Alginate-Edible Coatings for Application on Wild Andean Blueberries (Vaccinium meridionale Swartz): Effect of the Addition of Nanofibrils Isolated from Cocoa By-Products

Edible coatings and films are appealing strategies for the postharvest management of blueberries. In the current work, alginate and alginate/cellulose nanofibril (CNF) edible coatings crosslinked with calcium chloride were developed for application on Andean blueberry (a promissory wild blueberry). Cocoa by-products were valorized through the isolation of their CNFs, and these were incorporated in the edible coatings. Edible coating formulations were based on blends of alginate (2% w/v), CNFs (0%, 0.1%, or 0.3%), glycerol, and water. In addition, stand-alone films were prepared, and their light and water vapor barrier properties were studied before applying the coating on the fruit surface. The results show that the addition of CNFs caused a significant decrease in the transparency and the water vapor permeability of the alginate films. After applying on the Andean blueberry fruits, the alginate and alginate/CNF coatings enhanced the appearance and the firmness of the fruits. Moreover, they significantly reduced the respiration rate and the water loss of the Andean blueberries throughout the 21 days of refrigerated storage. Alginate and alginate/CNFs coatings may be considered a useful alternative for the delay of the postharvest deterioration of Andean blueberries.


Introduction
Blueberries are widely consumed small red fruits with high economic value and several health benefits [1]. Highbush blueberries (Vaccinium corymbosum) are the most commercial blueberries. However, there are several other wild shrubs of the genus Vaccinium with similar or higher biological activity, but with few commercial exploitations, such as the Vaccinium meridionale Swartz (Andean blueberry). The latter grows in the Andean region of South America at 2300-3300 m above sea level (m.a.s.l.) [2].

Isolation of Cellulose Nanofibrils (CNF) from Cocoa Byproducts
Cocoa shell was chemically treated following a modification of the KOH-5 procedure developed by Zuluaga et al. [26] to remove non-cellulosic components. First, the cocoa shell was vigorously stirred at room temperature for 14 h with 5 wt.% KOH solution. Then, the insoluble residue was delignified with 1 wt.% NaClO 2 at pH 5.0, and adjusted with 10 wt.% acetic acid, at 70 • C for 1 h. At each step of the different treatments, the insoluble residue was extensively washed with distilled water until the pH was neutral. Finally, the cellulosic material at a concentration of 2 wt.% was passed 30 times through grinder equipment (Masuko Sangyo, Supermasscolloider), according to the G30 procedure developed by Velásquez-Cock et al. [27].

Atomic Force Microscopy
The morphology of cellulose nanofibrils from cocoa shell was studied using atomic force microscopy (AFM). Samples were imaged in tapping mode using a Flex AFM (Nanosurf) equipped with a multimode head and operated with a resonance frequency of 183 kHz. It used a cantilever of 125 mm in length and 5-10 nm in tip radius. Nanocellulose samples were diluted in distilled water and sonicated (Elma Elmansonic P) for 15 min at room temperature to achieve a good dispersion of the nanocellulose. Subsequently, a fine layer of the sample was deposited on mica by using a spin-coater for 2 min at 2000 rpm. Before morphological analysis, samples were stored in a vacuum desiccator for 3 days.

Preparation of Film/Coating Solutions
Film/coating solutions with and without cellulose nanofibrils from cocoa shell were prepared. Alginate solutions were made by dissolving sodium alginate powder (2% w/v) in distilled water and heating at 70 • C under constant stirring until the mixture became clear. Glycerol (30% w/v dry weight basis) was added as a plasticizer to the sodium alginate solution and stirred for 15 min.
For the preparation of alginate/CNF blends, alginate solutions of two different concentrations of Cocoa CNFs (0.1% and 0.3% w/v) were added and homogenized at 20,000 rpm for 3 min using an Ultra Turrax T25 (IKA ® WERKE, Germany) homogenizer. The concentrations of sodium alginate, glycerol, and cocoa shell nanofibrils were chosen according to previous works [28].
All formulations were degassed using a vacuum pump and cooled to room temperature for later application on the fruits.

Formation and Characterization of Edible Films
The film-forming solutions were dispensed into polypropylene plates and dried at 50 • C for 24 h. Then, dried films were peeled from the plates, submerged in a gelling bath of calcium chloride solution (1% w/v) for 30 min, cleaned with distilled water, and air-dried at room temperature. All films were conditioned at room temperature into desiccators containing a supersaturated solution of sodium bromide (RH~57%) for 48 h prior to characterization studies.
The thicknesses of the films were measured using an electronic digital caliper. Water vapor permeability (WVP) tests were carried out at room temperature following the ASTM E96/ASTM E96M-16 method. Film samples were sealed over a circular opening of 4 × 10 −4 m 2 in a permeation cell, containing calcium chloride. Then, the cells were placed in desiccators conditioned with sodium chloride saturated solution (75% RH). Changes in the weight of the cell were recorded to the nearest 0.0001 g and plotted as a function of time, and the slope of each line was calculated by linear regression. WVP (g Pa −1 s −1 m −1 ) was calculated as follows: where WVTR is the water vapor transmission rate calculated as the ratio between the slope of the straight line (g/s) and the cell area (m 2 ); P is the saturation vapor pressure of water (Pa); RH is the relative humidity in the desiccator, and d is the film thickness (m). Film transparency was measured as reported by Piñeros-Hernandez, Medina-Jaramillo, López-Córdoba, & Goyanes, 2017 [29]. Films were cut into rectangles (50 mm × 10 mm) and placed on the inner side of a quartz spectrophotometer cell. The percent transmittance (% T) of light at 600 nm (T 600 ) was measured using a UV-visible spectrophotometer (X-ma 1200 Human Corporation, Loughborough, UK), and the transparency was calculated as the ratio between logT 600 and the thickness (mm) of each film.

Application of Edible Coatings
A total mass of 6 kg of Andean blueberries was randomly divided into four groups (control, alginate, alginate/CNFs 0.1%, alginate/CNFs 0.3%), each group containing 1.5 kg of fruit. The blueberries were dip-coated by immersion in the coating solutions for 90 s, drained of excess coating, submerged in a gelling bath of calcium chloride solution (1% w/v) for 30 min, cleaned with distilled water, and air-dried at room temperature. Control samples (without coating) were also prepared by immersion of fruits in distilled water and kept under the same storage conditions than the treated ones, for comparison.
The surface solid density (SSD) was estimated as an indicator of the coating's average thickness as follows [30,31]: where SSD is the surface solid density (g/m 2 ); M CA is the mass of coating solution adhered to the fruit surface (g); X S is the mass fraction of solid in the coating solution; and A S is the surface area of Andean blueberries (m 2 ). The average sample surface area (A s ) was estimated by considering blueberry as a sphere. Samples were weighed before and after coating to determine the mass of coating solution adhered to the fruit surface (M CA ). The non-coated sample was used as a control.

Evaluation of Quality Attributes of Andean Blueberries along Storage
The coated and uncoated Andean blueberries were packed in PET trays with perforated vents and stored for 21 days. Evaluations of quality attributes were performed at 1, 7, 14, and 21 days of refrigerated storage (4 • C and 90% RH). For every sampling time, three trays containing 125 g (~250 units) of Andean blueberries each were prepared.  [32]. Approximately 120 g of Andean blueberries were placed for 30 min at 25 • C inside hermetically sealed 2 L flasks. Then, the CO 2 concentration was determined using an infrared analyzer (LabQuest ® 2 Model LQ2-LE, Beaverton, OR, USA), and results were expressed as the rate of respiration (CO 2 ) in mg kg −1 s −1 .

Weight Loss
Weight loss of Andean blueberries during storage was determined by weighing all fruit trays at the beginning of the storage and every day of analysis. The weight loss (% W) was calculated with the following equation: where m f is the weight at each time and m 0 the initial weight of each sample.

Soluble Solids Content, pH, and Titratable Acidity (%)
The soluble solids content was measured in the fruit juice using an Atago refractometer model PR 101 (Atago CO., Tokyo, Japan) and expressed as Brix (AOAC 932.12). Fruit samples were crushed using a blender and filtered through filter paper to obtain the fruit juice.
The pH of the fruit samples was assessed using a digital pH meter (Oakton Instruments, Vernon Hills, IL, USA) (AOAC 981.12).
Titratable acidity (%) was determined by titration with 0.1 N NaOH up to pH 8.2, using 0.5 g of sample in 10 mL of distilled water (AOAC 942.15). The results were expressed in citric acid percentage.

Firmness Analysis
Firmness was determined using a digital Force Gauge PCE-FM200 (Southampton, UK) equipped with a 6 mm diameter stainless steel probe. Firmness was defined as the maximum force to disrupt the tissue at the penetration time used (5 s) [33]. The results were expressed as an average of at least five measurements.

Statistical Analysis
The statistical analysis was performed using Minitab v. 16 statistical software (State College, PA, USA). Analysis of variance (ANOVA) and Tukey's pairwise comparisons were carried out using a level of 95% confidence. The experiments were performed at least in triplicate, and the data were reported as mean ± standard deviation. Figure 1a shows some bundles with diameters around 300 nm and some individual CNFs. The morphology of individual CNFs is evidenced more clearly in Figure 1b, where nanofibers with diameters between 15 and 50 nm and several microns in length are observed. Therefore, AFM images presented in Figure 1a,b reveal that the cellulosic material isolated from cocoa shell has a fibrillar morphology and forms an entangled network of high-aspect materials, which makes it a suitable material to develop edible coatings and films with barrier properties and prevent the gaseous exchange.  Table 1 shows the thickness, transparency, and water vapor permeability of the alginate films with and without cellulose nanofibrils from cocoa shell. All films were clear enough to be used as see-through packaging. However, alginate/CNF 0.1% and alginate/CNF 0.3% films showed a decrease in the transparency of 27% and 40%, respectively, in comparison to the alginate ones. Martins et al. [34] also reported a higher opacity in starch/CNF films as more CNFs were added to the filmogenic matrix, and it was attributed to the dispersion of light by nano-sized fibrils.

Film Characterization
In addition, the addition of cocoa shell nanofibrils caused a decrease in the water vapor permeability of the films of 65%, compared to the films without nanofibrils, regardless of the concentration used (0.1% or 0.3%). This behavior could be attributed to the nanofibrils being able to act as a filler, causing a tortuous pathway that limits water vapor transmission through the alginate matrix. These results are in agreement with those reported in the literature [34]. Table 1. Thickness, water vapor permeability, and transparency of the alginate films with and without cellulose nanofibrils from cocoa shell.

Sample
Thickness (mm) Transparency (%) Different letters within the same column indicate statistically significant differences (p < 0.05). Figure 2 shows the appearance of Andean blueberries with and without coatings. It can be seen that the coated fruits were brighter than the uncoated ones. This behavior was attributed to the smoother surface of the coated fruit, compared to the fruit skin, and it caused a greater reflection of visible light [35]. The brightness is an important quality perceived attributed to blueberries because consumers commonly associate it with a fresh-like appearance [36].  Table 1 shows the thickness, transparency, and water vapor permeability of the alginate films with and without cellulose nanofibrils from cocoa shell. All films were clear enough to be used as see-through packaging. However, alginate/CNF 0.1% and alginate/CNF 0.3% films showed a decrease in the transparency of 27% and 40%, respectively, in comparison to the alginate ones. Martins et al. [34] also reported a higher opacity in starch/CNF films as more CNFs were added to the filmogenic matrix, and it was attributed to the dispersion of light by nano-sized fibrils. In addition, the addition of cocoa shell nanofibrils caused a decrease in the water vapor permeability of the films of 65%, compared to the films without nanofibrils, regardless of the concentration used (0.1% or 0.3%). This behavior could be attributed to the nanofibrils being able to act as a filler, causing a tortuous pathway that limits water vapor transmission through the alginate matrix. These results are in agreement with those reported in the literature [34]. Figure 2 shows the appearance of Andean blueberries with and without coatings. It can be seen that the coated fruits were brighter than the uncoated ones. This behavior was attributed to the smoother surface of the coated fruit, compared to the fruit skin, and it caused a greater reflection of visible light [35]. The brightness is an important quality perceived attributed to blueberries because consumers commonly associate it with a fresh-like appearance [36]. Besides, the surface solid density (SSD) was estimated as an indicator of the applied coating's thickness. It was observed that the addition of CNFs did not cause important changes in the SSD of the fruits, obtaining values of 0.52, 0.54, and 0.60 g.m −2 for the alginate, alginate/CNFs 0.1%, and alginate/CNFs 0.3% systems, respectively. SSD values between 1.0 and 1.85 g.m −2 were reported by Falcó et al. [31] for blueberries (Vaccinium corymbosum) coated with edible coatings based on carrageenan (κ−, ι−, and λ).

Behavior of Andean Blueberry Quality Parameters during Storage
The changes in the respiration rate of Andean blueberries with and without edible coatings during storage are shown in Figure 3. All edible coatings caused a significant decrease in the respiration rate of the Andean blueberries. It has been well documented that alginate edible coatings are suitable to work as a barrier to oxygen, retarding the respiration rate, and also browning reactions [9,37,38]. At the end of the storage, the control fruits increased their respiration rate, while the fruits coated with alginate and alginate/CNFs 0.3% showed a similar respiration rate than at the initial time. Unlike the other samples, the fruits coated with alginate/CNFs 0.1% showed a significant decrease in their respiration rate at the end of the storage. It has been reported that CNFs with a high aspect ratio can turn and entangle into a network structure, leading to a tortuous diffusion path, which prevents the gaseous exchange [39,40]. However, above a critical concentration, CNFs tend to form agglomerate held together by hydrogen bonding [34]. Weight loss is a primary factor in blueberry deterioration [41]. It is well known that the loss of water in fresh fruits is mainly associated with respiration and transpiration through the skin, and it causes sensory quality loss generally because of the fruit shrinkage. Here, all systems showed a gradual increase in weight loss during storage (Figure 4). However, in the case of the coated fruits, Besides, the surface solid density (SSD) was estimated as an indicator of the applied coating's thickness. It was observed that the addition of CNFs did not cause important changes in the SSD of the fruits, obtaining values of 0.52, 0.54, and 0.60 g.m −2 for the alginate, alginate/CNFs 0.1%, and alginate/CNFs 0.3% systems, respectively. SSD values between 1.0 and 1.85 g.m −2 were reported by Falcó et al. [31] for blueberries (Vaccinium corymbosum) coated with edible coatings based on carrageenan (κ−, ι−, and λ).
The changes in the respiration rate of Andean blueberries with and without edible coatings during storage are shown in Figure 3. All edible coatings caused a significant decrease in the respiration rate of the Andean blueberries. It has been well documented that alginate edible coatings are suitable to work as a barrier to oxygen, retarding the respiration rate, and also browning reactions [9,37,38]. At the end of the storage, the control fruits increased their respiration rate, while the fruits coated with alginate and alginate/CNFs 0.3% showed a similar respiration rate than at the initial time. Unlike the other samples, the fruits coated with alginate/CNFs 0.1% showed a significant decrease in their respiration rate at the end of the storage. It has been reported that CNFs with a high aspect ratio can turn and entangle into a network structure, leading to a tortuous diffusion path, which prevents the gaseous exchange [39,40]. However, above a critical concentration, CNFs tend to form agglomerate held together by hydrogen bonding [34]. Besides, the surface solid density (SSD) was estimated as an indicator of the applied coating's thickness. It was observed that the addition of CNFs did not cause important changes in the SSD of the fruits, obtaining values of 0.52, 0.54, and 0.60 g.m −2 for the alginate, alginate/CNFs 0.1%, and alginate/CNFs 0.3% systems, respectively. SSD values between 1.0 and 1.85 g.m −2 were reported by Falcó et al. [31] for blueberries (Vaccinium corymbosum) coated with edible coatings based on carrageenan (κ−, ι−, and λ).
The changes in the respiration rate of Andean blueberries with and without edible coatings during storage are shown in Figure 3. All edible coatings caused a significant decrease in the respiration rate of the Andean blueberries. It has been well documented that alginate edible coatings are suitable to work as a barrier to oxygen, retarding the respiration rate, and also browning reactions [9,37,38]. At the end of the storage, the control fruits increased their respiration rate, while the fruits coated with alginate and alginate/CNFs 0.3% showed a similar respiration rate than at the initial time. Unlike the other samples, the fruits coated with alginate/CNFs 0.1% showed a significant decrease in their respiration rate at the end of the storage. It has been reported that CNFs with a high aspect ratio can turn and entangle into a network structure, leading to a tortuous diffusion path, which prevents the gaseous exchange [39,40]. However, above a critical concentration, CNFs tend to form agglomerate held together by hydrogen bonding [34]. Weight loss is a primary factor in blueberry deterioration [41]. It is well known that the loss of water in fresh fruits is mainly associated with respiration and transpiration through the skin, and it causes sensory quality loss generally because of the fruit shrinkage. Here, all systems showed a gradual increase in weight loss during storage (Figure 4). However, in the case of the coated fruits, Weight loss is a primary factor in blueberry deterioration [41]. It is well known that the loss of water in fresh fruits is mainly associated with respiration and transpiration through the skin, and it causes sensory quality loss generally because of the fruit shrinkage. Here, all systems showed a gradual increase in weight loss during storage (Figure 4). However, in the case of the coated fruits, Polymers 2020, 12, 824 8 of 13 the weight loss was significantly lower than in the control ones, regardless of the presence of cocoa shell nanofibrils. At the end of storage, control fruits and coated fruits showed water loss around 10% and 5%, respectively (Figure 4). It has been suggested that postharvest treatments that restrict blueberry water loss to below 8% are likely to be beneficial in retaining acceptable blueberry freshness and firmness [4,41]. The ability to reduce weight loss of alginate coatings has been demonstrated in several fruits, including pears [42] and plums [9].
Polymers 2020, 12, x FOR PEER REVIEW 8 of 14 the weight loss was significantly lower than in the control ones, regardless of the presence of cocoa shell nanofibrils. At the end of storage, control fruits and coated fruits showed water loss around 10% and 5%, respectively ( Figure 4). It has been suggested that postharvest treatments that restrict blueberry water loss to below 8% are likely to be beneficial in retaining acceptable blueberry freshness and firmness [4,41]. The ability to reduce weight loss of alginate coatings has been demonstrated in several fruits, including pears [42] and plums [9].  The changes in Andean blueberry soluble solids content, titratable acidity, and pH are shown in Figures 5 and 6. Control fruits showed a slight increase in their soluble solids content during storage ( Figure 5). This behavior was probably due to both the water loss and the cell wall degradation caused by the fruit ripening. In the case of the coated samples, it was expected that the water loss caused an increase in the soluble solids contents. Therefore, it can be hypothesized that the edible coatings were able to prevent the cell wall degradation, avoiding the increase in the soluble solids content of the coated samples. The changes in Andean blueberry soluble solids content, titratable acidity, and pH are shown in Figures 5 and 6. Control fruits showed a slight increase in their soluble solids content during storage ( Figure 5). This behavior was probably due to both the water loss and the cell wall degradation caused by the fruit ripening. In the case of the coated samples, it was expected that the water loss caused an increase in the soluble solids contents. Therefore, it can be hypothesized that the edible coatings were able to prevent the cell wall degradation, avoiding the increase in the soluble solids content of the coated samples. Concerning the pH and the titratable acidity (%), no relevant differences were detected among the uncoated and the differently coated samples neither at the initial time nor during the storage period ( Figure 6). The behavior of the firmness of the Andean blueberries with and without edible coatings during storage is shown in Figure 7. All the coated fruits showed significantly higher firmness than the uncoated fruit throughout the storage. This behavior could be attributed to both the presence of a coating that provides structural rigidity to the surface of the fruit and to the use of calcium ions as a cross-linking agent, which could act as a textural enhancer, minimizing the softening [10,43]. In addition, the increase in the firmness of the samples throughout the storage could be associated with the increase in the water loss.
At the end of the storage, the firmness of coated fruits was 25% higher than that the control ones. Concerning the pH and the titratable acidity (%), no relevant differences were detected among the uncoated and the differently coated samples neither at the initial time nor during the storage period ( Figure 6).
The behavior of the firmness of the Andean blueberries with and without edible coatings during storage is shown in Figure 7. All the coated fruits showed significantly higher firmness than the uncoated fruit throughout the storage. This behavior could be attributed to both the presence of a coating that provides structural rigidity to the surface of the fruit and to the use of calcium ions as a cross-linking agent, which could act as a textural enhancer, minimizing the softening [10,43]. In addition, the increase in the firmness of the samples throughout the storage could be associated with the increase in the water loss. firmness than the other systems. This fact could be due to the good dispersion of Cocoa shell nanofibrils within the alginate matrix when it was used at lower concentrations. Then, at 7 and 15 days of storage, the two coatings added of Cocoa shell nanofibrils provided higher firmness to the Andean blueberries. However, at the end of the storage, the firmness of the fruits with alginate and alginate/CNFs was similar.

Conclusions
Agro-food byproducts such as cocoa shell show promise as a precursor of cellulose nanofibrils than can be used in the formulation of edible films and coatings for their potential application in the preservation of foods such as Andean blueberries.
Cellulose nanofibrils with diameters between 15 and 50 nm were successfully extracted from cocoa shells through chemical and mechanical treatment. When these cellulose nanofibrils were incorporated within calcium alginate matrices, they provoked a significant decrease in the water vapor permeability and the transparency of the films. Moreover, the alginate edible coatings were able to decrease the respiration rate and weight loss and to improve the firmness of Andean blueberries, regardless of the presence of cellulose nanofibrils. Therefore, it can be concluded that alginate and alginate/cellulose nanofibrils coatings were useful in the delay of the postharvest deterioration of Andean blueberries when compared to the fruits without coatings.  At the end of the storage, the firmness of coated fruits was 25% higher than that the control ones. At the beginning of storage, the fruits with alginate/CNF 0.1% edible coatings showed higher firmness than the other systems. This fact could be due to the good dispersion of Cocoa shell nanofibrils within the alginate matrix when it was used at lower concentrations. Then, at 7 and 15 days of storage, the two coatings added of Cocoa shell nanofibrils provided higher firmness to the Andean blueberries. However, at the end of the storage, the firmness of the fruits with alginate and alginate/CNFs was similar.

Conclusions
Agro-food byproducts such as cocoa shell show promise as a precursor of cellulose nanofibrils than can be used in the formulation of edible films and coatings for their potential application in the preservation of foods such as Andean blueberries.
Cellulose nanofibrils with diameters between 15 and 50 nm were successfully extracted from cocoa shells through chemical and mechanical treatment. When these cellulose nanofibrils were incorporated within calcium alginate matrices, they provoked a significant decrease in the water vapor permeability and the transparency of the films. Moreover, the alginate edible coatings were able to decrease the respiration rate and weight loss and to improve the firmness of Andean blueberries, regardless of the presence of cellulose nanofibrils. Therefore, it can be concluded that alginate and alginate/cellulose nanofibrils coatings were useful in the delay of the postharvest deterioration of Andean blueberries when compared to the fruits without coatings.