food packaging and shelf life 4 (2015) 2635

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Effects of a combination of antimicrobial silver low

density polyethylene nanocomposite films and
modified atmosphere packaging on the shelf life
of chicken breast fillets

Shafrina Azlin-Hasim a,d, Malco C. Cruz-Romero a, Michael A. Morris b,**,

Enda Cummins c, Joseph P. Kerry a,*
a
Food Packaging Group, School of Food & Nutritional Sciences, University College Cork, Cork, Ireland
b
Department of Chemistry, University College Cork, Cork, Ireland
c
Biosystems Engineering, School of Agriculture, Food Science and Veterinary Medicine, Agriculture and Food Science
Centre, University College Dublin, Belfield, Dublin 4, Ireland
d
Department of Food Science, School of Food Science and Technology, Universiti Malaysia Terengganu, 21030 Kuala
Terengganu, Terengganu, Malaysia

article info abstract

Article history: Low-density polyethylene (LDPE) nanocomposite films containing different concentrations
Received 20 November 2014 of silver (Ag) nanoparticles (NPs) (0.5 and 1% of polymer weight, w/w) were manufactured via
Received in revised form extrusion and subsequently characterised. The microbiological quality of chicken breast
24 January 2015 fillets wrapped with Ag/LDPE nanocomposite films, followed by modified atmosphere
Accepted 6 March 2015 packaging (using conventional laminates and employing a gas mix of 40% CO2:60% N2)
Available online 19 March 2015 were assessed. The tensile strength of Ag/LDPE nanocomposite films were significantly
lower ( p < 0.05) than control films (without Ag NPs), indicating that the presence of Ag NPs
Keywords: reduced the film strength. Independent of the concentration of Ag NPs used, Ag/LDPE
Antimicrobial packaging nanocomposite films extended the shelf life of the chicken breast fillets and significantly
LDPE nanocomposites ( p < 0.05) enhanced oxidative stability compared to control films. The results indicated that
Shelf life LDPE nanocomposite films containing Ag NPs could potentially be used as antimicrobial
Silver nanoparticles packaging for food applications.
# 2015 Published by Elsevier Ltd.

significant area of research over the past two decades, as the

1. Introduction
Retailer and consumer demands challenge suppliers to deliver
fresh products more quickly and cost-effectively. Consequent-
ly, the development of antimicrobial packaging has become a
incorporation of antimicrobial agents into or on antimicrobial
packaging materials could provide greater safety assurance,
shelf life extension and quality maintenance of food products
by inhibiting microbial spoilage and suppressing microbial
food-borne illnesses (Appendini & Hotchkiss, 2002). Since

* Corresponding author. Tel.: +353 0 21 4903798.

** Corresponding author. Tel.: +353 0 21 4902180.
E-mail addresses: M.Morris@ucc.ie (M.A. Morris), Joe.Kerry@ucc.ie (J.P. Kerry).
http://dx.doi.org/10.1016/j.fpsl.2015.03.003
2214-2894/# 2015 Published by Elsevier Ltd.
 food packaging and shelf life 4 (2015) 2635
microbial contamination of most food products occurs
predominantly at the surface, mainly due to post-process
handling, attempts have been made to improve safety and to
delay spoilage through the use of antibacterial sprays or dips;
however, limitations to their wider use include; neutralisation
of antimicrobial compounds on contact with the food surfaces
and diffusion of antimicrobial compounds into the food
product (Lopez-Rubio et al., 2004).
The successful development of antimicrobial packaging is
still challenging technology, and relatively few commercialised
products are available on the market today. This is most likely
due to the extremely strict safety and hygiene regulations
adhered to by regulatory authorities, particularly within the EU.
Unfortunately, these guidelines may be out of step with the pace
of technological innovation and information being generated by
researchers, and this will inevitably lead to consumer mistrust
in such technologies, lack of technological and commercial
development of antimicrobial packaging systems and perceived
high costs associated with such technologies through the under
development of commercial materials.
The use of silver (Ag) in antimicrobial packaging offers
numerous advantages including high thermal stability, ease of
incorporation into or onto numerous materials such as
polyester, polyamide, and polypropylene (PP), it has been
classified as GRAS (Generally Recognised as Safe) by the Food
and Drug Administration (FDA) to be used as food preserva-
tive; however, the European Food Safety Authority (EFSA)
classified it as one of a list of additives with general restrictions
(Martnez-Abad, Lagaron, & Ocio, 2012; Zapata et al., 2011).
Silver antimicrobial nanocomposite films have been used for
packaging applications with horticultural produce, such as;
Chinese jujube, lettuce, fresh fruit salad, asparagus and fresh
orange juice (An, Zhang, Wang, & Tang, 2008; Costa, Conte,
Buonocore, & Del Nobile, 2011; Emamifar, Kadivar, Shahedi, &
Soleimanian-Zad, 2010; Li et al., 2009; Martnez-Abad, Ocio,
Lagaron, & Sanchez, 2013), with the shelf life of these food
products was significantly longer than those packaged with
control films which did not possess Ag NPs.
Cushen, Kerry, Morris, Cruz-Romero, and Cummins
(2014a), Huang et al. (2011), and Panea, Ripoll, Gonzalez,
Fernandez-Cuello, and Albert (2014) reported low migration of
Ag from Ag/LDPE nanocomposite films into food products and
food simulants. Cushen et al. (2014a) also reported that the
exposure results fell well below than the exposure limits for all
extreme conditions of the scenarios tested, suggesting Ag/
LDPE nanocomposite films are suitable to be used as food
contact material with non-acidic foods such as chicken breast
fillets because they are less likely to facilitate migration.
Chicken meat is a popular source of protein worldwide,
high in nutritional value and relatively cheap (van Horne &
Bondt, 2013). However, it is very perishable and contains more
pathogenic bacteria than other types of meat (Mantilla et al.,
2012; Rodrguez-Calleja, Cruz-Romero, OSullivan, Garca-
Lopez, & Kerry 2012). Under aerobic chilled conditions the
shelf life of chicken breast fillets was determined to be not
more than 4 days (Balamatsia, Patsias, Kontominas, &
Savvaidis, 2007; Patsias, Badeka, Savvaidis, & Kontominas,
2008); however, under chilled and modified atmosphere
packaging (MAP) conditions the shelf life was extended up
to 7 days (Chouliara, Badeka, Savvaidis, & Kontominas, 2008;
27
Patsias et al., 2008). Any packaging systems that would provide
for longer product shelf life would be of significant interest to
poultry processors, retailers and consumers alike.
From an extensive review of the scientific literature, little is
known about the effects of LDPE nanocomposites containing
Ag NPs on protein-rich food products, such as fresh chicken
breast fillets. Therefore, the objectives of this study were to
manufacture LDPE nanocomposite films containing different
concentrations (0.5 or 1%) of Ag NPs (Ag/LDPE nanocompo-
sites) via extrusion and subsequently characterise the man-
ufactured materials, and investigate the effects of wrapping
chicken breast fillets with Ag/LDPE nanocomposite films
followed by MAP (using conventional laminates and employ-
ing a gas mix of 40% CO2:60% N2), on the physicochemical and
microbiological quality of chicken breast fillets during chilled
storage at 4 8C.
2. Materials and methods
2.1. Reagent and supply
Silver nitrate (AgNO3), poly(N-vinylpyrrolidone) (PVP,
MW = 40,000), ethanol 99.5% and acetone were purchased
from SigmaAldrich. All chemicals were used without further
purification. Low-density polyethylene pellets were supplied
by Boxmore plastics (Ireland), and skinless breasts of chicken
fillets of similar sizes were purchased from a local supplier
(Shannon Vale, Ireland). Stomacher bags were obtained from
Seward (UK) and Plate Count Agar (PCA) was purchased from
Merck (UK). Pseudomonas Agar Base with selective supple-
ment CFC (cetrimide, fucidin, cephaloridine) (SR0103E); Strep-
tomycin Thallous Acetate Actidione (STAA) supplemented
with Streptomycin Sulphate and Thallous Acetate (SR0151E);
de Man, Rogosa and Sharpe (MRS) agar, Maximum Recovery
Diluent (MRD) and Oxidase Touch Sticks were purchased from
Oxoid (Basingstoke, UK). Compact Dry-EC chromogenic plates
were obtained from Nissui Pharmaceutical (Co. Ltd. Japan).
2.2. Synthesis and characterisation of Ag nanoparticles
Silver NPs of 10 nm particle size was synthesised as outlined in
Cushen et al. (2014a). Images of Ag NPs were taken by scanning
electron microscopy (SEM, FEI Company, FEG Quanta 6700) at
5.0 kV. The stability, surface properties, and average size of Ag
NPs (0.1%, w/v) dissolved in absolute ethanol and ultrasoni-
cated (Cole-Palmer 8891) for 30 min was measured using the
Zetasizer (Zetasizer Nano ZS, Malvern, US). In order to confirm
the presence of the Ag NPs in the ethanolic solution,
spectrophotometric scanning at a wavelength range of 300
800 nm was carried out in a UVvisible spectrophotometer
(Varian Cary 300 Bio, USA) using ethanol as blank.
2.3. Manufacture of LDPE films
The Ag/LDPE nanocomposite used in this study was prepared on
a micro 27 labscale twin screw extruder (Leistritz, Nuernberg,
Germany) with a 27 mm screw diameter and a 38/1 length to
diameter ratio, using the screw geometry as previously outlined
by Cushen et al. (2014a). In the compounding process, the
28 food packaging and shelf life 4 (2015) 2635
temperature profile was increased from 160 8C at the hopper to
200 8C at the die with a screw speed of 120 rpm. Pure LDPE
pellets were used to manufacture control films (without the
presence of Ag NPs). To manufacture Ag/LDPE nanocomposites,
Ag NPs (0.5% or 1%, w/w) were added to LDPE pellets and mixed
thoroughly. In order to obtain a well distributed antimicrobial
Ag NPs in the LDPE polymer matrix, pellets with the appropriate
amount of Ag NPs were fed into the twin extruder at a rate of
60 rpm and the LDPE films formed were then shredded and
subsequently extruded to form the final Ag/LDPE nanocompo-
site films.
The mechanical properties of films (tensile strength (TS),
elongation at break (EAB), and Youngs Modulus (YM)) were
determined according to the ASTM-D882-02 (ASTM, 2002)
using a Mecmesin force and Torque software (Imperial 2500
instruments, Mecmesin Ltd., Slinfold, West Sussex, England).
Initial grip separation was set at 50 mm and cross-head speed
at 50 mm/min. Tensile strength or stress (s) was calculated by
dividing the maximum force and cross-sectional area. The
EAB or strain (e) was calculated by dividing the elongation at
the moment of rupture by the initial gauge length and
multiplying by 100. The YM was obtained by calculating the
slope of the first rising and linear part of stressstrain plot (s/e)
(Lee, Yam, & Piergiovanni, 2008).
2.4. Preparation and MAP treatment of chicken sample
To avoid cross contamination all utensils used including the
LDPE films (control and Ag nanocomposites) were sanitised with
ethanol. The excess fat and cartilage were trimmed from the
chicken breast fillets and chicken breasts were immediately
wrapped with LDPE control or Ag/LDPE nanocomposite films. A
wrapped chicken breast fillet was placed into a polystyrene/
EVOH/polyethylene white tray (203 mm  146 mm  60 mm;
Hochdorf, Switzerland). The trays were heat-sealed with
laminated barrier film (polyolefin), with an oxygen transmission
rate of 3 cm3/m2/24 h at STP, supplied by Cryovac, W.R. Grace
Europe Inc. (Lausanne, Switzerland). A packaging system type
VS 100 BS (Gustav Muller and Co., Bad Homburg 6, Germany) was
used to MAP-pack the samples with a gas ratio of 60% N2/40%
CO2 and stored at 4 8C. The whole test was repeated in two
independent experiments. Sampling was carried out every
three days and for each sampling day, two trays were randomly
selected for microbiological and physicochemical analysis.
2.5. Proximate analysis
Fat and moisture were determined using the CEM Analysis
System (CEM Corporation, Matthews, NC 28105, USA) (Bostian,
Fish, Webb, & Arey, 1985); protein content was determined
according to AOAC Procedures (1999) (method 981.10). The ash
content of the chicken breast fillets was determined by
incineration of the chicken meat samples in a furnace
(Nabertherm, Model L9/C6, Nabertherm, Germany) at 550 8C.
2.6. Gas, colour and pH measurement during chilled
storage
The concentrations of CO2 and O2 in the headspace of packages
during storage were determined using a PBI Dansensor
checkmate 9900 gas analyser (PBI-Dansensor, Ringsted,
Denmark). Each value represents the average gas concentration
from twelve readings (two independent experiments  two
samples  three readings).
The surface colour of the chicken breast fillets during
storage at 4 8C was monitored as outlined by Rodrguez-
Calleja et al. (2012) using a Minolta chromameter (CR-300,
Minolta Camera Co., Osaka, Japan). Ten random areas
were measured per sample on each measurement day
and the average values of fourty reading of CIE L-value
(lightness), a-value (redness) and b-value (yellowness) are
reported (two independent experiments  two samples  ten
readings).
The pH of the chicken breasts fillets was measured using a
digital pH metre (Mettler-Toledo GmbH, Schwerzenbach,
Switzerland) by inserting the glass probe directly into the
chicken flesh. Each value represents the average of twenty-
four readings (two independent experiments  two sam-
ples  six readings).
2.7. Microbiological analysis
In order to obtain a representative sample, 10 g of chicken
from both upper and bottom parts of the surface of the
chicken breast was taken aseptically and placed in a sterile
stomacher filter bag. Then, 90 ml of sterile MRD was added
aseptically into the stomacher bag and homogenised for
3 min using BA6021 stomacher 400 (Colworth, Bury St.
Edmunds, UK). The homogenate was then ten-fold diluted
and used for enumeration of total viable counts (TVC),
psychrotrophic bacteria, Pseudomonas spp., Brochothrix Ther-
mosphacta (B. thermosphacta), lactic acid bacteria (LAB), total
coliforms and Escherichia coli (E. coli).
The TVC and psychrotrophic bacteria were enumerated in
PCA plates after incubation for 48 h at 30 8C or for 7 days at 4 8C,
respectively. Enumeration of B. thermosphacta was carried out
after incubation aerobically at 25 8C for 48 using STAA agar base
added with supplement; Pseudomonas spp. was enumerated
after 2 days incubation at 30 8C on Pseudomonas agar base to
which CFC supplement was added. Oxidase test was performed
on randomly selected colonies and only oxidase-positive
colonies were counted as Pseudomonas spp. For the enumeration
of total coliforms and E. coli, 1 ml of the appropriate dilution was
placed in duplicate onto the centre of Compact Dry-EC
chromogenic plates and incubated at 37 8C for 24 h. Blue
colonies were counted as E. coli and the total coliforms
included red and blue colonies, as indicated by the manufac-
turer. For LAB count, overlaid MRS agar plates were incubated at
30 8C for 72 h. The bacterial numbers were converted to log10
colony-forming units per gram (CFU/g) prior to statistical
analyses.
2.8. Lipid oxidation
The lipid oxidation of chicken breast fillets was assessed by
the thiobarbituric-acid-reacting substances (TBARS) assay as
outlined by Siu and Draper (1978) and expressed as the mg
malonaldehyde (MDA)/kg of chicken breast fillets. Each value
represents the average of eight readings (two independent
experiments  two samples  two readings).
 food packaging and shelf life 4 (2015) 2635
Fig. 1 The SEM image of silver nanoparticles that were
incorporated into LDPE film.
2.9. Statistical analysis
All data was analysed for means and standard deviations, and
analysis of variance. One-way analysis of variance of data was
carried out using the SPSS 21 for Windows (SPSS Statistical
software, IBM Corp., Armonk, NY, USA) software package.
Differences between pairs of means was resolved by means of
confidence intervals using Tukeys test; the level of signifi-
cance was set at p < 0.05.
Fig. 2 The Zetasizer image and Z-average value of the silver nanoparticles.
29
3. Results and discussion
3.1. Characterisation of silver nanoparticles
Scanning electron microscopy image in Fig. 1 shows that the
Ag NPs used in this study had a primary average particle size of
10.10  0.60 nm. However, the average particle size of Ag NPs
dispersed in ethanol determined using the Zetasizer was
15.69 nm (Fig. 2). Yoksan and Chirachanchai (2010) reported
that the diameter of the NP measured using TEM analysis was
smaller than that determined by the Zetasizer. The differences
in the particle size obtained by SEM and Zetasizer may be due
to the high surface energy of the particles and the attractive
forces were dominant compared to repulsive forces, resulting
in unstable and agglomerated NPs dispersion (Jiang, Oberdor-
ster, & Biswas, 2008). The results (Fig. 2) also showed that the
particle size of the 2.3% of the particles present in ethanolic
solution had a particle size of 4145 nm, suggesting that
agglomeration of Ag NPs may have occurred. Rakcheev,
Philippe, and Schaumann (2013) reported that agglomeration
of NPs can occur in suspensions over time induced by
electrostatic attraction and high ionic strength.
The spectrophotometric scanning data showed a maxi-
mum absorption peak at 410 nm, which is attributed to the Ag
NPs surface plasmon excitation, indicating the presence of Ag
NPs (Fig. 3). Ghosh et al. (2010) reported that maximum
absorption peak of Ag NPs with an average particle size of 15
20 nm was 410 nm.
3.2. Physical and mechanical test of the LDPE
nanocomposites
The effect of Ag NPs on the mechanical properties of LDPE
films is presented in Table 1. The TS and EAB values of LDPE
control films were similar to those reported by Jokar, Abdul
30 food packaging and shelf life 4 (2015) 2635

Table 1 Tensile strength, elongation at break, and

Youngs modulus of LDPE control films and LDPE
nanocomposites films containing 0.5% or 1% (w/w) silver
nanoparticles.
Types of TS (MPa) EAB (%) YM (MPa)
packaging
films
Control 11.7  0.9 a 511.3  58.5 a 81.0  25.1 a
Ag/LDPE (0.5%) 9.6  1.3 b 204.3  47.7 b 75.2  16.4 a
Ag/LDPE (1%) 8.5  1.0 b 177.8  43.2 b 83.8  17.8 a
a,b
Mean values and standard deviations in the same column with
different superscripts are significantly different ( p < 0.05).

Fig. 3 The UVvis spectra of typical silver nanoparticle

solution.
Rahman, Ibrahim, Abdullah, and Tan (2010). The addition of
0.5 or 1% of Ag NPs into LDPE films significantly decreased
( p < 0.05) TS values by 17.9% and 27.4%, while EAB values were
60% or 65.2% lower compared to control films, respectively.
Chae and Kim (2005) reported that TS and EAB of PP
nanocomposites decreased when the concentration of Ag
NPs were >0.1 wt%. Kanmani and Rhim (2014) found that this
behaviour might be due to the discontinuity of the polymer
networks, reducing the stretching capability and mobility of
the Ag/LDPE nanocomposite films. Rong, Zhang, and Ruan
(2006) stated that hydrophilic Ag NPs and hydrophobic LDPE
polymers are incompatible materials, thus, resulting in poor
interfacial interaction of nanocomposite films. On the other
hand, the YM of the LDPE nanocomposites (independent of the
concentration of Ag used) did not change significantly
compared to control films. It has been reported that no
enhancement of YM values were observed in thermoplastic
polymers materials when nanosized particles formed aggre-
gates (Hanemann & Szabo, 2010).
3.3. Proximate analysis
Overall, independent of the packaging system used, no
significant difference in the proximate composition was
noticed during the chilled storage (Data not shown). The
initial proximate composition was 72.1, 24.4, 1.0, 1.2, and 1.3%
for moisture, protein, fat, ash, and carbohydrate, respectively.
Similar results were reported by Andres, Garci?a, Zaritzky, and
Califano (2006), Karakok, Ozogul, Saler, and Ozogul, 2010, and
Kurozawa, Park, and Hubinger (2009). Small differences with
data reported in the literature may be attributed to factors
such as diet, seasonality or type of chicken breed across
different poultry farms (Cortez-Vega, Pizato, & Prentice, 2012;
Karakok et al., 2010).
3.4. Physicochemical analysis during chilled storage
3.4.1. Headspace gas composition
The initial average concentrations of CO2 and O2 were 40.66%
and 0.07%, respectively (Data not shown). Independent of the
packaging system used, no significant changes on the
headspace gas composition were noticed on specific sampling
day. However, the concentrations of CO2 and O2 decreased
significantly ( p < 0.05) during storage time, reaching the
concentration of these gases at the end 12 days of storage,
were 35.60% and 0.01%, respectively. Similar results for
reduction of CO2 and O2 over time was reported by Al-
Nehlawi, Saldo, Vega, and Guri (2013). Rodrguez-Calleja et al.
(2012) proposed that the reduction of gases may due to
microbial growth, muscle tissue respiration, and dissolution of
CO2 in the chicken meat.
3.4.2. Colour
The effect of a specific packaging system on the colour of
chicken breast fillets is summarised in Table 2. During storage,
L-values significantly ( p < 0.05) increased independent of the
type of packaging used, indicating that chicken breast fillets
were lighter over time; however, at any specific sampling day,
no significant changes in L-values were noticed independent
of the packaging systems used, except for day 12. It was
also found that the highest value of lightness was obtained
on chicken breast fillets wrapped using 1% Ag/LPDE

Table 2 The colour changes of chicken breast fillets over storage time using different types of packaging films.
Day L-Value a-Value b-Value

Control 0.5% 1% Control 0.5% 1% Control 0.5% 1%

Ag/LDPE Ag/LDPE Ag/LDPE Ag/LDPE Ag/LDPE Ag/LDPE
0 52.3  3.3a,A 52.3  3.3a,A 53.5  3.3a,A 2.0  1.2a,A 2.0  1.2a,A 2.0  1.2a,A 2.8  1.8a,A 2.8  1.8a,A 2.8  1.8a,A
6 54.1  3.0a,B 54.4  3.2a,B 54.3  3.0a,A 1.6  1.0a,A 2.4  0.9b,A 2.2  1.6b,A 3.4  2.5a,A 3.5  2.3a,A 4.0  1.8a,A
12 55.1  2.5a,B 56.0  3.2ab,B 58.0  3.2b,B 1.9  0.9a,A 2.4  1.0a,A 2.1  1.3a,A 3.7  1.6a,A 3.8  1.7a,A 3.7  1.8a,A
a,b
Mean values in the same row with different superscripts are significantly different ( p < 0.05).
A,B
Mean values in the same column under same day with different superscripts are significantly different ( p < 0.05).
 food packaging and shelf life 4 (2015) 2635
nanocomposite films (Table 2). Angsupanich and Ledward
(1998) and Ledward (1998) suggested that changes in muscle
colour may be related to the denaturation of myofibrillar and
sarcoplasmic proteins. Das, Saxena, and Dwivedi (2009) and
Wang, Zhang, Zhang, and Li (2012) demonstrated that the
presence of Ag NPs can cause protein denaturation. Regarding
redness (a-values) and yellowness (b-values), no significant
differences were noticed for chicken breast fillets for inde-
pendent type of packaging films used. The overall trend in this
study is in agreement with data reported by Patsias et al. (2008)
who found that storage of chicken breast fillets under the MAP
conditions at 4 8C did not affect significantly the colour
parameters.
3.4.3. pH measurement
The initial average pH of chicken breast fillets wrapped with
control or Ag/LDPE nanocomposites (0.5 or 1%) packaging films
stored under MAP conditions was 5.7 (Data not shown).
Independent of the type of packaging used, the pH values of
the chicken breast fillets increased significantly throughout
the storage until day 9 (pH 5.9); however, a significant
( p < 0.05) reduction in pH values (pH 5.5) was noticed from
day 9 onwards. The increased pH might be due to the protein
denaturation and growth of psychrotrophic bacteria, which
produce volatile amine and ammonia, thus increasing the pH
of the chicken meat (Cortez-Vega et al., 2012). A decrease in the
pH correlated well with the microbiological spoilage of the
chicken breast fillets (maximum level of acceptability). The
results indicated that during storage, LAB was the main
spoilage microorganism in chicken breast fillets (see Section
3.5). It was reported that a decrease in pH values may also be
due to the production of lactic acid by LAB (Cortez-Vega et al.,
2012; Rodrguez-Calleja et al., 2012).
3.5. Microbiological analysis during chilled storage
From the microbiological point of view, the acceptable limit for
total viable count (TVC) of chicken meat products is 6
7 log CFU/g (EC, 2007). In this study, a TVC value of 6 log CFU/g
was set as the maximum limit of acceptability. Changes in
TVC, psychrotrophic bacteria, Pseudomonas spp., LAB, B.
thermosphacta, E. coli, and total coliform bacteria during chilled
storage of chicken breast fillets wrapped with control films or
Ag/LDPE nanocomposite films (0.5% or 1%) under MAP
conditions are shown in Fig. 4.
The initial TVC and psychrotrophic bacteria values were
3.2 log and 4.1 log CFU/g, respectively, indicating that the
chicken breast fillets were good microbiological quality.
Patsias et al. (2008) reported that chicken breast fillets with
TVC values <4 log CFU/g were considered good quality. During
storage and independent of the packaging system used TVC
and psychrotrophic bacteria growth increased over time. In
the present study, the limit of acceptability for chicken breast
fillets wrapped with control films was reached at day 7. A
similar shelf life for chicken breast fillets stored under MAP
conditions was reported by Rodrguez-Calleja et al. (2012). On
the other hand, the shelf life of chicken breast fillets wrapped
with antimicrobial Ag/LDPE nanocomposite films (0.5 or 1%)
significantly increased ( p < 0.05) to 8 days, and exhibited
16.67% slower growth of psychrotrophic bacteria compared to
31
chicken breast fillets wrapped with control films. For chicken
breast fillets wrapped with Ag/LDPE nanocomposite films,
similar shelf life of 8 days (TVC = 6 CFU/g) was obtained when
LDPE blended with Ag and ZnO NPs (5% and 10%, w/w) was
used (Panea et al., 2014) even though, the concentration of Ag
antimicrobial was 10-fold higher than the concentration of
Ag NPs used in this study (0.5%). It was also noticed that the
growth of psychrotrophic bacteria during the storage time was
higher compared to TVC. This may be due to the fact that the
chicken was stored at a chilled temperature which may have
encouraged the growth of psychrotrophic bacteria than
mesophilic bacteria (TVC) (Murphy, OGrady, & Kerry, 2013).
In general, the microbiological growth of bacteria was
delayed significantly ( p < 0.05) until day 6 (up to 22.5% of
reduction) when chicken breast fillets were wrapped with Ag/
LDPE nanocomposite compared to control films. However,
after day 6, the bacteria outgrow until the end of the storage. It
was reported that antimicrobial activity of Ag NPs can be
affected by food components (Ilg & Kreyenschmidt, 2011), and
low antimicrobial activity of Ag NPs may be due to the
presence of protein functional groups in the chicken breast
fillets that can potentially bind with Ag ion (Ag+) and reducing
the potency of antimicrobial activity of Ag NPs.
At the end of the shelf life (day 8), no significant differences
in the bacterial growth was observed in chicken breast fillets
wrapped with control or Ag/LDPE nanocomposite films. It was
also noticed that the main spoilage microorganisms at the end
of the shelf life were psychrotrophic bacteria, LAB, and B.
thermosphacta with values of 6.7, 6.6, and 6.2 log CFU/g,
respectively. Similar results were reported by Nowak, Rygala,
Oltuszak-Walczak, & Walczak (2012) where the major bacteria
able to grow on chicken meat under MAP were psychrotrophic
bacteria, LAB, and B. thermosphacta.
The initial count of LAB on the chicken breast fillets was
3 log CFU/g and this increased significantly during chilled
storage. LAB is a facultative anaerobic bacteria that can grow
in the presence or absence of O2 and that the presence of CO2
in MAP have little or no effect in LAB growth (Mantilla et al.,
2012; Patsias et al., 2008). Diez, Santos, Jaime, and Rovira (2009)
reported that the taste and odour of chicken meat becomes
sour when LAB count exceeded 8 log CFU/g. In the present
study, the LAB count was reached 8 log CFU/g after day 12,
regardless of the type of packaging film used. During storage, a
significant decrease on the pH values of chicken breast fillets
may have been related to the growth of LAB. Regarding B.
thermosphacta, a facultative anaerobic bacteria, the initial
count was 2.6 log CFU/g. In this study the growth of B.
thermosphacta on the chicken breast fillets were slower than
LAB and little effect against this microorganism was noticed
when the chicken breast fillets were wrapped with Ag/LDPE
nanocomposite packaging films.
Slower growth of Pseudomonas spp. was noticed in chicken
breast fillets wrapped with Ag/LDPE nanocomposite (1%) films
compared to chicken breast fillets wrapped with Ag/LDPE
nanocomposites (0.5%) or control films. Even though Pseudo-
monas spp. is considered an aerobic microorganism, higher
counts of Pseudomonas spp. (4.8 log CFU/g) were observed at
day 8. This may be due to the high initial numbers of
Pseudomonas spp. (3.2 log CFU/g) and that localised growth
may have occurred in remaining pockets of oxygen-containing
32 food packaging and shelf life 4 (2015) 2635

Fig. 4 The microbiological count of (a) TVC, (b) psychrotrophic bacteria, (c) LAB, (d) B. thermosphacta, (e) Pseudomonas spp., (f)
E. coli and (g) total coliform for chicken breast fillets during chilled storage under MAP conditions using (^) control (&) 0.5%
Ag/LDPE nanocomposite and (~) 1% Ag/LDPE nanocomposite packaging films.

atmospheres as MAP systems do not always facilitate
complete removal of oxygen (Rodrguez-Calleja et al., 2012).
In this study, the O2 concentration at day 0 and day 9 were
0.07% and 0.03%, respectively. Filiatrault, Picardo, Ngai,
Passador, and Iglewski (2006) were also reported that
Pseudomonas spp. can alternatively use external electron
acceptors when oxygen is limited or not available.
The initial counts for E. coli and total coliform were 0.9 and
1 log CFU/g, respectively. The established limits of acceptabil-
ity in raw chicken meat for E. coli set by the Commission
Regulation no. 1441/2007 is 5  102 (EC, 2007). Independent of
packaging systems used, no significant differences were
observed on E. coli up to day 6 of storage. However, higher
growth for E. coli numbers were noticed in control samples
compared to chicken samples wrapped with Ag/LDPE (0.5% or
1%). It was also observed that all of the chicken samples did
not exceed the limit of acceptability for E. coli set by the
Commission Regulation no. 1441/2007. In general, throughout
storage, independent of the packaging system used, no
significant differences were also observed on the total coli-
forms count on chicken breast fillets. Overall, results reported
in this study are similar to values reported by Rodrguez-
Calleja et al. (2012).
In general, lower counts of Gram-negative than Gram-
positive bacteria was observed. Fernandez, Picouet, and Lloret
(2010) and Azlin-Hasim et al. (2014) reported that in both, meat
 food packaging and shelf life 4 (2015) 2635
0.9
0.8
0.7
0.6
(mg MDA)/kg
0.5
0.4
b c
0.3 c
0.2 a a a
0.1
0.0
0 3
Fig. 5 Effects on TBARS values (mg MDA/kg) of chicken breast fillets using control ( ), 0.5% Ag/LDPE nanocomposite (
and 1% Ag/LDPE nanocomposite ( ) films during chilled storage under MAP conditions.
products and culture media, Gram-negative bacteria were
more susceptible to Ag NPs than Gram-positive bacteria. On
the other hand, Mantilla et al. (2012) and Patsias et al. (2008)
reported that the bacterial load of Gram-positive bacteria was
little or not affected by high concentrations of CO2 present in
MAP. Gram-positive bacteria posses a thicker peptidoglycan
layer making it difficult for antimicrobial substances to
penetrate into the cells wall (Shrivastava et al., 2007).
3.6. Lipid oxidation during chilled storage
Lipid oxidation is a major contributor to flavour deterioration
in meat and meat products (McMillin, 2008). The effects of
different types of packaging systems on the lipid oxidation
stability of chicken breast fillets during chilled storage under
MAP conditions are shown in Fig. 5. The initial TBARS values
of chicken breast fillets were 0.13 mg MDA/kg. These results
are in agreement with data previously reported for fresh
chicken breast fillets (Chouliara et al., 2008). During storage,
independent of the packaging system used, the TBARS values
increased significantly ( p < 0.05); however, at the end of
storage time the TBARS values of chicken breast fillets
wrapped with Ag/LDPE nanocomposite (1%) films were
significantly ( p < 0.05) lower than samples wrapped with
control films or Ag/LDPE nanocomposite (0.5%) films. These
results are in agreement with data reported by Panea et al.
(2014) where lower TBARS values were also observed in
chicken packaged with Ag and ZnO LDPE nanocomposite films
and stored under MAP at 4 8C.
4. Conclusion
Independent the concentration of Ag NPs used, Ag/LDPE
nanocomposite films extended significantly the shelf life of
chicken breast fillets. During storage, lipid oxidation values
measured as TBARS values were significantly lower in
chicken breast fillets wrapped with Ag/LDPE nanocomposite
films than in control samples. Previous studies conducted by
our group demonstrated that the incorporation of Ag NPs
(either commercial or laboratory produced) into LDPE films
33
f
g
h
d
de
e
6 9
Days
),
can be used in food contact materials due to the low migration
levels and exposure results that fell well below the exposure
limits, suggesting that Ag/LDPE nanocomposite films are
suitable for use with non-acidic foods such as chicken breast
fillets (Cushen et al., 2014a; Cushen, Kerry, Morris, Cruz-
Romero, & Cummins, 2014b). Overall, Ag/LDPE nanocomposite
films have the potential to be used as antimicrobial packaging
of food products.
Acknowledgements
The funding for this research was provided under the National
Development, through the Food Institutional Research Mea-
sure (FIRM) administered by the Department of Agriculture,
Fisheries and Food. The author, Azlin-Hasim, would like to
thank the Ministry of Education of Malaysia (MOE) for her PhD
scholarship.
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