International Journal of Biological Macromolecules 103 (2017) 721–732

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International Journal of Biological Macromolecules

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

Effects of plasticizers on the physicochemical properties of

kappa-carrageenan films extracted from Eucheuma cottonii
A.M. Ili Balqis a , M.A.R. Nor Khaizura b , A.R. Russly a,c , Z.A. Nur Hanani a,c,∗
a
Department of Food Technology, Faculty of Science and Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
b
Department of Food Science, Faculty of Science and Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
c
Institute of Halal Product Research, Universiti Putra Malaysia, Putra Infoport, 43400 UPM Serdang Selangor, Malaysia

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

Article history: The physicochemical properties of ␬-carrageenan films extracted from Eucheuma cottonii (E. cottonii)
Received 12 January 2017 incorporated with different concentrations and types of plasticizers were studied. Glycerol, sorbitol,
Received in revised form 2 May 2017 and polyethylene glycol-300 (PEG-300) in the range of 10–60% were used as plasticizers. The results
Accepted 16 May 2017
showed that the thickness and moisture content (MC) of films increased significantly (p ≤ 0.05) with the
Available online 18 May 2017
increase in plasticizer concentration. Sorbitol-plasticized films had the lowest values. Sorbitol-plasticized
films have better mechanical properties and the lowest water vapor permeability (WVP), solubility and
Keywords:
water uptake ratio (WUR) compared with glycerol and PEG-plasticized films (p ≤ 0.05). Fourier transform
Carrageenan
Eucheuma cottonii
infrared (FTIR) spectra showed the intermolecular reactions between ␬-carrageenan and the plasticizers
Plasticizer in the films. Scanning electron microscopy (SEM) observations indicated that sorbitol-plasticized films
have a compact structure, even at the highest concentration. The melting temperature (Tm) of films
decreased (p ≤ 0.05) with an increase in the plasticizer concentration. Here, the glycerol-plasticized films
had the lowest values. X-ray diffraction (XRD) showed broad and narrow peaks of the un-plasticized
␬-carrageenan film at 2␪ = 20.0◦ and 2␪ = 8.4◦ , respectively. The intensity of the broad peak increased and
the narrow peak disappeared as the concentration of plasticizers increased. In conclusion, films from
E. cottonii successfully produced with sorbitol as the plasticizer exhibited good physical properties as
packaging films.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction tions. Polysaccharides are biopolymers that have gained a high level
of interest in the packaging industry based on their unique colloidal
Consumers today are more concerned about the effects of nature, abundance, low cost and good film-forming property as well
petroleum-based synthetic packaging films on the environment as their moderate oxygen and moisture permeability [3–5]. Car-
rather than their benefits due to their poor biodegradability, sus- rageenan is a type of polysaccharide extracted from many species
tainability, and biocompatibility. Replacing synthetic polymers of red seaweeds (Rhodophyceae), which is an abundant and the
with biopolymers is one alternative to overcome these problems. most fundamental hydrocolloid after starch and gelatin [6]. Differ-
In recent years, biodegradable films have received great interest ent species of red seaweeds produce various types of carrageenan,
from the research community as they offer several advantages, such depending on the major components. For example, carrageenan
as good biodegradability, biocompatibility, environmental friend- extracted from species of Eucheuma cottonii contains the high-
liness, availability and even edibility [1]. In addition, these films est percentages of ␬-carrageenan and almost pure ␬-carrageenan,
have shown excellent physical and mechanical properties and are with less than 10% of ὶ-carrageenan [7]. Meanwhile, ὶ- and ␭-
highly effective barriers to oxygen, aroma, carbon dioxide, lipids carrageenan are found abundantly in seaweed species of Eucheuma
and flavors [2]. denticulatum and Gigartina pistillata, respectively.
Biodegradable films have been prepared from various biopoly- Carrageenan is an alternating copolymer of ␣-(1–3)-d-galactose
mers, such as polysaccharides, proteins, lipids, and their combina- and ␤-(1–4)-3,6-anhydro-d-galactose and is classified as kappa
(␬), iota (ὶ), and lambda (␭) based on the number of sulfate ester
groups and presence of 3,6-anhydro-d-galactose. The number of
∗ Corresponding author at: Department of Food Technology, Faculty of Food Sci- sulfate esters in ␬-carrageenan dimers is one, while the ὶ- and
ence and Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, ␭-carrageenan dimers possess two and three, respectively. The sol-
Malaysia. ubility temperature of carrageenan is influenced by the amount of
E-mail address: [email protected] (Z.A. Nur Hanani).

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722 A.M. Ili Balqis et al. / International Journal of Biological Macromolecules 103 (2017) 721–732

sulfate esters, where higher levels of sulfate ester groups mean a sorbitol, and PEG-300 were obtained from Sigma-Aldrich Co. (St
lower solubility temperature [8]. Moreover, the small amounts of Louis, MO, USA) and used as plasticizers.
sulfate esters cause the ␬-carrageenan to be the least negatively
charged, which allows less repulsion for intermolecular hydro-
2.2. Preparation of films
gen bonding. Thus, stronger films with better gelling properties
will form. The presence of 3,6-anhydro-d-galactose in ␬- and ␫-
Films of ␬-carrageenan were prepared by the casting method
carrageenan reduces the hydrophilicity of the sugar residue and
on circular polystyrene petri dish by which preliminary stud-
inverts the chair conformation. This allows carrageenan to undergo
ies were conducted to determine the appropriate concentration
conformational transitions, which are conducive to the gelation
of ␬-carrageenan for preparing films (1–3% concentration). Film
properties of carrageenan [9]. ␭-carrageenan does not gel due to the
forming solutions (FFSs) containing a 1% (w/v) concentration of ␬-
absence of 3,6-anhydro-d-galactose. As a sequence, ␬-carrageenan
carrageenan were easy to handle during stirring, and the resulting
has the potential to form a strong and rigid gel that enhances its
films were able to be peeled off easily.
good film-forming ability [10].
FFSs were prepared by dissolving ␬-carrageenan powder (1%,
Biopolymer films need a plasticizer to increase the intermolecu-
w/v) in deionized water by using a magnetic stirrer for 15 min
lar spacing and molecular mobility of the polymer chains. This will
at 80 ◦ C. Then, the plasticizer (glycerol, sorbitol, or PEG-300) was
increase the flexibility and stretchability of biopolymer films. With-
added separately at various concentrations (10, 20, 30, 40, 50, and
out the plasticizers, biodegradable films are brittle due to extensive
60%, w/w based on the weight of the ␬-carrageenan). The FFSs were
intermolecular forces involving polymer chain-to-chain interac-
stirred continuously for another 25 min and were cooled before
tions [11]. The ability of a plasticizer to form a good biopolymer
60 ml of FFSs was cast into the circular polystyrene petri dish plates
film depends on its compatibility with biopolymers, an appropri-
with the dimension of 140 mm length and 8 mm depth. FFSs were
ate amount for plasticization as well as its number of free hydroxyl
dried at 25 ± 2 ◦ C for two days. The process of film preparation
groups [12–14]. Therefore, the type and optimum concentration
is illustrated in Fig. 1. The dried films were manually peeled off
of the plasticizer are crucial to its successful use under various
and conditioned at a temperature of 25 ± 2 ◦ C and 50 ± 5% relative
conditions.
humidity (RH) prior to further characterization. One FFS without
Some studies on carrageenan highlighting E. cottonii were lim-
plasticizer was prepared as a control for comparison.
ited to the physicochemical properties of ␬-carrageenan powder
[15] and production of a semi-refined carrageenan [6,16]. Addi-
tionally, there have been some studies focused on carrageenan 2.3. Thickness
ester [17] and coatings [18]. However, the details on the origin and
species of the carrageenan used were not specifically indicated. The The film thickness was determined using a digital micrometer
incorporation of various plant oils on ␬-carrageenan films extracted (Mitutoyo, Serial No. 7301, Mitutoyo Corp., Kawasaki-shi, Japan).
from E. cottonii using glycerol as a plasticizer were determined The reported values are the average of 10 random locations for
recently [19]. each film.
Research on the effect of plasticizers on ␬-carrageenan films
is relevant despite a number of papers there have been pub-
2.4. Moisture content
lished on polysaccharide-based films [14,20–29]. This is because
carrageenan has a different chemical structure with different prop-
The moisture content (MC) of films was measured by the method
erties compared with other polysaccharides as it contains sulfate
of Kanmani and Rhim [2]. Films were cut into a 3 × 3 cm2 width,
esters and galactose. Different levels and positions of the sulfate
and their initial weights (W0 ) were recorded prior to drying at
esters influence the functional properties of the films produced.
110 ± 2 ◦ C for 24 h in a hot air oven. The final dry weights (W1 ) of
Moreover, the ability of gel formation is dependent on the shape
the film samples were determined and the MC (%) was calculated
(linear or branched) and sequence of residues. Starches with high
as follows:
amylose content will form stronger and firmer gels due to the linear
structure of amylose than the branched polymers, such as amy-
MC(%) = [(initialweight − finaldryweight)/initialweight] × 100
lopectin. Alginates with a linear sequence of mannuronic acid and
guluronic acid residues have higher gel strengths than an alternat-
The mean of three replicates of each film was used to calculate
ing sequence of residues. Haq et al. [20] have reported that glycerol
the moisture content.
exerts a strong influence towards a gum cordia film. Meanwhile,
some studies have reported that sorbitol was the preferred plas-
ticizer for rice starch-chitosan and composite manioc starch films 2.5. Water vapor permeability (WVP)
[29,30].
This study helps to determine the proper amount and type of WVP of films was measured by following the method of Nur
plasticizer that is compatible with ␬-carrageenan to form good Hanani, McNamara, Roos, and Kerry [11]. Deionized water (6 ml)
films and coatings. This will provide some information to other was placed in a crucible and a film sample was tightly covered
researchers and food industries to utilize this material under opti- over the crucible opening. The crucibles were maintained under
mum condition to suit their needs. Therefore, the aim of this study controlled humidity and temperature conditions (50 ± 5% RH and
was to investigate the effect of plasticizers on the physicochemical 25 ± 2 ◦ C). The weight of the crucibles was recorded at intervals of
properties of ␬-carrageenan films extracted from E. cottonii. 1 h over a 9 h period. The WVP of the film was calculated using this
formula:

2. Materials and methods WVP(g mm s−1 m−2 Pa−1 ) = w.␥.A−1 t−1 p−1

2.1. Materials where w is the weight loss of the crucible (g); ␥ is the film thickness
(mm); A is the exposed area of the film (m2 ); t is the time (s); and p
␬-carrageenan from E. cottonii farming from Sabah was pur- is the vapor pressure difference (3167.2 Pa at 25 ◦ C). Each sample
chased from Lucky Frontier Sdn. Bhd. (Sabah, Malaysia). Glycerol, was measured in triplicate.
 A.M. Ili Balqis et al. / International Journal of Biological Macromolecules 103 (2017) 721–732 723

Fig. 1. The steps of carrageenan film process.

2.6. Water resistance properties 2.8. Mechanical properties

The solubility of films in water was carried out in triplicate The mechanical properties of ␬-carrageenan films, tensile
according to the method of Rhim and Wang [17] with modifica- strength (TS) and elongation at break (EAB) were determined at
tions. Films were cut into 1 × 3 cm2 strips and dried at 110 ± 2 ◦ C 25 ◦ C by using the INSTRON 4302 Series IX machine following the
for 24 h. The dried samples were weighed as their initial dry weight methods of Shohajaee-Aliabadi et al. [18]. Prior to measuring, film
and immersed in 10 ml of deionized water in a test tube under strips (1.5 × 10 cm2 ) were conditioned for 2 days at 25 ± 2 ◦ C and
constant agitation at 25 rpm using shaking water bath (MEMMERT 50 ± 5% RH. The film strips were stretched with an initial grip sepa-
WNB 14) for 1 h at 25 ◦ C. Finally, all immersed samples were dried ration of 50 mm and cross-head speed of 50 mm/min with a tensile
at 110 ± 2 ◦ C for 24 h, and the final dry weight of samples was mea- load cell of 0.5 kn. The TS was calculated by dividing the peak load
sured. Solubility (%) was calculated as follows: with the cross-sectional area of the initial film strips. EAB is the
Solubility (%) = [(initial dry weight − final dry weight)/initial dry percentage of change in the length of film strips from the origi-
weight] x 100 nal length between the grips (50 mm). Each sample was tested in
The water uptake ratio (WUR) of films was identified following triplicate.
the method of Rhim and Wang [17]. The weight of rectangular films
(2.5 × 5 cm2 ) prior to immersion (mo ) was recorded. Then, the films
were immersed for 30 min in 25 ml of deionized water. Films were 2.9. Attenuated total reflectance-Fourier transform infrared
removed and weighed after removal of surface water with filter (ATR-FTIR)
paper (mt ). Triplicate samples were tested for each film. The WUR
was calculated as follows: The IR spectra of the films were determined following the
method described by Nur Hanani et al. [11] using a Spectrum
WUR(%) = [(mt −mo )/mo ] × 100. 100 spectrometer (Perkin Elmer, USA) equipped with a horizontal
attenuated total reflectance (ATR) ZnSe cell at room temperature.
A background spectrum reading using a clean crystal cell was
recorded prior to film analysis. Films were placed onto the crystal
cell and the cell was clamped into position on the FTIR spectrom-
2.7. Optical properties
eter. The spectra in the range of 800–4000 cm−1 with automatic
signal gain, collected over 16 scans with a resolution of 4 cm−1 , were
The lightness (L), redness (a) and yellowness (b) of the films were
recorded and compared against a background spectrum recorded
determined by using a spectrophotometer (RSEX mode, UltraScan
from the clean empty cell at room temperature.
PRO, HunterLab, U. S. A.). Prior to measuring the color of film sur-
face, the spectrophotometer was calibrated using a standard white
color plate. The color values were determined by an average of three
readings from each sample. 2.10. Scanning electron microscopy (SEM)
The opacity of the film was determined using a UV–vis spec-
trophotometer (Genesys 10-UV–vis Spectrophotometer, Thermo The morphology of the surface and cross-section of films were
Scientific) following the equation: observed using a scanning electron microscope (SEM) (JEOL, JSM-
6460LV, Tokyo, Japan). Samples for surface morphology were
mounted on a bronze stub by double-sided tape, while for cross-
Opacity = Abs600/␥ section, samples were cut into half using a razor blade and were
made to stand on double-sided tape using carbon ink. Then, all of
where Abs600 is the value of absorbance at 600 nm and ␥ is the the samples were coated with gold. The images were captured with
thickness of the film (mm). an accelerating voltage of 15 kV.
724 A.M. Ili Balqis et al. / International Journal of Biological Macromolecules 103 (2017) 721–732

2.11. Thermal analysis Table 1

Physical and water resistance properties of ␬-carrageenan films incorporated with
different concentration and type of plasticizers.
Thermal analysis of the films was performed using the method
of Abdorreza, Cheng and Karim [23] with a differential scanning Concentration (%) Plasticizer
calorimetry (DSC-Q100, TA Instruments, New Castle, DE, USA), Glycerol Sorbitol PEG-300
where 9–10 mg of the film was sealed in a 40-␮l aluminum cru-
Thickness (m)
cible using a crucible press balance. The film was heated from 30 0 31.53 ± 0.06f,1 31.53 ± 0.06g,1 31.53 ± 0.06g,1
to 220 ◦ C at a heating rate of 20 ◦ C/min. The tests were duplicated 10 36.70 ± 0.00e,1 35.40 ± 0.17f,2 36.63 ± 0.12e,1
with an empty crucible as a reference. 20 37.27 ± 0.31e,1 36.50 ± 0.10e,2 34.17 ± 0.06f,3
30 43.80 ± 0.17d,1 41.00 ± 0.00d,3 42.67 ± 0.21d,2
40 46.73 ± 0.06c,1 44.50 ± 0.27c,3 45.20 ± 0.10c,2
2.12. X-ray diffraction (XRD)
50 51.20 ± 0.20b,1 49.53 ± 0.38b,2 50.70 ± 0.35b,1
60 56.27 ± 0.38a,1 55.67 ± 0.12a,12 55.33 ± 0.40a,2
The XRD pattern of the ␬-carrageenan films was studied follow-
Moisture content (%)
ing the method established by Shankar, Reddy, Rhim, and Kim [31]. 0 8.48 ± 0.08e,1 8.48 ± 0.08a,1 8.48 ± 0.08d,1
The XRD spectra were acquired and normalized using Cu-K␣ radi- 10 2.61 ± 0.73g,1 1.50 ± 0.72c,1 1.34 ± 0.37e,1
ation at a wavelength of 0.1546 nm and a nickel monochromator 20 5.80 ± 0.85f,1 1.59 ± 0.81c,2 1.45 ± 0.25e,2
filtering wave at a voltage and current of 40 kV and 30 mA, respec- 30 10.97 ± 0.26d,1 3.09 ± 0.54c,2 10.13 ± 1.29d,1
40 18.82 ± 0.31c,1 5.18 ± 1.04b,3 13.72 ± 0.74c,2
tively. The diffraction angles used were between 5 and 65◦ (2␪)
50 30.03 ± 1.05b,1 8.06 ± 0.75a,3 21.69 ± 0.31b,2
with a scanning rate of 0.4◦ /min at 25 ◦ C. 60 35.41 ± 1.08a,1 8.95 ± 0.65a,3 24.92 ± 1.38a,2

Water vapor permeability (x 10−10 g mm s−1 m−2 Pa−1 )

2.13. Statistical analysis 0 7.61 ± 0.65a,1 7.61 ± 0.65a,1 7.61 ± 0.65c,1
10 6.57 ± 0.62b,1 3.41 ± 0.28c,2 4.12 ± 0.34e,2
The data from the experiments were statistically analyzed by 20 6.82 ± 0.44a,1 3.27 ± 0.26c,3 5.35 ± 0.63de,2
performing one-way analysis of variance (ANOVA) using Minitab 30 5.16 ± 0.36c,1 4.18 ± 0.86bc,1 5.73 ± 0.56d,1
40 5.24 ± 0.38c,12 4.35 ± 0.43bc,2 5.79 ± 0.37d,1
statistical software version 16. Tukey’s multiple-range tests were
50 6.52 ± 0.44b,2 4.40 ± 0.30bc,3 9.94 ± 0.65b,1
used to determine any significant differences among the treatments 60 7.60 ± 0.49a,2 5.02 ± 0.11b,3 13.12 ± 0.77a,1
at a 95% confidence level.
Solubility (%)
0 93.98 ± 6.29a,1 93.98 ± 6.29a,1 93.98 ± 6.29a,1
3. Results and discussion 10 82.29 ± 1.39b,2 88.87 ± 2.70a,1 85.49 ± 0.89b,12
20 82.36 ± 1.70b,1 81.14 ± 0.34b,1 84.21 ± 2.20b,1
3.1. Thickness 30 82.07 ± 0.76b,1 81.09 ± 3.41b,1 81.01 ± 0.59b,1
40 82.99 ± 2.09b,1 80.77 ± 0.90b,12 78.63 ± 1.60b,2
50 81.99 ± 0.48b,1 79.76 ± 1.96b,12 78.58 ± 1.04b,2
The thickness of packaging films is crucial for the protection of 60 79.12 ± 1.42b,1 76.89 ± 4.08c,2 78.29 ± 0.77b,1
food products. The gas permeability and pest resistance of films
Water uptake ratio (%)
may decrease as the thickness of the films decreases [32]. Table 1 0 3597.20 ± 14.30a,1 3597.20 ± 14.30a,1 3597.20 ± 14.30a,1
shows the effects of different types and concentrations of plas- 10 2994.40 ± 2.20b,2 2630.60 ± 5.30b,3 3272.50 ± 3.20b,1
ticizer on the thickness of ␬-carrageenan films. The thickness of 20 2599.50 ± 2.00c,2 2413.50 ± 54.00c,3 2857.10 ± 8.00c,1
plasticized films increased significantly (p ≤ 0.05) with the increase 30 2423.50 ± 1.60d,2 2278.10 ± 16.40d,3 2644.80 ± 31.90d,1
40 2121.90 ± 26.30e,2 2014.00 ± 59.20e,3 2457.90 ± 27.60e,1
in plasticizer concentration regardless of their type. This is due to
50 2079.20 ± 75.70f,2 1896.50 ± 25.50f,3 2274.30 ± 10.60f,1
the increase in molecular volume contributed by the plasticizers. 60 1950.80 ± 32.10g,2 1658.70 ± 20.30g,3 2189.10 ± 23.20f,1
Glycerol-plasticized films led to the thickest films due to the ten-
Values were given as mean ± standard deviation. Means with different superscript
dency of glycerol to absorb more moisture than films that contain alphabets in the columns and numbers in the rows of each analysis are significantly
other plasticizers [33]. These films swelled to a larger extent, thus different at p ≤ 0.05.
increasing their thickness [34]. The thickness range of plasticized
films obtained was similar to those of commercial packaging films
(polyethylene), which were between 25–75 ␮m [32].

3.2. Moisture content (MC)

The hydrophilic nature of ␬-carrageenan caused the un-

plasticized film to have a high MC (Table 1). However, the MC of
carrageenan films decreased as the plasticizers were introduced
due to a complete reaction between small amounts of the plasti-
cizer and the free sites of ␬-carrageenan. On the contrary, the MC
started to increase significantly (p ≤ 0.05) as more plasticizers were
added. This could be due to an excess of plasticizers available in the
network.
Fig. 2. Chemical structure of glycerol, sorbitol, and PEG-300.
In terms of plasticizer types, glycerol-plasticized films have the
highest MC, followed by PEG-300 and sorbitol-plasticized films. The
hydrophilicity of the plasticizer greatly influences the MC, where a absorb or release water [33]. Hence, sorbitol tends to maintain the
higher number of hydroxyl groups (OH) contained in the plasticizer MC of films, even at high concentrations. The MC of films also can
will increase the MC. However, although sorbitol possesses six OH, be explained based on the polarity of the plasticizers. Polarity gives
the MC of the sorbitol-plasticized film was the lowest compared to the plasticizer a charge and allows it to interact with water through
other films even though glycerol and PEG-300 contain only three hydrogen bonding, which is known as hydrophilicity. Glycerol has
and two OH, respectively (Fig. 2). This was probably due to the low the highest polarity, with a dielectric constant of 42.5, compared to
hygroscopicity of the sorbitol, which means that it has less ability to sorbitol (33.5) and PEG-300 (12.4).
 A.M. Ili Balqis et al. / International Journal of Biological Macromolecules 103 (2017) 721–732 725

Fig. 3. Effects of different concentration and type of plasticizers on (a) tensile strength and (b) elongation at break of ␬-carrageenan films. Error bars represent standard
error, n = 3.

3.3. Water vapor permeability (WVP) concentration. This can be explained due to the hygroscopicity
of the plasticizers, which increase the MC of films. This produces
The proper concentration and type of plasticizer must be an increase in the effective diffusion coefficient of water vapor
considered to develop food-packaging films with good barrier in the films, thus increasing the water vapor transfer rates [36].
properties in which the WVP should be low. According to Fick’s Meanwhile, the low hygroscopicity of sorbitol maintained the WVP
law, permeability should be inversely proportional to the film values even at the highest concentration.
thickness. However, this law cannot be applied in this study as The highest WVP was recorded for films that contained PEG. This
the WVP of plasticized films increased along with the increase was due to the higher molecular weight of PEG-300 (300 g mol−1 )
of thickness (Table 1). The WVP of the un-plasticized film compared to sorbitol (182 g mol−1 ) and glycerol (92 g mol−1 ),
was 7.61 × 10−10 g mm s−1 m−2 Pa−1 and decreased significantly which caused a widening of the voids (an increase in the free vol-
(p ≤ 0.05) as the plasticizers were introduced (Table 1). However, ume) in the polymer chains, thus increasing the diffusion rate of
the WVP starts to increase (p ≤ 0.05) with increasing plasticizer water molecules through the films. Cao, Yang and Fu [13] have
726 A.M. Ili Balqis et al. / International Journal of Biological Macromolecules 103 (2017) 721–732

Fig. 4. Effect of different concentration and type of plasticizers on FTIR spectrum of ␬-carrageenan films. (a) Un-plasticized and (b) plasticized films (-, 0%, -, 10%, -, 20%, -,
30%, -, 40%, -, 50%, -, 60%).

proved that the WVP of films were increased with an increase in commercial packaging films, such as PE films, which have a WVP
the molecular weight of PEG in gelatin films. Srinivasa, Ramesh and of 10−13 g mm s−1 m−2 Pa−1 . The ␬-carrageenan films produced in
Tharanathan [37] also reported that PEG-plasticized chitosan films this study were considered to be more permeable to water vapor
have higher WVP values compared to other plasticized films. Gen- compared to PE films by a factor of 103 .
erally, the WVP of biopolymer films is 102 –103 higher than that of
 A.M. Ili Balqis et al. / International Journal of Biological Macromolecules 103 (2017) 721–732 727

3.4. Water resistance properties Table 2

Optical and thermal properties of ␬-carrageenan films incorporated with different
concentration and type of plasticizers.
The water resistance properties of the ␬-carrageenan films stud-
ied were the solubility and water uptake ratio (WUR). The solubility Concentration (%) Plasticizer
of films was improved with the addition of plasticizers, where the Glycerol Sorbitol PEG-300
solubility of films decreased significantly (p ≤ 0.05) as plasticizers
L
were introduced (Table 1). The thinness of the un-plasticized film 0 85.43 ± 0.47b,1 85.43 ± 0.47a,1 85.43 ± 0.47a,1
can be one of the reasons for its highest solubility. The other rea- 10 85.34 ± 0.20b,1 85.18 ± 0.13a,1 85.55 ± 0.29a,1
son was due to the hydrophilicity of ␬-carrageenan, which is very 20 85.33 ± 0.33a,1 85.25 ± 0.31a,1 85.58 ± 0.03a,1
hydrophilic, thus absorbing water and swelling quickly [38]. The 30 85.42 ± 0.05a,1 84.82 ± 0.09a,2 85.65 ± 0.26a,1
40 85.55 ± 0.11a,1 84.72 ± 0.22b,2 85.63 ± 0.34a,1
solubility of films decreased insignificantly (p > 0.05) with increas- 50 86.65 ± 0.03a,1 84.60 ± 0.05b,3 85.65 ± 0.13a,2
ing plasticizer concentration regardless of the type. This is in 60 86.89 ± 0.40a,1 84.28 ± 0.09b,3 85.61 ± 0.24a,2
contrast with the results of other researchers who reported that
a
the solubility of biodegradable films increased with the increasing 0 -1.13 ± 0.04a,1 -1.13 ± 0.04a,1 -1.13 ± 0.04a,1
concentration of plasticizers [20,24–29,35,39,40]. 10 -1.14 ± 0.04a,1 -1.10 ± 0.04a,1 -1.13 ± 0.01a,1
Among the plasticizers used, sorbitol-plasticized films showed 20 -1.13 ± 0.02a,1 -1.10 ± 0.01a,1 -1.09 ± 0.04a,1
the lowest solubility compared to films containing glycerol and 30 -1.11 ± 0.04a,12 -1.08 ± 0.02a,1 -1.14 ± 0.01a,2
40 -1.11 ± 0.01a,1 -1.13 ± 0.00a,1 -1.10 ± 0.03a,1
PEG-300 (p ≤ 0.05). Haq et al. [20] mentioned that plasticizers
50 -1.11 ± 0.03a,1 -1.08 ± 0.00a,1 -1.12 ± 0.03a,1
would alter the interaction between polymer chains, which may 60 -1.10 ± 0.05a,1 -1.08 ± 0.01a,1 -1.11 ± 0.04a,1
increase or decrease the dissolution of the polymer. For example,
b
whey protein film containing sorbitol dissolved completely in only 0 3.56 ± 0.12a,1 3.56 ± 0.12a,1 3.56 ± 0.12a,1
7 h, while the same film plasticized with glycerol only showed 31% 10 3.25 ± 0.08b,1 3.30 ± 0.07b,1 3.37 ± 0.04a,1
solubility after 24 h [41], even though glycerol is more hydrophilic 20 3.22 ± 0.05b,2 3.40 ± 0.06ab,1 3.40 ± 0.04a,1
than sorbitol. Sorbitol can reduce the dissolution of ␬-carrageenan 30 3.30 ± 0.06b,1 3.31 ± 0.03b,1 3.35 ± 0.04b,1
40 3.34 ± 0.05b,1 3.40 ± 0.02ab,1 3.37 ± 0.06a,1
films due to it being less hygroscopic. The high solubility of ␬-
50 3.36 ± 0.06ab,1 3.32 ± 0.04b,1 3.38 ± 0.08a,1
carrageenan films can be considered for edible coatings and film 60 3.38 ± 0.10ab,1 3.30 ± 0.11b,1 3.54 ± 0.10a,2
applications.
Opacity
WUR is a measure of the water absorbed by films when 0 2.91 ± 0.10a,1 2.91 ± 0.10a,1 2.91 ± 0.10a,1
immersed in water for a period of time. The un-plasticized film has 10 2.56 ± 0.02b,2 2.61 ± 0.08b,2 2.91 ± 0.03a,1
a significantly higher WUR compared to plasticized films (p ≤ 0.05) 20 2.08 ± 0.06c,2 2.50 ± 0.08b,1 2.17 ± 0.07b,12
(Table 1). This was due to the availability of free ␬-carrageenan sites 30 2.06 ± 0.04c,2 2.28 ± 0.06c,1 2.07 ± 0.02b,2
40 1.88 ± 0.01cd,3 1.96 ± 0.03d,2 2.00 ± 0.02b,1
for binding water where more water can be absorbed. The results
50 1.73 ± 0.22d,1 1.76 ± 0.03e,1 1.80 ± 0.08c,1
showed a decrease (p ≤ 0.05) of WUR with the increasing concen- 60 1.74 ± 0.14d,1 1.73 ± 0.02e,1 1.63 ± 0.11c,1
tration of plasticizers. Increasing the concentration of plasticizers
Melting temperature (◦ C)
will cause the limited sites of ␬-carrageenan available to bind with
0 131.51 ± 1.65a,1 131.51 ± 1.65a,1 131.51 ± 1.65a,1
water as the polymers-plasticizer reaction is high, thus allowing 10 131.47 ± 1.54a,1 132.68 ± 1.27a,1 132.30 ± 0.78a,1
less water to be absorbed. 20 132.68 ± 1.80a,1 130.65 ± 0.21a,1 134.45 ± 0.23a,1
Films containing PEG-300 showed the highest WUR compared 30 130.98 ± 0.29a,1 132.33 ± 0.17a,1 129.48 ± 0.46b,1
40 126.07 ± 1.17b,2 130.67 ± 0.97a,1 127.86 ± 0.42b,2
with films containing glycerol and sorbitol. This was due to the
50 125.53 ± 0.04b,2 129.11 ± 0.21a,1 128.46 ± 0.51b,1
large molecular size of PEG-300, which was unable to be inserted 60 124.55 ± 1.84b,2 128.68 ± 0.61b,1 128.99 ± 1.17b,1
effectively between the polymers chains, thus leaving gaps where
Values were given as mean ± standard deviation. Means with different superscript
water will be filled. Films containing sorbitol showed the lowest alphabets in the columns and numbers in the rows of each analysis are significantly
WUR. Films with a low WUR will form with good integrity for different at p ≤ 0.05.
transportation and storage. There is a correlation between WUR
and solubility where films with a low WUR will also have low
solubility. where the opacity of films was reduced significantly (p ≤ 0.05) with
an increase in the plasticizer concentration (Table 2). Glycerol-
3.5. Optical properties plasticized films have the lowest opacity, meaning that the films
were highly transparent. The transparency of the films is a very
The optical properties of films are an important factor for important aspect for food-packaging industries to allow consumers
product acceptability to consumers where the oxidation deteri- to inspect food before purchasing, and at the same time, the appear-
oration of packaged food could be prevented by reducing the ance of the products can be better presented.
lightness of the film [42]. The concentration of plasticizers did
not greatly affect the lightness (L), redness (a), and yellowness
(b) values of ␬-carrageenan films (Table 2). However, the L val- 3.6. Mechanical properties
ues of glycerol and sorbitol-plasticized films were increased and
decreased significantly (p ≤ 0.05), respectively, with an increase in Tensile strength (TS) indicates the maximum tensile stress that
the plasticizer concentration. There was no significant (p > 0.05) the film can sustain. The un-plasticized film exhibited a TS value
difference in the L, a, and b values among the types of plas- of 69.69 MPa, which decreased significantly (p ≤ 0.05) with the
ticizers, as reported by Gennadios, Weller, Hanna and Froning increase in plasticizer concentration (Fig. 3a). This occurred due
[43]. In their study, they also observed no effect of the plasti- to the increased spatial distance between polymer chains, which
cizer type (glycerol, sorbitol, and PEG) on the color of egg albumen was contributed by plasticizers positioned between the polymer
films. molecules [44]. No significant difference (p > 0.05) was observed
Opacity was calculated based on the absorbance value at for TS between the various types of plasticizers. This was in con-
600 nm, which indicates the opaqueness of the films. The addi- trast with the results of Haq et al. [20], who showed that the TS
tion of plasticizers in ␬-carrageenan films improved the opacity, values of gum cordia films were dependent on the type of plasti-
728 A.M. Ili Balqis et al. / International Journal of Biological Macromolecules 103 (2017) 721–732

cizers (glycerol, sorbitol, PEG-200 and 400), where films with PEG 3.7. ATR-FTIR
had the highest TS.
Elongation at break (EAB) is the maximum change in the length Fig. 4a shows the FTIR spectrum of the ␬-carrageenan film with-
of a test specimen before breaking. This was found to be inversely out plasticizers. The broad band ranging from 3600 to 3000 cm−1
correlated with TS, where the EAB of ␬-carrageenan films increased refers to the stretch of bonded hydroxyl (O H) and that from 3000
significantly (p ≤ 0.05) with the increase in plasticizer concentra- to 2700 cm−1 describes the stretching of C H bonds. The band from
tion (Fig. 3b). Plasticizers increase the mobility of polymer chains, 1800 to 1500 cm−1 describes the stretch of carbonyl groups (C O)
which contributes to more stretchable and flexible films [45]. In contained in D-galactose, which is a monomer of ␬-carrageenan.
the same manner as TS, the EAB of plasticized films was not depen- The region between 1500 and 800 cm−1 is known as the finger-
dent on the type of plasticizer, except for films containing sorbitol. print region because it contains certain peaks that characterize
Sorbitol-plasticized films showed the lowest (p ≤ 0.05) EAB com- ␬-carrageenan. The peaks at 1228, 925 and 846 cm−1 are attributed
pared with other plasticized films. As stated before, sorbitol has to the sulfate ester (O = S O), 3,6-anhydrogalactose (C O C), and
a low ability to absorb water where water can act as a plasticizer galactose-4-sulfate (C O-S), respectively [2,47,48], and the peak at
to give flexibility to the films. Hence, the mobility of the polymers 1066 cm−1 was attributed to the stretch of the C O C glycosidic
was reduced, causing the flexibility of films to decrease. Biopolymer linkage [49].
films with an appropriate plasticizer at an adequate concentration The FTIR spectra of the plasticized films were similar to those
can be classified as having good mechanical properties (TS in the of the un-plasticized film, except for the bands indicating O H
range of 10–100 MPa and EAB >10%) [46]. and C H stretching (Fig. 4b). The O H stretching band was shifted

Fig. 5. SEM images of surface (1000 magnification) and cross-section (5000 magnification) of the un-plasticized and plasticized (10% and 60%) ␬-carrageenan films.
 A.M. Ili Balqis et al. / International Journal of Biological Macromolecules 103 (2017) 721–732 729

Fig. 5. (Continued)

from 3384 to 3360, 3378 and 3389 cm−1 when glycerol, sorbitol, firmed that glycerol was attracted and bonded to the carrageenan
and PEG-300 were added, respectively. The C H stretching bands matrix.
of glycerol and sorbitol-plasticized films were shifted from 2943 The intensity of the bands was increased as the concentration of
to 2929 and 2920 cm−1 , respectively, while films containing PEG- the plasticizers was increased (Fig. 4b). Glycerol-plasticized films
300 showed no shifting. This was attributed to the longer chain of showed a gradual increase in band intensity compared with films
PEG-300 (Fig. 2), where the chain itself is stable when reacted with containing sorbitol and PEG-300. This can be explained by the sig-
polymer chains. These shifted bands indicate that a chemical reac- nificant changes of the MC, thickness, WVP, solubility, and WUR of
tion occurred between plasticizers and ␬-carrageenan polymers the films. Haq et al. [20] and Zhang and Han [44] also reported that
through hydrogen bonding. The characteristic peak of glycerol that the concentrations of sorbitol and PEG did not show any changes
appears at 1456 cm−1 [50] was shifted to 1420 cm−1 , which con- to gum cordia and starch films, respectively. The intensity of the
730 A.M. Ili Balqis et al. / International Journal of Biological Macromolecules 103 (2017) 721–732

Fig. 6. Differential scanning calorimetry (DSC) thermogram of (a) components in ␬-carrageenan film and (b) ␬-carrageenan films contained 60% of plasticizers (glycerol,
sorbitol and PEG-300).

C H stretching band in films containing PEG-300 was the highest 3.8. Scanning electron microscopy (SEM)
compared with those of the other plasticized films. This is probably
due to the high molecular weight of PEG-300, which means that it The surface morphology of the un-plasticized film appeared to
contains a higher amount of C H bonding compared to sorbitol and be smooth (Fig. 5). However, the plasticized films were slightly
glycerol (Fig. 2). different and varied depending on the level of plasticizer added.
 A.M. Ili Balqis et al. / International Journal of Biological Macromolecules 103 (2017) 721–732 731

The similar structure of the 10% plasticized films to that of the

un-plasticized film was observed, despite some irregularities were
present on the films. On the contrary, rougher surfaces with larger
irregularities appeared when 60% plasticizer was incorporated into
the films. These results are due to the hydrophilicity of the plasti-
cizers where voids on the surface of the films could be formed due
to the absorption of moisture [28]. This explains the high WVP of
films at high concentrations of plasticizers.
Cross-sectional images revealed that un-plasticized and 10%
plasticized films have homogeneous and compact microstructures,
which are likely caused by strong cohesion forces that appear dur-
ing the slow drying of the films [51]. Meanwhile, films with a 60%
plasticizer concentration showed the obvious layer arrangement.
This layer showed that the film was not compact and was thus able
to retain more water. Hence, this condition increased the MC, WVP,
solubility, WUR, and flexibility of the films. By comparing the type of Fig. 7. X-ray diffractograms of un-plasticized and plasticized (60%) ␬-carrageenan
films. (-,un-plasticized, -, glycerol-plasticized, -, sorbitol-plasticized, and -, PEG-
plasticizer at 60% of the concentration, the sorbitol-plasticized film
plasticized films).
has the most compact structure, followed by films containing glyc-
erol and PEG-300. The compact structure of the sorbitol-plasticized
film caused the vapor to transfer through the film matrix at a slower there was a narrow peak at 2␪ = 8.4◦ , which indicated a crystalline
rate. Therefore, the WVP of sorbitol-plasticized films was the lowest region, that decreased after the plasticizers were incorporated. This
compared with the other plasticized films. Hence, the less-flexible shows that water alone cannot dissolve the crystalline structure
condition of sorbitol-plasticized films can also be influenced by this of the film and requires plasticizers. The intensity of the peak at
morphology. 2␪ = 20.0◦ , which increased with the addition of 60% of plasticizer
regardless of the plasticizer type, was contributed by the plasticiz-
3.9. Thermal analysis ers themselves.

Thermal analysis of ␬-carrageenan films was performed with 4. Conclusion

differential scanning calorimetry (DSC). The individual components
used in the preparation of ␬-carrageenan films were identified to The plasticizers used in this study exhibited compatibility with
investigate the effect of plasticizers. The ␬-carrageenan powder ␬-carrageenan extracted from local E. cottonii in the range of the
exhibited a glass transition temperature (Tg) at 84.72 ◦ C (Fig. 6a), concentrations used. The type and concentration of plasticizer
which is the temperature at which polymers change from a brittle significantly affected the physical properties of the films. Films
to rubbery material. This peak disappeared (Fig. 6b) when the pow- containing sorbitol exhibited promising effects, where the films
der was formed into films due to the reduction of the intermolecular had better water-related properties with low MC, WVP, solubility,
forces of polymers by water and plasticizers during film prepara- and WUR. These films were stronger and showed more compact
tion. Thus, Tg was decreased below the tested temperature range microstructure than other films. Additionally, sorbitol had less abil-
(30–220 ◦ C). Low Tg can be correlated to the decrease and increase ity to reduce the Tm, which explained the lower EAB value. These
of EAB and TS, respectively where the plasticizer spaced out the findings demonstrated that ␬-carrageenan obtained from E. cottonii
polymer chains and increased the free volume. Glycerol and sorbitol could be produced as films and coatings, thus having high potential
showed broad endothermic peaks at 140–180 ◦ C and 90–120 ◦ C, to be applied for food packaging and to prolong the shelf life of food
respectively. By contrast, PEG-300 did not show any peak and had products. Further research should be undertaken to establish the
a curve similar to that of ␬-carrageenan powder. Abdorreza et al. application of this type of carrageenan.
[23] obtained similar broad endothermic peaks at 165–220 ◦ C and
85–100 ◦ C for glycerol and sorbitol, respectively, which correspond Acknowledgements
to the degradation and melting temperature of those plasticizers
[41,52]. The authors are thankful to the Ministry of Higher Education
A significant decrease (p ≤ 0.05) of the film melting tempera- (MoHE) and Universiti Putra Malaysia for the financial support
ture (Tm) was observed along with an increase in the plasticizer through FRGS (62675-87513). They also thank Mr. Rafhayudi Jamro
concentration, except for films containing sorbitol (Table 2). The and Mr. Wilfred@Sylvester Paulus for the assistance in XRD analy-
Tm of sorbitol-plasticized films was the highest compared with sis.
other plasticized films due to the lower tendency of the film
to absorb water, where water acts as a mobility enhancer, thus
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