Development of antioxidant edible films based on mung bean protein enriched with pomegranate peel

Journal Pre-proof Development of antioxidant edible films based on mung bean protein enriched with pomegranate peel

Maryam Moghadam, Maryam Salami, Mehdi Mohammadian, Maryam Khodadadi, Zahra Emam-Djomeh PII:

S0268-005X(19)32771-7

DOI:

https://doi.org/10.1016/j.foodhyd.2020.105735

Reference:

FOOHYD 105735

To appear in:

Food Hydrocolloids

Received Date:

28 November 2019

Accepted Date:

02 February 2020

Please cite this article as: Maryam Moghadam, Maryam Salami, Mehdi Mohammadian, Maryam Khodadadi, Zahra Emam-Djomeh, Development of antioxidant edible films based on mung bean protein enriched with pomegranate peel, Food Hydrocolloids (2020), https://doi.org/10.1016/j. foodhyd.2020.105735

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Development of antioxidant edible films based on mung bean protein enriched with pomegranate peel

Maryam Moghadam1, Maryam Salami1*, Mehdi Mohammadian1, Maryam Khodadadi2, Zahra Emam-Djomeh1

1Department

of Food Science and Engineering, University College of Agriculture & Natural

Resources, University of Tehran, Karaj, Iran. 2Research

and Development, Eversea Inc., Prince Edward Island, Canada

*Corresponding author. Email address: [email protected]

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Abstract In this study, mung bean protein films enriched with different concentrations (0, 2.5, 12.5, and 25% w/w based on protein content) of pomegranate peel as a rich source of bioactives were prepared and characterized. The incorporation of pomegranate peel increased the thickness, water vapor permeability, water contact angle, tensile strength, and flexibility of the films, but decreased their solubility and moisture content. The color parameters (L*, a*, and b*) of mung bean protein film were significantly affected by the addition of pomegranate peel. The surface and cross-section of films were further studied using scanning electron microscopy (SEM). In addition, observations made by Fourier transform infrared (FT-IR) spectroscopy suggested the formation of hydrogen bonds in the film matrix. X-ray diffraction (XRD) analysis confirmed that the amorphous nature of mung bean protein film was not affected by enriching with pomegranate peel. The films enriched with pomegranate peel also showed higher total phenolic content, antioxidant activity (measured by ABTS and DPPH radical scavenging and reducing power), and antibacterial capacity compared to the control mung bean protein film. Generally, the results emphasized the potential use of mung bean protein and pomegranate peel as by-products of food industry to develop bio-functional edible films intended for packaging of food products. Keywords: Active packaging; Mung bean protein; Pomegranate peel; Bioactive compounds; Mechanical properties; Bio-functional attributes

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1. Introduction Population growth leads to increased consumption of plastic-based packaging and environment concerns about the disposal of these non-degradable materials. Nowadays, people have chosen to use renewable active packaging systems that are environmental friendly (Fathi, Almasi, & Pirouzifard, 2019). Bio-based active packaging systems are usually composed of biopolymers that include proteins, polysaccharides, lipids, or their mixture. These packaging systems can be a convenient replacement for the synthetic polymers such as plastics (Emam-Djomeh, Moghaddam, & Yasinin Ardakani, 2015). Protein-based edible films have better mechanical attributes, barrier characteristics, and nutritional-promoting properties in comparison with the polysaccharide and lipid-based counterparts (Abdelhedi et al., 2018). Legume seeds are low-cost sources of plant proteins with diverse nutritional characteristics that make them potent to be the main components of bio-based films (Ebrahimi, Koocheki, Milani, & Mohebbi, 2016). Mung bean (Vigna radiate L.) is a favorable green seed that belongs to the legume family that has been widely cultivated in Asian countries for many years (Budseekoad et al., 2018). Mung bean contains 25-28% protein is a source of bioactive compounds such as flavonoids, phenolic acids, organic acids, and polysaccharides (Chai et al., 2018). They possess with

antioxidant,

antidiabetic,

anticholesteromic,

anticancer

(Chandrasiri,

Liyanage,

Vidanarachchi, Weththasinghe, & Jayawardana, 2016), anti-tumor, and detoxifying properties (Du et al., 2018). There are three main mung bean products, vermicelli (glass noodles), flour, and sprout. Starch is the main constituent extracted from mung beans, and is utilized in vermicelli production; however, the remaining protein-rich by-product is treated as a waste stream and it could be used as a valuable source of various amino acids like lysine (Budseekoad et al., 2018). Therefore, valorization of this protein-rich by-product of starch production from mung beans 3

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would reduce the costs of waste disposal and protein manufacturing, and could provide the required raw ingredients for products such as edible films. Recently, researchers have focused on the incorporation of natural bioactive compounds like αtocopherol, phenolic compounds, and essential oils into packaging materials to produce active edible films to enhance the shelf-life of food products and maintain their safety and quality without the use of synthetic preservatives (Abdelhedi et al., 2018; Adilah, Jamilah, Noranizan, & Hanani, 2018). Fruit peels are rich sources of bioactive compounds and natural antioxidants that have been widely used in the formulation of biopolymer-based edible films to improve their biofunctionalities. Apple (Henriquez, Cordova, Lutz, & Saavedra, 2013), banana (Kamel, Elmessieh, & Saleh, 2017), pomelo (Wu et al., 2019), pomegranate (Hanani, Yee, & Nor-Khaizura, 2019), papaya and jackfruit (Hanani, Husna, Syahida, Khizura, & Jamilah, 2018), blood orange (Jridi et al., 2019), and potato (Borah, Das, & Badwaik, 2017) peels have been employed to boost the antioxidant and antimicrobial activities of edible films from biopolymers. Pomegranate (Punica granatum L.) is a super fruit, native to Iran (Emam-Djomeh et al., 2015). In addition to its well-known effects in prevention and treatment of cardiovascular diseases, this fruit also possesses significant health-promoting properties such as anticancer, antioxidant, and antimicrobial activity (Emam-Djomeh et al., 2015; Hanani et al., 2019). Pomegranate peel is a noteworthy source of phenolic compounds (ellagic acid, lignins, catechin, epicatechin, and ellagitannins) that account for 78% of pomegranate juice manufacturing waste stream. Pomegranate peels are often discarded without regard for their valuable properties (Ali et al., 2019). Therefore, this by-product could be an economic source of phenolic compounds that could be used to enrich mung bean protein-based edible films and increase their antioxidant properties. 4

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There is no research on the fabrication of active and antioxidant films based on mung bean protein. The purpose of current research was to produce biodegradable and bioactive edible packaging systems that used mung bean protein incorporated with different amounts of pomegranate peel as a low-cost source of bioactive compounds. Next, we investigated their physical, mechanical, optical, structural, antioxidant and antibacterial characteristics as a novel active packaging system. 2. Materials and methods 2.1. Materials Mung bean and pomegranate peel powder were obtained from a local market in Iran. 2,2'azinobis-(3-ethylbanzthiazo-line-6-sulfonate) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin-Ciocalteu, nutrient broth, and nutrient agar were purchased from Sigma-Aldrich (Sa. Louis, MO, USA). Glycerol was acquired from Merck (Darmstadt, Germany). All other chemicals were also obtained from Merck and Sigma-Aldrich with analytical grade. 2.2. Extraction of mung bean protein The mung bean protein isolate was prepared according to El-Adawy (2000) with minor modifications. For this purpose, mung bean seeds were peeled and ground to obtain mung bean flour. Next, 5% (w/v) dispersion of mung bean flour in deionized water was prepared and the pH of resulted solution was adjusted to 9.0 with 1.0 N NaOH, stirred for 1.0 h at room temperature, and centrifuged at 5000 g for 15 min. The obtained supernatants were collected and pH was adjusted to 4.5 with 1.0 N HCl followed by another centrifugation round at 5000 g for 15 min. The precipitate was collected, washed with distilled water, lyophilized, and kept at -20°C until further use. To determine the protein content of obtained powder, the Bradford method with 5

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bovine serum albumin as a standard was employed which showed a protein concentration of 87.62% (Bradford, 1976). 2.3. Preparation of films The mung bean protein solution (4% w/v) was prepared in distilled water. The pH of solution was adjusted to 8.0 with NaOH 5.0 N, and stirred overnight using a magnetic stirrer at room temperature for complete hydration. The obtained solution was heated at 85°C for 30 min to denature the protein fraction, followed by cooling down to 25°C. The pomegranate peel powder at concentrations of 0, 2.5, 12.5, and 25% w/w based on dry protein content was added to the protein solution, and stirred for 2 h. Ultrasound technique (100 W for 5 min) was utilized to improve the solubility of mixture, then the solution was centrifuged at 4000 rpm for 10 min to remove any large particles. Finally, the supernatant was collected and mixed with glycerol (50% w/w based on protein) as a plasticizer, followed by stirring and degasification. 15 mL of the prepared solution was cast on Petri dishes (10 cm diameter) and allowed to be dried at 40°C for 12 h. The dried films were peeled from the surface of the plates and conditioned for 24 h at 25°C and 53% relative humidity (RH). This equilibrium RH was provided by the saturated solution of magnesium nitrate. The resulting film samples are shown in Fig. 1. 2.4. Film thickness The thickness of the films (mm) was measured using a manual micrometer with a sensitivity of 0.01 mm. Each film sample was measured at ten random positions and the average value was reported. 2.5. Moisture content (MC)

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The MC of films was evaluated by the method of Carpine, Dagostin, Bertan, and Mafra (2015). The samples were weighed, placed into a pre-weighed aluminum capsule, and dried at 110°C for 24 h in an oven. To calculate the MC, the Eq. 1 was applied: 𝑀𝐶 (%) =

𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑓𝑖𝑙𝑚 𝑤𝑒𝑖𝑔ℎ𝑡 ― 𝑑𝑟𝑖𝑒𝑑 𝑓𝑖𝑙𝑚 𝑤𝑒𝑖𝑔ℎ𝑡 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑓𝑖𝑙𝑚 𝑤𝑒𝑖𝑔ℎ𝑡

× 100

[Eq. 1]

2.6. Water solubility (WS) The WS of films was measured using the procedure of Ebrahimi et al. (2016). For this purpose, 1 cm × 4 cm pieces of films were dried at 110°C for 24 h. The dried samples were immersed in 40 mL of distilled water and stirred for 24 h at room temperature. The remaining non-soluble parts of the films were separated and dried at 110°C in a laboratory oven, and their final weights were measured. The percentage of WS was obtained with Eq. 2:

𝑊𝑆 (%) =

𝑊𝑖 ― 𝑊𝑓 𝑊𝑖

× 100

[Eq. 2]

where Wi and Wf are the initial and final weight of films, respectively. 2.7. Contact angle The surface wettability of film samples was studied from the contact angle measurement between the surface of film and water using a video-based contact angle meter (Kruss G10 goniometer, Kruss, Germany) by the sessile drop method as described by Abdelhedi et al. (2018). For this purpose, 10 μL of distilled water was placed on the surface of each film and the angle between water drop and surface of films was measured on both sides of the water drop. 2.8. Water vapor permeability (WVP)

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The gravimetric method was employed to investigate the WVP of the film samples (Motedayen, Khodaiyan, & Salehi, 2013). The films were cut into suitable sizes and fixed onto the mouth of glass cups (internal diameter of 1.1 cm and a depth of 6.3 cm) which contained about 30 g anhydrous calcium chloride (CaCl2) with ~0% RH. Thereafter, the cups were transferred into a desiccator which was saturated with NaCl (75% RH) solution and kept for 24 h at 25°C. The changes in the weight of the cups were periodically recorded every 1 h during the first 8 h and finally after 24 h. The WVP (g m-1 s-1 Pa-1) was determined with the Eq. 3: ∆𝑤 × 𝑋

𝑊𝑉𝑃 = 𝑡 × 𝐴 × ∆𝑝

[Eq. 3]

where Δw is the weight change of cup (g), X is the average film thickness (m), t is the time (s), A is the area of the exposed film (m2), and Δp is the water vapor pressure difference across the two sides of the film (2376.39 Pa). 2.9. Mechanical attributes To determine the mechanical attributes including tensile strength (TS) and elongation at the break (EAB), a texture analyzer (TexturePro CT V1.8 Build 31, Brookfield, US) was employed. The prepared films were cut into strips of 20×80 mm and then fixed on the grips of the texture analyzer with an initial gap distance of 30 mm and a crosshead speed of 1.0 mm/s. 2.10. Scanning electron microscopy (SEM) The microstructure (surface and cross-section) of film samples was imaged with scanning electron microscope (VEGA-TESCAN, Czech Republic). The samples were dried at 40°C for 12 h, and then were fixed followed by coating with an appropriate gold layer. The micrographs were captured at an accelerating voltage of 15.0 kV. 8

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2.11. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy The chemical structure analysis of composite film samples was carried out with a FT-IR spectrometer (Bruker, MA, USA) equipped with an attenuated reflection accessory (ATR). A wavenumber of 4000 to 500 cm-1 was selected to record the FT-IR spectra. 2.12. X-ray diffraction (XRD) analysis The XRD was used to investigate the structural changes of the edible films using the X-ray diffractometer (PW1730, PANalytical, Netherlands) with Cu Kα radiation source working at 40 kV and 30 mA in the 2θ range of 10°-35°. 2.13. Color parameters and opacity To demonstrate the color attributes of mung bean protein-based films with different concentrations of pomegranate peel powder, a Minolta colorimeter (CR-300, Japan) was utilized that was calibrated using a standard white plate. Color parameters that included L*, a*, and b* were measured, respectively, showing the lightness (0 to 100), green/red (-60 to +60), and blue/yellow (-60 to +60) of the samples. The opacity of the film samples was also evaluated by a spectrophotometer. For this purpose, the absorbance of each film was read at 600 nm with a UV/vis spectrophotometer and the obtained values were divided by the film thickness (Abdalrazeq et al. 2019). 2.14. Total phenolic content (TPC) The Folin-Ciocalteu method was used to measure the phenolic compounds according to Liang and Wang (2018) with some modifications. For this purpose, 50 mg of films were dipped in 10 mL of distilled water and then stirred for 4 h at 25°C to prepare the film extract. Briefly, 0.5 mL 9

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of the film extract were mixed with 2 mL of Folin-Ciocalteu reagent (10% v/v) and kept for 3 min in a dark place. Then, 2.5 mL of sodium carbonate solution (7.5% w/v) was added to the mixture and kept for 2 h in the dark at room temperature. Finally, the absorbance of the solutions was read at 765 nm with a spectrophotometer. A standard curve (0-100 μg/mL) was plotted using gallic acid as the standard component. The results were reported as the milligram of gallic acid equivalent per unit gram weight of the film (mg GAE/g film). 2.15. Antioxidant properties To investigate the antioxidant properties, the extracts from films were obtained by immersing 50 mg of film samples in 10 mL of distilled water, stirred for 2 h followed by centrifugation at 4000 rpm for 5 min, and the supernatant was employed as extract for evaluating the following antioxidant properties. 2.15.1. ABTS radical scavenging activity The ABTS+ radical scavenging activity of the film extracts was performed according to Yu et al. (2018) with slight modification. In brief, the ABTS solution (7.4 mM) was prepared and mixed with potassium persulfate (2.6 mM). The mixture was transferred to a dark place and stored for 16 h at room temperature to form the ABTS radical. The absorbance of the mixture was adjusted to 0.700 ± 0.001 at 734 nm by diluting the stock solution with distilled water. Then, 100 μL of film extracts or 100 μL of distilled water as the control were mixed with 1.0 mL of ABTS solution, and kept in a dark place for 10 min. The absorbance of the samples was read at 734 nm and the scavenging activity was determined using Eq. 4:

𝐴𝐵𝑇𝑆 𝑟𝑎𝑑𝑖𝑐𝑎𝑙 𝑠𝑐𝑎𝑣𝑒𝑛𝑔𝑖𝑛𝑔 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (%) =

𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙 ― 𝐴𝑠𝑎𝑚𝑝𝑙𝑒 𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙

10

× 100

[Eq. 4]

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2.15.2. DPPH radical scavenging assay The activity of film extracts to scavenge the DPPH free radical was performed according to Parveen, Chaudhury, Dasmahapatra, and Dasgupta (2019) with some adjustments. The DPPH solution (0.1 mM) was prepared in ethanol. Then, 0.5 mL of each extract as well as the control sample (0.5 mL distilled water) was added to 1.5 mL of the DPPH solution. The absorbance was read at 517 nm and the DPPH scavenging activity was obtained using the Eq. 5: DPPH scavenging activity (%) = 1 ―

𝐴𝑠 𝐴𝑐

× 100

[Eq. 5]

where As and Ac are the measured absorbance of the sample and control, respectively. 2.15.3. Reducing power assay The ability of edible films to reduce ferric ion was tested. For this purpose, 0.5 mL of each film extract was mixed with 1.25 mL potassium ferricyanide (1% w/v) and 1.25 mL of phosphate buffer (0.2 M, pH 6.6) followed by incubating at 50°C for 20 min. The obtained solution was mixed with 1.25 mL of TCA (10%) and centrifuged at 4000 rpm for 10 min. At last, 1.25 mL of the supernatant was added to 1.25 mL distilled water that contained 250 μL FeCl3 (0.1%) and the absorbance was recorded at 700 nm (Abdelhedi et al., 2018). 2.16. Antimicrobial properties Qualitative antibacterial activity of the edible films was characterized by measuring the diameter of disk inhibition zone against Escherichia coli O157:H7 and Listeria monocytogenes following the procedure of Zhang et al. (2019). The films were cut into a round shape that had a 10 mm diameter, sterilized utilizing an ultraviolet lamp for about 20 min, and then placed on the surface of a solid culture medium, which had been inoculated with the above-mentioned pathogens. The 11

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samples were incubated for 24 h at 37°C. At the end of the incubation period, the diameters of the clear zones around discs were measured. 2.17. Statistical analysis SPSS software version 16 was employed to analyze the obtained results by subjecting to oneway analysis of variance (ANOVA). The significance of the differences between means was evaluated at p < 0.05 with Duncan’s multiple range tests. Experiments were performed with at least three replications. 3. Results and discussion 3.1. Film thickness The thickness of mung bean protein film without and with pomegranate peel is reported in Table 1. The thickness of the mung bean protein was notably increased after enrichment with pomegranate peel (p < 0.05). The increase of film thickness due to the addition of the polyphenol-rich compounds was attributed to the interactions between the functional groups of the biopolymer and the phenolic hydroxyl groups present in the bioactive compounds of pomegranate peel which can lead to form a film with a higher thickness (Riaz et al., 2018). Our results are agreed with those reported by Hanani et al. (2019) and Riaz et al. (2018) who investigated the effects of pomegranate peel powder and apple peel polyphenols on thickness of fish gelatin and chitosan films, respectively. 3.2. Moisture content and water solubility The MC of different film samples is presented in Table 1. The MC of mung bean protein film decreased slightly following the addition of pomegranate peel. This could be due to addition of 12

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pomegranate peel led to changes in the hygroscopic properties of the mung bean protein as a result of presence of hydrophobic components (Hanani et al., 2019). In harmony with our findings, Shams, Ebrahimi, and Khodaiyan (2019) also observed that the moisture level of whey protein-gelatin film decreased after enrichment with orange peel due to the increased film hydrophobicity which limited water absorption and retention ability of the films. WS of the film samples is shown in Table 1. The highest WS was related to the control sample (32.31%) and its solubility was significantly (p < 0.05) decreased by adding of pomegranate peel. However, in the case of peel-enriched sample, no significant change was observed in the solubility of films by increasing the pomegranate peel concentration. This decrease in the WS of mung bean protein film could be related to the formation of hydrogen bonds between the polyphenols of the pomegranate peel and protein molecules, which could limit the formation of interactions between protein hydrophilic groups and water molecules (Riaz et al., 2018). The lower WS of edible films that contained pomegranate peel can be due to the insolubilized parts of the pomegranate peel that remained at the end of experiment (Hanani et al., 2019). In accordance with these results, it was investigated that the solubility of gelatin/polyethylene bilayer films was reduced by enriching with different fruit (pomegranate and jackfruit) peels (Hanani et al., 2018). 3.3. Contact angle and WVP Contact

angle

measurement

is

broadly

used

to

determine

the

wettability

and

hydrophilic/hydrophobic properties of films. In simple terms, when the contact angle parameter is less than 90°, the surface of the film is considered hydrophilic and it is considered hydrophobic when the contact angle value is higher than 90° (Hannani et al., 2018). Table 1

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presents the water contact angle of mung bean protein-based films enriched with different concentrations of pomegranate peel. The pure mung bean protein film exhibited contact angle of 44.23° and the addition of pomegranate peel at a level of 2.5% had no significant effect on its contact angle value. However, the contact angle was increased significantly by adding 12.5 and 25% of pomegranate peel into the mung bean protein-based film. This decrease in hydrophilicity can be due to the occurring of new interactions and decreasing of free hydrophilic groups (Fathi et al., 2019) as a result of enriching with pomegranate peel. In fact, the significant changes in the contact angle through the addition of high concentrations of pomegranate peel can be attributed to the effect of pomegranate peel addition on the surface roughness, pore size, and heterogeneity (Hannani et al., 2018) which is supported by the SEM results. However, it should be noted that although the surface hydrophobicity of films increased by enriching with pomegranate peel, generally all of the mung bean protein films with incorporated pomegranate peel were still considered hydrophilic since their contact angle was lower than 90°. The WVP of mung bean protein films enriched with different amounts of pomegranate peel was assessed (Table 1). The WVP of the control mung bean protein film was 2.99 10-10 g m-1 s-1 Pa-1. The enriching of film with 2.5% of pomegranate peel had no significant effect on the WVP of mung bean protein film. However, the addition of pomegranate peel at the concentrations of 12.5 and 25% resulted in a significant increase in WVP. This could be due to the effect of the high concentrations of pomegranate peel on the morphology of film samples as evaluated by SEM. It was observed that the film samples with pomegranate peel were more heterogeneous than control film which can increase the water penetrability. In fact, the permeability of a film can be influenced by different factors such as the hydrophobic/hydrophilic nature of the materials used for the preparation of film, the presence of cracks or voids, and also the steric hindrance and 14

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tortuosity in the structure (Matta, Tavera-Quiroz, & Bertola, 2019). Therefore, according to the results of the SEM, it seems that the addition of pomegranate peel at high concentrations resulted in the formation of some voids and agglomerated particles on the surface of the mung bean protein films which can be accounted for the higher WVP. It seems that the void spaces between polymer chains were increased through the addition of pomegranate peel which can increase the WVP. Indeed, although the addition of pomegranate peel into the mung bean protein-based film increased its hydrophobicity according to the results of moisture content, water solubility, and contact angle measurements, it seems that the incomplete dissolution of pomegranate peel in the film matrix resulted in a more heterogeneous microstructure providing more space for water vapor to pass through the film. In this regard, Borah et al. (2017) reported that the utilization of the high concentrations of potato peel in the biopolymer film resulted in a higher WVP compared to the films with low concentration of peel due to the formation of a film matrix with larger pore size despite its denser structure. In accordance, Hanani et al. (2019) reported that the enrichment of gelatin films with pomegranate peel powder significantly increased their WVP due to their more heterogeneous microstructure resulted from the incomplete dissolution of pomegranate peel in the film matrix. Moreover, the presence of soluble fibers, pectin, and starch in the pomegranate peel may influence the WVP of mung bean protein films. The hygroscopicity of these compounds as well as their plasticization effects can increase the water absorption capacity of the protein-based films (Matta et al., 2019). These observations suggested that the mung bean protein films with high content of pomegranate peel are not appropriate candidates for the applications that need an excellent barrier to water vapor. 3.4. Mechanical properties

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The mechanical properties of film samples including TS and EAB describing the strength and flexibility (Ruan et al., 2019) of samples were evaluated (Table 1). The TS and EAB of mung bean protein-based film were changed significantly (p < 0.05) by adding different concentration of pomegranate peel. There were increases in TS and EAB with increased pomegranate peel concentration. The highest values of TS and EAB were related to the mung bean protein film with 25% of pomegranate peel. Therefore, both strength and flexibility of the mung bean protein film could be improved by enriching with pomegranate peel. It seems that the addition of pomegranate peel can increase the free volume, molecular mobility, and flexibility of the mung bean protein chains and therefore, increase TS and EAB. In fact, the mechanical properties of a composite film are dependent on the type and degree of interactions between the film components (Ebrahimi et al., 2016). Therefore, the changes in the mechanical attributes of mung bean protein film can be due to the possible interaction between the compounds present in pomegranate peel and the mung bean protein in the film matrix. The compounds present in pomegranate peel can form complexes with mung bean proteins through different interactions including hydrogen bonding, electrostatic interactions, and hydrophobic forces which can modify the mechanical properties of the resulting films (Emam-Djomeh et al., 2015). In this regard, polyphenols of the pomegranate peel can create crosslinks between proteins, which increase the strength of film, but also they can form polyphenol-protein links that interfere with proteinprotein linkages resulting in a film with lower strength and higher flexibility. The formation of these interactions and linkages in the present study appears to be in an optimal state which can lead to the formation of more flexible and cohesive film matrices. In agreement with our findings, it was investigated that the TS and EAB of gelatin films were increased by the addition of curcuma extract caused by forming of covalent cross-links (Bitencourt, Fávaro-Trindade,

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Sobral, & Carvalho, 2014). In addition to the polyphenols, other components present in the pomegranate peel such as soluble fibers, complex polysaccharides, pectin, and starch may influence the mechanical properties of the mung bean protein films due to their plasticizing effects which can improve the flexibility and stretch-ability of the film samples (Hanani et al., 2018). In addition to the plasticizing effect, the complex polysaccharides of pomegranate peel such as pectin can interact with the phenolic compounds of the pomegranate peel and improve the strength of the protein film which was also reported by Hanani et al. (2019). Generally, it seems that the mechanical properties of mung bean protein-based film including TS and EAB were improved by enriching with pomegranate peel due to the balance between interaction of different materials in the film matrix, including protein, polysaccharides, and phenolic compounds. 3.5. Film microstructure The surface and cross-sectional morphologies of different samples obtained by SEM are presented in Fig. 2. There were no cracks in the surface areas of any of the film samples and there was a continuous microstructure without any pores. A homogenous and smooth surface morphology was observed for the control mung bean protein film suggesting the formation of continuous network of mung bean protein molecules in the matrix of film. Addition of pomegranate peel at the concentration of 2.5% had no significant effect on the surface morphology of film. However, some small white dots were observed on the surfaces of films which were incorporated with pomegranate peel at the concentrations of 12.5 and 25%. These white dots can be related to the insoluble particles of pomegranate peel embedded in the films. Therefore, films with more heterogeneous microstructures were formed at the higher enriching levels of pomegranate peel which can be accounted for their higher permeability towards 17

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moisture. In agreement, Hanani et al. (2019) also observed these small particles on the surface of fish gelatin films enriched with pomegranate peel attributing to the presence of insoluble fibers in the peel. Riaz et al. (2018) also reported that when apple peel was added into chitosan films, white spots observed in the SEM images. Cross-section images showed that all of the samples had compact and continuous network integrity without any voids. However, some layers were detected for control mung bean protein film in contrast to those which were enriched by pomegranate peel. Therefore, it seems that films with more compact structures were formed in the presence of pomegranate peel in comparison with single-component protein-based film which can be due to the formation of intermolecular forces (such as hydrogen bonds and hydrophobic interactions) between the protein chains and the bioactive molecules present in the pomegranate peel which was also investigated by EmamDjomeh et al. (2015) in the case of sodium caseinate-based film enriched with the extract of pomegranate peel. 3.6. FTIR spectroscopy The infrared spectra of mung bean protein films with different amounts of pomegranate peel are shown in Fig. 3. A sharp peak at wavenumber of 1041 cm-1 was observed for all of the film samples attributing OH group of glycerol confirming that the matrix of film was successfully incorporated with glycerol as plasticizer (Chentir et al., 2019). The spectrum of control mung bean protein showed characteristic peaks at 1632 cm-1 and 1535 cm-1 corresponding to the occurrence of amide I (primarily related to the stretching vibrations of C=O) and amide II (attributed to N-H bending and C-N stretching vibrations), respectively (Kudre, Benjakul, & Kishimura, 2013). These peaks in the amide I and II regions are commonly used to study the

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secondary structural properties of proteins. Addition of pomegranate peel at the concentration of 2.5 and 12.5% had no significant effect on the position of these peaks. However, shifts from 1632 cm-1 to 1636 cm-1 and from 1535 cm-1 to 1540 cm-1 were observed after enriching of protein film with 25% of pomegranate peel. These observations suggested that the molecular organization of mung bean protein in the film matrix was changed through the incorporation of pomegranate peel especially at higher concentrations of peel which can be due to its interaction with the polypeptide chain of mung bean protein. Parveen et al. (2019) also similarly reported that the FT-IR spectrum of film made of cataractous eye protein isolate was altered by adding of gallic acid as a bioactive ingredient. The control mung bean protein film also showed characteristic peaks at 3285 cm-1 corresponding to the O-H stretching overlapping N-H stretching vibrations and 2936 cm-1 attributing to the symmetric and asymmetric C-H stretching vibrations (Chentir et al., 2019). It was observed that the positions of these two characteristic peaks were shifted to the lower wavenumbers after the incorporation of pomegranate peel. Changes in the position of these peaks could be associated with the formation of hydrogen bonds in the film matrix (Butnaru et al., 2019). Therefore, it seems that the above-mentioned peak displacements can be a result of hydrogen bonds formation between mung bean protein and polyphenols available in the pomegranate peel. In agreement, it was reported that the hydrophobic and hydrogen bonds play the most important roles in the interaction of proteins and phenolic compounds (Mohammadian et al., 2019). 3.7. XRD The XRD profiles of films made of mung bean protein and different concentrations of pomegranate peel are displayed in Fig. 4. A broad band peak at 2θ around 20° was observed for different film samples, which is usually attributed to the amorphous phase of proteins (Babaei, 19

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Mohammadian, & Madadlou, 2019). The enrichment of mung bean protein film with pomegranate peel did not influence on its amorphous character. In fact, the pomegranate peel did not impart any crystalline structure to binary films suggesting that the possible interaction between mung bean protein and the bioactive compounds of pomegranate took place in the amorphous phase. In accordance with our findings, Liang and Wang (2018) also indicated that the enriching of soy protein isolate film with the extract of cortex Phellodendron did not change its dominant amorphous nature with respect to the appearance of any new peak in the XRD pattern. Therefore, the XRD analysis suggested that there is a good compatibility between pomegranate peel and mung bean proteins for the fabrication of edible films. 3.8. Color properties and opacity of films The color parameters (L*, a*, b*) and opacity of edible films obtained from mung bean protein with different concentrations of pomegranate peel are presented in Table 2. The incorporation of pomegranate peel increased the values of a*, b*, and opacity of the mung bean protein film. These parameters were also significantly (p < 0.05) increased by increasing of the content of pomegranate peel in the matrix of film samples. However, the brightness (L* value) of the film samples was reduced by increasing the pomegranate peel content. Therefore, it seems that the darkness, yellowness, and redness of mung bean protein film were increased by adding of pomegranate peel which can be due to the presence of anthocyanins in the pomegranate peel. The anthocyanins are responsible for the orange, red, and purple colors of plants and fruits (Hanani et al., 2019). In accordance with our finding it was reported that the color properties of chitosan-pullulan (Kumar, Ojha, & Singh, 2019) and sodium caseinate (Emam-Djomeh et al., 2015) edible films were significantly affected through the incorporation of pomegranate peel extract. 20

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3.9. TPC and antioxidant activity The Folin-Ciocalteu method was used to measure the amount of phenolic compounds of the film samples (Table 3). The incorporation of pomegranate peel had a significant effect on the TPC of mung bean protein film. The TPC increased from 3.48 mg GAE/g film for the peel-free film to 13.88 mg GAE/g for the film sample that contained 25% of pomegranate peel. The low TPC observed for the control mung bean protein film could be due to the presence of amino acid residues such as tyrosine and histidine in the mung bean protein that had the ability to react with the Folin-Ciocalteu reagent (Wu et al., 2019). The higher TPC of film samples enriched with different concentrations of pomegranate peel in comparison with the control sample can be due to the fact that the pomegranate peel is a rich source of phenolic compounds such as catechins, punicalin, pedunculagin, punicalagin, gallic acid, and ellagic acid (Smaoui et al., 2019). A previous study confirmed that the phenolic activity of chitosan and pullulan edible films dramatically improved by enriching with the extract of pomegranate peel (Kumar et al., 2019). TPC is important because its higher content can produce a higher antioxidant film. The impact of pomegranate peel on the antioxidant properties of mung bean protein films was studied by measuring their ability to scavenge the free radicals of DPPH and ABTS, as well as their reducing power capacity (Table 3). The results indicated that the anti-radical activity and reducing power of the mung bean protein film was significantly (p < 0.05) increased as the pomegranate peel content increased. This increase could be associated with the higher TPC of the films and the higher content of the pomegranate peel. Generally, our findings showed that the antioxidant activity of the mung bean protein-based film greatly improved after enriching with pomegranate peel, which might be of benefit for certain commercial uses like packaging of food products that have high susceptibility to oxidation. Hanani et al. (2019) also indicated that the 21

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ABTS and DPPH scavenging activity of fish gelatin film was drastically enhanced by adding of pomegranate peel powder attributing to the presence of bioactive phenolic compounds such as punicalagin in the pomegranate peel which is a potent antioxidant. Similar results were also investigated by Kumar et al. (2019), who showed that the antioxidant property of chitosanpullulan edible films improved through enriching with the extract of pomegranate peel. 3.10. Antibacterial properties The antimicrobial property of films against Gram-positive (L. monocytogenes) and Gramnegative (E. coli) bacteria strains was assessed by determining the inhibition zones and results are reported in Table 3. The control mung bean protein film did not show any antibacterial activity against the tested strains; meanwhile, film samples that contained pomegranate peel exhibited better inhibition of L. monocytogenes and E. coli. Higher concentrations of the pomegranate peel showed increased antibacterial activity. The greatest inhibition zone was related to the mung bean protein film with 25% pomegranate peel. These observations agreed with Ali et al. (2019) who investigated that the antimicrobial activity of starch-based film against Staphylococcus aureus and Salmonella was improved by the addition of pomegranate peel. The antibacterial activity of pomegranate peel was attributed to the presence of tannins (especially punicalagins) and polyphenols (mainly ellagic acid) which can act as antibacterial agents through different mechanisms such as microbial enzyme inhibition, reduction of membrane fluidity or by membrane perforation (Mushtaq, Gani, Gani, Punoo, & Masoodi, 2018). The results indicated that the film samples with pomegranate peel were slightly more effective against L. monocytogenes compared to E.coli, which agreed with the previous studies indicating that the phenolic compounds were more effective against the Gram-positive bacteria compared to the Gram-negative bacteria (Hanani et al., 2019). Therefore, the films made of mung bean protein 22

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and pomegranate peel can be considered promising and effective candidates for developing antimicrobial packaging systems. 4. Conclusion In the present research, mung bean protein-based edible films were successfully enriched with different concentrations of pomegranate peel as a source of bioactive compounds to develop an antioxidant and antibacterial packaging system. Enrichment with pomegranate peel increased the thickness, contact angle, and WVP of the mung bean protein films, but decreased their MC and WS. The mechanical properties, reducing power, anti-radical activity, and antibacterial attributes of mung bean protein films were considerably improved by incorporating of pomegranate peel. The optical and microstructural attributes of mung bean protein film were changed by enriching with pomegranate peel. These changes were more noticeable at the higher concentrations of pomegranate peel. SEM, FT-IR, and XRD observations also showed that the compatibility between pomegranate peel and film matrix was quite good. Generally, this study suggested that mung bean protein, as a novel plant protein could be employed as a promising film-forming biopolymer. Pomegranate peel, a low-cost by-product of the food industries, also has a worthy potential to be incorporated into mung bean protein film to improve its bio-functional properties. Acknowledgment This research was carried out by the support of University of Tehran. References

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Abdalrazeq, M., Giosafatto, C. V. L., Esposito, M., Fenderico, M., Di Pierro, P., & Porta, R. (2019). Glycerol-plasticized films obtained from whey proteins denatured at alkaline pH. Coatings, 9(5), 322.

Abdelhedi, O., Nasri, R., Jridi, M., Kchaou, H., Nasreddine, B., Karbowiak, T., Debeaufort, F., & Nasri, M. (2018). Composite bioactive films based on smooth-hound viscera proteins and gelatin: Physicochemical characterization and antioxidant properties. Food Hydrocolloids, 74, 176-186.

Adilah, A. N., Jamilah, B., Noranizan, M. A., & Hanani, Z. N. (2018). Utilization of mango peel extracts on the biodegradable films for active packaging. Food Packaging and Shelf Life, 16, 1-7.

Ali, A., Chen, Y., Liu, H., Yu, L., Baloch, Z., Khalid, S., Zhu, J., & Chen, L. (2019). Starchbased antimicrobial films functionalized by pomegranate peel. International Journal of Biological Macromolecules, 129, 1120-1126.

Babaei, J., Mohammadian, M., & Madadlou, A. (2019). Gelatin as texture modifier and porogen in egg white hydrogel. Food Chemistry, 270, 189-195.

Bitencourt, C. M., Fávaro-Trindade, C. S., Sobral, P. J. D. A., & Carvalho, R. A. D. (2014). Gelatin-based films additivated with curcuma ethanol extract: Antioxidant activity and physical properties of films. Food Hydrocolloids, 40, 145-152.

24

Journal Pre-proof

Borah, P. P., Das, P., & Badwaik, L. S. (2017). Ultrasound treated potato peel and sweet lime pomace based biopolymer film development. Ultrasonics Sonochemistry, 36, 11-19.

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(12), 248-254.

Budseekoad, S., Yupanqui, C. T., Sirinupong, N., Alashi, A. M., Aluko, R. E., & Youravong, W. (2018). Structural and functional characterization of calcium and iron-binding peptides from mung bean protein hydrolysate. Journal of Functional Foods, 49, 333-341.

Butnaru, E., Stoleru, E., Brebu, M. A., Darie-Nita, R. N., Bargan, A., & Vasile, C. (2019). Chitosan-based bionanocomposite films prepared by emulsion technique for food preservation. Materials, 12(3), 373.

Carpine, D., Dagostin, J. L. A., Bertan, L. C., & Mafra, M. R. (2015). Development and characterization of soy protein isolate emulsion-based edible films with added coconut oil for olive oil packaging: Barrier, mechanical, and thermal properties. Food and Bioprocess Technology, 8(8), 1811-1823.

Chai, W. M., Ou-Yang, C., Huang, Q., Lin, M. Z., Wang, Y. X., Xu, K. L., Huang, W. Y., & Pang, D. D. (2018). Antityrosinase and antioxidant properties of mung bean seed proanthocyanidins: Novel insights into the inhibitory mechanism. Food Chemistry, 260, 27-36. 25

Journal Pre-proof

Chandrasiri, S. D., Liyanage, R., Vidanarachchi, J. K., Weththasinghe, P., & Jayawardana, B. C. (2016). Does processing have a considerable effect on the nutritional and functional properties of Mung bean (Vigna radiata)?. Procedia Food Science, 6, 352-355.

Chentir, I., Kchaou, H., Hamdi, M., Jridi, M., Li, S., Doumandji, A., & Nasri, M. (2019). Biofunctional gelatin-based films incorporated with food grade phycocyanin extracted from the Saharian cyanobacterium Arthrospira sp. Food Hydrocolloids, 89, 715-725.

Du, M., Xie, J., Gong, B., Xu, X., Tang, W., Li, X., Li, C., & Xie, M. (2018). Extraction, physicochemical characteristics and functional properties of Mung bean protein. Food Hydrocolloids, 76, 131-140.

Ebrahimi, S. E., Koocheki, A., Milani, E., & Mohebbi, M. (2016). Interactions between Lepidium perfoliatum seed gum–Grass pea (Lathyrus sativus) protein isolate in composite biodegradable film. Food Hydrocolloids, 54, 302-314.

El-Adawy, T. A. (2000). Functional properties and nutritional quality of acetylated and succinylated mung bean protein isolate. Food Chemistry, 70(1), 83-91.

Emam‐Djomeh, Z., Moghaddam, A., & Yasini Ardakani, S. A. (2015). Antimicrobial activity of pomegranate (Punica granatum L.) peel extract, physical, mechanical, barrier and antimicrobial

26

Journal Pre-proof

properties of pomegranate peel extract‐incorporated sodium caseinate film and application in packaging for ground beef. Packaging Technology and Science, 28(10), 869-881.

Fathi, N., Almasi, H., & Pirouzifard, M. K. (2019). Sesame protein isolate based bionanocomposite films incorporated with TiO2 nanoparticles: Study on morphological, physical and photocatalytic properties. Polymer Testing, 105919.

Hanani, Z. N., Husna, A. A., Syahida, S. N., Khaizura, M. N., & Jamilah, B. (2018). Effect of different fruit peels on the functional properties of gelatin/polyethylene bilayer films for active packaging. Food Packaging and Shelf Life, 18, 201-211.

Hanani, Z. N., Yee, F. C., & Nor-Khaizura, M. A. R. (2019). Effect of pomegranate (Punica granatum L.) peel powder on the antioxidant and antimicrobial properties of fish gelatin films as active packaging. Food Hydrocolloids, 89, 253-259.

Henríquez, C., Córdova, A., Lutz, M., & Saavedra, J. (2013). Storage stability test of apple peel powder using two packaging materials: High-density polyethylene and metalized films of high barrier. Industrial Crops and Products, 45, 121-127.

Jridi, M., Boughriba, S., Abdelhedi, O., Nciri, H., Nasri, R., Kchaou, H., Kaya, M., Sebai, H., Zouari, N., & Nasri, M. (2019). Investigation of physicochemical and antioxidant properties of gelatin edible film mixed with blood orange (Citrus sinensis) peel extract. Food Packaging and Shelf Life, 21, 100342.

27

Journal Pre-proof

Kamel, N. A., El-messieh, S. L. A., & Saleh, N. M. (2017). Chitosan/banana peel powder nanocomposites for wound dressing application: Preparation and characterization. Materials Science and Engineering: C, 72, 543-550.

Kudre, T. G., Benjakul, S., & Kishimura, H. (2013). Comparative study on chemical compositions and properties of protein isolates from mung bean, black bean and bambara groundnut. Journal of the Science of Food and Agriculture, 93(10), 2429-2436.

Kumar, N., Ojha, A., & Singh, R. (2019). Preparation and characterization of chitosan-pullulan blended edible films enrich with pomegranate peel extract. Reactive and Functional Polymers, 144, 104350.

Liang, S., & Wang, L. (2018). A Natural antibacterial-antioxidant film from soy protein isolate incorporated with cortex phellodendron extract. Polymers, 10(1), 71.

Matta, E., Tavera-Quiroz, M. J., & Bertola, N. (2019). Active edible films of methylcellulose with extracts of green apple (Granny Smith) skin. International Journal of Biological Macromolecules, 124, 1292-1298.

Mohammadian, M., Salami, M., Momen, S., Alavi, F., Emam-Djomeh, Z., & MoosaviMovahedi, A. A. (2019). Enhancing the aqueous solubility of curcumin at acidic condition through the complexation with whey protein nanofibrils. Food Hydrocolloids, 87, 902-914.

28

Journal Pre-proof

Motedayen, A. A., Khodaiyan, F., & Salehi, E. A. (2013). Development and characterisation of composite films made of kefiran and starch. Food Chemistry, 136(3-4), 1231-1238.

Mushtaq, M., Gani, A., Gani, A., Punoo, H. A., & Masoodi, F. A. (2018). Use of pomegranate peel extract incorporated zein film with improved properties for prolonged shelf life of fresh Himalayan cheese (Kalari/kradi). Innovative Food Science & Emerging Technologies, 48, 25-32.

Parveen, S., Chaudhury, P., Dasmahapatra, U., & Dasgupta, S. (2019). Biodegradable protein films from gallic acid and the cataractous eye protein isolate. International Journal of Biological Macromolecules, 139, 12-20.

Riaz, A., Lei, S., Akhtar, H. M. S., Wan, P., Chen, D., Jabbar, S., Abid, M., Hashim, M. M., & Zeng, X. (2018). Preparation and characterization of chitosan-based antimicrobial active food packaging film incorporated with apple peel polyphenols. International Journal of Biological Macromolecules, 114, 547-555.

Ruan, C., Zhang, Y., Wang, J., Sun, Y., Gao, X., Xiong, G., & Liang, J. (2019). Preparation and antioxidant activity of sodium alginate and carboxymethyl cellulose edible films with epigallocatechin gallate. International Journal of Biological Macromolecules, 134, 1038-1044.

29

Journal Pre-proof

Shams, B., Ebrahimi, N. G., & Khodaiyan, F. (2019). Development of Antibacterial Nanocomposite: Whey Protein-Gelatin-Nanoclay Films with Orange Peel Extract and Tripolyphosphate as Potential Food Packaging. Advances in Polymer Technology, 1973184.

Smaoui, S., Hlima, H. B., Mtibaa, A. C., Fourati, M., Sellem, I., Elhadef, K., Ennouri, K., & Mellouli, L. (2019). Pomegranate peel as phenolic compounds source: Advanced analytical strategies and practical use in meat products. Meat Science, 107914.

Wu, H., Lei, Y., Zhu, R., Zhao, M., Lu, J., Xiao, D., Jiao, C., Zhang, Z., Shen, G., & Li, S. (2019). Preparation and characterization of bioactive edible packaging films based on pomelo peel flours incorporating tea polyphenol. Food Hydrocolloids, 90, 41-49.

Yu, Z., Sun, L., Wang, W., Zeng, W., Mustapha, A., & Lin, M. (2018). Soy protein-based films incorporated with cellulose nanocrystals and pine needle extract for active packaging. Industrial Crops and Products, 112, 412-419.

Zhang, C., Wang, Z., Li, Y., Yang, Y., Ju, X., & He, R. (2019). The preparation and physiochemical characterization of rapeseed protein hydrolysate-chitosan composite films. Food Chemistry, 272, 694-701.

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We confirm that the authors of this manuscript have no conflict of interest with anyone.

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Credit author statement Maryam Moghadam : Data curation, Methodology, Writing- Original draft preparation Maryam Salami : Conceptualization, Methodology, Visualization, Reviewing and Editing Mehdi Mohammadian : Data curation, Writing- Original draft preparation Maryam Khodadadi: Visualization, Reviewing and Editing Zahra Emam-Djomeh: Visualization,

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Figure captions

Fig. 1 Scheme explaining the fabrication process of edible films made of mung bean protein enriched with different amounts of pomegranate peel. Fig. 2 SEM images of the surface (1000 × magnification) and the cross-section (500 × magnification) of mung bean protein films incorporated with 0 (PP-0), 2.5 (PP-2.5), 12.5 (PP12.5), and 25% (PP-25) of pomegranate peel. Fig. 3 FT-IR spectra of mung bean protein films incorporated with 0 (PP-0), 2.5 (PP-2.5), 12.5 (PP-12.5), and 25% (PP-25) of pomegranate peel. Fig. 4 XRD patterns of mung bean protein films incorporated with 0 (PP-0), 2.5 (PP-2.5), 12.5 (PP-12.5), and 25% (PP-25) of pomegranate peel.

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Figures

Fig. 1

Fig. 2

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Fig. 3

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Fig. 4

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Highlights



Active films were prepared consisting mung bean protein and pomegranate peel (PP).



The presence of PP improved the mechanical properties of mung bean protein film.



The functional properties of mung bean protein films were modified by adding PP.



The antioxidant and anti-bacterial activity were improved by enriching with PP.

Tables

Table 1 Thickness, moisture content (MC), water solubility (WS), contact angle, water vapor permeability (WVP), tensile strength (TS), and elongation at break (EAB) of mung bean protein films containing different concentrations of pomegranate peel (PP).

PP (%)

Thickness (mm)

MC (%)

WS (%)

Contact angle (°)

WVP ( × 10-10 g m-1 s-1 Pa-

TS (MPa)

EAB (%)

)

1

0

0.082 ± 0.005c

24.61 ± 0.13a

32.31 ± 1.55a

44.23 ± 0.42c

2.99 ± 0.08c

3.33 ± 0.32d

81.18 ± 1.90c

2.5

0.085 ± 0.004c

24.07 ± 0.23ab

27.64 ± 1.93b

45.34 ± 0.50c

3.09 ± 0.34c

4.11 ± 0.26c

97.60 ± 1.08b

12.5

0.110 ± 0.008b

23.44 ± 0.31bc

27.60 ± 0.64b

49.30 ± 0.72b

3.61 ± 0.20b

5.16 ± 0.08b

163.96 ± 7.22a

25

0.145 ± 0.006a

22.82 ± 0.55c

27.16 ± 0.73b

53.50 ± 1.13a

4.30 ± 0.19a

5.84 ± 0.19a

172.96 ± 12.05a

Means with different letters within a column are significantly different (p < 0.05).

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Table 2 Color parameters and opacity of mung bean protein films containing different concentrations of pomegranate peel (PP). PP (%)

L*

a*

b*

Opacity (A600/mm)

0

89.47 ± 0.78a

-2.92 ± 0.41d

15.50 ± 0.84d

0.65 ± 0.07c

2.5

80.88 ± 1.67b

1.44 ± 0.49c

28.77 ± 1.98c

0.90 ± 0.07b

12.5

72.15 ± 1.46c

3.81 ± 0.44b

46.86 ± 0.88b

1.05 ± 0.02ab

25

63.11 ± 1.37d

11.66 ± 0.29a

53.89 ± 1.26a

1.18 ± 0.04ab

Means with different letters within a column are significantly different (p < 0.05).

Table 3 Total phenolic content (TPC), radical (ABTS and DPPH) scavenging activity, reducing power, and antibacterial activity of mung bean protein films containing different concentrations of pomegranate peel (PP). Diameter of inhibition zone (mm)

TPC

ABTS

DPPH

Reducing power

(mg GAE/g film)

scavenging (%)

scavenging (%)

(Abs 700 nm)

L. monocytogenes

E. coli

0

3.48 ± 0.08d

28.72 ± 0.83d

5.00 ± 0.74d

0.067 ± 0.017d

0.00 ± 0.00d

0.00 ± 0.00d

2.5

4.22 ± 0.04c

33.76 ± 2.44c

13.84 ± 2.10c

0.118 ± 0.006c

6.00 ± 0.10c

3.50 ± 0.50c

12.5

7.59 ± 0.10b

90.18 ± 3.86b

52.65 ± 2.18b

0.515 ± 0.014b

11.66 ± 1.52b

8.33 ± 1.52b

25

13.88 ± 0.13a

97.82 ± 0.24a

65.22 ± 2.02a

0.763 ± 0.028a

15.33 ± 1.52a

14.33 ± 1.52a

PP (%)

Means with different letters within a column are significantly different (p < 0.05).