Chitosan-zinc oxide nanoparticle composite coating for active food packaging
applications

Laila Al-Naamani, Sergey Dobretsov, Joydeep Dutta

PII: S1466-8564(16)30458-1
DOI: doi: 10.1016/j.ifset.2016.10.010
Reference: INNFOO 1657

To appear in: Innovative Food Science and Emerging Technologies

Received date: 23 March 2016

Revised date: 28 September 2016
Accepted date: 11 October 2016

Please cite this article as: Al-Naamani, L., Dobretsov, S. & Dutta, J., Chitosan-zinc
oxide nanoparticle composite coating for active food packaging applications, Innovative
Food Science and Emerging Technologies (2016), doi: 10.1016/j.ifset.2016.10.010

This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.
 ACCEPTED MANUSCRIPT

T
Chitosan-zinc oxide Nanoparticle Composite Coating for Active Food Packaging
Applications

P
RI
Laila Al-Naamania, Sergey Dobretsova b*, Joydeep Duttac**

SC
NU
a
Department of Marine Science and Fisheries, Sultan Qaboos University, 123 Al-Khodh, Oman

b
Center of Excellence in Marine Biotechnology, Sultan Qaboos University, 123 Al-Khodh,
MA
Oman

c
Functional Materials Division, Materials and Nano Physics Department, ICT School, KTH
Royal Institute of Technology, SE-164 40, Kista, Stockholm, Sweden
ED

* Corresponding author email: sergey@squ.edu.om

PT

** Corresponding author email: joydeep@kth.se

CE
AC

1
 ACCEPTED MANUSCRIPT

Abstract

In this study antimicrobial properties of chitosan and chitosan-zinc oxide (ZnO) nanocomposite

T
coatings on PE films were studied. Oxygen plasma pretreatment of PE films led to increased
adhesion by 2% of chitosan and the nanocomposite coating solutions to the packaging films.

P
Scanning Electron Microscopy (SEM) revealed uniform coatings on PE surfaces. Incorporation

RI
of ZnO nanoparticles into the chitosan matrix resulted in 42% increase in solubility; swelling
decreased by 80% while the water contact angle (WCA) increased from 60° to 95°compared to

SC
chitosan coating. PE coated with chitosan-ZnO nanocomposite films completely inactivated and
prevented the growth of food pathogens, while chitosan-coated films showed only 10-fold
decline in the viable cell counts of Salmonella enterica, Escherichia coli and Staphylococcus

NU
aureus after 24-h incubation compared to the control.
MA
Industrial relevance: One of the greatest challenges of food industry is microbial
contamination. The present study suggests that PE coating with chitosan-ZnO nanocomposite is
a promising technique to enhance antimicrobial properties of the films. Chitosan-ZnO
nanocomposite coatings improved antibacterial properties of PE by inactivating about 99.9% of
ED

viable pathogenic bacteria. Hence, our results show the effectiveness of the nanocomposite
coating in the development of active food packaging in order to prolong the shelf life of food
products.
PT
CE

Keywords: Polyethylene, chitosan, ZnO nanoparticle, nanocomposite, active food packaging.

AC

Chemical compounds used in this article:

Acetic acid (PubChem CID: 176); Chitosan (PubChem CID: 71853); Ethanol (PubChem CID:
702); ZnO (PubChem CID: 14806).

2
 ACCEPTED MANUSCRIPT

1. Introduction

Microbial contamination is one of the most common problems in food products which
can lead to quality deterioration and reduce shelf life of products (Ding, Fu & Smith, 2013).

T
Packaging provides some level of protection to food products from external and internal

P
unfavorable conditions (Mihindukulasuriya & Lim, 2014). Antimicrobial packaging offers

RI
enhanced protection to products by the incorporation of antimicrobial agents into the packaging
film in order to inhibit microbial growth and extend the shelf-life of the product (Soares et al.,
2009; Rocha, Ferreira, Souza & Prentice, 2013). In this technology packaging materials are

SC
coated with antimicrobial agents leading to the death or inhibition of microbial growth on food
surfaces. The incorporation of antimicrobial agents into the packaging material could enhance its

NU
functional properties by retarding microorganism growth and extending shelf life of food
products (Rocha, Ferreira, Souza & Prentice, 2013; Sirelkhatim et al., 2015).

Among the polymers used in food packaging, polyethylene (PE) is most popular due to
MA
its lower cost, high impact strength and reasonable chemical resistance (Franklin et al., 2007;
Carrion, Sanes & Bermudez, 2007). PE has relatively high gas permeability, lower sensitivity to
oils but poor odour resistance and does not possess antimicrobial properties (Bag, Ghosh &
Maiti, 1998; Munteanu et al., 2014). Antimicrobial PE films can be prepared either by the
ED

incorporation and immobilization of antimicrobial agents to the polymer films or by the

modification of film surface with coatings. Incorporation of antimicrobial agents into the PE
matrix prior to film fabrication is limited due to the stability of the antimicrobial agents and their
PT

incompatibility with the PE polymer matrix (Theapsak, Watthanaphanit & Rujiravanit, 2012).
Therefore, PE surface modification and coating are normally used. Coating of plastic films with a
CE

polymer based solution is an inexpensive method often leading to a higher stability and
adhesiveness of attached antimicrobial molecules (An, Kim, Lee, Paik & Lee, 2000).

Chitosan (1–4 linked 2-amino-deoxy-β-D-glucan) is a well-known biopolymer that could

AC

inhibit the growth of wide varieties of fungi, pathogenic bacteria and spoilage microorganisms
(Ravi Kumar, 2000; Dutta, Tripathi, Mehrotra & Dutta, 2009). It has widespread application in
biomedical, chemical and food industries due to its antimicrobial activity, biocompatibility,
biodegradability, high water permeability, low toxicity and susceptibility to chemical
modifications (Kaushik et al., 2008; Munteanu et al., 2014). Chitosan is a partially deacetylated
polymer of acetyl glucosamine that is a naturally occurring linear cationic polysaccharide.
Chitosan is obtained through alkaline deacetylation of chitin, which can be extracted from a
variety of crustacean shells, fungal cell walls and other biological materials (Coma et al., 2002,
Kim et al., 2011). Antimicrobial activity of chitosan depends on the concentration, molecular
weight and deacetylation degree (Shahidi, Arachchi & Jeon, 1999; Chi, 2004). Chitosan
solutions in various organic acids can be prepared which upon drying form flexible, clear and
tough films that have been shown to be good oxygen barriers (Caner, Vergano & Wiles, 1998;
Bourtoom, 2008). In 2001, chitosan was classified as safe by the US FDA (Sagoo, Board &
Roller,2002) and is thus considered safe to be used as a food preservative (Friedman & Juneja,
3
 ACCEPTED MANUSCRIPT

2010). In food industries, chitosan has been extensively used for direct surface coating of meat
and fruit products to reduce food deterioration and water loss, as well as delay in the ripening of
fruits (Hernández-Muñoz, Almenar, Ocio & Gavara, 2006; Aranaz et al., 2009).

T
Antimicrobial activity and stability of chitosan can be enhanced by the incorporation of

P
conducting polymers, metal nanoparticles and oxide agents (Dhillon, Kaur & Kaur Bra, 2014).

RI
Though silver nanoparticles have proven antimicrobial effects, it is still not clear whether it is
safe to use silver containing products in direct human exposure (Nowack, Krug, & Heigh, 2011,
Echegoyen & Nerín, 2013; Cushen, Kerry, Morris, Cruz-Romero & Cummins, 2014). Previous

SC
reports demonstrated that chitosan composite with inorganic metal oxides has increased stability
and antibacterial activity (Applerot et al., 2009; Haldorai & Shim; 2013).

NU
Amongst the metal oxides, zinc oxide (ZnO) is one of the most widely applied materials
in various fields due to its remarkable antimicrobial and photocatalytic properties (Vasilache,
Popa, Fiolte, Creto & Benta, 2011). ZnO nanoparticles in comparison to other metal oxide
MA
nanoparticles are regarded as safe materials for human beings, and have been used as food
additives, packaging materials and in water purification (Stoimenov, Klinger, Marchin,
Klabunde, 2002; Chaudhry et al., 2008; Bradley, Castle & Chaudhry, 2011). ZnO has been
introduced into a number of food packaging coatings to maintain food colors and avoid spoilage
ED

and improve packaging material properties, including mechanical strength, barrier properties and
stability (Shi et al., 2014; Sirelkhatim et al., 2015). PE with chitosan- ZnO nanocomposite can
enhance antimicrobial properties of active packaging but there are very few reports on the use of
PT

chitosan and ZnO nanocomposite for antimicrobial food packaging (de Azeredo, 2012; Sanuja,
Agalya & Umapathy, 2015)
CE

In this study, antimicrobial activity of low density PE films coated with chitosan and
ZnO-chitosan nanocomposite was investigated. The specific aims of this study were to: 1) coat
PE with chitosan and ZnO-chitosan nanocomposite coatings; 2) characterize films using Fourier
AC

Transformation Infrared (FTIR) spectroscopy, water contact angle (WCA) measurements and
Scanning Electron Microscopy (SEM); 3) test antibacterial effect of the coatings against food
pathogens Salmonella enterica, Escherichia coli and Staphylococcus aureus.

2. Material and methods

2.1 Materials

Low density polyethylene (PE, 5x8x2MIL) was purchased from Cole-Parmer Instrument
Co, Oman. Medium molecular weight chitosan with 110 cps viscosity and 95.6 % deacetylation
degree was purchased from Tru-Nutra Nutraceuticals LLC, India. Acetic acid, ethanol and ZnO
nanoparticles were purchased from SIGMA Aldrich, USA.

4
 ACCEPTED MANUSCRIPT

2.2 Methods

T
2.2.1 Characterization of Commercial ZnO nanoparticles

P
Particle size distribution of commercial ZnO powder (35-45 nm) was determined using CILAS

RI
NanoDS (France) dynamic light scattering (DLS) based particle size analyzer. Further
characterization was done by High Resolution Transmission Electron Microscopy (HRTEM)

SC
(JEOL JEM-2100F, Japan).

NU
2.2.2 Preparation of Chitosan and Chitosan-ZnO nanocomposite Coating

Preparation of Chitosan-ZnO nanocomposite solution

MA
2 g of chitosan powder was dissolved in 0.5 % acetic acid (SIGMA Aldrich, USA) to
prepare 2 % chitosan solution. Solution was kept for 24 h until powder was totally dissolved. 0.1
% solution of commercial ZnO nanoparticles in ethanol was added with rigorous stirring to the
previously prepared chitosan solution to obtain chitosan-ZnO nanocomposite. The optimum ZnO
ED

nanoparticle concentration was tested (data not shown) and the lowest concentration of ZnO
nanoparticles that gave the best gel consistency of the nanocomposite solution and antimicrobial
activity was used. The concentration of ZnO nanoparticles in the solution was selected after
PT

preliminary experiments (data not shown) in order to use the lowest concentration of ZnO
nanoparticles showing acceptable antimicrobial properties. The nanocomposite solution was
CE

analyzed using UV/VIS Spectroscopy (PerkinElmer Lambda 25, USA) in the wavelength range
of 250- 600 nm. For comparison, 2 % chitosan solution in 0.5 % acetic acid solution was used to
form chitosan coatings.
AC

Packaging Coating Application

Polyethylene (PE) films were cleaned with 70 % ethanol solution and dried at room
temperature (26 °C). The dried PE films were then treated using a plasma instrument
(MiniFlecto, Germany) (Pressure: 0.2 mBar, O2: 3-4 standard cm3 per minute (SCCM), Power:
50 %). Plasma treatment was used to provide a hydrophilic property to the PE surface in order to
result in a better attachment of chitosan to the PE surface. After plasma treatment, 6 ml of
previously prepared chitosan-ZnO nanocomposite solution was sprayed on the PE surface (10 x
15 cm) and allowed to dry at room temperature (26 °C). PE films coated with 2 % chitosan were
used for comparison, and uncoated PE was used as a control. More than 20 coated films were
prepared. The testing and characterization (see below) was done on randomly cut samples from
the larger films. The experiments were repeated at least 5 times for definitive confirmation of
results.

5
 ACCEPTED MANUSCRIPT

2.2.3 Characterization of Coated Packaging Material

T
2.2.3.1 FTIR, SEM and EDS

P
Fourier Transform Infra-red (FTIR) spectroscopy was used to identify the chemical
structure of the composite films and the possible interactions between their components. The

RI
coated and uncoated PE films were analyzed using Frontier (FTIR) spectrometer (PerkinElmer
USA), in a spectral range from 4000 to 500 cm-1. Surface morphology of all samples were

SC
characterized by JEOL JSM-7200 (Japan) field emission scanning electron microscope (FESEM)
working at 20 kV. Energy Dispersive Spectrometry (EDS) was used to determine the elemental
composition of the coatings.

NU
2.2.3.2 Water contact angle (WCA) measurements
MA
To determine film hydrophobicity, the static contact angles of the coatings with chitosan
and composite coatings and uncoated PE films were measured using a Theta Lite attension
tensiometer (Biolin Scientific, Sweden) using sessile drop technique (Al-Fori, Dobretsov, Myint
ED

& Dutta, 2014). The measurements were conducted by averaging over five different locations of
the surface of the films and the mean WCA was reported for each coating.
PT

2.2.3.3 Swelling and Solubility

CE

Film swelling and solubility in water was determined according to the methods reported
by Casariego et al. (2009) and Zhong, Song & Li (2011) with minor modifications. Small pieces
(2 cm × 4 cm) were cut from each film and dried in an oven at 70 °C for 24 h (to a constant
AC

weight) to determine the initial dry weight (M1).Then the dry films were placed in 50 mL of de-
ionized water at 30 °C under constant agitation. After 24 h of immersion, the wet weight (M2) of
the samples were measured immediately after drying superficially with filter paper (Whatman
No. 1), to remove excess water. The film samples were dried again in an oven at 70 °C for 24 h
(to a constant weight) to determine the final dry weight (M3). Three replicates were taken from
each film sample and the weights were taken to the nearest of 0.0001 g. Film swelling and
solubility percentages were calculated using the following equations:

Swelling ratio (%) = (( −  )/ )  100

Film solubility (%) = (( −  )/ ) 100

2.2.3.4 Antibacterial Assay

6
 ACCEPTED MANUSCRIPT

The antimicrobial effect of coated and uncoated PE films on the bacterial growth was
evaluated by suspension culture medium according to Leceta, Gurrero, Ibarburu, Dueñas & Caba
(2013) with some modifications. Antibacterial activity of the coated and uncoated PE films was

T
tested against pathogenic bacteria, Escherichia coli (ATCC 25922), Salmonella enterica serovar
Typhimirium (ATCC 14028) and Staphylococcus aureus (ATCC 6538) obtained from the Sultan

P
Qaboos University culture collection. This test is an end point assay and gives information about

RI
inhibition of microbial growth at a specific time point and it is more reliable than the agar
diffusion method, Kirby–Bauer antibiotic testing (Bauer, Perry & Kirby, 1959), which is affected

SC
by the nature of the coating. Prior to the assay, all bacterial strains were cultivated in nutrient
broth (SIGMA Aldrich, USA) for 24 h at 37 °C. A volume of 2.5 ml of nutrient broth was added
to each well of a 24- well plate (SIGMA Aldrich, USA). PE (size 2 cm2) coated with chitosan or

NU
chitosan-ZnO nanocomposite was placed into each well. In the control wells, uncoated PE (size 2
cm2) were used. Twenty µl of fresh bacterial culture was added to each well and incubated at 37
°C for 24 h. Uncoated PE was used as control. The initial concentrations of bacterial cells in each
MA
well before incubation was about 4.6x106 CFU/ml, 3.2x106 CFU/ml and 3.9x106 CFU/ml for
S,enterica, S.aureus and E.coli respectively. All samples in the experiment were tested in
triplicate and the test was repeated at least 5 times to confirm the results. After incubation for 24
h, the number of bacterial colony forming units per ml (CFU/ml) was calculated in each sample.
ED

2.2.4 Statistical Analysis

PT

Differences between the data were analyzed using the Analysis of Variance (ANOVA).
Before the analysis, assumption of normality of the data was verified using the Shapiro-Wilk test
CE

(Shapiro & Wilk, 1965). Tukey HSD post hoc test was used to test the significance of differences
between different treatments. In all cases the significance level was p=0.05.
AC

3. Results and Discussion

3.1 Particle size of Commercial ZnO Nanoparticles

Figure 1(a) shows the transmission electron micrograph (TEM) of commercial ZnO
nanoparticles. The particle size distribution shows that most of the ZnO nanoparticles were in the
range of 50- 60 nm with an average particle diameter of ~ 55 nm (Figure 1b).

Figure 1

3.2 UV/VIS Spectroscopy of coating solutions

7
 ACCEPTED MANUSCRIPT

Figure 2 presents UV/VIS absorbance spectra of chitosan and chitosan/ZnO (0.1 %)

composite ZnO suspension. The results indicate the incorporation of ZnO nanoparticles in a
chitosan matrix as an absorption band was observed at about 367 nm. The obtained results are in

T
accordance with those reported by Abdelhady (2012) as a small absorption peak at 360 nm was
observed that can be attributed due to the formation of nanoparticles of a larger size. A shift in

P
the adsorption peak to 350 nm was observed when the ZnO concentration was increased to 1 %

RI
(Abdelhady, 2012).

Figure 2.

SC
NU
MA
3.3 Characteristics of Coated PE Films

3.3.1 Fourier Transform Infrared (FT-IR)

The ATR-FTIR spectra of uncoated PE, and PE coated with chitosan and chitosan-ZnO
ED

nanocomposite are shown in Figure 3. The LDPE spectrum observed in this study was similar to
one reported previously (Munteanu et al., 2014). For the chitosan coated PE films, the
characteristic peaks of chitosan were observed at 3329 cm-1 (N−H and O−H stretching) and at
PT

1649 and 1562 cm-1 (amide I and amide II) (Figure 3c). A peak at 1035 cm-1 was attributed to the
stretching vibration of C–O–C (Haldorai & Shim, 2013). In comparison, the spectrum of
CE

chitosan-ZnO nanocomposite coating has a slight shift of the bands corresponding to hydroxyl,
amino, and amide groups towards lower spectral ranges (Figure 3b). This is attributed to the
interaction between chitosan and ZnO nanoparticles (Haldorai & Shim, 2013).
AC

Figure 3.

3.3.2 Microstructure and composition of films

Chitosan-ZnO nanocomposite and chitosan coatings were transparent and did not show
any change in color of the PE film. The Scanning Electron micrographs (SEM) for coated and
uncoated PE are given in Figure 4. The SEM results confirmed the agglomeration of ZnO
nanoparticles to about 500 nm in the case of nanocomposite coating (Figure 4c).

Figure 5 illustrates EDS spectrum of chitosan-ZnO nanocomposite coating. The EDS

profile was acquired on a single spot on the spherical particles on the coating. As peaks of zinc
were shown in the spectra, it proves that ZnO nanoparticles were successfully incorporated in the
chitosan matrix.

8
 ACCEPTED MANUSCRIPT

Figure 4.

Figure 5.

P T
3.3.3 Water contact angle (WCA)

RI
Water contact angle is an indicator of the relative hydrophobicity or hydrophilicity of a
substrate (Rivero, García & Pinotti, 2012). It is defined as the angle between the substrate

SC
surface and the tangent line at the point of contact of the liquid droplet with the substrate. It
determines the wettability of a surface. High contact angle indicates poor wetting which reflect
its hydrophobic property (Shafrin & Zisman, 1960). Figure 6 shows a dramatic decrease in the

NU
WCA from approximately 100 ° (untreated PE) to about 60 ° (1s plasma treated PE) which
demonstrates that the plasma treatment increased the hydrophilicity of the PE film. It was
reported earlier that non- thermal plasma treatment can generate radicals and excited species
MA
which are able to initiate chemical and physical modifications within the depth of few
nanometers on polymer film resulting in the dramatic improvement in the surface free energy
and hydrophilicity of films (Riccardi et al., 2003; Shao et al., 2010). The lowest WCA value (40
ED

°) was found after 3 s of treatment and then WCA increased for a longer treatment. Thus, 3 s
plasma treatment was selected for further film coatings.

Chitosan and ZnO nanocomposite coating exhibits contact angle value of 95 ° which is
PT

higher than the value of chitosan coating (ANOVA, HSD, p < 0.05, Figure 7). This can be
attributed to change in hydrophobicity of coatings by the addition of ZnO nanoparticles. It was
CE

reported that treatment of chitosan films or its blending with other materials changed film
wettability (Rivero, García & Pinotti, 2013). Chitosan cross-linked with tannic acid decreased
WTA from 88.4 °, which is attributed to the hydrophilic nature of tannic acid (Rivero, García &
AC

Pinotti, 2013). Similarly, increasing hydrophobicity of chitosan films was observed when
blended with Halloysite nanoclay (Sarkar, 2015).

Figure 6.

Figure 7.

3.3.4 Swelling ratio and film solubility

The swelling behavior of chitosan, as well as chitosan/ZnO nanocomposite coating on PE films,

is shown in Table 1. Film solubility of chitosan ZnO nanocomposite coating was higher than
chitosan coating. In contrary, the swelling ratio of chitosan decreased dramatically when ZnO
nanocomposite coating was made (Table 1). Similar decrease in swelling of chitosan after the
incorporation of ZnO was observed in an earlier work (Liu &Kim, 2012). It was recently

9
 ACCEPTED MANUSCRIPT

reported that the incorporation of ZnO strongly reduce water uptake by forming a three
dimensional ZnO network in chitosan matrix (Das, Maiti & Li, 2014). Swelling degree of
polymers is dependent on the nature and amount of intramolecular chain interaction (Pierro et al.,

T
2006). Reicha, Sarhan, Abdel-Hamid & El-Sherbiny (2012) addressed a reduction in swelling
ratio when silver nanoparticles were incorporated into chitosan matrix and a continuous

P
reduction in swelling values was observed with the increase in silver concentrations. This

RI
behavior was attributed to silver acting as a crosslinker between the chitosan chains reducing the
mobility of the latter and consequently lowering the degree of swelling of the chitosan films.

SC
Table 1.

NU
MA
3.3.5 Antibacterial Assay

The inhibitory activity of the coated and uncoated PE films was investigated using two
ED

Gram negative bacteria, S.enterica and E.coli, and one Gram-positive bacterial strain, S.aureus
(Figure 8). The results showed that both chitosan and nanocomposite coatings significantly
(ANOVA, HSD, p<0.05) inhibited bacterial growth Chitosan films had a high antimicrobial
PT

activity against all tested bacteria showing 1.3, 1.6 and 1.4-log reduction in S.enterica and E.coli,
and S.aureus viability respectively. However, chitosan-ZnO nanocomposite showed a complete
inhibition of bacterial growth after 24 h incubation (Figure 8).
CE

Antimicrobial activity of chitosan against microorganisms, such as E.coli, S.aureus,

Listeria monocytogenes, Bacillus cereus, Salmonella Typhimurium, was reported previously
AC

(Coma et al., 2002; Pareda, Ponce, Marcovich, Ruseckaite & Martucci, 2011; Lago et al., 2014;
Leceta,Gurrero, Ibarburu, Dueñas & Caba, 2013; Hromis et al., 2014). Antimicrobial activity of
chitosan is due to its positively charged amine groups which form electrostatic interactions with
anionic groups of microbial cell membranes, leading to cell damage by blockage of nutrient
intake of the cell or causing leakage of proteinaceous cytoplasmic fluid (Tsai & Su, 1999; Rabea,
Badawy, Stevens, Sagghe & Steurbaut, 2003; Alisashi & Aider, 2012). The antimicrobial effect
of chitosan coatings diminish over time (Leceta, Gurrero, Ibarburu, Dueñas & Caba, 2013), thus
it is necessary to use nanoparticles or other additives to improve chitosan activity.

The chitosan metal complexes enhance the stability of the coating and increase
antimicrobial activity 10- to 20-fold (Kong, Chen, Xing, & Park 2010). For example, a thiourea
chitosan-silver ion complex overcame the instability of Ag+ and increased antibacterial activity
by 20-fold compared to chitosan alone (Chen, Wu & Zeng 2005). While chitosan ZnO
nanoparticle nanocomposites are slightly less effective than silver nanoparticle composites (Li et

10
 ACCEPTED MANUSCRIPT

al. 2010; Dhillon, Kaur & Kaur Bra 2014), they are less toxic to humans and environmentally
friendly (Reddy et al. 2007). Haldorai & Shim (2013) found that 99.92% of viable bacteria of
E.coli are inactivated after 24 h incubation with chitosan-ZnO nanocomposite. The antibacterial

T
effect of chitosan ZnO nanocomposite against B.subtilis, Enterococcus faecalis, Pseudomonas
aeruginosa, S.typhimurium, S.aureus and E.coli was reported by Sivakumar, Muralidharan,

P
Santhosh, Rajendran & Sivakumar (2014). Chitosan ZnO composite films showed enhanced

RI
antibacterial activities against B. subtilis, E. coli and S. aureus (Li, Deng, Deng, Liu& Li, 2010).
Similar observations for S. aureus and Micrococcus luteus were reported in a study of Dhillon,

SC
Kaur & Kaur Bra (2014). The enhanced antibacterial activity of nanocomposite coatings is
possibly due to synergistic antibacterial activities of chitosan and ZnO nanoparticles. The
antimicrobial activity of ZnO nanoparticles is most likely due to disruption of the bacterial cell

NU
membranes by Zn ions and oxidative stress because of photocatalytic production of reactive
oxygen species (ROS) (Xie, He, Irwin, Jin & Shi, 2011). ROS eventually leads to the damage of
bacterial proteins, DNA and lipids leading to cell death (Sawai, 2003; Shi et al., 2014).
MA

Figure 8.
ED

4. Conclusions
PT

In conclusion, our study provide a method to prepare PE films coated with chitosan and
chitosan ZnO nanocomposites. The results prove the successful interaction of chitosan and ZnO
CE

nanoparticles which was confirmed by the formation of spherical ZnO nanoparticles in the
nanocomposite coating. Incorporation of ZnO nanoparticles into the chitosan matrix improved its
water affinity. While chitosan coating provided an effective antimicrobial defense against
AC

S.enterica, E.coli and S.aureus, chitosan-ZnO nanocomposite coatings fully inhibited growth of
the pathogens after 24h incubation. From the data obtained, it can be concluded that PE coating
with chitosan-ZnO nanocomposite is a promising technique to prevent microbial growth and it
can be used as an active food packaging to prolong shelf-life of packaged food.

Acknowledgements

The authors would like to acknowledge financial support from the Chair in
Nanotechnology, The Research council (TRC) of Oman. LN’s work was supported by the SQU
PhD fellowship. SD’s work was supported by an internal SQU grant (IG/AGR/FISH/15/02). The
authors would also like to acknowledge Dr. Karthik Laxman for his assistance in nanoparticle
characterization.

11
 ACCEPTED MANUSCRIPT

5. References

AbdElhady M. M. (2012). Preparation and characterization of chitosan/zinc oxide nanoparticles

for imparting antimicrobial and UV protection to cotton fabric. International Journal of

T
Carbohydrate Chemistry, 1-6.

P
Al-Fori, M., Dobretsov, S., Myint, M. & Dutta, J. (2014) Antifouling properties of zinc oxide

RI
nanorod coatings, Biofouling: The Journal of Bioadhesion and Biofilm Research, 30(7), 871-
882.

SC
Alisashi, A. & Aïder, M. (2012). Applications of chitosan in the seafood industry and
aquaculture: a review. Food and Bioprocess Technology, 5(3), 817-830.

NU
An, D. S., Kim, Y. M., Lee, S. B., Paik, H. D. & Lee, D. S. (2000). Antimicrobial low density
polyethylene film coated with bacteriocins in binder medium. Food Science and
Biotechnology, 9, 14-20.
MA
Applerot, G., Lipovsky, A., Dror, R., Perkas, N., Nitzan, Y., Lubart, R. &Gedanken, A.
(2009).Enhanced antibacterial activity of nanocrystalline ZnO due to increased ROS-
mediated cell injury. Advanced Functional Materials, 19, 842-852.
ED

Aranaz, I., Mengíbar, M., Harris, R., Paños, I., Miralles, B., Acosta, N., Galed, G. & Heras A.
(2009) Functional characterization of chitin and chitosan. Current Chemical Biology, 3(2),
PT

203-230.

Bag, D. S., Ghosh, S. N. & Maiti, S. (1998). Surface modification and evaluation of polyethylene
CE

film. European Polymer Journal, 34(5-6), 855-861.

Bauer, A. W., Perry, D. M., & Kirby, W. M. (1959). Single-disk antibiotic-sensitivity testing of
staphylococci: An analysis of technique and results. AMA archives of internal medicine,
AC

104(2), 208-216.

Bourtoom, T. (2008). Review Article: edible films and coatings: characteristics and properties.
International Food Research Journal, 15(3), 237-248.

Bradley, E. L, Castle, L. & Chaudhry, Q. (2011).Applications of nanomaterials in food

packaging with a consideration of opportunities for developing countries. Trends in Food
Science and Technology, 22, 604-610.

Caner C., Vergano P. J., & Wiles, J. L. (1998). Chitosan film mechanical and permeation
properties as affected by acid, plasticizer, and storage. Journal of Food Science, 63, 1049-
1053.

Carrion, F. G., Sanes, J. & Bermudez, M. D. (2007). Influence of ZnO nanoparticle filler on the
properties and wear resistance of polycarbonate. Wear, 262, 1504.

12
 ACCEPTED MANUSCRIPT

Casariego, A., Souza, B., Cerqueira, M., Teixeira, J., Cruz, L., Di´az, R. & Vicente, A. A. (2009)
Chitosan/clay films’ properties as affected by biopolymer and clay micro/nanoparticles’
concentrations. Food Hydrocolloids, 23, 1895-1902.

T
Chaudhry, Q., Scotter, M., Blackburn, J., Ross, B., Boxall, A., Castle, L., Aitken, R. & Watkins

P
R. (2008). Applications and implications of nanotechnologies for the food sector. Food

RI
Additives and Contamination, A. 25(3), 241-258.

Chi, S. (2004). Development and characterization of antimicrobial food coatings based on

SC
chitosan and essential oils. Master thesis, University of Tennessee, Knoxville.

Chen, S.P., Wu, G.Z., Zeng, H.Y. (2005). Preparation of high antimicrobial activity thiourea-

NU
chitosan Ag+ complex. Carbohydrate Polymer, 60, 33-38.

Coma, V., Martial-Gros, A., Garreau, S., Copinet, A., Salin, F. & Deschamps, A. (2002). Edible
antimicrobial films based on chitosan matrix. Journal of Food Science, 67(3), 1162-1169.
MA
Cushen, M., Kerry, J., Morris, M., Cruz-Romero, M., Cummins, E. (2014). Evaluation and
simulation of silver and copper nanoparticle migration from polyethylene nanocomposites to
food and an associated exposure assessment. Journal of Agricultural and Food Chemistry,
ED

62(6), 1403-11.

Das, K., Maiti, S. & Li, D. (2014). Morphological, mechanical and thermal study of ZnO
PT

nanoparticle reinforced chitosan based transparent biocomposite films. Journal of the

Institution of Engineers (India), 95(1), 35-41.
CE

Dhillon, G. S., Kaur, S. & Kaur Bra, S. (2014). Facile fabrication and characterization of
chitosan-based zinc oxide nanoparticles and evaluation of their antimicrobial and antibiofilm
activity. International Nano Letters, 4, 107.
AC

Ding, H., Fu, T-J. & Smith M. A. (2013). Microbial contaminationin sprouts: How effective is
seed disinfection treatment? Journal of Food Science, 78(4), 459-501.

Dutta, P. K., Tripathi, S., Mehrotra, G. K., & Dutta, J. (2009). Perspectives for chitosan based
antimicrobial films in food applications. Food Chemistry, 114, 1173-1182.

Echegoyen, Y. & Nerín, C., (2013), Nanoparticle release from nano-silver antimicrobial food
containers. Food Chemistry and Toxicology, 62, 16-22.

Franklin, N. M., Rogers, N. J., Apte, S. C., Batley, G. E., Gadd, G. E. & Casey, P. S. (2007).
Comparative toxicity of nanoparticulate ZnO, bulk ZnO, and ZnCl2 to a fresh water
microalga; Pseudokirchneriellasubcapitata: The importance of particle solubility.
Environmental Science and Technology, 41 (24), 8484-8490.

13
 ACCEPTED MANUSCRIPT

Friedman, M. & Juneja, V.K. (2010). Review of antimicrobial and antioxidative activities of
chitosans in food. Journal of Food Protection, 73, 1737-1761.

Haldorai, Y. & Shim, J. (2013). Chitosan-Zinc Oxide hybrid composite for enhanced dye

T
degradation and antibacterial activity. Composite Interfaces, 20(5), 365-377.

P
De Azeredo, H. M. C. (2012) Antimicrobial Activity of Nanomaterials for Food Packaging

RI
Applications (N. Cioffi and M. Rai, Eds.). Chapter 13, Nano-Antimicrobials. Springer-Verlag
Berlin Heidelberg.

SC
Hernández-Muñoz, P., Almenar, E., Ocio, M. & Gavara, R. (2006). Effect of calcium dips and
chitosan coatings on postharvest life of strawberries (Fragaria x ananassa). Postharvest

NU
Biology and Technology, 39(3), 247-253.

Hromiš N. M, Lazić V. L , Markov S. L, Vaštag Z. G, Popović S. Z, Šuput D. Z, Džinić N. R.

(2014). Improvement of antioxidant and antimicrobial activity of chitosan film with caraway
MA
and oregano essential oils. APTEFF; 45, 1-283.

Kaushik, A., Khan, R., Solanki, P. R., Pandey, P., Alam, J., Ahmad, S. & Malhotra B. D. (2008).
Iron oxide nanoparticles-chitosan composite based glucose biosensor. Biosensors and
ED

Bioelectronics, 24:676-683.

Kim, K.W., Min, B. J., Kim, Y. T., Kimmel, R. M., Cooksey, K. & Park, S. I. (2011).
PT

Antimicrobial activity against foodborne pathogens of chitosan biopolymer films of different

molecular weights. LWT - Food Science and Technology, 44(2), 565-569.
CE

Kong, M., Chen, X. G., Xing, K. & Park, H. J. (2010). Antimicrobial properties of chitosan and
mode of action: A state of the art review. International Journal of Food Microbiology,
144:51-63.
AC

Lago, M. A., Sendón, R., Rodríguez-Bernaldo de Quirós, A., Sanches-Silva, A., Costa, H. S.,
Sánchez-Machado, D. I., Soto Valdez, H., Angulo, I., Aurrekoetxea, G. P., Torrieri, E.,
López-Cervantes, J. & Paseiro P. (2014). Preparation and characterization of antimicrobial
films based on chitosan for active food packaging applications. Food Bioprocess Technology,
7(10), 2932-2941.

Li, L. H., Deng, J. C., Deng, H. R., Liu, Z. L., Li, X. L. (2010). Preparation, characterization and
antimicrobial activities of chitosan/Ag/ZnO blend films. Chemical Engineering Journal, 160,
378-382.

Leceta, I., Gurrero, P., Ibarburu, I., Dueñas, M. T. & Caba, K. (2013). Characterization and
antimicrobial analysis of chitosan-based films. Journal of Food Engineering; 116(4), 889-899.

14
 ACCEPTED MANUSCRIPT

Liu, Y. & Kim, H. (2012). Characterization and antibacterial properties of genipin-crosslinked

chitosan/poly (ethylene glycol)/ZnO/Ag nanoparticles. Carbohydrate Polymers, 89(1), 111-
116.

T
Mihindukulasuriya, S. D. F. & Lim, L-T. (2014). Nanotechnology development in food

P
packaging-a review. Trends in Food Science & Technology, 40(2), 149-167.

RI
Munteanu, B. S, Paslaru, E., Zemljic, L. F., Sdrobis, A., Pricope, G. M. & Vasile, C. (2014).
Chitosan coating applied to polyethylene surface to obtain food packaging materials.

SC
Cellulose Chemistry and Technology, 48 (5-6), 565-575.

Nowack, B., Krug, H. F. & Height, M. (2011). 120 Years of Nanosilver History: Implications for

NU
Policy. Environmental Science and Technology, 45, 1177-1183.

Pierro, P., Chico, B., Villalonga, R., Mariniello, L., Damiao, A. E. & Masi P. (2006).Chitosan-
whey protein edible films produced in the absence or presence of transglutaminase: Analysis
MA
of their mechanical and barrier properties. Biomacro-molecules, 7 (3), 744-749.

Pareda, M., Ponce, A. G., Marcovich, N. E., Ruseckaite, R. A., Martucci, J. F. (2011).Chitosan-
gelatin composites and bi-layer films with potential antimicrobial activity. Food
ED

Hydrocolloids, 25(5), 1372-1381.

Rabea, E. I., Badawy, M. E. T., Stevens, C. V., Sagghe, G. & Steurbaut, W. (2003). Chitosan as
PT

antimicrobial agent: applications and mode of action. Biomacro-molecules, 4(6), 1457-1461.

Ravi Kumar, M. N. V. (2000). A Review of chitin and chitosan applications. Reactive and
CE

Functional Polymers, 46(1), 1-27.

Reicha, F. M., Sarhan, A., Abdel-Hamid, M. I. & El-Sherbiny, I. M. (2012). Preparation of silver
AC

nanoparticles in the presence of chitosan by electrochemical method. Carbohydrate Polymers,

89(1), 236-244.

Reddy, K.M., Feris, K., Bell, J., Wungett, D.G., Hanley, C. & Punnoose, A. (2007). Selective
toxicity of zinc oxide nanoparticles to prokaryotic and eukaryotic systems. Appl Phys Lett
213902

Riccardi, R., Barni, R., Selli, E., Mazzone, G., Massafra, M. R., Marcandalli, B. & Poletti, G.
(2003). Sufarce modification of poly(ethylene terephthalate) fibers induced by radio
frequency air plasma treatment. Applied Surface Science, 211(1-4), 386-397.

Rivero, S., García, M. A. & Pinotti, A. (2012). Heat treatment to modify the structural and
physical properties of chitosan-based films. Journal of Agricultural and Food Chemistry,
60(1), 492-499.

15
 ACCEPTED MANUSCRIPT

Rivero, S., García, M. A. & Pinotti, A. (2013). Physical and chemical treatments on chitosan
matrix to modify film properties and kinetics of biodegradation. Journal of Materials Physics
and Chemistry, 1(3), 51-57.

T
Rocha, M., Ferreira1, F. A., Souza1, M. M. & Prentice, C. (2013). Microbial pathogens and

P
strategies for combating them: science, technology and education (A. Méndez-Vilas, Ed.).

RI
Antimicrobial films: a review (pp. 23-31). Formatix research center.

Sagoo, S., Board, R., & Roller, S. (2002). Chitosan inhibits growth of spoilage micro-organisms

SC
in chilled pork products. Food Microbiology, 19, 175-182.

Sanuja, S., Agalya, A. & Umapathy, M. J. (2015). Synthesis and characterization of zinc oxide–

NU
neem oil–chitosan bionanocomposite for food packaging application, International Journal of
Biological Macromolecules, 74, 76-84.

Sarkar, P. (2015). Properties affected by different shape and different weight percentages
MA
nanoparticles, embedded in chitosan polymer thin film. European Journal of Advances in
Engineering and Technology, 2(1), 91-100.

Sawai, J. (2003). Quantitative evaluation of antibacterial activities of metallic oxide powders

ED

(ZnO, MgO and CaO) by conductimetric assay. Journal of Microbiological Methods, 54(2),
177-182.
PT

Shahidi, F., Arachchi, J.K. V. & Jeon,Y. J.(1999). Food applications of chitin and chitosans.
Trends in Food Science & Technology, 10(2), 37-51.
CE

Shafrin, E. G. & Zisman, W. A. (1960).Constitutive relations in the wetting of low-energy

surfaces and the theory of the retraction method of preparing monolayers. Journal of Physical
Chemistry, 64, 519-524.
AC

Shapiro, S. S. & Wilk, M. B. (1965). An analysis of variance test for normality (complete
samples). Biometrika, 52(3-4), 591-611.

Shao, T., Zhang, C., Long, K., Zhang, D., Wang, J., Yan, P. & Zhou, Y. (2010). Surface
modification of polyamide films using unipolar nanosecond-pulse DBD in atmospheric air
Applied Surface Science, 256(12), 3888-3894.

Shi, L. E., Li, Z. H.,Zheng, W., Zhao, Y. F., Jin Y. F.& Tang, Z. X. (2014). Synthesis,
antibacterial activity, antibacterial mechanism and food applications of ZnO nanoparticles: a
review, Food Additives & Contaminants, A, 31(2), 173-86.

Sirelkhatim, A., Mahmud, S., Seeni, A., Kaus, N. H. M., Ann, L. C., Bakhori, S. K. M., Hasan,
H.& Mohamad. D. (2015). Review on Zinc Oxide Nanoparticles: Antibacterial Activity and
Toxicity Mechanism. Nano-Micro Letters, 7(3), 219-242.

16
 ACCEPTED MANUSCRIPT

Sivakumar P., Muralidharan N. G., Santhosh V., Rajendran P., Sivakumar P. (2014). Synthesis
of Cs-ZnO-Ag nanocomposite from crustacean shells and its antibacterial activity.National
Conference on Green Engineering and Technologies for Sustainable Future. Journal of

T
Chemical and Pharmaceutical Sciences, ISSN: 0974-2115.

P
Soares, N., Silva, C. A. S., Santiago-Silva, P., Espitia, P. J. P, Gonc¸ alves, M. P. J. C., Lopez,

RI
M. J. G., Miltz, J., Cerqueira, M. A., Vicente, A. A. & Teixeira, J. (2009). Engineering
Aspects of Milk and Dairy Products .J.A.T. Jane Selia dos Reis Coimbra ed., Active and
Intelligent Packaging for Milk and Milk Products (pp. 155-174).CRC Press.

SC
Stoimenov, P.K., Klinger, R.L., Marchin, G. L. &Klabunde, K. J. (2002). Metal oxide
nanoparticles as bactericidal agents. Langmuir. 18(17), 6679-6686.

NU
Theapsak S., Watthanaphanit A., and Rujiravanit R. (2012). Preparation of chitosan-coated
polyethylene packaging films by DBD plasma treatment. American Chemical Society,
MA
Applied Materials and Interfaces, 4(5), 2474-2482.

Tsai, G. J. & Su, W.H. (1999). Antibacterial activity of shrimp chitosan against Escherichia coli.
Journal of Food Protection, 62(3), 239-243.
ED

Vasilache V., Popa C., Fiolte C., Creto M. & Benta M. (2011). Nanoparticles applications for
improvingthe food safety and food processing. 7th International Conference on Materials
Science and Engineering, 12 No. 1(31), 77-81.
PT

Zhong, Y., Song, X. & Li, Y. (2011). Antimicrobial, physical and mechanical properties of
kudzu starch-chitosan composite films as a function of acid solvent types. Carbohydrate
CE

Polymer, 84(1), 335-342.

AC

17
 ACCEPTED MANUSCRIPT

Figure and Table legends

Figure 1. (a) High Resolution transmission electron micrograph of the commercial ZnO
nanoparticles (35-45 nm) and (b) particle size analysis made from photocorrelation spectroscopy

P T
Figure 2. UV/VIS spectra of chitosan and ZnO nanocomposite and chitosan (chitosan/ZnO)
solutions. Insert - Zn2+ absorption peak.

RI
Figure 3. FTIR spectra of polyethylene (PE) films coated with chitosan and chitosan-ZnO

SC
nanocomposite compared to uncoated PE: (a) uncoated PE, (b) PE coated with chitosan-ZnO
nanocomposite, (c) PE coated with chitosan.

Figure 4. SEM images of PE films: (a) uncoated PE (1000X), (b) PE coated with chitosan

NU
(1000X), (c) PE coated with chitosan-ZnO nanocomposite (20000X).

Figure 5. Energy Dispersive Spectrometry (EDS) spectrum of PE surface coated with

MA
Chitosan/ZnO nanocomposite. Each peak represents different elements.

Figure 6. The effect of plasma treatment time on WCA of plasma treated PE films. The data are
means ± standard deviations of five replicates.
ED

Figure 7. Measurement of static contact angle of a water droplet on coated and uncoated
polyethylene (PE) films: (a) Uncoated plasma treated PE, (b) PE coated with chitosan; and PE
coated with chitosan-ZnO nanocomposite (ANOVA, HSD, p < 0.05). The data are means ±
PT

standard deviations of five replicates.

Figure 8. Antibacterial activity of polyethylene (PE) films, PE films coated with chitosan and
CE

chitosan-ZnO nanocomposite. There was no growth of bacteria in the presence of nanocomposite

coating. The data are mean + standard deviations of three replicates.
AC

Table 1. Swelling ratio and solubility properties of chitosan coating and chitosan/ZnO composite
coating on PE.

18
 ACCEPTED MANUSCRIPT

Figures

(a)

PT
RI
SC
(b) NU
MA
ED
PT
CE
AC

Figure 1.

19
 ACCEPTED MANUSCRIPT

PT
RI
SC
NU
MA

Figure 2.
ED
PT
CE
AC

Figure 3

20
 ACCEPTED MANUSCRIPT

PT
RI
SC
Figure 4
NU
MA
ED
PT
CE
AC

Figure 5

21
 ACCEPTED MANUSCRIPT

110

T
100

P
Water Contact Angle (degree)

90

RI
80

SC
70

60

50
NU
MA
40

30
0 1 2 3 4 5
Plasma Treatment Time (s)
ED

Figure 6
PT
CE
AC

Figure 7

22
 ACCEPTED MANUSCRIPT

P T
RI
SC
NU
MA
ED

Figure 8
PT
CE

Table 1.
AC

Swelling ratio Film solubility

Coating
% SD % SD

Chitosan 1.51a ±0.43 0.42a ±0.06

Chitosan/ZnO 0.29b ±0.21 0.73b ±0.10

Different letters (a,b) indicate significant differences between treatments (ANOVA, HSD,
p<0.05).

23
 ACCEPTED MANUSCRIPT

Highlights:

· A chitosan - ZnO nanocomposite coating on polyethylene films was developed.

· Solubility increase and reduction in swelling of the coatings by 42% and 80%,

T
respectively, were observed upon the incorporation of ZnO nanoparticles into chitosan

P
matrix.

RI
· Viability of E.coli, S.enterica and S.aureus cultures was reduced by 99.9% upon using
the nanocomposite coating.

SC
· Nanocomposite coatings are a promising method for active food packaging.

NU
MA
ED
PT
CE
AC

24