Food Hydrocolloids 46 (2015) 208e215

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Development of a chicken feet protein film containing essential oils

Ji-Hyun Lee, Jihyeon Lee, 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: To prepare an edible film, after extraction of protein from chicken feet, chicken feet protein (CP) films
Received 9 August 2014 were prepared using various types of plasticizers, and their physical properties were evaluated. Incor-
Received in revised form porating more sorbitol than glycerol as a plasticizer in the CP film made the film more rigid, and a 3:2
2 December 2014
ratio (w/w) of glycerol-sorbitol was optimal. To render the film antimicrobial and antioxidative, mar-
Accepted 20 December 2014
joram (MA), coriander (CO), and clove bud oil (CL) were incorporated. The type of essential oil affected
Available online 10 January 2015
the mechanical properties of the CP film, and the film containing CL had the most desirable physical
properties: a tensile strength of 6.49 MPa, an elongation at break of 20.91%, and a Young's modulus of
Keywords:
Chicken feet
313.49 MPa. The antimicrobial and antioxidant activity was the highest for the film containing CL. When
Protein film applying the CP film during food packaging, sliced cheddar cheese was wrapped with a CP film con-
Essential oil taining CL. Consequently, using a CP film containing CL inhibited microbial growth and delayed lipid
Antimicrobial activity oxidation in the sliced cheddar cheese during storage. Therefore, a CP film containing CL can be used in
antimicrobial and antioxidative packaging for food products.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction
Biodegradable and edible films have been studied as alterna-
tives to plastic packaging materials (Rhim, Park, & Ha, 2013). Pro-
teins, carbohydrates, and lipids can be used to prepare
biodegradable films; in particular, proteins have good film-forming
abilities, making them ideal base materials for these applications. In
general, the physical properties of protein-based films can be
improved depending on the type of plasticizer or cross-linking
agent. However, some limitations, such as poor thermal resis-
tance, mechanical property, and high costs (Song et al., 2013) can be
by incorporating the appropriate plasticizers into the films and
utilizing inexpensive protein sources, such as chicken feet.
As a byproduct of poultry industry, most chicken feet produced
worldwide are disposed off except in some Asian countries where
chicken feet are cooked and consumed. With the increase of
chicken consumption, disposed chicken feet also have increased
and caused environmental concern. Therefore, there have been
studies on developing foods such as jelly and gelatin using collagen
isolated from chicken feet by a Brazilian research group (Almeida,
Calarge, & Santana, 2013; de Almeida, de Araújo, & Santana,
2012). Chicken feet consist of 85% protein, which is mainly
* Corresponding author. Tel.: þ82 42 821 6723; fax: þ82 42 825 2664.
E-mail address: kbsong@cnu.ac.kr (K.B. Song).
collagen, and 2.7% fat (Almeida & Lannes, 2013). Collagen from
chicken feet mainly consists of type I and type II and includes 30%
glycine, 11.7% proline, and 10e12.7% alanine and glutamic acid (Liu,
Lin, & Chen, 2001). Collagen is a fibrous insoluble protein with a
triple helix structure, and collagen, which is insoluble, can be
converted to gelatin, which is soluble, through partial hydrolysis
(Go mez-Guillen, Gimenez, Lo pez-Caballero, & Montero, 2011). To
prepare gelatin, collagen is normally heated in water warmer than
45
C, breaking the hydrogen and covalent bonds and resulting in a
helix-to-coil transition (Go mez-Guillen et al., 2011). It has been
reported that gelatin films have good mechanical properties
depending on the gelatin composition and conditioning condition.
Wu et al. (2013) reported that silver carp skin gelatin film (4%
protein and 1 g glycerol) had TS of 25.25 MPa and elongation of
48.09% after conditioning at 22
C and 60% relative humidity (RH).
In addition, fish skin gelatin film (3.5% protein and 0.7 g glycerol)
conditioned at 25
C and 50% RH had TS of 49.09 MPa and elon-
gation of 9.61% (Tongnuanchan, Benjakul, & Prodpran, 2012).
Therefore, the gelatin extracted from chicken feet should be a
suitable base material when preparing edible films.
Concern about microbial contamination in processed food has
been increasing. Listeria monocytogenes can grow in various raw
food products, including dairy products, at refrigerated tempera-
tures, resulting in listeriosis. Salmonella Enteritidis also causes a
foodborne disease. Listeria monocytogene and S. Enteritidis
contamination has been reported in cheese (Smith-Palmer, Stewart,

http://dx.doi.org/10.1016/j.foodhyd.2014.12.020
0268-005X/© 2014 Elsevier Ltd. All rights reserved.
 J.-H. Lee et al. / Food Hydrocolloids 46 (2015) 208e215
& Fyfe, 2001) and to prevent these pathogens, antimicrobial
packaging used for cheese.
Essential oils, such as clove and coriander oils, have phenolic
compounds with antimicrobial activity, and these substances can
be added to edible films as an antimicrobial agent (Burt, 2004).
Therefore, the objective of this study was to prepare an antimi-
crobial and antioxidant film using the protein extracted from
chicken feet and to use the film to package sliced cheddar cheese.
2. Materials and methods
2.1. Materials
The chicken feet and sliced cheddar cheese were purchased
from a local market (Daejeon, Korea). The ammonium sulfate and
plasticizers (glycerol, fructose, sorbitol, and sucrose) were pur-
chased from SigmaeAldrich Chemical Co. (St. Louis, MO, USA). The
marjoram oil (MA), coriander oil (CO), and clove bud oil (CL) were
obtained from The Certification Academy for Holistic Aroma-
therapy (Seoul, Korea). Minimum inhibitory concentration (MIC) of
MA, CO, and CL were 0.25, 0.25, and 0.25% (v/v) against Escherichia
coli and 0.5, 0.25, and 0.25% (v/v) against Staphylococcus aureus
(Hammer, Carson, & Riley, 1999).
2.2. Extraction of chicken feet protein
The chicken feet protein was extracted according to the method
reported by Almeida and Lannes (2013) with a few modifications.
The frozen chicken feet were thawed under running tap water
overnight. Ten volumes of 20% ethanol (v/w) were added at 4
C to
defat the chicken feet. After 24 h, the chicken feet were soaked in 10
volumes of 5% HCl (v/w) at 20
C for 24 h to swell gelatin of the
chicken feet; afterwards, they were rinsed with tap water, and
stirred at 70
C for 3 h after adding 2 volumes of distilled water (v/
w). The supernatant was obtained by centrifugation at 10,000 g
for 1 h, and the extracted protein was precipitated using 70%
ammonium sulfate. After centrifugation, the pellet was desalinated
through dialysis with a 3500 cut-off membrane and freeze-dried.
2.3. Film preparation
First, to determine the optimal concentration of protein and
plasticizer for CP film, the mechanical properties of the films made
at several concentrations of protein (3, 5, and 7%) and plasticizer
(30, 40, and 50% of the protein content) were determined. Among
them, 5% CP and 40% plasticizer had the best physical properties
such as tensile strength and elongation at break. Therefore, 5% CP
and 40% plasticizer were used to prepare the CP film. CP (5 g) was
mixed with 100 mL of distilled water, and the mixture was stirred
for 30 min. To the film-forming solution, 2 g plasticizers (glycerol,
sorbitol, and their blends), 1 g essential oil, and 0.25 g emulsifier
(Tween #20) were then added. After complete dissolution, the
solution was homogenized using a homogenizer at 13,400 rpm for
5 min and sonication for 8 min. The solution was degassed for 5 min
under vacuum and stored in a water bath at 70
C for 30 min. After
filtration, the filtrate (80 mL) was poured onto a Teflon-coated plate
and dried at room temperature for 18 h. The dried films were
peeled off and conditioned in an environmental chamber at 25
C
and 50% RH for 48 h.
2.4. Mechanical and optical properties
The film thickness was measured using a micrometer (Mitutoyo,
Model No. 2046-08, Tokyo, Japan). Instron (M250-2.5 CT, The Tes-
tometric Company Ltd., Lancashire, UK) was used to measure the
209
tensile strength (TS), elongation at break (E), and Young's modulus
(YM) of the CP films. The films were stored at 25
C and 50% RH for
48 h and were cut into rectangular shapes (2.54  10). The TS, E, and
YM of the films were evaluated. The initial grip distance was 5 cm,
and the cross-head speed was 50 cm/min. Five replicates were used
when testing each film.
The color of the CP film was evaluated with a colorimeter
(Minolta, CR-400, Tokyo, Japan). A Hunter Lab system was used, and
the measurements were performed in triplicate for each film. The
total color difference (DE) was obtained using the following
equation.
h 2  2  2 i0:5
DE ¼ DL* þ Da* þ Db*
where DL*, Da*, and Db* are the differences between the color
values of CP film without essential oils and those of the CP films
containing essential oils.
The opacity of the CP films was measured according to the
method of Pena-Serna and Lopes-Filho (2013). The films were cut
into 2.5  1 cm, and the absorbance at 600 nm was measured using
a spectrophotometer (UV-2450, Shimadzu Corporation, Kyoto,
Japan). The opacity was calculated based on the following equation.
Opacity ¼ Abs600 =x
where Abs600 is the absorbance at 600 nm and x is the film
thickness.
2.5. Water vapor permeability (WVP) and moisture content (MC)
To measure the WVP of the CP films, a polymethylacrylate cup
containing 18 mL of distilled water was covered with the film
(2  2 cm), stored in an environmental chamber at 25
C and 50%
RH, and the weight of the cup was measured every hour (ASTM
Method ;E96-95, 1995).
The CP film was cut into 2  2 cm pieces and dried in an oven at
110
C for 24 h to measure the MC. After drying, the dry weight of
the film was determined, and the MC was calculated using the
following equation:
MCð%Þ ¼ ðinitial sample weight  dry sample weightÞ=
initial sample weight  100
2.6. Thermo-gravimetric analysis (TGA)
TGA was performed using a Mettler Toledo DSC 1 (Mettler
Toledo, Columbus, OH, USA). The samples (5.5 ± 0.1 mg) were
heated at 10
C/min from 25 to 500
C. Nitrogen was used as a purge
gas at 50 mL/min.
2.7. Antimicrobial activity of the CP film
Disc agar diffusion and viable cell count tests were performed to
determine the antimicrobial activity of the CP films that contained
essential oils. E. coli O157:H7 (NCTC 12079) and S. aureus (KCTC
1621) were cultured in 25 mL of Tryptic Soy Broth (TSB, Bacto/Difco,
Becton Dickinson, Sparks, MD, USA), and L. monocytogenes (ATCC
19111) and S. Enteritidis (KCTC 2930) were cultured in 25 mL of
Brain Heart Infusion (BHI, Bacto/Difco, Becton Dickinson, Sparks,
MD, USA) at 37
C for 24 h.
For the disc agar diffusion test, the inoculums of each microor-
ganism (0.1 mL) were placed in Tryptic Soy Agar (TSA, Difco) and
Brain Heart Infusion Agar (BHIA, Difco). The CP films were cut into
discs 10 mm in diameter, placed onto the inoculated medium and
210 J.-H. Lee et al. / Food Hydrocolloids 46 (2015) 208e215
incubated at 37
C for 24 h. A Digimatic caliper (Model 500-181-20,
Mitutoyo Corp., Kawasaki, Japan) was used to evaluate the inhibi-
tion zone.
The viable cells were counted according to the method of Rhim,
Lee, and Hong (2011). The cultured broths were centrifuged at
20,000 g for 15 min, and 100 mL of sterile TSB and BHI broth were
added to the pellet. The inoculated broth was then diluted with
peptone water (106 CFU/mL), and 50 mL of diluted broth was added
to a conical flask. The film sample (5  5 cm) was placed in the flask
and incubated at 37
C for 12 h. The broth without a film sample
was used as a control. The changes in the populations of each
microorganism were assessed every 3 h.
2.8. Antioxidant activity of the CP film
The antioxidant activity of the CP films was evaluated using
DPPH and ABTS as radical scavengers. The film extract solution was
prepared by mixing a film sample (25 mg) with distilled water
(5 mL) and stirred for 30 min (Jouki, Mortazavi, Yazdi, & Koocheki,
2014). The DPPH radical scavenging activity was measured using
the method of Brand-Williams, Cuvelier, and Berset (1995). The film
extract solution (0.1 mL) was mixed with 3.9 mL of a DPPH solution
and stored in a dark room for 60 min. Afterwards the absorbance at
517 nm was determined and expressed as a percentage of the DPPH
radical scavenging activity. The ABTS radical scavenging activity
was determined based on the method of Re et al. (1999). Prior to the
test, an ABTS solution (7 mM) was added to 2.45 mM potassium
persulfate (2:1, v/v) and stored in the dark room for 16 h. The
mixture was diluted to an absorbance of 0.7 at 734 nm with
ethanol. The film extract solution (60 mL) was added to the ABTS
solution (2940 mL) and stored in the dark at 37
C for 10 min. After
centrifugation at 3000 rpm for 5 min, the supernatant was
collected, and the absorbance at 734 nm was measured. The result
was expressed as a percentage of the ABTS radical scavenging ac-
tivity and every test was performed in triplicate.
2.9. Application of the CP film to packaging for sliced cheddar
cheese
After the surface of sliced cheddar cheese (10 g) was sterilized
under UV light for 10 min, L. monocytogenes and S. Enteritidis (106
log CFU/g) were inoculated onto each side of the cheese surface and
dried for 30 min. Afterwards, the cheese samples were wrapped
with the CP film or the CP film containing CL (CP-CL), which made
direct contact with the samples. The control samples were only
kept in polyethylene terephthalate boxes, and all samples were
stored at 10
C. The microbial enumeration and lipid oxidation
determination were performed every three days during storage.
2.10. Microbial analysis of sliced cheddar cheese
The sliced cheddar cheese (10 g) was added to 0.1% peptone
water and was homogenized using a stomacher (MIX 2, AES Lab-
oratoire, Combourg, France) for 3 min. Afterwards, a serial dilution
was performed, and the diluted sample (0.1 mL) was plated onto
BHIA and TSA and incubated at 37
C for 24 h. The microbial counts
were expressed as log colony-forming units (CFU). The experiments
were performed in triplicate.
2.11. Lipid oxidation
The thiobarbituric acid (TBA) and peroxide values (POV) were
measured to assess the lipid oxidation of the sliced cheddar cheeses
during storage. According to the method of Vyncke (1970), the TBA
values was evaluated. The cheddar cheeses samples (2 g) were
homogenized with 10 mL of 7.5% trichloroacetic acid for 8 min
using a stomacher. After filtration through cheesecloth, the filtrate
(5 mL) was mixed with the TBA reagent (0.02 M 2-thiobarbituric
acid in distilled water, 5 mL) and boiled for 45 min. The mixture
was then cooled with water and the absorbance was determined at
539 nm. The results were expressed as mg malonaldehyde (MDA)
per kg of sample (mg MDA/kg sample).
For the POV, the AOAC Official Method 965.33 was used. A
mixture of acetic acid/chloroform (3:2, v/v, 30 mL) was added to the
cheese sample (2 g), and the mixture was stirred for 30 min. The
filtrate was added to a saturated potassium iodide solution and
stored in a dark room for 5 min. After adding 30 mL of distilled
water, the solution was titrated against 0.01 N sodium thiosulfate.
Starch (1%) was used as an indicator, and the POV was expressed as
meq peroxides/kg of sample.
2.12. Statistical analysis
To perform an analysis of variance and Duncan's multiple range
tests (p < 0.05), SAS version 8.0 (SAS Institute, Inc., Cary, NC, USA)
was used. The data are represented as the means ± the standard
deviations (SD).
3. Results and discussion
3.1. Physical properties of the CP films
As a plasticizer, glycerol and sorbitol were used for the CP films
and their mechanical properties were assessed. In addition, the
mechanical properties of the films containing different ratios of
glycerol and sorbitol were compared to optimize the type of plas-
ticizer. The CP film containing glycerol had the highest E value
(13.69%), and the film containing sorbitol had the highest TS
(3.38 MPa). However, the films containing only glycerol or sorbitol
only was too weak or rigid; consequently, a blend of glycerol and
sorbitol was added to the CP film-forming solution. Table 1 shows
the effects of altering the ratio of glycerol and sorbitol on the
physical properties of the CP films. The CP films containing 1:2 (w/
w) glycerol-sorbitol had the highest TS (22.60 MPa), while the
highest YM (227.73 MPa) was observed when using a 2:3 (w/w)
mixture of glycerol-sorbitol. When the ratio of sorbitol increased
from 1:0 to 1:1 and 1:2, the TS and YM of the film increased.
Because the TS and YM represent the rigidity and stiffness of the
film, increasing the sorbitol content strengthened the CP film.
These results agreed with the report by Song et al. (2013): the TS of
chicken feather protein films increased when the amount of sor-
bitol, which was used as a plasticizer, increased. In contrast, the E
value increased from 10.39 to 12.69% when the ratio of sorbitol
decreased from 1:1 to 2:1 (w/w) glycerol-sorbitol. Similarly,
Thomazine, Carvalho, and Sobral (2005) also reported that the TS of
protein film increased with the increase of sorbitol ratio in the
glycerol-sorbitol blends. This increase might be due to the differ-
ence in molecular weight, size, number of oxygen atoms, and hy-
drophilicity of the plasticizer (Sothornvit & Krochta, 2001). In
contrast, the plasticizing effect of glycerol is stronger than that of
sorbitol because molecular size of glycerol is smaller than that of
sorbitol (Sothornvit & Krochta, 2001). Therefore, the CP films
containing more glycerol had higher E values, while adding sorbitol
increased the TS. In addition, mixtures of glycerol and sorbitol are
better plasticizers than glycerol or sorbitol alone. Similarly, Song
et al. (2013) also reported that the TS of chicken feather protein
film increased compared to the film incorporated with glycerol or
sorbitol alone after adding a blend of glycerol and sorbitol.
The WVP and MC of the CP films depended on the concentra-
tion and type of plasticizers (Table 1). The WVP of the CP film
 J.-H. Lee et al. / Food Hydrocolloids 46 (2015) 208e215 211

Table 1
Mechanical properties, WVP, and moisture content of the CP films with different plasticizers.

G:S ratio* (w/w) Tensile strength (MPa) Elongation (%) Young's modulus (MPa) Moisture content (%) WVP (109 g m/m2 s Pa)

1:0 1.61 ± 0.03f 13.69 ± 0.01b 41.52 ± 1.70e 15.57 ± 2.59b 3.44 ± 0.10ab
1:1 14.11 ± 0.61b 10.39 ± 0.94d 64.71 ± 4.64d 14.82 ± 0.35b 3.00 ± 0.04bcd
1:2 22.60 ± 0.73a 4.18 ± 0.39e 111.66 ± 9.87c 10.17 ± 0.57c 2.69 ± 0.16d
2:1 8.82 ± 0.33c 12.69 ± 0.71c 35.94 ± 3.69f 19.31 ± 0.58a 3.38 ± 0.21abc
2:3 9.14 ± 0.62c 11.39 ± 0.01d 227.73 ± 20.72a 12.01 ± 1.32c 3.60 ± 0.14a
3:2 7.13 ± 0.36d 21.78 ± 1.66a 147.30 ± 14.41b 17.66 ± 1.41a 3.49 ± 0.46ab
0:1 3.38 ± 0.02e 1.01 ± 0.01f 5.12 ± 1.89g 7.81 ± 1.04d 2.90 ± 0.22cd

Means ± S.D.
*
Total amount of plasticizer was 2 g.
aeg
Any means in the same column followed by different letters are significantly (p < 0.05) different by Duncan's multiple range test.

containing 1:2 (w/w) glycerol-sorbitol was the lowest
(2.69  109 g m/m2 s Pa), while the CP films containing 2:1 and
3:2 (w/w) glycerol-sorbitol had the highest MC. The differences in
the WVP and MC of the films might be due to the different in-
teractions between the plasticizers and the water molecules
(Thomazine et al., 2005). In particular, the CP film containing
glycerol only had higher WVP and MC than those of the CP film
containing sorbitol only. And the WVP and MC of the CP film
tended to increase with the increase of glycerol content. Similar to
our results, Thomazine et al. (2005) also reported that the WVP
and MC of gelatin film increased with the increase of glycerol
content in the plasticizer blend due to higher hygroscopicity of
glycerol. In addition, the MC affected the TS and E of the CP film,
resulting in the decrease of TS and increase of E value because of
plasticizing effect by the increase of MC (Sothornvit & Krochta,
2001). Based on the mechanical properties, the WVP, and MC of
the CP films (Table 1), a 3:2 (w/w) ratio of glycerol-sorbitol was
chosen when preparing the CP films.
3.2. Physical and optical properties of the CP films containing
essential oils
When using a blend of glycerol-sorbitol (3:2, w/v) as a plasti-
cizer, CP films containing various essential oils were prepared, and
the physical properties of the resultant films were determined
(Table 2). The TS of the CP films containing marjoram oil (MA,
7.59 MPa) and coriander oil (CO, 10.00 MPa) increased, while that of
the CP film containing clove bud oil (CL, 6.49 MPa) was lower than
that of the control (7.13 MPa). This difference might occur due to the
structural characteristics of the phenolic compounds (MA, CO, and
CL). Adding essential oil affects the molecular interactions in the CP
films. The TS increased with the incorporation of CO due to mo-
lecular rearrangement in the protein network, resulting in the in-
crease of resistance in the CP films (Atare s, De Jesús, Talens, &
Chiralt, 2010). Ojagh, Rezaei, Razavi, and Hosseini (2010) also re-
ported that incorporation of cinnamon oil resulted in an increase of
TS and decrease of E value due to the structural change between
chitosan networks. In contrast, the TS of the CP films containing CL
decreased. CL contains larger phenolic compounds (gallic acid and
eugenol) than MA or CO (limonene or terpinen-4-ol) (Shan, Cai,
Sun, & Corke, 2005; Vagi, Sim andi, Suhajda, & Hethelyi, 2005).
Therefore, the decrease in the TS of the CP film containing CL is
mainly due to the breakdown of molecular interactions in the film,
resulting in a discontinuous film compared to the control film
(Benavides, Villalobos-Carvajal, & Reyes, 2012). Similarly, Song, Lee,
Al Mijan, and Song (2014) also reported that a chicken feather
protein film containing CL had decreased TS values because protein
molecules were replaced by CL in the film matrix. The YM values
also increased after adding essential oils, indicating that the films
were more rigid due to the interactions between the essential oils
and the CP molecules. Pereda, Amica, and Marcovich (2012) also
suggested that the YM value increased after incorporating olive oil
into a chitosan film. In contrast, the E of the CP films containing
essential oils decreased compared to the control film except the CP
film containing CL. This change might have occurred because strong
interactions formed between the protein molecules and essential
oils, decreasing the flexibility and mobility of the films (Hosseini,
Razavi, & Mousavi, 2009). The type and amount of phenolic com-
pounds present in CO, MA, and CL cause different binding and in-
teractions between protein molecules and essential oil. Therefore,
the CP films containing CO, MA, and CL had different mechanical
properties.
The MC of the CP films containing essential oils remained largely
unchanged except for that containing MA. The increased MC of the
films containing MA might be related to the increase of water
molecules between polymer chains by hydrogen bonding (Hosseini
et al., 2009). In addition, the WVP decreased after incorporating the
essential oils into the CP film-forming solution. After adding the
essential oils, the hydrophilic portion of the film decreased,
changing the water vapor transfer. There was no significant dif-
ference among the essential oils, but the CP-MA film had the lowest
WVP. In general, the small particle size of the essential oils are well
distributed within the protein matrix, lowering the WVP (Atare s
et al., 2010). Therefore, these results might be due to the small
particle size of MA (Shan et al., 2005; Vagi et al., 2005). According to
Benavides et al. (2012), the WVP of alginate film decreased after
adding oregano oil because the hydrophilicity of the films had
changed.

Table 2
Mechanical properties, WVP, and moisture content of the CP films with different essential oils.

Tensile strength (MPa) Elongation (%) Young's modulus (MPa) Moisture content (%) WVP (109 g m/m2 s Pa)
bc a c b
Control 7.13 ± 0.36 21.78 ± 1.66 147.30 ± 14.41 17.66 ± 1.41 3.49 ± 0.46a
CP-MA* 7.59 ± 0.62b 11.92 ± 1.44b 230.82 ± 28.19b 20.19 ± 0.90a 2.59 ± 0.11b
CP-CO 10.00 ± 1.06a 6.43 ± 1.37c 227.71 ± 22.33b 18.00 ± 0.85b 2.71 ± 0.22b
CP-CL 6.49 ± 0.72c 20.91 ± 1.75a 313.49 ± 6.96a 17.89 ± 0.03b 2.93 ± 0.15b

Means ± S.D.
*
The amount of essential oil was 1 g.
aec
Any means in the same column followed by different letters are significantly (p < 0.05) different by Duncan's multiple range test.
212 J.-H. Lee et al. / Food Hydrocolloids 46 (2015) 208e215

Table 3
Optical properties of the CP films with different essential oils.

L* a* b* DE Opacity

Control 95.17 ± 0.33a 0.80 ± 0.10a 4.63 ± 0.46c e 3.49 ± 0.33a

CP-MA 94.99 ± 0.56a 0.96 ± 0.11b 6.10 ± 0.21b 1.70 ± 0.16b 3.05 ± 0.17b
CP-CO 94.36 ± 0.59b 0.95 ± 0.13b 6.29 ± 0.54b 1.86 ± 0.11b 2.91 ± 0.04b
CP-CL 94.18 ± 0.36b 3.40 ± 0.10c 11.22 ± 0.30a 7.25 ± 0.54a 1.86 ± 0.11c

Means ± S.D.
aec
Any means in the same column followed by different letters are significantly
(p < 0.05) different by Duncan's multiple range test.

Table 3 shows the optical properties of the CP films containing

essential oils. The CP films containing essential oils exhibited a
slight decrease in lightness (L*) and redness (a*), while the yel-
lowness (b*) increased. Salarbashi et al. (2014) also reported
decreased L* values in soybean polysaccharide films containing
Zataria multiflora Boiss and Mentha pulegium essential oils. The
reason for the color change in the CP films containing essential oils
Fig. 1. TGA graph of CP film containing various essential oils.
might occur because the phenolic compounds of essential oils
absorb light (Jouki et al., 2014). In particular, the CP film containing
CL had the highest b* and DE values due to the yellowish color of the TGA profile of the CP films containing essential oils were lower,
CO. In addition, the opacity decreased after adding the essential demonstrating the decreased thermal stability of the films. This
oils. The CP film containing CL was the most transparent compared change might occur because the intermolecular interactions in the
to the control film. Sanchez-Gonza lez, Gonza lez-Martínez, Chiralt,
CP film matrix were weakened after adding the essential oils
and Cha fer (2010) reported that a chitosan film containing tea-
(Tongnuanchan et al., 2014). In addition, the residual masses of the
tree oil had a reduction in opacity primarily due to the effects of CP films with essential oils were slightly lower (25.48, 25.25, and
the essential oils on the protein chains. 25.56%) than the control film (26.30%), which is most likely due to
the weak film networks in the CP films formed after adding the
3.3. Thermal properties of the CP films containing essential oils essential oils (Tongnuanchan et al., 2012). Therefore, incorporating
essential oils might affect the thermal stability of the CP films.
The thermal properties of the CP films containing essential oils
were evaluated using TGA, and their degradation temperature (Td)
3.4. Antimicrobial and antioxidant activities of the CP films
and weight loss (Dw) values are shown in Table 4 and Fig. 1. Four
major weight losses occurred for the CP films containing essential containing essential oils
oils. The first weight loss (Dw1: 1.86e3.70%) appeared at
34.99e43.62
C (Td1). The weight loss at these temperature ranges The antimicrobial activity of the CP films containing essential
oils was evaluated using disc agar diffusion tests and viable cell
is primarily attributed to the evaporation of low molecular weight
count method. The inhibition zones of the CP films with or without
compounds, such as water or flavor compounds (Wu et al., 2013).
essential oils are shown in Table 5. The control film had no
After incorporating the essential oils, the water content of the CP
films decreased, increasing their degradation temperatures versus
the control film (Tongnuanchan, Benjakul, & Prodpran, 2014).
Table 5
Similar results were reported: gelatin films containing bergamot Antimicrobial activities of the CP films containing various essential oils against
and lemongrass oil had higher first degradation temperatures than pathogenic bacteria.
the control film (Ahmad, Benjakul, Prodpran, & Agustini, 2012). The
Inhibition zone (mm)
second weight loss (Dw2: 22.29e24.71%) was observed at
95.22e110.29
C (Td2) and was primarily related to the evaporation E. coli O157:H7 S. Enteritidis L. monocytogenes S. aureus

of low molecular weight protein or plasticizer. Wu et al. (2013) also Control 0.00 ± 0.00Ab 0.00 ± 0.00Ac 0.00 ± 0.00Ac 0.00 ± 0.00Ac
reported similar results in a gelatin film made from silver carp skin. CP-MA 13.33 ± 0.05Aa 12.51 ± 0.78Ab 13.53 ± 0.19Ab 13.58 ± 0.92Ab
± 1.26Aa ± 0.58Aa ± 1.22Aa ± 0.51Aa
The third weight loss (Dw3: 34.45e38.34%) was observed at CP-CO 14.77 15.04 15.86 16.32
CP-CL 13.49 ± 0.42Ba 13.55 ± 0.35Bab 14.55 ± 0.35Bb 16.61 ± 1.30Aa
261.08e266.10
C (Td3) for the CP films, which was related to the
degradation of large size molecules, such as strongly interacting Means ± S.D.
aec
Any means in the same column followed by different letters are significantly
proteins (Tongnuanchan et al., 2014). The fourth weight loss (Dw4: (p < 0.05) different by Duncan's multiple range test.
10.44e12.78%) was observed at 342.92e356.57
C (Td4). The AeB
Any means in the same row followed by different letters are significantly
degradation temperatures of the second, third, and fourth range in (p < 0.05) different by Duncan's multiple range test.

Table 4
Thermal degradation temperatures (Td,
C) and weight loss (Dw, %) of CP films containing various essential oils.

D1 D2 D3 D4 Residue (%)

Td1 Dw1 Td2 Dw2 Td3 Dw3 Td4 Dw4

Control 34.99 2.63 110.29 22.29 262.08 38.34 356.57 10.44 26.30
CP-MA 42.94 1.86 95.22 24.71 264.64 35.52 344.17 12.43 25.48
CP-CO 40.40 3.70 107.49 23.82 261.08 34.45 344.04 12.78 25.25
CP-CL 43.62 2.11 99.97 23.89 266.10 35.93 342.92 12.51 25.56
 J.-H. Lee et al. / Food Hydrocolloids 46 (2015) 208e215
antimicrobial activity against pathogens, such as E. coli O157:H7, S.
Enteritidis, L. monocytogenes, and S. aureus. The phenolic com-
pounds in CO (linalool), CL (eugenol), and MA (terpinen-4-ol)
exhibited antimicrobial activity against pathogens (Shan et al.,
2005; Vagi et al., 2005). The leakage of ions into the cell mem-
brane might occur due to the hydrophobic essential oils, disturbing
the proton motive force and electron flow (Burt, 2004). The CP films
containing essential oils were more effective against
L. monocytogenes and S. aureus (Gram-positive) than E. coli O157:H7
and S. Enteritidis (Gram-negative). Burt (2004) reported that the
outer membrane of Gram-negative bacteria inhibited the diffusion
of essential oils into the cell, decreasing the bacterial growth in-
hibition. The CP film containing CO displayed the best inhibition
against all of the pathogenic bacteria. The different compositions of
the essential oils might affect the antimicrobial activity of the CP
films (Hosseini et al., 2009).
The antimicrobial activity of the CP films containing essential
oils was also evaluated in liquid media (Fig. 2). S. Enteritidis and
L. monocytogenes were incubated with CP films containing various
essential oils. The initial populations of S. Enteritidis and
L. monocytogenes were 6.24 and 6.84 log CFU/mL, respectively. The
control film exhibited no antimicrobial activity against either
pathogen. However, the CP films containing MA, CO, and CL
exhibited antimicrobial activity against both Gram-negative and
-positive bacteria. In addition, the antimicrobial activity changed
slightly depending on the type of essential oil. For S. Enteritidis, the
Fig. 2. Antimicrobial activity of the CP film containing various essential oils against (a)
S. Enteritidis and (b) L. monocytogenes.
213
Fig. 3. Changes in POV of sliced cheddar cheese packaged with CP film containing CL
during storage.
populations decreased to 4.72, 5.14, and 4.50 log CFU/mL after 12 h
for the CP films containing MA, CO, and CL, respectively. In addition,
the populations of S. Enteritidis slightly increased for 3 and 9 h for
the CP films with MA and CO, whereas the populations of
L. monocytogenes continuously decreased. The CP films containing
CO or CL were also more effective against the growth of
L. monocytogenes than CP-MA film. The type and concentration of
the phenolic compounds in the essential oils incorporated into the
CP film affect the antimicrobial ability. Moreover, the antimicrobial
activities of the essential oils against Gram-negative and Gram-
positive bacteria were slightly different. The cell wall lipopolysac-
charides and outer membranes of the Gram-negative bacteria can
protect the diffusion of phenolic compounds from the essential oils
(Ahmad et al., 2012). Our results indicate that the antimicrobial
activities against both Gram-negative and -positive bacteria were
the highest in the CP films containing CL (Fig. 3).
The antioxidant activity of the CP films containing essential oils
was assessed (data not shown). The control film without essential
oils had no antioxidant activity, while the CP film containing CL had
the highest DPPH (15.22%) and ABTS radical scavenging activities
(93%). In addition, the CP film containing MA, CO, and CL had
stronger ABTS radical scavenging activity than DPPH radical scav-
enging activity. Tongnuanchan et al. (2012) reported that the ABTS
radical scavenging activity was higher than that for DPPH or FRAP
in a film from fish skin gelatin containing lemon, lime, bergamot,
and kaffir lime oils. The antioxidant activity of the essential oils is
primarily affected by the types of phenolic compounds and their
concentration. Therefore, the CP film with CL had the highest
antimicrobial and antioxidant activity because its phenolic com-
pound content is higher than that of CO and MA (Li, Miao, Wu,
Chen, & Zhang, 2014); consequently, CL was used for packaging of
sliced cheddar cheese.
3.5. Antimicrobial and antioxidant activities of the CP films
containing CL on sliced cheese
To assess the antimicrobial activity of the CP film containing CL,
L. monocytogenes and S. Enteritidis were inoculated on the sliced
cheddar cheese. The initial populations of L. monocytogenes and S.
Enteritidis were 5.50 and 5.30 log CFU/mL, respectively (Table 6).
The population of L. monocytogenes in the control and sliced cheese
wrapped in the CP film increased continuously during storage over
214 J.-H. Lee et al. / Food Hydrocolloids 46 (2015) 208e215

Table 6
Changes in the populations of pathogenic bacteria in sliced cheddar cheese during storage.

Storage time (d)

0 3 6 9 12 15
Da CDa CDa BCa Ba
L. monocytogenes Control 5.50 ± 0.31 5.76 ± 0.14 5.87 ± 0.23 6.12 ± 0.13 6.46 ± 0.15 7.04 ± 0.14Aa
CP 5.50 ± 0.31Ca 5.65 ± 0.16Ca 5.74 ± 0.30Cab 6.04 ± 0.12Ba 6.26 ± 0.12Bab 6.96 ± 0.24
CP-CL 5.50 ± 0.31BCa 5.36 ± 0.10CDb 5.09 ± 0.16Db 5.77 ± 0.27ABa 6.00 ± 0.04Ab 5.86 ± 0.34ABb
S. Enteritidis Control 5.30 ± 0.05Ca 5.20 ± 0.17Ca 5.22 ± 0.06Ca 6.39 ± 0.36Ba 6.89 ± 0.01Aa 6.98 ± 0.03Aa
CP 5.30 ± 0.05Da 5.16 ± 0.28Da 5.00 ± 0.02Db 6.00 ± 0.01Cab 6.69 ± 0.01Ba 7.04 ± 0.06Aa
CP-CL 5.30 ± 0.05Aa 5.00 ± 0.01Aa 4.98 ± 0.03Ab 5.35 ± 0.49Ab 5.20 ± 0.17Ab 5.39 ± 0.12Ab

Means ± S.D.
aeb
Any means in the same column followed by different letters are significantly (p < 0.05) different by Duncan's multiple range test.
AeD
Any means in the same row followed by different letters are significantly (p < 0.05) different by Duncan's multiple range test.

15 days, while the populations in the samples wrapped in the CP-CL
film decreased up to 6 days before increasing. After 15 days of
storage, the population of L. monocytogenes in the control sample
was 7.04 log CFU/mL, while that in the sample packaged with CP-CL
was 5.86 log CFU/mL, revealing a 1.18 log CFU/mL reduction. Similar
to L. monocytogenes, the population of S. Enteritidis in the cheese
wrapped in the CP film and the control sample changed slightly
over the first 6 days of storage before increasing. In contrast, the CP-
CL film showed an increase of only 0.09 log CFU/mL over 15 days of
storage. On day 15, the population of S. Enteritidis in the cheese
wrapped in the CP-CL film had a 1.59 log CFU/mL reduction
compared to the control. The antimicrobial activity is primarily
attributed to the phenolic compounds, such as eugenol and
carvacrol, in the clove bud oil (Hosseini et al., 2009; Shan et al.,
2005).
POV and TBARS values were determined to evaluate the anti-
oxidant activity of the CP films containing CL on the sliced cheese
(Figs. 3 and 4). The initial POV was 0.3 meq peroxide/kg sample, and
the POV of the control sample increased over the 15 days of storage.
In contrast, the POV of the cheese wrapped with the CP films with
and without CL decreased slightly over the first 6 days before
increasing. The POV of the CP films containing CL was the lowest
among the samples. On day 15, the POV of the control was 2.3 meq
peroxide/kg, while that of the cheese wrapped in the CP film con-
taining CL was 0.91 meq peroxide/kg. In addition, the TBARS of the
control increased continuously from 0.22 to 2.15 mg malonalde-
hyde/kg sample during storage. In contrast, the cheese wrapped in
the CP or CP-CL film had a significantly lower TBARS value than the
control. The difference between the control and the CP film occurs
because the film creates an oxygen barrier, blocking the diffusion of
oxygen onto the surface of the cheese (Jeon, Kamil, & Shahidi,
2002). Similar to our results, Song et al. (2014) suggested that
smoked salmon packaged with a chicken feather protein film
containing clove bud oil decreased the POV and TBARS over 12 days
of storage. Therefore, the phenolic compounds in CL are might
impart antimicrobial and antioxidant activity to the CP film.
4. Conclusions
The proteins extracted from chicken feet can be used as a pre-
cursor to edible films. The mechanical properties and WVP of the
film were changed depending on the ratio of glycerol and sorbitol
used as a plasticizer. A 3:2 (w/w) ratio of glycerol-sorbitol was the
optimal plasticizer, and essential oils such as MA, CO, and CL can be
incorporated to impart antimicrobial and antioxidant properties.
The physical, optical, and heat resistance properties varied
depending on the type of essential oils. Among the essential oils
used in this study, the CP film containing CL had the highest anti-
microbial and antioxidant activity. Therefore, the CP film contain-
ing CL can be used when packaging sliced cheddar cheese;
consequently, the populations of Gram-positive bacteria and
-negative bacteria, POV, and TBARS in the cheese decreased
significantly during storage compared to the control. The CP films
containing CL might be an active packaging material that ensures
microbial safety and quality in food products.
Acknowledgments

This research was supported by Basic Science Research Program

through the National Research Foundation of Korea funded by the
Ministry of Education (2013-1074).

References

Ahmad, M., Benjakul, S., Prodpran, T., & Agustini, T. W. (2012). Physico-mechanical
and antimicrobial properties of gelatin film from the skin of unicorn leather
jacket incorporated with essential oils. Food Hydrocolloids, 28, 189e199.
Almeida, P. F., Calarge, F. A., & Santana, J. C. C. (2013). Production of a product similar
to gelatin from chicken feet collagen. Engenharia Agrícola, 33, 1289e1300.
de Almeida, P. F., de Araújo, M. G. O., & Santana, J. C. C. (2012). Collagen extraction
from chicken feet for jelly production. Acta Scientiarum Technology, 34, 345e351.
Almeida, P. F., & Lannes, S. C. D. S. (2013). Extraction and physicochemical charac-
terization of gelatin from chicken by-product. Journal of Food Process Engi-
neering, 36, 824e833.
ASTM. (1995). Standard test methods for water vapor transmission of material
(E96e95). In Annual book of ASTM standards. Philadelphia, PA: American Society
for Testing and Materials.
s, L., De Jesús, C., Talens, P., & Chiralt, A. (2010). Characterization of SPI-based
Atare
edible films incorporated with cinnamon or ginger essential oils. Journal of Food
Engineering, 99, 384e391.
Benavides, S., Villalobos-Carvajal, R., & Reyes, J. E. (2012). Physical, mechanical and
Fig. 4. Changes in TBARS of sliced cheddar cheese packaged with CP film containing CL antibacterial properties of alginate film: effect of the crosslinking degree and
during storage. oregano essential oil concentration. Journal of Food Engineering, 110, 232e239.
 J.-H. Lee et al. / Food Hydrocolloids 46 (2015) 208e215
Brand-Williams, W., Cuvelier, M. E., & Berset, C. L. W. T. (1995). Use of a free radical
method to evaluate antioxidant activity. LWT-Food Science and Technology, 28,
25e30.
Burt, S. (2004). Essential oils: their antibacterial properties and potential applica-
tions in foodsda review. International Journal of Food Microbiology, 94,
223e253.
Go mez-Guille n, M. C., Gime nez, B., Lo
pez-Caballero, M. A., & Montero, M. P. (2011).
Functional and bioactive properties of collagen and gelatin from alternative
sources: a review. Food Hydrocolloids, 25, 1813e1827.
Hammer, K. A., Carson, C. F., & Riley, T. V. (1999). Antimicrobial activity of essential
oils and other plant extracts. Journal of Applied Microbiology, 86, 985e990.
Hosseini, M. H., Razavi, S. H., & Mousavi, M. A. (2009). Antimicrobial, physical and
mechanical properties of chitosan-based films incorporated with thyme, clove
and cinnamon essential oils. Journal of Food Processing and Preservation, 33,
727e743.
Jeon, Y. J., Kamil, J. Y., & Shahidi, F. (2002). Chitosan as an edible invisible film for
quality preservation of herring and Atlantic cod. Journal of Agricultural and Food
Chemistry, 50, 5167e5178.
Jouki, M., Mortazavi, S. A., Yazdi, F. T., & Koocheki, A. (2014). Characterization of
antioxidanteantibacterial quince seed mucilage films containing thyme
essential oil. Carbohydrate Polymers, 99, 537e546.
Li, J. H., Miao, J., Wu, J. L., Chen, S. F., & Zhang, Q. Q. (2014). Preparation and char-
acterization of active gelatin-based films incorporated with natural antioxi-
dants. Food Hydrocolloids, 37, 166e173.
Liu, D. C., Lin, Y. K., & Chen, M. T. (2001). Optimum condition of extracting collagen
from chicken feet and its characteristics. Asian Australasian Journal of Animal
Sciences, 14, 1638e1644.
Ojagh, S. M., Rezaei, M., Razavi, S. H., & Hosseini, S. M. H. (2010). Development and
evaluation of a novel biodegradable film made from chitosan and cinnamon
essential oil with low affinity toward water. Food Chemistry, 122, 161e166.
Pena-Serna, C., & Lopes-Filho, J. F. (2013). Influence of ethanol and glycerol con-
centration over functional and structural properties of zeineoleic acid films.
Materials Chemistry and Physics, 142, 580e585.
Pereda, M., Amica, G., & Marcovich, N. E. (2012). Development and characterization
of edible chitosan/olive oil emulsion films. Carbohydrate Polymers, 87,
1318e1325.
Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999).
Antioxidant activity applying an improved ABTS radical cation decolorization
assay. Free Radical Biology and Medicine, 26, 1231e1237.
Rhim, J. W., Lee, S. B., & Hong, S. I. (2011). Preparation and characterization of agar/
clay nanocomposite films: the effect of clay type. Journal of Food Science, 76,
N40eN48.
215
Rhim, J. W., Park, H. M., & Ha, C. S. (2013). Bio-nanocomposites for food packaging
applications. Progress in Polymer Science, 38, 1629e1652.
Salarbashi, D., Tajik, S., Shojaee-Aliabadi, S., Ghasemlou, M., Moayyed, H.,
Khaksar, R., et al. (2014). Development of new active packaging film made from
a soluble soybean polysaccharide incorporated Zataria multiflora Boiss and
Mentha pulegium essential oils. Food Chemistry, 146, 614e622.
S
anchez-Gonza lez, L., Gonza
lez-Martínez, C., Chiralt, A., & Cha fer, M. (2010). Phys-
ical and antimicrobial properties of chitosanetea tree essential oil composite
films. Journal of Food Engineering, 98, 443e452.
Shan, B., Cai, Y. Z., Sun, M., & Corke, H. (2005). Antioxidant capacity of 26 spice
extracts and characterization of their phenolic constituents. Journal of Agricul-
tural and Food Chemistry, 53, 7749e7759.
Smith-Palmer, A., Stewart, J., & Fyfe, L. (2001). The potential application of plant
essential oils as natural food preservatives in soft cheese. Food Microbiology, 18,
463e470.
Song, N. B., Jo, W. S., Song, H. Y., Chung, K. S., Won, M., & Song, K. B. (2013). Effects of
plasticizers and nano-clay content on the physical properties of chicken feather
protein composite films. Food Hydrocolloids, 31, 340e345.
Song, N. B., Lee, J. H., Al Mijan, M., & Song, K. B. (2014). Development of a chicken
feather protein film containing clove oil and its application in smoked salmon
packaging. LWT-Food Science and Technology, 57, 453e460.
Sothornvit, R., & Krochta, J. M. (2001). Plasticizer effect on mechanical properties of
b-lactoglobulin films. Journal of Food Engineering, 50, 149e155.
Thomazine, M., Carvalho, R. A., & Sobral, P. J. (2005). Physical properties of gelatin
films plasticized by blends of glycerol and sorbitol. Journal of Food Science, 70,
172e176.
Tongnuanchan, P., Benjakul, S., & Prodpran, T. (2012). Properties and antioxidant
activity of fish skin gelatin film incorporated with citrus essential oils. Food
Chemistry, 134, 1571e1579.
Tongnuanchan, P., Benjakul, S., & Prodpran, T. (2014). Structural, morphological and
thermal behaviour characterisations of fish gelatin film incorporated with basil and
citronella essential oils as affected by surfactants. Food Hydrocolloids, 41, 33e43.
Vagi, E., Simandi, B., Suhajda, A., & Hethelyi, E. (2005). Essential oil composition and
antimicrobial activity of Origanum majorana L. extracts obtained with ethyl
alcohol and supercritical carbon dioxide. Food Research International, 38, 51e57.
Vyncke, W. (1970). Direct determination of the thiobarbituric acid value in tri-
chloroacetic acid extract of the fish as a measure of oxidative rancidity. Euro-
pean Journal of Lipid Science and Technology, 72, 1084e1087.
Wu, J., Chen, S., Ge, S., Miao, J., Li, J., & Zhang, Q. (2013). Preparation, properties and
antioxidant activity of an active film from silver carp (Hypophthalmichthys
molitrix) skin gelatin incorporated with green tea extract. Food Hydrocolloids,
32, 42e51.