Shelf Life of Blackberry Fruits (Rubus fruticosus) with Edible Coatings Based on Candelilla Wax and Guar Gum

Abstract

Blackberries are very perishable with a limited shelf life due to a high metabolic activity and susceptibility to mechanical damage and microbial attack. The effect of edible coatings (EC) based on candelilla wax (CW) and guar gum (GG) on the quality, shelf life, bioactive-compounds content and antioxidant activity of blackberries stored at 25 ± 2 °C for 6 days, was evaluated. All EC contained GG (0.4%) and gallic acid (GA)(0.06%); EC1 contained CW (0.2%), glycerol (GL)(0.2%); EC2 contained CW (0.4%) and GL (0.2%); EC3 contained CW (0.2%) and GL (0.3%) and EC4 contained CW (0.4%) and GL (0.3%). The decay percentage (Decay%), physiological weight loss (%PWL), firmness, pH, total soluble solids (TSS), titratable acidity (TA), total phenolic content (TPC), total anthocyanin content (TAC) and antioxidant activity were analyzed. EC3 showed potential for prolonging the shelf life of blackberry by reducing Decay% (47%) and %PWL (64%) on day 6, while 86% of uncoated blackberries were decayed on day 4. Fruit coated with EC3 maintained pH, gradually reduced firmness, increased the TSS and decreased the TA. In addition, EC3 maintained the TAC and decreased the TPC in blackberries, while preventing a rapid increase in antioxidant capacity. Thus, EC3 showed to be a promising alternative in prolonging shelf life and preserving the quality of blackberries.

1. Introduction

Rubus fruticosus is a perennial plant belonging to the Rosaceae family, whose worldwide production and consumption of fruits have increased in recent decades [1]. Blackberry fruits are rich in carbohydrates, dietary fiber, vitamins and minerals [2], and are widely appreciated due to their aroma, color, flavor and texture, in addition to their potent antioxidant activity, attributed to the high level of phenolic compounds (e.g., phenolic acids, anthocyanins) [3]. Blackberry phytochemical content has been shown to positively influence human health [4], exerting anti-inflammatory, anticancer, antimicrobial, antihypertensive and analgesic effects, among others [5].

Fruits of blackberry are highly perishable due to their high respiration rate and lack of a protective shell, which makes them susceptible to postharvest mechanical damage and microbial attack [6], mainly by pathogenic fungi such as Botrytis cinerea, Rhizopus stolonifer, Mucor mucedo, Colletotrichum acutatum and Peronospora corda [7,8]. Technological alternatives have recently been investigated to ensure the quality of fresh fruits and vegetables during transport and postharvest storage [9]. In this context, the development of edible coatings (EC) has been proposed. EC form a thin layer of natural, edible, and biodegradable polymeric matrix directly on a food surface [10,11]. EC can lower fruit respiration rate and maintain quality characteristics (e.g., color, texture, aroma, and nutritional content) [12], acting as a semi-permeable barrier to the loss of water, oxygen (O₂), carbon dioxide (CO₂) and ethylene, which prevents dehydration and physiological processes associated with postharvest deterioration [13]. Additionally, EC are used as the vehicle for the incorporation of bioactive compounds that serve to improve fruit quality and protect food products from microbial contamination and spoilage [13,14]. Thus, EC could be applied to extend blackberries shelf life [15], and their formulation has been reported from different biopolymers, mainly polysaccharides, such as cassava starch and chitosan [16], guar gum [17], and alginate [18]; lipids such as beeswax [19] and essential oils (e.g., limonene) [18]; and combinations of these with proteins [20,21].

In this sense, the application of EC based on candelilla wax has been used to prolong the shelf life of fruits and vegetables [22]; however, its application in berries has been little studied. Candelilla wax is a natural plant wax derived from Euphorbia antisyphilitica Zucc. grown in the arid regions of northern Mexico and is a promising substance for use in many food applications. The functional properties and physicochemical characteristics of candelilla wax-based biopolymers depend significantly on the inclusion of different functional ingredients, such as antimicrobials, antioxidants, nutrients, flavorings, and colorants to improve food quality, stability and safety [22]. Thus, glycerol can be added as a plasticizer and surfactant [23,24]. Gallic acid is an antioxidant and antimicrobial compound [25], and it can improve the mechanical properties of some formulations as well [26]. Guar gum is a natural hydrophilic polymer, which has the ability to be a good oxygen, carbon dioxide and lipid barrier [27,28]; it can also be added as an emulsifier and thickener [29].

Oregel-Zamudio et al. [30] applied EC based on candelilla wax added with gallic acid, guar gum and glycerol in strawberry fruits, showing a reduction in weight loss and decay, without significant changes in the fruits’ physicochemical characteristics; besides controlling postharvest rot caused by Rhizopus stolonifer, these results revealed the potential of candelilla wax and guar gum edible coatings for preservation and shelf-life extension in berries. Therefore, the objective of this work was to evaluate the effect of edible coatings based on candelilla wax and guar gum on the quality, shelf life, content of bioactive compounds and antioxidant activity of blackberry fruits.

2. Materials and Methods

2.1. Blackberry Fruits

Fruits of Rubus fruticosus cv. Catherine were donated by regional producers (Zamora, Michoacán, Mexico), and harvested in field at the complete five maturation stage (reddish-black color) according to the scale proposed by Padilla et al. [31]. After harvesting, fruits were selected with uniform size, shape, weight, and color, without physical damage and apparent infection by microorganisms.

2.2. Materials

Refined candelilla wax was supplied by Ceras Naturales Mexicanas, S.A. de C.V. (Saltillo, Coah., Mexico). Guar gum, glycerol and gallic acid were purchased from Golden Bell products Inc (Orange, CA, USA).

Ethanol, hydrochloric acid (HCl), gallic acid and cyanidin-3-glucoside standards, ABTS+• (2,2′-azino-di (3-ethyl benzthiazoline-6-sulphonic acid), DPPH• (1,1-diphenyl-2-picrylhydrazyl) and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.3. Experimental Design, Preparation and Application of Edible Coatings Formulations

The edible coatings were prepared using Candelilla wax, guar gum, glycerol and gallic acid as described previously by Oregel-Zamudio et al. [30]. The Design-Expert^® software (version 13, Stat-Ease) was used to develop a factorial design of two factors: ingredients (candelilla wax and glycerol), and two concentration levels (%): candelilla wax (0.2 and 0.4%) and glycerol (0.2 and 0.3%) resulting in 4 formulations (

Table 1. Edible coating (EC) formulations based on candelilla wax and guar gum.

Formulations	Ingredients (%)
	Guar Gum	Candelilla Wax	Glycerol	Gallic Acid
EC1	0.4	0.2	0.2	0.06
EC2	0.4	0.4	0.2	0.06
EC3	0.4	0.2	0.3	0.06
EC4	0.4	0.4	0.3	0.06

.)

Candelilla-wax-based edible coatings were applied on fresh blackberries, according to the method used by Potma da Silva et al. [32] with some modifications. Fruits were dipped in the emulsion at 25 °C for 2 s and were allowed to dry at 25 ± 2 °C for 10 min. Afterwards, the fruits were packed on sterile closed plastic boxes, which allowed ventilation. The set was kept over a bench, in the laboratory, without sunlight exposition, at 25 ± 2 °C and 77% relative humidity (RH). Boxes containing 9 berries each, were allocated for shelf life and quality monitoring; each box was an experimental unit and there were three experimental units. The experiments were performed in duplicate.

2.4. Shelf-Life Assay

The treatments were control without coating, and the four edible coating previously designed (EC1, EC2, EC3 and EC4). Fruits were evaluated for decay and physiological weight loss (%PWL) at 2, 4 and 6 d of storage at room temperature (25 ± 2 °C).

Decay percentage (Decay%) was defined as the percentage of unacceptable fruits, and fruits were considered unacceptable when showing one or more of the following conditions under a visual appearance evaluation: discoloration, spots on the surface, softening and fluid secretion.

Fruit weight was recorded (PA214, Ohaus Pionner, Pine Brook, NJ, USA) and the percentage of lost weight was calculated by the following formula:

% Physiological weight loss = (Wi − Wf )/Wi × 100,

where Wi is day 0 weight and Wf is weight in the evaluation time.

2.5. Quality Measurements

Fruit firmness was measured with a Fruit Pressure Tester FT 011 (QA Supplies LLC, Norfolk, VA, USA) (0–5 Kgf), on two opposite equatorial points of each fruit, and the average firmness per fruit was registered.

Blackberry juice was obtained by squeezing the fruits with cheesecloth. The pH was measured with a pH meter (HI 2211, HANNA Instruments, Boston MA, USA). The total soluble solids (TSS) were measured by a manual refractometer (ATC-1E, ATAGO Co., Minato-Ku, Tokyo, Japan), and expressed as degree Brix (°Bx). The titratable acidity (TA) was defined as the % of malic acid. Both determinations were carried out according to AOAC methods [33].

For the above quality measurements, three fruits were taken from each of the experimental units (n = 3). All determinations were made at 0, 2, 4 and 6 d of storage at room temperature (25 ± 2 °C).

2.6. Extracts Preparation

The fruits were freeze dried (Lyophilizer, LABCON, Kansas City, MO, USA), and the freeze-dried tissue was pulverized with a pestle and mortar and stored at −20 °C until use. Extracts were prepared as reported by Bernal-Gallardo et al. [34] with some modifications. A total of 1 g of samples was extracted with 5 mL of ethanol (85): HCl 1N (15) (v/v), agitated with vortex, and the pH was adjusted to 1 with HCl. Extraction was continued using an ultrasonic processor (ULTRAsonik, DENSTPLY, NEYTECH, Yucaipa, CA, USA) at 55 ± 5 Hz for 30 min at room temperature. The extracts were vacuum filtered through a 0.45 µm cellulose filter, and 1.25 mL were stored in 1.5 mL microtubes at −20 °C.

2.7. Total Phenolic Content

Total phenolic content in the extracts were quantified by UV-Visible spectrophotometry as described by Spinardi et al. [35]. The absorbance was measured at 700 nm using a spectrophotometer (PowerWave HT, Biotek, Biotek Instruments, VT, USA). Gallic acid was used as standard (0.2 a 2.0 mg/mL). The results were expressed in milligram gallic acid equivalents per gram of dry weight (mg GAE/g DW). The quantification was performed at 0 and 6 d of storage at room temperature (25 ± 2 °C) in triplicate.

2.8. Total Anthocyanin Content

The total anthocyanin content in extracts was determined by UV-Visible spectrophotometry according to Abdel-Aal and Hucl [36]. The absorbance was read at 535 nm using a spectrophotometer PowerWave HT, Biotek (Biotek Instruments, Winooski, VT, USA). The quantification was performed at 0 and 6 d of storage at room temperature (25 ± 2 °C) in triplicate. The total anthocyanin content was expressed as cyanidin-3-glucoside equivalent (mg CGE/g DW), and was calculated by the following formula:

$$Total anthocyanin content=A535 nm/\mathrm{25,965} \times \left(volume extract\right)/1000 \times 449 \times 1/\left(sample weight\right) \times {10}^6,$$

and expressed as cyanidin-3-glucoside equivalent per gram of dry weight (mg CGE/g DW). The assay was performed in triplicate.

2.9. Antioxidant Activity by ABTS Assay

The antioxidant activity of the extracts was determined by the ABTS method described by Samaniego et al. [37] with some modifications. The radical cation (ABTS‚+) was produced by reacting 7 mM ABTS stock solution with 2.45 mM potassium persulfate under dark conditions at room temperature for 16 h before use. The ABTS solution was diluted with deionized water to an absorbance of 0.70 ± 0.10 at 734 nm. After the addition of 20 μL of extract to 280 μL of diluted ABTS solution, absorbance readings were taken after incubation for 6 min at room temperature. The absorbance of the mixture was measured at 734 nm in a microplate reader (PowerWave HT, Biotek, Biotek Instruments, VT, USA). The standard curve was linear with concentrations of 0–600 μmol of Trolox (µMET = −0.0952(Abs_734nm) + 1.301, r² = 0.9991). The antioxidant capacity was expressed as micromole Trolox equivalents per gram on dry weight (μmol TE/g DW). The assay was performed at 0 and 6 d of storage at room temperature (25 ± 2 °C) in triplicate.

2.10. Antioxidant Activity by DPPH Assay

The DPPH radical scavenging activity method was performed according to the procedure by Silva et al. [38] with some modifications. The antioxidant capacity of the extracts was expressed as µmol TE/g DW using a calibration curve (µmol TE = −0.0793 [A_515nm] + 0.2178, r² = 0.9577) with concentrations of 0–600 µmol of Trolox. The assay was performed at 0 and 6 d of storage at room temperature (25 ± 2 °C) in triplicate.

2.11. Statistical Analysis

All data were expressed as mean ± standard deviation. An analysis of variance (ANOVA) was conducted followed by Tukey’s test (p < 0.05) using the R Studio software Version 4.0.3.

3. Results and Discussion

3.1. Shelf Life

Decay

The decay percentage (% Decay) increased during storage in both uncoated and coated fruits with all EC (Figure 1). Coated fruits had a significantly lower decay than the uncoated fruits both, on day 4 (1.2–2.2 times), and on day 6 (1.3–1.9 times).

The uncoated fruits reached a decay of 100% on day 6, while the lowest Decay% values were found in coated fruits with EC2, EC3 and EC4 (60.0, 53.3 and 60.0%, respectively). It should be noted that EC3 showed a similar behavior to that of the edible coating reported by Oregel-Zamudio et al. [30] formulated with guar gum (0.8%), candelilla wax (0.2%), glycerol (0.3%) and gallic acid (0.15%) and applied in strawberry. The authors observed on day 4 of storage at 25 ± 2 °C a decay close to 30% in coated fruits, and on day 6, a decay of coated fruits close to 50%. In the present study, the decay of blackberries coated with EC3 was 38.1% on day 4, and 53.1% on day 6. However, these results differ from those reported by Gol et al. [39], who evaluated chitosan, alginate, and carboxymethylcellulose coatings on Indian blackberry (Syzygium cumini L.), stored at 10 °C. All the assayed coatings protected fruits against decomposition up to day 4 of storage, but on day 12, the uncoated fruits showed a Decay% of 95%, while the coating with chitosan at 1.5% showed the least Decay% (7.78%). In the case of EC applied on strawberry, the generation of microenvironments with conditions that inhibit the growth of microorganisms causing postharvest deterioration was suggested by Peretto et al. [40], which could also be favored with the coatings applied to blackberries in this study, due to the combination of functions and benefits provided by the ingredients. Thus, candelilla wax acts mainly as a barrier to mass transfer (e.g., water, oxygen or carbon dioxide) and provides protection against various factors that affect the quality of food products; in addition, antimicrobial properties have been suggested for candelilla wax [41]. Guar gum, despite having low resistance to water loss, given its hydrophilic nature [42], has oxygen and carbon dioxide barrier properties that allow creating a modified atmosphere inside the food, resulting in a longer shelf life [43]. For its part, glycerol, by acting as a plasticizer, reduces the activity of water, which can limit microbial growth [44].

3.2. Physiological Weight Loss

Physiological weight loss (%PWL) in blackberry (Rubus fruticosus) fruits increased during storage at 25 ± 2 °C (Figure 2). Uncoated and coated blackberries with EC1 and EC2 showed a tendency to increase the %PWL on day 4, while on day 6, the increase was significant (49.39, 30.90 and 39.81%, respectively).

On the other hand, coated blackberries with EC3 and EC4 significantly increased the %PWL from day 4 (15.33 and 14.37%, respectively). The average %PWL of the coated blackberries was 33% on day 6, being that EC1 and EC4 were the coatings that showed the lowest %PWL, and this was significantly lower (1.6 and 1.8 times) than that of uncoated fruits. These results, were similar to those reported by Oliveira et al. [45], who found physiological weight losses between 5% and 30% in blackberry fruits treated with a coating based on cassava starch and water kefir grains, stored for 12 days, even when the storage temperature was 10 °C. In this sense, Oliveira et al. [16] observed that a higher storage temperature promoted greater %PWL. Villegas and Albarracín [9] found significant differences in the %PWL of blackberry (Rubus glaucus Benth.) treated with coatings formulated with hydroxypropyl methylcellulose and high concentrations of beeswax (40–60%) on day 10 of storage at 4 °C. Tumbarski et al. [46] found significant differences in this variable with celery pectin coatings singly and in combination with a Bacillus methylotrophicus BM47 bacteriocin in blackberries (Rubus spp.) stored at 4 °C. Similar results have been reported by Cortés-Rodríguez et al. [21] with coatings based on cassava starch, in Andean blackberry (Rubus glaucus Benth) stored at 4 °C, and by Gol et al. [39] in Indian blackberry (Syzygium cumini) with biodegradable coatings of chitosan, alginate, and carboxymethylcellulose, stored at 10 °C. The fact that the uncoated fruits showed a greater physiological weight loss (p ≤ 0.05) than the coated fruits with EC1 and EC4 is indicative of EC physiological weight loss prevention, which is one of the purposes of using them, since they modify the exchange of gases between the fruit and the external environment, by reducing the respiration rate [47]. It is known that solid lipids form coatings with better water vapor barrier properties [48]. In this regard, Villegas and Albarracín [9] reported a higher respiratory index in uncoated blackberries than that found in blackberries coated with EC containing hydroxypropyl methylcellulose and beeswax. In this study, candelilla wax contributed mainly to the barrier properties of the tested formulations, promoting a lower %PWL in coated fruits [22].

3.3. Firmness

At the beginning of the trial, blackberry firmness was found in a range of 2.17 to 2.53 Kgf (Figure 3). The firmness of uncoated and coated fruits decreased during storage. The softening in blackberry fruits is associated with metabolic changes that occur during fruit ripening, such as the starch to sugar conversion, biosynthesis of volatile organic compounds responsible for aroma, and, moreover, the structural changes in the cell wall due to the degradation of various polysaccharides (e.g., pectin, cellulose and hemicellulose) [49].

Uncoated blackberries decreased firmness from day 4, maintaining it on day 6 (1.50 Kgf). The firmness of fruits coated with EC1 and EC4 decreased from day 2 to 1.53 and 1.87 Kgf, (29.5 and 17.6% respectively), in addition, fruits coated with EC1 registered another significant reduction to 1.2 Kgf on day 6. Fruits coated with EC2, EC3 and EC4 (1.63, 1.67 and 1.47 Kgf, respectively) equaled the firmness of uncoated fruits on day 6; however, fruits coated with EC3 maintained their firmness from day 0 to day 4, the date on which they exceeded the firmness of uncoated fruits by 22%. In this sense, it was shown that various EC act as barriers that delay certain physiological processes such as transpiration and respiration in strawberries [50,51], which delay the polymer degradation in the cell wall that adhere to peptic acids, preventing softening [47]. Oliveira et al. [16], reported that the application of EC of chitosan or starch did not affect the firmness of blackberries, since the firmness of coated and uncoated decreased during storage at 10 °C; and Bambace et al. [52], found that the application of EC with sodium alginate, inulin, oligofructose, and probiotics did not influence the firmness of blueberries during refrigerated storage. It is important to point out that EC3 formulation can be optimized to overcome the firmness of uncoated fruits at the end of storage, as suggested by other authors [53].

The influence of the assayed EC on firmness can be attributed to the formulation of the coatings, which includes candelilla wax and a gum of vegetable origin, as was previously reported in apples with coatings formulated with candelilla wax and gum Arabic [54,55]. In this sense, Cortés-Rodríguez et al. [21] reported the decrease in the loss of firmness of Andean blackberry (Rubus glaucus Benth.) fruits coated with an EC based on cassava starch, suggesting that this formulation acts as a barrier and reduces physiological processes of the blackberry, delaying the degradation of cell wall polymers. [56,57]. A similar behavior was reported in different berries such as blackberries (Rubus sp.) coated with cassava starch [45], blackberries (Rubus sp.) cv. Tupi coated with chitosan, cassava starch and water kefir grains (at 0 and 10 °C) [16].

3.4. pH

The pH values of the fruits at the beginning of the experiment ranged between 2.6 and 3.6 (Figure 4A), similar to those reported by Carvalho et al. [58] in Andean blackberry (Rubus glaucus Benth.) (pH 2.8–2.9), and Bersanetti et al. [53] in Rubus spp. cv. Tupy (pH ≈ 3.5). Uncoated fruits significantly increased in pH on day 4 and on day 6, while the fruits coated with EC1, EC2 and EC4 also increased in pH from day 4, maintaining it until day 6. It should be noted that the coating EC3 maintained the pH of fruits throughout the storage period.

In general, coated and uncoated fruits showed increases in pH during storage, which may be due to the union of pectin fragments with polyphenols during ripening [19], and are indicative of deterioration [59]; in this sense, the application of EC favored the preservation of the fruits, since the pH in uncoated fruits increased from 2.60 to 3.83 (47%) during storage, while fruits coated with EC3 showed the lowest pH increase from 3.60 to 3.90 (8%). Oliveira et al. [45] observed in blackberries (Rubus sp.) cv. Tupy coated with a matrix of kefir grains, stored for 12 days at 10 °C, an increase in pH from 2.95 to 3.37, while in uncoated fruits, the increase in pH was from 2.83 to 3.53. In blackberry (Rubus sp.) cultivars, Comanche and Brazos [60] reported initial pH values of 3.59 and 3.39 and, after 12 days of storage, values of 3.94 and 4.09; this represented increases of 0.40 and 0.70.

It was also reported that the pH of fruits changes slightly during storage, thus, Bersanetti et al. [53] observed that the pH values in blackberries coated with cassava starch, glycerol and nystose were maintained around 3.5 for 20 days at 4 °C, a behavior shown by blackberries coated with EC3 as well. Likewise, Cortés-Rodríguez et al. [21] reported slight pH changes (from 3.0 to 3.1) in Andean blackberries coated with an EC based on cassava starch, after 12 days of storage at 4 °C. On the other hand, these results differ from those reported by Gol et al. [39], who observed that the pH of the fruits of Indian blackberry (Syzygium cumini L.), without coatings, was higher (4.5) than that of the fruits coated with chitosan and carboxymethylcellulose (4.1) stored for 12 days at 10 °C.

3.5. Total Soluble Solids

Regarding the total soluble solids (TSS) expressed in °Bx, which are indicative of the amount of sugar in fruits, values between 11.6 and 12.6 were observed in coated and uncoated fruits on day 0 (Figure 4B). Carvalho and Betancur [58] reported values close to 9 °Bx in Andean blackberry (Rubus glaucus Benth), while values of 7.0 to 10.0 °Bx were reported in Rubus sp. cv. Tupy [16,53].

The differences in TSS values obtained in this study for Rubus fruticosus cv. Catherine, may be due to a genetic variety effect as reported by Gündogdu et al. [2], who studied 11 blackberry cultivars cultivated in Eastern Turkey, and found significant differences in the content of sugars and organic acids, among other compounds, determining that the biochemical characteristics of the fruits were strongly influenced by factors, such as cultivar and genotype.

The TSS increased during storage (Figure 4B) on day 6 for uncoated fruits, and fruits coated with EC1, EC2, EC3 and EC4 showed a significant increase compared to day 0 (4.5, 2.0, 3.4, 3.7 and 3.9 °Bx, respectively). Only the fruits coated with EC1 showed a significantly lower increase in TSS than uncoated fruits, while the rest of EC equaled the uncoated fruits in the content of TSS at the end of the storage period.

Although the decrease in TSS is caused more rapidly under storage conditions with high temperatures [61], and this phenomenon occurs naturally during storage, some authors reported an increase during the first days of storage, followed by reduction. A similar behavior was reported by Cortés-Rodríguez et al. [21] in Andean blackberry (Rubus glaucus Benth.) fruits coated with a matrix based on cassava starch, showing an increase in TSS until day 8, after which they decreased. The increase in TSS was mainly attributed to evaporation caused by the difference in chemical potential of water (driving force of mass transfer) between the fruit and the environment [19,47,62], in addition to the conversion of organic acids into sugars during the physiological processes of blackberries. The downward trend in TSS during storage could be attributed to the consumption of sugars and organic acids in respiration during senescence, and weight loss by leaching due to enzymatic reactions developed within the fruit [62].

3.6. Titratable Acidity

The average titratable acidity (TA) was 1.8% at the beginning of the trial (Figure 4C), which agrees with that reported by Potma da Silva et al. [32], in fruits of Rubus sp. cv. Arms. The TA was significantly reduced in uncoated fruits on day 6, while in fruits coated with EC2, EC3 and EC4, the TA was reduced from day 4, showing values of 1.3, 0.63 and 1.17%, respectively. Although EC acts as a barrier to gases and can control metabolic reactions and respiration, generating a modified atmosphere inside the fruit [63], the physiological processes continue and changes in the concentrations of organic acids may occur [19,32]. In this regard, Cortés-Rodríguez et al. [21] observed a decrease in the TA of blackberries coated with a coating based on cassava starch and chitosan after 8 days of storage at 4 °C. While Potma da Silva et al. [32] applied EC of micro fibrillated nanocellulose and lemongrass essential oil in blackberry cv. Brazos and found a decrease in TA after 3 and 6 days of storage at 25 °C; Gol et al. [39], reported a significant reduction in the TA values of Indian blackberry (Syzygium cumini L.) during the storage period (16 days) at 10 °C, both in uncoated and fruits coated with chitosan, alginate and carboxymethylcellulose. The minimum TA values were observed in uncoated fruits, while the highest TA values were obtained in all coated fruits. This behavior appears to be linked to a delayed ripening. On the other hand, Bersaneti et al. [53] reported that the TA remained almost constant for 15 days of storage at 4 °C in blackberry fruits coated with EC of starch and nystose, as occurred in this work in fruits with EC1, which maintained the TA during the entire storage period. On day 6, they had a TA twice as high as that of the uncoated fruits. On the other hand, Tumbarski et al. [46] observed an increase in the AT of blackberry fruits (Rubus fruticosus) with edible coatings based on celery pectin and bacteriocin from Bacillus methylotrophicus BM47 for 16 days of storage at 4 °C. The variation was from 1.09 on day 0 to 1.74% on day 16. In this study, uncoated fruits showed a significantly lower TA (0.77%) than the fruits with EC1, EC2, EC3 (1.07, 1.05 and 1.40%, respectively), during the entire storage period.

3.7. Total Phenolic Content

The average initial total phenolic content (TPC) was 25 mg GAE/g DW (

Table 2. Effects of candelilla wax and guar gum edible coatings on the total phenolic and anthocyanin contents of fresh blackberries during storage at 25 ± 2 °C.

Coatings	Total Phenolic Content (mg GAE/g DW)	Total Anthocyanins Content (mg CGE/g DW)
	Day 0	Day 0
Ctrl	25.17 ± 0.06 cd	5.53 ± 0.15 cd
EC1	25.10 ± 0.17 cd	6.93 ± 0.15 ab
EC2	25.27 ± 0.25 cd	5.47 ± 0.23 cd
EC3	26.64 ± 0.64 bc	6.20 ± 0.56 bc
EC4	21.08 ± 2.61 f	7.23 ± 0.06 a
	Day 6	Day 6
Ctrl	22.90 ± 0.10 def	4.13 ± 0.25 e
EC1	24.05 ± 0.15 cde	7.13 ± 0.47 a
EC2	29.47 ± 0.59 b	6.90 ± 0.35 ab
EC3	21.50 ± 0.10 ef	5.82 ± 0.45 c
EC4	35.55 ± 1.67 a	4.73 ± 0.06 de

Ctrl = uncoated; edible coatings = EC1, EC2, EC3 and EC4; GAE = gallic acid equivalents; CGE = cyanidin-3-glucoside equivalents; DW = dry weight. The mean ± standard deviation is shown. Different letters in a column indicate statistically significant differences by Tukey’s test (p < 0.05).

), which was in the TPC range of 23.40 to 63.00 mg GAE/g DW, reported by Mertz et al. [64] and from 9.56 to 89.76 mg GAE/g DW reported by Kaume et al. [65], although it differed from what was reported in fruits of Andean blackberry cultivars: Brazos, Colombiana, Castilla and Andimora (31.59, 45.18, 44.68, 46.19 mg GAE/g DW, respectively) [37]. The differences with the values obtained in fruits of Rubus fruticosus cv. Catherine in this study, could be attributed to the effect of genetic variety pointed out by Gündogdu et al. [2]. On the other hand, the uncoated fruits maintained the TPC until day 6; this behavior was shown in fruits coated with EC1, unlike fruits coated with EC3, whose TPC was significantly reduced (19.6%) at the end of the trial, although it remained the same as that in uncoated fruits. This coincided with the findings of other authors., Gol et al. [62] evaluated uncoated strawberries and strawberries treated with coatings of carboxymethylcellulose, hydroxypropyl methylcellulose and chitosan, finding that the TPC decreased progressively throughout storage, but uncoated fruits had a significantly greater decrease. In blackberry (Rubus fruticosus) uncoated fruits and fruits coated with pectin and bacteriocin based coatings, Tumbarski et al. [46] reported a decrease in TPC. Additionally, Cortés-Rodríguez et al. [21] observed a similar behavior in Andean blackberries coated with EC based on cassava starch, where the authors found the maximum TPC value up to day 6 of storage at 4 °C, which subsequently decreased, while the uncoated fruits reached the maximum value of TPC up to day 8 of storage and remained stable until day 10.

The decrease in the content of phenolic compounds is associated with the decomposition of cellular structures caused by senescence processes in the fruits. However, EC2 and EC4 were able to protect and even promote the accumulation of phenolic compounds, significantly increasing TPC on day 6 (17.4 and 68.6%, respectively). These results coincided with those reported by Gol et al. [39] in fruits of Indian blackberry (Syzygium cumini L.), with biodegradable coatings of chitosan, alginate and carboxymethylcellulose, since the initial TPC increased in coated fruits was on day 12 and the decrease in uncoated fruits was on day 4.

3.8. Total Anthocyanin Content

The average total anthocyanin content (TAC) at the beginning of the trial was 6.3 mg CGE/g DW (Table 2), which was lower than that reported by Samaniego et al. [37] in Andean Blackberry Cultivars: Brazos, Colombiana, Castilla and Andimora (8.63, 12.26, 10.89, 9.26 mg CGE/g DW, respectively), the differences could be due to the genetic load effect of the species and cultivars as it was suggested by Gündogdu et al. [2]. On day 6, uncoated fruits reduced their anthocyanin content (25.3%), as well as the fruits coated with EC4 (34.6%). This was similar to that observed by Tumbarski et al. [46], who reported that coatings of pectin and bacteriocin did not prevent the decrease in TAC in coated and uncoated blackberries (Rubus fruticosus) stored for 16 days at 4 °C. In the same way, Cortés-Rodríguez et al. [21] reported that the TAC in Andean blackberry fruits coated with films based on cassava starch decreased with storage time. This was attributed to the pigment instability during processing and storage, which generated colorless and insoluble derivatives of these [3,66]. Numerous factors can contribute to the degradation of anthocyanins, such as sugars (especially fructose), which accelerate the browning process, but a pH between 1 and 3.5 gives greater stability [16]. On the other hand, the fruits covered with EC1 and EC3 maintained the TAC during storage, and fruits coated with EC1 reached the highest TAC (7.13 mg CGE/g DW), being 1.7 times higher than that found in uncoated fruits. Interestingly, the fruits coated with E2 significantly increased the TAC (26.1%) on day 6 compared to that found on day 0. These results coincided with what was observed by Chiabrando et al. [67] in blueberries (Vaccinium corymbosum L.) with alginate and chitosan coatings. The fact that the uncoated fruits presented a lower TAC than the fruits coated with EC1, EC2, EC3 could be due to a protective effect of EC by reducing the fruits metabolic activity, as suggested by Bersaneti et al. [53], in fruits coated with a matrix of starch and nystose.

3.9. Antioxidant Activity

The antioxidant activity of blackberries fruits by the ABTS method was found in the range of 504.90 and 516.81 µmol TE/g DW (

Table 3. Effects of candelilla wax and guar gum edible coatings on antioxidant activity of fresh blackberries during storage at 25 ± 2 °C.

Coatings	DPPH µM TE/g DW	ABTS µM TE/g DW
	Day 0	Day 0
Ctrl	43.86 ± 0.96 f	504.9 ± 2.7 c
EC1	45.12 ± 0.18 def	505.18 ± 3.1 c
EC2	46.28 ± 0.63 bc	516.81 ± 2.1 a
EC3	47.75 ± 0.48 cde	513.45 ± 1.11 a
EC4	45.75 ± 0.66 ef	511.2 ± 2.53 ab
	Day 6	Day 6
Ctrl	56.05 ± 0.95 a	507.84 ± 1.06 bc
EC1	46.49 ± 0.36 cdef	507 ± 3.18 bc
EC2	49.96 ± 0.73 cd	508.82 ± 1.26 bc
EC3	52.69 ± 1.19 ab	503.08 ± 2.46 c
EC4	46.91 ± 1.64 cd	504.48 ± 1.06 c

Ctrl = uncoated; edible coatings = EC1, EC2, EC3 and EC4; TE = trolox equivalents; DW = dry weight. The mean ± standard deviation is shown. Different letters in a column indicate statistically significant differences by Tukey’s test (p < 0.05).

), which falled in the range (344.42–658.28 µmol TE/g DW) reported by Samaniego et al. [37] in Andean blackberry fruit cultivars, Brazos, Colombiana, Castilla and Andimora.

The antioxidant capacity (DPPH method) increased during the storage (Table 3), which is consistent with the increase in antioxidant capacity while ripening, reported for Rubus adenotrichus Schltdl., from a light red to dark bluish purple surface color [68], since in this work, the blackberries used were reddish-black in color. The blackberry has been reported as a non-climacteric fruit [69,70], but Burdon and Sexton [71] found that ethylene production depended on the cultivar, ranging from no increase during development, to a doubling of ethylene production upon color development. It could be that when the color develops, some reactions involving the production of antioxidants take place in the fruit. In this sense, Mahmood et al. [72] reported an increase in antioxidant activity while ripening in mulberry fruit of different species. The EC application showed an effect on the antioxidant activity since coated fruits showed a lower increase in antioxidant capacity on day 6 compared to day 0, as the greatest increase was 4 µM TE/g DW (EC3) while in uncoated fruits, it was three times higher (12 µM TE/g DW). The EC are semipermeable to oxygen, thus, a lower oxygen concentration would prevent oxidation; therefore, it would be not necessary for antioxidant substances to be produced. Additionally, the antioxidant activity measured by the ABTS method showed a similar behavior, although less pronounced, and in fruits coated with EC2, EC3 and EC4, the antioxidant capacity even decreased.

Based on the results shown above, at the end of the shelf-life assay, after 6 days of storage at 25 ± 2 °C, the EC3 resulted in a promising formulation for extending the shelf life of blackberries, since it reduced %PWL (64%) and Decay% (47%), favoring an increasing of 2 days from day 4 of storage, when 86% of uncoated fruits were decayed. Fruits coated with EC3 equaled the firmness, pH and TSS of uncoated fruits on day 6. However, fruits coated with EC3 maintained their firmness from day 0 to day 4, and then from day 4 to day 6 showed a gradual softening. In addition, the pH of blackberries was maintained throughout the complete storage period. Furthermore, EC3 allowed fruits to increase TSS, despite the decrease in AT. In the same way, EC3 maintained the TAC in fruits, but decreased the TPC. Interestingly, EC3 prevented an increase in the antioxidant capacity (DPPH) or even diminished (ABTS).

4. Conclusions

Edible coating formulations containing candelilla wax and guar gum applied on blackberries provided a barrier to reduce physiological weight loss, generally reduced fruit decay, and had no effect on firmness, pH, TSS and AT; in addition, they prevented a rapid increase in antioxidant capacity and preserved or even increased the content of phenolic and/or anthocyanin during storage at room temperature storage for 6 days. It was possible to find an edible coating formulation based on candelilla wax and guar gum, EC3 (0.4% GG, % CW, 0.3% GL, 0.06% GA) that would increase the shelf life of blackberries by 2 days. However, the results suggest that new formulations should be designed and tested to optimize the edible coating benefits for blackberry fruits. The present study demonstrated that the application of edible coatings based on candelilla wax and guar gum is a promising alternative to preserve blackberry quality and has the potential for extending fruit shelf life.