LWT - Food Science and Technology 57 (2014) 453e460

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LWT - Food Science and Technology

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Development of a chicken feather protein lm containing clove oil and

its application in smoked salmon packaging
Nak-Bum Song, Ji-Hyun Lee, Mohammad Al Mijan, Kyung Bin Song*
Department of Food Science and Technology, Chungnam National University, Daejeon 305-764, Republic of Korea

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 14 November 2013
Received in revised form
17 January 2014
Accepted 9 February 2014

Chicken feathers, a by-product of the poultry industry, were utilized as a lm base material after
extraction of chicken feather protein (CFP). Composite lms of CFP and gelatin were prepared, and their
mechanical properties were investigated. The tensile strength and elongation at break of the CFP/gelatin
composite lm signicantly (p < 0.05) increased as the gelatin content in the lm increased. As a crosslinking agent, 0.5% cinnamaldehyde further improved the lms mechanical properties. Incorporation of
clove oil into the composite lm resulted in strong inhibition zones against Escherichia coli O157:H7 and
Listeria monocytogenes compared with the lm without clove oil. Packaging smoked salmon with the
composite lm containing 1.5% clove oil resulted in a decrease in the populations of E. coli O157:H7 and
L. monocytogenes by 1.41 and 1.34 log CFU/g, respectively, compared with the control during storage at
4
C for 12 days. Furthermore, the peroxide value and thiobarbituric acid reactive substances value
decreased by 28 and 36%, respectively, in the smoked salmon packaged with the composite lm containing 1.5% clove oil compared with the control during storage. These results suggest that a CFP/gelatin
composite lm with 1.5% clove oil can be used as an active packaging material for smoked salmon.
2014 Elsevier Ltd. All rights reserved.

Keywords:
Composite lm
Physical property
Antimicrobial
Lipid oxidation

1. Introduction
Biodegradable packaging is one of few alternative strategies that
can be considered to reduce the use of environmentally harmful
synthetic polymers in the food packaging industry. Biodegradable
packaging materials can be degraded into water, carbon dioxide,
and inorganic substances without producing any toxic components
(Siracusa, Rocculi, Romani, & Rosa, 2008). Moreover, biodegradable
packaging lms are suitable for food preservation because they can
act as a barrier to moisture, oxygen, and volatile substances (Kim &
Ustunol, 2001). For the development of biodegradable packaging,
protein-based materials are considered attractive sources because
of their excellent lm-forming properties (Cao, Fu, & He, 2007;
Souza, Cerqueira, Teixeira, & Vicente, 2010). A number of protein
sources, such as soy protein, sunower protein, chicken feather
protein, barley bran protein, and whey protein, have been studied
recently for the manufacture of edible packaging lms (Guerrero,
Stefani, Ruseckaite, & de La Caba, 2011; Salgado, Molina Ortiz,
Petruccelli, & Mauri, 2010; Song, Shin, & Song, 2012; Song et al.,
2013). Chicken feathers are one of the potential sources of edible

* Corresponding author. Tel.: 82 42 8216723; fax: 82 42 8252664.

E-mail address: kbsong@cnu.ac.kr (K.B. Song).
http://dx.doi.org/10.1016/j.lwt.2014.02.009
0023-6438/ 2014 Elsevier Ltd. All rights reserved.

lm material because they are abundant, cheap, and a rich source of

protein (Martelli, Moore, & Laurindo, 2006).
Chicken feathers are a by-product of poultry industry and are
mostly disposed of without any pretreatment, causing severe
environmental pollution (Moore, Martelli, Gandolfo, Sobral, &
Laurindo, 2006). The major components of chicken feathers are
proteins (91%), lipids (1%), and water (8%) (Kock, 2006). The protein
portion of chicken feathers is mainly composed of a structural
protein called keratin (Schrooyen, Dijstra, Oberthur, Bantjes, &
Feijen, 2001). Keratin has high amounts of cysteine and halfcysteine, which can be distinguished from collagen or other structural proteins (Tonin et al., 2007). Reportedly, the cysteine residues
present in keratin can be oxidized to form intra- and intermolecular
disulde bonds through cross-linking, resulting in water insolubility
and improved mechanical properties for edible lms (Schrooyen
et al., 2001). Thus, as an abundant source of keratin, chicken
feather protein (CFP) can be utilized as a potential edible lm material. In general, CFP lms are too fragile, and a plasticizer is needed
to increase the strength and exibility of the lm (Yamauchi,
Yamauchi, Kuisnoki, Kohda, & Konishi, 1996). Recently, Song et al.
(2013) reported that the use of plasticizers in CFP lm preparation
resulted in an improvement in the lms mechanical properties.
Gelatin has been incorporated into edible lms to improve the
mechanical properties of the lms. Gelatin, which is obtained

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N.-B. Song et al. / LWT - Food Science and Technology 57 (2014) 453e460

through the partial degradation of collagen, has excellent lmforming properties because of its unique thermo-reversibility at a
melting point close to body temperature, which is a crucial factor in
edible lm application (Achet & He, 1995). Although the high cost of
gelatin inhibits its application as a base material for edible lm
preparation, previous experimental results have revealed that the
incorporation of gelatin into soy protein isolate and barley bran
protein lms remarkably improved the mechanical properties of
the lms (Cao et al., 2007; Song et al., 2012).
Cold smoked salmon is a ready-to-eat food, which is generally
consumed without cooking and may provide serious health threats
to consumers due to its contamination with pathogenic bacteria,
such as Escherichia coli and Listeria monocytogenes (Rotariu, Thomas,
Goodburn, Hutchison, & Strachan, 2014). In addition, the polyunsaturated fatty acids present in salmon are easily oxidized and lead
to spoilage of the salmon during storage (Bell et al., 2001). Therefore,
preservation of smoked salmon with an edible lm containing antimicrobials and antioxidant substances may be an effective strategy.
Clove oil is a potent antimicrobial substance that has been
proven to be very effective against E. coli and L. monocytogenes
(Cressy, Jerrett, Osborne, & Bremer, 2003; Mytle, Anderson, Doyle, &
Smith, 2006). In addition, clove oil has nonpolar phenolic compounds with antioxidant properties (Atars, Bonilla, & Chiralt, 2010).
Moreover, a recent study demonstrated that a sunower seed meal
protein lm containing clove oil had a very high level of antimicrobial and antioxidant properties in the preservation of sh patties
(Salgado, Lpez-Caballero, Gmez-Guilln, Mauri, & Montero, 2013).
Therefore, the aim of the present study was to develop a composite
lm from CFP and gelatin and to apply the CFP/gelatin composite
lm containing clove oil in the smoked salmon preservation.
2. Materials and methods
2.1. Materials
The white chicken feathers used in this study were supplied by a
local company (Harim Corporation, Iksan, Jeonbuk, Republic of Korea). Sorbitol, gelatin from the pork, and cinnamaldehyde were purchased from SigmaeAldrich Chemical Co. (St. Louis, MO, USA). Clove
oil was purchased from The Certication Academy for Holistic
Aromatherapy (Seoul, Republic of Korea). Smoked salmon (salmon,
97%) was obtained from a local company (Daejeon, Republic of Korea).
2.2. Extraction of chicken feather protein
Chicken feathers were cleaned and dried at room temperature for
72 h. The feathers were cut into small laments of 75e700 mm. CFP

2.3. Preparation of the CFP/gelatin composite lm

Five grams of CFP was dispersed in 100 mL of distilled water.
Two grams of sorbitol was chosen as a plasticizer. To prepare the
CFP/gelatin composite lms, various amounts of gelatin (0.5, 1,
1.5, and 2 g) were dispersed with the sorbitol (2 g) into the CFP
solution and stirred for 1 h, followed by ultra-sonication for
8 min. The solution was then heated using a hot water bath at
75
C for 30 min. In addition, clove oil (0.5, 1.0, and 1.5 g) and
cinnamaldehyde (0.5, 1.0, and 1.5 g) were incorporated into the
lm-forming solution with 0.25 g of Tween 20, and the samples
were then cooled to 40
C for 20 min. After straining through the
cheesecloth, each lm-forming solution was poured onto a at
Teon-coated glass plate (24  30 cm) and dried at 25
C for
24 h. The uniformity of the lms thickness was maintained by
pouring a constant amount of solution onto each plate. The lms
were removed intact from the casting surfaces and stored for
analyses.
2.4. Film conditioning and lm thickness
For proper testing, the lm samples were conditioned in
an environmentally controlled chamber (25
C and 50% RH)
for 48 h. The thickness of the lms was measured at four
random points of each lm using a digital micrometer
(Mitutoyo Co., Tokyo, Japan), and the average thickness was
calculated.
2.5. Tensile strength and elongation at break
The tensile strength (TS) and elongation at break (E) values of
the lms were determined using an Instron Universal Testing
Machine (Model 4484, Instron Co., Canton, MA, USA). Rectangular strips (2.54  10 cm) were cut and xed in the self-aligning
grips of the device. The grip distance was 5 cm, and the lm
stretching speed was 50 cm/min. TS was calculated as the
maximum stress the lm endured before breaking, and E was
expressed as the percentage of change of the initial length of a
specimen at the point of breaking. Five replicates of each lm
were tested.
2.6. Moisture content
The moisture content of lm pieces (2  2 cm) was determined
by measuring the weight losses of the lms upon drying in an oven
at 110
C. Three replicates of each lm were tested. The moisture
content (%) was calculated as follows:

Moisture content % initial sample weight  dry sample weight=initial sample weight  100

was extracted according to the method of Nomura et al. (2006) with a

modication. The chicken feathers (500 g) were immersed in water
(1:1, w/w) and blended for 1 h. After blending, 300 mL of a 1 M NaOH
solution was added to the mixture, and the mixture was homogenized at room temperature for 24 h. The mixture was centrifuged at
10,000  g for 1 h, followed by ltering through a cheesecloth and
neutralization with 1 N HCl. Dialysis of the ltrate was conducted
using Spectra/Por dialysis membranes of regenerated cellulose
(MWCO, 3.5 kDa, Spectrum Laboratories, Inc., Rancho Dominguez,
CA, USA) for 72 h, and then the samples were freeze-dried.

2.7. Film water solubility

The lm water solubility (FWS) was determined according to the
method of Shen, Wu, Chen, and Zhao (2010). Film pieces (2  2 cm)
were dried to a constant weight at 105
C. Each sample was placed
into a 50-mL beaker containing 20 mL of distilled water and was
shaken at room temperature (25
C) for 24 h. After collecting the
undissolved lm, its dry weight was determined after drying in an
oven at 105
C for 24 h. The FWS of each lm was calculated as
follows:

N.-B. Song et al. / LWT - Food Science and Technology 57 (2014) 453e460

FWS % initial dry weight

 final dry weight=initial dry weight  100

455

was the mean of three determinations, and the counts were

expressed as the log CFU/g.
2.12. Measurement of lipid oxidation

2.8. Measurement of water vapor permeability

The water vapor permeability (WVP) was determined according to the method described by Hong, Lim, and Song (2009).
Deionized water (12 mL) was added to each polymethylacrylate
cup (12 mL), and a lm specimen was xed onto the opening of
each cup. The thickness of the lms was 0.067  0.002 mm, and
the exposed area of the permeability cups was 16 mm in diameter. The cups were stored in a room with constant temperature
(25
C) and humidity (50% RH). The weight loss of the cup was
monitored over an 8 h period with an interval of 1 h. The measurement was performed to account for variability from temperature or barometric pressure. Three replicates of each lm
type were prepared, and the slope was calculated using the linear
regression analysis.
2.9. Disk diffusion test for antimicrobial activity
E. coli O157:H7 (NCTC 12079) and L. monocytogenes (ATCC 19111)
were cultured at 37
C for 24 h in 50-mL conical tubes containing
25 mL of LuriaeBertani (LB) broth (Difco, Detroit, MI, USA) or Listeria enrichment broth (Oxoid Ltd., Basingstoke, UK). E. coli
O157:H7 culture (0.1 mL) was plated onto tryptic soy broth Bacto
agar (Difco), and L. monocytogenes culture (0.1 mL) was plated onto
Oxford medium base (Difco). Discs (10 mm in diameter) were cut
from the lms and placed onto the inoculated plates. After 30 min
at 4
C to allow the clove oil to diffuse, the E. coli O157:H7 plates
were incubated at 37
C for 24 h, and the L. monocytogenes plates
were incubated at 37
C for 48 h. Each microbial count was determined as the mean of three replicates, and the inhibition zone was
measured in millimeters using a Digimatic caliper (Model 500-18120; Mitutoyo Corp., Kawasaki, Japan).

The evaluation of lipid oxidation was conducted using two

different methods: peroxide value (PV) and thiobarbituric acid
reactive substances (TBARS) assays. The PV was determined according to the method described by Pereira de Abreu, Paseiro
Losada, Maroto, and Cruz (2010). Each sample (2 g) was placed
into a 250-mL Erlenmeyer ask, and 30 mL of a mixture of acetic
acid/chloroform (3:2, v/v) was added. The solution was stirred to
completely dissolve the lipids. After adding the potassium iodide
solution, the mixture was allowed to stand for 5 min in a dark room.
Then, distilled water (30 mL) was added and titrated against 0.1 N
sodium thiosulfate with 1% starch suspension as an indicator. The
PV value was expressed as meq O2 per kg of lipid, and each sample
was determined as the mean of three replicates. The TBARS was
determined according to the method described by Vyncke (1970).
The sample (2 g) was mixed with 10 mL of 7.5% trichloroacetic acid
and homogenized for 3 min using a Stomacher (MIX 2; AES Laboratoire, Combourg, France). The suspension was ltered, and 5 mL
of the ltrate was added to 5 mL of TBARS reagent (0.02 M 2-thiobarbituric acid in distilled water). The mixture was immersed in a
boiling water bath at 95
C for 45 min, cooled with water, and the
absorbance was read at 539 nm using a spectrophotometer (UV2450, Shimadzu Corporation, Kyoto, Japan). The TBARS value was
recorded as mg malonaldehyde (MDA) per kg sample, and each
sample was determined as the mean of three replicates.
2.13. Statistical analysis
Analysis of variance and Duncans multiple range tests were
performed to analyze the data using the SAS version 8.1 (SAS
Institute, Inc., Cary, NC, USA). Differences at p < 0.05 were
considered signicant. All the results are expressed as the
mean  standard deviation.

2.10. Inoculation of pathogens on smoked salmon

3. Results and discussion

E. coli O157:H7 and L. monocytogenes were incubated at 37
C

in LB and Listeria enrichment broth, respectively, until they
reached a population of 106 CFU/mL. E. coli O157:H7 and
L. monocytogenes (1 mL) were spread evenly on the smoked
salmon surface with a sterile glass rod and allowed to rest for
30 min. The smoked salmon samples (10 g) were classied into
three groups: the control without the lm packaging, samples
packed with CFP/gelatin composite lm without clove oil, and
samples packed with CFP/gelatin composite lm containing 1.5%
clove oil. Each sample packed in a sterile polyethylene bag
(TwirlEM sampling bags, Lab Plas, Quebec, Canada). All the
samples were stored at 4  1
C for 12 days and removed for
measuring microbial counts every 3 days.

3.1. Mechanical properties of the CFP/gelatin composite lm

2.11. Microbiological analysis

The smoked salmon samples (10 g) were homogenized for 3 min
using a Stomacher (MIX 2; AES Laboratoire, Combourg, France),
ltered through sterile cheesecloth, and diluted with 0.1% peptone
water. Serial dilutions were performed in triplicate on each selective agar plate. Diluted samples of E. coli O157:H7 were plated onto
MacConkey agar (BBL), and L. monocytogenes were plated onto
Oxford Medium Base agar (Difco). For determination of E. coli
O157:H7 and L. monocytogenes populations, each plate was incubated at 37
C for 24 h and 48 h, respectively. Each microbial count

The extracted protein from the chicken feather was mainly

composed of beta-keratin which is a major structural brous protein (Song et al., 2013). Composite lms were prepared by adding
various amounts of gelatin (0.5, 1.0, 1.5, and 2.0%) into the CFP lmforming solution. Table 1 shows the effect of gelatin content on the
mechanical properties of the CFP composite lms. CFP lms containing gelatin had signicantly higher TS and E and lower WVP
than the control. The TS of the lms showed an increasing trend
with the increasing amount of gelatin. Noticeably, TS increased
from 1.35 to 15.31 MPa as the inclusion of gelatin into the lms
increased from 0 to 2%. There was a substantial increase in the E
value upon the incorporation of gelatin. However, a low concentration (0.5%) of gelatin increased the E substantially, whereas the E
value displayed a decreasing trend as the gelatin concentration
increased. WVP was reduced upon the addition of gelatin, and the
highest reduction was found in the lm containing 2% gelatin,
representing 22% reduction compared with the control.
The above increase in TS and E indicated the enhancement of
the strength and exibility of the CFP/gelatin composite lm. In
the present study, the brous tertiary structure of gelatin might
help form an organized network during the lm-forming process
(Chambi & Grosso, 2006). In addition, gelatin molecules facilitated the protein/protein interactions by increasing the hydrogen

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N.-B. Song et al. / LWT - Food Science and Technology 57 (2014) 453e460

Table 1
Mechanical properties, moisture content, and water solubility of the CFP lm containing various amounts of gelatin.
CFP (g)

Gelatin (g)

Tensile strength (MPa)

5
4.5
4.0
3.5
3.0

0.0
0.5
1.0
1.5
2.0

1.35
2.40
5.20
7.63
15.31







0.07d
0.13d
0.99c
0.20b
0.75a

WVP (109 g m/m2 s Pa)

Elongation (%)
22.10
158.23
120.67
100.01
98.51







1.06c
22.74a
17.25b
31.83b
15.66b

2.67
2.58
2.55
2.36
2.13







0.02a
0.01a
0.07a
0.04b
0.17c

Moisture content (%)

10.83
10.14
10.21
9.46
9.02







0.36a
0.11ab
0.57ab
1.10ab
1.39b

Film water solubility (%)

100
100
83.45
77.13
71.74







0.00a
0.00a
1.59b
2.32c
1.51d

Mean values  SD.

aed
Values in a column followed by different superscript letters are signicantly different (p < 0.05).

bonding and electrostatic interactions among the protein polymers (Zou et al., 2012). However, higher concentrations (>0.5%)
of gelatin decreased the elongation (E) of the lms. Similar results
were reported by Harper, Barbut, Lim, and Marcone (2013),
where the increase in gelatin content (>0.4%) lowered the elongation of alginate lm. These results could possibly be the
consequence of the increased polymer density and decrease in
water molecules by gelatin incorporation into the lm network
(Rattaya, Benjakul, & Prodpran, 2009). The decrease in the WVP
values can be justied by the fact that the presence of gelatin in
the CFP lm resulted in the increased compactness of protein
molecules, which caused the lm to become more hydrophobic
and less soluble in water (Denavi et al., 2009). According to the
study of Cao et al. (2007), TS and E increased when gelatin was
blended with soy protein isolate (SPI), due to the creation of
intermolecular interactions between SPI and gelatin molecules.
Similarly, Li, Kennedy, Jiang, and Xie (2006) reported a signicant
increase in TS and E in the konjac glucomannan/gelatin composite lm as a result of gelatin incorporation. It was suggested
that these results can be attributed to the formation of hydrogen
bonds between the protein polymers.
Moisture content and water solubility of the CFP lm decreased
signicantly (p < 0.05) with the incorporation of gelatin. As illustrated in Table 1, the moisture content and water solubility were the
highest for the control CFP lm and the lowest for the CFP lm with
2% added gelatin. In particular, the moisture content in the CFP lm
blended with 2% gelatin was reduced by 16% compared with the
control. Gelatin incorporation into the lm might increase the
density of the protein polymers interacting with keratin molecules
present in CFP, which contributes to the reduction of intermolecular spaces, and eventually led to a decrease of water molecules in
the lm structure. Notably, a CFP lm without gelatin was 100%
soluble in water, whereas the solubility decreased by 28% for the
CFP lm containing 2% gelatin. It was suggested that the lower
solubility of the protein lms might result from the increased aggregation of protein molecules through the formation of disulde
bonds (Hernndez-Muoz, Villalobos, & Chiralt, 2004). It can be
assumed that the gelatin incorporation increased the proteine
protein interactions in the lm, which led to higher protein aggregation to make the lm less soluble in water. Thus, the above
results suggested that CFP (3 g) blended with gelatin (2 g) displayed
the best mechanical properties and was selected for the targeted
composite lm preparation in this study.

As a cross-linking agent, cinnamaldehyde was added into the

CFP/gelatin composite lm to further improve the lms mechanical properties. At a low concentration (0.5% of the lm-forming
solution), cinnamaldehyde increased the TS to 29 MPa from 15 MPa
(Table 2). Further increases in the concentration (>0.5%) of cinnamaldehyde caused the reduction of TS signicantly (p < 0.05). With
0.5% cinnamaldehyde, the E value of the composite lm was
signicantly lower (44%) than the control (99%), whereas WVP was
not considerably changed by the incorporation of cinnamaldehyde.
In addition, with 0.5% cinnamaldehyde, the moisture content of the
lm was not changed signicantly, whereas the water solubility
(61%) was substantially lower than that of the control (72%)
(Table 2). Therefore, 0.5% cinnamaldehyde of the lm-forming solution was chosen as an optimum concentration for the CFP/gelatin
composite lm preparation.
3.2. Mechanical properties of the CFP/gelatin composite lm
containing clove oil
The mechanical properties of the CFP/gelatin composite lms
containing various amounts of clove oil are presented in Table 3. In
general, the addition of lipids affects the integrity of the protein
matrix in a lm resulting in the modication of its mechanical
characteristics such as strength and stretchability (Chen, 1995). In
this study, the addition of clove oil resulted in the signicant
decrease of the TS of the composite lms (p < 0.05). The composite
lm without clove oil had a TS of 29.39 MPa, whereas TS was
reduced to 13.57 MPa as the proportion of clove oil increased to
1.5%. Similarly, decreases in the TS of the whey protein isolate and
wheat gluten upon the increase of lipid concentrations have been
reported by previous studies (Gontard, Duchez, Cuq, & Guilbert,
1994; Shellhammer & Krochta, 1997). These results were also
consistent with the ndings of Altiok, Altiok, and Tihminlioglu
(2010), who reported that increasing the concentration of thyme
oil decreased the TS of chitosan lm. Cagri, Ustunol, and Ryser
(2004) concluded that except for the cross-linking agent, incorporation of any additive generally lowered the TS value of protein
lms. It was stated that the incorporation of essential oils interfered
with the lm structure by partially replacing the protein polymers
in the lm (Atars et al., 2010). Yang and Paulson (2000) stated that
the afnities between polar polymer molecules were much stronger than those between polar polymers and nonpolar lipid molecules. Therefore, the decrease in the TS value of the CFP/gelatin

Table 2
Mechanical properties, moisture content, and water solubility of the CFP (3 g)/gelatin (2 g) composite lm containing various amounts of cinnamaldehyde.
Cinnamaldehyde (g)
0.0
0.5
1.0
1.5

Tensile strength (MPa)

15.31
29.39
14.31
11.64






0.75
0.21a
1.75b
1.03c

Elongation (%)
98.51
44.06
67.05
73.48






15.66
5.18c
12.66b
10.63b

WVP (109 g m/m2 s Pa)

2.13
2.28
2.37
2.38






0.17a
0.13a
0.11a
0.17a

Mean values  SD.

aec
Values in a column followed by different superscript letters are signicantly different (p < 0.05).

Moisture content (%)

9.02
10.17
10.33
11.52






1.39b
0.39ab
0.51ab
0.53a

Film water solubility (%)

71.74
61.04
67.78
68.90






1.51a
0.08c
1.31b
0.96b

N.-B. Song et al. / LWT - Food Science and Technology 57 (2014) 453e460

457

Table 3
Mechanical properties, moisture content, and water solubility of the CFP (3 g)/gelatin (2 g) composite lm containing various amounts of clove oil.
Clove oil (g)

Tensile strength (MPa)

0.0
0.5
1.0
1.5

29.39
15.90
15.28
13.57






0.21a
0.23b
1.91b
0.82b

WVP (109 g m/m2 s Pa)

Elongation (%)
44.06
46.25
46.33
53.64






5.18a
8.01a
3.56a
5.51a

2.28
2.29
2.47
2.54






0.13a
0.05a
0.09a
0.26a

Moisture content (%)

10.17
10.22
10.28
10.41






0.39a
1.08a
0.23a
0.66a

Film water solubility (%)

61.04
72.39
73.85
74.08






0.08b
0.33a
1.63a
0.85a

Mean values  SD.

aeb
Values in a column followed by different superscript letters are signicantly different (p < 0.05).

composite lm in this study could possibly be due to the replacement of the protein molecules by clove oil in the lm matrix.
Both the E and WVP values slightly increased as a result of clove
oil incorporation into the lm. The CFP/composite lm without
clove oil had E and WVP values of 44% and 2.28  109 g m/m2 s Pa,
respectively. The highest E (53.54%) and WVP (2.54 g m/m2 s Pa)
values were found for the lm containing 1.5% clove oil. The
interaction of clove oil with the protein polymers resulted in a
greater extensibility of the lm, resulting in the increase of E value.
Similar to this result, Monedero, Fabra, Talens, and Chiralt (2009)
reported an increase in E after the addition of oleic acid.
Zinoviadou, Koutsoumanis, and Biliaderis (2009) also found that
incorporation of oregano oil into whey protein isolate lms made it
more extensible lms. In accordance with our study, Ahmad,
Benjakul, Prodpran, and Agustini (2012) reported that the WVP of
unicorn leatherjacket lm slightly increased with a higher concentration (5%) of bergamot oil. Similarly, Pranoto, Salokhe, and
Rakhsit (2005) reported that the incorporation of a larger amount
of garlic oil (0.03e0.4%) led to increase in WVP of alginate-based
lms. It can be hypothesized that the clove oil present among the
protein molecules in the lm weakened the intermolecular forces
and increased the mobility of the polypeptide chains, facilitating
the migration of water molecules through the lm.
In contrast, incorporation of clove oil did not noticeably affect
the moisture content of the lms (Table 3). Nevertheless, as the
concentration of clove oil increased, a slight (p < 0.05) increase of
moisture content in the CFP/gelatin composite lm was observed.
This small increase of water content may be due to the breaking up
of the lm network by clove oil, allowing the increase of water
molecules between the polymer chains. This nding corresponded
with a recent report in which the moisture content of quince seed
mucilage lm was not markedly affected by the addition of oregano
essential oil (Jouki, Yazdi, Mortazavi, & Koocheki, 2014). The water
solubility of the CFP composite lm was largely inuenced
(p < 0.05) by the addition of clove oil (Table 3). The CFP/gelatin
composite lm without clove oil exhibited the lowest solubility
(61%) in water, whereas the solubility was highest with the addition
of 1.5% clove oil into the lm. It is plausible that the lower solubility
of the protein lms might result from the proteineprotein interactions that stabilize the lm network (Hernndez-Muoz et al.,
2004). Therefore, it can be assumed that incorporating clove oil into
the composite lm increased the lipideprotein interactions against
Table 4
Antimicrobial properties of the CFP/gelatin lm containing clove oil.
Inhibition zone (mm)
Clove oil (g)

E. coli O157:H7

0
0.5
1
1.5

13.50
13.61
20.01
22.23






0.28c
0.11c
0.07b
0.28a

L. monocytogenes
12.55
16.50
22.01
26.67






0.74d
0.08c
0.31b
0.60a

Mean values  SD.

aed
Values in a column followed by different superscript letters are signicantly
different (p < 0.05).

proteineprotein interactions, destabilizing the lm network. Pires

et al. (2013) found that the water solubility of the hake protein
edible lms signicantly increased because of the addition of
essential oils, and they hypothesized that the essential oils had
inhibitory effects on proteineprotein and proteineglycerol (plasticizer) interactions. Likewise, the addition of thyme and clove
essential oils into the gelatinechitosan composite lm resulted in
the increase of its water solubility (Gmez-Estaca, Bravo, GmezGuilln, Alemn, & Montero, 2009). It should be noted that the
lms with high solubility may be advantageous as a packaging
material, as it can release the antimicrobial substances to the food
surface gradually. In addition, it should be considered that incorporation of clove oil reduces TS and increases WVP of the CFP
composite lm, resulting in inferior physical property of the lm as
a possible negative effect.
3.3. Antimicrobial activity of the CFP/gelatin composite lm
containing clove oil
The results of antibacterial activity assays of the CFP/gelatin
composite lm containing various amounts of clove oil determined
with disc diffusion method are shown in Table 4. All lm samples in
this study displayed inhibitory effects against L. monocytogenes and
E. coli O157:H7. The inhibitory activity of the control lm against the
bacteria was mostly due the presence of cinnamaldehyde. A previous investigation reported that a cellulose-based lm containing
cinnamaldehyde had strong inhibitory effects against E. coli and
L. monocytogenes (Sanla-Ead, Jangchud, Chonhenchob, & Suppakul,
2012). However, the antibacterial properties of the lms incorporated with clove oil were signicantly higher (p < 0.05) than the
control. As shown in Table 4, the inhibition zones of the lms
against both types of bacteria increased with the increasing amount
of clove oil. Antibacterial activities were the lowest in the composite lm without clove oil and the highest in the lm containing
1.5% clove oil. In particular, the control lm had inhibition zones of
14 and 13 mm, whereas the lm with 1.5% clove oil had inhibition
zones of 22 and 27 mm against E. coli and L. monocytogenes,
respectively. The above results indicated very strong antimicrobial
activities of clove oil against E. coli (Gram-negative) and
L. monocytogenes (Gram-positive). It should also be noted that clove
oil had higher inhibition against the Gram-positive bacteria than
the Gram-negative bacteria. The literature suggests that the efcacy
of essential oils against Gram-positive food spoilage and foodborne bacteria is higher than against Gram-negative bacteria
(Rasooli, 2007). The higher resistance of Gram-negative bacteria to
clove oil is probably due to the existence of an additional outer
membrane of the bacteria, which limits the dispersion of the hydrophobic substances through the cytoplasmic membrane
(Sharma, Jain, Yadav, & Sharma, 2010). Instead of clove oil used in
the present study, the hydrolyzate of channel catsh bone can be
used as an antibacterial agent for biodegradable lms with antimicrobial activity (Ren et al., 2012). The antimicrobial activities of
clove oil are attributed to the presence of functional components
(eugenol and carvacrol), and these hydrophobic compounds are

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N.-B. Song et al. / LWT - Food Science and Technology 57 (2014) 453e460

Table 5
Change in the populations of E. coli O157:H7 inoculated in smoked salmon during storage at 4
C (log CFU/g).
Sample

Storage time (day)

0

Control
CFP composite lm without clove oil
CFP composite lm containing clove oil

3
Ba

6.43  0.27
6.43  0.27Aa
6.43  0.27Aa

6
Aa

6.91  0.01
6.13  0.06ABb
5.72  0.08Bc

9
Ba

6.49  0.17
5.92  0.03BCb
5.30  0.19Bc

12
BCa

6.21  0.23
5.59  0.28Cb
4.80  0.18Dc

5.98  0.11Ca
5.13  0.19Db
4.64  0.15Dc

Mean values  SD.

aec
Values in a column followed by different superscript letters are signicantly different (p < 0.05).
AD
Values in a row followed by different superscript letters are signicantly different (p < 0.05).

capable of penetrating the bacterial cell membrane to disrupt the

cell structure (Burt, 2004). Ahmad et al. (2012) found higher
inhibitory activities of gelatin lm for both Gram-positive and
Gram-negative bacteria when incorporated with essential oils.
Furthermore, Hosseini, Razavi, and Mousavi (2009) revealed that
with 1.5% clove oil, chitosan lm was very effective against several
bacterial strains, such as E. coli and L. monocytogenes. Similarly, in
the present study, clove oil had the highest inhibitory activity at the
concentration of 1.5% in the composite lm. Therefore, 1.5% clove oil
was chosen as the optimum concentration to be incorporated into
the CFP/gelatin composite lm for use in smoked salmon antimicrobial packaging.
3.4. Quality changes of salmon packaged with the CFP/gelatin
composite lm containing clove oil
Considering the strong antimicrobial properties of the CFP/
gelatin composite lm containing clove oil obtained from the disc
diffusion test, its efcacy was evaluated in the preservation of
smoked salmon. Tables 5and 6 represents the changes in the populations of E. coli O157:H7 and L. monocytogenes in the smoked
salmon packaged with the CFP/gelatin composite lm containing
1.5% clove oil during storage at 4
C for 12 days. On the rst day of
storage, the total counts of E. coli O157:H7 and L. monocytogenes
inoculated on smoked salmon were 6.64 and 6.43 log CFU/g,
respectively. It is noticeable that the populations of E. coli O157:H7
displayed an increasing trend during the rst 9 days of storage and
slightly decreased afterward (Table 5). After 12 days of storage, the
population of E. coli O157:H7 in the control sample was
8.02 log CFU/g, whereas the population in the sample packed with
the CFP/gelatin composite lm containing clove oil was
6.61 log CFU/g, indicating a 1.41 log CFU/g reduction. These results
are supported by the ndings of Song et al. (2012), where barley
bran proteinegelatin composite lm with 1.2% grapefruit seed
extract had stronger efcacy against E. coli O157:H7 proliferation on
salmon than the control during 15 days of storage at 4
C.
Similar to the trend of E. coli O157:H7, the populations of
L. monocytogenes in the smoked salmon increased during the rst 9
days of storage and decreased to a small extent thereafter (Table 6).
CFP/gelatin composite lm with 1.5% clove oil displayed a strong
inhibitory effect against L. monocytogenes in smoked salmon. After

12 days of storage, the population (4.64 log CFU/g) of

L. monocytogenes in the smoked salmon packaged with the composite lm containing 1.5% clove oil was 1.34 log CFU/g lower than
that of the control (5.98 log CFU/g). Similarly, Miladi et al.
demonstrated that the clove oil (2%) incorporation could reduce the
growth of L. monocytogenes in fresh-cut salmon by 2.35 log CFU/g
compared with the control during storage for 15 days (Miladi,
Chaieb, Ammar, & Bakhrouf, 2010). Having a high degree of water
solubility, the lm containing clove oil can absorb more water and
become swollen, which allowed the easy migration of the antibacterial substances such as eugenol and carvacrol of clove oil
present in the lm into the surface of smoked salmon to exert their
inhibitory actions against the microbial growth (Hosseini et al.,
2009; Miladi et al., 2010).
The antioxidant properties of the CFP/gelatin composite lm
containing 1.5% clove oil as assessed by PV and TBARS values in the
smoked salmon during 12 days of storage are shown in Figs. 1 and 2.
Until 9 days of storage, the PV signicantly increased in the control
and in the salmon packaged with the CFP composite lm without
clove oil and demonstrated a decreasing trend afterward (Fig. 1).
However, the PV of the salmon packaged with the CFP composite
lm containing clove oil did not increase and remained the lowest
among the smoked salmon samples throughout the entire storage
period. At the end of the storage time, the PV of the smoked salmon
packaged with the CFP composite lm containing clove oil was 28%
lower than that of the control. Moreover, during storage, TBARS had
been the lowest for the salmon packaged with the CFP composite
lm containing clove oil. The TBARS assay in this study measured
the amount of malonaldehyde production as a secondary product
from lipid oxidation, providing the information on the status of
lipid oxidation on the smoked salmon. The malonaldehyde level
was initially 1.28 mg MDA/kg in all samples and increased during
storage. On the nal day of storage, the TBARS value in the control
sample was 4.26 mg MDA/kg, whereas the malonaldehyde production in the smoked salmon packaged with the CFP composite
lm containing clove oil was 2.74 mg MDA/kg, indicating a 36%
reduction of malonaldehyde compared with the control. A signicant reduction of malonaldehyde in the sh patties stored with
clove oil-containing lm was recently reported by Salgado et al.
(2013). Song et al. (2012) also reported the reduction of PV and
TBARS of the salmon packaged with barley bran proteinegelatin

Table 6
Change in the populations of L. monocytogenes inoculated in smoked salmon during storage at 4
C (log CFU/g).
Sample

Storage time (day)

0

12

Control
CFP composite lm without clove oil
CFP composite lm containing clove oil

6.64  0.10Da
6.64  0.10Ba
6.64  0.10Ba

7.67  0.10Ca
6.99  0.09Ab
6.62  0.09Bc

8.00  0.08Ba
7.11  0.19Ab
6.77  0.04ABc

8.66  0.09Aa
7.21  0.04Ab
6.89  0.04Ac

8.02  0.05Ba
7.13  0.12Ab
6.61  0.23Bc

Mean values  SD.

aec
Values in a column followed by different superscript letters are signicantly different (p < 0.05).
AD
Values in a row followed by different superscript letters are signicantly different (p < 0.05).

N.-B. Song et al. / LWT - Food Science and Technology 57 (2014) 453e460

459

considered to be benecial for the migration of phenolic compounds

into the product. Moreover, the antimicrobial properties of the
composite lm were signicantly improved by the addition of clove
oil. In the storage experiment, the proliferation of E. coli O157:H7 and
L. monocytogenes on the smoked salmon were substantially inhibited
by the CFP/gelatin composite lm containing clove oil. Furthermore,
the oxidative substances, such as PV and TBARS, were reduced to a
great extent in the smoked salmon by the CFP/gelatin composite lm
containing clove oil. Therefore, the CFP/gelatin composite lm containing 1.5% clove oil can be considered a potential active packaging
material for smoked salmon preservation.
Acknowledgment
This research was supported by Basic Science Research Program
through the National Research Foundation of Korea funded by the
Ministry of Education (2013-1074).
Fig. 1. Change in PV of smoked salmon during storage at 4
C. C, Control; B, CFP lm
without clove oil; ;, CFP lm containing clove oil.

composite lm containing grapefruit seed extract. It is well known

that the antioxidant capacities of essential oils are attributed to
their total and individual phenolic compounds content (Shan, Cai,
Sun, & Corke, 2005). In the present study, the remarkable increase of antioxidant property of the CFP/gelatin composite lm
upon the incorporation of clove oil suggested the existence of a
fraction of clove oil in a free form in the lm network, which did not
interact with the protein matrix during the lm formation and
might migrate to the surface of the smoked salmon during storage
to prevent its oxidation. To summarize, the above results indicate
that the CFP/gelatin composite lm containing clove oil could be a
very effective antioxidant packaging for smoked salmon.
4. Conclusion
In this study, a composite lm was successfully prepared by
incorporating gelatin (2%) into a CFP lm. Gelatin incorporation
resulted in higher strength and elasticity of the lm. In addition, the
water vapor barrier property of the CFP lm was signicantly
improved. The incorporation of 1.5% clove oil into the lm did not
noticeably affect its mechanical properties. However, small increases
in the lm water solubility due to the addition of clove oil were

Fig. 2. Change in TBARS value of smoked salmon during storage at 4
C. C, Control; B,

CFP lm without clove oil; ;, CFP lm containing clove oil.

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