Edible film based on corn zein containing dill extract and essential oil/β‐cyclodextrin inclusion complex: Shelf life enhancement of common carp fillet

Abstract The aim of this study was to examine the impacts of corn zein edible film (Z) fortified with dill leaves extract (DE) and encapsulated dill essential oil with β‐cyclodextrin (nDEO) on the quality of refrigerated common carp fillet. Gas chromatography/mass spectrometry (GC/MS) analysis showed that the most frequent substances of DEO were apiol (35.1%) and carvone (31.4%), respectively. Designated treatments were as follows: (1) Control (C), (2) Z, (3) Z‐DE, (4) Z‐DEO, (5) Z‐nDEO, (6) Z‐DE‐DEO, and (7) Z‐DE‐nDEO. The physicochemical properties (thickness, moisture percent, tensile strength, elongation at break, Young's modulus, color, morphology, functional groups, and thermal resistance) of the activated films significantly improved (p ≤ .05). The total viable counts, lactic acid bacteria, Enterobacteriaceae, and psychrotrophic bacteria significantly decreased in all wrapped fillets compared to the unwrapped ones (p ≤ .05). Throughout storage period, the wrapped fillets exhibited lower changes in pH, thiobarbituric acid reactive substances, and total volatile‐based nitrogen values than the unwrapped fillets. According to the sensory findings, incorporating DE and nDEO in the zein films created significantly desirable aroma and flavor in the wrapped samples during storage time (p ≤ .05). Encapsulation of DEO with β‐cyclodextrin significantly fortified preservative effects of the films in fish fillets during storage period (p ≤ .05). In conclusion, the designated composite zein edible film containing DE and nDEO can be introduced as an active edible packaging in the shelf life improvement of common carp fillets during cold storage.

been recently preferred by consumers compared to synthetic approaches. This can be due to economic, health, and environmental issues. Zein protein is derived from corn and due to its unique features, such as filmmaking, significant thermal stability and gas barrier are used in food packaging. Zein edible films and coatings create a transparent yellowish, brilliant, and elastic appearance and show exceptional preservative features in food packaging (Barkhordari & Bazargani-Gilani, 2021;Cui et al., 2020).
The inhibitory effects of edible films can be fortified by the incorporation of antimicrobial and antioxidant agents into them. Plant extracts and essential oils have been recently considered as natural preservatives. Dill with the scientific name of Anethum graveolens L.
is an annual fragrant plant from Umbelliferae family and belongs to West Asia and Mediterranean. Previous studies demonstrated the presence of many unique antioxidant, antimicrobial, fragrant, and flavoring components in dill products. They found strong antimicrobial and antioxidant activities in dill essential oil and extract, respectively. Due to the unique sensory features (taste and aroma) of dill herb as well as its significant biochemical properties, simultaneous usage of DE and DEO can create a perfect and complete food additive (Singh et al., 2005;Tavakkoli et al., 2020).
However, the active compounds of volatile essential oils can be easily neutralized in the presence of environmental agents, such as oxygen, light, temperature, etc. (Dias Antunes et al., 2017;Rakmai et al., 2017). Furthermore, vigorous taste and aroma of essential oils may inappropriately affect the organoleptic features of foods. Also, fast evaporation and high water insolubility can limit the contact of essential oils with pathogens. Encapsulation by cyclodextrins can increase the stability and solubility of the essential oils in foods (Dias Antunes et al., 2017). Cyclodextrin molecules composed of the cyclic glucopyranosyl oligosaccharides linked by α-(1,4) bonds have two unique structures consisting of a hydrophilic area and hydrophobic calyx, which can assemble inclusion complex with many substrates.
Compatible cavity structure with common substrates, availability, and reasonable cost has caused β-cyclodextrin to have the highest consumption among cyclodextrins (Dias Antunes et al., 2017;Rakmai et al., 2017).
Therefore, due to the long-standing interest of Asians to consume dill plant along with common carp fillets, we intended to investigate the efficiency of zein edible film enriched with dill extract and essential oil/β-cyclodextrin inclusion complex in the shelf life improvement of common carp fillet under refrigerated (4 ± 1°C) storage in this study.

| Extraction and analysis of DEO
Anethum graveolens L. seeds were provided by the local grocery of Hamedan. The seeds were powdered by a homemade mill (Hardston).
The powdered seeds were extracted by hydro-distillation method using Clevenger apparatus (Simax, Pyrexfan) for 4 h (Tavakkoli et al., 2020). The extracted DEO was collected in closed vials containing anhydrous sodium sulfate for dehydration and stored at refrigeration conditions until the next use. The ingredients of DEO were identified by a gas chromatograph (GC) equipped with a mass spectrometer (MS; Hewlett-Packard).

| Provision of β -CD/DEO inclusion complex
Encapsulation of DEO by β-CD was performed based on the method of Dias Antunes et al. (2017). After dissolving 2 g of β-CD in distilled water (50 mL), 1.5 g of DEO was mixed with the solution under heating (35°C) and stirring (3 h). Then, the resulting solution was stored in the refrigerator for 24 h. The obtained precipitate in the solution was filtered through a Whatman No. 1 filter paper and then rinsed with ethanol (95%) and dried at 40°C for 24 h. The β-CD/DEO inclusion complex was stored in the refrigerator (4 ± 1°C) until the next use.

| Encapsulation efficiency
Encapsulation efficiency was determined based on the method of Bae and Lee (2008). Fifty milliliters of hexane was mixed with 2 g of IC and shaken for 2 min to extract free DEO. The obtained solution was then filtered through a Whatman No. 1 filter paper. After rinsing the remained powder with 20 mL of hexane solvent three times, it was dried at 60°C. The free DEO content was calculated by the weight difference of the inclusion complex powder before and after washing with hexane solvent. EE was computed by the following equation:

| Extraction of DE
Anethum graveolens L. leaves were procured from the greengrocery.
After washing and rinsing with potable water, the samples were dried in the shade at ambient temperature for 2 weeks. After grinding the dried leaves with the grinder (Hardston), the ratio of 1:10 of the sample to solvent [ethanol 70% (W/W)] was provided by the immersion method under shaking at 250 rpm for 24 h. After separating the solid and liquid phases by filtration through Whatman No. 1 filter paper, the liquid phase was concentrated by rotary evaporator apparatus (Lab Tech) at 40°C. Then, the remained solvent was evaporated by vacuum oven at 40°C. Then, the obtained extracts were stored at −18°C for the next tests (Singh et al., 2005).

| Thickness
Five different sites of the films were considered for thickness measurement using a digital micrometer (IP65 Alpa Exacto).

| Color
The color of the zein films was analyzed by a colorimeter (Minolta CR300 Series). The color indexes included L* (brightness/ darkness), a* (red/green), and b* (yellow/blue).

| Scanning electron microscopy
The morphological properties of the prepared films were determined by a scanning electron microscope (SEM; JEOL JSM-840). The studied films were covered with gold and images were taken at 30 kV with a magnification of ×10,000.

| Moisture
The moisture content of the films was evaluated by drying small pieces of the films at 105°C for 24 h. The weight of the samples was considered before (W0) and after (W1) drying in the oven . Moisture percent was computed as the weight difference of the samples before and after drying as follows: 2.6.6 | Fourier transform infrared spectroscopy Fourier transform infrared spectrometer (FTIR; Helios) was used to identify functional groups and molecular interactions among zein, DE, DEO, and nDEO. A scan range from 4000 to 500 cm −1 at a resolution of 4 cm −1 was considered for the film analysis.
2.6.7 | Thermogravimetric analysis and differential scanning calorimetry Thermal resistance of the studied films was determined by thermogravimetric analyzer (TGA) and differential scanning calorimetry (DSC; STA449F3, Netzsch). The temperatures were considered in the range of 25-500°C with a flow rate of argon at 20 mL/min and heating speed of 10°C/min .

| Preparation of the treatments
Fresh common carp fillets were provided by a local fish processing unit immediately after hunting and shipped to the laboratory in the containers containing ice packs. Fish fillets were cut into 10 g pieces and wrapped in the prepared films by heat stitching (Figure 1). The treated fillets were packaged in polyethylene zip packs and stored in refrigerator (4 ± 1°C) for the next analyses. Microbial, chemical, and sensory analyses were carried out at 3-day intervals to evaluate the shelf life and quality of the fish fillets for 12 days. The following treatments were studied: (1) Control (unwrapped fillets), (2) Z (wrapped fillets by zein films), (3) Z-DE (wrapped fillets by zein films F I G U R E 1 View of the prepared zein edible film (left side) and wrapped common carp fillet by zein film (right side).

| Microbial analysis
Total viable count, psychrotrophic bacteria, lactic acid bacteria (LAB), and Enterobacteriaceae populations of the samples were evaluated during storage days.
Fish fillet (10 g) was mixed with 90 mL of 0.1% sterile peptone water (Merck) in a sterile stomacher bag and stomached for 1 min.

| pH value
The pH value of the fillets was determined using a pH meter (Jenway). After homogenizing 5 g of the fish fillet with 25 mL of distilled water for 30 s, pH values of the obtained solutions were recorded (Brannan, 2008).

| TBARS value
Colorimetric technique was used for TBARS value determination based on the modified method of Pikul et al. (1989). TBARS value was recorded as milligram of malondialdehyde/kilogram of the sample. 1, 1, 3, 3-tetraethoxypropane (TEP) was used for drawing a calibration curve.

| Total volatile basic nitrogen value
The distillation method using a Kjeldahl set (Pyrexfan) was used for total volatile basic nitrogen value (TVB-N) value determination of the fish fillets based on the method of Fernández et al. (2009).
TVB-N values were calculated in milligram of nitrogen/100 g of the sample.

| Sensory analysis
The sensory evaluation was performed using the 5-point hedonic scale. Totally, 20 students of the Department of Food Hygiene and Quality Control participated in this test. They evaluated the odor, color, texture, and overall acceptability of the samples in each interval. Score 5 was considered an extremely liked sample and Score 1 was considered extremely disliked (Bazargani-Gilani & Pajohi-Alamoti, 2020; Tavakkoli et al., 2020).

| Scanning electron microscopy
The scanning electron microscopy (SEM) graphs of the studied films are illustrated in Figure 3a-f. A smooth, homogeneous, and compact appearance was observed in the Z film (Figure 3a). The changes in the morphology of the films can be linked to the miscibility of the TA B L E 2 Mechanical (stress, elongation at break, and young's modulus) and color (L*, a*, b*) properties of the studied films.

| Fourier transform infrared (FTIR) spectroscopy
Fourier transform infrared (FT-IR) was used to identify the functional groups and the type of chemical reactions or bonding in the considered films. According to Figure 2c, zein films showed a broad peak at 3404 and two sharp peaks at 3027 and 2877 cm −1 , attributed to the stretching vibration of N-H, O-H and H-C groups, respectively Dong et al., 2017); two broad bands at 1646 and 1525 cm −1 were also linked to stretching vibration of C=O (amide I) and C-N functional groups (amide II), respectively Dong et al., 2017;Ghamari et al., 2022). The bands situated at 1465 and 1377 cm −1 are ascribed to paired blending vibration with stretching one of CH 2 and CH 3 , respectively, while the peaks at 1065 and 1045 cm −1 revealed the stretching vibration of ether C-O and primary alcohol C-O groups, respectively Ghamari et al., 2022). Generally, peaks around 500-1500 cm −1 can be related to the existence of a functional group of polyphenols present in DE and DEO (Liu et al., 2021;Nunes et al., 2021;Zhou et al., 2021). As illustrated in Figure 2c,

| Thermogravimetric analysis
Thermal features of the studied films were analyzed by thermogravimetric analysis (TGA; Figure 2d) and DSC (Figure 2e).
According to Figure 2d, the weight loss of all films happened at three steps: first, at 100-150°C due to the moisture, solvent, and volatile compounds' loss in the zein films; second, at 150-270°C due to the decomposition of small functional groups with weak bounds; and third, at 270-400°C due to the complete destruction of the films. According to the graph, Z-DE-DEO film showed lower weight loss compared to the others at the same temperature and Z-DE-nDEO, Z-DEO, Z-nDEO, Z-DE, and Z were in the next ranks, respectively. In other words, the simultaneous addition of DE, DEO, or nDEO to zein polymer caused the superior thermal resistance to free zein film. This phenomenon can be due to the potent interactions and intermolecular bonds among Z, DE, DEO, and nDEO and also higher boiling points of the molecules present in DE and DEO, resulting in higher thermal resistance of the studied films (Fazli et al., 2016;Surendhiran et al., 2020).

| Differential scanning calorimetry
Thermal behavior of the considered films was also shown using differential scanning calorimetry (DSC). In order to further study the variation of crystallinity, the DSC method was utilized to obtain the relevant thermal energy as a function of temperature. Generally, in DSC graph, an exothermic peak indicates an exothermic reaction caused by crystallization and the endothermic peak refers to an endothermic reaction by melting . The created exothermic peaks in the thermographs of the films cab were due to their transitions or reactions during the heating process. DSC thermograms of the studied films are illustrated in Figure 2e. According to the obtained results, by increasing the temperature, all the studied films showed an exothermic peak in certain temperatures that can be related to the crystallization phenomenon, and then graphs descended in the higher temperatures which can be due to the melting initiation.
The exothermic peak of Z-DEO treatment appeared at the highest temperature (241°C) among the others and Z-DE-nDEO (240°C), Z-DE-DEO (229°C), Z-DE (227°C), Z (216°C), and Z-nDEO (215°C) treatments were in the next ranks, respectively. It seems that adding the DEO and DE to zein film led to the formation of the proper molecular interaction and increasing crystallinity, following more thermal stability of them in comparison with the free zein films (Hu et al., 2015;Surendhiran et al., 2020). TGA and DSC findings demonstrated the excellent thermal stability up to 400°C of the combined films containing Z, DE, and DEO blends. In agreement with FT-IR results, it seems that this formulation could fortify zein film structure.  Z-DEO, Z-DE, Z, and control were in the next ranks during cold storage, respectively. β-CD/DEO inclusion complex significantly (p ≤ .05) exhibited stronger antibacterial activities than free DEO which can be attributed to its high stability due to the protective effect of β-CD, less evaporation, and controlled release compared to the free DEO (Dias Antunes et al., 2017). Previous research showed that carvone and limonene of DEO possessed significant antibacterial and antifungal activities. Also, DE, rich in linoleic acid, anethole, and dill apiole displayed a broad spectrum of antibacterial effects against the types of positive and negative gram bacteria (Singh et al., 2005).

| Sensory analysis
The sensory characteristics (odor, color, texture, and overall acceptability) of the fish fillets are illustrated in Figure 6a-

| CON CLUS ION
It can be concluded that adding DE and DEO to zein edible film not only improved its mechanical features, thermal stability, and appearance but also successively upgraded the preservative effects (antioxidant and antimicrobial activities) of zein film in the shelf life enhancement of refrigerated common carp fillets. Also, the designated formulation created palatable sensory properties, including overall acceptability, odor, texture, and color in common carp fillets compared to pure zein films. Physicochemical activities of β-CD/DEO inclusion complex significantly increased in comparison with free DEO by less evaporation, low movement into the food or bonding with food particles, and controlled release (p ≤ .05). Although the simultaneous addition of DE and nDEO to the designated Z film decreased its optimum mechanical properties, preservative effects of Z-DE-nDEO in shelf life enhancement of common carp fillet were very impressive. Therefore, considering the consumer demands for natural products, Z-DE-nDEO was introduced as the most promising biodegradable active film for food packaging, but in order to improve the mechanical properties of this film, using other techniques such as nanoemulsion for using DE and DEO in the films and other types of film production techniques, such as electrospinning method for the fabrication of this film is proposed for the future studies. Finally, releasing rates of DE, DEO, and nDEO from zein film need to be assessed.

ACK N O WLE D G E M ENTS
The present research was supported by the Faculty of Veterinary Sciences at Bu-Ali Sina University, Hamedan, Iran.

FU N D I N G I N FO R M ATI O N
This research received no specific grant from any funding source.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author.

E TH I C S S TATEM ENT
This study does not involve any human or animal testing.