Characterization of banana powder-based film reinforced with CNF and Bio-mediated ZnO nanoparticles for potential food applications

The formulation of a multifunctional nanocomposite packaging material with potential against agents of food deterioration, such as free radicals and microorganisms, has emerged as a solution for shelf-life extension and food security. This study developed banana powder (BP) film infused with cellulose nanofiber (CNF) and ZnO-PPW and ZnO-PSW nanoparticles (NPs) at different concentrations for food applications. The BP/CNF/ZnO films were characterized using UV–vis spectroscopy, XRD, FT-IR, and SEM techniques. The analyses confirmed the successful infusion of ZnO NPs into the BP/CNF matrix, leading to significant changes (p < 0.05) in color and appearance, enhanced UV–vis barrier properties, and increased thickness and flexibility of the films. Furthermore, the presence of ZnO in the base matrix influenced the moisture content (19%–29%), film solubility (68%–74%), and oil permeability significantly more than the control BP/CNF film. Adding ZnO significantly improved the UV barrier properties compared to the control. The nanocomposite BP/CNF/ZnO films demonstrated concentration-based antioxidant and good antimicrobial activity against five selected food pathogens (Escherichia coli, Enterococcus faecalis, Listeria monocytogenes and Staphylococcus aureus). Similarly, good antioxidant properties were reported in different assays, proving superior to the control BP/CNF. These key findings, especially those of the BP/CNF/ + 0.6% ZnO NPs films, showed that these films possess great potential for application as food packaging materials with antioxidant and antimicrobial properties.


Introduction
Food packaging is crucial for food security, as it acts as a barrier against physical, chemical, and biological factors that can lead to spoilage and contamination [1].This protection preserves the quality and safety of food items, reducing the risk of foodborne illnesses and thereby contributing to overall food security [1,2].Traditionally, materials like plastic have been favored for food packaging due to their ease of processing, cost-efficiency, and convenience [1].However, the non-renewable, non-biodegradable, carcinogenic, and environmentally hazardous nature of these materials has raised significant global concerns [3,4].Consequently, there is a growing need for safer, natural, and more environmentally friendly materials with a better cost-benefit ratio, convenience and potential for shelf life extension [5].This shift has led to the adoption of biopolymer-based materials, which offer desirable properties that address concerns associated with traditional packaging materials.These properties include biodegradability, biocompatibility, optical clarity, mechanical strength, gas barrier capabilities, and specific biological functionalities [6].
Most biopolymer-based materials are derived from lipids, proteins or polysaccharides [7][8][9][10][11][12], and have been engineered to incorporate additional functional properties beyond basic packaging tasks, such as shelf-life extension [5].Technology advancements have introduced enhanced packaging features like moisture resistance, airtight seals, and temperature control, significantly reducing food losses, increasing availability, and mitigating scarcity [1].These improvements are achieved by incorporating additives with antimicrobial, antioxidant, or oxygen-scavenging properties into the biopolymer matrices, thus enhancing the quality and marketability of fresh produce or food products [5,13].One notable application of biopolymers in food preservation is their use in films, which act as effective carriers for active ingredients, strengthening their role in extending the shelf life of fresh produce or food products.
Interest in biodegradable films has grown due to the need for effective preservation and safety during food transportation [14].Additional factors include the demand for aesthetically pleasing and hygienic products.However, environmental concerns from the non-biodegradable nature of most of these materials continue to pose significant challenges [14].As a result, nature-based biopolymeric materials like cellulose acetate, gelatin, and chitosan have become valuable alternatives, offering benefits like edibility, non-toxicity, environmental sustainability, and biodegradability [14].Likewise, starch-based biopolymers from sources like bananas are widely utilized in nanocomposite film production for packaging due to their availability, affordability, biodegradability, and renewable nature [15].According to the literature, banana powder, a starch-based biopolymer, exhibits excellent film-forming and oxygen barrier capabilities [16,17].However, despite these advantages, banana powder's poor water barrier and mechanical properties and high solubility limit its use in food packaging [17].While certain materials can increase hydrophobicity when mixed with other biopolymers [18,19], the inherently hydrophilic nature of many biopolymer-based materials may not fully meet the necessary properties for direct-contact applications like food packaging and preservation.This situation could promote microbial growth, potentially leading to food spoilage and illnesses [20].Consequently, despite their numerous benefits, some starch, cellulose, or chitosan-based biopolymers remain prone to microbial growth because of their hydrophilic nature [20].Thus, particularly for applications involving wet produce and material contact, there is a need to develop materials with enhanced hydrophobic properties while retaining essential intelligent/active packaging features.
Furthermore, literature reports suggest that issues such as poor water barrier, mechanical properties and hydrophilicity can be addressed with natural fillers like cellulose nanofibers (CNF) [21].CNF is known for its inherent properties, including high strength, substantial water uptake capacity, low gas permeability, biofunctional attributes, and insolubility in common solvents [21,22].However, while the incorporation of CNF may improve various physicochemical properties, these films might still fail to protect against microbial infestation, a critical aspect of active food packaging design.
Metal-based nanoparticles have emerged as useful biological agents, offering antimicrobial inhibitions, radical scavenging and antiproliferative properties [1].ZnO nanoparticles, in particular, have gained significant attention due to their non-toxicity and classification by the Food and Drug Administration (FDA) as Generally Recognized as Safe (GRAS) [23].They offer multiple valuable properties for packaging materials, such as a large surface area, UV-blocking capabilities, and strong antimicrobial attributes [24,25].Another notable advantage of ZnO nanoparticles is their ease of synthesis from bioresources like plant extracts, making their production cost-effective and environmentally sustainable [1].The incorporation of ZnO as an additive into packaging materials has been reported to enhance packaging mechanical strength, water resistance, and UV barrier properties [26].Moreover, ZnO-CNF film composites are considered promising for food packaging due to their mechanical properties, including flexibility, UV light responsiveness, and antimicrobial activity [22,27,28].Therefore, ZnO and CNF are ideal for reinforcing films because of their crystallinity, amorphous structure, abundance, cost-effectiveness, and environmental sustainability [29].
Building on our prior work where ZnO nanoparticles (NPs) were synthesized from pomegranate peel and seed wastes [30], this study further explores the development of films derived from banana powder (BP), reinforced with cellulose nanofibers (CNF) and various concentrations of the biologically synthesized ZnO NPs.A range of characterization techniques was employed to elucidate the physicochemical properties of these composite films.Additionally, the efficacy of BP/CNF/ZnO film composites, with varying ZnO nanoparticle concentrations, as active packaging materials was demonstrated through antioxidant (DPPH, ABTS, and FRAP) and antimicrobial studies against selected foodborne pathogens.

Synthesis of ZnO
NPs from pomegranate wastes and the formulation of banana-powered nanocomposite films (BP/CNF/ZnO) The ZnO NPs were synthesized using a method reported by Leta et al [30].Extracts from the peels and seeds of pomegranate fruit wastes were used as mediating agents to react with Zn(NO 3 ) 2 •6H 2 O in an alkaline environment.Once a residue was obtained, it was washed, dried, and calcinated inside a furnace at 400 °C for 3 h.The resulting off-white powders were labelled ZnO-PPW and ZnO-PSW nanoparticles, corresponding to their origin from peel or seed extracts wastes.These powders were subsequently used in varying concentrations to fabricate the films.
The film formulation employed the casting method, as illustrated in figure 1.Initially, three grams of banana powder (BP) was dissolved in 100 ml of distilled water and heated at 50 °C for 30 min.Subsequently, a blend of 1 ml each of Tween 80 and canola oil was introduced to enhance the film's flexibility.Glycerol was then added as a plasticizer at 40% v/w of BP and the mixture was stirred for an additional 15 min.Afterwards, 1% CNF (w/v) was added, and the mixture was further heated and stirred for 20 min at 50 °C.Different concentrations of the previously synthesized ZnO NPs (0, 0.2, 0.4, and 0.6% relative to dry BP) were mixed for 30 min.The solutions were then homogenized and sonicated for 1 h to remove bubbles.Finally, 30 ml of the solution was dispensed into a Petri dish (85 mm), and the dishes were placed in the oven at 50 °C for 72 h.The composition of the prepared films and controls is summarized in table 1.

Colour
Film colour evaluation was carried out using a colorimeter (Konica Minolta Chroma Meter CR-400, Osaka, Japan), which had been previously calibrated with a standard calibration tile.The parameters L * (indicating lightness or brightness), a * (denoting red to green spectrum), and b * (representing yellow to blue spectrum) were measured in triplicates to assess the colour attributes of the film samples.The total colour difference, defined as ΔE, was calculated using the following equation (1): Where ΔL, Δa, and Δb represents the difference between each colour value of standard colour control film [31] 2.4.Moisture content (MC) MC was determined using a method described by Amjadi et al [3].Dried strips of the nanocomposite films were cut into 2 cm × 2 cm fragments in triplicates.The initial mass was recorded using a measuring scale (RADWAG Electronic PS 4500.R2.M, Poland, 0.01 accuracy).Samples were then dried in an oven at 100 ± 1 °C for 24 h.Afterwards, the final mass of each sample was recorded.MC was measured in triplicate using equation (2): and M 1 represent the initial mass before drying and the final mass of the film in grams after drying, respectively.

Water solubility (WS)
Water solubility was determined following the method described by Ahmadi et al [29], with slight modifications.Film sections were cut into three 2 cm × 2 cm pieces in triplicate and dried at 50 °C for 24 h.After drying, the samples' initial weights (Winitial) were measured using a measuring scale (RADWAG Electronic PS 4500.R2.M, Poland, 0.01 accuracy).These sections were then immersed in 30 ml of deionized water and left overnight at 50 °C with occasional stirring.After immersion, the samples were filtered using Whatman filter paper to collect the non-solubilized fragments.These fragments were then dried in an oven at 100 °C for 2 h, and their final weights were recorded.The WS percentage was calculated using equation (3): and W final represent the films' initial and final mass, respectively.Mean values were recorded in triplicates.

Water vapor permeability (WVP)
The WVP attributes of the films were investigated using a method outlined by Ghanbarzadeh et al [32], with slight modifications.Dry film sections were cut into 8 cm × 8 cm squares in triplicates and mounted onto measuring cups, each filled with 25 ml of deionized water.The cups were tightly sealed with parafilm wrap to create a watertight environment.With initial weights recorded using a measuring scale (RADWAG Electronic PS 4500.R2.M, Poland, 0.01 accuracy), the cups were stored in an oven at 50 °C and a relative humidity (RH) of 50%.Weight changes for each cup were measured at hourly intervals for 8 h.The Water Vapor Transmission Rate (WVTR) and WVP were calculated using equations (4) and (5): Where WVTR (g/mm hr) is described as the slope (g/h) and divided by the total surface area (mm 2 ); X (mm) represents the Thickness of the nanocomposite film; (Δp = p (R2 − R1), R2 = 50% and R1 = 0%).Analysis was carried out in triplicates.

Oil permeability (OP)
The oil permeability of the films was evaluated using a method adapted from Li et al [33], with slight modifications.Film samples were cut into 4 cm × 4 cm squares and carefully positioned on top of small polyethylene tubes, each filled with 5 ml of canola oil.The tubes were then inverted and sealed with pre-weighed Whatman filter paper using a measuring scale (RADWAG Electronic PS 4500.R2.M, Poland, 0.01 accuracy).The setup was then placed in an oven at 25 °C for 72 h.Afterwards, the weight of the Whatman filter paper was recorded to determine the amount of canola oil that had permeated through the film samples.The oil permeability of the films was calculated using equation (6): ΔM represents the change in mass (mg) of the Whiteman filter paper, X represents the Thickness of the film (mm), and A is ascribed to the surface area (mm 2 ) and t storage period time (hours).
2.8.Mechanical properties 2.8.1.Thickness of the films Film thickness was measured in triplicates using a digital micrometre (±0.001 mm accuracy).Measurements were conducted at five distinct, randomly selected points near the center of the film, including the midpoint.The average thickness of each film (n = 5) was recorded [3].

Tensile strength (TS)
Tensile strength of the film samples was assessed in triplicate using a texture analyzer (GÜSS-FTA, Strand, South Africa).The nanocomposite films were cut into 4 cm × 8 cm strips, followed by subjecting each strip to puncture using an 8 mm probe.The force at which each film exhibited maximum puncture resistance force was recorded.Tensile strength was calculated by dividing the puncture resistance force by the cross-sectional area of the film strips, according to equation (7) (Ghanbarzadeh et al [32]. Where F max represents the maximum puncture resistance force (N), L represents the length (cm), and W represents the width (cm) of film samples.

Light transmission and opacity
The optical barrier properties of the films were assessed using a UV spectrophotometer (Spectrum instruments, SP-UV 300, shanghai, China) over a wavelength range of 400-800 nm.For the testing, the nanocomposite films were cut into 1 cm × 3 cm strips, positioned perpendicularly in a plastic cuvette, and scanned against an empty cuvette as a reference to measure light transmittance through the samples, as described by Aloui et al [34].Opacity was calculated by dividing the absorbance at 600 nm by the thickness of the film strips, as outlined in equation (8): A 600 represents the absorbance measured at 600 nm, and X represents the thickness (mm) of the film samples.

X-ray diffraction (XRD)
The crystallinity of the nanomaterials in the film was assessed using an x-ray diffractometer system (PANalytical Empyrean, XPERT-PRO, USA) at room temperature with a scanning rate of 2 min-1 and 2θ ranging from 20°to 80°with Cu Kα radiation (λ= 1.5418 Å) scanning at room temperature (25 °C).For testing, the nanocomposite films were cut into circular shapes with a diameter of 3.5 cm, and each film sample was individually placed on the x-ray exposure stage (Spinner PW3064).The intensity of the diffracted x-rays from the film samples at various 2θ angles was plotted and indexed at the corresponding diffraction peaks, aligning with standard crystallographic planes [31].

Morphological analysis
The morphological surface of the film samples was examined using scanning electron microscopy (SEM) (Tescan Vega 3, Borno, Czech Republic).Each film was cut into small fragments, dried, and gold-coated.Images were then collected at three magnifications using VEGAS TESCAN in transmission mode [33].

Fourier transform infrared spectroscopy (FT-IR) analysis
The structural interactions among the banana powder, cellulose nanofiber, and the bio-synthesized ZnO nanoparticles were investigated using an FT-IR spectrophotometer (Thermo Scientific Smart iTR, (Attenuated Total Reflectance), Thermo Fisher Scientific Inc., USA), operating within a spectral range of 4000-400 cm −1 .Each strip was individually placed on the ray exposure stage.This analysis used two film samples, each of specific dimensions, [35].

Film antioxidant properties 2.13.1. DPPH radical scavenging assay
The radical scavenging capacity of the films against 2, 2-diphenyl-1-picrylhydrazyl (DPPH) was evaluated using a method by Ahmadi et al [29].Film fragments (50 mg) were immersed in 10 ml of distilled water in a test tube.This mixture was vigorously vortexed for 5 min, then centrifuged at 5000 rpm for another 5 min at 4 °C.The resultant supernatant (3 mL) was mixed with 1 ml of methanolic DPPH and incubated for 30 min in the dark at room temperature.Absorbance values were measured at 517 nm using a UV spectrophotometer (Spectrum instruments, SP-UV 300).The inhibition percentage of DPPH was quantified using equation (9):

ABTS + radical scavenging activity (RSA) assay
The ABTS+ radical scavenging activity of the films was evaluated using a method adapted from Medeiros Silva et al, with slight modifications [17].A reaction mixture of 7 mM ABTS and 2.45 mM potassium persulfate was prepared and left to react in the dark at room temperature for 12-16 h.The resulting mixture was diluted (1:60) with 96% absolute methanol to absorb 0.700 ± 0.02 at 734 nm.Film samples (50 mg) were immersed in 10 ml of distilled water, vortexed for 5 min, and centrifuged at 5000 rpm for 5 min at 4 °C.The supernatant (3 ml) was mixed with 1 ml of the methanolic ABTS solution and incubated for 30 min in the dark at room temperature.The absorbance was measured at 734 nm using a UV spectrophotometer (Spectrum instruments, SP-UV 300, shanghai, China).The radical scavenging activity was calculated using equation (10)

= -Ábs
ABTS is the absorbance of the solution containing methanol and ABTS, while Abs ABTS/sample is the absorbance of ABTS solution with film samples.

Ferric reducing antioxidant power (FRAP) assay
The Ferric Reducing Antioxidant Power (FRAP) of the films was assessed according to Rufino et al [36], with slight modifications.A fresh FRAP reagent was prepared by mixing 10 mM 2, 4, 6-tripyridyl-s-triazine (TPTZ) and 20 mM ferric chloride in 0.25 M acetate buffer, pH 3.6.in a 10:1:1 ratio.Film fragments (50 mg) were immersed in 10 ml of distilled water in a test tube.This mixture was vigorously vortexed for 5 min, then centrifuged at 5000 rpm for another 5 min at 4 °C.The resultant supernatant (90 μl) was combined with 270 μl of distilled water, followed by adding 2.7 ml of freshly prepared FRAP solution.Subsequently, this mixture was incubated in the dark at room temperature for 30 min.Absorbance values were measured at 517 nm using a UV spectrometer (Spectrum Instruments, SP-UV 300, shanghai, China).The results were expressed as Trolox (mM) equivalents per milligram weight for the film samples (mM TE/ mg films).

Antimicrobial activity of the nanocomposite films
Antimicrobial properties of the nanocomposite film samples were evaluated using the agar well disc diffusion method against selected foodborne pathogens (Gram-negative: Escherichia coli ATCC 25922; Gram-positive: Listeria monocytogenes ATCC 19114, Staphylococcus aureus ATCC 25923).In a sterile environment, film discs of 10 mm diameter were placed on the surface of Mueller Hinton agar plates inoculated with freshly cultured bacteria.Subsequently, the plates were incubated for 24 h at 37 °C.The zones of inhibition (ZOI) around the films were measured in duplicate [3].

Statistical analysis
Collected data were subjected to one-way analysis of variance (ANOVA) using STATISTICA software (STATISTICA 14.0, TIBCO, Tulsa, OK, United States) using Duncan's multiple range test to indicate the significant difference (p < 0.05).Graphical illustrations were constructed using Microsoft Excel (Microsoft cooperation v13.0,Washington, DC, USA).

Film characterization 3.1.1. Colour and visual appearance
The colour attributes and total colour difference (ΔE) of the nanocomposite films are detailed in table 2 presents, and figure 2 visually illustrates the distinct influence of ZnO NPs on their surface colour.The control film (BP/CNF) exhibited a high L * value (lightness level) of 80.92, which was significantly higher (p < 0.05) than the ZnO NPs-enriched films.Previous studies have reported lower L * values in banana peel flour and neat banana films, indicating reduced lightness [17,37].A trend of decreasing lightness with increasing ZnO NP concentration was observed, with the highest concentration of ZnO NPs (0.6%) resulting in the lowest L-values (ZnO-PSW = 64.70 and ZnO-PPW = 64.28).However, the b * value, which represents blue/yellow colouration, increased with higher ZnO NP content, attributable to the colour properties of the banana powder.The total colour difference (ΔE) between the BP/CNF films and those with added ZnO NPs was significant, with BP/ CNF + 0.6% ZnO-PSW showing the highest ΔE (20.49).A similar pattern has been observed in other studies [27,37] involving different nanoparticle additions, suggesting that higher nanoparticle concentrations enhance the photoprotective properties of the films [38].These findings are essential for sensory evaluation and consumer acceptance of these films [17].MC in packaging materials is crucial for preserving food quality by preventing undesirable moisture transfer that can promote microbial growth and food deterioration [39].With the incorporation of ZnO NPs, a significant (p < 0.05) increase in MC was observed (table 3).Notably, films enriched with 0.2% ZnO-PSW NPs had the highest MC (29.54%), indicating increased hydrophilicity at lower nanoparticle concentrations.However, an increase in the concentration of ZnO-PSW to 0.4% and 0.6% led to gradual reductions in MC, indicating a complex interaction between nanoparticle concentration and film hydrophilicity [4].These variations suggest that incorporating ZnO nanoparticles alters the hydrophilic properties of the films, likely due to the nanoparticles' influence on the structural arrangement within the film matrix [4].This structure adjustment can modify the film's ability to absorb and retain moisture.According to Roy et al 2021 [4], the observed decrease in MC at higher nanoparticle concentrations may be attributed to reduced hydrophilicity facilitated by the nanoparticles acting as a nanofiller within the matrix.Similar behaviors have been reported in other studies.For instance, gelatin-based films containing ZnO NPs [29], where the addition of nanoparticles influenced moisture content dynamics.Moreover, Zhang et al [16] reported higher MC in chitosan films incorporated with 4% banana peel extract compared to neat BP/CNF films, underlining the impact of biopolymer composition on moisture interaction.In packaging applications, a lower MC value generally indicates reduced hydrophilicity, which can be advantageous in reducing the risk of microbial growth and spoilage [27].

Water solubility (WS)
Soluble nanocomposite films are favored for fresh-cut food products due to their easy dissolution in water [40].
The water solubility for the prepared films ranged from 66.17 to 79.28%, as presented in table 3. Additionally, introducing biosynthesized ZnO NPs decreased the WS of the films in water.The BP/CNF films with 0.6% ZnO NPs significantly (p < 0.05) reduced the solubility from 79.28 to 66.17% (ZnO-PPW) and 79.28 to 67.60% (ZnO-PSW) compared to the control film (BP/CNF).Additionally, ZnO-PSW NPs significantly (p < 0.05) reduced WS more effectively than ZnO-PSW.Furthermore, ZnO-PSW NPs significantly (p < 0.05) reduced WS better than ZnO-PSW.Banana powder is highly soluble; therefore, incorporating nanofillers such as cellulose nanofiber or glycerol improves tensile strength and reduces solubility and moisture resistance.This reduction is likely due to significant interactions between these additives and the polymeric chains of the biopolymer, which diminish the biopolymer's affinity for water molecules [29,41,42].This finding is similar to a report that showed a decrease from 47.8% to 37.4% in water solubility properties of gelatin plus cellulose nanofiber films upon adding ZnO NPs [27].Films with low water solubility and moisture content are therefore suitable for packaging food items sensitive to moisture [38].

Water vapour permeability (WVP)
WVP is crucial for examining the movement of water vapor between packaging material and the atmosphere, representing a key attribute of food packaging materials [43].Nanocomposite films with low WVP can minimize the amount of water reaching the food, thereby decreasing water loss and extending the food's shelf life [44].For most formulations, the incorporation of ZnO NPs led to an increase in WVP values; however, a notable decrease was observed at the highest nanoparticle concentration (0.6% NPs).The incorporation of ZnO NPs probably created a complex network within the polymer matrix, potentially forming barriers that alter the pathway of water vapor molecules and increase the overall water vapor barrier properties of the BP/CNF films [45].
However, this phenomenon is true at higher nanoparticle concentrations, regardless of the source (PPW or PSW), where the nanoparticle aggregation led to increased matrix density and reduced porosity, significantly impacting the WVP.Similar effects have been reported in the literature, such as in rice starch-gelatin films and other biopolymer matrices, where the presence of ZnO NPs altered the structural dynamics of the films [46].These findings suggest the importance of optimizing nanoparticle content to balance the mechanical properties and barrier functions of biopolymer-based packaging films [47].

Oil permeability (OP)
OP is a crucial measure for evaluating the efficacy of packaging films in preventing the transmission of oil-based substances, which can affect the freshness and integrity of packaged food [43].This study assessed the OP of BP/ CNF films with ZnO NPs, as detailed in table 3. The control film (BP/CNF) exhibited an OP of 11.69 ± 0.02 mg.mm/m 2 .day(table 3); however, the incorporation of 0.2% ZnO-PPW resulted in increased and highest permeability of 13.11 ± 0.03 mg.mm/m 2 .dayfor BP/CNF + 0.2% ZnO-PPW and 12.18 ± 0.04 mg.mm/m 2 .dayfor BP/CNF + 0.2% ZnO-PSW (table 3).Conversely, increasing ZnO NP concentrations to 0.6% ZnO-PPW and 0.6% ZnO-PSW in BP/CNF films led to a significant (p <0.05) reduction in OP to 9.44 ± 0.01 and 10.56 ± 0.02 mg.mm/m 2 .day,respectively.This trend suggests that higher concentrations of ZnO NPs enhance the films' barrier properties by increasing their compactness and structural integrity [46].These findings support the hypothesis that ZnO nanoparticles function as crosslinkers within the nanocomposite matrix, contributing to a denser, less permeable film structure.The increased hydrophobicity associated with the nanoparticle addition also likely plays a crucial role in reducing oil permeability [43].This enhancement in barrier properties is consistent with the observed reduction in oil transmission rates, particularly at higher nanoparticle concentrations.These results indicate that optimal incorporation of ZnO NPs into BP/CNF films can significantly improve their oil barrier properties, making them more suitable for packaging applications where oil resistance is necessary.thickest films (89.33 μm) being BP/CNF + 0.6% ZnO-PSW, while the thinnest was BP/CNF at 47.66 μm.This increase in thickness may be attributed to the surface roughness evident in SEM images (figure 4) and the higher solids content due to the ZnO NPs [48].Moreover, BP/CNF + ZnO-PSW nanocomposite films were denser than BP/CNF + ZnO-PPW films across all tested ZnO NP concentrations (0.2%, 0.4%, and 0.6%).The average thickness of the prepared nanocomposite films was similar to the observation of Arquelau et al [49] for banana peel flour films (0.059 mm to 0.085 mm).

Thickness and tensile strength (TS) of nanocomposite films
Ensuring sufficient mechanical strength in nanocomposite films is crucial for protecting food during transportation and sale, with this strength quantified as tensile strength (TS) [40].Upon the addition of ZnO NPs, TS values decreased from 15.63 to 13.31 MPa (table 4), suggesting that while ZnO NPs can enhance certain physical properties like barrier functions and thickness, they may adversely affect the film's tensile strength by disrupting polymeric matrix.This reduction could result from the degradation of structural integrity due to the uneven distribution of ZnO NPs, which create stress concentration points and diminish cross-linking within the matrix [50][51][52].Such dynamics contrast with observations in other studies, such as those involving Gelatin/ CNF films, where TS was proportionally increased with higher ZnO NP concentrations [29] Understanding these dynamics is crucial as they influence the film's flexibility and overall structural integrity, which are essential factors in maintaining the protective quality of the packaging throughout production and transit processes.These insights are critical for advancing the design of nanocomposite films to optimize their protective properties and mechanical integrity in real-world applications [29].

Transmittance
Transparency is crucial as it influences the external appearance and quality of the perceived packaged fresh produce [29].The light transmission of the banana powder-based nanocomposite films was assessed through a UV-vis spectrophotometer across the 400-800 nm wavelength range (table 5).Light transmittance decreased with increasing concentrations of ZnO NPs due to their absorption of visible light and the consequent reduction in photon-free path [29].A higher transmittance value signifies greater transparency, allowing more visible light (at 800 nm) to pass through the film.The light transmission of the control (BP/CNF) composite film ranged from 10.65% to 18.76%, as shown in table 5. Light transmission of BP/CNF/ZnO-PPW and BP/CNF/ZnO-PSW nanocomposite films ranged from 0.00%-6.86%and 0.00%-13.23%,respectively, across the NP concentrations.These findings indicate that incorporating higher concentrations of ZnO NPs into BP/CNF films significantly (p < 0.05) reduced UV-vis transmission, enhancing the film's screening ability due to light scattering by the nanomaterials embedded within the film matrix [46].This is consistent with [53], where increasing ZnO NP concentrations (0.2%, 0.4%, and 0.8%) in PVA films decreased UV absorbance.Additionally, the observed light transmittance result in our study agrees with the other literature report on films in which ZnO NPs were incorporated into the base coating, such as CNF/grape seed extract [4], gelatin [54], Carrageenan-based [55] and gelatin/cellulose nanofiber films [29].

Opacity
Opacity was measured at 600 nm using a UV-vis spectrophotometer.BP/CNF composite film exhibited the lowest opacity at 12.43%, significantly (p < 0.05) lower than those embedded with nanomaterials.However, the highest opacity was recorded for BP/CNF + 0.4% ZnO-PSW NPs at 20.61%.Significant differences (p < 0.05) in opacity were observed between BP/CNF + ZnO-PPW and BP/CNF + ZnO-PSW films at all concentrations, with BP/CNF + ZnO-PSW consistently exhibiting higher opacity.Lower opacity may result from the absence of a creamy solution during the addition of synthesized nanomaterials to the banana powder solution in film formation, potentially reducing light transmittance [29].Similarly, the addition of nano ZnO increased the opacity in konjac glucomannan/chitosan [56], chitosan [57], and gelatin/cellulose nanofiber matrices [29].The suitability of nanocomposite films for food packaging is attributed to their UV-screening capabilities.This suggests that ZnO NPs play a critical role in blocking UV radiation from penetrating the films, considering UV's potential as a damaging factor [4,53].

Microstructure of the nanocomposite films
The SEM micrographs, which illustrate the microstructural characteristics of the banana powder-based nanocomposite films, are displayed in figure 4. The control film exhibited marked homogeneity, characterized by a smooth surface with minimal or no visible cracking.Conversely, films containing various concentrations of ZnO NPs from PPW and PSW demonstrated surface roughness (marked by a yellow arrow), wrinkle formations (highlighted with a red circle), irregularities, and agglomerations, which increased with the nanoparticle concentration.The roughness and wrinkle formations were more pronounced in films containing ZnO-PPW than those with ZnO-PSW.This influence is reflected in the thickness data presented in table 4. Notably, the observed agglomeration is a commonly reported phenomenon in previous studies [58].Additionally, the images show that a significant portion of ZnO-PSW was integrated into the banana powder matrix, particularly at the 0.2% and 0.4% concentrations, as evidenced by minimal agglomerations.This is consistent with literature based on including ZnO NPs in gelatin + cellulose nanofiber matrices [29].Additionally, gelatin-based films incorporating ZnO NPs have exhibited rough surfaces, similar to those reported in our study, as noted by Shankar et al, [54].Moreover, ZnO NPs have been shown to enhance the compactness and thickness of gelatin films by reducing pores and cracks, indicating a strong affinity between gelatin and ZnO NPs [3].Blending 4% banana peel extract with a polymer like chitosan has been shown to create a more uniform film and form small white spots similar to those seen in the BP/CNF film [16].
3.  1451.These results confirm that ZnO nanoparticles were successfully integrated and maintained their crystalline structure within the BP/CNF matrix, as evident from the identified peaks [59].As expected, no peaks corresponding to the diffraction pattern of ZnO were detected in the BP/CNF films.These findings are consistent with reports on the formulation of gelatin-based films embedded with ZnO NPs [31].
3.7.FTIR analysis of the films FTIR spectroscopy was employed to assess the interactions between banana powder, cellulose nanofiber, and ZnO nanoparticles, as depicted in figure 6.A consistent spectral pattern was observed across most samples, except for the BP/CNF and the BP/CNF + 0.2% ZnO-PPW films.Prominent bands observed between 3700 and 3100 cm −1 could be attributed to hydroxyl groups linked to hydrogen bonds of water and phenolic compounds from banana powder and CNF [29].The band around 1600 cm −1 corresponds to the amide I of the proteins, predominantly involving C=O stretching of carbonyl groups or conjugated C-C groups of phenolic compounds in the base structure [17].The peak around 1550 cm −1 is ascribed to N-H bending and stretching of amide II, and the peak at 1450 cm −1 to the bending vibration of the hydroxyl groups [29].The minor peak at 1390 cm −1 corresponds to the amide III of vibrations of the C-N groups [17].The band around 1050 cm −1 resulted from interactions involving the hydroxyl (O-H) groups of glycerol and banana powder [54].Peaks in the 1300 to 900 cm −1 range indicate C-C, C-O, and C-OH stretching within the banana powder [17].Minor peak formation occurred by adding bio-mediated ZnO nanoparticles (figure 4(b)) to the film samples, but no peak position shift was detected [27].Overall, the obtained spectra reveal that the neat BP/CNF film spectra are nearly identical to those of various BP/CNF/ZnO films.The addition of ZnO nanoparticles does not seem to alter the band positions.However, subtle differences were noted with the addition of ZnO-PPW and ZnO-PSW, which became more marked as ZnO concentrations increased.A notable observation was the diminishing vibration band linked with the out-of-plane C-O bending, which reduced with increasing ZnO concentration, suggesting a possible interaction between the BP and ZnO NPs.

Antioxidant capacity of the films
The antioxidant activity of nanocomposite films is expected to protect oxidation-prone fresh produce, thus extending their shelf life [4].In this study, the antioxidant capacity of banana-based films was assessed using radical scavenging assays (RSA) of ABTS+ and DPPH, as well as Ferric-reducing antioxidant power (FRAP) (figures 7(A)-(C)).The control films (BP/CNF) exhibited the lowest antioxidant properties across all assays.Specifically, films with 0.6% ZnO NPS, regardless of the source (PPW or PSW), showed a higher percentage inhibition than the control in ABTS + and DPPH.The antioxidant activity of films ranged from 65.19 to 76.14% for ABTS + (figures 7(A)) and 58.69 to 75.60% for DPPH (figure 7(B)), with the antioxidant activity of films with ZnO NPs being significantly higher (p < 0.05) than in BP/CNF films.This study also showed that stabilization of unstable free radicals in ABTS assay occurs more rapidly and extensively than in DPPH assay, a trend also noted in films made from cellulose nanofiber, grape seed extract, chitosan, and zinc oxide nanoparticles [4].These results agree with previous studies where a scavenging ability of 69.82% was observed for chitosan-ZnOantioxidant bamboo leaves (CS/ZnO/AOB I) films in DPPH assays [57].Similarly, the same study reported scavenging activities of about 87.93% in CS/ZnO/AOB III films in the ABT+ assay.Similar to this report, a study on gelatin/CNF/ZnO/Selenium films demonstrated that these materials exhibit superior radical scavenging properties compared to neat films lacking nanomaterials [29].Consistent with the aforementioned findings, the addition of ZnO nanoparticles resulted in a concentration-dependent increase in antioxidant activity.Furthermore, as depicted in figure 7(C), the FRAP values indicated that increasing concentrations of ZnO NPs enhanced the ferric-reducing antioxidant power, with 0.6% ZnO-PPW achieving the highest FRAP at 2.35 mM TE/ml DM.The data showed that all films containing ZnO nanoparticles exhibited enhanced antioxidant activities, ranging from 2.05 to 2.35 mM TE/ml DM, compared to the control films, which showed 2.00 mM TE/ml DM.Similarly, films made from banana peel and 2% loquat leaf extract demonstrated outstanding antioxidant activity, as measured by FRAP [17].Medeiros et al [17] also noted that the high antioxidant activity is attributable to the phenolic compounds present in banana powder.
3.9.Antimicrobial activity study of film samples Microbial activities significantly contribute to food deterioration and spoilage.Therefore, it is crucial to develop packaging materials that can inhibit or halt these detrimental activities to ensure food safety and prolong storage.Film samples with increasing concentrations of bio-mediated ZnO nanoparticles were tested for their antimicrobial efficacy against common foodborne pathogens, including Gram-negative E. coli and Grampositive E. faecalis, L. monocytogenes, and S. aureus, using the disk diffusion method.The zones of inhibition (ZOI) against the microbes are listed in table 6 and representative images of the plates are presented in figure 8 for visual illustration.The BP/CNF films showed no antimicrobial activity against any of the selected microbes except E. faecalis (figure 8).Banana powder is reported to possess antimicrobial properties against L. monocytogenes and E. coli [60].However, the BP/CNF + 0.6% ZnO-PPW film exhibited the highest zones of inhibition against all tested Gram-negative microbes, with approximately 30.62, 15.06, and 10.7 mm, respectively.The susceptibility pattern of the bacteria to the films was in the order of E. faecalis (G +ve) > L. monocytogenes (Gr+ve) > S. aureus (G+ve) > E. coli (G−ve).Furthermore, consistent with other studies, these films demonstrated greater antimicrobial efficacy against Gram-positive bacteria than Gram-negative ones [29].This effect is attributed to the carotenoid content in Gram-positive S. aureus, which enhances antioxidant protection against the oxidative stress induced by nanoparticles [29].Other studies have similarly reported the impact of incorporating ZnO into films for food packaging, showing that ZnO-enriched films significantly reduce cell viability in Gram-negative E. coli and Gram-positive L. monocytogenes [52].Average values and their respective ± standard deviation (n = 3).Numerical data with distinct letters indicate significant differences in their respective column conducted using the Duncan's multiple range test (p < 0.05).BP: Banana powder.CNF: Cellulose Nanofiber.ZnO: Zinc Oxide.PPW: Pomegranate peel waste.PSW: Pomegranate seed waste.The species ATCC: American Type Culture Collection is in a bracket.

Conclusion
Incorporating biosynthesized ZnO nanoparticles significantly impacted the properties of the formulated nanocomposite films.X-ray diffraction patterns confirmed the presence of biosynthesized ZnO nanoparticles within the film matrix, contributing to surface roughness as observed in SEM morphological studies.Spectroscopic analysis identified key functional groups and metal oxides in the nanocomposite films.The addition of varying concentrations of ZnO nanoparticles significantly enhanced the film's water barrier, mechanical, and UV-vis barrier properties.Films functionalized with ZnO nanoparticles exhibited improved UV barrier potential, demonstrating their suitability as packaging materials over control films.A concentrationdependent increase in antioxidant activity was noted for ZnO nanoparticles synthesized from PPW and PSW.Consequently, films containing 0.6% ZnO nanoparticles showed higher inhibition percentages than the control in both ABTS+ and DPPH assays and exhibited superior FRAP activity compared to the control films (BP/ CNF).Similarly, the films containing 0.6% ZnO NPs exhibited the most effective antimicrobial activities against all tested food pathogens, highlighting their promise as packaging materials for fresh produce.

Figure 1 .
Figure 1.Graphical illustration for formulation process of the active nanocomposite films of BP/CNF/ZnO using the casting method.
is the absorbance of the Methanolic DPPH solution, while Abs DPPH/sample is the absorbance of the reaction mixture (methanolic DPPH solution with the film samples).

Figure 2 .
Figure 2. Appearance of banana powder films reinforced with cellulose nanofiber and varying concentrations of ZnO NPs.ZnO: Zinc Oxide.PPW: Pomegranate peel waste.PSW: pomegranate seed waste.

Figure 3
illustrates the flexibility and physical integrity of the developed nanocomposite films.The mechanical properties of the films, as shown in table 4, indicate that film thickness ranged from 47.66 to 87.00 μm.The incorporation of the ZnO NPs significantly (p < 0.05) increased the thickness of nanocomposite films, with the

Table 1 .
Composition of banana powder films reinforced with cellulose nanofiber and varying concentrations of ZnO NPs.

Table 2 .
Colour values and total colour difference (ΔE) of banana powder films reinforced with cellulose nanofiber and varying concentrations of ZnO NPs.

Table 3 .
Moisture content (MC), water solubility (WS), water vapour permeability (WVP) and oil permeability (OP) of banana powder films reinforced with cellulose nanofiber and varying concentrations of ZnO NPs.

Table 4 .
Thickness and tensile strength (TS) of banana powder films reinforced with cellulose nanofiber and varying concentrations of ZnO NPs.Average values and their respective ± standard deviation (n = 3).Numerical data with distinct letters indicate significant differences in their respective column conducted using Duncan's multiple range test (p < 0.05).BP: Banana powder.CNF: Cellulose Nanofiber.ZnO: Zinc Oxide.PPW: Pomegranate peel waste.PSW: Pomegranate seed waste.

Table 5 .
Light transmittance (%) and opacity (600 nm) of BP/CNF film incorporated with different concentrations of ZnO NPs.
bAverage values and their respective ± standard deviation (n = 3).Numerical data with distinct letters indicate significant difference in their respective column conducted using the Duncan's multiple range test (p < 0.05).BP: Banana powder.CNF: Cellulose Nanofiber.ZnO: Zinc Oxide.PPW: Pomegranate peel waste.PSW: Pomegranate seed waste.