The synergistic effects of zinc oxide nanoparticles and fennel essential oil on physicochemical, mechanical, and antibacterial properties of potato starch films

Abstract The purpose of this study was to evaluate the effects of a combination of zinc oxide (ZnO‐N) nanoparticles and fennel essential oil (FEO) on the functional and antimicrobial properties of potato starch films. Films based on potato starch containing a combination of ZnO‐N (1, 3, and 5%(w/w)) and FEO (1, 2, and 3% (w/w)) produced by casting method and water solubility, water absorption capacity (WAC), barrier properties, mechanical properties, color indexes, and antimicrobial activity of the films against Staphylococcus aureus, Escherichia coli, and Aspergillus flavus were studied. The combination of ZnO‐N and FEO had a significant decreasing effect on solubility, WAC, water vapor and oxygen permeability, elongation, and L* index. These additives had an increasing impact on tensile strength, Yang's modulus, and a* and b* indexes (p < .05). By increasing the concentration of ZnO‐N and FEO, the antimicrobial activities of bionanocomposite films significantly increased (p < .05). Both ZnO‐N and FEO had a significant effect in this respect, although the effects of ZnO‐N were more significant. In conclusion, an excellent synergistic effect of ZnO‐N and FEO was observed in potato starch films.


| INTRODUC TI ON
Today, the demand for packaging films based on biopolymers has increased because, unlike synthetic polymers, they are environmentally friendly (Toscano Ávila et al., 2020;Xue Mei et al., 2020). These biopolymers include polysaccharides, proteins, and lipids (Islam et al., 2020;Jahdkaran et al., 2021). Starches are one of the most important biopolymers used to make biocomposites. They are nontoxic, available, biodegradable, and renewable and have low prices (Esfahani et al., 2020). Potato starch contains high amounts of amylopectin, and its granule size is large compared to other starches. This starch offers several advantages in paste clarity, transparency, and extensibility (Alcázar-Alay & Meireles, 2015; Aminian et al., 2013).
Since biopolymer-based films usually have poor barrier and mechanical properties, to overcome these problems, either these polymers are used in combination, or plasticizers, fillers, and/ or cross-linking agents are used in biocomposite films. The use of nanoparticles in packaging films is one of the best ways to improve physicochemical and functional characteristics such as antioxidant and antimicrobial activity (Ahmadi et al., 2019;Jafarzadeh et al., 2021). Metal and metal dioxide nanoparticles are most widely used in packaging films due to their optical characteristics, good flexibility, impermeability to gases, and antimicrobial activity (Jafarian et al., 2020).
ZnO is a metal nanoparticle with antimicrobial activity approved by the Food and Drug Administration (FDA) (Li et al., 2019). These nanoparticles have good biocompatibility and thermal stability and are nontoxic (Huang et al., 2017). Researchers have shown that ZnO nanoparticles show strong and significant antibacterial activity against both gram-positive and gram-negative bacteria (Shahvalizadeh et al., 2021).
Plant essential oils (EOs) extracted from different parts of plants have a complex combination of various active and aromatic compounds (Chang et al., 2021;Plant et al., 2019). EOs often demonstrate significant antimicrobial and antioxidant activities and can be used as a substitute for synthetic preservatives in the food industry (Moslehi et al., 2021;Mousavian et al., 2021). Sun et al. (2020), in an investigation of the effect of ZnO nanoparticles and mulberry extract on the chitosan/konjac glucomannan films, demonstrated that the use of these additives in film samples improves barrier, mechanical, thermal stability, optical properties, and antimicrobial activity. In another study, it was found that titanium dioxide and Zataria multiflora essential oil had synergistic effect on each other and improved the basic and antimicrobial activity of chitosan/whey protein-based bionanocomposite films (Gohargani et al., 2020).
Fennel, with the scientific name of Foeniculum vulgare L., is a plant of the Apiaceae family that is widely cultivated all over the world due to its aromatic seeds and leaves. The essential oil of fennel is an important source of medicinal and active compounds such as antioxidants and antimicrobial compounds (Chang, Mohammadi Nafchi, et al., 2016). The antimicrobial activity of fennel essential oil has been reported in different studies (Chang, Abbaspour, et al., 2016;Gonçalves et al., 2020).
There is no report on the effect of ZnO nanoparticles and fennel essential oil on potato starch edible films that is yet available to the best of our knowledge. Therefore, the purpose of this study was to develop an active bionanocomposite film based on starch potato containing a combination of ZnO nanoparticles and fennel essential oil and to investigate the physicochemical, mechanical, and antimicrobial activity of obtained bionanocomposite films.

| Preparation of potato starch/ ZnO-N/fennel essential oil bionanocomposite films
To properly distribute the ZnO nanoparticles in the film structure, these nanoparticles were first dispersed uniformly in water, and then, this water was used to prepare potato starch dispersion. To prepare nanosolutions with concentrations of 1, 3, and 5% (nanoparticle weight to dry weight of starch), first, a suitable amount of ZnO nanoparticles was poured into 100 ml of distilled water. To ensure the homogeneity of the nanosolutions, they were homogenized in an ultrasonic bath (BANDELIN SONOREX Digitec, Germany) for 20 min. Then, 4 g of potato starch was added to the nanosolutions and mixed. After that, 2 g glycerol (50% w/w) was added as a plasticizer and stirred. While stirring, the suspension was heated to 90℃ and kept at this temperature for 45 min to complete gelatinization of the starch. While the mixture was cooling, a suitable amount of fennel essential oil (1, 2, and 3%) was added to the film solution at 45℃ temperature and became homogenous for approximately 30 min.
Then, about 90 g of the final mixture was poured on the Plexiglas and placed in a specific oven (Memmert, Germany) at 25℃ and 50% relative humidity for 24 hr to dry the film. Finally, the dried film was separated from Plexiglas and placed desiccator that relative humidity was fixed between 50% and 60% until the films were tested (Akbariazam et al., 2016;Teymourpour et al., 2015).

| Determination of the thickness of the films
The thickness of the starch films was measured by a caliper (QLR IP54, America) with an accuracy of 0.001 mm. Measurements were performed at three points on the films, and their average was used in calculations related to physicochemical properties tests such as water vapor permeability.

| Determination of water solubility
Initially, pieces of films were cut and stored in a desiccator with calcium chloride (0% relative humidity) and in an oven for 1 day.
The samples were then weighted and mixed in a beaker containing 100 ml of deionized water, then covered with aluminum foil, and placed at room temperature (20-25℃) for 24 hr. Meanwhile, the samples were gently stirred every 4 hr for 10 min. The remaining pieces of films were filtered with filter paper and placed in an oven at 30℃ to stabilize the weight. Finally, the water solubility percentage of the films was calculated through Equation (1) (Ekramian et al., 2021a): where w 1 was the dry film weight (g), and w 2 was the swollen film weight (g).

| Determination of water absorption capacity (WAC)
To determine the water capacity of the films, pieces of film were first cut and placed in a desiccator with calcium chloride and kept in an oven at 30℃ for one day. The film samples were then stored in a desiccator containing deionized water for 24 hr. After that, the film pieces were removed from the desiccator and weighted again.
Finally, the water absorption capacity of the films was obtained through Equation (2)

| Determination of oxygen permeability (OP)
In order to evaluate the oxygen permeability of the film samples,

| Determination of mechanical properties
The

| Agar diffusion method (Static test)
The film samples were cut into 5 mm diameter disks, and the obtained disks were placed on brain-heart infusion medium under sterile conditions. Before placing the disks on the surface of the culture medium, surface culture was performed using 0.1 ml of liquid culture of each of the tested microorganisms (10 5 -10 6 CFU/ml) such as E. coli, S. aureus, and A. flavus. The plates were then incubated at 37℃ for 24 hr. Finally, the inhibition zones area was measured using a caliper with an accuracy of 0.02 mm (Alebooyeh et al., 2012).

| Shake flask method (dynamic test)
The antimicrobial activity of film samples was also studied according to the shake flask method. In this method, bacteria and mold grown in 100 ml of Mueller Hinton Broth were used. 2 × 10 5 CFU/ml of each microorganism were added to 100 ml of Mueller Hinton Broth medium and incubated at 37℃ for 12 hr. Every 2 hr, the absorbance was recorded by spectrophotometer UV/Visible (Shimadzu, model UV-1700, Japan) at a wavelength of 600 nm, and microbial growth was obtained. The test was repeated 3 times, and the average values were used (Sadeghnejad et al., 2014).

| Statistical analysis
Using the SPSS 22.0 software, the mean of each tested parameter was analyzed by one-way analysis of variance (ANOVA). Differences between treatments were assessed using Duncan's multiple range test at a 5% level of probability (p <.05).

| Effect of ZnO-N and FEO on the thickness of starch films
The thickness values of the bionanocomposite films containing a combination of ZnO-N and FEO are compared in Figure 1. They show

| Effects of ZnO-N and FEO on the solubility of starch films
The combined effect of ZnO nanoparticles and FEO on potato starch-based films is given in Figure 2.

| Effect of ZnO-N and FEO on water absorption capacity (WAC) of starch films
In Figure

| Effect of ZnO-N and FEO on water vapor permeability (WVP) of starch films
Permeability is a process of solubility and diffusion in which  The addition of nanoparticles by dispersing in the film bed and compacting the film structure reduces the passage space of F I G U R E 2 Effects of nano-ZnO and fennel essential oil on water solubility of (%) potato starch films F I G U R E 3 Effects of nano-ZnO and fennel essential oil on water absorption capacity (g/g dried film) of potato starch films water molecules and makes their penetration path more difficult.
Therefore, the penetrating molecules have to travel a long distance to cross the film, thus reducing the rate of transmission and penetration (Bourtoom, 2008;Müller et al., 2008). The decrease in WVP due to the addition of different levels of FEO is probably because water penetration in the film is done through their hydrophilic parts, while essential oils have a hydrophobic nature and can reduce the water vapor permeability by increasing the ratio of hydrophobic areas to hydrophilic (Hernandez, 1994).

| Effect of ZnO-N and FEO on oxygen permeability (OP) of starch films
One of the important parameters for a food packaging material is its resistance to gasses. Excessive transfer of oxygen from the environ- Other researchers have reported improved oxygen permeability of polymer films by the addition of nanoparticles Teymourpour et al., 2015). also showed that the addition of grape seed extract to chitosan films reduced the strength of films.

| Effect of ZnO-N and FEO on color properties of starch films
The effect of a combination of ZnO-N and FEO on color indexes of starch-based bionanocomposite films is shown in Table 2 due to the addition of nanoparticles has also been reported by other researchers (Kanmani & Rhim, 2014;Rhim et al., 2013;Zolfi et al., 2014). Gonçalves et al. (2020) also observed that due to the addition of sweet fennel essential oil to the cellulose acetate films, the amount of L* decreased, but a* and b* increased. Sun et al. (2020) also demonstrated a significant reduction in lightness intensity of konjac glucomannan/chitosan films due to the incorporation of nano-ZnO and mulberry extract.

| Effect of ZnO-N and FEO on antimicrobial activity of starch films
The antimicrobial activity of starch films containing a combination of ZnO-N and FEO was investigated by the agar disk diffusion, and the obtained results are given in Table 3

TA B L E 3
Comparison of Inhabitation zone (mm 2 ) of potato starch films containing nano-ZnO and fennel essential oil Several mechanisms have been proposed for the antimicrobial action of metal nanoparticles, the most important of which include the catalytic activity of oxidation and reduction, which affects the active site of enzyme, DNA, and the activity of ribosomes and disrupts metabolic activity (Bruna et al., 2012). Induction of reactive oxygen species such as hydrogen peroxide, hydroxyl radicals, and superoxide (Emamifar et al., 2010) and cell wall damage are also suggested mechanisms for the antimicrobial activity of nanoparticles (Li et al., 2011).
One of the important properties of essential oil and its effective compounds is its hydrophobicity, which enables them to destroy the cell wall of bacteria or the wall of mitochondria and cause the destruction of the cellular structure and greater permeability of cells. Migration of ions and other cellular contents can also occur, which depletes the contents of the microbial cell and release their sensitive substances. The essential oils that have the strongest antimicrobial activity against food poisoning microorganisms contain high levels of active phenolic compounds usually. Hence, their mechanism of action seems to be similar to other phenolic compounds. These active compounds usually disrupt the function of the cell membrane and break and disrupt the proton kinetic force, electron current, and the active transfer and coagulation of cell contents. Essential oil compounds also affect proteins in the cytoplasmic membrane (Oraki et al., 2011). Researchers have stated that FEO has desirable antimicrobial activity, which is due to the presence of flavonoids, terpenoids, carotenoids, and coumarins in the essential oil of this plant (Singh et al., 2006). There are more than 30 types of terpene or terpenoid compounds in fennel essential oil (Guillén & Manzanos, 1996

| CON CLUS ION
The results of this study demonstrated that the incorporation of ZnO-N and FEO combination to potato starch-based nanocomposite films significantly improved the tensile strength and barrier properties of starch films against oxygen and water vapor.
Bionanocomposite films had higher moisture resistance than control films. However, they were darker in color. ZnO-N and FEO showed significant antimicrobial activity in starch films so that by increasing the concentration of these additives, the antimicrobial activity of bionanocomposite films was improved significantly. In general, the best characteristics, as well as antimicrobial performance, were observed in the starch film containing 5% ZnO-N and 3% FEO.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

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 from the corresponding author, upon reasonable request.