Application of a Multi-Component Composite Edible Coating for the Preservation of Strawberry Fruit

Abstract

The rapid perishability of strawberries due to factors such as fungal decay, mechanical damage, and respiration significantly limits their shelf life. In this study, a novel multi-component edible coating composed of bacterial cellulose, chitosan, and gellan gum (BChG) was developed to enhance the postharvest quality and extend the shelf life of strawberries. The coated fruits were evaluated over a 15-day storage period for key parameters such as weight loss, total soluble solids (TSS), titratable acidity (TA), enzymatic activity, color retention, antioxidant activity, and microbiological analysis. The results demonstrated that coated strawberries exhibited significantly lower weight loss, reduced cellulase activity, and higher retention of TSS and TA compared to uncoated controls. The evaluation of microbial quality indicated that coatings, particularly those with higher concentrations of chitosan, control the growth of total mesophilic aerobic bacteria (TMAB) and molds and yeasts (MY), due to the antimicrobial properties of chitosan. This contributed to extending the shelf life of the fruit by preventing spoilage and reducing the risk of toxic compound formation. Additionally, the BChG coatings also preserved the characteristic red color of the fruit and maintained higher antioxidant activity, with BChG-4 being the most effective formulation. The inclusion of chitosan in the coatings was found to play a crucial role in reducing respiration, delaying ripening, and enhancing the fruit’s resistance to oxidative damage. Overall, multi-component coatings, particularly those with higher chitosan concentrations, offer a promising method for extending the shelf life of strawberries, reducing postharvest losses, and preserving fruit quality under ambient storage conditions.

1. Introduction

Strawberries are highly valued for their vibrant color, pleasant taste, and notable nutritional benefits. They are a rich source of essential vitamins such as vitamin C, vitamin E, thiamine, riboflavin, niacin, and vitamin B₆, along with amino acids, minerals, phenolic compounds, flavonoids, carotenoids, and antioxidant enzymes [1]. However, strawberries are also highly perishable, being particularly susceptible to fungal decay and mechanical damage, and due to their naturally soft texture, all of which significantly limit their shelf life [2]. One widely used method to preserve fruit quality and reduce microbial activity is rapid cooling immediately after harvest, followed by storage at low temperatures (0–4 °C). Despite these measures, the shelf life of strawberries is typically limited to less than five days [3]. While cold storage offers short-term benefits, the associated costs and potential taste deterioration from prolonged exposure to elevated CO₂ levels underscore the need for alternative preservation strategies

In addition to low-temperature storage, various approaches have been employed to reduce postharvest losses and extend the shelf life of fresh strawberries, including active packaging and edible coatings [4]. Within the fruit and vegetable industry, edible coatings have emerged as a promising postharvest technique for maintaining product quality. Edible coatings are primarily composed of thin layers of edible materials uniformly applied to the food surface [5]. Certain coatings have been shown to effectively maintain the quality of coated fruits and fresh products, such as guava [6], apples, and mangoes [7]. For instance, Vakili-Ghartavol et al. [8] studied coatings containing different concentrations of Mentha × piperita L. essential oil on strawberries’ postharvest quality and shelf life. Their findings demonstrated that these coatings preserved strawberries’ postharvest quality, reduced spoilage, and extended both the shelf life and nutritional value of the fruit during cold storage. Similarly, Al-Hilifi et al. [9] developed a coating composed of hyaluronic acid, chitosan, and gelatin, finding that the inclusion of hyaluronic acid enhanced the antioxidant properties of coated strawberries. Li et al. [10] developed an edible alginate-based coating containing lactic acid bacteria, which improved the quality of strawberries during storage. However, despite these advances, coatings for strawberry preservation have traditionally incorporated bacteria, plant-derived extracts, or oils without fully exploring the unique potential offered by certain hydrocolloids.

One example is bacterial cellulose, also known as bio-cellulose, which is produced by bacteria such as Gluconacetobacter and Acetobacter. This cellulose has a chemical formula (C₆H₁₀O₅) identical to that of plant cellulose [11] but offers the advantage of greater purity due to the absence of lignin, pectin, and hemicellulose. As a result, bacterial cellulose has excellent physical properties [12] that can be exploited for the preservation of food matrices. Chitosan (β-(1,4)-2-amino-2-deoxy-D-glucose) is another natural biopolymer, derived from the exoskeletons of marine invertebrates and insects, as well as from fungi, algae, and yeast [13]. It is the second most abundant polysaccharide in nature after cellulose and is likely the only natural polysaccharide with antimicrobial activity against bacteria, molds, and yeasts [14]. Gellan gum is an industrially significant polymer frequently used in coating formulations due to its biodegradability, biocompatibility, and ability to form non-toxic gels with various textural properties. It is an anionic linear heteropolysaccharide composed of 1,3-β-D-glucose, 1,4-β-D-glucuronic acid, 1,4-β-D-glucose, and 1,4-α-L-rhamnose [15]. Gellan gum was recognized as a natural food additive by regulatory authorities as early as the last century due to its safety and excellent gelling properties. To ensure food safety, it is essential to select coating materials classified as GRAS (Generally Recognized as Safe) by the FDA. In this context, bacterial cellulose, chitosan, and gellan gum are environmentally sustainable materials with favorable mechanical, antimicrobial, and biocompatible properties and are safe for consumption. Thus, they are excellent candidates for use in developing multi-component coating. These properties can be leveraged to produce a coating for application on economically important, perishable food matrices, such as strawberries. Accordingly, this study aims to investigate the effects of a novel multi-component composite coating—comprising bacterial cellulose, chitosan, and gellan gum—on extending the postharvest shelf life of strawberries.

2. Materials and Methods

2.1. Fruit Material

Strawberries (Fragaria × ananassa) were obtained from a local market at the stage of commercial ripeness, indicated by 75% red surface coverage. The fruits were carefully harvested and promptly transported to the laboratory. Selection criteria included uniformity in shape, size, and color, as well as the absence of visible damage, pests, or disease. The selected strawberries were thoroughly washed to remove any surface contaminants, then disinfected by immersion in a 1% sodium hypochlorite solution for 1 min, followed by rinsing with distilled water.

2.2. Production of Bio-Cellulose

Acetobacter aceti (2% inoculum) was introduced into a fruit waste medium with an initial pH of 5.8. The medium was prepared by blending orange, kiwi, and guava peels (33.3 g each) in 200 mL of distilled water. The resulting mixture was strained and centrifuged at 2000× g for 10 min to obtain a clarified solution. Following inoculation, the culture was incubated at 30 °C for seven days to synthesize bio-cellulose. The cellulose pellicle formed was initially centrifuged at 9000× g for 10 min at 4 °C to separate it from bacterial cells. The supernatant was then poured into chilled isopropyl alcohol to precipitate the bio-cellulose. Subsequently, the bio-cellulose was centrifuged again at 9000× g for 20 min to collect the bio-cellulose pellets, which were then boiled in a 2% (w/v) NaOH solution to remove any remaining attached cells. Finally, the pellets were filtered and rinsed several times with distilled water to neutralize the bio-cellulose’s pH [16].

2.3. Preparation and Application of Composite Coating

The composite solution was prepared by dissolving gellan gum (0.5 g/100 mL), chitosan (2 g/100 mL in 2% v/v acetic acid), and bacterial cellulose in distilled water, as shown in

Table 1. Experiment design 2³⁻¹ employed in the development of edible coating.

Coating Name	Bacterial Cellulose (% p/v)	Chitosan (% p/v)	Gellan Gum (% p/v)
BChG-1	25	0.4	0.3
BChG-2	4	0.4	0.8
BChG-3	4	2.0	0.3
BChG-4	25	2.0	0.8

. Various combinations were prepared, and the solution was mixed using a homogenizer (IKA T25 digital Ultra-Turrax, Staufen, Germany) at 3000 rpm for 10 min to ensure uniform distribution. The experimental design for the composite solutions is detailed in Table 1. Strawberries were thoroughly washed with distilled water to remove any dust or soil particles. After air drying at room temperature, the coating was applied to the strawberries by dipping. The strawberries were divided into groups, each consisting of 40 fruits, and each group was immersed in the coating solution for 2 min. After coating, the fruits were placed in a sterile air cabinet at room temperature for two hours to allow the coating to dry. Quality parameters were analyzed throughout the storage period at 20 °C and 80% relative humidity (RH).

2.4. Soluble Solids, Titrable Acidity (TA), and Weight Loss

The fruits were crushed using a mortar, and the soluble solids (SS) content was measured by placing four drops of the macerated fruit onto the prism of a refractometer (Fischer, Extech Model 2132, Extech Instruments, Nashua, NH, USA, EEUU). Results were recorded in degrees Brix (°Brix). To determine acidity, the mash was filtered and titrated with 0.1 N NaOH until the solution reached a pH of 8.3. Strawberry weight loss was recorded at the beginning of the experiment and subsequently during storage using a digital balance. Measurements were taken on days 0, 3, 6, 9, 12, and 15, and weight loss was calculated using Equation (1).

$$Weightloss\left(\%\right)={\displaystyle \frac{W_0-W_t}{W_0}}\times 100$$

(1)

where W₀ is the initial weight of the fruit and W_t represents the weight at each sampling time.

2.5. Determination of Color Change (ΔE)

The surface color of the fruit was evaluated using a CR-20 colorimeter (Konica Minolta, Tokyo, Japan). Measurements were taken at five distinct points on each fruit’s surface. The CIELAB color parameters—L* (Lightness), a* (Green-Red), and b* (Blue-Yellow)—were recorded on the initial and final days of sampling.

$$∆E=\sqrt{{\left({∆L}^{*}\right)}^2+{\left({∆a}^{*}\right)}^2+{\left({∆b}^{*}\right)}^2}$$

(2)

2.6. Cellulase Activity

Approximately 0.2 g of strawberry fruit tissue was combined with 0.02 M potassium phosphate buffer at pH 6.8 and 4 °C. The mixture was then cold-centrifuged at 15,000× g for 20 min, and the supernatant was collected for enzyme activity measurement. After incubating 50 μL of supernatant with 200 μL of 2.5 mg/mL cellulose in phosphate buffer at pH 7 and 42 °C for 1 h, the reaction was stopped by adding 750 μL of a 2:1 ethanol–acetone solution. The mixture was then centrifuged at 4000× g for 15 min, and the absorbance was measured at 550 nm [17].

2.7. Antioxidant Activity

A 50 μL extract was prepared using a methanol–water mixture (methanol, 8:2) and combined with 950 μL of a 2,2-diphenyl-1-picrylhydrazyl (DPPH) solution (0.025 g/100 mL in 85% methanol). The preparation was incubated in the dark for 60 min at room temperature, and the absorbance at 517 nm was measured. Trolox was used as a reference standard, and the DPPH radical scavenging activity was expressed in millimoles of Trolox equivalents per 100 g (mmol TE/100 g).

2.8. Microorganism Count

Strawberries (11 g) were homogenized in sterile peptone solution, and total mesophilic aerobic bacteria (TMAB) as well as yeast and mold counts were assessed on days 0, 3, 6, 9, 12, and 15 of storage. TMAB counts were determined using the deep plate method on plate count agar (PCA, Merck, Darmstadt, Germany) after incubation for 48 h at 37 °C. Yeast and mold counts were evaluated on potato dextrose agar (PDA, Merck, Darmstadt, Germany) after incubation for 5 days at 25 °C. Microbiological counts were expressed as log colony-forming units (cfu) per gram of sample.

2.9. Statistical Analysis

All analyses were conducted in triplicate, and results are presented as the mean ± standard deviation. Treatment comparisons were evaluated using a one-way analysis of variance (ANOVA) at a 95% confidence level. Post hoc multiple comparisons were conducted using the least significant difference (LSD) test.

3. Results and Discussion

3.1. Soluble Solids, Titrable Acidity (TA), and Weight Loss

Total soluble solids (TSS) are a key parameter for evaluating fruit quality, as they indicate the sweetness level of strawberries, a factor that directly influences consumer preferences [18]. Figure 1a shows the changes in TSS during storage for both control and coated fruits. Across all samples, TSS increased until the sixth day of storage. This increase can be attributed to the breakdown of starch into soluble sugars by α- and β-amylases within the chloroplast, the hydrolysis of cell wall polysaccharides, and the synthesis of sucrose-by-sucrose phosphate synthase in the cytosol during the ripening process [19]. However, after the sixth day of storage, a significant decrease (p < 0.05) was observed, particularly in the control fruits. This trend was similarly reported by Li et al. [20] in strawberries coated with alginate and lactic acid bacteria. The authors attributed this to the antimicrobial effect of the coating, which effectively delayed the degradation of TSS and maintained higher levels in strawberries. TSS are the primary target of consumption during the respiratory process [21]. Among the samples, the control group showed the most significant decrease (p < 0.05) in TSS at the end of storage (15 days) with 6.34 °Brix, followed by strawberries coated with BChG-2 and BChG-1, with 7.94 and 8.35 °Brix, respectively. In contrast, the BChG-3 and BChG-4 coatings showed the highest TSS content, with 8.69 and 8.64 °Brix, respectively. These latter two coatings have in common higher chitosan concentrations, a polymer with antimicrobial properties that can protect strawberries from external microbial damage, thus delaying nutrient loss [22]. This indicates that these types of coatings are effective in preserving higher TSS levels and delaying TSS degradation in strawberries.

Titratable acidity (TA) is closely linked to the concentration of organic acids in fruits and serves as an indicator of changes in the fruit’s intrinsic acidity over time. In strawberries, citric acid is the most abundant acid, followed by malic acid. The metabolic activities of strawberries consume organic acid, which in turn can lead to strawberry aging. Titratable acidity (TA) levels in strawberries reflect the balance between sugar and acidity, and changes in these levels can indicate the metabolic state of the strawberries, which, in turn, reflects their quality [19]. The variation in titratable acidity in strawberries stored for 15 days is shown in Figure 1b, where it can be observed that as the strawberries ripened, the titratable acidity content decreased. Compared to the control group, coated strawberries exhibited a significantly slower rate of acidity decline, especially those coated with BChG-3 (0.78%) and BChG-4 (0.87%). In contrast, the control strawberries had the lowest acidity value, reaching 0.26%, followed by strawberries coated with the formulations containing the lowest chitosan concentration (0.4% w/v). The films’ ability to modify the internal atmosphere around the strawberries by producing a high CO₂ environment reduces the respiratory intensity of strawberries during storage, thereby decreasing titratable acid consumption as a respiratory substrate and slowing the rate of acidity decline [23]. Based on these results, it can be inferred that the inclusion of chitosan in edible coatings may reduce the respiration rate of coated strawberries, thereby decreasing the release of organic acids and extending their shelf life.

Fruits such as strawberries are particularly susceptible to degradation and weight loss over time, mainly due to their thin, highly permeable skin, which increases the risk of moisture loss and, consequently, reduces fruit quality [24,25]. Figure 1c shows that the weight loss percentage of uncoated and coated strawberries over 15 days of storage followed an upward trend, with the control group (uncoated fruits) showing the highest values, reaching a 20.15% weight loss. This is attributed to the fruit’s respiration and transpiration processes [25]. In contrast, coated fruits had significantly lower weight loss values (p < 0.05) compared to uncoated fruits. This reduction is due to the semi-permeable barrier properties of the polymer-based coatings, which effectively slow moisture diffusion through the strawberries’ stomata, impeding water vapor passage generated by the fruit’s respiration through the film and its condensation on the film and fruit surfaces [26]. Similarly, Al-Hilifi et al. [8] reported weight loss values close to 5.5% in strawberries coated with chitosan and gelatin. Based on this trend, it can be concluded that multi-component coatings help reduce weight loss and preserve strawberry quality during the 15-day storage period.

3.2. Color Change (ΔE) Analysis

Color remains a critical visual determinant for strawberry appeal, directly influencing its market acceptability. The color changes in both control and coated strawberries are shown in Figure 1d, which illustrates that ΔE gradually increases over time. Similar results were reported by Li et al. [10] in strawberries coated with gellan gum and sodium alginate/sodium carboxymethyl cellulose; these authors observed ΔE values exceeding 60 after 14 days of storage. In our findings, the highest ΔE value (21.55) was recorded in uncoated fruits at the end of the storage period. This significant change may be attributed to anthocyanin degradation triggered by hydrolase activity, causing color loss during postharvest storage through the disruption of glycosidic linkages in the molecule [27]. For the coated strawberries, significant differences (p < 0.05) were observed among all samples at the end of the storage period, highlighting that bacterial cellulose, chitosan, and gellan gum have a pronounced effect on preserving strawberry color. The least color change at the end of storage was observed in strawberries coated with BChG-4 (0.47), followed by BChG-3 (0.88); again, coatings with a higher concentration of chitosan (2.0% w/v) showed a greater effect on postharvest preservation. These findings suggest that the application of multi-component coatings, especially those containing chitosan, can delay the aging process of the fruit. These results align with those of Van et al. [28], who reported similar findings in strawberries coated with sodium carboxymethyl cellulose, cellulose nanofibers, and various concentrations of mandarin oil.

3.3. Enzymatic Activity

The results of this study demonstrated that the coated fruits exhibited significantly lower cellulase activity (p < 0.05) compared to the control group, as shown in Figure 2a. These findings align with previous research by Wani et al. [24], who reported a reduction in cellulase activity in strawberries and pears treated with edible coatings compared to untreated controls. By the end of the storage period (15 days), the highest enzymatic activity was observed in the uncoated fruits, with a value of 2.68 U/min/g FW. Among the coated fruits, those treated with the BChG-4 coating showed the lowest enzymatic activity (1.43 U/min/g FW), followed by fruits coated with BChG-3 (1.56 U/min/g FW). Similarly, Vakili-Ghartavol et al. [8] found that cellulase activity was higher in control strawberries than in those coated with different concentrations of Mentha × piperita L. essential oil. The high concentrations of chitosan in these coatings seem to play a role in reducing cellulase activity in strawberries.

3.4. Antioxidant Activity

Antioxidant activity is a critical factor in determining the quality of fruits and vegetables [29], as it helps protect the human body from damage caused by reactive oxygen species (ROS) [30]. Figure 2b presents the DPPH activity of different coatings over the 15-day storage period. A significant decrease (p < 0.05) in antioxidant levels was observed in uncoated strawberries compared to those with coatings. By the end of the storage period, fruits coated with BChG-4 exhibited the highest antioxidant activity (71.02%), followed by those coated with BChG-3 (66.11%). The coated fruits demonstrated a greater DPPH radical scavenging capacity compared to the uncoated control, likely due to the antioxidant properties of the amino and hydroxyl functional groups present in chitosan chains [31]. Similarly, Vakili-Ghartavol et al. [8] observed higher antioxidant activity in strawberries coated with Mentha × piperita L. essential oil (MEO) and solid lipid nanoparticles containing MEO. The enhanced preservation of antioxidant activity in coated fruits may be attributed to the free-radical scavenging properties of the edible coating [9]. In contrast, control fruits exhibited the lowest antioxidant activity, reaching only 27.55% by the end of storage, likely due to the degradation of phenolic compounds and ascorbic acid as ripening progressed. Overall, the BChG-3 and BChG-4 coatings provided greater protection against maturation and decay compared to uncoated controls, consistent with findings by Mohammadi et al. [32] and Perumal et al. [29], who noted that edible coatings form a protective barrier on the fruit’s surface, limiting oxygen penetration and thereby reducing the enzymatic oxidation of phenolic compounds.

3.5. Microbiological Quality

The microbial quality assessment of strawberries was conducted by monitoring total mesophilic aerobic bacteria (TMAB) and mold and yeast (MY) counts during the storage period, given that strawberries are highly susceptible to tissue damage and infections from various phytopathogenic fungi and bacteria [33]. The microbial counts for both uncoated and coated strawberries during storage are shown in Figure 3a,b. The initial TMAB count for strawberries ranged from 0.11 to 0.38 log CFU/g. The control sample had the highest TMAB value at the end of storage, reaching 6.20 log CFU/g, while strawberries with coatings exhibited the lowest values. For instance, strawberries coated with BChG-4 and BChG-3 displayed the lowest TMAB levels, with values of 0.19 and 1.48 log CFU/g, respectively. This result is attributed to the antimicrobial properties of chitosan, wherein the cationic groups of chitosan interact with the anionic peptidoglycan in the microbial cell wall, causing the wall to collapse and release intracellular fluid, leading to cell death [14].

Regarding MY counts, a positive correlation with storage duration was observed in both control and coated strawberries. Notably, a decrease in MY counts was evident at the end of storage, likely due to the stationary phase of these microorganisms caused by nutrient depletion and microbial competition. The lowest MY counts at the end of storage were recorded for the BChG-3 (3.41 log CFU/g) and BChG-4 (2.48 log CFU/g) coatings, while BChG-1 and BChG-2 coatings showed counts of 4.72 and 5.3 log CFU/g, respectively. These counts are close to the reference value of 10⁶ CFU/g, which is used to estimate the shelf life of strawberries [34]. This threshold was maintained for up to 15 days in strawberries coated with multi-component coatings, particularly those with a higher concentration of chitosan. In contrast, control strawberries presented values of 9.42 log CFU/g. Exceeding this microbial population limit can lead to the production of toxic substances under light exposure, as noted by Howard and Dewi [35].

4. Conclusions

This study demonstrates the effectiveness of multi-component edible coatings, particularly those with high chitosan concentrations, in preserving the quality and extending the shelf life of strawberries during postharvest storage. The coated fruits exhibited significantly lower weight loss, reduced cellulase activity, and enhanced retention of total soluble solids (TSS) and titratable acidity (TA) compared to the uncoated controls. The coatings also effectively delayed color changes, preserving the characteristic red hue of strawberries for a longer period, and provided superior antioxidant protection by maintaining higher DPPH radical scavenging activity throughout the storage period, likely due to the antioxidant properties of chitosan. The enhanced antioxidant preservation helped reduce oxidative stress, contributing to better maintenance of fruit quality and potential health benefits. Finally, the microbial quality assessment showed that strawberries coated with BChG-4 and BChG-3 had the lowest microbial counts, both in total mesophilic aerobic bacteria (TMAB) and mold and yeast (MY), due to chitosan’s antimicrobial properties. These coatings effectively inhibited microbial growth, helping to extend the fruit’s shelf life by preventing spoilage and reducing the risk of toxic compound formation. Overall, the results indicate that multi-component coatings, especially those containing higher concentrations of chitosan, effectively enhance the shelf life and quality of strawberries during storage, offering a promising strategy for postharvest fruit preservation.