lowest measurement.

Table 3. Summary of the analysis of variance (ANOVA) of pH of the treatments.

Source of variation	Degrees of freedom	MS
		Day 0	Day 2	Day 4	Day 6	Day 8	Day 10	Day 12	Day 14
Total	26
Treatment	8	0,05*	0,03*	0,03*	0,06*	0,06*	0,07*	8,7*	9,28*
Aloe vera gel (A)	2	0,14*	0,09*	0,1*	0,15*	0,17*	0,024*	34,82*	37,10*
Carnauba gel (B)	2	0,02*	0,02*	0,01*	0,05*	0,04*	0,02*	0,009*	0,01*
Interaction (A*B)	4	0,01*	0,002*	0,006*	0,013*	0,01*	0,002*	0,0032*	0,007*
Error	18	0,0009	0,001	0,0006	0,0005	0,0006	0,01	0,0005	0,0008

 * Significant at 5% probability by the F test.

However, at day 14 (D14), T4 had the highest values of pH (p < .05), whereas T6, T7, T8, and T9 had lower values than T4 and T5, demonstrating that in the long term, the increase of AVG concentration to 45% does not produce any extra control in pH, but the maximum percentage of CWP (0.4%) is necessary to protect the fruit against the increase of this parameter. In spite of increases in pH, all treatments were within the acceptable range. This could be due to the effect that Aloe Vera and Carnauba Wax had on the fruit, as these EC create a semi-permeable layer over the surface of the fruit, modifying the internal atmosphere and controlling the respiration rate which causes a delay in ripening, thereby decreasing the degradation of organic acids, liberation of glucose and accumulation of CO₂ within the package (Sophia et al., 2015).

Similar findings were observed by Qamar et al. (2018) when measuring the pH of strawberries after 12 days of storage at 5–7°C, found that treatments coated with AVG had the lowest measurements of pH compared to the strawberries without coat and coated with chitosan and sodium alginate.

Ripening Index

Strawberries were selected based on level 4 state of the ripening scale proposed by Villagrán et al. (2012); hence, at day 0 (D0) and 2 (D2), there were not significant differences between ripening index values (°Brix/Titratable acidity, refer to tables 5-8 for values and statistical analysis of each variable) for each treatment, as seen in Figure 5 (See Table 10 for datails). Ripening index increased over time for all treatments, as expected; however, by day 4 (D4), T1 had the highest value (20.91 ± 0.52) (p < .05, Table 9). This is due to the absence of an edible cover, which allowed the fruit to increase its respiration rate, accelerating the release of ethylene and increasing total soluble solids because of the conversion of organic acids to starch during ripening (Sophia et al., 2015).

Table 5. Summary of the analysis of variance (ANOVA) of the percentage of Brix degrees of the treatments.

Source of variation	Degrees of freedom	MS
		Day 0	Day 2	Day 4	Day 6	Day 8	Day 10	Day 12	Day 14
Total	26
Treatment	8	0,08 *	0,5 *	2,19 *	3,05 *	5,7 *	6,34 *	73,4 *	80,3 *
Aloe vera gel (A)	2	0,16 *	1,26 *	7,26 *	8,6 *	19,15 *	21,32 *	290,3 *	318,62 *
Carnauba gel (B)	2	0,06 *	0,35 *	1,14 *	2,14 *	2,75 *	3,6 *	1,9 *	1,52 *
Interaction (A*B)	4	0,05 *	0,18 *	0,2 *	0,74 *	0,44 *	0,23 *	0,71 *	0,45 *
Error	18	0,009	0,03	0,06	0,03	0,09	0,05	0,02	0,01

 * Significant at 5% probability by the F test.

Table 6. Brix degrees of the treatments.

Treatment/Days	Day 0	Day 2	Day 4	Day 6	Day 8	Day 10	Day 12	Day 14
T1	$6,58\pm 0,08$ $AB$f	$7,97\pm 0,15$ $A$e	$9,60\pm 0,36$ $A$d	$10,67\pm 0,35$ $Ac$	$11,37\pm 0,25$ $Ab$	$12,10\pm 0,36$ $Aa$	-	-
T2	$6,76\pm 0,05$ $Ad$	$7,27\pm 0,15$ $Bd$	$8,77\pm 0,32$ Bc	$9,00\pm 0,10$ $Bc$	$10,83\pm 0,15$ $ABb$	$11,40\pm 0,36$ $Ba$	-	-
T3	$6,50\pm 0,10$ $ABCd$	$7,07\pm 0,12$ $BCc$	$8,60\pm 0,20$ $Bb$	$8,73\pm 0,15$ $Bb$	$10,60\pm 0,20$ $ABa$	$11,00\pm 0,20$ $Ba$	-	-
T4	$6,77\pm 0,15$ Ae	$7,00\pm 0,20$ BCDe	$8,27\pm 0,31$ Bd	$8,20\pm 0,20$ $Cd$	$10,40\pm 0,40$ $Bc$	$10,93\pm 0,12$ $Bbc$	$11,53\pm 0,12$ $Aab$	$11,73\pm 0,12$ $Aa$
T5	$6,43\pm 0,06$ $BCe$	$7,00\pm 0,00$ $BCDe$	$8,13\pm 0,12$ BCd	$8,07\pm 0,12$ $Cd$	$9,07\pm 0,64$ $Cc$	$10,00\pm 0,20$ $Cb$	$10,53\pm 0,23$ $Bab$	$11,00\pm 0,20$ $Ba$
T6	$6,47\pm 0,12$ $BCg$	$7,00\pm 0,20$ $BCDfg$	$7,47\pm 0,31$ $CDef$	$8,00\pm 0,20$ $Cde$	$8,47$ $\pm 0,31$ $CDcd$	$9,07\pm 0,31$ $Dbc$	$9,60\pm 0,20$ $Cab$	$10,20\pm 0,20$ $Ca$
T7	$6,40\pm 0,10$ BCf	$6,80\pm 0,20$ BCDf	$7,40\pm 0,20$ $De$	$7,93\pm 0,12$ $CDd$	$8,33\pm 0,12$ $CDd$	$8,87\pm 0,12$ $DEc$	$9,33\pm 0,23$ $Cb$	$9,93\pm 0,12$ $Ca$
T8	$6,37\pm 0,06$ BCf	$6,73\pm 0,12$ CDf	$7,13\pm 0,12$ De	$7,47\pm 0,12$ $DEe$	$7,93\pm 0,12$ $Dd$	$8,33\pm 0,23$ $EFc$	$8,87\pm 0,12$ $Db$	$9,47\pm 0,12$ $Da$
T9	$6,30\pm 0,10$ Ce	$6,53\pm 0,23$ De	$7,07\pm 0,12$ Dd	$7,33\pm 0,12Ed$	$7,80\pm 0,20$ $Dc$	$8,07\pm 0,12$ $Fc$	$8,53\pm 0,12$ $Db$	$9,00\pm 0,20$ $Ea$

Means followed by at least one capital letter in the columns do not differ from each other, by Tukey’s test at 95% confidence.

Means followed by at least one lowercase letter in the rows do not differ from each other, by Tukey’s test at 95% confidence.

The response variable was determined each day and when it exceeded the established specification, the analysis was stopped for the following days, which is why cells with a hyphen appear.

Table 7. Summary of the analysis of variance (ANOVA) of acidity of the treatments.

Source of variation	Degrees of freedom	MS
		Day 0	Day 2	Day 4	Day 6	Day 8	Day 10	Day 12	Day 14
Total	26
Treatment	8	0,003*	0,005*	0,002*	0,0003*	0,003*	0,003*	0,2*	0,10*
Aloe vera gel (A)	2	0,007*	0,012*	0,006*	0,0008*	0,0001*	0,0004*	0,42*	0,41*
Carnauba gel (B)	2	0,0002*	0,003*	0,0003*	0,005*	0,003*	0,003*	0,0002*	0,00004*
Interaction (A*B)	4	0,004*	0,001*	0,0001*	0,003*	0,004*	0,004*	0,00009*	0,0001*
Error	18	0,0004	0,001	0,0002	0,0001	0,0001	0,00002	0,0001	0,00004

 * Significant at 5% probability by the F test.

Table 8. Acidity of the treatments.

Treatment/Days	Day 0	Day 2	Day 4	Day 6	Day 8	Day 10	Day 12	Day 14
T1	0,56 ± 0,01 ABa	0,48 ± 0,01 ABCb	0,46 ± 0,01 ABCbc	0,44 ± 0,02 Bcd	0,43 ± 0,0 ABd	0,42 ± 0,0 Ad	-	-
T2	0,5 ± 0,04 Ca	0,5 ± 0,06 ABCa	0,47 ± 0,01 ABa	0,36 ± 0,02 Db	0,32 ± 0,02 Db	0,32 ± 0,0 Db	-	-
T3	0,57 ± 0,01 ABa	0,51 ± 0,0 ABb	0,47 ± 0,0 ABCc	0,47 ± 0,01 Ac	0,43 ± 0,0 Ad	0,43 ± 0,0 Ad	-	-
T4	0,55 ± 0,01 ABCa	0,48 ± 0,01 ABCb	0,47 ± 0,0 ABCb	0,43 ± 0,01 BCc	0,37 ± 0,02 Ccd	0,37 ± 0,0 Cd	0,37 ± 0,03 Ad	0,39 ± 0,01 Ad
T5	0,59 ± 0,03 Aa	0,54 ± 0,004 Aab	0,49 ± 0,04 Ab	0,4 ± 0,01 Cc	0,4 ± 0,01 ABCc	0,39 ± 0,01 Bc	0,39 ± 0,01 Ac	0,38 ± 0,01 Ac
T6	0,53 ± 0,01 ABCa	0,52 ± 0,0 ABa	0,47 ± 0,01 ABb	0,42 ± 0,01 BCc	0,39 ± 0,01 BCd	0,39 ± 0,01 Bd	0,38 ± 0,0 Ad	0,37 ± 0,0 ABd
T7	0,5 ± 0,01 Ca	0,44 ± 0,01 BCb	0,42 ± 0,0 Cbc	0,4 ± 0,01 Ccd	0,38 ± 0,01 Cde	0,37 ± 0,0 Ce	0,36 ± 0,01 Ae	0,35 ± 0,01 Ce
T8	0,5 ± 0,02 Ca	0,42 ± 0,06 Cb	0,42 ± 0,01 Cb	0,41 ± 0,01 BCb	0,38 ± 0,02 Cb	0,38 ± 0,0 BCb	0,37 ± 0,0 Ab	0,36 ± 0,0 BCb
T9	0,51 ± 0,01 BCa	0,47 ± 0,02 ABCb	0,43 ± 0,01 BCc	0,41 ± 0,01 BCcd	0,4 ± 0,01 ABCcde	0,39 ± 0,01 Bdef	0,37 ± 0,01 Aef	0,36 ± 0,0 BCf

Means followed by at least one capital letter in the columns do not differ from each other, by Tukey’s test at 95% confidence.

Means followed by at least one lowercase letter in the rows do not differ from each other, by Tukey’s test at 95% confidence.

The response variable was determined each day and when it exceeded the established specification, the analysis was stopped for the following days, which is why cells with a hyphen appear.

Table 9. Summary of the analysis of variance (ANOVA) of the ripening index of the treatments.

Source of variation	Degrees of freedom	MS
		Day 0	Day 2	Day 4	Day 6	Day 8	Day 10	Day 12	Day 14
Total	26
Treatment	8	1,99*	5,11n.s.	6,92*	19,92*	58,97*	62,73*	531,96*	579,10*
Aloe vera gel (A)	2	1,77*	6,88n.s.	19,00*	37,73*	129,91*	133,58*	2088,98*	2303,1*
Carnauba gel (B)	2	0,43*	8,5n.s.	7,42*	21,09*	41,47*	58,10*	22,79*	8,78*
Interaction (A*B)	4	2,89*	2,53n.s.	0,64*	10,43*	32,25*	29,62*	8,04*	2,28*
Error	18	0,33	1,29	0,59	0,54	2,15	0,71	0,78	0,39

* Significant at 5% probability by the F test.

n.s. not significant at 5% probability by the F test.

Table 10. Treatment ripening index.

Treatment/Days	Day 0	Day 2	Day 4	Day 6	Day 8	Day 10	Day 12	Day 14
T1	11,75 ± 0,26 BCf	16,74 ± 0,49Ae	20,91 ± 0,52 Ad	24,39 ± 1,05 Ac	26,73 ± 0,8 BCb	28,85 ± 0,73 Ba	-	-
T2	13,67 ± 1,17 Ad	14,74 ± 1,5 ABCd	18,66 ± 0,44 Bc	24,88 ± 1,07 Ab	33,53 ± 2,32 Aa	35,56 ± 1,35 Aa	-	-
T3	11,51 ± 0,49 BCd	13,82 ± 0,15 ABCc	18,48 ± 0,55 BCb	18,47 ± 0,34 BCb	24,71 ± 0,44 BCDa	25,54 ± 0,61 Ca	-	-
T4	12,34 ± 0,37 ABCd	14,62 ± 0,59 ABCcd	17,72 ± 0,5 BCDbc	19,3 ± 0,16 BCb	28,22 ± 2,84 CDEa	29,85 ± 0,65 Ba	31,6 ± 2,27 Aa	30,14 ± 0,44 Aa
T5	10,89 ± 0,66 Ce	12,94 ± 0,89 Ce	16,68 ± 1,42 BCDc	20,12 ± 0,62 Bc	22,73 ± 1,21 DEc	25,67 ± 0,81 Cb	27,31 ± 0,51 Bab	28,83 ± 1,06 ABa
T6	12,15 ± 0,46 ABCf	13,37 ± 0,29 BCf	15,94 ± 0,11 CDe	19,18 ± 0,62 BCd	21,65 ± 1,14 DEc	23,56 ± 1,14 CDbc	25,18 ± 0,69 BCDab	27,37 ± 0,68 Ba
T7	12,69 ± 0,45 ABg	15,5 ± 0,41 ABCf	17,44 ± 0,52 BCDe	19,78 ± 0,99 BCd	21,95 ± 0,58 DEc	24,08 ± 0,51 CDb	25,98 ± 0,55 Bb	28,19 ± 1,11 BCa
T8	12,88 ± 0,63 ABf	16,27 ± 2,6 ABe	16,83 ± 0,64 BCDe	18,43 ± 0,59 BCde	20,78 ± 1,1 DEcd	22,05 ± 0,77 DEbc	24,12 ± 0,14 CDab	26,23 ± 0,45 CDa
T9	12,46 ± 0,17 ABCf	13,85 ± 0,96 ABCf	16,41 ± 0,63 CDe	17,85 ± 0,58 Cde	19,46 ± 0,84 Ecd	20,73 ± 0,65 Ec	23 ± 0,91 Db	25,05 ± 0,56 Da

Means followed by at least one capital letter in the columns do not differ from each other, by Tukey’s test at 95% confidence.

Means followed by at least one lowercase letter in the rows do not differ from each other, by Tukey’s test at 95% confidence.

The response variable was determined each day and when it exceeded the established specification, the analysis was stopped for the following days, which is why cells with a hyphen appear.

Figure 5. Ripening index for strawberry samples coated with different EC compositions of aloe vera gel and carnauba wax microparticles.

However, by day 12 (D12), T1, T2, and T3 had a rapid degradation, which ended their life span. This showed that the absence of AVG causes a rapid increase in the ripening of strawberries. On the other hand, T4 had the highest ripening value (31.60 ± 2.27), being statistically different (p < .05) from T5 through T9, which presented the lowest indexes (Table 10). This demonstrated that the combination of AVG + CWP provided greater protection against ripening than AVG or CWP alone; nevertheless, more than 30% AVG and 0.3% CWP did not result in greater preservation (Table 9). This can be due to the fact that CWP droplets get embedded in the AVG, giving no extra protection. This effect could only be seen until D12, since, by day 14 (D14), treatments were not significantly different, ending with an approximate ripening index of 27.636. The protection conferred by the edible coating could be potentiated by the presence of antioxidants in AV leaves, such as gallic acid, catechin, and chlorogenic acid, that prevent the rapid liberation of free radicals; thus, delaying ripening. Furthermore, CW is known to have antioxidant activity due to the content of phenols (flavonoids and flavanols) (da Silva Andrade et al., 2018).

Chrysargyris et al. (2016) investigated the effect of different AVG concentrations (0%, 5%, 10%, 15% and 20%) on the ripening index of tomatoes. After 7 days of storage, in the treatment with 10% AVG the ripening index decreased to 6.25, while the rest of the treatments, including the control, exhibited a more pronounced ripening.

Microbiological Assay: Botrytis cinerea Severity Index

The optimum temperature range for B. cinerea to grow is 15–22°C, under 70% humidity (Koike and Bolda, 2016), reason why, all the samples for microbiological assays were kept at 20°C (at 4°C the strawberries did not show any sign of B. cinerea growth after 14 days of storage). In addition to the control, treatments T5, T6, T8, and T9 were used for this assay since they had the best results in controlling the physicochemical parameters previously analyzed. As time progressed, the senescence of the fruit started, which led to fungal colonization in different areas of the fruit, determining the shelf life of each treatment. If consumed or inhaled by allergenic precipitants of asthma and hypersensitivity pneumonitis, B. cinerea can produce pneumonia; thus, if the fruit shows signs of mold on its surface, it must be immediately discarded. The control sample (T1) started showing gray mold since day 2, having the shortest lifespan (<2 days). The absence of EC in the control sample caused an acceleration of its senescence due to the loss of water, high cellular metabolic activities, and easier colonization of the fungus (Qamar et al., 2018). Consequently, by the end of day 7, the strawberry had a severity index of 51–100%.

On the other hand, T5, T6, T8, and T9 had a life span of <4 days; however, T5 treatment with the least amount of AVG and CWP of the four, showed a higher contamination by the fungus on its surface (11–25%). Therefore, it had a higher severity index by day 7. For T6 and T8, their behavior was similar throughout the days, ending up with a severity index of 26–50%. Aloe Vera contains anti-fungical compounds, such as phenols and quinones that prevent B. cinerea growth. Specifically, anthraquinones (aloin and aloe-emodin) and the protein of 14 kDa from the Aloe Vera leaf gel are the compounds that could have the greatest suppression effect against molds and yeasts. Moreover, it contains the polysaccharide acemannan that has anti-bacterial activity (Qamar et al., 2018). Carnauba Wax contains gallic acid, catechin, and chlorogenic acid, also known to have anti-fungi activity (da Silva Andrade et al., 2018). This explains why the treatment that had the lowest severity index after 7 days of storage was the one with the maximum amount of AVG and CWP (T9). The outcome showed that the application of AVG + CWP protects the fruit against B. cinerea, allowing the strawberry to have 2 more days of shelf life compared to the control sample when stored at 20°C.

Navarro et al. (2011) found that after 6 days of storage at 25°C, the application of AV significantly reduced the fungus infection by Botrytis, Rhizopus, and Penicillium compared to untreated nectarines. Ruiz-Martínez et al. (2020) confirmed this, finding that a coat composed of Candelilla wax, whey protein, and glycerol had 23, 28, and 3% more inhibition activity of B. cinerea, C. gloeosporioides and F. oxysporum, respectively, than tomatoes without coat.

Sensory Analysis

The goal of using edible coatings for fruits is not only to increase its shelf life, but to avoid the consumer from distinguishing any difference in the fruit’s organoleptic characteristics due to its addition (Oliveira et al., 2018). As shown in Figure 6, there was no significant difference between the treatments in relation to any attribute (p > .05). Sample 581 had 6 days of storage, which led to a decrease of 1.26% of weight due to water loss. However, the loss percentage was less than 5%; thus, no negative changes on the strawberries’ surface were produced (FAO, 2011), reason why all samples were in the category like slightly (6). Despite the microbiological and physicochemical alterations generated by the storage of the fruits, until day 6, the judges did not detect any significant changes in odor or flavor between the samples and the control. Both attributes obtained a score of like slightly (6). Chiumarelli and Hubinger (2012) mentioned that EC made of polysaccharides are tasteless and odorless, which corroborated the obtained for the coat made of AVG. Regarding overall acceptance, there was no significant difference between treatments. Details on specifc values and statistical analysis of the data are presented in Tables 11-13.

Figure 6. Sensory attributes of strawberries with different coating formulations.

Table 11. Summary of the analysis of variance (ANOVA) of the global perception and attributes of the treatments.

Source of variation	Freedom degrees	CM
		Appearance	Smell	Taste	Global
Total	247
Treatments	3	6,63*	3,68n.s.	3,88n.s.	4,03n.s.
Blocks	61	5,20*	6,56*	4,80*	3,57*
Experimental error	183	2,62	2,36	3,08	1,91

* Significant at 5% probability by the F test.

n.s. not significant at 5% probability by the F test.

Table 12. Appearance of the treatments.

Sample	Code	Appearance
Control	149	6,71 ± 2,20 ab
T9 (0)	342	7,29 ± 2,07 a
T9 (3)	267	6,94 ± 1,78 ab
T9 (6)	581	6,53 ± 1,83 b

Means followed by at least one lowercase letter do not differ from each other, at a 5% probability by Tukey’s test.

Table 13. Global perception and attributes of the treatments.

Sample	Code	Global	Smell	Taste
Control	149	7,31 ± 1,55	6,45 ± 1,74	6,90 ± 1,91
T9 (0 days)	342	6,59 ± 1,57	6,35 ± 1,92	6,48 ± 1,85
T9 (3 days)	267	6,76 ± 1,28	6,17 ± 1,84	6,38 ± 1,62
T9 (6 days)	581	6,79 ± 1,66	6,76 ± 1,88	6,38 ± 2,14

Since all attributes had a score above like slightly (6), with a tendency to like moderately (7), the maximum combination of AVG + CWP not only did not affect negatively the appearance, odor, flavor or overall acceptance of the strawberry, but also helped to increase its shelf life. Taking into consideration the usage of a hedonic scale of nine points, if the rating for an attribute is above category 5, the fruit is acceptable for commercial purposes (Chen et al., 2019). Thus, the strawberries used in this research were in condition to be commercialized.

Conclusion

The present research assessed the effect of Aloe vera gel and Carnauba wax (a component that had not been previously used for strawberries) on the effectiveness of edible coatings to extend the fruit’s shelf life. The different coating formulations (AVG and CWP concentrations) were homogeneously distributed throughout the fruit’s surface, a factor that had a significant role in improving its efficacy. Moreover, both coatings, AVG and CWP, were important for reducing changes in the physicochemical properties of strawberries undergoing a ripening process, in comparison to the uncoated fruits. The treatments that had a higher concentration of both components (AVG + CWP) not only caused the least change in weight loss, pH values, and ripening index, but also provided the greatest protection against B. cinerea contamination. Furthermore, during the sensory analysis, this treatment was equally accepted as the fruit without coat. Therefore, the application of these coats is promising for industrial extrapolation and application to avoid important losses commonly found in strawberry’s value chain.

Acknowledgments

The researchers want to thank Karen Herrera and Antonio León from USFQ Food and Agricultural Biotechnology Laboratory, for their support in the microbiological part of this investigation.

Disclosure Statement

No potential conflict of interest was reported by the author(s).

Data Availability Statement

The data to support the results and conclusions of this study is presented within the article. Detailed data is available upon request.