Application of essential oil of Pistacia atlantica Gum, polypropylene and silica nanoparticles as a new milk packaging

Abstract The aim of current study was to investigate the antimicrobial effect of gum essential oil of Pistacia atlantica (wild pistachio) tree (GEO) and design a new film based on polypropylene polymer coated with silica nanoparticles and GEO. The antimicrobial activity of the packaging film was evaluated with or without milk on Staphylococcus aureus, Salmonella enterica, Escherichia coli, and Listeria monocytogenes during 35 days. The results showed that GEO has significant antibacterial properties. It was most effective on Salmonella enterica, while its effect on Listeria monocytogenes was the weakest. Antimicrobial activity of the film without milk showed no significant differences among the different sizes of nanoparticles used (0.05, 0.025, and 0.051 g) (p ≥ .05). It can be concluded that polypropylene incorporated with GEO and silica nanoparticles active film had antimicrobial properties up to 35 days, while using with milk or without milk. Therefore, this type of packaging is effective to enhance the shelf life of milk.

growth of microorganisms, hence prevent the spread of various foodborne diseases (Abdoul-Latif et al., 2010). Studies conducted in this regard have been indicated that using antimicrobial films is much more effective than direct addition of antimicrobials into foodstuff as these active compounds could be released slowly from the packaging to the surface of food and remained in the required concentration to prevent microbial growth (Appendini & Hotchkiss, 2002;Mohajeri et al., 2018;Rahimi et al., 2019;Silveira et al., 2007).
Essential oils (EO S ) vary in function and composition depending on the type of plant, climatic conditions, and drying and processing methods. Mediterranean countries have a strong background in using EOs as antioxidants and antimicrobials (Akrami et al., 2015;Rahimi et al., 2019). Pistacia atlantica tree (Pistacia atlantica subsp. Kurdica) is a wild pistachio plant from the Anacardiaceae family (Sharifi & Hazell, 2011). Pistacia atlantica tree is grown mainly in western and less in the central and eastern part of Iran which has been used by local people as ingredients in some food, jams, and also gum (Hatamnia, Abbaspour, & Darvishzadeh, 2014). The essential oil derived from gum contains α-thujene, α-pinene, camphorene, sabinene, β-pinene, ∆3-carene limonene, and also the alpha-trapezoid neol, armandander, and caprylic acid (Sharifi & Hazell, 2011).
The aim of this study was to design a new polypropylene package for milk, which is coated by nano-scale of GEO, and investigate its antimicrobial properties in vitro and in a food model.

| Preparation of suspensions of nanoparticles and Essential oil
GEO was obtained from Pistacia atlantica tree by Clevenger's apparatus. Silica nanoparticles (Merck, Germany) with approximate size of 100 nm were used for further stabilization of the GEO molecules and prevent its rapid evaporation. Three concentrations (0.05, 0.025, 0.001 g) of silica nanoparticles separately mixed with 500 μl of nondiluted GEO in an acid-resistant polyethylene container. It was heated and mixed intensely on the shaker for 10 min. The achieved concentrated suspension was used for the following tests.

| Preparation of active packaging
For each treatment, a piece of 6 × 6 cm sheet of polypropylene polymer was used and 10 tiny pores of about 100 µm made manually in one side of label by needle before sterilization with 70% ethanol.
Then, 4 layers of 3 × 3 cm Whatman cellulose paper were placed between the 2 layers of polypropylene films. Three edges of the films were sealed by heat. Then, each suspension contains specific concentration of GEO-silica nanoparticle was injected into the Whatman paper layer with a sterile syringe. Finally, the free (fourth) edge of the package was sealed by heat .

| In vitro antimicrobial active packaging
Antibacterial activity of the package was investigated by vapor diffusion method. The films were placed on inner surface door of Mueller-Hinton agar (Merck) plates which surface inoculated with 10 6 CFU/ml of microorganisms. The plates were incubated at 37°C for 24 hr. For each of the above samples, a control medium, coated with paraffin, was run, too. Inhibition of microorganisms' growth was studied by counting the number of colonies compare to control group. The experiment was operated in triplicate for 4 samples (Adel, Abedian Amiri, Divband, Safari, & Khalili, 2015;Akrami et al., 2015).

| Antimicrobial activity of active packaging in milk
For the antibacterial activity test in milk, Staphylococcus aureus, Salmonella enterica, Escherichia coli, and Listeria monocytogenes cultures obtained from the culture collection of Yazd University were used. The cultures of bacterial were grown on nutrient agar slants and kept at 4°C. A portion of 700 μl of the suspension containing 10 6 CFU/ml each bacterium was inoculated into sterile containers containing 70 ml of commercial low-fat sterile milk obtained from a local market. In the next step, the prepared package with GEO and 0.001 g silica nanoparticles was put inside the milk container after sterilization with alcohol, and the milk container placed in refrigerator at 4°C. The colonies were counted on days 1, 3, 7, 14, 21, 28, and 35 (Adel et al., 2015).

| Scanning electronic microscopy
Surface and cross-sectional imaging of the packages were performed by scanning electron microscopy (SEM) (EM3200 model, KYKY Corporation) at various magnifications. In order to determine the structure of nanoparticles, the 26 KW SEM was used. To have cross-sectional image of the surfaces, a small slice of films was placed on the aluminum base in the liquid nitrogen with the help of a silver adhesive, and the bases were coated in the gold coating/disperser device (Jouki, Mortazavi, Yazdi, & Koocheki, 2014;Salarbashi et al., 2013).

| Statistical analysis
Data of different groups were collected and analyzed using one-way ANOVA by the SPSS software. In all calculations, p-value of <.05 was considered statistically significant.

| In vitro antimicrobial activity of the active packaging
Antibacterial activity of the new package containing GEO and silica nanoparticles with various concentrations (0.05, 0.025, 0.001 g) of silica nanoparticles against Staphylococcus aureus, Salmonella enterica, Escherichia coli, and Listeria monocytogenes is shown in Figure 1. Colony numbers reduced slightly with decrease in the amount of silica nanoparticles, although this reduction was not significant (p ≥ .05). In general, GEO with silica nanoparticle incorporated films determined similar antimicrobial activity against all the tested bacteria (p ≥ .05). These findings showed that 0.001 g silica nanoparticle has higher inhibitory effect, which is suitable for making an antimicrobial packaging. Therefore, this concentration selected for further experiments. The growths of

Staphylococcus aureus, Salmonella enterica, Escherichia coli, and
Listeria monocytogenes in the presence of 0.001 g silica nanoparticles and GEO active packaging in volatile phase without milk are compared.
According to Figure 2, GEO has a strong inhibitory effect on Salmonella. However, no significant difference was found in the counts of bacteria (p ≥ .05).

| Antimicrobial activity of the active packaging in milk
The colony numbers grown in the presence of milk were evaluated on days 1, 3, 7, 14, 21, 28, and 35 ( Figure 3). New milk packaging was able to reduce the growth of all tested bacteria during each storage times (p < .05).

| Microstructure of the nanomaterial packaging
Microstructures of cellulose without and with silica nanoparticles and GEO are shown in Figures 4 and 5, respectively. As shown in Figure 5, the silica nanoparticles and GEO are distributed finely in the smooth and continuous form, which increased the maintenance and stability of GEO.  (Salarbashi et al., 2013). Incorporating essential oils in packaging is limited due to their volatile nature and organoleptic acceptance.

| D ISCUSS I ON
Therefore, applying the essential oils in nano-scale could solve these problems (Salarbashi et al., 2013). As reported in our previous study, α-pinene (92.08%) is the main ingredient of Pistacia atlantica tree's gum essential oil (Ellahi et al., 2019), which is considered as an antimicrobial agent. Although, other compounds, such as ß-pinene, sabinene, and camphene, in GEO may contribute to the bacterial inhibitory effect (Akrami et al., 2015;Ellahi et al., 2019).
The inhibitory effect of new packaging containing GEO and 0.001 g silica nanoparticles against the four selected bacteria is shown in Figure 2. Some studies reported that decreasing the silica nanoparticle concentration resulted in higher inhibitory activity due to more release of essential oil. According to our results, the best amount of nanoparticle that had the most antibacterial activity was 0.001 g. The greater inhibitory effect of the active packaging without applying in food system was observed on S. enterica.
Akrami et al. investigated the antimicrobial activity of active packaging containing 2%, 4%, and 6% w/w of Zataria multiflora essential oil against gram-positive and gram-negative bacteria. They concluded that growth of S. aureus, Pseudomonas sp, E. coli, S. enterica, and Listeria innocula strains was inhibited by using 4% and 6% w/w of Zataria multiflora essential oil, while the effective concentration of Zataria multiflora essential oil against S. enterica and S. aureus was 2% (Akrami et al., 2015). Rodriguez (Rodriguez et al., 2007).
In recent years, Nanoparticles of clay, chitosan, and starch have been