Development of antimicrobial/antioxidant nanocomposite film based on fish skin gelatin and chickpea protein isolated containing Microencapsulated Nigella sativa essential oil and copper sulfide nanoparticles for extending minced meat shelf life

Fish skin gelatin and chickpea protein isolated (G-CP) edible blend films incorporated with 0.25 and 0.5% copper sulfide nanoparticle (CuSNP) and microencapsulated Nigella sativa essential oil (MNEO) (0.015 and 0.03%, w/w of protein) were prepared and optimized by the response surface methodology based on the central composite design (RSM-CCD). Antimicrobial activity, infrared spectroscopy (FTIR), x-ray diffraction (XRD), morphological characteristics and thermal attributes of composite films were examined. In general, the effect of CuSNPs and MNEO on the properties of blended films, besides their inherent nature, is related to their interactions with the protein matrix and the synergistic effect on each other. As authenticated by the FTIR and XRD, the simultaneous use of CuSNPs and MNEO because of the synergistic effect of CuSNPs on the antibacterial attributes of MNEO and raising the content of antimicrobial components in the blend film expressed the highest antimicrobial functionality against E. coli. and S. aureus. Also, the results of microbiological and chemical tests of packaged minced meat revealed that the simultaneous use of MNEO and CuSNP in the film has a positive synergistic effect in increasing the storage life of minced meat, as compared to the other samples.


Introduction
To prolonging the shelf life and storage life of food products and decreasing the hazard of food-borne ailments, wrapping plays an essential role in preventing and limiting physical damage and microbial pollution [1]. Petroleum-based plastics because of their good mechanical attributes and the satisfying barrier properties against gases and water are the most widely used food packaging material. However, the accumulation of nonbiodegradable plastics leading to serious environmental issues [2]. In the past few decades, developing edible film using natural compounds including flavonoids, polyphenols, proteins, fats and polysaccharides or the simultaneous use of these ingredients has been considered as a promising alternative to petroleum-based plastics [2,3].
Among these biopolymers, proteins, because of their abundance, suitable barrier characteristics against gases and volatile compound and good film-forming ability, and diversity of amino acid composition which can lead to a wide range of interactions and chemical modification reactions, have been extensively utilized for the improvement of properties of edible wrapping films [4]. The high price of animal protein has pushed the researchers to be in search of cheap sources of protein for the fabrication of edible film packaging. Considering this issue, the isolated plant proteins such as chickpea protein and the gelatin isolated from maritime protein 2. Materials and methods

Preparation of fish skin
Fresh bigeye snapper (P. tayenus), off-load approximately 48 h after capture. Fresh fishes were stored in ice and transported within 3 h to the Department of Food Technology of Urmia University. The thawed fishes were washed and cleaned with running tap water just after arrival and fish skins were then scraped and removed. The removed fish skins were cut into small pieces (1×1 cm 2 ), and refrigerated at −20°C until gelatin extraction.

Extraction of fish skin gelatin
Gelatin was extracted from bigeye snapper fish skin according to the technique reported by Rattaya et al [2] with a minor alteration. The washed skins were immersed in 0.025 M NaOH with a fish skin/solution ratio of 1:10 (w/v) with continuous gentle stirring at ambient temperature. To take off non-collagenous protein and pigments the alkaline solution was switched every 3 h. NaOH-treated skins were then washed by tap water until the neutral wash water (pH of washed water <7.5) was achieved. Then the fish skins were immersed in 0.05 M CH 3 COOH with a skin/solution ratio of 1:10 (w/v). The solution was switched every 3 h with a mild stirring to swell the collagenous ingredient of fish skins. CH 3 COOH-treated skins were washed as previously described. To extract gelatin, the swollen fish skins were immersed distilled water (45°C) with a skin/distilled water ratio of 1:10 (w/v) for overnight with continuous gentle stirring. The combination was then filtered using two layers of cheesecloth and freeze-dried.

Chickpea protein isolate (CPI) extraction
The chickpea protein extraction was prepared according to the methodology described by Mousazadeh et al [5] with slight modifications. Firstly, for defatting chickpea flour, it was mixed with hexane with continuous stirring two times for 2 h at ambient temperature (1:5 [w/v], chickpea flour: hexane). In the second step, the defatted chickpea powder was suspended in doubly distilled water (1:10 [w/v]) than the pH was adjusted at 10 using 1 N NaOH with subsequent stirring at 1200 rpm for 2 h at room temperature (25°C). The mixture was centrifuged at 9000×g for 30 min and the obtained supernatant was used for the next step. Finally, to precipitate the protein, the pH of the obtained supernatant adjusted at 4 (isoelectric point of CPI) using 1 N HCl and was centrifuged at 8500×g for 15 min. The precipitated CPI protein was washed with doubly distilled water two more times and was centrifuged again to remove the non-protein components and freeze-dried.

Encapsulation of nigella sativa essential oil
Sodium caseinate (SC) was used as the wall materials to microencapsulate Nigella sativa essential oil (MNEO). SC was dispersed in deionized water (60°C) using magnetic agitation and kept overnight at 4°C for complete hydration. Nigella sativa essential oil was gradually added to the solution to prepare a coarse emulsion. Afterward, the mixture was pre-homogenized using a homogenizer (Silverson L4R, Buckinghamshire, England) for 10 min at 5000 rpm. Finally, to provide a fine emulsion, the coarse emulsion was prepared whit a highpressure homogenizer (AH100D, ATS Engineering Inc., Canada) at 300 and 250 MPa, and spray dried to provide the dried powder of MNEO.

Preparation of nanocomposite films
The composite edible films were made utilizing a casting methodology. First, 2.5 g of gelatin and 2.5 g of CPI powder were added to 100 ml of pure water. The pH of the produced dispersion was adjusted to 8 with 0.1 N NaOH and, the blend was stirred on a magnetic stirrer for 20 min at 90°C to ensure its denaturation. Afterward, the encapsulate MNEO (0%-0.5%) was added to the dispersion and a homogenizer device (Avestin Inc., Ottawa, Canada) was utilized to combine the dispersion for 10 min at 10000 rpm. Then the CuSNPs (0%-0.03% w/v) were added to the dispersion and it was combined by a magnetic stirrer for 15 min and then settled in an ultrasonic bath for 15 min to ensure homogeneous distribution of CuSNPs. Finally, glycerol (30% w/w dry matter) was added, and the dispersion was mixed by a stirrer for 30 min. The achieved blend was ventilated for 10 min and 40 ml of it was poured into the center of the petri dish and was dried at room temperature for 1 days. The dried films were equilibrated at ambient temperature and 50% relative humidity (RH) until further analysis.

Thickness
The films thickness was determined by a digital micrometer (QLR digit-IP54, China) with 0.001 mm of accuracy at ten random locations of each blend film sample. Average values were used for other calculations [18].

DPPH radical-scavenging activity (RSA)
The antioxidant activity of the composite digestible films was ascertained utilizing the DPPH methodology [18]. In Brief, 50 mg of blend film samples were dissolved in 10 ml of water. After that, 0.2 ml of film extracts solutions were united to 7.8 ml of the DPPH solution (0.1 M methanol solution), and then, the solutions were saved in the dark at 25°C for 1 h. Finally, the absorbance was measured against pure methanol at 517 nm. The percentage of DPPH radical-scavenging activity was estimated by the following formula: where A blank is the absorbance of the control, and A s ample is the absorbance of the test material.

Moisture content
The blend films moisture content was estimated by measuring the lose-weight of preconditioned edible films after drying in an oven at 103±2°C to the point that reaching a constant weight [19]: where, M 1 is the preconditioned film sample weight and M 2 is dry film sample weight.

Water vapor permeability (WVP)
The composite films WVP was evaluated utilizing the standard gravimetrical methodology of E96 [20], with little modifications according to the equations of Chavoshizadeh [21]. Composite film samples were sealed tightly with parafilm on the top of the cups, which were filled with approximately 35 g of anhydrous CaCl 2 to provide relative humidity (RH) of 0%. Thereafter, the glass cups were stored in a saturated NaCl solution desiccator (75% RH) at ambient temperature. The difference in RH of the two sides of the films corresponds to a driving force of 1753.55 Pa, expressed as water vapor partial pressure. The weighing was performed every 1 h until 8 h and then every 8 h until 48 h. The increase in weight of cups was plotted against time and the Slopes were determined by linear regression. The WVP was calculated in accordance with the following equation and expressed as g m −1 s −1 Pa −1 .
where Δm/Δt is the weight of moisture gain per unit of time (g/s), X is the average composite film thickness (m), A is the area of the exposed composite film surface (m 2 ), and Δp is the partial vapour pressure difference between the two sides of the composite film (Pa).

Color parameters
A colorimeter (D25-9000, Hunterlab, U.S.) was utilized to assess the color values of composite film samples.
Edible film samples were located on a white standard plate (L * =95.49, a * =−0.30 and b * =−0.08) and values of L * (brightness), a (redness-greenness) and b (yellowness-blueness) of films were determined. The total color difference (ΔE) was measured by the following equation [22].

Mechanical properties
The preconditioned blend films mechanical behavior including tensile strength (TS) and elongation at break (EAB%) were estimated utilizing Texture Analyzer (Stable Micro System, Surrey, UK) according to ASTM standard technique D882 [23]. Initial grips separation and cross head speed were set to 40 mm and 10 mm min −1 , respectively. TS and EAB were calculated as follows: where, F (N) is the maximum stress that the blend film samples can withstand; A (m 2 ) is the cross-sectional area of the film samples (thickness×width); ΔL is the increase in length at the breaking point; and L is the initial length between the grips.

Evaluation of antimicrobial activity of films
In order to estimate the antibacterial activity of the blend films was evaluated utilizing an agar disk diffusion technique, according to the methodology reported by Mehmood et al, [24]. The composite film samples were cut into 2×2 cm 2 pieces and settled on a special plate of Mueller-Hinton Agar plates (Merck), which was inoculated with 0.1 ml of broth cultures comprising approximately 10 5 -10 6 CFU ml −1 of E. coli O157: H7 and S. aureus bacteria. The agar plates were incubated at 36±2°C for 1 day. The area of inhibition zones (mm) around the composite film pieces was estimated taking into account the primary diameter of the blend films.

Fourier transform infrared spectroscopy (FT-IR) analysis
To determine the chemical composition, preliminary structures and possible interaction between the components of the blend films, the FTIR spectra of composite films were registered by the FT-IR spectrophotometer (EQUINX55, Brucher, and Germany). The FTIR spectra were recorded over at the wavenumber range from 600 to 4000 cm −1 with 4 cm −1 resolutions [25].

X-ray diffraction (XRD) analysis
To assess the crystalline structures of the blend film samples, there x-ray patterns were measured utilizing an XRD diffractometer (RINT2000, Tokyo, Japan). The composite film samples were scanned at the diffraction range (2θ) of 10°to 80° [26].

Morphological characterization (SEM)
The morphological characteristics (surface and cross-sectional) of the composite film samples were analyzed by a scanning electron microscope (S-4800, Hitachi, Japan) at a magnification of 5000×and acceleration voltages of 10 kV [27].
2.17. Differential scanning calorimetry (DSC) DSC of blend film samples was done on a thermogravimetric analyzer (Shimadzu Scientific Instruments, 154 Kyoto, and Japan) in an Argon atmosphere. In this analysis, the blend film samples were heated (25 to 300°C) with a heating rate of 10°C min −1 .

Preparation of minced meat
The minced beef meat was provided from a local butchery and moved to refrigerator condition (4°C) within 1 h. Then, the minced meat samples (50 g) were afforded under aseptic positions and wrapped with edible composite films. Following that, the wrapped minced meat samples were located in permeable polyethylene bags and stored in the refrigerator at 4°C for 14 days. The minced meat sample without composite film was applied as a control sample [17].

Physicochemical analyses of minced meat
2.19.1. pH value 5 g of the various minced meat samples were mixed with 45 ml of distilled water and the pH of the mixture was measured utilizing an electronic pH-meter (pH-30 sensor; Corning, Lisboa, Portugal) [28].

Determination of 2-thiobarbituric acid (TBA)
2-Thiobarbituric acid (TBA) (mg malonaldehyde/kg (MDA/kg) minced meat) was determined according to the methodology defined by Kirk and Sawyer [29] with minor alteration. Briefly, minced meat (10 g) was homogenized with 20 ml of TBA solution (15% trichloroacetic acid, 0.375% TBA). The compound was boiled for 1 h in boiling water (90°C) and then cooled with running water at ambient temperature and centrifuged at 4200×g for 15 min. The absorbance of the final reaction solution was recorded at 532 nm applying a UV-1601 spectrophotometer (Shimadzu, Kyoto, Japan). TBA values of the minced meat were estimated from the standard curve of malondialdehyde per kg of meat (mg MDA/kg meat). Three replicate was performed for each minced meat sample.

Determination of total volatile basic nitrogen (TVB-N)
The total volatile base nitrogen (TVB-N) content of minced meat samples was measured according to the methodology of AOAC [30]. Ultimately, TVB-N contents were reported as mg nitrogen/100 g of minced meat samples. Three replicates were accomplished for each sample.

Bacteriological analysis of minced meat
At 1, 4, 8, and 14 days of storage, 20 g of each minced meat sample were mixed with 180 ml of 0.1% peptone water in a stomacher-400 (Seward Ltd, Worthing, UK) for 1 min. Ten-fold serial dilution was performed and cultured in Selective media. Then, the plates were incubated under certain conditions. Selective media, incubation temperature and time were as follows: Enterobacteriaceae in Violet Red Bile Glucose (VRBG) agar (30°C for 1 day), Psychrotrophic bacteria in plate count agar (6°C for 10 days), S. aureus in Baird Parker agar (38°C for 2 days). Microbiological enumeration outcomes were reported as logarithms of Colony Forming Units (cfu/g) minced meat [16].

Statistical analysis
The statistical measure was done in 2 segments. In the first part; The MNEO concentration (in 3 levels) and CuSNPs concentration (in 3 levels) were considered as independent parameters to study their effect on the thickness, RSA, WVP, moisture content, and color properties of edible composite films applying a central composite design (CCD) (table 1). Statistical equations, data analysis (at 95% confidence level) and drawing of diagrams were performed by Design Expert 11.0.0 program. In the second part; a completely randomized factorial design was used to investigate the effect of MNEO and CuSNPs on the different properties of composite films (table 1) Table 2 explains the mathematical equations that represent the relationships among MNEO and CuSNP on thickness, RSA, WVP, moisture content, and color properties of the gelatin-chickpea protein (G-CP) based film. Figure 1 and 2 shows the 3D response surface plots of the effect of MNEO and CuSNP on the thickness, RSA, WVP, moisture content and color properties of G-CP-based film. As shown in figure 1, the thickness of G-CP films incorporated with MNEO increased significantly with the rising level of MNEO, this is maybe due to the increase in the solids content of the films. In contrast, the addition of CuSNP to the composite film reduces the thickness of the blend film, so that the edible film that has the highest level of MNEO and the lowest amount of CuSNP has the highest thickness. The findings were in accordance with the result reported by Asdagh et al [31], which revealed that by raising the ratio of coconut essential oil, the thickness of nanocomposite films based on  whey protein/copper oxide nanoparticles increased. Also, Sani et al, [3], reached similar findings, with the increase in the level of Melissa officinalis essential oil, the thickness of chitosan composite films increased. Lipid oxidation is a major problem in fatty foods such as meat, fish, and dairy products, so the application of films containing antioxidants compounds like MNEO and CuSNP can be very effective in solving this problem. Antioxidative activity of G-CP based films in the different levels of MNEO and CuSNP is presented in figure 1. In the present study, the DPPH was used to evaluate the antioxidant activities of the bioactive films. In general, essential oils have high phenolic compounds, which means their high antioxidant power. Kadam et al, [32], and Akloul et al, [33] studied the antioxidative effect of Nigella sativa essential oil. The incorporation of MNEO significantly raised the DPPH radical scavenging activity of G-CP films. The antioxidant effect of MNEO might be due to the presence of an aromatic nucleus containing polar functional groups in its components (including thymoquinone, carvacrol, trans-anethole, and 4-terpineol) [34]. On the other hand, the DPPH radical scavenging activities of G-CP films containing CuSNP were higher stronger than G-CP films containing MNEO, this shows that CuSNP could enhance the antioxidant activity of films by accepting or donating electrons. From the results obtained on the antioxidant properties of film samples containing MNEO and CuSNP, it can be concluded that produced active films are a suitable option for protecting the wrapped food against free radicalinduced oxidation [35].
WVP is one of the most important parameters in the determination of the amount of water transmission of the edible films. An important performance of biopolymeric films is to decrease the exchange of moisture between the wrapped food and outside packaging environment, hence the water loss of product can be decreased [36]. The results of the WVP values of the films are shown in figure 1. The effect of MNEO and CuSNP on the WVP of the G-CP films shows a contradicting effect at the different levels studied. The results indicate a rise in the values of WVP of G-CP-MNEO blended films compared to G-CP-CuSNP films. The WVP of the films increased with a raise in MNEO levels, which probably due to the interactions of −OH and COO − groups of NSE with the active sites (amino groups) in G-CP. These interactions, in turn, may have led to diminished interactions between G and CP in the film matrix, thus reducing film integrity and, consequently, reducing the WVP values. A similar phenomenon was observed in chitosan-based films containing Nigella sativa [32]. Initially, the WVP decreases with increasing CuSNP levels but increased with further increases content of CuSNP. The reduced WVP values of the active blended films at low levels of CuSNP was maybe because of the tortuous path of moisture diffusion created by the well-distributed moisture vapor impermeable CuSNP [14]. Nevertheless, the increased WVP values of the bioactive films at the high percentage of CuSNP was mainly due to the agglomeration of the CuSNP, and interaction between CuSNPs has decreased the number of SH-bonds between active groups of protein matrix (G-CP) [37].
Low moisture content composite films facilitated and accelerated the wrapping of water sensitive food materials [38]. The moisture content of G-CP based films is presented in figure 2. Our results indicated that with raising MNEO levels, the moisture content of composite films reduced. This may be because of the interactions of −OH and COO − groups of MNEO with the −OH and −NH 2 in the G-CP polymeric matrix. This interaction, in turn, may have led to decreased interactions between G-CP and moisture molecules. These results were similar to those achieved by Kadam et al [32] for chitosan-based films. Similarly, reduction in moisture contents of the CuSNP-containing films with increasing CuSNP levels could be explained by the arrangement effect made between the copper atom and −OH/−NH 2 groups in the G-CP matrix, which also limited the interactions between hydrophilic groups in G-CP and water molecules. A similar result was observed in the chitosan film incorporated with silver nanoparticles and purple corn extract [39].
The colorimetric results in figure 2 show that the color parameters of the G-CP films are strongly affected by incorporation CuSNP, but the addition of MNEO does not effect on the color of the produced G-CP-based films. The brightness parameter L * reduces with a raising of the level of CuSNP. Also, as CuSNP content increased, a * and b * parameters were enhanced. Additionally, the total color difference (ΔE) of the G-CP films was enhanced with an increased percentage of CuSNP. This observation is in agreement with those reported by Roy et al [14] for the agar-based-CuSNP-containing film.

Mechanical properties
An edible film with desirable properties must have high tensile strength (TS) and elongation at break (EAB). The type of polymeric matrix and type and degree of interactions between ingredients influence on the mechanical behavior of blend film [22]. The TS and %EAB of G-CP-based blend films are shown in table 3. The addition of MNEO decreased the TS and increased %EAB. This indicated molecular interactions (electrostatic interactions and ester linkages) between MNEO and G-CP chains, these, in turn, affect the protein-protein chain interactions and provide the flexible domains within the composite films with the lesser tensile strength [32]. After incorporation of CuSNP in G-CP composite film, TS and EAB% decreased (table 3). This can be attributed to the aggregation of the nanoparticles, as observed in the SEM results (figure 3(C)) [40]. Due to the synergistic effect of MNEO and CuSNP, G-CP-MNEO 0.5−CuSNP 0.03 film exhibited the highest TS than other active films (G-CP-MNEO 0.5 and G-CP-CuSNP 0.03) (p<0.05).

Antimicrobial property
The antimicrobial attributes of the G-CP-based blend films were examined versus both Gram-positive (S. aureus) and Gram-negative (E. coli) microorganisms, and findings are shown in table 3. Results of the inhibition zone revealed that, in general, blending G-CP edible film with the MNEO and CuSNP caused effective antibacterial activity versus the S. aureus and E. coli. As anticipated, the G-CP composite film did not show any antibacterial activity, but G-CP films containing MNEO and CuSNP showed varied antibacterial activity depending on the species of bacteria. The GCP-CuSNP 0.03 composite films demonstrated stronger antibacterial activity against E. coli than S. aureus. A comparable effect of antimicrobial activity was observed in alginate-based composite films blended with CuSNP [40]. The various antibacterial action of CuSNP depending on the type of bacteria is maybe because of the different cell wall structures and morphological differences of these bacteria [14]. The Gram-negative organisms are composed of complex cell wall structure with a thin peptidoglycan layer surrounded by an outer phospholipidic membrane, on the contrary, the Gram-positive organisms have a thick cell wall structure with a multilayer of peptidoglycan [14]. Although until now antibacterial activity of CuSNP has not been clearly outlined yet and not clearly understood, however, it is believed that free copper ions (Cu++) be able to interact with the negatively charged microorganism cells membrane protein and demolish the microorganism cell wall [40]. Another possible mechanism is the antioxidant defense or interaction antioxidant defense of CuSNP with bacterial, which activates intracellular reactive oxygen species-mediated oxidative damage to antioxidant resistance and damages bacterial cell membranes, leading to microbial cell death [14,41]. The results of the antibacterial activity of films containing MNEO show that S. aureus is more sensitive against MNEO than E. coli. As mentioned earlier, the reason for this difference could be allocated to the morphological distinction among the bacteria, as a result, Gram-negative organisms like E. coli due to having an outer layer of phospholipid membrane that carrying the lipopolysaccharide ingredients makes the cell wall strong and impenetrable to lipophilic compounds like MNEO [42,43]. The antimicrobial mechanism of the essential oils (EOs) is also attributed to the disabling of the cytoplasmic membrane and disrupting of the cellular energy metabolisms [42]. Notably, G-CP/MNEO/CuSNP film exhibited the strongest antibacterial property (p<0.05), indicating that the synergistic effect of MNEO and CuSNP has improved the antimicrobial properties of the film and this active film could be utilized as antibacterial wrapping material in the food industry.

FTIR
FTIR analysis was used to evaluate the chemical structure and possible interactions between nanocomposite films different components. FTIR spectra of all components individually and FTIR spectra of different produced nanocomposite films were shown in figure 3(A). As can be seen in figure 3(A). (a and b) the absorption band at 1242 cm −1 , 1544 cm −1 , and 1664 cm −1 was attributed to the presence of amide-III (C-N and N-H stretching), amide-II (N-H bending), and Amide-I (C=O stretching) functional groups in gelatin and chickpea protein structure, respectively. Also, the wide absorption band at 3600-3100 cm −1 was attributed to O-H and N-H vibration stretching [5,36]. The spectrum of MNEO was shown in figure 3(A).  spectrum can be observed in the spectrum of the nanocomposite film incorporated with MEO ( figure 3(A). (f and h)). However, some peaks were shifted to a higher or lower frequency or their amplitude changed which is attributed to the conformational changes of functional groups as a result of components' different interactions [36]. As can be seen in figure 3(A) (g) there are no notable changes in active groups by incorporation of CuSNP. These findings demonstrated that no new chemical interaction takes place among the other ingredients and CuSNP and the changes in peak intensities could be because of van der Walls or H-bonding among CuSNP and blend film matrix [14].

XRD
The XRD patterns of the control film (G-CP) showed a broad peak at 2θ of 34°, typical of protein materials which illustrate its amorphous structure (figures 3(B)-(a)) [45]. . The mentioned peaks were also can be seen in the XRD pattern of nanocomposite film containing both MNEO and CuSNPs (figures 3(B)-(d)) which confirm that no new chemical interaction carried out between the other components and CuSNPs [14]. On the other hand, the broad peak at the XRD pattern of control film (G-CP) approximately disappeared in the XRD pattern of this sample which indicated the perfect homogeneous distribution of CuSNP in the proteinous matrix of nanocomposite film.

SEM
The SEM surface micrographs of prepared nanocomposite films were shown in figures 3(C)-(a). A smooth and homogenous surface without any observable crack was observed for the control film (G-CP). The observed homogenous surface structure indicates that fish skin gelatin and chickpea protein have great compatibility to be mixed. As can be seen in figures 3(C)-(b) the surface morphology of nanocomposite film became slightly rough by the incorporation of MNEO. The homogeneity of mentioned roughness confirms the homogeneous distribution of MNEO in the film matrix. A similar observation was reported by Kadam et al, [32]. As can be seen in figures 3(C)-(c) some granular protrusions were appeared by the incorporation of CuSNP, which decreased the uniformity of the nanocomposite film surface. The appearance of granular protrusions was most likely because of the aggregation of some CuSNP [1,14]. The mentioned effects of incorporation of MNEO and CuSNP (figures 3(C)-(d)) were also observable in the surface micrograph of nanocomposite film containing both MNEO and CuSNP. showed that the addition of MNEO and CuSNP in G-CP film significantly increases the melting temperature (Tm) and glass transition temperature (Tg) of the produced films. Also, the simultaneous use of MNEO and CuSNP has a significant synergistic effect in improving the Tm of composite films. This increase in Tm and Tg was probably due to the inherent nature of MNEO and CuSNP, changes in the degree of crystallinity with incorporation of MNEO and CuSNP in protean matrix and increased CuSNP/MNEO interactions with G-CP matrix [32,40]. G-CP displayed a Tm of 88°C whereas the all composites showed higher Tm.

Physicochemical analyses of minced meat
Changes in pH values of fresh minced meat wrapped with G-CP protein-based films during refrigerated storage for 14 days are presented in table 5. The initial (1 day) pH value of the control film (G-CP) increased from 5.85 to 6.35 after storage for 14 days. Generally, pH of all samples slightly raised, when the storage time increased (P<0.05). Such a raise in pH indicates a degree of minced meat spoilage through higher bacterial growth and microbial enzymatic actions leading to the formation and accumulation of alkaline compounds such as ammonia and amines, etc [46]. Samples packaged in G-CP films containing MNEO and CuSNP alone and in combination with together has lower pH compared to control film (G-CP) throughout the storage period (P<0.05), showing the protective effects of the MNEO and CuSNP against substrate decomposition. Also, the pH value in the packaged sample with G-CP composite film enriched with MNEO and CuSNP were less than other minced meat samples wrapped with G-CP-MNEO 0.5 and G-CP-CuSNP 0.03 active films (p<0.05), that this indicates the positive synergistic effect of MNEO and CuSNP in the packaging film to reduce meat spoilage. These findings confirm the results of other physicochemical and bacterial analyses of wrapped minced meat and are by other studies [15,46]. The effect of G-CP film containing MNEO and CuSNP on the changes of TVB-N of minced meat during the 14 days' storage at refrigerated temperature are shown in table 5. The primary TVB-N of 7.00 mg N/100 g values were incremented progressively in all samples (P<0.05), and reached to 37 mg N/100 g for G-CP (control sample). Previous studies have reported that the increase of TVB-N with storage period is likely because of spoilage bacteria and the formation of compounds such as dimethylamine, methylamine, ammonia and trimethylamine [47]. As it was observed, TVB-N values in both samples of meat packed in MNEO-containing films (G-CP-MNEO 0.5 and G-CP-MNEO 0.5-CuSNP 0.03) were less than the other samples, although this value in the sample wrapped in G-CP-CuSNP 0.03 film was significantly lower than in the control sample (P<0.05) [48], indicating antibacterial properties of CuSNP. Generally, the lower and acceptable TVB-N values in treated minced meat samples could be associated with the antibacterial activities of examined preservative agents. MNEO and CuSNP remarkably reduced the bacterial population of the meat sample than G-CP and subsequently decreased the oxidative deamination and accumulation of non-protein nitrogen and other volatile compounds [49].
The changes of TBA values during storage at refrigerated temperature of minced meat wrapped with G-CP or G-CP-MNEO 0.5, G-CP-CuSNP 0.03 and G-CP-MNEO 0.5−CuSNP 0.03 films are presented in table 5 to evaluate the impact of these films on minced meat lipid oxidation. TBA value has been extensively used to assess the extent of fat oxidation in meat, meat products and meat by-product [50]. The primary TBA value of all treatments was 0.4 mg MDA/kg sample. Generally, TBA values of all minced meat treatments raised regularly up to 14 days of storage (p <0.05). However, the minced meat samples wrapped with G-CP-MNEO 0.5, G-CP-CuSNP 0.03 and G-CP-MNEO 0.5-CuSNP 0.03 films had much lower TBA values, tan sample wrapped with control G-CP film. These findings may be related to the high capacity of the MNEO and CuSNP on preventing the oxidation of fatty acids of meat through antioxidant properties and inhibition of microbial growth. Since fat oxidation can be initiated, extended, inhibited or reduced through control of several mechanisms including the enzymatic and non-enzymatic generation of free radicals, production of singlet oxygen and active oxygen [51], the strategy of using MNEO and CuSNP in packaging film has been very effective. Also, during the storage time, the TBA values were lower in G-CP active film containing both MNEO and CuSNP than in the other ones, this effect confirms the positive synergy of these two compounds for preventing the increase of TBA value due to various reactions, in G-CP based film.

Bacteriological analysis of minced meat
One of the most important parameters limiting the shelf life of meat is microbial growth [46]. As meat bacterial spoilage is initially considered typically a superficial phenomenon, minced meat becomes more sensitive to microbial spoilage via an enlargement in the surface and also the ease of bacterial entrance from the surface following spreading. Table 5 shows the changes of L. monocytogenes, S. aureus, Enterobacteriaceae and Pseudomonas spp. the population of minced meat packaged by G-CP film incorporated with MNEO and CuSNP during refrigerated storage. Generally, all films in this study represented significant inhibitory effects (p<0.05) against studied bacteria than the control sample (G-CP). It should be noted that, in all treatments, the bacterial population (S. aureus, Enterobacteriaceae and Pseudomonas spp. population) were considerably increased, except L. monocytogenes population, which is decreasing for meat sample wrapped with active films (G-CP-MNEO 0.5, G-CP-CuSNP 0.03 and G-CP-MNEO 0.5-CuSNP 0.03) over 14 days of storage at different rates (p<0.05). The highest and lowest bacterial population were found for untreated (G-CP film) and treated samples with G-CP containing both MNEO 0.5 and CuSNP 0.03 (G-CP-MEO 0.5-CuSNP 0.03) respectively. The following sequence inhibition effect on all studied bacterial groups was found in selected films: G-CP-MNEO 0.5-CuSNP 0.03>G-CP-MNEO 0.5>G-CP-CuSNP 0.03>G-CP. These findings suggest that MNEO and CuSNP could inhibit bacterial growth. Also, the simultaneous existence of MNEO and CuSNP in the matrix of the blended film more suppressed the growth of all tested bacterial groups in comparison with the other active films (P0.05). This, as mentioned earlier, shows the antimicrobial synergistic effect of MNEO and CuSNP. The antimicrobial effect of MNEO is possibly to be related to the effect of a bacterial membrane and leads to changes in the permeability of cations and eventually cell death [42]. Generally, EOs coagulate the cytoplasm, denaturation of cellular proteins and enzymes, and inhibition of DNA, RNA and protein synthesis of bacterial cells and also, disrupt the electron motive force and proton [15]. Also, the adding of CuSNP into the G-CP-based films prevents microbial growth through inhibition of cytoplasmic membrane function of bacteria [40] and interfering and altering with energy metabolisms of bacteria [14]. It should be noted that the function and effectiveness of incorporated MNEO and CuSNP in the G-CP based films through indirect and direct displacement from the film to minced meat depends on the nature of the antibacterial ingredients. Nonvolatile ingredients like CuSNP transfer through diffusion and need direct touch among the films and the minced meat. In contrast, volatile ingredients like MNEO release to the headspace, tan penetrate the minced meat surface and are adsorbed into its [15]. Therefore, it can be concluded that this is one of reasons for the greater antimicrobial action and effectiveness of MNEO compared to CuSNP. These results are in agreement with those reported by Gomez-Estaca et al [52] and Ahmad et al [53] who observed an antibacterial effect of essential oils to extending the shelf life of the fish and lemongrass essential oil to extend the shelf-life of the sea bass slices, respectively.

Data availability statement
All data that support the findings of this study are included within the article (and any supplementary files).