Food Packaging and Shelf Life 22 (2019) 100389

Contents lists available at ScienceDirect

Food Packaging and Shelf Life

journal homepage: www.elsevier.com/locate/fpsl

Active intelligent packaging film based on ethylene vinyl acetate T

nanocomposite containing extracted anthocyanin, rosemary extract and
ZnO/Fe-MMT nanoparticles
⁎ ⁎
Somayeh Mahmoudi Eskandarabadia, Mehdi Mahmoudiana, , Kave Rahmani Farahb, ,
Arezoo Abdalia, Ehsan Nozada, Mojtaba Enayatic
a
Nanotechnology Research Institute, Urmia University, Urmia, Iran
b
Artemia and Aquaculture Institute, Urmia University, Urmia, Iran
c
Department of Food Science, College of Agriculture and Life Sciences, Cornell University, Ithaca 14853, NY, USA

A R T I C LE I N FO A B S T R A C T

Keywords: A new strategy to prevent food deterioration is the use of intelligent-active food packaging. Intelligent-active
Intelligent-active packaging film packaging is defined as a barrier film which prevents or delays the spoilage of food or reveals the deterioration
Nanocomposite process in food by different mechanisms. In this study, the intelligent-active property was imbedded into food
Antibacterial activity packaging films made of ethylene-vinyl acetate copolymer an abundant, flexible, and non-toxic polymer blend
Ethylene-Vinyl acetate copolymer
by incorporation of different additives. Anthocyanin extracted from red cabbage was stabilized on montmor-
illonite and added to the film as a time-temperature indicator. Other materials such as ZnO nanoparticles,
rosemary extract, and modified montmorillonite were used to achieve antimicrobial, antioxidant, and air barrier
activity. The additives and prepared films were characterized by FTIR, XRD, FESEM, TGA, and mechanical
analysis. The performance of the prepared films was investigated by antioxidant capacity test by DPPH, in vitro
test, minimum bactericidal concentration (MBC), minimum inhibition concentration (MIC), antibacterial test
and capacity of oxygen absorbance. Results showed that films which contain rosemary extract, ZnO, and
modified montmorillonite exhibited remarkable antibacterial and antioxidant activity compared to pristine EVA
film.

1. Introduction
In recent years, there has been a growing demand for food products
that can maintain their quality for a long time, resulting in a decrease in
economic loss and health problems caused by food spoilage. Although
the addition of chemical preservatives increases shelf-life of foods, this
is not perceived as healthy or safe and is undesirable by consumers
(Vasile et al., 2017). Conventional packaging methods only slightly
reduce the rate of these deterioration reactions, so new technologies
seek to increase food stability while providing healthier food products.
New food packaging technologies have been developed in this field and
have made significant advances thus far. Different materials are used
for packaging, which are typically polymers. Synthetic polymers are
advantageous compared to biopolymers when used as the main mate-
rial in packaging films due to their higher resistance against environ-
mental conditions, physical forces, moisture, and gas passage. Up to
now, various polymers such as polyethylene, polypropylene (Jo et al.,
2018), polyvinyl alcohol (Yu, Li, Chu, & Zhang, 2018), and poly(ethy-
lene terephthalate) have been used in the food packaging. While most
of these polymers are processed in harsh conditions such as high tem-
perature and pressure, ethylene vinyl acetate (Ganiari, Choulitoudi, &
Oreopoulou, 2017) is readily processed at room temperature in atmo-
spheric pressure, and shows increased flexibility with respect to other
polymers used in food packaging (Wattananawinrat, Threepopnatkul, &
Kulsetthanchalee, 2014).
Although the development of packaging industry guaranteed the
quality of food, the package cannot provide information about food
storage history such as temperature fluctuation, environmental condi-
tions, packaging stability, and food spoilage (Ghoshal, 2018). There-
fore, in recent years, efforts have been made to develop a system that
can inform consumers about the quality and safety of the food (Rezaei,
2018). The use of intelligent and active (intelligent-active) packaging
systems is one of the newest methods developed for this purpose.
Active function in food packaging films is achieved by adding

⁎
Corresponding authors.
E-mail addresses: m.mahmoudian@urmia.ac.ir (M. Mahmoudian), k.rahmani@urmia.ac.ir (K.R. Farah).

https://doi.org/10.1016/j.fpsl.2019.100389
Received 13 September 2018; Received in revised form 15 August 2019; Accepted 19 August 2019
2214-2894/ © 2019 Elsevier Ltd. All rights reserved.
S.M. Eskandarabadi, et al.
certain compounds to the film formulation which can interact with the
contents of the package and, consequently, eliminate the factors that
cause food deterioration, creating favorable conditions in terms of
freshness and food safety (Cruz, Alves, Khmelinskii, & Vieira, 2018). In
this field, materials such as silver ions and its nanoparticles, chlorine
dioxide, metal oxides (zinc oxide, copper oxide), antibiotics, organic
acids, and essential oils have been incorporated as antibacterial ad-
ditives in the packaging matrix (Prasad & Kochhar, 2014). The use of
oxygen absorbers and antioxidant molecules in the packaging for-
mulation prevents the oxidation of food by eliminating free radicals
(Pereira, de Arruda, & Stefani, 2015); iron and iron salts, ascorbic acid
and glucose oxidase enzymes are among these substances (Lo’ay & El-
Khateeb, 2018). Furthermore, extracted polyphenols from natural plant
sources can be used as antibacterial and antioxidant agents in active
food packaging systems (Chang-Bravo, Lopez-Cordoba, & Martino,
2014; Wrona, Nerín, Alfonso, & Caballero, 2017).
On the other hand, intelligent packaging allows monitoring and
recording of critical parameters for food quality during packaging and
storage. This property is usually provided through the inclusion of
equipment or materials inside or outside the package. The addition of
indicators such as temperature detectors, gas or pH sensors, and bio-
sensors (Kerry, O’Grady, & Hogan, 2006) into the packaging system is a
technique to prepare intelligent packaging films. In systems based on
indicators, qualitative information is usually detected by visual
changes, such as a shift in color upon pH changes (Pereira et al., 2015).
The performance of these indicators is based on the detection of any
chemical changes in the composition of the food that occurs as a result
of microbial growth in food (Jideani & Vogt, 2016).
Anthocyanins, which are natural pigments and have a wide range of
colors in a variety of plants, fruits, and vegetables, have been con-
sidered as a suitable pH indicator in intelligent food packaging due to
their ability to change color at different pH. Some reports showed the
ability of anthocyanin to increase the shelf life of food in addition to
other applications in the food industry (Bassolino et al., 2013;
Grotewold, 2006).
Addressing active and intelligent functions concurrently, the food
packaging industry can reveal and prevent the process of deterioration
in the food, yet not so much work in this field has been reported. In this
study, a particular type of packaging for food is fabricated, which shows
intelligent and active functions simultaneously. Ethylene vinyl acetate
was used as a polymer matrix due to advantages such as transparency,
flexibility, availability, and low price. Extracted anthocyanin from red
cabbage as a natural pigment was stabilized on montmorillonite and
utilized as an effective sensor such that its color changes in the presence
of alkaline materials like ammonia, a by-product of food deterioration
(intelligent function). Moreover, to create the active function in the
prepared packaging films, antibacterial and antioxidant agents, as well
as oxygen adsorbents (ZnO nanoparticles, Fe stabilized montmorillonite
and rosemary) were used in combination with pH sensor (anthocyanin)
in polymer matrices. The performance of prepared films was studied by
several experiments including antioxidation activity, antibacterial test,
minimum inhibition concentration (MIC) measurement with macro
dilution technique, minimum bactericidal concentration (MBC) mea-
surement with macro dilution technique, and oxygen absorbance ca-
pacity. Results showed the nanocomposite films as intelligent-active
packaging exhibited improved performance.
2. Experimental
2.1. Materials
Ethylene vinyl acetate (Ganiari et al., 2017), chloroform, FeSO4,
NaBH4, methanol, montmorillonite (MMT), Zn(NO3)2 0.6H2O, PVA
(Mw 89,000–98,000, 99% hydrolyzed), 2,2-diphenyl-1-picrylhydrazyl
(DPPH), brain-heart broth, Muller Hinton agar, ethanol, and HCl were
purchased from Merck with high purity and used without further
2
Food Packaging and Shelf Life 22 (2019) 100389
purification. The red cabbage was prepared from the local market; ro-
semary extract was purchased from Idearaye Pishgam Co (Iran).
2.2. Synthesis of ZnO nanoparticles
In order to synthesize ZnO nanoparticles, 0.6 g of Zn(NO3).6H2O
was dissolved in 20 ml of distilled water in a beaker at room tem-
perature under stirring, which was named solution A. In another
beaker, 0.3 g of PVA as stabilizer was dissolved in 80 ml distilled water
at 50 °C under stirring which was named solution B. Afterwards, both
solutions A and B were mixed together and 1 M solution of KOH was
added dropwise to the prepared solution until the pH reached 12 and
the precipitation was complete. The resulting precipitate was dried at
90 °C for 18 h. The dried precipitate was calcinated at 500 °C for 1 h.
2.3. Synthesis of Fe-MMT
For the preparation of iron-fixed montmorillonite (Fe-MMT), 0.53 g
of FeSO4 was dissolved in 1.8 ml of a solution of ethanol and water and
with a volumetric ratio of 1:2. Then 0.12 g of MMT was dispersed in the
above solution by magnetic stirring. In a glass vial, 0.25 g of NaBH4 was
dissolved in 6.6 ml of distilled water and was used to reduce the FeSO4
on the MMT surface. Finally, the black precipitate was filtered on filter
paper and washed with ethanol several times, then dried at 45 °C under
vacuum.
2.4. Extraction of anthocyanin
Chopped red cabbage (180 g) was added to 80 ml of a solution of
water and ethanol (with a volumetric ratio of 3:7), and the pH was
adjusted at 2.0 with a 1.0 M solution of HCl. The mixture was kept away
from light and maintained at 5 °C for 12 h. Then, the solution was fil-
tered out, and the filtrate was centrifuged at 2000 rpm for 10 min.
Afterward, the pH of the obtained substance was increased to 7 by
washing with a 2.5 M NaOH solution (Pereira et al., 2015).
2.5. Preparation of Anthocyanin-MMT
In a glass beaker, 0.5 g of MMT was added to 1.0 g of anthocyanin
solution (5 wt%), then the resultant mixture was agitated with a mag-
netic stirrer for 24 h at room temperature. Afterward, the MMT con-
taining adsorbed anthocyanin was filtered and dried for 12 h at room
temperature to obtain the anthocyanin-MMT powder (Ogawa, Takee,
Okabe, & Seki, 2017).
2.6. Preparation of nanocomposite films containing different additives
To fabricate the nanocomposite films, EVA was dissolved in
chloroform to obtain a 10 wt% solution. Then, ZnO and Fe-MMT na-
noparticles (1 wt%) were added to the above solution under ultrasonic
agitation for 2 min. Required amounts of 1 wt% of anthocyanin-MMT
and 3 wt% of rosemary extract were added to the resultant mixture
subsequently. Eight samples with different formulation have been
prepared according to Table 1. Selected amounts for additives included
optimal values for maximum transparency in the prepared films. All
mixtures were stirred for an extra 10 min to provide a homogeneous
dispersion of materials inside the EVA solution. Finally, the prepared
mixture was spread out on a smooth and cleaned glass plate and was
dried for 12 h at room temperature.
2.7. Characterization
To characterize the structure of additives and possible variations in
films, Fourier transform infrared (FTIR) spectroscopy was used. The
analysis was performed using an FTIR JASCO 4100 instrument in the
range of 500 to 4000 cm−1. The crystalline structure of ZnO, Fe-MMT
S.M. Eskandarabadi, et al. Food Packaging and Shelf Life 22 (2019) 100389

Table 1 2.10. Minimum inhibition concentration (MIC) measurement with macro

Formulation components for fabricated films. dilution technique
Sample code ZnO Nano- Fe-MMT Anthocyanin- Rosemary (%
particles (% Nano- MMT (% wt) wt) In order to study the MIC by alcohol extraction, solutions with 12.5,
wt) particles (% 25, 50, 100, 200, 400 mg ml−1 concentrations of each additive in
wt) Mueller-Hinton agar medium were prepared. A bacterial suspension
M1 0 0 0 0
with 1.5 × 106 CFU. ml−1 concentration was added into each of the
M2 0 0 1 0 solutions. Positive control test tubes (a culture medium containing
M3 1 0 1 0 bacteria without extract) and negative control test tubes (culture
M4 0 0 1 3 medium without bacteria) were also prepared. After 24 h incubation at
M5 1 0 1 3
37 °C, the tubes were investigated for opacity (grown or not grown).
M6 0 1 1 0
M7 1 1 1 0 The last tube which exhibits no opacity is considered to be the MIC (da
M8 1 1 1 3 Silva Dannenberg, Funck, Mattei, da Silva, & Fiorentini, 2016).

2.11. Minimum bactericidal concentration (MBC) measurement with macro

and MMT nanoparticle powders and their dispersion in the polymeric
matrix was analyzed with XRD (X’PertPro, Panalitycal.co, Holland) in
the 2θ range of 5° to 80°. The morphology of nanoparticles and fabri-
cated films was investigated with field emission scanning electron mi-
croscopy (FESEM) technique by using a Hitachi S4160 instrument.
Thermal behavior and stability of EVA nanocomposite films containing
ZnO, Fe-MMT, and their mixture were analyzed using thermal gravi-
metric analysis (SANAF TGA, STGA-1000). The measurement was car-
ried out under nitrogen atmosphere, and the samples were heated from
25 °C to 500 °C at a rate of 20 °C min−1. Tensile strength tests of the
fabricated films were carried out using a Santam STM-150 machine,
with the maximum 10 kN capacity and extension resolution of
0.05 mm. Prepared film samples were cut into tape-shaped (2.5 × 4 cm)
samples. The rotation speed was adjusted at 1 mm sec−1.
2.8. Antioxidation activity
DPPH test was used to investigate the antioxidation activity of
fabricated films. In a glass vial, 0.05 g of the prepared film was added to
5 ml of methanol and kept for 20 min to extract the antioxidant out of
the formulation. In another vial, 4.0 mg of DPPH was added to 100 ml
of methanol and was stirred for 30 min. 1 ml of the antioxidant con-
taining solution was added to 3 ml of the DPPH solution under vigorous
stirring. The resultant mixture was kept in a dark place at room tem-
perature for 30 min, and its absorbance was measured at 515 nm. The
absorbance of the solution was also measured at 515 nm, and the an-
tioxidation activity was calculated based on Eq. (1) as below:
I % = [(Ablank − Asample )/ Ablank ] × 100
In this equation, I is the antioxidation activity (in percent), and
Ablank and Asample are the absorbance of the blank and real sample,
respectively.
2.9. Antibacterial test
The antibacterial activity of the prepared films was evaluated by E.
Coli and Staphylococcus Ureus bacteria. 50 ml of brain-heart broth so-
lution was prepared and placed in an autoclave at 121 °C for 15 min.
The bacteria with 0.5 McFarland concentration was added to the above
solution and incubated for 24 h. 50 ml of Mueller-Hinton agar was
prepared and placed in an autoclave at 121 °C for 15 min. The agar
solution was poured into plates, and the bacteria solution was im-
pregnated onto the agar growth medium by bacterial lawn technique
(Caldwell & Caldwell, 2015. The prepared films with 1 × 1 cm di-
mensions were placed in culture medium containing bacteria and in-
cubated for 24–48 hrs at 37 °C. Finally, the area of inhibition zone was
measured.
dilution technique
For determination of the MBC, 100 ml of each MIC tube was cul-
tured on Mueller-Hinton agar culture medium using surface culture
method. The minimum concentration of additive, in which the bacteria
was not grown, was defined as MBC.
2.12. The oxygen absorbance capacity
The oxygen absorbance capacity of Fe and its composites was as-
sessed by measurement of the oxygen concentration in a closed medium
at specified times. 0.01 g of Fe-MMT powder and the films containing
this additive were placed in a 40-ml glass container fitted with a
septum. Another vessel that contains 1 ml of water was also inserted
inside the main container to maintain 100% humidity. The EVA film
was considered as the blank sample. Then, the containers were sealed
and kept at room temperature for 24 h. In these containers, atmosphere
correction was not done; nonetheless, the percentage of oxygen was
measured with an oxygen sensor instrument (Regensburg, Germany) in
different periods. Each experiment was repeated three times, and the
averages were reported as oxygen scavenging activity.
2.13. Performance of the indicator (anthocyanin-MMT)
For the investigation of the indicator performance, 0.01 g of the
indicator was added to aqueous media with pH of 2, 4, 6, 8, 10, and 12,
and the color changes were observed in acidic and alkaline environ-
ments. The films were exposed to hydrogen chloride and ammonia
vapors, and the observed colors were recorded.
(1)
2.14. The reaction of intelligent film with ammonia
The interaction of the film containing anthocyanin with ammonia
vapors is measurable as a sensible change in color, according to a
previously reported method (Kuswandi, Jayus, Abdullah, Heng, &
Ahmad, 2012). The desired film was cut to 5 × 1 cm rectangles and
exposed to ammonia vapors for a specified period of time (1 h). At 10-
min intervals, the absorbance of the film was measured at 580 nm.
3. Results and discussion
3.1. Characterizatin of additives and prepared films
To achieve the concept of active food packaging, various additives
were added to EVA film. For the preparation of intelligent packaging
films, stabilized anthocyanin was incorporated into the formulation.
The chemical structures and functional groups of materials used in the
preparation of the films were characterized by FTIR. Fig. 1a shows the
spectra of the pristine additives that were used to formulate the
packaging films, and Fig. 1b shows the spectra of selected films, which
contain these additives. In the FTIR spectrum of zinc oxide

3
S.M. Eskandarabadi, et al. Food Packaging and Shelf Life 22 (2019) 100389

Fig. 1. FTIR of a) additives and materials used in the preparation of the packaging films, b) the produced films containing additives.

nanoparticles, Zn-O peaks appear in the area below 1000 cm−1, and the
peaks around 3350 and 1560 cm−1 are associated to hydroxyl groups
and adsorbed water (Zare et al., 2018). In the spectrum of Fe-MMT, the
observed peak at 3495 cm−1 is related to hydroxyl groups. Further-
more, the peaks at 1127 and 873 cm−1 are attributed to Si – O – Si and
Si – O – Al, respectively. The presence of two peaks at 780 and
625 cm−1 are due to the stretching vibration of Fe-O bonds (Fig. 1a).
Rosemary characteristic peaks include 3400, 1752, and 1515 cm−1,
which are associated with stretching vibrations of OeH, C]O and C]C
groups, respectively. The characteristic peaks related to the aromatic
and phenolic structure of the anthocyanin can be seen at 3250 cm−1,
1400-1600 cm−1, and 1300-1400 cm−1.
In the EVA films (M1, Fig. 1b), the peak of the carbonyl group at
1739 cm−1 is visible, and other characteristic peaks are observed at
2923, 1242 and 1020 cm−1 which can be related to CeH and CeO
bonds. The spectra of films containing additives are mainly similar to
that of the original EVA film (probably because of the lower amount of
additives), and the only significant difference is the increase in peak
intensity in composite films (especially in the film containing ZnO na-
noparticles). The presence of additives in the films can be verified by
the appearance of the corresponding peaks of the additives.
The XRD pattern of Fe-MMT, ZnO nanoparticles, and the nano-
composite films are shown in Fig. 2. The ZnO nanoparticles show a
typical XRD pattern, which is consistent with synthesized samples with
hexagonal crystal symmetry (Khorsand Zak, Razali, Abd Majid, &
Darroudi, 2011). The size obtained for ZnO nanoparticles is about
38 nm using the Sherer relationship (Patterson, 1939). XRD analysis of
Fe-MMT (Jia & Wang, 2012) shows characteristic peaks at
2θ = 25, 30, 35 and 43°. The appearance of a new peak at 2θ = 35
compared to the MMT may be related to the formation of FeOOH and
proves the successful insertion of Fe onto MMT plates (Guo & Zhang,
2016). The calculated size for these particles is about 15 nm. Fig. 2. XRD pattern of Fe-MMT and ZnO nanoparticles and some of the fabri-
Furthermore, XRD patterns observed for the prepared packaging cated packaging films that contain these additives.
films (pristine and nanocomposite EVA films) are approximately iden-
tical to each other with a characteristic peak at 2θ = 23°. As can be seen polymeric chain rearrangement in the presence of additives. The effect
in Fig. 2, the presence of ZnO nanoparticles increases the intensity of of simultaneous incorporation of ZnO and montmorillonite nano-
this peak, and the addition of Fe-MMT reduces its intensity. This is due particles is much more severe, and the crystalline structure of EVA is
to the changing crystalline structure of EVA polymer in the presence of almost eliminated.
additives (Ghanipour & Dorranian, 2013). The presence of nano- FESEM images of nanoparticles and prepared films are represented
particles and additives in films is confirmed by the appearance of very in Figs. 3 and 4, respectively. It is clearly observed in Fig. 3 that ZnO
poor peaks at 2θ = 35°, and the low intensity of these peaks can be nanoparticles are spherical and have a diameter of about 40 nm, which
attributed to the low percentage of additives in the film. The presence is in good agreement with XRD results. The layered structure of the
of both nanoparticles in the EVA formulated film (M7) has a severe MMT is also seen in Fig. 3. While the incorporation of anthocyanin on
effect on the crystallinity of the EVA resin. This is probably due to MMT does not change its morphology dramatically, the stabilization of

4
S.M. Eskandarabadi, et al. Food Packaging and Shelf Life 22 (2019) 100389

Fig. 3. FESEM micrographs of nanoparticles used in the preparation of the packaging films.

Fe on the MMT plates is recognizable as needle-shaped bumps (Fig. 3). additives have decreased surface smoothness. In all cases, nanoparticles
FESEM images of the fabricated packaging film surfaces were in- are observable on the surface of the composite films as very fine grains.
vestigated to study their morphology. The pristine EVA film has a Based on the FESEM images of the composite films, it is obvious that
smooth surface, depicted in Fig. 4, while films that contain different there are no cracks on the surface of the fabricated films, which

Fig. 4. FESEM micrographs of prepared films.

5
S.M. Eskandarabadi, et al. Food Packaging and Shelf Life 22 (2019) 100389

Fig. 5. TGA Thermograms of selected prepared films.

confirms the integrity and mechanical strength of the products.
In order to study the thermal stability of the fabricated films,
thermal gravimetric analysis (TGA) was performed on selected samples
(Fig. 5). As can be seen in Fig. 5, the pristine EVA film begins to degrade
at 280 °C, and weight loss occurs continuously in two steps up to 450 °C.
The thermal stability of composite films has increased compared to
pristine EVA film. Films containing ZnO nanoparticles (M3 and M7)
have the highest stability (up to 330 °C), and the thermal stability of the
film containing MMT has also increased by about 20 °C.
To evaluate the mechanical strength of the films, we also performed
strain-stress tests of representative packaging films. The resulting dia-
gram is shown in Fig. 6. All specimens show the typical behavior of
flexible plastic. However, films containing ZnO and Fe-MMT (M3 and
M6, respectively) exhibit higher tensile strength (86 and 70.2 MPa)
compared to pure EVA film (55 MPa). Moreover, Young's modulus of
these films shows a similar trend (Fig. 6). The M7 film, which contains
both types of nanoparticles has decreased tensile strength, which may
be due to the change in crystallinity of the polymer owing to the entry
of nanoparticles into the polymer structure. This change in crystallinity
was also observed in the XRD studies (Fig. 2).
3.2. Investigation of the performance of prepard filmes
3.2.1. Oxygen absorption
Oxygen absorption by Fe-MMT powder and the films containing this
additive has been studied, and results are represented in Fig. 7.
Fig. 7. Oxygen absorption by Fe-MMT powder and the prepared films con-
taining this additive.
Previous studies (Busolo & Lagaron, 2012; Foltynowicz, Bardenshtein,
Sängerlaub, Antvorskov, & Kozak, 2017) have shown the effect of
moisture on oxygen absorption, so the relative humidity of the en-
vironment was adjusted to 100%. The highest observed oxygen ab-
sorption belongs to Fe-MMT powder, which is almost double the op-
timal absorption by the tested films.
The absorption of oxygen in films containing Fe nanoparticles is
caused by both active and passive mechanisms (Anthierens et al.,
2011). The passive absorption of oxygen is carried out by the MMT due
to its layered structure, which causes low oxygen absorption. However,
active oxygen scavenging in higher quantities was done by stabilized Fe
nanoparticles on the MMT surface, which occurs in the presence of
moisture according to the following reaction. (Busolo and Lagaron,
2012)
4Fe + 3O2 + 6H2O → 4Fe(OH)3 → 2Fe2O3.3H2O
Among the formulated films tested, M8 containing nanomaterials
and additives shows the highest oxygen absorption, and films M6, M7,
and M1 show decreasing oxygen adsorption capacity, respectively. It is
practically seen that the EVA pure film shows a slight absorption of
oxygen. Interestingly, it is observed that the adsorption capacity in-
creased with an increasing rate up to the tenth day, reaching a plateau,
which is probably due to oxygen saturation of the film.
3.2.2. Antioxidant activity
Antioxidants are microconstituents that can scavenge free radicals
by terminating oxidizing chain reactions (Avşar, Özler, Berber, &
CİVek, 2016). DPPH has been widely applied to evaluate the radical
scavenging ability of different antioxidants (Nagajyothi et al., 2014;
Phang, Malek, & Ibrahim, 2013). Table 2 shows the results of DPPH
radical scavenging activity with all formulated samples as well as pure

Table 2
DPPH-free radical inhibitory effect.
Sample EVA Film containing Ia %

M1 Pristine EVA 8.8 ± 7.64

M2 Anthocyanin-MMT 35.67 ± 4. 04
M3 ZnO/Anthocyanin-MMT 30.00 ± 6.56
M4 Anthocyanin-MMT/ Rosemary 31.33 ± 6.66
M5 ZnO/Rosemary/Anthocyanin-MMT 34.00 ± 7.94
M6 Fe-MMT/Anthocyanin-MMT 31.00 ± 6.08
M7 Fe-MMT/ZnO/Anthocyanin-MMT 28.67 ± 8.08
M8 Fe-MMT/ZnO/Anthocyanin-MMT/Rosemary 23.00 ± 9.54

Fig. 6. Mechanical properties of selected packaging films. a

Inhibition of DPPH-free radical.

6
S.M. Eskandarabadi, et al. Food Packaging and Shelf Life 22 (2019) 100389

Fig. 8. antibacterial activity of the ZnO nanoparticles and the fabricated packaging films.

Table 3 There are some reports of antimicrobial activity by different antho-

MICs and MBCs of ZnO powder, rosemary extract, and anthocyanin against the cyanins (Liu et al., 2009) and rosemary (Salehi et al., 2018) extracts.
microorganism. The results showed that anthocyanin and rosemary extract films had
S. Ureus + E. Coli - higher antimicrobial activities than the ZnO film (Fig. 8). This may be
described with the lower penetration rate of ZnO compared to a volatile
MBC MIC mg ml−1 MBC MIC mg ml−1 material such as anthocyanin and rosemary extract.
200 100 Anthocyanin 200 100 Anthocyanin
200 100 ZnO 400 200 ZnO
400 200 Rosemary 400 200 Rosemary 3.2.4. MIC and MBC determination
The ZnO nanoparticles, anthocyanin, and rosemary extract were
evaluated for antimicrobial activity against Gram-positive (S. aureus)
EVA film. Pristine EVA film revealed poor antioxidant activity, while and Gram-negative (E. coli) bacteria. The result showed the high anti-
other treatments showed notable increases in antioxidant activity. The microbial activity of ZnO, anthocyanin, and rosemary extract (Table 3),
highest antioxidant activity is detectable in samples with anthocyanin- and anthocyanin was found to be the most active against both bacterial
MMT and rosemary additives (M2 and M5 films). The combination of strains. The MICs for the ZnO, anthocyanin, and rosemary extract
anthocyanin and rosemary with other additives decreased antioxidant ranged from 100, 200 and 200 (mg ml−1) for E. Coli test microorgan-
activity (except film M5, which is probably due to the synergistic effect isms, and the MICs for S. Ureus ranged from 100, 100 and 200, re-
of the simultaneous presence of ZnO and rosemary additives). The spectively. MBC values of the three samples were similar or even higher
strong antioxidant activity of anthocyanin extract (Gabrielska & than their corresponding MIC values. These differences in susceptibility
Oszmiański, 2005), rosemary extract (Ganiari et al., 2017) and ZnO of the microorganisms to the test samples could be attributed to the
nanoparticles (Zare, Pourseyedi, Khatami, & Darezereshki, 2017) has variation in the rate of samples’ penetration through the cell wall and
been reported previously. cell membrane structures (Cox, Betts, Jones, Spall, & Totterdell, 2000).
One of the more exciting results in this study was the higher anti-
microbial effect of rosemary extract compared to ZnO (Table 3).
3.2.3. Antibacterial activity
However, the higher MIC and MBC of the ZnO containing film, despite
To investigate the antibacterial activity of the fabricated packaging
the significant anti-microbial properties of ZnO nanoparticles alone,
films, selected films were subjected to E. Coli and Staphylococcus Ureus
can be explained by the lower availability of these nanoparticles in the
bacterial culture medium. As demonstrated in Fig. 8, pristine EVA film
EVA matrix. The antimicrobial effect could not be attributed to any
did not show any antimicrobial activity, but all of the nanocomposite
particular active components because it can be significantly affected in
films that contained additive showed antibacterial activity against both
a mixture of different chemical compounds (Salehi et al., 2018).
Gram-positive and Gram-negative bacteria. The maximum antibacterial
Our experimental treatments showed that gram-negatives bacteria
activity was observed against S. Ureus (9 mm for ZnO powder), (0 mm
consisting of E. Coli is more susceptible than gram-positive bacteria (S.
For ZnO nanocomposites), (17 mm Rosemary nanocomposites) and
Ureus). This is in accordance with previously reported results (Jiang
(13 mm for anthocyanin). Lower antibacterial activity was depicted
et al., 2011) who evaluated the antimicrobial effect of rosemary and
against E. coli (8 mm anthocyanin). The size of the inhibition zone was
other results that reported ZnO antimicrobial effect (Nagajyothi et al.,
different according to the type of pathogens, synthesis method, shape,
2014).
size, and concentration of antibacterial materials (Nagajyothi et al.,
2014; Zhang, Ding, Povey, & York, 2008). Release of Zn2+ ions, which
damages the cell membrane and interacts with intracellular contents, 3.2.5. Investigation of the color change behavior of anthocyanin
describes ZnO antibacterial activity (Li, Xing, Li, Jiang, & Ding, 2010). To investigate the intelligent function of prepared films, color

7
S.M. Eskandarabadi, et al. Food Packaging and Shelf Life 22 (2019) 100389

Fig. 9. The color change of anthocyanin-MMT in different pHs.

Fig. 10. The color change in the films containing anthocyanin-MMT.

change behavior of anthocyanin additive and the films containing this

indicator at different pHs was studied. For this purpose, the color of the
mixture containing stabilized anthocyanin on MMT was evaluated
qualitatively at different pHs, and the related images are shown in
Fig. 9. A range of colors from red to yellow results from acidic and
alkaline conditions, respectively, highlighting the high sensitivity of
anthocyanin to pH change. This change in color at different pHs is
easily distinguished and detected by the naked eye. Also, the color of
the films containing anthocyanin is also sensitive to pH variations,
showing pink and blue color in acidic and alkaline conditions, respec-
tively. These color changes in the films are shown in Fig. 10. Since these
films are originally designed to show meat deterioration, this dete-
rioration can be observed through the detection of released ammonia,
which changes the color of the packaging film from red (acidic color) to
yellow (basic color).
To show the efficiency of films over time, the absorption of am-
monia vapors by the M2 film was investigated over 60 min. As shown in
Fig. 11, the absorption rate is almost constant over time with a slight
decrease. The constant rate of absorption of ammonia over time in- Fig. 11. The absorbance of ammonia by the M2 film. Absorbance at 580 nm is
dicates that the produced films maintain their effectiveness during measured every 10 min.
storage time and can monitor food deterioration.
containing ZnO nanoparticles, montmorillonite decorated with iron
4. Conclusion nanoparticles, and rosemary extract with anthocyanin as an indicator
have been utilized in the fabrication of an effective intelligent-active
Considering the increase in global population, the need for appro- food packaging film. Based on the analyses and results presented here,
priate packaging systems that can maintain the quality of food for a these packaging films display improved thermal stability and mechan-
long time is a real concern. In this study, in addition to increasing the ical properties compared to pristine EVA copolymer film. Antioxidant
shelf-life of food by creating active packaging, an intelligent system was activity measurements show that film samples that contain anthocyanin
designed in food packaging films using a natural pigment in order to and rosemary exhibit significant improvement. All of the films were
detect food deterioration. Ethylene vinyl acetate nanocomposites

8
S.M. Eskandarabadi, et al. Food Packaging and Shelf Life 22 (2019) 100389

analyzed with antibacterial and antimicrobial tests, and results reveal heterogeneous photo-Fenton catalytic properties. RSC Advances, 6(4), 2537–2545.
that the maximum antibacterial activity belongs to the film containing Jia, H., & Wang, C. (2012). Comparative studies on montmorillonite-supported zero-valent
iron nanoparticles produced by different methods: Reactivity and stability, Vol. 34.
rosemary extract. Oxygen adsorption capacity measurements of films Jiang, Y., Wu, N., Fu, Y.-J., Wang, W., Luo, M., Zhao, C.-J., et al. (2011). Chemical
were conducted, and the ability to absorb oxygen by films with Fe composition and antimicrobial activity of the essential oil of Rosemary. Environmental
nanoparticles has been proven. The ability to change the color of an- Toxicology and Pharmacology, 32(1), 63–68.
Jideani, V. A., & Vogt, K. (2016). Antimicrobial packaging for extending the shelf life of
thocyanin in the film was confirmed by subjecting the films to different bread—A review. Critical Reviews in Food Science and Nutrition, 56(8), 1313–1324.
pHs, revealing the film’s ability to visually display the deterioration of Jo, Y., Garcia, C. V., Ko, S., Lee, W., Shin, G. H., Choi, J. C., et al. (2018). Characterization
foodstuff. and antibacterial properties of nanosilver-applied polyethylene and polypropylene
composite films for food packaging applications. Food Bioscience, 23, 83–90.
Kerry, J. P., O’Grady, M. N., & Hogan, S. A. (2006). Past, current and potential utilisation
References of active and intelligent packaging systems for meat and muscle-based products: A
review. Meat Science, 74(1), 113–130.
Kuswandi, B., Jayus, R., Abdullah, A., Heng, L. Y., & Ahmad, M. (2012). A novel col-
Khorsand Zak, A., Razali, R., Abd Majid, W. H., & Darroudi, M. (2011). Synthesis and
orimetric food package label for fish spoilage based on polyaniline film. Food Control,
characterization of a narrow size distribution of zinc oxide nanoparticles.
25(1), 184–189.
International Journal of Nanomedicine, 6, 1399–1403.
Lo’ay, A. A., & El-Khateeb, A. Y. (2018). Antioxidant enzyme activities and exogenous
Anthierens, T., Ragaert, P., Verbrugghe, S., Ouchchen, A., De Geest, B. G., Noseda, B.,
ascorbic acid treatment of ‘Williams’ banana during long-term cold storage stress.
et al. (2011). Use of endospore-forming bacteria as an active oxygen scavenger in
Scientia Horticulturae, 234, 210–219.
plastic packaging materials. Innovative Food Science & Emerging Technologies, 12(4),
Nagajyothi, F., Weiss, L. M., Zhao, D., Koba, W., Jelicks, L. A., Cui, M.-H., et al. (2014).
594–599.
High fat diet modulates trypanosoma cruzi infection associated myocarditis. PLoS
Avşar, C., Özler, H., Berber, İ., & CİVek, S. (2016). Phenolic composition, antimicrobial
Neglected Tropical Diseases, 8(10), e3118.
and antioxidant activity of Castanea sativa mill. Pollen Grains from Black Sea Region of
Ogawa, M., Takee, R., Okabe, Y., & Seki, Y. (2017). Bio-geo hybrid pigment; clay-an-
Turkey, 23.
thocyanin complex which changes color depending on the atmosphere. Dyes and
Bassolino, L., Zhang, Y., Schoonbeek, H., Kiferle, C., Perata, P., & Martin, C. (2013).
Pigments, 139, 561–565.
Accumulation of anthocyanins in tomato skin extends shelf life. The New Phytologist,
Patterson, A. L. (1939). The scherrer formula for X-Ray particle size determination. The
200(3), 650–655.
Physical Review, 56(10), 978–982.
Busolo, M. A., & Lagaron, J. M. (2012). Oxygen scavenging polyolefin nanocomposite
Pereira, V. A., de Arruda, I. N. Q., & Stefani, R. (2015). Active chitosan/PVA films with
films containing an iron modified kaolinite of interest in active food packaging ap-
anthocyanins from Brassica oleraceae (Red Cabbage) as Time–Temperature
plications. Innovative Food Science & Emerging Technologies, 16, 211–217.
Indicators for application in intelligent food packaging. Food Hydrocolloids, 43,
Caldwell, G. A., & Caldwell, K. A. (2015). Chapter 6 use of caenorhabditis elegans to
180–188.
model human movement disorders. In M. S. LeDoux (Ed.). Movement disorders (pp.
Phang, C.-W., Malek, S. N. A., & Ibrahim, H. (2013). Antioxidant potential, cytotoxic
97–116). (second edition). Boston: Academic Press.
activity and total phenolic content of Alpinia pahangensis rhizomes. BMC
Chang-Bravo, L., Lopez-Cordoba, A., & Martino, M. (2014). Biopolymeric matrices made
Complementary and Alternative Medicine, 13(1), 243.
of carrageenan and corn starch for the antioxidant extracts delivery of Cuban red
Prasad, P., & Kochhar, A. (2014). Active packaging in food industry. IOSR Journal of
propolis and yerba mate. Reactive & Functional Polymers, 85, 11–19.
Environmental Science, Toxicology and Food Technology, 8(5), 1–7.
Vasile, C., Sivertsvik, M., Carmen, A., Mitelu, T., Brebu, M. A., Stoleru, E., et al. (2017).
Rezaei, A. (2018). Food safety: The farmer first health paradigm. One Health (Amsterdam,
Comparative analysis of the composition and active property evaluation of certain
Netherlands), 5, 69–73.
essential oils to assess their potential applications in active food packaging. Materials.
Salehi, B., Valussi, M., Jugran, A. K., Martorell, M., Ramírez-Alarcón, K., Stojanović-
Cox, P., Betts, R., Jones, C., Spall, A., & Totterdell, J. (2000). Acceleration of global
Radić, Z. Z., et al. (2018). Nepeta species: From farm to food applications and phy-
warming due to carbon-cycle feedbacks in a coupled model, Vol. 408.
totherapy. Trends in Food Science & Technology.
Cruz, R. M. S., Alves, V., Khmelinskii, I., & Vieira, M. C. (2018). Chapter 2 - New food
Wattananawinrat, K., Threepopnatkul, P., & Kulsetthanchalee, C. (2014). Morphological
packaging systems A2 - Grumezescu, alexandru Mihai. In A. M. Holban (Ed.). Food
and thermal properties of LDPE/EVA blended films and development of antimicrobial
packaging and preservation (pp. 63–85). Academic Press.
activity in food packaging film. Energy Procedia, 56, 1–9.
da Silva Dannenberg, G., Funck, G. D., Mattei, F. J., da Silva, W. P., & Fiorentini, Â. M.
Wrona, M., Nerín, C., Alfonso, M. J., & Caballero, M.Á. (2017). Antioxidant packaging
(2016). Antimicrobial and antioxidant activity of essential oil from pink pepper tree
with encapsulated green tea for fresh minced meat. Innovative Food Science & Emerging
(Schinus terebinthifolius Raddi) in vitro and in cheese experimentally contaminated
Technologies, 41, 307–313.
with Listeria monocytogenes. Innovative Food Science & Emerging Technologies, 36,
Li, X. H., Xing, Y. G., Li, W. L., Jiang, Y. H., & Ding, Y. L. (2010). Antibacterial and
120–127.
physical properties of poly(vinyl chloride)-based film coated with ZnO nanoparticles.
Foltynowicz, Z., Bardenshtein, A., Sängerlaub, S., Antvorskov, H., & Kozak, W. (2017).
Food Science and Technology.
Nanoscale, zero valent iron particles for application as oxygen scavenger in food
Liu, Y., He, L., Mustapha, A., Li, H., Hu, Z. Q., & Lin, M. (2009). Antibacterial activities of
packaging. Food Packaging and Shelf Life, 11, 74–83.
zinc oxide nanoparticles against Escherichia coli O157:H7. Journal of Applied
Gabrielska, J., & Oszmiański, J. (2005). Antioxidant activity of anthocyanin glycoside de-
Microbiology.
rivatives evaluated by the inhibition of liposome oxidation, Vol. 60.
Yu, Z., Li, B., Chu, J., & Zhang, P. (2018). Silica in situ enhanced PVA/chitosan biode-
Ganiari, S., Choulitoudi, E., & Oreopoulou, V. (2017). Edible and active films and coatings as
gradable films for food packages. Carbohydrate Polymers, 184, 214–220.
carriers of natural antioxidants for lipid food, Vol. 68.
Zare, E., Pourseyedi, S., Khatami, M., & Darezereshki, E. (2017). Simple biosynthesis of
Ghanipour, M., & Dorranian, D. (2013). Effect of Ag-Nanoparticles doped in polyvinyl
zinc oxide nanoparticles using nature’s source, and it’s in vitro bio-activity. Journal of
alcohol on the structural and optical properties of PVA films. Journal of
Molecular Structure, 1146, 96–103.
Nanomaterials, 2013, 10.
Zare, M., Namratha, K., Byrappa, K., Surendra, D. M., Yallappa, S., & Hungund, B. (2018).
Ghoshal, G. (2018). Chapter 10 - Recent trends in active, smart, and intelligent packaging
Surfactant assisted solvothermal synthesis of ZnO nanoparticles and study of their
for food products A2 - Grumezescu, alexandru Mihai. In A. M. Holban (Ed.). Food
antimicrobial and antioxidant properties. Journal of Materials Science & Technology,
packaging and preservation (pp. 343–374). Academic Press.
34(6), 1035–1043.
Grotewold, E. (2006). The genetics and biochemistry of floral pigments. Annual Review of
Zhang, L., Ding, Y., Povey, M., & York, D. (2008). ZnO nanofluids – A potential anti-
Plant Biology, 57, 761–780.
bacterial agent. Progress in Natural Science, 18(8), 939–944.
Guo, S., & Zhang, G. (2016). Green synthesis of a bifunctional Fe–montmorillonite
composite during the Fenton degradation process and its enhanced adsorption and