International Journal of Biological Macromolecules 51 (2012) 774–782

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International Journal of Biological Macromolecules

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Effect of phenolic compounds on protein cross-linking and properties

of film from fish myofibrillar protein
Thummanoon Prodpran a,∗ , Soottawat Benjakul b , Suttirug Phatcharat b
a
Department of Material Product Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand
b
Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand

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

Article history: The effects of several phenolic ocmpounds (caffeic acid, catechin, ferullic acid and tannic acid) at various
Received 18 April 2012 concentrations (1, 3 and 5% based on protein) on cross-linking and properties of film from myofibril-
Received in revised form 21 June 2012 lar proteins of bigeye snapper (Priacanthus tayenus) were investigated. Among all phenolic compounds
Accepted 9 July 2012
used, tannic acid exhibited the highest cross-linking ability on myofibrillar protein as evidenced by higher
Available online 21 July 2012
decrease in free amino groups with coincidentally lower band intensity of myosin heavy chain (MHC).
In addition, the extent of protein cross-linking increased with increasing concentration of phenolic com-
Keywords:
pounds. Addition of phenolic compounds could enhance mechanical properties of the resulting films.
Film
Myofibrillar proteins
As phenolic compounds content increased, Young’s modulus (E) and tensile strength (TS) of the films
Phenolic compounds increased, while their elongation at break (EAB) decreased (P < 0.05), suggesting stronger and stiffer film
Cross-linking structure. At the same concentration used, tannic acid rendered the film with higher mechanical prop-
Physico-mechanical properties erties, compared to others. Phenolic compounds decreased film transparency and affected color of the
films differently, depending on types and concentrations used. Films from myofibrillar proteins with
and without polyphenol generally had the excellent barrier properties to UV light at the wavelength of
200–800 nm. Therefore, it could potentially be used as inner packaging material for high-fat foods to
prevent the lipid oxidation and thus prolonging the shelf-life of foods during storage.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction range of functional properties, especially a high intermolecular

The non-biodegradability of most synthetic polymer-based
packaging as well as the increasing environmental concern by
consumers and government bodies have paved the way for alter-
native approaches [1]. This awareness has led to a focus on
eco-friendly packaging materials derived from naturally occurring
polymer in order to reduce environmental pollution and ecological
related problems caused by non-biodegradable plastic packaging
[2]. Currently, there has been an interest in edible film made
from renewable and natural polymers such as proteins, polysac-
charides and lipids. Among them, protein-based edible films are
the most attractive due to impressive gas barrier properties, com-
pared with those prepared from lipids and polysaccharides [3]. The
properties of protein films are determined by their microstruc-
ture, which significantly varies depending on the protein structure
and intermolecular interaction between polypeptide chains [4]. The
functional properties of protein-based edible films are better than
those of polysaccharide and fat-based films due to the unique struc-
ture of proteins (20 different monomers), which confer a wider
∗ Corresponding author. Tel.: +66 7428 6357; fax: +66 7455 8866.
E-mail address: thummanoon.p@psu.ac.th (T. Prodpran).
binding potential [5]. It has been known that protein based-films
have good oxygen, carbon dioxide and lipid barrier properties [6,7].
Protein-based film potentially used for coating or packaging could
improve shelf-life and maintain the quality of foods during stor-
age, by serving as selective barrier to moisture transfer, oxygen
uptake, light transmission, losses of volatile aroma compounds [8].
Food packaging system using plastic packaging materials typically
encounters several problems involving mass transfer phenomena;
atmospheric oxygen penetration into foods causes oxidation of
food ingredient; inks, solvents and monomeric additives in plastic
packaging materials can migrate into foods. Therefore, biopolymer-
based materials including protein films and coatings may wrap
these food products or be located between heterogeneous parts of
food products to prevent these migration phenomena and preserve
food quality [9]. Protein-based films have been applied for coating
nuts or used for bakery products [10].
Among protein from different sources, myofibrillar and sar-
coplasmic proteins from fish muscle have been used as film forming
material [11–15]. Thailand is one of the largest surimi producers
in Southeast Asia. Bigeye snapper (Priacanthus spp.) is one of fish
species commonly used for surimi production owing to its excellent
gel-forming ability [11]. Apart from gelation, the appropriate devel-
opment of myofibrillar protein film from bigeye snapper muscle

0141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijbiomac.2012.07.010
 T. Prodpran et al. / International Journal of Biological Macromolecules 51 (2012) 774–782 775

Table 1
Molecular structures and some physical and chemical properties of phenolic compounds used in this study.

Phenolic compounds Molecular structure Physical state Solubility in water Melting temp. (◦ C) Acidity (pKa )

Catechin Yellowish crystals 1.76–2.75 g/L 177 8.45–8.91

Caffeic acid Yellowish crystal Soluble in hot water 223–225 8.69–8.69

(soluble in alcohol)

Ferulic acid Light yellowish solid Soluble in cold water 168–172 4.52–9.36
powder 0.9 g/L

Tannic acid Yellowish brown powder 2850 g/L 200–210 ca. 10

should be an alternative promising means to obtain the nutritional
and biodegradable film. Chinabhark [15] prepared films from big-
eye snapper, threadfin bream and goat fish surimi plasticized with
50% glycerol based on protein, and found that films from bigeye
snapper exhibited the highest tensile strength and elongation at
break compared to those of threadfin bream and goat fish. Chin-
abhark et al. [11] reported that bigeye snapper myofibrillar protein
films prepared from film-forming solution (FFS) containing protein
of 2% (w/v) at acidic pH (pH = 3) had better mechanical properties
than those prepared from 1% protein FFS at alkaline pH (pH = 11).
However, the poor mechanical properties of protein-based films
especially when exposing to wet and humid conditions as well
as the higher cost of protein compared to conventional oil-based
polymers limit its application in food packaging [13–16]. Hence,
the structure modifications of protein are required to enhance its
mechanical strength and water-resistant properties. In order to
improve the properties of protein-based film, different approaches
have been used including chemical, physical and enzymatic mod-
ifications [17]. Chemical cross-linking of natural proteins has a
long history in the development of protein-based materials for
food processing, packaging, coating formulations and pharmaceu-
tical/medical applications [18].
The study of phenol–protein interactions has been carried out
for more than half a century. Phenolic compounds derived from
various sources have been reported to be interactive or reactive
with proteins, and resulted in improved gel or film properties for
gelatin-based materials [19]. Polyphenols are known to react under
oxidizing conditions with side chain amino groups of peptides,
leading to the formation of protein cross-links [20]. Ferulic acid
can cross-link with protein and polysaccharides by producing a
resonance-stabilized free radical intermediate [21]. Oxidized fer-
ulic acid can react with amino and thiol groups in protein [22].
Additionally, free radical formed from ferulic acid can react with
tyrosine and with itself to form diferulic acid, which acts as a
bridge between protein molecules [22]. Tannic acid also showed
the ability to bind proteins [23]. Covalent bonds between phenolic
compounds and proteins yielded the cross-links, which are more
rigid and thermally stable than other interactions. Nevertheless,
information regarding the effect of phenolic compounds on cross-
linking of fish myofibrillar protein (FMP) and on its film properties
is very scarce. Thus, the study aimed to investigate the effect of
different phenolic compounds, including ferulic acid, tannic acid,
catechin and caffeic acid on the properties of films from bigeye
snapper (Priacanthus tayenus) muscle proteins.
2. Materials and methods
2.1. Chemicals
High molecular weight protein markers, 2,4,6-
trinitrobenzenesulfonic acid (TNBS), 5,5 -dithiobis (2-nitrobenzoic
acid) (DTNB), ferulic acid, tannic acid and ␤-mercaptoethanol
(␤-ME) were obtained from Sigma (St. Louis, MO, USA). Caffeic acid
and catechin were purchased from Fluka (Buchs, Switzerland).
Glycerol and tris (hydroxymethyl) aminomethane were obtained
from Merck (Darmstadt, Germany). Sodium dodecyl sulfate (SDS),
Coomassie Blue R-250 and N,N,N ,N -tetramethylethylenediamine
(TEMED) were procured from Bio-Rad Laboratories (Hercules, CA,
USA). All chemicals were of analytical grade. Table 1 shows molec-
ular structures as well as some physical and chemical properties
of phenolic compounds used in this study.
2.2. Fish sample
Bigeye snapper (P. tayenus) was used as source of myofibril-
lar protein for film preparation. Bigeye snapper is white muscle
fish species which is in huge quantity on the market in Southern
Thailand. This fish also has lower price compared to other white
muscle fish species and it is mainly used in surimi industry. More-
over, this fish is particularly rich in protein, typically containing
about 93–96% protein (dry-basis weight). Like other white mus-
cle fish species, bigeye sanapper muscle possesses a large amount
776 T. Prodpran et al. / International Journal of Biological Macromolecules 51 (2012) 774–782
of myofibrillar protein fraction of high molecular weight and less
low-molecular weight sacroplasmic protein fraction, yielding
higher strength film [11,15].
Fresh bigeye snapper (400–500 g/fish) were purchased from a
local market in Hat Yai, Songkhla province, Thailand. Fish were
kept in ice with a fish/ice ratio of 1:2 (w/w) and transported to
the Department of Material Product Technology, Prince of Songkla
University within 30 min. Upon the arrival, fish were immediately
washed, fileted and minced to uniformity, using a mincer with a
hole diameter of 0.5 cm.
2.3. Preparation of myofibrillar protein
Myofibrillar protein from bigeye snapper muscle was prepared
according to the method of Benjakul et al. [24] with a slight modifi-
cation. Mince was homogenized with five volumes of 50 mM NaCl
using an IKA Labortechnik homogenizer (Selangor, Malaysia) at
a speed of 11,000 rpm for 2 min, followed by centrifuging using
a Beckman Model Avanti J-E centrifuge (Beckman Coulter, Inc.,
Fullerton, CA) at 9600 × g at 4 ◦ C for 10 min. The washing process
was repeated twice. Washed mince containing 95.94% protein (dry
basis weight) as determined by Kjeldahl method was stored on ice
until used for analysis or for film preparation.
2.4. Preparation of film-forming solution
Film-forming solution (FFS) was prepared as described by Shiku
et al. [25] with a slight modification. Washed mince was mixed
with the distilled water to obtain the final protein concentration of
1.5% (w/v). This protein concentration of FFS was used in order to
avoid the entrapped air bubble which was encountered at higher
protein concentration of FFS. The mixture was homogenized at
13,000 × g for 1 min. Subsequently, glycerol at 50% (w/w) based on
protein content was added as a plasticizer and the mixture was
stirred gently for 30 min at room temperature. The pH of filmo-
genic solution was adjusted to 11 with 1 M NaOH. Then, different
phenolic compounds (caffeic acid, ferulic acid, tannic acid and cat-
echin) at different concentrations (1, 3 and 5% of protein content)
were added into FFS and stirred at room temperature for 1 h with
continuous oxygen purging to oxidize the phenolic compound into
quinone (oxidized form), a reactive specie to interact with amino
group of protein at alkaline pH [19,20]. FFS obtained was subjected
to analyses and used for film casting.
2.5. Analyses of FFS
2.5.1. Determination of free amino group content
Free amino group content was determined following the
method of Benjakul and Morrissey [26]. Properly diluted samples
(125 ␮l) were mixed thoroughly with 2.0 ml of 0.2125 M phosphate
buffer, pH 8.2, followed by the addition of 1.0 ml of 0.01% TNBS solu-
tion. The mixtures were then placed in a temperature controlled
water bath at 50 ◦ C for 30 min in the dark. The reaction was termi-
nated by adding 2.0 ml of 0.1 M sodium sulfite. The mixtures were
cooled at room temperature for 15 min. The absorbance was mea-
sured at 420 nm using a double beam spectrophotometer (Model
UV-1800, Shimadzu, Kyoto, Japan). Free amino group content was
determined from the standard curve using l-leucine and expressed
as mM l-leucine.
2.5.2. Determination of sulfhydryl group content
Sulfhydryl group content was determined, using 5,5 -dithiobis
(2-nitrobenzoic acid) (DTNB) according to the method of Ellman
[27] as modified by Benjakul et al. [28] with a slight modifica-
tion. To 1.0 ml of sample solution, 9 ml of 0.2 M Tris–HCl buffer, pH
6.8, containing 8 M urea, 2% SDS and 10 mM ethylenediaminete-
traacetic acid (EDTA) were added. To 4 ml of the mixture, 0.4 ml
of 0.1% DTNB was added and incubated at 40 ◦ C for 25 min. The
absorbance at 412 nm was measured using a double beam spec-
trophotometer (model UV-1800, Shimadzu, Kyoto, Japan). A blank
was conducted by replacing the sample with 0.6 M KCl. Sulfhydryl
group content was calculated using the extinction coefficient of
13,600 L/(mol cm) and was expressed as mol/g protein.
2.6. Film casting, drying and conditioning
FFS (4 g) was cast onto a rimmed silicone resin plate
(5 cm × 5 cm) and air blown for 12 h at room temperature prior to
further drying in an environmental chamber (WTB Binder, Tuttlin-
gen, Germany) at 25 ◦ C and 50% relative humidity (RH) for 24 h. The
resulting films were manually peeled off and subjected to analyses.
Prior to testing, film samples were conditioned for 48 h at 50 ± 5%
relative humidity (RH) at 25 ± 0.5 ◦ C. All film samples obtained had
similar average thickness of 0.037 ± 0.006 mm (P > 0.05).
2.7. Analyses of films
2.7.1. Mechanical properties
Young’s modulus (E), tensile strength (TS) and elongation at
break (EAB) of film samples were determined as described by
Iwata et al. [29] using the Universal Testing Machine (Lloyd Instru-
ment, Hampshire, UK). The test was performed in the controlled
room at 25 ◦ C and ∼50 ± 5% relative humidity. Ten film samples
(2 cm × 5 cm) with the initial grip length of 3 cm were used for
testing. The film samples were clamped and deformed under ten-
sile loading using a 100 N load cell with the cross-head speed of
30 mm/min until the samples were broken. The maximum load
and the final extension at break were used for calculation of TS
and EAB, respectively. Young’s modulus was determined from the
initial slope of stress–strain diagram.
2.7.2. Color measurement
The color of films was measured by a Hunter lab color meter
(Color Flex, Hunter Lab Inc., Reston, VA, USA). L*, a* and b*
parameters indicating lightness/brightness, redness/greenness and
yellowness/blueness, respectively, were recorded. The colorimeter
was warmed up for 30 min and calibrated with a white standard.
2.7.3. Light transmission
Light transmission of films against ultraviolet (UV) and visible
light was measured at wavelengths between 200 and 600 nm, using
a UV–Visible spectrophotometer (model UV-160, Shimadzu, Kyoto,
Japan) according to the method of Jongjareonrak et al. [8].
2.7.4. SDS–polyacrylamide gel electrophoresis (SDS–PAGE)
Protein patterns of FFS and the films were analyzed by
SDS–PAGE according to the method of Laemmli [30] using a 10%
running gel and 4% stacking gel. To solubilize the films, the sam-
ples were mixed with a solution containing 5% SDS, homogenized
at 13,000 rpm for 1 min using an IKA homogenizer and stirred
continuously for 24 h at room temperature. The supernatants
obtained after centrifugation at 3000 × g for 5 min were subjected
to SDS–PAGE analysis under reducing conditions as described by
Yarnpakdee et al. [31].
2.8. Statistical analyses
Experiments were performed in triplicate and a completely
randomized design (CRD) was used. Data were presented as
means ± standard deviation and a probability value of <0.05 was
 T. Prodpran et al. / International Journal of Biological Macromolecules 51 (2012) 774–782
Table 2
Free-amino group and sulfhydryl group contents of film-forming solution (FFS) from fish myofibrillar protein added with various phenolic compounds at different
concentrations.
Treatment Free-amino group (mM)
Control 4.78 ± 0.07g
Caffeic acid (1%) 1.42 ± 0.02d
Caffeic acid (3%) 1.14 ± 0.02b
Caffeic acid (5%) 0.98 ± 0.04a
Ferulic acid (1%) 4.73 ± 0.04g
Ferulic acid (3%) 4.58 ± 0.10f
Ferulic acid (5%) 4.30 ± 0.11e
Tannic acid (1%) 1.25 ± 0.04bc
Tannic acid (3%) 0.99 ± 0.03a
Tannic acid (5%) 0.93 ± 0.02a
Catechin (1%) 1.47 ± 0.11d
Catechin (3%) 1.39 ± 0.02d
Catechin (5%) 1.27 ± 0.11c
Values are given as mean ± SD (n = 3). Different letters in the same column indicate significant differences (P < 0.05).
considered significant. Analysis of variance (ANOVA) was per-
formed and the mean comparisons were done by Duncan’s multiple
range tests. Statistical analysis was performed using the Statistical
Package for Social Sciences (SPSS for Windows, SPSS Inc., Chicago,
IL, USA).
3. Results and discussion
3.1. Free amino group content of FFS
Changes in free amino group content of fish myofibrillar protein
in FFS added with various phenolic compounds at different con-
centrations are depicted in Table 2. Continuous decreases in amino
group content of all samples were noticeable when the levels of
phenolic compounds increased from 1 to 5% (P < 0.05). Neverthe-
less, no changes in amino group content were observed between
the control and samples added with 1% ferulic acid (P > 0.05).
This result suggested that ␣- or ␧-NH2 groups of amino acids
from muscle proteins covalently attached to cross-linkers to form
phenol–proteins complex to a greater extent, particularly at higher
concentrations. FFS added with all oxidized phenolic compounds
had lower free amino group content than that of control (P < 0.05).
Quinone, an oxidized form of phenolic compounds, is reactive in
attacking amino group [19,20,32]. Among phenolic compounds
tested, tannic acid exhibited higher protein cross-linking ability
than did caffeic acid, catechin and ferulic acid, respectively, as evi-
denced by the lowest content of free amino group in FFS added with
tannic acid (P < 0.05). This was associated with the higher number
of hydroxyl groups present in the tannic acid, compared with oth-
ers. The hydroxyl groups were presumably converted to quinone,
which functioned as cross-linkers [20,33]. Additionally, remain-
ing hydroxyl groups might interact with amino groups via weak
bonds/interactions [33]. The interactions between hydroxyl groups
of phenolic compounds and the nitrogen or oxygen of some amino
acids were reported by Prigent [34]. Hydrophobic interactions may
occur between phenolic compounds and amino acids [34].
3.2. Total sulfhydryl group content of FFS
Total sulphydryl group content of fish myofibrillar proteins in
FFS added with different oxidized phenolic compounds at vari-
ous concentrations is shown in Table 2. FFS added with different
oxidized phenolic compounds had lower sulphydryl group con-
tent than that of the control (P < 0.05). Phenolic compound might
induce the formation of preferable conformation, in which inter-
action between SH group of protein and OH group of phenolic
777
Sulfhydryl group (×10−5 mole/g protein)
7.06 ± 0.21e
0
0
0
6.67 ± 0.08d
6.23 ± 0.05c
5.73 ± 0.14b
0
0
0
0.23 ± 0.02a
0
0
compounds was formed. Moreover, oxidation of sulfhydryl group
to disulfide bond could be enhanced when phenolic compounds
was added, as evidenced by the lowered sulfhydryl group content.
Additionally, quinone could interact directly to sulfhydryl group
[20]. As a result, these sulfhydryl groups could be masked by those
quinones. A decrease in total sulfhydryl group content was reported
to be due to the formation of disulfide bonds through oxidation
of sulfhydryl groups or disulfide interchanges [35,36]. Among all
phenolic compounds used, caffeic acid and tannic acid at all lev-
els caused the greater decrease in sulfhydryl group content. The
formation of disulphide bonds might contribute to the improved
strength and stiffness of the films added with the oxidized phenolic
compounds, as shown in Table 3.
3.3. Protein pattern of FFS
Protein pattern of FFS incorporated with various oxidized phe-
nolic compounds at different concentrations under non-reducing
and reducing conditions are shown in Fig. 1. For the washed mince,
myosin heavy chain (MHC) was the dominant protein, followed
by actin, tropomyosin and troponin, respectively. MHC has been
reported as the most dominant protein in fish muscle [37]. Under
non-reducing condition, the decrease in MHC band intensity was
found in all FFS as the concentration of oxidized phenolic com-
pounds increased from 1 to 5%. This suggested the formation of
MHC cross-link. Covalent modification of proteins by phenolic oxi-
dation products generated at alkaline pH has been reported [38].
The results were in agreement with the decrease in free-amino
group and sulfhydryl group contents (Table 2). When protein pat-
terns were determined under reducing condition, MHC band was
more recovered, compared with that observed under non-reducing
condition. However, the band intensity tended to be lower as
the concentration of oxidized phenolic compounds increased. The
results suggested that disulfide bond played a role in network
stabilization in both control film and those added with oxidized
phenolic compounds. Nevertheless, the addition of oxidized phe-
nolic compounds was associated with the increased formation of
non-disulfide covalent bonds as indicated by the less retained MHC
band in the presence of ␤ME. Moreover, no significant changes in
actin band intensity were observed. From the results, no differ-
ences in MHC band intensity among the control and those added
with ferulic acids at different concentrations under both reducing
and non-reducing conditions were noticeable. This suggested that
non-disulphide covalent bonds induced by oxidized ferulic acid
might be formed at low level. It was noted that actin and tro-
ponin were more retained when oxidized ferulic acid at all levels
778 T. Prodpran et al. / International Journal of Biological Macromolecules 51 (2012) 774–782

Table 3
Young’s modulus (E), tensile strength (TS) and elongation at break (EAB) of fish myofibrillar protein films added with various phenolic compounds at different concentrations.

Treatment E (MPa) TS (MPa) EAB (%)

Control 163.91 ± 2.93a 3.88 ± 0.23a 92.65 ± 2.80g

Caffeic acid (1%) 202.84 ± 7.85cd 4.23 ± 0.17c 66.29 ± 3.23e

Caffeic acid (3%) 213.94 ± 6.36e 4.45 ± 0.07d 56.42 ± 3.43d
Caffeic acid (5%) 239.82 ± 5.33gh 4.60 ± 0.10de 54.08 ± 2.91d

Ferulic acid (1%) 172.12 ± 9.14a 3.95 ± 0.05ab 94.75 ± 2.22g

Ferulic acid (3%) 185.02 ± 2.82b 4.15 ± 0.12c 77.69 ± 2.50f
Ferulic acid (5%) 205.08 ± 3.76de 4.20 ± 0.14c 68.90 ± 2.73e

Tannic acid (1%) 225.81 ± 2.31f 4.50 ± 0.10de 57.00 ± 1.79d

Tannic acid (3%) 231.29 ± 7.17fg 4.68 ± 0.10e 47.90 ± 3.44c
Tannic acid (5%) 241.64 ± 4.74h 4.90 ± 0.03f 28.30 ± 1.30a

Catechin (1%) 193.75 ± 1.76bc 4.11 ± 0.10bc 74.81 ± 4.17f

Catechin (3%) 211.56 ± 2.79de 4.44 ± 0.04d 59.08 ± 4.11d
Catechin (5%) 224.49 ± 5.74f 4.61 ± 0.13de 42.13 ± 3.40b

Values are given as mean ± SD (n = 3). Different letters in the same column indicate significant differences (P < 0.05). Film samples containing moisture content in the range
of 10–12% were tested at 25 ± 2 ◦ C and 50 ± 5% RH.

Fig. 1. Protein pattern of film-forming solutions (FFS) added with various phenolic compounds at different concentrations under non-reducing (a) and reducing (b) conditions.
MHC: myosin heavy chain.
 T. Prodpran et al. / International Journal of Biological Macromolecules 51 (2012) 774–782
Fig. 2. Protein pattern of FMP films added with various phenolic compounds at different concentrations under reducing conditions. MHC: myosin heavy chain.
was added, when determined under non-reducing and reducing
conditions. The results indicated that those proteins were less sus-
ceptible to cross-linking induced by oxidized ferulic acid. Under
reducing condition, most of proteins in samples added with oxi-
dized caffeic acid and tannic acid had very low band intensity. This
indicated that both oxidized phenolic compounds at high concen-
tration effectively induced the formation of cross-links stabilized
by non-disulfide covalent bonds. Thus, bondings involved in pro-
tein cross-linking were governed by types and concentrations of
phenolic compounds used.
3.4. Protein pattern of films
Protein patterns of FMP films incorporated with various phe-
nolic compounds at different concentrations under reducing
conditions are shown in Fig. 2. Decrease in MHC as well as actin
band intensity was found in all film samples as the concentration
of phenolic compounds increased from 1 to 5%. This suggested the
formation of cross-link of FMP proteins, which was stabilized by
non-disulfide covalent bond, especially during film drying process.
Also, it was suggested that MHC was more susceptible to cross-
linking induced by phenolic compounds. Protein patterns of all
films incorporated with phenolic compounds exhibited lower band
intensity, especially for MHC band, as compared to the correspond-
ing protein patterns observed in FFS (Fig. 1). This suggested that
protein cross-linking induced by phenolic compounds was more
pronounced upon film drying process. Among phenolic compounds
used, incorporation of ferulic acid resulted in the lowest decrease
in band intensity, especially for actin band, suggesting the lowest
cross-linking efficiency. This was in accordance with that found in
FFS (Fig. 1). In general, non-disulfide covalent bond, disulfide bond
as well as other weak bonds most likely contributed to the film
strengthening caused by the addition of phenolic compounds as
shown in Table 3. Thus, the amount and type of bonding involved in
protein cross-linking could be varied with types and concentrations
of phenolic compounds used, which contributed to strengthening
of the resulting films.
3.5. Mechanical properties of film
Tensile strength (TS), elongation at break (EAB) and Young’s
modulus (E) of fish myofibrillar protein (FMP) films added with
various phenolic compounds at different concentrations are shown
in Table 3. Young’s modulus (E) significantly increased when
phenolic compounds were incorporated in all FMP films at increas-
ing concentrations (Table 3). Control films showed the lowest
779
Young’s modulus (163.91 MPa). However, no difference in E was
noted between control and FMP films added with ferulic acid
at 1% (P > 0.05). TS of the films also increased, when pheno-
lic compounds at the level of 1–5% were incorporated (P < 0.05).
The increase in strength and stiffness (i.e., E) of films incorpo-
rated with phenolic compounds was due to the cross-linking
involving non-disulfide covalent bonding and disulfide bonding
as well as non-covalent bonding, as evidenced by the decreases
in amino group and sulfhydryl group contents (Table 2) as well
as the protein patterns in films (Fig. 2). However, no increase
in TS was noted for films added with caffeic acid, ferulic acid
and catechin at the concentration of 3–5% (P > 0.05). This might
be due to the similar cross-linking degree in those films as
suggested by the similar band intensity observed in the corre-
sponding films added with 3-5% phenolic compounds (Fig. 2). On
the other hand, the lowered EAB was obtained with the addi-
tion of phenolic compunds at levels of 1–5% (P < 0.05), while
addition of 3–5% caffeic acid had no effect on EAB (P > 0.05).
The results indicated that incorporation of phenolic compounds
at higher concentration resulted in the increased film rigidity. As a
consequence, the film had less stretchability. Phenolic compounds
incorporated were contributable to development of a stronger and
stiffer film structure, via enhancing the interaction between pro-
tein chains. The interaction between protein molecules led to the
increased rigidity with less extensibility. The result suggested that
under alkaline pH, phenolic compounds were oxidized to quinone.
The quinone as a protein-crosslinker could interact with nucle-
ophilic amino group of protein molecules [20]. Quinones react
with amino or sulfhydryl side chains of polypeptides to form
covalent C N or C S bonds [20]. Oxidized phenolic compounds
induced the formation of non-disulfide covalent bond of protein,
as suggested by protein pattern results (Figs. 1 and 2), which con-
tributed in the improvement of E and TS of resulting film. As a
result, inter-connection between protein molecules was more pro-
nounced. Furthermore, hydroxyl group of polyphenol was able to
combine with hydrogen acceptor in protein molecules by hydrogen
bonds. Rattaya et al. [16] also reported the increased mechani-
cal properties of gelatin film incorporated with seaweed extracts
through protein–polyphenol hydrophobic interactions and hydro-
gen bonds.
At the same level of phenolic compounds used, FMP films added
with tannic acid had the highest E and TS, followed by those added
with caffeic acid, catechin and ferulic acid, respectively. This was
mostly due to the more cross-linking efficiency of tannic acid com-
pared to other phenolic compounds tested, which was evidenced
from the protein patterns (Figs. 1 and 2) and free amino group
780 T. Prodpran et al. / International Journal of Biological Macromolecules 51 (2012) 774–782

Fig. 3. Photographs of FMP films added with various phenolic compounds at different concentrations (white paper was used as background).

content results (Table 2). This suggested that different structures
of phenolic compounds affected the protein–phenol interaction
differently. Different molecular structures and high number of
hydroxyl groups are necessary to form hydrogen bonds with
carbonyl groups of protein molecules and modify the network of
film [19,20]. Use of different phenolic compounds to strengthen
various protein-based films, via protein–polyphenol interactions
has been reported [19,39]. Increases in TS were reported in ferulic
acid treated gelatin films at pH 7 and tannic acid treated films at
pH 9 [19], tannic acid, caffeic acid and ferulic acid treated porcine-
plasma protein films at pH 7–10 [39] and ferulic acid treated soy
protein isolate films [40].
3.6. Color and light transmission
Colors of fish myofibrillar protein-based films added with var-
ious phenolic compounds at different concentrations are shown
in Fig. 3 and Table 4. Color of films was affected by the types
and levels of phenolic compounds incorporated (Fig. 3). In gen-
eral, the control film was rather clear, while the films added with
phenolic compounds were yellow-green in color. The intensity
of color was increased with increasing concentration of phe-
nolic compounds added. Decreased L*-values (lightness) with
the coincidental increases in a*-values (greenness) and b*-values
(yellowness) were found in films incorporated with 1–5% phe-
nolic compunds, compared to the control films (P < 0.05). No
difference in L*- and a*-values was noted for films incorporated
with 3 and 5% ferulic acid (P > 0.05). Films added with phenolic
compounds had increased b*-value as the concentration of phe-
nolic compounds increased from 1 to 5%, except for that added
with 5% catechin, where the decrease in b*-value was observed
(P < 0.05). These results suggested that color of FMP films was
affected mostly by the type and amount of phenolic compounds
added in the films, attributable to different chemical moieties
and type and amount of pigments in different phenolic com-
pounds. Changes in color of film were also observed in gelatin
film incorporated with seaweed extracts due to pigments in the
seaweed extracts [16]. Porcine-plasma protein-based film incor-
porated with oxidized caffeic acid showed increase in b*-value
[39].

Table 4
Color parameters of fish myofibrillar protein films added with various phenolic compounds at different concentrations.

Treatment L* a* b*

Control 90.7 ± 0.21a −1.47 ± 0.04c 2.15 ± 0.04 l

Caffeic acid (1%) 80.12 ± 0.56d −0.95 ± 0.13d 23.45 ± 1.13g

Caffeic acid (3%) 70.65 ± 0.67f 2.37 ± 0.27f 38.96 ± 0.81e
Caffeic acid (5%) 59.69 ± 0.39h 7.87 ± 0.12g 52.83 ± 0.12c

Ferulic acid (1%) 89.78 ± 0.11a −2.03 ± 0.10b 4.81 ± 0.10k

Ferulic acid (3%) 88.57 ± 0.07b −2.99 ± 0.13a 9.58 ± 0.30j
Ferulic acid (5%) 87.85 ± 0.26b −3.26 ± 0.11a 11.59 ± 0.58i

Tannic acid (1%) 85.46 ± 0.11c −1.92 ± 0.10b 18.70 ± 0.41h

Tannic acid (3%) 80.12 ± 0.48d −1.38 ± 0.02c 34.58 ± 1.58f
Tannic acid (5%) 75.41 ± 0.85e 1.37 ± 0.63e 45.66 ± 1.94d

Catechin (1%) 64.07 ± 0.40g 24.36 ± 0.49h 34.34 ± 0.31f

Catechin (3%) 44.25 ± 0.49i 51.79 ± 0.22i 66.47 ± 0.43a
Catechin (5%) 38.04 ± 0.44j 55.07 ± 0.20j 55.54 ± 2.36b

Values are given as mean ± SD (n = 3). Different letters in the same column indicate significant differences (P < 0.05).
 T. Prodpran et al. / International Journal of Biological Macromolecules 51 (2012) 774–782 781

CATECHIN CAFFEIC ACID

100 100

80 80
%Transmittant

%Transmittant
60 60

40 1%CAT 40 1%CAF
20 3%CAT 20 3%CAF
5%CAT 5%CAF
0 0
CONTROL CONTROL
-20 -20
0 100 200 300 400 500 600 700 800 900 0 100 200 300 400 500 600 700 800 900
Wavelength (nm) Wavelength (nm)

FERULLIC ACID TANNIC ACID

100 100
80 80
%Transmittant

%Transmittant
60 60
40 1%FER 40 1%TAN
20 3%FER 3%TAN
20
5%FER 5%TAN
0
0
CONTROL CONTROL
-20
-20
0 100 200 300 400 500 600 700 800 900
0 100 200 300 400 500 600 700 800 900
Wavelength (nm) Wavelength (nm)

Fig. 4. Light transmittance of FMP films added with various phenolic compounds at different concentrations.

Transmission of UV and visible light at wavelength range of 4. Conclusion

200–800 nm of FMP films added with various phenolic compounds
at different concentrations is depicted in Fig. 4. The transmission
of UV light was very low at 200–280 nm for all films, regardless
of types and concentrations of phenolic compounds used. Films
incorporated with phenolic compounds at higher levels had lower
transmission in UV range. The result suggested that all phenolic
compounds effectively prevented the transmission of UV and vis-
ible light of FMP films. This decrease in light transmission can be
beneficial for food preservation since UV light has been known to
induce deleterious change, particularly lipid oxidation, in foods.
Light transmission in visible range (490–600 nm) for control films
was in the range of 80–90%. In visible range, control film showed
higher light transmission, compared with those incorporated with
phenolic compounds. Optical properties of films are an impor-
tant attribute, which influences its appearance, marketability, and
their suitability for various applications [4]. Clear edible films are
typically desirable with higher applicability and acceptability in
food packaging systems. Lowered visible light transmission was
observed when the concentration of phenolic compounds incor-
porated increased. The result suggested that phenolic compounds,
especially at higher amount, with high light transmission barrier,
more likely contributed to limited light transmission of films at vis-
ible ranges. Phenolic compounds therefore showed varying effects
on transmission of visible light, depending on the levels of phenolic
compounds incorporated. Light transmission more likely depended
on amount and distribution of phenolic compounds in the film
matrix as well as the interaction between FMP molecules medi-
ated by phenolic compound added. This led to the differences in
film matrix morphology with different light transmission. There-
fore, incorporation of different types and concentration of phenolic
compounds had an impact on the color and light transmission or
transparency of resulting films from fish myofibrillar proteins.
Incorporation of phenolic compounds could effectively induce
protein cross-linking or interaction of FMP, which significantly
influenced properties of the resulting FMP films. The phenolic com-
pounds incorporated FMP films at different concentrations showed
the increased stiffness and strength. Different phenolic compounds
affected the film properties differently due to the different modes
of action determined by compounds. Films incorporated with tan-
nic acid exhibited higher TS, due to the greater protein cross-linking
efficiency. However, phenolic compounds added affected color and
light transmission of the FMP films. Films added with phenolic com-
pounds exhibited increased transmission barrier in UV and visible
light, which could be beneficial for prevention of lipid oxidation in
foods. Therefore, physico-mechanical properties of FMP films can
be modified by selected phenolic compounds.
Acknowledgements
The authors would like to express their sincere thanks to the TRF
research scholar program (Grant No. MRG4880020) and the Grad-
uate School, Prince of Songkla University for the financial support.
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