Valorisation of pomegranate processing waste for the synthesis of ZnO nanoparticles: antioxidant and antimicrobial properties against food pathogens

The sustainable management of food waste is a pressing concern, with fruit waste valorisation emerging as a viable strategy to address this challenge. This study investigated the potential of pomegranate peel waste (PPW) and pomegranate seed waste (PSW) as mediating agents for the biosynthesis of zinc oxide (ZnO) nanoparticles (NPs); ZnO-PPW and ZnO-PSW, respectively, for potential utilization as additives in various polymer matrices for food packaging materials. The resulting physicochemical characteristics were ascertained using Ultraviolet visible spectroscopy (UV–vis), x-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), x-ray diffraction (XRD), Scanning electron microscope (SEM) and Energy Dispersive x-Ray Analysis (EDX). The obtained indexed diffractogram from the XRD analysis for both ZnO-PPW and ZnO-PSW confirmed the wurtzite crystalline structure of ZnO NPs. The observed morphology from the TEM and SEM analysis showed a similar spherical shaped structure, with agglomerations. However, ZnO-PSW, had a smaller size (58 nm) in comparison to ZnO-PPW (59 nm). Total phenolic content (TPC) for ZnO-PPW and ZnO-PSW ranged from 16.87–54.4 μg GAE/g DM, respectively. Also, the estimated minimum inhibitory concentration at 50% (IC50) for both DPPH and ABTS are 2.97 and 2.57 mg ml−1 for ZnO-PPW; and 3.43 and 3.33 mg ml−1 for ZnO-PSW, respectively. Moreover, due to its smaller size, ZnO-PSW demonstrated superior antimicrobial activity against five foodborne microorganisms. These findings suggest that pomegranate waste derived ZnO NPs could be beneficial for developing active food packaging materials.


Introduction
In recent decades, the demand for food has witnessed a significant increase, driven by various factors such as population growth and improved economic status [1].This has prompted the food industry to evolve, becoming increasingly mechanized to meet the market demand for 'Ready-to-use' and minimally processed food products, spanning fresh, dried, frozen, and canned varieties [2,3].However, the quest to maximize food production has significantly increased the generation of food waste (FW) [4].Current estimates suggest that approximately 1.3 billion tonnes of FW are generated worldwide per year, a figure projected to double to 2.6 billion tonnes by 2025 [5], with households responsible for 42% of this waste, while industries that manufacture food and provide food services contribute 39% and 14%, respectively [6].A large volume of FWs that are not utilized (i.e., for animal feed) are mostly thrown into municipal landfills where they generate greenhouse gas (GHG) and methane that may result in severe environmental impact [7].Among these FWs is pomegranate (Punica granatum L.), which is considered one of the most valuable edible fruits of the Punicaceae family [8].
Punica granatum L. fruit extracts have an array of bioactive compounds such as anthocyanin, flavanols, and hydrolysable tannins, which include pedunculagin, punicalin, ellagitannins, gallotannins, organic acids and phenolic acids and found in the fruit peel, seed and aril juice [8][9][10][11].Furthermore, the health benefits of these compounds have contributed to the surge in global demand for pomegranate fruit and products [9].In 2015, the production of pomegranate fruit in South Africa witnessed a tremendous growth of about 56%, while the total exports grew by 31% [11].The juice industry uses the edible pulp surrounding the seeds and considers the byproducts, mainly the rind and seed, as processing waste.These by-products constitute approximately 54% of the fruit's total weight [12].Many studies have shown that the fruit peel and seeds possess vital nutrients and phytochemicals [13] such as amino acids, alkaloids, enzymes, phenolics, proteins, polysaccharides, terpenoids, and vitamins which can be found in the aqueous extracts of various parts of the plant (fruit, leaves, peel, roots, seeds, stem and flowers) which suggest their potentials as mediating agent in the green biosynthesis of nanomaterials [14][15][16][17].
As an alternative to chemical synthesis, plant-based biosynthesis of metal and metal oxide nanomaterials has gained popularity and other approaches due to the 'greenness' of the process, its simplicity, low cost, speed, and the rich biodiversity of the plant kingdom with different varieties of phytochemical compounds [13].Although this approach has received considerable attention in recent years, with different propositions on the mechanism of synthesis, due to the diverse nature of phytochemical compounds, the exact mechanism behind the biosynthesis remains unclear [18].Nevertheless, numerous phenolic compounds have been linked to the bioreduction of metal salt ions into 0 valence metal nanomaterials [19].Thus, these bioactive compounds are fundamental reducing and capping agents [5,20].Therefore, since many food wastes contain a significant number of bioactive compounds, they have been employed in the bio-fabrication of metal and metal oxide nanomaterials such as Ag, Au, CuO, MgO, and ZnO due to safe handling and availability [16,21].For instance, the synthesis of super paramagnetic iron-oxide nanoparticles (SPION) through microwave-assisted commercial chemical processes costs €130 per 10 g of SPION [22].In contrast, producing iron NPs through green synthesis using plant extracts costs around €0.5-10 per gram, depending on the plant species and purity [22].Additionally, the energy consumption for the chemical synthesis approach (54.99 KW h −1 ) was notably higher than that for green synthesis techniques (52.88 KW h −1 ) [13].
Amongst the class of nanomaterials that have been extensively studied, ZnO remains at the forefront due to its safety profile, which has led to its diverse application in various areas such as medicine, food, cosmetics, water purification, and agriculture [23].It is a semiconducting nanomaterial with a band gap of around 3.1-3.3eV [24].It also possesses a binding energy of 60 meV [21], low electron conductivity and excellent heat resistance [24,25].Due to being a multifunctional compound, ZnO NPs have been utilized in many industries as antimicrobial [26,27], anticancer [28], antiviral [29], and antioxidant [30] agents.Furthermore, the Food and Drug Administration (21CFR182.8991) in the United States has declared ZnO to be generally recognized as safe (GRAS) when used in food products and dietary supplements in accordance with the approved guidelines [31].Recent studies have synthesized ZnO nanomaterials from various plant extracts such as Acacia caesia [32], Hibiscus sabdariffa [16], Mentha pulegium L. [33], Thymbra Spicata L. [34], and Punica granatum L. (pomegranate) [35].Specifically, for pomegranates, It has been established in literature that the concentration of phenolic, flavonoid, anthocyanin and hydrolysable tannins compounds present in pomegranate peels from different regions varies based on the cultivar, maturity of the fruit, extraction processes and storage conditions [8,11].These pomegranates peel from different regions, thus influencing the physicochemical properties such as crystallographic structure, morphology, size, purity, and quantity of the resulting nanoparticles.For instance, in a report by Alnehia et al [36], pomegranate fruit peels in the summer of 2021 were obtained from Saadah farms in Yemen after two weeks of harvest and used for the bio-mediated synthesis of ZnO nanoparticles.At different concentrations of the peel extracts, varying sizes between 18-30 nm were observed, which increases as the plant extract increases in each case.In a similar report according to Shaban et al [37], a size range of 20-40 nm was obtained for ZnO NPs mediated using pomegranate peel extracts from Egypt at almost similar conditions, thus indicating a variation emerging from protocols and possibly, origin, cultivars, or maturity.These variations are significant because, according to several reports in literature, physicochemical properties such as size play a role in the resulting antimicrobial properties observed [32,38].Moreover, Zinc oxide nanomaterials have been reported to inhibit different Gram-positive and Gram-negative microorganisms, including foodborne pathogens, which are the cause of many foodborne illnesses [39], by either impeding bacterial growth or killing them [36,37,40,41].According to the World Health Organization (WHO), contaminated food causes approximately 600 million cases of foodborne illness and 420,000 deaths caused by microbes [15].Hence, due to their already established antioxidant and antimicrobial properties and safety profile, they are now being embedded into the fibre matrices of food packaging materials as active agents for food preservation [42].
Therefore, this study sought to harness the beneficial characteristics of ZnO nanoparticles and the abundant availability of pomegranate waste in South Africa, particularly fruit peel and seeds.It focused on using these waste materials as mediators to fabricate ZnO nanomaterials.The study assessed the properties of the biosynthesized ZnO-PPW and ZnO-PSW nanoparticles through various characterisation methods.Additionally, it investigated the potential of these ZnO nanomaterials for creating active food packaging by conducting biological screening using assays such as DPPH, ABTS, and antimicrobial studies.Notably, this research marks the first evaluation of ZnO-PPW and ZnO-PSW nanoparticles against foodborne pathogens.

Collection of pomegranate fruits and chemicals
The individual processed Punica granatum L. fruit peel and seeds waste was collected from Ubali Pomegranate Farm, Kameelfontein, Johannesburg, in the 2022 season.The pomegranate fruit waste was transported to the postharvest and agro-processing research centre at the University of Johannesburg.All the reagents used in this study were purchased from Sigma-Aldrich, South Africa and used without further purifications.These include chemicals such as zinc nitrate hexahydrate utilized as a precursor for the biosynthesis of ZnO nanoparticles, 2,2azino-bis [3-ethylbenzothiazoline-6-sulphonic acid] ABTS+, hydrochloric acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin-Ciocalteu's phenol reagent, ferric chloride, 6-hydroxy-2,5,7,8-tetramethyl-chroman-2carboxylic acid (Trolox), ascorbic acid (AA), gallic acid, methanol (absolute), Mueller-Hinton (MH) agar, sodium carbonate, sodium acetate.The Clinical isolates of bacterial strains (table 1) were obtained from the Department of Food Science, University of Stellenbosch, South Africa.These strains were maintained on nutrient agar at 4 °C and stored for 24 h.The saturated cultures were diluted and adjusted by adding MH Broth until an absorbance reading (at 600 nm) of 0.400-0.600abs was obtained (Eloff, 1998).The following bacterial species and strain numbers used in this study are listed in table 1.

Extraction of peel and seed waste of pomegranate fruit
The collected pomegranate fruit waste was cleaned with deionized water and then disinfected with infected sodium hypochlorite (0.01% NaClO).The fruit peel or seeds were dried at 50 °C for 3 days in an oven and ground to powder form.Then, peel or seed ground powder (20 g) was boiled with 200 ml of distilled water at 70 °C for 10 min.The resulting mixture was filtered using Whatman no.1 filter paper for further use.

Biosynthesis of ZnO nanomaterials using peel and seed extracts of pomegranate fruit
The biosynthesis of ZnO nanomaterials followed a reported procedure by [43] with slight modification.In this procedure, 100 ml of 1 mM Zn(NO 3 ) 2 •6H 2 O was added to 200 ml of the respective fruit peel or seed extracts at an alkaline condition.Subsequently, the solution was heated to 70 °C and stirred for 2 h until a white-yellow mixture ensued.The mixture was centrifuged at 8000 rpm for 15 min, and the sedimented particles were washed with distilled water and 100% methanol.The paste collected was transferred into a crucible and oven dried at 50 °C for 12 h.After the residue had been adjudged dry, it was then calcined in a furnace for 3 h at 400 °C.The resulting off-white powder from peel or seed extracts was assigned ZnO-PPW and ZnO-PSW nanoparticles, respectively.

Structural and morphological of biosynthesized nanomaterials
The crystallinity of the biosynthesized nanomaterials was characterized using x-ray diffractometer system XPERT-PRO at room temperature with a scanning rate of 2°min −1 and 2θ ranged from 4 to 90°with Cu Kα radiation (λ = 1.5418Å).The functional groups present in the pomegranate waste extracts that served as reducing and stabilizing agents were studied using FTIR spectrophotometer (FT/R-4100typeA) with a spectrum range of 4000-400 cm −1 .Moreover, the optical properties of prepared nanomaterials were carried out using UV-visible spectrophotometer (UV-vis spec.2450, Shimadzu) analysis with a range of 300 to 800 nm [16].Information regarding the structure, size and shape of the biosynthesized nanomaterials was carried out using TEM (JEM-2100 electron microscope).The surface morphology, microstructure and elemental composition based on the energy released by the bombardment of x-rays of the prepared nanomaterials were done using SEM analysis operating at 20 kV and EDX (AMETEK).2.5.Evaluation of phytochemical content and biological property studies 2.5.1.Evaluation of Total Phenolic Content (TPC) Total phenolic content in the biosynthesized nanomaterials was evaluated following the method reported by [44] with slight modification.Diluted nanomaterials (50 μl) were mixed with 450 μl of 50% methanol in the 25 ml test tube, followed by adding 500 μl Folin-C reagent into the mixture and incubated for 10 min.Furthermore, 2.5 ml of sodium carbonate solution was introduced into the mixture, vortexed, and incubated for 40 min at room temperature (25 °C) in the dark room.A spectrophotometer was used to measure the absorbance of the samples at 725 nm.The gallic acid (0-0.8mg ml −1 ) was used to construct a standard curve.The results were presented as mg gallic acid equivalent (GAE) per ml extract.

DPPH radical scavenging activity (RSA)
The antioxidant activity of the prepared nanomaterials was carried out according to [30] with some modifications.The method utilized to calculate the percentage scavenging capacity of the biosynthesized ZnO nanomaterials.A 10 mg ml −1 stock solution was prepared with 100% methanol and sonicated for 30 min at 25 °C to obtain a clear dispersion.This was then serially diluted (0.625, 1.25, 2.5, 5.0, 10 mg ml −1 ).The nanomaterials (15 μl) were diluted with 100% methanol (735 μl) in glass cuvettes, followed by the addition of methanolic DPPH solution (750 μl) and the positive control (ascorbic acid) was prepared in the same concentration range.After 30 min of incubation, the resulting mixtures were determined using a UV-visible spectrophotometer set at 517 nm and blanked with 100% methanol.The test was conducted in quadruplicates.
The inhibition concentration at 50% (IC 50 value) for the nanomaterials and ascorbic acid was determined using the standard formula in equation (1 Where Abs DPPH is the absorbance of the control solution (containing methanol and DPPH, except the prepared nanomaterials), while Abs DPPH/sample is the absorbance of the control solution with the prepared nanomaterials.
2.5.3.ABTS + radical scavenging activity ABTS radical scavenging activity from the biosynthesized nanomaterials was conducted using a method reported by [30].In brief, stock ABTS (7.0 mM) and potassium persulfate (2.4 mM) were prepared, mixed to react stoichiometrically at 1:1 ratio, and stored in the dark at room temperature for 12 h to generate incomplete oxidation of ABTS radical cation (ABTS •+).About 1 ml of the mixture was diluted with 100% methanol to obtain an absorbance of 0.704 ± 0.001 units at 734 nm.The assay was carried out by taking 70 μl of each concentration (0.625, 1.25, 2.5, 5.0, 10 mg ml −1 ) of the nanomaterials into a centrifuge tube.Then, 1425 μl of the ABTS solution was added and vortexed.The mixture was incubated for 7 min in the dark.The absorbance values were recorded at 734 nm and blanked against the methanol.Ascorbic acid was prepared in the same concentration range and used as a positive control.All tests were carried out in quadruplicates.The percentage (%) inhibition of ABTS radical scavenging activity was calculated using the standard formula in equation (2): Where Abs ABTS is the absorbance of the control solution (containing methanol and ABTS, except the prepared nanomaterials), while Abs ABTS/sample is the absorbance of the control solution with the prepared nanomaterials.

Ferric Reducing Antioxidant Power (FRAP) Assay
Briefly, 0.25 M acetate buffer, pH 3.6, 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) and 20 mM ferric chloride were combined to create a fresh FRAP reagent.Different nanomaterial concentrations (150 μl each) were added to the test tubes in quadruplicates before 2850 μl of the FRAP working solution was added.The test tubes were then incubated for 30 min in the dark.UV-vis spectrophotometer was used to measure the absorbance at 595 nm.
Results were expressed as Trolox (mM) equivalents per ml sample (mM TE/ml sample).

Evaluating the antimicrobial activity of biosynthesized ZnO nanomaterials
The prepared nanomaterials biosynthesized from pomegranate peel and seed wastes were investigated for their antimicrobial activities using the MH agar well disc diffusion method [45] and tested against common food pathogenic bacterial strains such as Escherichia coli

Results and discussion
3.1.Identification of phytochemicals and biosynthesis of ZnO Nanomaterials Punica granatum L. fruit has been reported to contain significant amounts of bioactive compounds, such as flavanols, anthocyanins and hydrolysable tannins [10].Furthermore, these phytochemicals have been reported to possess the ability to adequately reduce zinc salts into metallic [8,46].The graphical representation of the synthetic pathway has been summarized in figure 1.This study employed FTIR spectroscopy to analyze the phytochemical constituents of pomegranate peel and seeds and the respective annealed ZnO-NPs after the calcination process.The obtained FTIR spectra of the peel and the seed waste powder and biosynthesized nanomaterials are presented in figure 2, which showed that broad peaks detected between 3800 and 3600 cm −1 present in all samples corresponding to the vibrational band of the symmetric and asymmetric intermolecular hydrogen-bonding suggesting that pomegranate peel and seed consists of polyphenols and alcoholic functional groups with various hydrogen bonds [39].The peaks identified between 3000 and 2800 cm −1 represent the C-N stretching vibrations [21].The Other bands around 2356 ± 10 cm −1 correspond to aromatic -C=Cbond or N-H bending vibrations [47].Furthermore, the peaks observed between 1500 and 1400 cm −1 have been attributed to the C-H bending vibration [21].Major peaks detected between 1100 and 1000 cm −1 have been ascribed to the C-O stretching and -OH deformation of tertiary alcohols due to the water molecules.Other peaks in both the ZnO PPW and ZnO PSW peak at about 470 cm −1 have been attributed to Zn-O bond [43].
According to [21], the band below 500 cm −1 indicates the formation of ZnO due to bioactive compounds (polyphenol) in the pomegranate fruit wastes through oxidation or reduction mechanism.These bands are absent in the spectra of the extracts of a and c presented in figure 1.It has been reported that pomegranate fruit peel and seeds have a variety of bioactive compounds such as anthocyanin, ascorbic acid, gallic acid, phenolic acid, protocatechuic acids, terpenoids and amides [44].These bioactive compounds have been reported to carry out the bio-reduction of Zn 2+ ions through the transfer of electrons from NADH through an extracellular enzyme called NADH reductase, acting as an electron carrier resulting in the formation of ZnO NPs [48].
Specifically, the presence of polyphenols, amide, amino and carboxyl groups have been implicated to possess capping and stabilizing properties to prevent aggregation and agglomeration of biosynthesized ZnO nanomaterials [49].

Total phenolic content (TPC)
The bioactive compounds (i.e., flavonoids, terpenoids, phenolics) present in P. granatum fruit peel and seeds have been reported to have redox properties responsible for reducing and capping agents of metal ions during NP biosynthesis [50].In accordance with existing literature, pomegranate fruit peels have been found to contain significantly higher concentrations of polyphenols compared to seeds [11,51].Specifically, the phenolic content in pomegranate peels was quantified at 249.4 mg g −1 (tannic acid equivalents), while seeds contained only 24.4 mg g −1 [52].This substantial difference in phenolic content provides a rational explanation for the observed yield disparities in a higher yield (14.3 g) achieved with pomegranate peels compared to 10.2 g when seeds were used.Furthermore, the total phenolic content (TPC) present in biosynthesized ZnO nanomaterials is presented in figure 3. TPC in both samples (ZnO-PPW and ZnO-PSW) increased with concentration, and the values were recorded to be in the range of 16.87-54.4μg GAE/g DM), which indicates promising antioxidant activity [53].This result agrees with a recent report on three different cultivars of pomegranate fruit peel and seeds aqueous extracts, which found that the fruit peel extracts produced significantly higher extraction yields than seed extracts regardless of the cultivar and the extraction solvent and were richer in TPC [54].
Based on the formula, D represents the average crystalline particle size of the biosynthesized ZnO nanomaterials, Scherrer constant (k) is equivalent to 0.93, λ is the x-ray wavelength employed (1.5406 Å), and the full width at half-maximum (FWHM) of the peak denoted as β and θ is the Bragg angle.From this equation, the average crystallite sizes of ZnO nanomaterials were calculated to be 41.51 nm (ZnO-PPW) and 51.7 nm (ZnO-PSW), respectively.Similar to other reports in literature, these observed average crystalline sizes correspond to the biosynthesized ZnO from Acacia caesia bark extract was found to be 32.32 nm [32] and those mediated using Mentha pulegium leaf extract found at about 44.94 nm [33].

Optical properties of the biosynthesized ZnO nanomaterials
The optical characteristics of the prepared nanomaterials were analyzed utilizing a UV-vis spectrophotometer ranging between 300 and 800 nm.The UV-vis spectra exhibited excitonic absorption peaks at 369 and 372 nm for ZnO-PPW and ZnO-PSW, respectively, as seen in figure 5.These peaks are caused by the Surface Plasmon Resonance (SPR) characteristic of the biosynthesized ZnO nanomaterials [55].According to [21] the absorption peak could be detected due to the intrinsic band gap found in ZnO nanomaterials since the excited electrons are from the valence band migrate to the conduction (O 2p = > Zn 3d ).These results are supported by [21], who also reported a band gap at 378 nm using pomegranate fruit peel, whereas [33] reported a similar band gap at 370 nm using leaf extract of Mentha pulegium (L.).

Microscopic and elemental composition studies of the biosynthesized ZnO nanomaterials
The morphological characteristics such as shape, size and roughness of nanomaterials have been reported to influence their applications.It is thus crucial to examine their microstructure to determine the nature of a nanomaterial [43].The scanning electron microscope (SEM) at low and high magnifications was used to investigate morphological characteristics of the prepared nanomaterials.Figure 6 shows the synthesized nanoparticles at different magnifications.The obtained micrographs for both ZnO-PPW and ZnO-PSW nanomaterials showed the agglomeration of several numbers of densely packed nanoparticles aggregating in a non-repetitive pattern like other reports by [47,56].On the other hand, the transmission micrographs (TEM) showed a more detailed morphological property of the as-prepared materials.As shown in figure 6, the morphological surface of both ZnO-PPW and ZnO-PSW nanomaterials was spherical, tending towards a rodlike morphology.The agglomeration in the prepared materials may have been caused by calcination or the presence of bioactive components of the pomegranate fruit extracts wrapped around the nanomaterials, causing the particles to clump together [39].ImageJ, origin software was used to determine the mean size of the nanomaterial's software and the histogram distribution curve are shown in figures 6(E)-(F), were 59.48 and 57.72 nm for ZnO-PPW and ZnO-PSW, respectively.These results agree with those of [21] who also reported a spherical shaped morphology with agglomeration.The energy dispersive x-ray (EDX) analysis was used to investigate the elemental composition of the nanomaterials and their purity.The collected spectra for both ZnO nanomaterials and the percentages of the constituting elements of the materials are presented in figure 7 (A and B) and table 2, respectively, showing the presence of carbon, oxygen, and zinc.The carbon peaks were due to the carbon tape that was used to hold the nanoparticles onto the holder [57].Additionally, no contaminants were found in any of the spectra, demonstrating the purity of the nanomaterials produced through biosynthesis.

Antioxidant activities (ABTS + , DPPH Radical Scavenging Activity and FRAP)
Reactive Oxygen Species (ROS) are chemically reactive oxygen-containing molecules, such as hydrogen peroxide (H 2 O 2 ), hydroxyl radicals ( • OH) and superoxide anion radicals (O 2 •− ) [58].ROS can be formed during the natural metabolic processes in living organisms, including food items, or through external factors like exposure to air, light, and high temperatures [58].ROS play a significant role in food deterioration through lipid oxidation, protein oxidation, and enzymatic browning [59].While ROS can lead to food deterioration, their impact can be mitigated by the presence of antioxidants in foods or antioxidant packaging materials.Antioxidants neutralize ROS, preventing or slowing down the oxidation processes and, preserving the quality attributes and prolonging the shelf life of food products.Hence, evaluating the antioxidant potentials of the prepared ZnO nanomaterials for a possible application in food packaging materials is important.In this study, ascorbic acid was compared to the antioxidant activities of the biosynthesized ZnO-PPW and ZnO-PSW nanomaterials.The collected data from the DPPH and ABTs assays of the percentage inhibition at a different concentration range between 10-0.625 mg are thus summarily presented in tables 3 and 4.This data showed a concertation-dependent profile within the used concentrations in both assays.Thus, in the DPPH assays, the best scavenging activity in the biosynthesized ZnO nanomaterials and ascorbic acid (AA) was found at 10 mg ml −1 in all the concentrations.Also, the ascorbic acid had better scavenging activity than the biosynthesized ZnO-PPW and ZnO-PSW in the lower concertation profile.However, as the concentration increased, the percentage inhibition of ZnO-PPW (68.5%) was almost parallel to ascorbic acid (72.5%).Furthermore, the antioxidant activity was assessed regarding minimum inhibition concentration at 50% (IC 50 value) of the prepared nanomaterials required to scavenge 50% DPPH free radicals.Hence, the minimum inhibition concentration at 50% (IC 50 values) of the biosynthesized ZnO-PPW and ZnO-PSW nanomaterials was found to be 2.98 mg ml −1 and 3.43 mg ml −1 , respectively, which is comparable to Ascorbic acid (2.31 mg ml −1 ), as seen in table 3. The obtained result thus  indicates that the biosynthesized nanomaterials, especially the ZnO-PPW, possess a comparative scavenging activity, such as ascorbic acid.Furthermore, the observed higher activity in the ZnO-PPW can be attributed to the higher concentration of the phytochemicals present in the pomegranate fruit peel extracts, as shown in the screening study.Another report in which a comparable IC 50 value between the standard ascorbic acid used as a positive control (4.96 mg ml −1 ) and a ZnO nanomaterials (8.99 mg ml −1 ) (mediated using Dovyalis caffra) has been made in literature [43].Moreover, an IC 50 of 182.63 μg ml −1 have been documented in literature for the scavenging activities of ZnO mediated using fruit extract of Myrica esculenta [5].Hence, our study here shows that the prepared ZnO nanomaterials have the potential to be added to the design of active food packaging,  consistent with recent literature results showing that the activity of produced ZnO nanomaterials and ascorbic acid is equivalent.The antioxidant assay (ABTS + ) generated a radical monocation of ABTS •+ by oxidation with potassium persulphate [60].The assay is influenced by the concentration of the biosynthesized ZnO nanomaterials.Like the DPPH assay, the ABTS assay revealed that ascorbic acid had a better radical scavenging potential than the biosynthesized ZnO nanomaterials in a dose-dependent manner, as displayed in table 4. Nevertheless, the ABTS radicals scavenging percentage of ZnO-PPW at 10 mg ml −1 (78.71%), which was the used highest concentration, was the closest to ascorbic acid (99.24%) between the two prepared ZnO NPs.The IC 50 values for ZnO-PPW and ZnO-PSW were 2.29 mg ml −1 and 3.33 mg ml −1 , respectively.Ascorbic acid (positive control) had a lower IC 50 (1.41mg ml −1 ).Like the DPPH assay, the observed higher radical scavenging activity in the ZnO-PPW is attributed to the high TPC present in the PPW extracts.Our findings are similar to previous research in which an IC50 value of 600 μg ml −1 was reported using Caesalpinia crista seed extracts as a mediating agent [61].Thus, the ZnO nanomaterials synthesized in this study demonstrate promising attributes for use as additives in the development of biodegradable food packaging materials.
The relationship between redox reactions and food spoilage is essential to understanding and controlling food preservation and safety.Food manufacturers and consumers often use antioxidant additives and airtight packaging to prevent spoilage and minimize oxygen exposure [62].The properties of these antioxidant additives are usually examined using the FRAP assay which is based on single electron transfer (SET) reactions, alongside other antioxidant techniques to assess the antioxidant potential of various substances [63].Furthermore, the FRAP assay measures the capability of a substance to reduce ferric ions to ferrous ions, indicating its capacity to donate electrons and act as a reducing agent to counteract free radicals, which damage biological systems [63].Therefore, its application in food packaging technology has also proven useful in measuring the antioxidant properties of the packaging material of interest or other additives, like nanomaterials, incorporated into the packaging.As with the other antioxidant assays, a concentration-dependent activity was found [64], and the obtained data (figure 8).Based on the collected data, the biosynthesized ZnO-PPW showed a stronger reducing property than the ZnO-PSW (figure 8).The highest concentration of the nanomaterials had the highest 0.28 and 0.26 (mM TE/ml sample) for ZnO-PPW and ZnO-PSW, respectively.Comparable concentration-dependent behaviour has been reported in other studies involving different biological sources for ZnO nanomaterial synthesis.For instance, good reducing power has been observed with ZnO nanomaterials prepared using Saccharomyces cerevisiae [48], Spinacia oleraceae [65], and Myrica esculenta [5].The crucial role of phenolic content in the biosynthesis of nanomaterials, as shown in the TPC analysis (in figure 3), may have accounted for the observed FRAP activities.This result thus indicates that both materials can be good additives to any safe polymer matrix in food packaging applications.

Antimicrobial activity study
Microbial activities play a crucial role in food deterioration and spoilage.Various microorganisms, such as bacteria, yeasts, moulds, and other fungi, can grow on and in food items, leading to changes that can render the food unsafe, unpalatable, or unappealing.Food packaging materials, called active packaging materials, have been designed with antimicrobial properties to modulate the impact of microbial actions by incorporating antimicrobial agents such as metal oxide nanomaterials [42].These antimicrobial agents help to maintain quality and prolong the shelf life of food products, minimize the risk of spoilage, and enhance food safety.The antimicrobial properties of ZnO-PPW and ZnO-PSW are presented in table 4. Antimicrobial activities with inhibitory zones greater than 6 mm are considered active [66].Although no clearly defined trend was observed, ZnO-PSW has the highest activity against the Gram-negative bacteria.On the other hand, for the Gram-positive bacteria, a comparable activity was observed for ZnO-PPW and ZnO-PSW (table 5).This observed trend can be attributed to the size of the nanoparticles as already established in literature, which, in turn, aids the penetration into the bacterial cell to inject Zn 2+ ions, resulting in disruption of cell processes such as active transport, enzyme activity, metabolism, ultimately causing cell death [32,38] (ZnO-PSW has a smaller size than ZnO-PPW as indicated in TEM analysis  Table 5. Antimicrobial activity of the biosynthesized ZnO-PPW and ZnO-PSW nanomaterials against selected food microbes.

Bacterial name
Zone of inhibition (mm) was slightly lower compared to E. coli (ATCC 35218) in ZnO-PPW.However, the results indicated that tetracycline was more effective against all tested microorganisms except against E. faecalis (ATCC 29212), where the biosynthesized nanomaterials demonstrated superior antimicrobial activity.Furthermore, the higher diameter observed for the Gram-positive might be due to thick peptidoglycan layers in the cell wall, cell physiology and metabolism, making them more susceptible to ZnO penetrations [49].The results here compare well with other literature reports [18,45].Our findings suggest that the prepared materials have the potential to inhibit or delay the proliferation of bacterial organisms on food substances.

Conclusion
This study demonstrates the potential utilization of pomegranate peel and seed waste for the biosynthesis of functional ZnO nanomaterials that could be used as additive materials in food packaging polymer matrices.The physicochemical properties of the biosynthesized ZnO nanomaterials from pomegranate peel (ZnO-PPW) and seeds (ZnO-PSW) were characterized using XRD, UV, TEM, SEM, and EDX.The results align well with existing literature, confirming a wurtzite crystalline structure of ZnO nanomaterials.The antioxidant activities of the biosynthesized ZnO nanomaterials were carried out and compared to ascorbic acid using three assays: DPPH, ABTS, and FRAP.ZnO-PPW exhibited better antioxidant activity than ZnO-PSW in all the assays but slightly less than ascorbic acid, as indicated by the estimated IC 50 values due to the high TPC.Additionally, the evaluation of the antimicrobial properties of ZnO-PPW and ZnO-PSW against common food pathogenic bacteria (Gram-positive and Gram-negative) showed that ZnO-PPW and ZnO-PSW demonstrated good antimicrobial activity, with ZnO-PSW showing higher activity against all the used bacteria due to its smaller nanoparticle size, which enhanced its penetration into bacterial cells; however, the ZOI were less than tetracycline.These results suggest that the biosynthesized ZnO nanomaterials possess antioxidant and antimicrobial properties, making them promising candidates for incorporation into food packaging materials.However, further research and testing are necessary to fully understand their efficacy, safety, and potential interactions with food products before considering their application in food packaging materials.
ATCC 25922 and ATCC 35218), Enterococcus faecalis (ATCC 29212), Listeria monocytogenes (ATCC 7644) and Staphylococcus aureus (ATCC 25923).Various concentrations of nanomaterials (10, 30 and 50 mg ml −1 ) were prepared by dissolving them in an organic solvent dimethyl sulfoxide (DMSO).The resultant mixture was then sonicated at 4 • C for a few minutes.Fifty micro litre (50 μl) of the biosynthesized nanomaterials were dispensed into each well (diameter of 8 mm) of the plates and left for 30 min in the biosafety cabinet to allow the ZnO nanomaterials to settle into the MH agar and incubated at 37 • C for 24 h.The diameter (mm) of the zones of inhibition (ZOI) was recorded.

Figure 1 .
Figure 1.Schematic representation of the biosynthesized ZnO nanomaterials from Punica granatum L. fruit peel and seed wastes.

Figure 3 .
Figure 3.Total phenolic content of the biosynthesized ZnO-PPW (pomegranate peel waste) and ZnO-PSW (pomegranate seed waste).Error bars indicate the standard error (SE) of the average samples (n = 3).Distinct letters on each bar indicate significant differences conducted using Duncan's multiple range test (p < 0.05).GAE = gallic acid equivalence.

Figure 7 .
Figure 7. EDX spectrum of biosynthesized ZnO nanomaterials from pomegranate fruit peel (a) and seeds (b) displaying the weight % of element composition.

Figure 8 .
Figure 8. Histogram plot of ZnO-PPW and ZnO-PSW in Ferric reducing antioxidant power assay.Error bars indicate the standard error (SE) of the average (n = 4).Distinct letters present on each bar indicate significant differences conducted using Duncan's multiple range test (p < 0.05).TE = Trolox equivalence, PPW = pomegranate peel waste and PSW = Pomegranate seed waste.

Table 1 .
List of food borne pathogens used to evaluate the antimicrobial properties of biosynthesized nanomaterials. ).