Physical, Chemical, and Antioxidant Characterization of Nano-Pomegranate Peel and Its Impact on Lipid Oxidation of Refrigerated Meat Ball

Pomegranate peel (CPP), enriched with bioactive constituents, had potent antioxidant features. &erefore, it is worth finding out functional and antioxidant features of the nanoscale pomegranate peel. &e nanoscale of pomegranate peel was prepared by ultrafine grinding in a ball mill for 45min (NPP45) and 90min (NPP90).&e physical (SEM, TEM, FTIR, and XRD) and chemical characteristics (phenolics, flavonoids, DPPH scavenging activity, FRAP, and reducing power) of nanoparticles were studied. &e quality aspects of cold stored (5± 2°C) meatballs formulated with 0.5% (W/W) of nano-peel powder were evaluated. Similarly, FTIR spectra and XRD patterns were recorded for nano and crude pomegranate peel samples. Generally, grinding the crude peel for 45 and 90min enhanced its scavenging activity, reducing power, FRAP, total phenolic, and flavonoid by a range of 12.58 to 20.37 and 20.57% to 35.18%, respectively. &e addition of crude/nanosized peel to the meat ball diminish (p< 0.05) formation of thiobarbituric acid (TBARS), peroxide (PV), and volatile nitrogen and kept the sensory attributes up to 9 days of cold storage. No significant differences were noticed in PV and TBARS of meatballs formulated with 0.5% NPP90 and 0.1% BHT, which suggests the potential use of nanoscale pomegranate peel as natural substitutes to BHT in meat products.


Introduction
e pomegranate (Punica granatum L.), originating in the Middle East, is one of the rising tree crops grown worldwide. Its peel constitutes about 50 of a whole fruit's weight and remains a waste product in the pomegranate juice production process. Pomegranate peel extract, enriched with phenolic and flavonoid constituents, had potent antioxidant features [1,2]. Even though the pomegranate peel's antioxidant and health benefits were upheld, scientific research should be done to improve the peel extract's effectiveness. erefore, it is necessary to improve the efficiency of pomegranate peel extract via modern emerging food processing technologies, i.e., superfine grinding [3] and nanotechnology [2]. Over the past few decades, nanotechnology increasingly attracted most researcher in the food processing sector such research achieved a promising result in food preservation which might cause a revolution in food processing and preservation [4]. Decreasing particle sizes might increase the particle surface area and consequently release bioactive compounds [5]. erefore, it is worth finding out the functional characteristics and antioxidant features of nanoscale pomegranate peel. Meatballs have a short shelf life due to lipid oxidation, protein decomposition, and microbial contamination during storage [6]. Lipid oxidation results in comprehensive changes in flavor, color, and structure, which minimize sensory traits and consumer acceptability of meat products. Such oxidative rancidity could be eliminated by using synthetic or natural antioxidants [7]. Although synthetic antioxidants have been effectively applied to prevent meat products oxidation, it has negative health effects. erefore, increased demand for natural antioxidants has been noticed in the recent decades [8]. Pomegranate peel extracts displayed extraordinary antioxidant properties with high competence in free radical scavenging and lipid oxidation suppression activity [9]. e antioxidant impact of pomegranate peel extracts has been scrutinized in cooked chicken products [10], in ground pork meat [11], beef sausage [12], and white shrimp [13]. On the other side, powdered pomegranate peel [14] as well as nanopomegranate peel [15] has been used as a promising natural antioxidant to prolong the shelf life of meat products.
Although diverse investigations were carried out on the bioactive compounds and health merits of pomegranate peel, there was little research focused on the impact of the nanosizing of pomegranate peel on its physicochemical properties and antioxidant activity, furthermore, the impact of using nano-pomegranate peel as a preservative to prolong the shelf life of meat products. erefore, the present study aimed to evaluate the effect of nanosized pomegranate peel (by mill ball grinding) on its physicochemical and antioxidant activities as well as its impact on retarding the lipid oxidation of cold-stored meat ball.

Materials.
Pomegranate (Punica granatum L.) fruits were purchased from the local market in Taif City, Kingdom of Saudi Arabia. All chemicals and reagents used in this investigation were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Preparation and Characterization of Pomegranate Peel
Nanoparticles.

Physical Characterization of Nano-Pomegranate Peel Particles
(1) Particle Size. e particle size of NPP45 and NPP90 were measured in nanosuspension using Zetasizer (Malvern, Model: Zetasizer nano-series, Nano ZS, United Kingdom). A dispersion of 20 mg NPP45 or NPP90 in 4 ml deionized water and 0.5 ml dimethyl sulfoxide was prepared and homogenized by stirring for 30 min, then centrifuged for 15 min at 5000 rpm. Serial concentrations were prepared by diluting the supernatant in deionized water. Samples were measured after 5 min and equilibration at 25°C based on electrophoretic mobility under an electric field. e dynamic laser scattering angle was 173°, size ranged between 0.6 and 6000 nm, zeta potential ranged between −200 and 200 mV: the average of runs was at 16 and 10 seconds intervals [17].
(2) Scanning Electron Microscope (SEM). e images of scanning electron microscopy (SEM) were taken by using SU8020 SEM system (Hitachi, Japan). e samples (PP, NPP45, and NPP90) were mounted onto a specimen holder (under reduced pressure) with gold sputtered.
(3) Fourier-transform Infrared Spectroscopy (FTIR). Fourier transform infrared (FTIR) spectroscopy was used to disclose the active groups of pomegranate peel nanoparticles. A defined weight (A 0.002 gm) of the powder samples (CPP, NPP45, and NPP90), blended with 0.02 gm KBr and fully grinded, then turned to pellet via intense compress in a spatial mound and put in the apparatus to accomplish the FTIR spectra.

Chemical Characterization
(1) Total Phenolic and Total Flavonoids. Total polyphenols and flavonoids were quantified as previously described by Slinkard and Singleton [18] using Folin-Ciocalteu and Dowd method [19].

Formulation of Meatballs.
Raw beef (top round) and fat were supplied from the Faculty of Agriculture farm (Shebin El-Kom, Egypt) and slaughtered (in the same frame slaughterhouse), cleaned, and deboned following standard commercial procedures. e meat (15% fat) was conveyed to the laboratory in an ice box to the laboratory, and then minced for 5 min. e meatballs were formulated by mixing 86.75% minced beef (∼15% fat content) with 5.5% breadcrumbs, 3.25% onion powder, 0.5% garlic powder, 0.25% black pepper, 0.5% cumin, 0.5% coriander, 1.75% salt, and 1.0% water [15]. e previous ingredients were mixed well to form a homogeneous mixture, and then divided into 5 portions.

Quality Traits of Meatballs Formulated with Nano-Pomegranate Peels
(1) Peroxide Value (PV). Peroxide value of cold-stored meatballs formulated with different nano-pomegranate peel was determined according to the IDF method [23]. e results were expressed as milliequivalents of peroxide/kg fat. (2) iobarbituric Acid Reactive Substances (TBARS). TBARS were determined spectrophotometrically as described by Vyncke [24] and expressed as mg of malonaldehyde (MDA)/kg sample.

(3) Total Volatile Base Nitrogen (TVB-N).
e TVB-N contents were determined by the method of Egan et al. [25]. e results were expressed as mg·N/100 g of the sample.

Sensory Evaluation.
Uncooked samples were placed randomly in coded covered cups. Fifteen semi-trained panelists were asked to evaluate the refrigerated stored meatballs for rancid odor three times repeatedly for 15 s with an interval of 30 s in the analysis on days 0, 3, 6, 9, and 12. en, the color attributes were evaluated using the same manner [26].

Statistical Analysis.
e data were analyzed in one way using SPSS version 19.0 (Chicago, IL, USA) to test the variance by one-way analysis of variance (ANOVA). Tukey's honest (HSD) test was followed to determine the differences in relative abundance. e results were expressed as the mean ± SEM, and p < 0.05 was considered statistically significant.

Particle Size Distribution.
e mean values of nanopomegranate milled for 45 min (NPP45) was 373 nm these mean values decreased to 125 nm with increasing the milling time to 90 min ( Figure 1).

Transmission Electron Microscopy (TEM).
High resolution transmission electron microscopy (HRTEM) is a sophisticated technique used to characterize the shape, size, and distribution of nanoscale materials [27]. e nanoparticle scale of pomegranate peel showed uniform distribution and smaller in size was detected in the NPP90 than those obtained in NPP45 with a prevalent spherical morphological shape (Figure 2).

Scanning Electron Microscopy (SEM).
e microphotographs of nano-pomegranate peel powder showed the morphology of CPP, NPP45, and NPP90 nanoparticles ( Figure 3). Milled pomegranate peel particles depicting the granular mechanical damage. Increasing the ball milling time increased the broken particles into smaller fractions, and the presence of various pomegranate peel particle shapes might be resulted from the combination of flattening, aggregation, and fracture of particles. At the beginning of crack proliferation and fracture, the aggregation of pomegranate peel particles might lead to an increase in size. e morphology of ball milled peel considerably changed, which might have a remarkable impact on physicochemical properties. e SEM images of CPP, NPP45, and NPP90 confirm well with that detected by XRD and maintain the particle's random amorphous structure. Similar trend was detected by Zhong et al [3] for superfine particles of pomegranate peel.

FTIR Spectra.
e FTIR spectra of crude (CPP) and nanoparticles (NPP45 and NPP90) of pomegranate peel powder are illustrated in Figure 4. Similar patterns of structures were detected for different pomegranate peel nanoparticles. However, a decrease in the transmittance intensity of bands was recorded by increasing the size of particles. No significant changes in FTIR spectra of six particle-sized pomegranate peel powders [3]. e relatively high number of bands might be correlated with the complexity of the peel constituents (proteins, carbohydrates, cellulose, and pectin) which contains different types of bonds. Similar results were detected by Zhong et al. [3] who found that grinding of pomegranate peel to different fine and superfine powder did not affect their chemical composition and consequently their FTIR spectra. Complex nature with wide variety of compounds in pomegranate peel powder was previously proved by Salih et al. [28]. Also, many active groups, which are mostly aldehyde compounds, ketones, amines, amides, alcohols, or aromatic or phenolic compounds were detected in pomegranate peel [29,30].

X-Ray Diffraction (XRD)
. X-ray diffractometry (XRD) was applied to assess the crystallinity features of crude and nano-pomegranate peel ( Figure 5). e diffractograms represented typical beaks for crude and nanosized pomegranate samples. e peak broadening revealed no crystallinity of crude and nano-size samples and maintain the random amorphous structure. ese results suggesting the inhomogeneous composition of the tested peels which is matching with the results previously stated by Vinay et al. [31] for pomegranate peel silver nanoparticles.

Antioxidant Characteristics and Phenolic and Flavonoid
Contents. Reducing power, ferric reducing power, and DPPH scavenging activity as well as, phenolic and flavonoid contents of crude and nano-pomegranate peel ethanolic extracts compared with BHT are illustrated in Table 1. Generally, decreasing the particle size of pomegranate peels by milling for 45 min (NPP45) and 90 min (NPP90) enhanced their DPPH-scavenging activities by 15.77% and 20.57%, respectively, and their FRAP by 20.37% and 35.18%, respectively. ese results maintained by the noticed increase in total phenolic (12.58% and 35.17%) and total flavonoids (14.12% and 24.51%) in the same table. is might be due to the decrease in particle size which increased the surface area via decreasing the size into nanoscale and NPP90 NPP45   consequently facilitate releasing of phenolic and flavonoids [2,3]. e DPPH-scavenging activity of crude pomegranate peel was lower than that of 0.1% BHT. Meanwhile, decreasing the pomegranate peel size into nanoscale enhanced its DPPH radical inhibition efficiency compared with BHT. On the other side, all pomegranate peels (crude and nanoscale) had a lower FRAP and reducing power compared to 0.1% BHT.

Peroxide Values.
Initial oxidation of oils and fats were usually expressed by peroxide values (PV). Both nanopomegranate addition and storage time significantly (p < 0.05) affected the PV of stored meatballs (Table 2). e highest (p < 0.05) initial PV was detected in the control samples (without pomegranate peel or BHT) followed by that formulated with crude pomegranate peel, whereas the lowest initial oxidation value was detected in meatballs formulated with 0.5% NPP45, NPP90, and 100 ppm BHT. e PV of the control sample reached to the threshold limit value on day 6 and decreased after that. Such decrease might be due to the degradation of the formed hydroperoxides and formation of secondary lipid oxidation derivatives. No significant differences were noticed among the stored meatballs formulated with 0.5% NPP45, NPP90, and 0.1% BHT, which indicated that progression of initial oxidation and the subsequent peroxides degradation was delayed with retardation of further oxidation progress. On the other side, significant (p < 0.05) increase in the mean of PV was detected by increasing the storage days. Similar trends were noticed in meatballs formulated with 1% crude pomegranate peel [30] and 1% nano-pomegranate peel [15].

Lipid Peroxidation (TBARS).
Secondary lipid oxidation products, the main entrepreneur of oxidative rancidity, were determined by the TBARS assay. Generally, BHT and crude and nano-pomegranate peel as well as the storage time affected (p ≤ 0.05) TBARS values of the formulated meatballs (Table 3). e TBA value of all formulated meatball samples was almost the same at zero time of the storage period. e TBA means of all formulated meatball samples significantly increased (p < 0.05) by increasing the storage period. e TBA value of meatball control was higher than other meatball samples at all storage periods. No significant differences (P > 0.05) were noticed in the TBA among the stored meatballs formulated with 0.5% NPP45, NPP90, and 0.1% BHT. ese results confirm well with the results of the peroxide value (Table 1), which might maintain that the nanoscales pomegranate peel might delay the peroxide degradation and retard further oxidation progression that reduce the rancid odor. Morsy et al. [15] detected a lower TBA value in meatball samples formulated with 1.5% lyophilized pomegranate peels nanoparticles compared to control and BHT. As illustrated in our results, both negative and positive control reached the upper TBARS quality standard limit by the eighth day of storage. (TVN) is the main proof of meat protein degradation. e mean TVN value of freshly prepared meatballs was 4.21 mg·N2/100 gm and increased significantly (p < 0.05) by increasing the storage periods and reached to 18.65 mg·N2/ 100 gm after 12 day of cold storage (Table 4). e highest (p < 0.05) mean TVN value was noticed in control (14.27 mg·N2/100 gm) meatball samples followed by both that formulated with 0.5% crude peel (10.06 mg·N2/100 gm) and 0.1% BHT (10.20 mg·N2/100 gm). e lowest (p < 0.05)

Sensory
Properties. e mean score of color and odor of meatballs formulated with 0.5% of nano-pomegranate particles are illustrated in Table 5. Generally, both sensory properties were affected (p < 0.05) by the cold storage period and added nano-pomegranate peel (NPP45 and NPP90) types. No difference (p > 0.05) was observed among all treatments at the beginning of the storage period. Color attribute was deteriorated (p < 0.05) by increasing (up to 3 days) the storage time in all the formulated samples, whilst   e meat ball formulated with NPP90 and BHT kept their odor acceptability till the 9th of storage, whilst the other samples showed unacceptable odor from the 6th day of storage. ese results indicated that the addition of nano-pomegranate particles (specially NPP90) might significantly inhibit the oxidation process of lipids and proteins and prolong the shelf life of cold stored meatballs. Morsy et al. [15] stated that the nano-pomegranate peel had remarkable effect to retard the lipid and protein oxidation and consequently prolong the shelf life of meatballs. Jouki and Khazaei [26] reported that oxidative rancidity (TBARS) and sensory quality of cold stored (4°C) camel meat can be easily enhanced by controlling the packaging atmosphere.

Conclusions
Using of crude pomegranate peel powder as a source of natural antioxidant have been previously studied. However, in the present study, crude peel was grinded in a planetary ball mill for 45 min (NPP45) and 90 min (NPP90). e physical and chemical characteristics of crude and nanosized pomegranate peel were evaluated and applied as a natural antioxidant to preserve the cold stored beef meatballs. Changing the pomegranate peel into nanoscale had no impact on its physical criteria where XRD diffractogram and FTIR showed a similar pattern. Meanwhile, nanosizing the peel improved the DPPH radical scavenging activity (even higher than BHT), FRAP, reducing power, and enhanced the efficiency of its phenolic and flavonoids to retard the lipid and protein oxidation in meat products, which could both satisfy consumer requests for natural food ingredients and add commercial value to pomegranate by-products. erefore, nanoscales of pomegranate peels (especially NPP90) could be successfully added to meatball products as a promising natural substitute of the synthetic antioxidants. For more transparency of safety issues, further mandatory testing of using nano-pomegranate peel as to retard lipid and protein oxidation in meat products is needed.

Data Availability
e data presented in this study are available on request from the corresponding author.