Application of Buckwheat Starch Film Solutions as Edible Coatings for Strawberries: A Proof-of-Concept Study

Abstract

The present study serves as a proof-of-concept of our previous work, as the buckwheat (BW) starch film solutions are applied as edible coatings on strawberries and as film packaging materials for strawberry preservation. The BW starch film solution was modified with citric acid (CA) for cross-linking and chitosan nanoparticles (CNP) and by ultrasound application. We tested four formulations for coating: uncoated (negative control), BW starch only (positive control), BW starch with CA and CNP, and ultrasonicated BW starch with CA and CNP. Results demonstrated that BW starch coating, with or without modifications, had positive effects in preserving strawberry quality during 14 days of refrigerated storage at 4 ± 1 °C and 82 ± 1% RH. Coating with only BW starch better suppressed weight loss; a 16% reduction in weight loss was observed compared to the uncoated counterpart at day 14. On the other hand, modifications of coating formulation played a role in preserving different fruit quality parameters. BW starch with CA and CNP had improved textural properties and reduced signs of decay. A 56% reduction in the decay index (DI) was observed in the coated fruits compared to the control. Starch coating restricted chemical changes and maintained total phenolic content (TPC) during storage. TPC in ultrasound-treated solution-coated fruits was the highest, 1.3 mg GAE/g, at the end of the storage. As packaging materials, BW starch films effectively reduced moisture loss from packaged strawberries. The future scope of the study lies in optimizing film solutions for various applications and in understanding enzymatic activities in BW starch-coated fruits.

1. Introduction

Buckwheat, an underutilized pseudocereal, is an excellent source of starch. Buckwheat starch has been regarded as a promising biodegradable packaging material [1,2]. However, compromised mechanical and barrier properties restrict its widespread use [3]. To improve the performance of starch-based films, starch molecules are cross-linked using different chemicals, such as citric acid, sodium trimetaphosphate, glutaraldehyde, ferulic acid, and boric acid [2]. Citric acid, which is a tricarboxylic organic acid, creates covalent intermolecular linkages between the carboxyl (COOH) group and the hydroxyl (OH) group, resulting in cross-links. Besides working as a cross-linking agent, citric acid acts as a plasticizer [2,4]. Improved mechanical and thermal properties and reduced moisture absorption and water vapor permeability have been reported for citric acid-treated starch-based films [4,5]. Moreover, better performance from biodegradable packaging material can be achieved using nanotechnology [6]. Nanoparticles act as fillers in a film matrix, improving the mechanical and barrier properties of the film. Moreover, nanoparticles provide added functionalities, such as antioxidant and antimicrobial activities, to the packaging materials [6]. Further, ultrasound treatment could promote homogeneous film structure by improving starch solubility and reducing agglomerated starch granules, which in turn could improve the tensile strength of starch films [7].

Strawberry (Fragaria × ananassa) is a non-climacteric fruit that is harvested at the full maturity stage for maximum marketing quality [8]. Strawberries are popular due to their unique taste and flavor. Strawberries are an excellent source of vitamin C, vitamin E, β-carotene, and phenolic compounds that make them beneficial to consumer health [9]. However, the shelf life of strawberries is very short, around 2 weeks if kept at 0 °C and only up to 3–4 days when kept at room temperature (~20 °C) [9]. Its high respiration rate, susceptibility to mechanical injury, and microbial infection cause its shorter shelf life after harvest [8]. Traditionally, strawberries are treated with different fungicides to control fungal decay, which have adverse effects on humans and the environment. Moreover, to extend the shelf life of strawberries, various postharvest treatments, such as edible coating, UV-C irradiation, ultrasonic treatment, controlled atmosphere packaging, etc., have been investigated [8]. Due to the environmental issues regarding the disposal of synthetic packaging materials, naturally occurring coating materials have become a topic of interest to consumers and researchers. Edible coatings perform a similar job to conventional packaging by reducing moisture loss, gas exchange, and microbial spoilage. Moreover, they carry bioactive compounds such as antioxidants and antimicrobial compounds [10,11]. However, the performance of an edible coating largely depends on its permeability and mechanical properties [12]. Such natural polymeric or starch films generally offer a good barrier against O₂ and CO₂ but have poor water vapor permeability. Also, edible films and coatings exhibit weak mechanical properties [13]. However, strawberries packaged in pea starch/soy protein isolate films loaded with nanoparticles showed reduced moisture and hardness loss [6].

Various innovative approaches, such as citric acid cross-linking/esterification, chitosan nanoparticles, and ultrasound application, have been combined to modify/improve starch for better film properties [14] and eventually for application as edible coatings. In a recent study [14], the effects of different strategies, such as citric acid (CA) cross-linking, the inclusion of chitosan nanoparticles (CNP), and ultrasonication of CA and CNP film solutions, on the properties of BW starch films were studied. The results indicated that films modified with CA and CNP had reduced water vapor permeability (WVP), and the CA and CNP-treated film with ultrasonic treatment exhibited improved mechanical properties, especially tensile strength. Moreover, modifications also resulted in improved resistance to moisture sorption compared to unmodified BW starch films.

Table 1. Summary of relevant film data (WVP, tensile strength, and moisture sorption) collected in previous work [14].

Film Samples	WVP (g mm/mm2 h kPa)	Tensile Strength (MPa)	Moisture Sorption (%) After 6 h
T2	2.73 × 10−10 ± 2.26 × 10−11	4.41 ± 0.83	77.32 ± 0.64
T3	2.76 × 10−11 ± 1.64 × 10−11	3.48 ± 0.57	65.33 ± 0.47
T4	2.13 × 10−10 ± 1.04 × 10−11	10.89 ± 6.65	73.88 ± 4.88

T2—BW starch, T3—BW starch + 5% CA + 1% CNP and T4—ultrasonicated BW starch + 5% CA + 1% CNP. BW: buckwheat; CA: citric acid; CNP: chitosan nanoparticle. The results are expressed as mean ± SD. Note: Films conditioned at room temperature (22 ± 1 °C) and 50 ± 2% RH were immediately evaluated. The texture analysis was performed at 50 ± 2% RH under ambient conditions. For the WVP test, the cups were placed in an incubator with constant temperature (25 °C) and relative humidity (25 ± 1%) [14].

provides a summary of the data collected from previous work [14]. Overall, CA, nanoparticles, and ultrasound applications could improve starch functionality for various purposes, including food coating films or packaging. Modified biodegradable films were applied as coatings on cherry tomatoes, and a promising preservation effect of the film solution was observed [15]. To our knowledge, this is the first time that BW starch has been blended with citric acid for cross-linking/esterification and chitosan nanoparticles and ultrasonication to formulate edible coatings for fresh produce. Moreover, the present work serves as an application or follow-up of previously published research, which makes it a novel step/concept, as BW starch film solutions are applied as edible coatings on strawberries and as film packaging materials for strawberry preservation. The world starch market has been dominated by corn, potato, sweet potato, and cassava [16], and BW starch can be considered a non-conventional source of starch. Overall, there are limited data on BW starch films, and to our knowledge, there is no study dedicated to the application of BW starch (film solutions) as edible coatings. Therefore, the aim of this work is to apply different BW starch film solutions as edible coatings and to study the effect of the coating treatments on strawberry quality. Moreover, BW starch films were used for strawberry packaging to understand their effect on strawberry weight loss.

2. Materials and Methods

2.1. Materials

The materials used in this experiment consisted of fresh strawberries collected from a local farm in West Virginia, USA. The strawberries were from the Malwina variety, with an average diameter of approximately 35 mm, firm, and ready-to-eat maturity. Organic buckwheat (BW) flour (100%, prepared from BW groats) was obtained from anthonygoods.com (Anthony’s Goods, Glendale, CA, USA). Starch was extracted from BW flour following a previously described protocol [14]. The amylose content was found to be (using iodine-binding-based colorimetry) 31.45 ± 0.73% of total starch. Chitosan nanoparticles (>99.9% purity, 100–200 nm particle size, 161 g/mol molecular wt., and 90.22% degree of deacetylation) were purchased from Nanochemazone (Chemazone Inc., Leduc, AB, Canada). Citric acid was purchased from Lab Alley (Austin, TX, USA). Glycerol and all other chemicals were of analytical grade.

2.2. Preparation of Film Solutions, Strawberry Samples, and Coating Application

Strawberry samples dipped into distilled water were served as a negative control (T1). Four film solutions were prepared as treatment groups: BW starch only (positive control/T2), BW starch with CA and CNP (T3), and ultrasonicated BW starch with CA and CNP (T4). 5% (w/v) BW starch film solution was prepared by adding 5 g of starch to 100 mL of distilled water at room temperature. A total of 1.2 mL of glycerin (30 wt % of starch, each mL of glycerin weighs 1.26 g) was added as a plasticizer. The mixture was heated on a hot plate at 90 °C with continuous stirring till gelation appeared. The film solution thus prepared served as a positive control (T2). Then, 5% (w/w of starch) CA and 1% (w/w of starch) CNP were dispersed in the film solution (starch–glycerin) and mixed with a standard laboratory stirrer at 1000 rpm for 15 min, and solutions with CA and CNP (T3) were heated in a similar manner as the positive control. The solution mixture with CA and CNP was placed in an ice bath and subjected to ultrasonication at 40% amplitude and 20 kHz [8] for 10 min using a Q700 Sonicator (QSonica, Newtown, CT, USA) with a calculated energy density [17] of 0.08 kJ/cm³, followed by heating at 90 °C for gelatinization (T4). The viscosity of the film solutions was measured in a previous study and reported as 0.78, 1.18, and 0.96 Pa.s for T2, T3, and T4 treatments, respectively [14]. The gelatinized starch solution was brought to room temperature (22 ± 1 °C) while continuously stirring before being applied as edible coatings. Mechanical force was applied to break the long glucose chains and reduce viscosity while cooling down to 22 ± 1 °C.

Strawberries of commercial maturity, uniform size (~35 mm average diameter), and without any damage or decay were selected and transported to the laboratory immediately. The strawberries were first washed with tap water to remove any adhering dirt and soil. After washing with tap water, strawberries were sanitized with 0.05% (w/v) sodium hypochlorite solution and then again rinsed with tap water. The cleaned strawberries were placed on a metal wire mesh and dried with the help of a fan. After being dried completely, strawberry samples were dipped into different BW starch film solutions for 5 min, drained, and air-dried at room temperature (22 ± 1 °C) using a fan until the coating layer was dry. Then, the control and coated fruits were subjected to packaging and storage.

Table 2. Treatments/film-forming solutions.

Treatments	Only Distilled Water	Buckwheat Starch	Citric Acid (CA) and Chitosan Nanoparticles (CNP)	Ultrasound Treatment Before Gelatinization
T1 (negative control)	+	−	−	−
T2 (positive control)	−	+	−	−
T3	−	+	+	−
T4	−	+	+	+

displays detailed information on the treatments/film solutions. Coated strawberries were packaged in clamshells (5 fruits/box, 3 boxes per treatment) and stored at 4 ± 1 °C and 82 ± 1% RH in an environmental chamber (Heratherm, Thermo Scientific, Waltham, MA, USA). To evaluate the effect of coating treatments on the shelf life and quality of strawberries, physicochemical studies were carried out on each sampling day—days 0, 4, 7, 10, and 14. Figure 1 shows the visual appearance of coated strawberries over the storage period.

2.3. Physicochemical Quality Study

2.3.1. Weight Loss

The weight of each clamshell box containing the control and coated strawberries was recorded (OHAUS Scout STX6201, Hogentogler & CO. Inc., Columbia, MD, USA) at the beginning of storage (day 0). On each sampling day, three replicate boxes per treatment group were reweighed. The weight loss (WL) was then calculated as a percentage of the initial weight on day 0:

$$\mathrm{WL} \left(\%\right)={\displaystyle \frac{{\mathrm{W}}_{\mathrm{a}}-{\mathrm{W}}_{\mathrm{b}}}{{\mathrm{W}}_{\mathrm{a}}}}\times 100$$

(1)

where $\mathrm{WL}$ is the weight loss expressed as a percentage rate, ${\mathrm{W}}_{\mathrm{a}}$ is the initial weight in g, and ${\mathrm{W}}_{\mathrm{b}}$ is the weight in g on the sampling day. The weight loss from all three replicates was averaged to record the $\mathrm{WL}\left(\%\right)$ from a particular level of treatment on a sampling day.

2.3.2. Texture Profile Analysis (TPA) and Firmness

The TPA test of the strawberries was performed using a Texture Analyzer (TA.XTExpress, Stable Micro Systems Ltd., Hamilton, MA, USA) equipped with a 2 Kg load cell. Ten fruits per treatment were evaluated for the TPA test. The strawberries underwent a compression test at the equator zone (the point of maximum diameter) with a 2 mm diameter cylindrical probe. TPA settings were as follows: pre-test speed of 2.0 mm/s, test speed of 5.0 mm/s, post-test speed of 5.0 mm/s, and trigger force of 0.05 N. The strawberries were centered and compressed at a distance of 3 mm, and then the hardness, chewiness, and gumminess values were calculated with the force–time curve using Exponent Connect Lite Express v.8.0.9.0 software (Stable Micro Systems Ltd.).

The internal firmness of strawberries was recorded to understand the actual fruit firmness without the coating layer. The internal firmness of strawberries was recorded using a penetrometer (FT 02, QA Supplies, Norfolk, VA, USA). Six fruits per treatment were subjected to the firmness test. A 5 mm disk of the fruit skin was removed with a sharp knife, and then a 3 mm cylindrical probe tip was forced into the fruit to a depth of 3 mm at a uniform speed. The force (N) required to puncture the strawberry flesh was recorded and reported as the mean ± standard deviation.

2.3.3. Color

Color changes in the strawberries were observed using a Konica Minolta CR-400 colorimeter (Konica Minolta, Tokyo, Japan). The color values of the fruits were expressed as L* (lightness), a* (greenness [−] to redness [+]), and b* (blueness [−] to yellowness [+]). Prior to the experiment, the colorimeter was calibrated using a standard white tile. To determine the color changes of the fruits at each sampling day, eight random fruits per treatment were picked, and reflectance spectra were evaluated for the values of L*, a*, and b* color components. All the observations were then averaged and reported as the mean ± standard deviation.

2.3.4. pH, Total Soluble Solids (TSS), and Titratable Acidity (TA)

All the fruits were homogenized with a blender and filtered with cheesecloth. The filtrate thus obtained was used for the chemical tests. The pH of the strawberry juice was measured using a digital pH meter (Orion Star A215, Thermo Fisher Scientific, Waltham, MA, USA), and the values were recorded. TSS was recorded using a digital pocket refractometer (PAL-1, ATAGO, Tokyo, Japan) and expressed as %. Titratable acidity (TA) was determined using an automatic titrator (Orion Star T910, Thermo Fisher Scientific, Waltham, MA, USA) by titration with 0.1 N NaOH up to the pH endpoint of 8.1 and expressed as % citric acid.

2.3.5. Decay Index (DI)

The proportion of the decay index (DI) was evaluated following the method outlined in [18] and [19] using the following scores: 0 = no area decay, 1 = 0–10% area decay, 2 = 11–30% area decay, 3 = 31–50% area decay, and 4 = 51–100% area decay. The fruit DI was calculated for the total fruit (n = 15) per treatment at each sampling time, as per the following equation:

$$\mathrm{DI}={\displaystyle \sum }\left({\displaystyle \frac{\left(\mathrm{d}\times \mathrm{f}\right)}{\left(\mathrm{N}\times \mathrm{D}\right)}} \right)\times 100$$

(2)

where d is the category of decay intensity scored on the fruit, and f is its frequency, N is the total number of examined fruits, and D is the highest category of decay intensity that occurred on the severity scale.

2.3.6. Antioxidants Assay

Antioxidant activity (AA) was analyzed using a DPPH (2,2-diphenyl-1-picryhydrazyl) assay, as previously described by Sarker et al. [17,20], with modifications. Briefly, 70% acetone was used to macerate the samples for about 24–48 h at 4 ± 1 °C. Thereafter, the samples were vortexed, centrifuged at 2000× g, filtered, and stored at −4 °C for further analyses. Trolox was used to generate a calibration curve (0–100 μg/mL). The DPPH⁺ scavenging capacity of strawberry extracts was demonstrated by plotting against the Trolox antioxidant standard curve. The experiment was conducted in triplicate, and data were expressed as μMol Trolox equivalents (TE)/g fresh fruit.

2.3.7. Total Phenolic Content (TPC) Assay

Fresh strawberry fruits on each sampling day were homogenized using a standard food blender. The liquid/solid ratio was 30:1. The extracting solvent, 70% acetone, was used to macerate the samples for about 24 h at −4 °C. Thereafter, the samples were vortexed, centrifuged at 8350 rpm, and filtered with a Whatman #1 filter paper. The gallic acid standard curve was plotted with 20 mg/L to 200 mg/L concentrations. The experiment was conducted in triplicate, and data were expressed as mg GAE/g fresh fruit.

2.4. Application as a Film Packaging Material

Apart from applying the film solutions (T2, T3, and T4) as edible coatings, the films prepared after casting and drying the solutions at 35 °C for 24 h [14] were used as packaging materials for strawberry preservation. Strawberries were divided into four groups and transferred to plastic cups, and the mouths were covered with film samples T2, T3, and T4, respectively. T1 was grouped as a control with uncovered strawberries. There was a total of 9 fruits per group, 3 fruits/cup with 3 replications. Fruits were stored in a stability chamber for 3 days, set at 25 °C and 50% RH [6]. Weight loss was monitored and recorded every day from day 0 to 3.

2.5. Experimental Design and Statistical Analysis

The proposed experiment was laid out in a completely randomized design (CRD) with three replications. There were four levels of treatment (T1—distilled water as negative control, T2—BW starch solution as positive control, T3—BW starch solution with CA and CNP, and T4—ultrasonicated BW starch solution with CA and CNP. There were five (5) storage (sampling) days (d) (0, 4, 7, 10, 14). Therefore, the total experimental units (5 strawberries/box) was 3 (replications) × (treatments) × 5 (days of storage) = 60. A total of 5 × 60 = 300 strawberry fruits were used in this experiment. Treatment levels were randomly assigned to the experimental units. A two-way analysis of variance (ANOVA) was performed to understand the effect of treatment, storage duration, and their interactions on the studied dependent variables. For within-day treatment comparison, a one-way ANOVA was performed. For all analyses, R statistical software [21] version 3.5.2 was used. Sample standard deviation was calculated using the sd () function. A Tukey HSD test with a 95% confidence interval was applied to evaluate differences among means.

3. Results and Discussions

3.1. Weight Loss

Weight loss was significantly (p < 0.05) affected by the treatment and storage duration, with no significant interaction between these two independent variables (

Table 3. Two-way analysis of variance (ANOVA) results of the % weight loss, color (L* a* b*) changes, TPA, and firmness.

		Df	SS	MS	F	Pr (>F)
% Weight loss (n = 3)	Trt.	3	24.36	8.121	4.9838	0.0125 *
	Day	3	551.06	183.686	112.7233	5.674 × 10−11 ***
	Trt × Day	9	28.01	3.112	1.9097	0.1241
	Residuals	16	26.07	1.630
L* (n = 8)	Trt	3	115.57	38.52	1.7098	0.1689
	Day	4	2105.79	526.45	23.3660	3.747 × 10−14 ***
	Trt × Day	9	273.11	30.35	1.3469	0.2208
	Residuals	115	2591.00	22.53
a* (n = 8)	Trt	3	530.75	176.917	25.9433	6.856 × 10−13 ***
	Day	4	398.63	99.658	14.6140	1.111 × 10−9 ***
	Trt × Day	9	111.61	12.401	1.8184	0.0721
	Residuals	115	784.23	6.819
b* (n = 8)	Trt	3	516.61	172.203	15.6502	1.336 × 10−8 ***
	Day	4	270.88	67.720	6.1545	0.0001595 ***
	Trt × Day	9	166.19	18.466	1.6782	0.1020587
	Residuals	115	1265.38	11.003
Hardness (n = 10)	Trt	3	30.608	10.2026	14.2888	4.97 × 10−8 ***
	Day	4	6.357	1.5893	2.2258	0.07015
	Trt × Day	9	6.505	0.7228	1.0122	0.43414
	Residuals	121	86.397	0.7140
Springiness (n = 10)	Trt	3	33.35	11.1155	2.1360	0.09921
	Day	4	16.23	4.0571	0.7796	0.54054
	Trt × Day	9	81.09	9.0099	1.7314	0.08892
	Residuals	121	629.68	5.2040
Chewiness (n = 10)	Trt	3	0.04088	0.0136254	1.7678	0.15693
	Day	4	0.10406	0.0260154	3.3753	0.01175 *
	Trt × Day	9	0.01346	0.0014954	0.1940	0.99445
	Residuals	121	0.93262	0.0077076
Firmness (n = 6)	Trt.	3	48.99	16.3299	12.5856	5.906 × 10−7 ***
	Day	3	4.421	1.4736	1.1357	0.3390
	Trt × Day	4	4.937	1.2341	0.9512	0.4383
	Residuals	91	118.073	1.2975

Statistical significance is denoted by asterisks: * (p < 0.05), and *** (p < 0.001).

). As shown in Figure 2, the weight loss of the strawberries increased with increasing storage. T2, i.e., the BW starch-coated fruits, had the least weight loss throughout the storage period. However, T1 with no coating treatments, as well as T3 and T4 with BW starch, CA, and CNP, and ultrasonicated BW starch, CA, and CNP, respectively, had consistently higher weight loss during storage. At the end of the storage duration, T4 strawberries had significantly higher (p < 0.05) weight loss compared to the T2-treated fruits (Figure 2). BW starch-coated fruits (T2) had 16% reduced weight loss compared to the uncoated counterpart (T1) on day 14.

The primary purpose of the coating treatment is to reduce fruit weight loss and moisture loss, which generally contributes to the weight loss of stored produce [22]. As shown in Table 1, even though CA and CNP inclusion in the BW starch film matrix improved the moisture barrier properties of films [14], T3 and T4 film solutions, as applied as coatings, could not reduce the moisture loss from fresh produce (strawberries). Moreover, film flexibility is one of the critical factors for packaging applications. According to previous research [14], the % elongation of T2, T3, and T4 films was 107.28, 87.67, and 32.55, respectively. Therefore, reduced flexibility might have played a role in increased water loss from strawberries as T3 and T4 film solutions applied as coatings. A brittle coating formulation may result in microcracks and pores with localized areas of increased water loss. The water pressure difference between the fruit tissue and the storage atmosphere determines the rate of produce moisture loss [22,23,24]. In this study, T2, i.e., BW starch’s superior performance in restricting moisture loss, could be due to the desirable water barrier properties of starch-based coating [6,18]. In contrast, despite being coated, T3 and T4 strawberries suffered from the highest water loss, even higher than the control. Osmotic dehydration could have played a role in accelerated moisture loss from T3 and T4-coated fruits. Osmotic dehydration occurs when fresh food with high moisture content encounters a solution with a high solute concentration. In this case, the increase in the film solutions’ concentration due to added CA and CNP resulted in an increased concentration gradient between the solution and the intracellular fluid, drawing water out of the fresh produce [25,26,27]. Strawberry skins are highly permeable to water and also very susceptible to osmotic uptake or loss due to transpiration [28]. T1 fruits with no coating treatments should have suffered from transpiration-related water loss, and T3 and T4 might have been affected by osmotic water loss. Similar data were reported by Sarker et al. [27] for cucumbers coated with a higher concentration of aloe vera gel. Moreover, increased weight loss was reported for tomatoes coated with a higher concentration of gum arabic coating. However, the increased weight loss was attributed to the thick coatings generating heat and end products due to anaerobic fermentation [29]. In the present study, for fresh produce, such as strawberries, the coating concentrations in T3 and T4 (with added CA and CNP) may be beyond optimum, resulting in thicker coating formulations and brittleness. Considering the hydrophilic nature of starch, incorporation of a hydrophobic substance is usually suggested for coating formulations to better control moisture loss [16]. Overall, further studies are needed with reduced coating concentrations to overcome the possible osmotic loss of water, the addition of a hydrophobic additive for reduced water permeability, or an increased concentration of plasticizer for improved flexibility.

3.2. Color Change

As shown in

Table 4. L*, a*, and b* changes in control and BW starch-coated fruits during storage at 4 ± 1 °C.

Treatments a	Days of Storage at 4 ± 1 °C
	0	4	7	10	14
L*
T1	19.19 ± 4.13 a	20.85 ± 4.19 a	24.85 ± 5.31 a	22.93 ± 7.66 a	12.26 ± 3.41 b
T2	19.19 ± 4.13 a	18.07 ± 2.73 a	26.31 ± 4.30 a	23.28 ± 3.86 a	12.28 ± 6.70 b
T3	19.19 ± 4.13 a	17.66 ± 4.73 a	23.88 ± 2.59 a	24.07 ± 6.14 a	20.15 ± 5.14 a
T4	19.19 ± 4.13 a	17.92 ± 3.08 a	25.99 ± 4.05 a	21.45 ± 2.43 a	14.63 ± 6.89 ab
a*
T1	23.01 ± 3.36 a	24.66 ± 2.78 a	27.55 ± 1.84 a	24.83 ±3.93 a	24.86 ± 2.36 a
T2	23.01 ± 3.36 a	22.59 ± 2.10 a	26.55 ± 1.99 a	22.89 ± 1.94 ab	22.47 ± 2.18 ab
T3	23.01 ± 3.36 a	19.12 ± 2.36 b	22.25 ±3.27 b	20.57 ±3.00 b	21.04 ± 3.11 b
T4	23.01 ± 3.36 a	19.27 ± 1.74 b	25.51 ± 2.56 ab	19.14 ± 2.70 b	17.46 ± 2.12 c
b*
T1	19.18 ± 5.02 a	15.55 ± 5.18 a	15.76 ± 4.54 a	15.00 ± 2.95 a	14.91 ± 1.85 a
T2	19.18 ± 5.02 a	14.75 ± 1.97 a	14.76 ± 2.87 a	14.26 ± 3.78 a	12.28 ± 3.03 ab
T3	19.18 ± 5.02 a	10.18 ± 1.53 a	13.29 ± 4.79 a	11.07 ± 2.77 ab	10.33 ± 2.95 b
T4	19.18 ± 5.02 a	10.88 ± 1.84 a	16.42 ± 3.38 a	8.82 ± 2.00 b	9.82 ± 2.78 b

T1—distilled water as control, T2—BW starch, T3—BW starch + 5% CA + 1% CNP, and T4—ultrasonicated BW starch + 5% CA + 1% CNP. Data (Mean ± SD) in the same column followed by different letters (a–c) are significantly different in terms of the treatment factor. On d-0, treatments have equal means, since data were recorded for fresh, untreated strawberries before storage.

, BW starch coating treatment did not significantly (p > 0.05) influence the lightness (L*) of strawberry samples until day 10. At the end of the storage period, both control and BW starch-treated fruits, except T3, exhibited decreased L* values, indicating darkness of the fruit samples. On the other hand, all starch-coated samples exhibited reduced redness (a*) compared to the control, and the effect was especially significant (p < 0.05) for T3 and T4. The control sample had a significantly (p < 0.05) higher blue (b*) value than the T3 and T4 samples, which correlates with the darker control sample as the storage duration progressed. As per two-way ANOVA (Table 3), the treatment and storage duration had significant effects (p < 0.05) on strawberry color changes.

Color is one of the most important factors affecting consumers’ purchase of strawberry fruit [8]. Decreased brightness of uncoated strawberries was attributed to unrestricted gas exchange and respiration rate [30]. Higher respiration rate causes faster senescence or degradation, leading to dark brown tissues in strawberries [30]. In the present research, coating treatment delaying fruit senescence might have preserved the lightness of strawberry fruits [31]. Similarly, brighter strawberries were reported for aloe vera–glycerol-coated fruits [32].

3.3. Texture Profile Analysis (TPA) and Firmness

Coated and uncoated strawberries were analyzed to evaluate their texture profile.

Table 5. Texture profile analysis (TPA) of control and BW starch-coated fruits during storage at 4 ± 1 °C.

Treatments a	Days of Storage at 4 ± 1 °C
	0	4	7	10	14
Hardness (N)
T1	2.00 ± 0.67 a	1.45 ± 0.85 b	1.17 ± 0.60 b	1.16 ± 0.46 a	1.43 ± 0.35 b
T2	2.00 ± 0.67 a	2.24 ± 0.67 ab	2.27 ± 0.61 a	2.10 ± 0.48 a	1.90 ± 0.99 b
T3	2.00 ± 0.67 a	2.87 ± 1.14 a	2.52 ± 0.89 a	2.31 ± 1.68 a	3.26 ± 0.84 a
T4	2.00 ± 0.67 a	2.76 ± 0.80 a	2.20 ± 0.96 a	2.41 ± 0.74 a	1.72 ± 0.95 b
Springiness
T1	4.02 ± 0.98 a	3.07 ± 0.37 a	3.29 ± 0.48 a	3.83 ± 0.73 a	3.29 ± 0.48 a
T2	4.02 ± 0.98 a	3.59 ± 0.65 a	3.28 ± 0.34 a	3.56 ± 0.52 ab	3.08 ± 0.51 a
T3	4.02 ± 0.98 a	3.28 ± 0.66 a	3.22 ± 0.41 a	2.29 ± 1.18 b	2.50 ± 1.94 a
T4	4.02 ± 0.98 a	3.44 ± 0.83 a	3.52 ± 0.67 a	3.85 ± 0.65 a	3.71 ± 1.59 a
Chewiness (N)
T1	0.05 ± 0.02 a	0.07 ± 0.06 a	0.07 ± 0.03 a	0.13 ± 0.04 a	0.11 ± 0.07 a
T2	0.05 ± 0.02 a	0.07 ± 0.03 a	0.07 ± 0.06 a	0.09 ± 0.04 a	0.15 ± 0.09 a
T3	0.05 ± 0.02 a	0.11 ± 0.08 a	0.10 ± 0.11 a	0.12 ± 0.12 a	0.18 ± 0.20 a
T4	0.05 ± 0.02 a	0.08 ± 0.05 a	0.09 ± 0.14 a	0.14 ± 0.06 a	0.15 ± 0.11 a

T1—distilled water as control, T2—BW starch, T3—BW starch + 5% CA + 1% CNP, and T4—ultrasonicated BW starch + 5% CA + 1% CNP. Data (Mean ± SD) in the same column followed by different letters (a,b) are significantly (p < 0.05) different for the treatment factor. On d-0, treatments have equal means, since data were recorded for fresh, untreated strawberries before storage.

displays the hardness, springiness, and chewiness of the analyzed fruits on different sampling days. All BW starch-coated fruits, primarily T3, exhibited significantly (p < 0.05) higher hardness than their uncoated counterparts. Moreover, at the end of the storage duration, the hardness of T3 fruits was higher than all other BW starch-coated fruits. However, hardness did not significantly (p > 0.05) differ across storage durations, and there was no significant interaction between treatment and storage duration (Table 3). On the other hand, the springiness and chewiness of control and coated fruits did not differ significantly (p > 0.05). As shown in Table 3, independent variables and their interactions are not significant for springiness, whereas for chewiness, only the effect of storage duration was significant (p < 0.05).

The reduced hardness of control fruits may be attributed to accelerated water loss, ripening, and senescence [27,33]. The reduced moisture loss resulting from the barrier properties of BW starch could be attributed to the hardness of coated fruits [11]. Even though T3 fruits had more moisture loss than T2 fruits, T3 fruits possessed higher hardness. This could be due to reduced ripening and/or a thicker coating material. Hardness is the peak force of the first compression that indicates the strength of the structure [22]. The BW starch coating, especially T3, with added CA and CNP, might have formed a stronger and thicker layer on the strawberry surface, contributing to its hardness. Nonetheless, at the end of storage, T4 fruits’ reduced hardness compared to T2 may be attributed to increased water loss (Figure 2), resulting in reduced cell turgor. Chewiness is positively correlated with hardness and springiness and indicates the mastication energy required to process a solid food in the mouth [22]. Therefore, looking into the hardness and springiness data, the higher chewiness in the coated fruits, especially in T3 samples, may be primarily due to the higher hardness attributed to the coating material. The TPA results suggest that microbial spoilage and the atmospheric composition inside the packages may have affected the quality of the control fruits [34].

As shown in Figure 3, the internal/flesh firmness of BW starch-coated fruits was higher compared to the uncoated fruits, and generally, there was an increment in firmness throughout the storage period. Nevertheless, the firmness among the treatments did not vary significantly (p > 0.05) at the end of storage. As per two-way ANOVA (Table 3), only treatment had a significant (p < 0.05) effect on internal firmness. Storage duration and interaction effects were not significant (p > 0.05).

Firmness is one of the critical sensory attributes indicating crispiness and juiciness and influences consumer acceptance [27,35]. Water loss has been linked to loss in fruit texture and firmness [36]. Contrary to weight loss data, there was an increase in internal firmness. This observation can be explained in two ways: either excessive water loss from fruits might have led to dehydration-related hardness and stiffness due to the loss of cell turgor pressure [37] or delayed ripening due to the restricted gas exchange, slowing down the respiration rate and enzyme activity [27], especially in BW-starch-coated fruits. During ripening, pectin substances start to break down due to increased cellulase enzyme activity, leading to reduced firmness [38,39]. For a better understanding of the firmness data, future research should aim to elucidate pectin methylesterase and cellulase activity [40].

3.4. Chemical Changes

Table 6. Two-way analysis of variance (ANOVA) results of the pH, TSS, % TA, AA, TPC, and % DI.

		Df	SS	MS	F	Pr (>F)
pH (n = 3)	Trt.	3	0.06876	0.022922	3.0434	0.04118 *
	Day	4	0.62238	0.155595	20.6592	6.442 × 10−9 ***
	Trt × Day	10	0.12754	0.012754	1.6934	0.12039
	Residuals	36	0.27113	0.007531
TSS (n = 3)	Trt.	3	3.6384	1.2128	515.44	2.2 × 10−16 ***
	Day	4	17.1448	4.2862	1821.64	2.2 × 10−16 ***
	Trt × Day	9	11.4783	1.2754	542.03	2.2 × 10−16 ***
	Residuals	36	0.0800	0.0024
% TA (n = 3)	Trt	3	0.013972	0.004657	636.87	2.2 × 10−16 ***
	Day	4	0.156258	0.039065	5341.99	2.2 × 10−16 ***
	Trt × Day	9	0.080491	0.008943	1223.00	2.2 × 10−16 ***
	Residuals	36	0.000249	0.000007
AA (n = 3)	Trt	3	1.5336	0.51118	7.2086	0.0007515 ***
	Day	4	9.2857	2.32143	32.7362	4.62 × 10−11 ***
	Trt × Day	9	3.3691	0.37434	5.2788	0.0001806 ***
	Residuals	33	2.3401	0.07091
TPC (n = 3)	Trt	3	0.82126	0.273752	20.4468	9.447 × 10−8 ***
	Day	4	0.10462	0.026154	1.9535	0.124
	Trt × Day	9	1.14524	0.127249	9.5044	4.685 × 10−7 ***
	Residuals	34	0.45521	0.013388
% DI (n = 2)	Trt	3	322.4	107.45	2.0753	0.1439
	Day	3	5598.7	1866.23	36.0423	2.394 × 10−7 ***
	Trt × Day	9	218.0	24.23	0.4679	0.8754
	Residuals	16	828.5	51.78
% Weight loss_film package (n = 3)	Trt	3	1480.83	493.61	60.9564	1.230 × 10−11 ***
	Day	1	242.57	242.57	29.9558	1.101 × 10−5 ***
	Trt × Day	2	103.67	51.84	6.4014	0.005691 **
	Residuals	25	202.44	8.10

Statistical significance is denoted by asterisks: * (p < 0.05), ** (p < 0.01) and *** (p < 0.001).

shows that both the treatment and storage duration had significant effects (p < 0.05) on strawberry pH, TSS, TA, and AA. Moreover, the interaction of these two independent variables was found to be significant for all dependent variables, except pH changes. Figure 4 shows that there was a rapid increase in pH levels in all samples until day 10, followed by a sharp decline at the end of the storage, except for T2. The control sample exhibited the lowest pH (p < 0.05) at day 14, while the T2 samples were the most stable. However, pH values among the BW starch-coated samples were non-significant (p > 0.05). As far as the TA is concerned, there was a gradual increase in TA in the T2 and T4 samples during storage. On the other hand, the TA of the control and T3 samples fluctuated, with the control samples possessing the highest TA (p < 0.05) at the end of the storage. Like TA, the TSS of T2 and T4 samples increased during storage, while it was unstable for T1 and T3 samples. The TSS of the control samples was significantly (p < 0.05) lower after the 10th day of storage. Overall, the BW starch-coated samples, especially T2 and T4, had better control over the chemical changes in strawberries during storage. The highest level of AA was observed in T3 samples until day 10, which was followed by a reduction at the end of storage. However, ultrasound treatment appeared to have a negative influence on the AA of strawberries; T4 samples, which were the ultrasonicated counterparts of T3, possessed reduced AA compared to T3. All BW-starch-coated fruits followed a similar trend of reduced AA after 14 days. However, the control samples demonstrated a steady increment in AA throughout storage.

After harvest, organic acids are used up as substrates in the respiration and ripening process, resulting in the depletion of TA or an increment in pH. On the other hand, the conversion of sugar into acids could also decrease the pH [22,41]. BW starch coating reduces the respiration rate by creating a modified atmosphere around the fruit, slowing down the fruit’s aging and conserving TA during storage [42]. The reduced TSS might be attributed to senescence, leading to a reduction in TSS and conversion of soluble sugars into acid [43] in the control samples, which explains the remarkable drop in pH after 14 days of storage. The BW starch-based film solution (T2) might have provided the optimum barrier properties as a packaging material to restrict the ripening or senescence and conserve the TSS of strawberries. Further, the superior performance of T2 fruits might be attributed to reduced water loss (Figure 2), since excessive water loss has been linked to increased senescence and cell wall degradation by activating pectin-degrading enzymes [36]. CA and CNP naturally possess antioxidant activity [2,44], which might have contributed to the enhanced AA of the T3 samples. However, its ultrasonicated counterpart exhibited reduced AA, which was in line with recent work on stand-alone films and was attributed to the increased hydrophobicity of starch films due to ultrasound application [14] and a negative effect of ultrasound application on phytochemicals responsible for AA [17]. The hydrophobic nature of ultrasound-treated film solutions [7] might have reduced the solubility of polyphenols (with a substantial number of polar groups) responsible for the AA [45]. Further, a positive correlation between ultrasound energy density and AA has been reported for food samples [46]. It is likely that acoustic cavitation with a low energy density (0.08 kJ/cm³) might have affected the bioactive potential of starch and CA and CNP, and, hence, reduced AA [17]. AA of strawberries continues to increase during the ripening process, followed by a decline at the fully ripe stage [47]. Continued aging or senescence in control fruits might have contributed to their increased AA even after the end of storage.

3.5. Total Phenolic Content (TPC)

Changes in the TPC of strawberry fruits were observed during the storage period and are shown in Figure 5. TPC was significantly (p < 0.05) affected by treatment, but the storage duration effect was not significant (p > 0.05); however, their interaction was significant (p < 0.05) (Table 6). TPC in starch-coated fruits exhibited an increase in storage. On the other hand, control fruits had a significantly (p < 0.05) reduced TPC starting at day 10 and reached 0.44 mg GAE/g at the end of storage. Treated fruits did not significantly (p > 0.05) differ until day 10, and at the end of storage, T4 samples had a significantly (p < 0.05) higher TPC (1.3 mg of GAE/g) than T3 and control fruits.

As fresh fruits mature and ripen, they continue to synthesize phenolic compounds as secondary metabolites [48]. Similar to the present study, rice starch-coated plum fruits had higher phenolic content than uncoated fruits, which showed reduced phenolic content [48]. The decline in phenolic content was attributed to the breakdown of cell structural components at the verge of senescence and the activities of phenol oxidase and peroxidase during storage [48,49]. Retention of phenolic content in coated fruits may be due to the modified atmosphere created by the starch layer, slowing down the aging process.

3.6. Decay Index

As shown in Figure 6, during the first four days of storage, there were no signs of decay in any fruits. Fruits started to decay on day 7; however, the BW starch-coated strawberries exhibited reduced % DI compared to the control fruits in all storage conditions. For T2 and T4 samples, % DI was not significantly lower (p > 0.05) than the control. However, at the end of storage, T3 fruits had a significantly (p < 0.05) reduced % DI compared to the control. A 56% reduction in DI was observed in T3-coated fruits compared to the control. As shown in Table 6 (two-way ANOVA), treatment had no significant effect on % DI; however, the storage duration had a significant effect (p < 0.05). The interaction between treatment and storage duration had no significant effect (p > 0.05) on % DI.

The cleaning and sanitation process followed during the sample preparation step might have restricted the decay process in all samples during the first few days [18]. Reduced senescence in BW starch-coated fruits might have slowed down the decay process. Fruits and vegetables become susceptible to pathogenic organisms on the verge of senescence due to the loss of cellular structure and weakening of tissues [18]. The reduced % DI in T3-coated samples could be attributed to the antimicrobial activity of CA [50] and CNP [51].

3.7. Application as a Film Packaging Material

Figure 7a shows the physical appearance of the films as it relates to film transparency. Ultrasonicated film (T4) appears to be more transparent than other films. CA and CNP increased the film (T3) opacity. Figure 7b shows the appearance of strawberries on day 0 and day 3, which demonstrates clear signs of decay in every treatment except T3. Nevertheless, as shown in Figure 7c, exposed strawberries had significantly greater moisture loss than samples packaged with BW starch films. In fact, the packaged samples had >10% moisture loss after 3 days of storage. The moisture loss of strawberries with or without the BW starch film package was significantly (p < 0.05) affected by treatment, storage duration, and their interaction (Table 6).

The results indicate that BW starch packaging could be a viable option to restrict moisture loss from fresh produce. The signs of decay and mold could be due to the limited water vapor transfer through the BW starch film packaging, as observed in our previous study [14]. Moisture is one of the respiration products of fresh produce; trapped moisture creates a humid environment, which could lead to mold growth, especially for packaged samples. Unlike other samples, strawberry samples packaged with T3 films did not have noticeable mold growth, which can be attributed to the antimicrobial activity of CA and CNP. These data are also in agreement with what was observed and discussed regarding the % decay index (Section 3.6).

4. Conclusions

Results presented in this study demonstrated that buckwheat starch coating had a positive effect on preserving strawberry quality during 14 days of storage. However, the type of starch coating formulation played a role in preserving different fruit quality parameters. The coating with only BW starch better suppressed weight loss, which was 16% lower than the uncoated counterpart at the end of storage. On the other hand, coating treatment incorporating CA and CNP suffered from increased water loss attributed to film brittleness (observed in previous research) and osmotic loss due to thicker coating formulations. However, treatment with BW starch with CA and CNP improved textural properties, such as hardness and firmness. Moreover, strawberries treated with BW starch with CA and CNP exhibited a 56% reduction in the decay index. Starch coating, especially with BW starch only, restricted pH, TSS, and % TA changes during storage. Moreover, coating treatment helped maintain phenolic contents during storage. When applied as packaging materials, BW starch films effectively reduced moisture loss from strawberries, accounting for >10% after 3 days of storage. This study serves as a proof-of-concept for a previous study on BW starch films’ effectiveness as edible coatings for fresh produce. However, further studies are required to optimize film solutions for a wide range of applications and to understand the enzymatic mechanisms involved in the delay in the ripening process or firmness retention of strawberries coated with BW starch and additives, such as CA and CNP.