International Journal of Biological Macromolecules 79 (2015) 934–942

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International Journal of Biological Macromolecules

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

Development and characterization of an LDPE/chitosan composite

antimicrobial film for chilled fish storage
K.V. Reesha a , Panda Satyen Kumar b , J. Bindu c,∗ , T.O. Varghese a
a
Centre for Bio-Polymer Science and Technology (CBPST), FACT Township, Eloor, Udyogamandal P.O., Cochin 683501, India
b
Quality Assurance and Management Division, Central Institute of Fisheries Technology, Cochin 682029, Kerala, India
c
Fish Processing Division, Central Institute of Fisheries Technology, Cochin 682029, Kerala, India

a r t i c l e i n f o a b s t r a c t

Article history: An antimicrobial packaging material was developed by uniformly embedding 1, 3 and 5% chitosan (w/w)
Received 4 March 2015 in low density polyethylene matrix using maleic anhydride grafted LDPE as a compatible agent. The
Received in revised form 9 June 2015 materials were mixed by compounding and blown into monolayer films via blown film extrusion. The
Accepted 12 June 2015
developed films showed good barrier properties against oxygen. Characterization of the composite films
Available online 16 June 2015
with Fourier transform infrared spectroscopy revealed that chitosan and LDPE interacted well with each
other. Overall migration showed better release of chitosan adduct from the LDPE matrix which enhanced
Keywords:
the antibacterial properties of the films. The interaction between the LDPE/CS and maleic anhydride
Chitosan
LDPE
grafted LDPE had a decreasing effect on the tensile strength and heat sealing properties. Investigation
Packaging film on antimicrobial properties of LDPE/CS films showed 85–100% inhibition of Escherichia coli. Efficacy of
Tilapia LDPE/CS films was evaluated by using them as packaging material for chilled storage of Tilapia (Ore-
Chilled storage ochromis mossambicus). Analysis of storage quality indices (peroxide value, free fatty acid, total volatile
base nitrogen and aerobic plate count) revealed good antibacterial property and extension of shelf life of
Tilapia in the chitosan incorporated novel composite films compared to virgin LDPE film.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction in the packaging material that can prevent proliferation of spoilage

Packaging plays a very important role in today’s commerce
and trade. The aim of packaging is to maintain the unique whole-
someness to produce right from manufacturing to storage, without
altering the inherent characteristics of the product. Packaging has
a significant impact on international trade of fish and fishery prod-
ucts which are important source of nutritional components like
protein, essential vitamins, minerals and polyunsaturated fatty
acids [1,2]. Fish is highly perishable due to the presence of high
moisture and protein. It is vulnerable to various microbial and bio-
chemical forms of deterioration throughout the production chain,
due to free amino acids and highly oxidizable polyunsaturated
fatty acids (PUFA) [3]. In order to extend shelf life, packaging has
to be either combined with refrigeration to further reduce the
growth of microorganisms or by antimicrobial agents incorporated
∗ Corresponding author. Tel.: +91 484 2666845; fax: +91 484 2668212; mobile:
+91 9447648921.
E-mail addresses: satyenpanda@gmail.com (P. Satyen Kumar),
bindujaganath@gmail.com (J. Bindu).
microorganisms.
Packaging materials are available mainly in the form of plas-
tic films. They should have flexibility, good mechanical and barrier
properties, and also should be easily recycled. In addition to this,
they should be capable of heat sealing and withstand the strain of
transportation and of low cost. Polyethylene packaging as sheets
or films is suitable to wrap, seal and protect consumer goods.
Polyethylene is considered superior to any other material because
of its cheap available nature and its use is widespread in the flexi-
ble packaging market. Polyethylene polymers are both durable and
resistant to environmental hazards.
For the preservation of fresh fish, active packaging technologies
can be used. The technology offers several benefits over standard
passive packaging materials and can be designed to protect product
quality such as freshness and storage stability. Antimicrobial pack-
aging is a promising form of active packaging since it extends the lag
phase and reduce growth phase of microorganisms to extend shelf
life of product [4]. While preparing packaging materials, antimi-
crobial components can be either incorporated or coated on to the
surface of packaging materials.
Biodegradable packing materials can be produced from natural
polymers such as chitosan, collagen, elastin and starch. Chitosan

http://dx.doi.org/10.1016/j.ijbiomac.2015.06.016
0141-8130/© 2015 Elsevier B.V. All rights reserved.
 K.V. Reesha et al. / International Journal of Biological Macromolecules 79 (2015) 934–942
is an amino polysaccharide biopolymer which demands an impor-
tant role in the world economy since it is an edible, biodegradable,
antimicrobial compound with film forming ability. Chitosan (CS),
the linear and partly acetylated (1–4)-2-amino-2-deoxy-<beta>-d-
glucan, is easily obtained from chitin [5]. Chitosan is a weak base
and is insoluble in water, but soluble in dilute aqueous acidic solu-
tions like acetic acid and propionic acid. The direct combination
of synthetic materials with biodegradable materials is one of the
easier and economic ways for biomaterial production [6] and also
enhances the expansion and functional properties of the mate-
rials. Extrusion and press moulding techniques are used in the
production of chitosan and low density polyethylene (LDPE) films,
which allow chitosan to perform antimicrobial activity on different
bacterial strains [7,8]. Chitosan incorporation in a LDPE matrix or
application by coating improves the barrier properties of LDPE and
also confers antimicrobial characteristics, which makes it a very
promising packaging material [9]. Chitosan film is used as an edi-
ble coating to prolong shelf life and preserve quality of fresh fish
due to the antimicrobial action between the positively charged chi-
tosan molecules and negatively charged microbial cell membranes
[10].
This study was undertaken to develop a chitosan incorporated
antimicrobial polyethylene film by blown film extrusion process
and to optimize the chitosan concentration and mechanical prop-
erties of the packaging films for studying the antimicrobial activity
of the films against Escherichia coli. The ultimate aim was to use the
films for packaging of fresh Tilapia steaks and to determine its shelf
life during chilled storage.
2. Materials and methods
2.1. Film preparation
2.1.1. Matrix
Low density polyethylene (LDPE) resin grade (Lotrene FD 0474)
was supplied by Qatar Petrochemical Company (QAPCO). The den-
sity of the polymer was 0.923 g/cm3 as determined by ASTM D
792-08 method [11] and temperature of melting was 140–150 ◦ C.
Melt flow rates of granules were determined by melt flow indexer
(M/s. Saumya machineries Pvt. Ltd., Ahmedabad) according to
ASTM D 123-04c method [12]. After conditioning at laboratory at
23 ± 2 ◦ C and 50 ± 5% relative humidity, the different concentrated
LDPE/chitosan (CS) granules were adopted to procedural condition-
ing of 190 ◦ C/2.16 kg/20 s. The apparatus consisted of a small die of
2 mm diameter inserted into the extruder. A small amount of gran-
ules were taken in sample enclosure. Proper packing of material
inside the barrel was ensured to avoid formation of air pockets. The
samples were preheated for specific time. A piston was introduced
which applied pressure on to the molten granules and caused extru-
sion. The combined action of shear and pressure made the molten
material to flow throughout the die. The melt samples were col-
lected after desired period of time and weighed accurately. Melt
flow index (MFI) was expressed as grams of polymer/10 min of flow
time.
2.1.2. Filler and processing aid
Low viscosity chitosan used as the filler material had a viscos-
ity of 74 cps. The average moisture content was 3.04% and degree
of deacetylation was 80.92%. Degradation temperature of chitosan
was above 190 ◦ C. Glycerol (glycerine C3 H8 O3 ) procured from Sisco
Research Laboratory (SRL) Pvt. Ltd. was used as plasticizer.
2.1.3. Compatibilizer
Maleic anhydride grafted LDPE grade, MA-g-LDPE (Commercial
name – OPTIM-E142) was purchased from Pluss Polymers Pvt. Ltd.
935
It had a melt flow of 2.52 g/10 min (190 ◦ C/2.16 kg) as determined
by ASTM D 1238-04c method [12].
2.2. Blend preparation and film production
Pre-drying of chitosan, LDPE and MA-g-LDPE was done in a hot
air oven at temperature of 80 ◦ C for 2 h and further pre-mixing
of weighed chitosan, LDPE, MA-g-LDPE with glycerol was done at
room temperature. The compounding of LDPE/chitosan was done
using a co-rotating twin screw extruder (ZV20). Composite blend
formulations were prepared by mixing low density polyethylene
as matrix material and low viscosity chitosan (74 cps) as filler
material. MA-g-LDPE was used to improve compatibility between
filler and matrix. For ease of processing glycerol was also used as
plasticizer. Three different combinations of blends were prepared
incorporating 1, 3 and 5% chitosan by w/w of LDPE.
The compounding process was carried out at a maximum speed
of 90 rpm and melt temperature of 185 ◦ C. The extrudates were pel-
letized separately using a pelletizer machine for each formulation.
Slight variations of processing temperature and torque occurred
for the different combinations. For LDPE/CS compounding the feed
zone was maintained at 103, 107 and 99 ◦ C for 1%, 3% and 5% com-
pounded mixture, respectively. The temperatures maintained in
the compression zones 1 and 2 were 103 ◦ C and 132 ◦ C for 1%,
107 ◦ C and 129 ◦ C for 3%, 99 ◦ C and 138 ◦ C for 5% LDPE/CS blends. The
melt temperature ranged from 176 to 184 ◦ C and the melt pressure
maintained was between 5 and 6 bar.
2.3. Blown film manufacture
The compounded pellets were fed into a twin screw extruder
(Konark Plastomech Pvt. Ltd.) by a gravimetric hopper which was
maintained at a melt temperature of 184 ◦ C and a melt pressure of
20 bar.
2.4. Film characterization
2.4.1. Thickness
Thicknesses of all four types of blown films were measured as
per IS: 2508 method [13] at 23 ◦ C and 64% saturated sodium nitrite
RH. The thickness was determined at five positions of each sample
using a gauge meter (Mitutoyo, Model no: 2046-08 Japan).
2.4.2. Transparency of film
The percentage of transparency of the films was determined by
a haze meter as per ASTM D 1003 method [14].
2.4.3. Surface morphology of film
Surface morphology was investigated by atomic force
microscopy (AFM) performed at room temperature on a PARK
systems XE 100 (Schaefer technologies GmbH) setup. Topographic
and amplitude images, obtained over an area from 5 ␮m2 for each
sample were recorded by using non-contact mode with silicone
tip.
2.4.4. Tensile and heat strength
The tensile strength elongation at break and heat seal strength of
different concentrated LDPE/CS films were done using Lloyd instru-
ments UK; model TA plus according to ASTM D 882-02 method [15].
Film pieces were stored in a desiccator at 23 ◦ C with 50% relative
humidity for at least 24 h. Samples were cut in lengthwise and cross
direction 50 mm × 15 mm. For tensile test, a load range and appro-
priate grip separation was used and the test specimens were placed
in the grip of testing machine. The extensometer measured the load
versus extension. The testing speed was at 500 mm/min with a load
cell of 500 N. The distance between the two anchorages was 50 mm.
936 K.V. Reesha et al. / International Journal of Biological Macromolecules 79 (2015) 934–942
The following formula was used in calculating the tensile strength.
maximum load
Tensile strength (TS) =
width × thickness
The same tensile strength test procedure was adopted for heat
seal strength. Unsealed film samples were heat sealed and speci-
men dimensions same as tensile test were used. The only difference
was that the heat sealable portion of the film was placed in the
centre of the grip.
2.4.5. Water vapour transmission rate (WVTR)
The water vapour transmission rate was performed by a Lyssy
water vapour permeability tester (PBI Dan sensor Denmark, Model
L80-5000) as per ASTM E 398-03 method [16].
2.4.6. Oxygen transmission rate (OTR)
The oxygen transmission rate was determined by using oxy-
gen transmission tester (OPT-5000) (PBI Dansensor, Denmark). The
samples were prepared according to ASTM F 2622-08 method [17].
2.4.7. Overall migration test
The overall migration of different LDPE/CS films was done as
per the USFDA 176:170 test procedure [18]. The film pouches were
filled with 250 mL stimulating solvent (water) and conditioned at
49 ◦ C at 24 h. After exposure for specified duration, the solvent was
transferred, concentrated and evaporated to dryness to measure
the residues.
Overall migration residue (OMR) in mg/L
Mass of residue (mg) × 1000
=
Volume of stimulant (mL)
2.4.8. Fourier transform infrared (FTIR) spectroscopy
Fourier transform infrared spectra of different concentrated
LDPE/CS films were carried out on a Thermo Nicolet, Avatar 370
FTIR spectrometer, at STIC (Sophisticated Test and Instrumenta-
tion Centre, CUSAT, Kochi). Scanning was performed at 4 cm−1
resolution. The measurements were recorded between 4000 and
400 cm−1 .
2.4.9. Film antimicrobial test
Antimicrobial efficacy of virgin LDPE and LDPE/CS films were
evaluated by exposing the films to E. coli. Virgin LDPE and LDPE/CS
films were cut to pieces of 5 cm × 6 cm (length × breadth) sizes and
sterilized overnight in an UV chamber. E. coli (ATCC 25922) was
inoculated in BHI broth (Hi-media) and incubated for 18–24 h at
35 ± 2 ◦ C. The overnight grown culture was centrifuged, washed
with sterile phosphate buffer and re-suspended in 9 mL of phos-
phate buffer. From this culture 100 ␮L was spread on to both films
by using sterile disposable spreader. The initial inoculum level was
5.2 × 108 cfu/cm2 . The films were kept in separate petridishes and
incubated at 35 ± 1 ◦ C. Films were drawn at 24, 72 and 120 h to
analyze load of E. coli. The load of E. coli was estimated as per
AOAC 991.14 [19] (Dry Rehydratable Film Method, PetrifilmTM
E. coli/Coli form Count Plate). Briefly, both control and inocu-
lated films were suspended in 100 mL phosphate buffer, vortexed
and serially diluted. One milliliter of inoculum from each dilution
was placed on E. coli petrifilms. The petrifilms were incubated at
35 ± 1 ◦ C for 24 h. Number of blue colonies with entrapped gas
bubbles were taken as positive and multiplied with corresponding
dilution to obtain final E. coli count.
2.5. Raw materials
Live Tilapia (Oreochromis mossambicus) fishes were procured
from a fish farm, washed and transported in ice to the laboratory
for further processing. The fishes were washed, dressed, cleaned,
cut into steaks and kept in iced condition before packing in the chi-
tosan (1, 3 and 5%) incorporated LDPE films and heat sealed. Virgin
LDPE pouches were also prepared for packing the control samples.
The samples were then iced separately in different insulated boxes
and kept in chill room at 2 ◦ C.
2.6. Biochemical and microbiological analysis
The chilled steaks were drawn at periodic intervals to deter-
mine the biochemical and microbiological qualities. Total volatile
base nitrogen (TVBN) content [20], free fatty acid content [21], per-
oxide value [22] and aerobic plate count (APC) [23] of Tilapia steaks
packed in control and LDPE/CS films were estimated.
2.7. Statistical analysis
Statistical analysis was performed using SPSS Statistics ver-
sion 20 (IBM, Armonk, NY, USA). Post hoc analysis using pair wise
comparison of Tukey’s test was carried out for microbial and film
property analysis.
3. Results and discussions
3.1. Optimization of process for film development
3.1.1. Compounding and extrusion of LDPE/chitosan
Three different concentrations (1%, 3% and 5%) of chitosan with
LDPE and other compounds were compounded in twin screw
extruder. The processing of LDPE/CS was difficult with increase in
the concentration of chitosan mainly due to the viscoelastic char-
acteristics of the component. According to Liang [24] the flow of
polymer introduces a structural deformation or aggregation during
extrusion process, which makes the viscoelastic properties of the
blend more complicated, resulting in variation in the volume, thick-
ness of the extruded material due to presence of aggregates and
separation of blend components. Hence in this study MA-g-LDPE
was used as a compatibilizer, and glycerol as plasticizer. According
to Quiroz-Castillo et al. [25] 5% weight of MA-g-LDPE is necessary
to increase the ductility of the LDPE/CS blend since this combina-
tion gives a positive effect to the blend and the glycerol/chitosan
concentration kept constant at 2 g of glycerol per 100 g of chitosan.
The compounding of the LDPE/CS was carried out in a twin
screw extruder at temperatures varying from 103 to 185 ◦ C in the
different zones. Slight variations could have occurred due to the
differences in chitosan concentration. The extruded pellets were
smooth, bubble-free and opaque in nature. The colour of the com-
pounded granules differed for each concentration because of the
chitosan adduction. The immiscible blends are characterized by
two or more phase that are separated by interfaces and when
interfacial tension goes to zero, the blend become miscible [26].
This is achieved by the addition of compatibilizer which has both
hydrophilic and hydrophobic molecules. In this reaction MA-g-
LDPE react with hydrophobic non polar LDPE and the other polar
end reacts with hydrophilic chitosan. In similar way, Liu et al. [27]
have reported that the miscibility between granular corn starch
and LDPE was improved by the addition of a commercially avail-
able compatibilizer, MA-g-LDPE. This was attributed to a chemical
reaction between hydroxyl groups in starch and anhydride groups
in MA-g-LDPE and the physical interaction between the PE in MA-
g-LDPE and LDPE.
 K.V. Reesha et al. / International Journal of Biological Macromolecules 79 (2015) 934–942 937

Table 1 also increased. Mechanical properties of the films are also associ-
Density and melt flow index (MFI) of the chitosan incorporated extruded LDPE and
ated with thickness of the film. Morphological differences due to
virgin LDPE granules.
the domain structure of immiscible polymer blend gave rise to films
Types of granules Density (g/cc) MFI value (g/10 min) with different thickness [29].
Virgin LDPE 0.923 ± 0.03 3.57 ± 0.01
1%LDPE/CS 0.945 ± 0.01 2.94 ± 0.04
3%LDPE/CS 0.950 ± 0.04 2.58 ± 0.02
3.2.2. Transparency
5%LDPE/CS 0.951 ± 0.03 2.37 ± 0.08 The transparency level for each formulation in LDPE/CS blends
is shown in Table 2. The control of transparency is very impor-
tant in order to achieve the desired visual effect. The percentage
The density of the extruded granules increased with chitosan of transparency decreased with incorporation of chitosan. The
adduction percentage (Table 1). By increasing the filler concentra- transparency got reduced because the higher amount of chitosan
tion the filler particles get attached to the matrix and form a blend. distributed in the packing blend system making it more disordered
The extruded granules showed increased density, which resulted in and rough or uneven with higher free volume, thus more light could
increased molecular weight of granules and decreased MFI value. diffuse back or get reflected. Hence, the transparency of the film was
Results showed that there was no significant difference in density found to be dependent on composition, mixing and the processing
of extruded granules (P > 0.05) in 3% LDPE/CS and 5% LDPE/CS. conditions of the film.

3.1.2. Melt flow Index of extruded granules

3.2.3. Tensile strength and elongation at break
The MFI values of LDPE/CS composites decreased as the content
Variations of tensile strength and elongation at break of LDPE/CS
of chitosan increased (Table 1). Reduction in MFI values indicated
composite films are shown in Table 2. Tensile strength and elon-
increase in the viscosity of the composite. The virgin LDPE granules
gation at break of LDPE/CS blends decreased with increase in
had an MFI value of 3.57 g/cc, whereas it decreased with the addi-
chitosan content. The tensile properties of films are influenced by
tion of chitosan. The MFI was respectively 2.94, 2.58 and 2.37 g/cc
the processing conditions (mixing, type and number of screws,
for 1, 3 and 5% chitosan incorporated LDPE. Similar findings have
rotor temperature and speed, and resident time) and the processing
been reported in most filled thermoplastics and Rosa et al. [28]
variables during blowing and cooling [30] apart from material char-
have reported a decrease in MFI of starch composite with increase
acteristics. Similar findings have been reported by Quiroz-Castillo
in filler loading. In the present study, the reduction in MFI value
et al. [25] where tensile strength as well as elongation at break
could be due to the chitosan granules that still retained their shape
decreased with increase in chitosan content. The decrease in the
and functioned as rigid particulate fillers when processed. With
tensile strength can be due to the uneven dispersion of chitosan in
increase in chitosan content the interaction between the granules
the LDPE matrix [31]. The tensile strength of 1% LDPE/CS decreased
increased and this contributed to the higher viscosity. For higher
as compared to virgin LDPE. Similar to tensile strength, decrease of
loading of chitosan, the spaces between particles to particle were
elongation at break occurred because of the weak interfacial adhe-
small. The matrix molecules become trapped in filler particles as
sion between LDPE and chitosan. In synthetic polymer blends, the
the size of agglomerates rise and flow becomes confined. Analy-
addition of the immiscible component to a ductile matrix gener-
sis of MFI variability of different LDPE/CS granules indicates that
ally decreases the elongation properties at break. The elongation
chitosan incorporated granules can be processed like virgin LDPE,
would therefore depend on the state of the interface [32]. When
however, with slight modification in process conditions such as
stress is applied on materials with chitosan blends, loss of adhe-
temperature and pressure.
sion between the inter phase components takes place that results
in formation of pores due to the unfolding of matrix fibres [30]. This
3.2. Film formation and properties
is due to the immiscibility of chitosan (particulate filler) with LDPE.
Thus, mechanical performance of a filled polymer depends on the
The three different LDPE/CS compounded and virgin LDPE gran-
strength and the filler module, which further explains the lower
ules were blown into a tubular film using a monolayer extruder.
tensile strength shown by the extruded films in the present study.
Films were extruded at 140–195 ◦ C. The uniform mixing and con-
trolled blowing determined the chitosan distribution and thickness
of the film. The LDPE and LDPE/CS films varied in thickness and 3.2.4. Heat seal strength
morphology. All films showed good processability, except the 5% All LDPE/CS film showed decreased heat sealing strength when
LDPE/CS film, because of increased amount of filler. As compared compared to virgin LDPE film (Table 2). Since LDPE itself is a good
with 5% LDPE/CS, 3% LDPE/CS films showed good distribution due heat sealable film, addition of hydrophilic chitosan to LDPE film
to proper interaction between the components. affected its sealing strength. Rahman et al. [33] observed similar
results in starch films where it was shown that hydrophilic nature
3.2.1. Thickness of starch was not compatible with the hydrophobic nature of syn-
The difference in thickness is given in Table 2. The results show thetic polymers that resulted in weakness of interfacial adhesion
that with increase in chitosan concentration the thickness of films and led to reduction in mechanical properties.

Table 2
Properties of LDPE/CS and virgin LDPE films.

Properties of films Virgin LDPE 1% LDPE/CS 3% LDPE/CS 5% LDPE/CS

Film thickness (mm) 0.13 ± 0.20 0.16 ± 0.70 0.20 ± 0.01 0.24 ± 0.01
Transparency (%) 87.00 ± 0.12 85.00 ± 0.20 74.60 ± 0.07 71.05 ± 0.23
Tensile strength (MPa) 9.62 ± 0.12 3.73 ± 0.12 2.26 ± 0.12 1.24 ± 0.01
Elongation at break (%) machine direction 279.79 ± 0.23 243.03 ± 0.20 157.60 ± 0.21 111.96 ± 0.21
Heat seal strength (MPa) 8.38 ± 0.11 3.17 ± 0.11 1.71 ± 0.22 1.26 ± 0.32
Oxygen transmission rate (mL/m2 /day) 2838 ± 0.32 2343 ± 0.20 2189 ± 0.12 2487 ± 0.03
Water vapour transmission rate (g/m2 day) 2.43 ± 0.01 3.59 ± 0.32 2.88 ± 0.01 4.19 ± 0.02
Overall migration residue (mg/L) 3.68 ± 0.01 7.14 ± 0.05 8.80 ± 0.01 8.85 ± 0.01
938 K.V. Reesha et al. / International Journal of Biological Macromolecules 79 (2015) 934–942
Fig. 1. Surface morphology of (a) virgin LDPE and (b) 1% LDPE/CS film.
3.2.5. Oxygen transmission rate (OTR)
Virgin LDPE films showed a higher OTR value when compared
to LDPE/CS films (Table 2). OTR value of virgin LDPE film was found
to be around 2838 mL/m2 /day, similar to earlier reports [34]. The
incorporation of chitosan decreased the oxygen transmission rate,
to 2343 mL/m2 /day in 1% LDPE/CS film and 2189 mL/m2 /day in 3%
LDPE/CS film. Kurek et al. [35] observed that application of chi-
tosan coatings on PE films resulted in more than two-order decrease
in oxygen permeability. When compared to each composition, 3%
LDPE/CS film had lower oxygen transmission rate than 1 and 5%
LDPE/CS.
3.2.6. Water vapour transmission rate (WVTR)
Polyethylene films are known to be highly hydrophobic and
relatively not very permeable to water vapour. The WVTR value
of pure LDPE film was 2.43 ± 0.01 g/m2 /day and showed good
barrier property. By increasing the chitosan content permeability
increased to 2.88 ± 0.01, 3.59 ± 0.32 and 4.19 ± 0.02 g/m2 /day for
1, 3 and 5% LDPE/CS films, respectively (Table 2). In the chitosan
incorporated films the hygroscopic chitosan layer acted as a water
reservoir on the PE surface, thus significantly promoting its water
vapour permeability [35].
3.2.7. Overall migration rate (OMR)
The overall migration (OMR) of virgin LDPE and LDPE/CS films
was within the stipulated upper limit of 60 mg/L as shown in
Table 2. The virgin LDPE had value of 3.68 mg/L, a very low value,
when compared to other films. By increasing chitosan concentra-
tion the values of migration increased to 7.14, 8.80 and 8.85 mg/L
in 1, 3 and 5% LDPE/CS films, respectively. Considering the low
migration rate, these films can be suitably used for food contact
applications. If the chitosan migration further increases then it will
enhance the antimicrobial properties of the films.
3.2.8. Surface morphology of virgin LDPE and LDPE/CS blown
films
The surface morphology of virgin LDPE and 1% LDPE/CS com-
posite films are shown in Fig. 1(a) and (b). Comparison of the
topography of surfaces shows less troughs and valleys in the virgin
LDPE films. The average roughness of LDPE film was 0.681 nm due
to the defects formed during processing stage. The 1% LDPE/CS films
showed much more troughs and valleys indicating more roughness.
The images revealed the distribution and dispersion of chitosan
in the matrix material. Chitosan was distributed in matrix with
surface roughness of 2.490 nm. Surface roughness was due to the
incorporation of hydrophilic chitosan into a hydrophobic matrix.
Surface roughness enhanced the antimicrobial nature of film pro-
viding adherence of bacterial cell wall.
3.2.9. FTIR analysis
FTIR spectra of virgin LDPE and LDPE/CS films in the wave num-
ber range of 4000–500 are given in Fig. 2. Virgin LDPE showed
accentuated peaks at 2915–2848 cm−1 for (CH) stretching. The
peaks of 722 cm−1 and 1464 represented the skeletal vibrations
of CH2 .
In general, absorption peaks of chitosan at 3440 cm−1 showed
the stretching vibration of (NH2 ) and (OH) as well as inter
and intra molecular hydrogen bonding. Peaks around 1082 and
1366 cm−1 were due to saccharide structure and around 1637 cm−1
was due to carbonyl groups, respectively [31]. The peak at
1637 cm−1 represented acetylated amino group of chitin, which
indicated incomplete deacetylation of the sample.
In case of 1% LDPE/CS film, absorption spectra shows widening
between 3750 and 3000 cm−1 . The virgin LDPE film shows sharp CH
stretching at 2915 and 2848 cm−1 , CH3 bending at 1643 cm−1 which
is almost the same as in the absorption spectra of 1% LDPE/CS film.
Additionally, appearance of 1041 cm−1 corresponds to the vibration
of C O C groups in chitosan since chitosan also belongs to the
aliphatic ethers.
The 3% LDPE/CS spectra is much broad and intense in the range
between 3750 and 3000 cm−1 because of the increase in chitosan
particles. As compared to 1% LDPE/CS, 3% LDPE/CS showed less
intense characteristic peak of CH3 bending at a range of 1465 cm−1 ,
which may be due to the reactions of MA-g-LDPE on both LDPE and
chitosan. In the same way skeletal vibration peak of 721 cm−1 was
less intense when compared to 1% LDPE/CS.
The 5% LDPE/CS films showed broadened and small sharpen-
ing in the range of 3750–3000 cm−1 because of chitosan OH &NH
stretching and bending. The overall spectra band of 5% LDPE/CS
films showed that there was not much difference with 3% LDPE/CS
incorporated films.
 K.V. Reesha et al. / International Journal of Biological Macromolecules 79 (2015) 934–942
Fig. 2. FTIR spectra of virgin LDPE and LDPE/CS films.
3.3. Determination of antimicrobial properties of films against
E. coli
Antimicrobial activity of virgin LDPE & LDPE/CS films against E.
coli is shown in Table 3. Growth and survival of E. coli was effected
with increasing concentration of chitosan. E. coli got reduced by
79.20% after 96 h exposure on virgin LDPE films, whereas it reduced
to 84.83% of initial count in 1% chitosan incorporated films. There
was 100% reduction of E. coli in both 3% and 5% chitosan–LDPE films
after 96 h. Chitosan films exhibiting antimicrobial activity against
E. coli and Lactobacillus plantarum have been reported [36]. Simi-
larly, complete inhibition of E. coli has been reported in 1.4% and
2.1% chitosan lactate impregnated LDPE films after 12 h exposure
[7].
3.4. Changes in quality indices of Tilapia steaks during storage in
ice
3.4.1. Changes in TVB-N values
The initial TVB-N value of chilled fish was 0.70 ± 0.10 mg/100 g
of fish (Fig. 3) which slowly increased during the storage period
and reached 18 ± 0.20 mg/100 g of fish. For LDPE/CS 1%, 3%, 5%
samples, the initial TVB-N value ranged between 0.77 ± 0.01 mg
and 0.80 ± 0.30 mg/100 g sample and reached to a final value of
9.30 ± 0.12, 8.50 ± 0.31 and 14.50 ± 0.61 mg/100 g sample, respec-
tively after 30 days of storage. TVBN content of LDPE/CS 3% was
significantly lower (P < 0.05) than other films on 15th and 30th day
of chilled storage. Maximum TVBN content of 35–40 mg% is usually
regarded as limit of acceptability [37,38]. However, in the present
939
study TVB-N values for all the samples were within the suggested
limit throughout the storage period. TVB-N are products of bacte-
rial spoilage and their contents are often used as an index to assess
the keeping quality and shelf life of fish products [39].
3.4.2. Changes in peroxide value (PV)
In the present study, the peroxide values increased progres-
sively in all samples during the storage period (Fig. 4). A value
of 15.01 ± 0.30 milli equivalents (meq) per kg was reported on
30th day of storage for Tilapia packed in virgin LDPE pouches.
Similarly, for the fish samples packed in LDPE/CS 1%, 3%, 5%,
the initial PV values were 0.52 ± 0.04 meq/kg which increased to
7.70 ± 0.21, 6.50 ± 0.10, 14.00 ± 0.01, respectively on 30th day of
storage. PV content of LDPE/CS 3% was significantly lower (P < 0.05)
than other films throughout the storage duration. PV value in the
range 18–20 meq/kg of fish sample is usually taken as the limit
of acceptability. The gradual increase in PV is due to the break-
down of unsaturated fat into primary products of lipid oxidation
which further changes to secondary lipid oxidation products like
malonaldehyde [40].
3.4.3. Changes in free fatty acid value
The initial FFA content of samples packed in virgin LDPE
was 2.00 ± 0.01 mg % oleic acid and it gradually increased to
15.20 ± 0.01 mg % oleic acid at the final day of storage (Fig. 5). The
presence of free fatty acids is due to the oxidation and hydrolysis of
lipids and is undesirable since the fatty acids may be converted to
odorous volatiles [40]. The FFA content of all the samples increased
during the storage period. In LDPE/CS 1%, 3% and 5% packed
940 K.V. Reesha et al. / International Journal of Biological Macromolecules 79 (2015) 934–942

Table 3
Percentage reduction of Escherichia coli on virgin LDPE and LDPE/CS films.

Duration (h) Reduction (%) of Escherichia coli

Virgin LDPE 1% LDPE/CS 3% LDPE/CS 5% LDPE/CS

24 10.07 ± 0.55 11.82 ± 1.26 12.91 ± 0.96 11.34 ± 0.33

48 53.84 ± 1.36 58.72 ± 2.63 60.74 ± 1.95 62.18 ± 1.12
72 62.18 ± 1.98 67.80 ± 3.12 69.67 ± 3.11 71.10 ± 2.56
96 79.20 ± 2.63 84.83 ± 2.12 100.00 ± 0.00 100.00 ± 0.00

Fig. 3. Changes in TVBN values of Tilapia during storage in LDPE and LDPE–chitosan films.

Fig. 4. Changes in free peroxide value (PV) of Tilapia during storage in LDPE and LDPE–chitosan films.

samples the initial FFA content ranged from 1.00 ± 0.50 mg % oleic and 30th day of chilled storage. The values observed for
acid which reached 7.00 ± 0.01, 6.00 ± 0.01and 11.00 ± 0.01 mg chilled fish samples in the present study were less than the
% oleic acid during the storage period. FFA content of LDPE/CS reported values for different fishes under chilled storage condition
3% was significantly lower (P < 0.05) than other films on 15th [41,42].
 K.V. Reesha et al. / International Journal of Biological Macromolecules 79 (2015) 934–942 941

Fig. 5. Changes in free fatty acid content (FFA) of Tilapia during storage in LDPE and LDPE–chitosan films.

3.4.4. Changes in aerobic plate count
The aerobic plate count of chilled Tilapia packed in various films
was carried for 15 days of storage. A significant difference (P < 0.05)
was observed in APC of Tilapia packed in different films on 7 and 15
days of chilled storage (Fig. 6). A linear increase in APC was observed
in sampled packed with virgin LDPE films, where it increased from
an initial 4.87 log10 cfu/g to 6.34 log10 cfu/g at the end of 15 days of
chilled storage. Similarly, APC of Tilapia packed in 1% LDPE/CS films
crossed 106 cfu/g on 15th day of storage life. However, there was
moderate increase in APC values of Tilapia packed in 3% and 5%
LDPE/CS films and samples remained acceptable even beyond 15
days. Although there was no significant difference in APC values of
virgin LDPE, LDPE/CS 1% and LDPE/CS 3% on 15th day, the lower APC
values of 5.89 and 4.12 log10 cfu/g obtained in 3% and 5% LDPE/CS
samples must be due to antimicrobial action of released chitosan
on the spoilage microflora. Similar antimicrobial activity of chitosan
has been reported by [7], where total viable log populations on fresh
red meat applied with chitosan-incorporated LDPE film were lower
than control, but no significant difference was observed. Coating
consisting of a blend of chitosan dissolved in acetic acid and gelatine
exerting inhibitory effect on the Gram-negative flora of fish patties
has been reported [43]. Tsai et al. [44] have reported that a 3 h
pre-treatment of fish fillets (Oncorhynchus nerka) with 1% chitosan
solution could retard the increase in mesophilic count.

Fig. 6. Changes in aerobic plate count of Tilapia during storage in LDPE and LDPE–chitosan films.
942 K.V. Reesha et al. / International Journal of Biological Macromolecules 79 (2015) 934–942
4. Conclusions
The study revealed that LDPE/CS antimicrobial blown films can
be prepared by process optimization and incorporation of chitosan
up to 3% level since good processability was found only up to
3% chitosan addition into the LDPE film. The extruded granules
showed increased density with increasing chitosan concentration
but 5% LDPE/CS granules did not show significant variation with
3% LDPE/CS films. The films showed varying processing character-
istics which brought about changes in the mechanical properties.
LDPE/CS film shows good oxygen permeability properties. But in
the case of water vapour transmission rate the hydrophilic chitosan
negatively influenced the barrier properties of the LDPE films. The
overall migration rates of films were within the limit of contact
applications and the slight increase in the migration was due to
increase in the chitosan concentration which enhanced the antimi-
crobial properties of the film. The antimicrobial assay against E. coli
proved that LDPE/CS films were highly efficient than virgin LDPE
films. Virgin LDPE and 1%, 3% and 5% LDPE/CS films tested as pack-
aging films for chill stored tilapia showed that samples packed in
LDPE films were rejected by 7th day whereas fish packed in 1%, 3%
were remained acceptable up to 15 days. The study revealed that
3% LDPE/CS films had better physical and antimicrobial property
and enhanced the keeping quality of Tilapia steaks during chilled
storage when compared to the other films used in the study.
Acknowledgements
The authors gratefully acknowledge the laboratory facilities
provided by Director, Central Institute of Fisheries Technology,
Cochin, India for film testing and the Officer-In-Charge, Centre for
Bio-Polymer Science and Technology, CIPET, Cochin India for film
extrusion and characterization.
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