Applied Clay Science 97–98 (2014) 174–181

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Applied Clay Science

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

Research paper

Preparation and characterization of carrageenan-based nanocomposite

films reinforced with clay mineral and silver nanoparticles
Jong-Whan Rhim ⁎, Long-Feng Wang
Department of Food Engineering and Bionanocomposite Research Institute, Mokpo National University, 61 Dorimri, Chungkyemyon, Muangun, 534-729 Jeonnam, Republic of Korea

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

Article history: Antimicrobial bionanocomposite films were prepared with κ-carrageenan and silver nanoparticles (AgNPs) and
Received 18 February 2014 organically modified clay mineral (Cloisite® 30B). AgNP-included nanocomposite films showed characteristic
Received in revised form 14 May 2014 plasmonic effect of the AgNPs with the maximum absorption at 420 nm and they also showed high UV barrier
Accepted 27 May 2014
properties. Depending on the type of nanofillers, tensile strength (TS) of the nanocomposite films increased by
Available online 12 June 2014
14–26% and water vapor permeability (WVP) decreased by 12–27% compared to those of the neat carrageenan
Keywords:
film. The nanocomposite films exhibited characteristic antimicrobial activity against Gram-positive (Listeria
Carrageenan monocytogenes) and Gram-negative (Escherichia coli O157:H7) pathogenic bacteria. While AgNP-included
Silver nanoparticles nanocomposite film showed strong antimicrobial activity against Gram-negative bacteria, the clay-included
Clay mineral nanocomposite films showed strong antimicrobial activity against Gram-positive bacteria. On the contrary, the
Nanocomposite nanocomposite film with combined use of both nanofillers showed strong antimicrobial activity against both
Antimicrobial activity Gram-positive and Gram-negative bacteria.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction with various types of natural or synthetic biopolymers such as proteins,

One of the most challenging subjects of research in the food packag-
ing sector is to develop environment-friendly packaging materials with
additional function such as antimicrobial activity to fulfill the sustain-
able development and to secure the food safety and to extend the
shelf-life of food (Duncan, 2011; Silvestre et al., 2011; de Azeredo,
2009; Sozer and Kokini, 2008; Sorrentino et al., 2007). As one of the
most promising technologies to achieve such goals, bionanocomposites
(BNCs) with antimicrobial activity have emerged due to their excellent
and unique properties, environment-friendliness, and functionality
(Armentano et al., 2013; Falguera et al., 2011; Ojijo and Ray, 2013;
Raquez et al., 2013; Reddy et al., 2013; Xie et al., 2013). BNC is a class
of hybrid materials composed of biopolymer matrix and nanoscale rein-
forcement fillers such as a fiber, platelet, or particles in which one or
more dimensions are in the order of 100 nm or less (Ojijo and Ray,
2013; Reddy et al., 2013). Owing to the nanometer-sized particles dis-
persed in the biopolymer matrix, these nanocomposites exhibit not
only controlled biodegradability but also markedly improved mechani-
cal, gas barrier and optical properties when compared with the pure
polymer with very low filler loading (typically b5 wt.%). Various types
of nanoparticles such as clay minerals, nanometals or metal oxides,
and cellulose nanofibers have been tested to prepare BNC by blending
⁎ Corresponding author. Tel.: +82 61 450 2423; fax: +82 61 454 1521.
E-mail address: jwrhim@mokpo.ac.kr (J.-W. Rhim).
polysaccharides, and polylactide (PLA) (Raquez et al., 2013; Rhim, 2013;
Rhim et al., 2009). In addition, a variety of antimicrobial agents such as
natural or synthetic antimicrobial agents (nisin, carvacrol, allyl isothio-
cyanate, antibiotics, propionic, benzoic, sorbic acids), enzymes (lyso-
zyme), nanometals (silver, copper), metal oxides (TiO2, ZnO, MgO),
and organically modified clay minerals have been used to develop anti-
microbial packaging materials (Appendini and Hotchkiss, 2002; Hong
and Rhim, 2008; Rhim et al., 2013). The combined use of more than
one antimicrobial agent is also probable to expect combined or syner-
gistic effect of their antimicrobial activity. Antimicrobial agents based
on organic materials are often not usable under conditions where ther-
mal or chemical durability is required. On the contrary, antimicrobial
agents based on inorganic materials are superior to those of organic an-
timicrobial materials in durability, heat stability, toxicity, and selectivity
(Malchová et al., 2009). Antimicrobial nanocomposite packaging sys-
tems are particularly effective because of the high surface-to-volume
ratio and enhanced surface reactivity of the nanosized antimicrobial
agents (Llorens et al., 2012), which are highly useful to prevent microbi-
al contamination for extending shelf-life and securing food safety.
Among the inorganic antimicrobial materials, silver nanoparticles
(AgNPs) have attracted considerable attention for packaging applica-
tions with their unique chemical and physical properties including
antibacterial activity and high thermal stability as well as low toxicity
(Incoronato et al., 2010; Llorens et al., 2012; Yoksan and Chirachanchai,
2010). AgNPs are prepared by various routes consisting of complex syn-
thesis or reduction procedures such as UV irradiation, laser ablation,

http://dx.doi.org/10.1016/j.clay.2014.05.025
0169-1317/© 2014 Elsevier B.V. All rights reserved.
 J.-W. Rhim, L.-F. Wang / Applied Clay Science 97–98 (2014) 174–181
sonochemical and photochemical reduction, and reduction by biological
systems such as bacteria, fungi, yeasts, and plant extracts (Rai et al.,
2009; Yoksan and Chirachanchai, 2010). However, AgNPs have a tenden-
cy to agglomerate when AgNPs are used alone, which causes deteriora-
tion of their chemical activities and decreases their antimicrobial
properties (Ahmad et al., 2009a; Kim et al., 2007). To overcome this
problem, preparation of silver/clay mineral nanocomposites, in which
AgNPs are supported within interlamellar spaces of clay mineral or on
its external surfaces, has been suggested by many researchers (Ahmad
et al., 2009a, 2009b; Carja et al., 2009; Kim et al., 2007; Malchová
et al., 2009). Montmorillonite (Mt) as-layered clay mineral has interca-
lation, swelling and ion exchange properties; its interlayer space has
been used for the synthesis of silver/clay mineral nanocomposite
materials. In addition, the silver/clay mineral nanocomposites are
known to have long term antimicrobial effects (Malchová et al., 2009;
Ahmad et al., 2009a, 2009b). Moreover, some organically modified
clay minerals such as Cloisite® 30B are known to have a strong antimi-
crobial activity due to the quaternary ammonium salt of the organically
modified Mt (Hong and Rhim, 2008).
Various polysaccharides have been used not only as carriers of active
antimicrobial nanoparticles but also as polymer matrices for biodegrad-
able films. As one of such biopolymers, carrageenan has been used
frequently to prepare biodegradable packaging films and hydrogels
since it forms strong gels with high water holding capacity (Cha et al.,
2002; Rhim and Wang, 2013). Carrageenan has also been used as a
completely biodegradable polymer matrix and stabilizing agent for the
nanoparticles such as nano-sized metals, metallic oxides, and clay
minerals (Kanmani and Rhim, 2014a; Martins et al., 2013; Rhim, 2012).
The main objective of the present study was to prepare environ-
ment-friendly nanocomposite films with carrageenan, AgNPs and clay
mineral to test their combined effect on the antimicrobial activity and
physico-chemical film properties. The nanocomposite films were char-
acterized using SEM, XRD, FT-IR, and TGA analysis. Apparent color,
transmittance, mechanical and water vapor barrier properties as well
as antimicrobial activity of the nanocomposite films were evaluated.
2. Materials and methods
2.1. Materials
Food grade κ-carrageenan was purchased from Hankook Carragen
Co., Ltd., (Whasoon, Jeonnam, Korea). Organically modified montmoril-
lonite (Cloisite® 30B) was obtained from Southern Clay (Gonzales, TX,
USA) and glycerol was obtained from Daejung Chemicals & Metals Co.,
Ltd. (Siheung, Gyeonggi-do, Korea). Silver nitrate (AgNO3) and sodium
citrate were of analytical grade and used without further purification.
2.2. Preparation of films
Carrageenan films were prepared by a solution casting method.
Three grams of carrageenan powder was dissolved in 150 mL distilled
water with 0.9 g of glycerol as plasticizer while mixing vigorously for
about 20 min at 95 °C using a magnetic stirrer and cast evenly onto a
leveled Teflon film (Cole-Parmer Instrument Co., Chicago, IL, USA) coat-
ed glass plate (24 × 30 cm), then dried for about 24 h at room temper-
ature. Carrageenan/Cloisite® 30B nanocomposite films were prepared
using a solution intercalation method (Rhim et al., 2011). First, precisely
weighed clay mineral (150 mg, 5 wt.% of carrageenan) was dispersed
into distilled water (150 mL) and stirred using a magnetic stirrer
for 24 h to get a completely swollen clay mineral. The fully hydrated
clay mineral solution was homogenized using a high shear mixer (T25
basic, Ika Labotechnik, Janke & Kunkel Gmbh & Co., KG Staufen,
Germany) at 20,500 rpm for 10 min followed by sonication for 10 min
using a High Intensity Ultrasonic Processor (Model VCX 750, Sonics &
Materials Inc., Newtown, CT, USA) with a medium size tip (2 cm diam-
eter). Three grams of carrageenan and 0.9 g of glycerol were then
175
dissolved into the clay mineral dispersions and heated for about
20 min at 95 °C with vigorous mixing using a magnetic stirrer, cast
onto the glass plate followed by the same procedures, as described
above. For the preparation of AgNP-loaded nanocomposite films (2 wt.%
of AgNPs), silver nanoparticles were synthesized by a modified chemical
reduction method (Rhim et al., 2013). Firstly, AgNO3 stock solution was
prepared by dissolving 4.724 g AgNO3 (63.5% of Ag) in 100 mL distilled
water boiling for 1 h, then the stock solution contains 0.03 g Ag per mL.
Secondly, to reduce AgNO3, 2 mL of the stock solution was dissolved
into 150 mL distilled water with 2 mL of 1% sodium citrate solution, and
the solution was kept boiling for 1 h. In this process, the sodium citrate so-
lution was used as an environment-friendly ‘green’ reducing agent (Rhim
et al., 2013). The silver sol prepared was greenish yellow. And lastly, 3 g of
carrageenan and 0.9 g of glycerol were then dissolved into the silver sol
and heated for about 20 min at 95 °C with vigorous mixing using a mag-
netic stirrer, cast onto the glass plate followed by the same procedures, as
described above.
2.3. Color and transparency
Surface color of the films was measured using a Chroma meter
(Konica Minolta, CR-400, Tokyo, Japan). A white standard color plate
(L = 97.75, a = − 0.49 and b = 1.96) was used as a background for
color measurements. Hunter color (L, a, and b) values were averaged
from five readings from each sample. The total color difference (ΔE)
was calculated as follows:
h i
2 2 2 0:5
ΔE ¼ ðΔLÞ þ ðΔaÞ þ ðΔbÞ ð1Þ
where ΔL, Δa, and Δb are the difference between each color values of
standard color plate and film specimen, respectively.
The optical properties of the films were tested by measuring UV/vis
absorption and transmission spectrum of the films. UV/vis absorption
and transmission measurements were performed in the range of 200–
700 nm using a UV/vis spectrophotometer (Model 8451A, Hewlett
Packard Co., Santa Alara, CA, USA). Transparency of the film samples
was expressed as the percent transmittance measured at 660 nm.
2.4. Characterization of film
The morphology of carrageenan and carrageenan/AgNP/clay mineral
films was observed using FE-SEM (Field Emission Scanning Electron
Microscope, S-4800, Hitachi Co., Ltd., Matsuda, Japan) operated at accel-
erating voltage (Vacc) of 1 kV and current (Ie) of 10 μA.
Structures of carrageenan and AgNP/clay mineral incorporated
carrageenan films were evaluated with XRD measurements using a
PANalytical Xpert pro MRD diffractometer (Amsterdam, Netherlands)
operated at 40 kV and 30 mA, equipped with Cu Kα radiation at a wave-
length of 0.1546 nm. The samples were scanned over the range of dif-
fraction angle 2θ = 0–90° with a scanning rate of 0.5°/min at room
temperature. From the XRD patterns, the crystallite size (D) of silver
particles was calculated by using the Scherrer equation (Borchert
et al., 2005).
D ¼ kλ=β cosθ ð2Þ
where k is a constant and generally equal to 1, λ is the X-ray wavelength
(0.1546 nm), θ is the Bragg's angle, and β is the full width at half
maximum (FWHM) in degrees.
Fourier transform infrared (FT-IR) spectrum of the nanocomposite
film samples was analyzed using FT-IR spectroscopy (TENSOR 37 spec-
trophotometer with OPUS 6.0 software, Billerica, MA, USA) operated at
a resolution of 4 cm−1. For this, samples were placed on the ray expos-
ing stage and the spectrum for each sample was recorded between the
wave number ranges of 500–4000 cm−1.
176 J.-W. Rhim, L.-F. Wang / Applied Clay Science 97–98 (2014) 174–181
2.5. Mechanical properties
The mechanical properties of the films were analyzed by measuring
the tensile strength (TS), elongation at break (EB), and elastic modulus
(EM) according to the standard ASTM method D 882-88 using an
Instron Universal Testing Machine (Model 5565, Instron Engineering
Corporation, Canton, MA, USA) equipped with a 0.5 kN load cell. Rectan-
gular strips (2.54 × 15 cm) were cut from individually prepared film
using a precision double blade cutter (model LB.02/A, Metrotec, S.A.,
San Sebastian, Spain). Initial grip separation was set at 50 mm and
cross-head speed at 50 mm/min. The TS (MPa) was determined by di-
viding the maximum load (N) by the initial cross sectional area (m2)
of the film sample; the EB (%) was determined by dividing the extension
at rupture of the film by the initial length of the film (50 mm) multiplied
by 100, and the EM was determined from the slope of the initial straight
line of stress–strain curve.
2.6. Water vapor permeability (WVP) and water contact angle (CA)
Water vapor transmission rate (WVTR) of the film sample was
determined gravimetrically at 25 °C under 50% RH conditions using
water vapor transmission measuring cups in accordance with the
ASTM E96-95 standard method. Then the WVP (g·m/m2·s·Pa) of the
film was calculated using the following equation:
WVP ¼ ðWVTR  LÞ=Δp
where WVTR was the measured water vapor transmission rate (g/m2·s)
through a film, L was the mean film thickness (m) and Δp was the
partial water vapor pressure difference (Pa) across the two sides of
the film.
The contact angle of water in air on the film surface was measured
using a CA analyzer (Model Phoenix 150, Surface Electro Optics Co., Ltd.,
Kunpo, Korea) after a water drop of ca. 10 mL was placed on
the surface of the film using a microsyringe. All the film samples
(3 × 10 cm) were glued on the movable sample stage (black Teflon
coated steel, 7 × 11 cm) leveled horizontally before measurement. The
CA on both sides of the drop was measured to assume symmetry and
horizontal level.
2.7. Thermal stability
The thermal stability of the film samples was tested with thermogra-
vimetric analysis (TGA; Hi-Res TGA 2950 thermogravimetric analyzer,
TA Instruments, New Castle, DE, USA). For this, 5 mg of each sample
was heated from room temperature to 600 °C at a heating rate of
10 °C/min under a nitrogen flow (50 cm3/min). A derivative form of
TGA (DTG) was obtained using differentials of TGA values, which was
calculated using a central finite difference method as follows:
 
DTG ¼ wtþ Δt −wt−Δt =2Δt
where wt + Δt and wt − Δt are the residual weight of sample at
time t + Δt and t − Δt, respectively, and Δt is the time interval
for reading residual sample weight (Rhim, 2012).
2.8. Antibacterial activity
The antibacterial activity of the film samples was evaluated by the
agar disk diffusion method with determination of inhibition zones
(Kanmani and Rhim, 2014b). Two food-borne pathogenic bacteria,
Gram-positive bacterium (Listeria monocytogenes ATCC-19111) and
Gram-negative bacterium (Escherichia coli O157:H7 ATCC-11775)
were used as testing organisms. The Gram-positive and negative bacte-
ria were incubated separately in brain heart infusion (BHI) broth (Difco
Laboratories Inc., Detroit, MI, USA) at 37 °C and in tryptic soy broth
(TSB) (Difco Lab., Inc.) at 30 °C, respectively, under aerobic conditions
for 16 h. Old cultured broths (0.1 mL) from each bacterial strain were
aseptically transferred to Eppendorf tube containing 0.9 mL sterile
water and were serially diluted to two-fold. 1 mL of cells from diluted
broths was uniformly spread on TSB and BHI agar plate. Film samples
prepared in the form of a round disk of 5 mm diameter were placed
carefully on the surface of agar medium and subsequently incubated
at 37 °C for 24 h. Zone of inhibitions was estimated by measuring the
diameter of the bacterial growth inhibition zone around the disk.
2.9. Statistical analysis
Film properties were measured with individually prepared films in
triplicate as the replicated experimental units and the results were pro-
vided with mean ± SD (standard deviation) values. One way analysis of
variance (ANOVA) was performed, and the significance of each mean
property value was determined (p b 0.05) with the Duncan's multiple
range test of the statistical analysis system using the SPSS computer
program (ver. 12.0, SPSS Inc., Chicago, IL, USA).
3. Results and discussion
3.1. Apparent color and optical properties
ð3Þ Free standing flexible films were formed with κ-carrageenan and its
nanocomposite films. Apparently, κ-carrageenan film was transparent
without any color tint, but the nanocomposite films became translucent
with the development of brown or pale brown surface colors. As shown
in Table 1, surface color values of the carrageenan films were greatly
influenced depending on the type of nanofillers. While the lightness
(L-value) of the carrageenan films blended with the clay mineral did
not change significantly (p b 0.05), those blended with AgNPs de-
creased by a factorN2 compared with the neat κ-carrageenan film. In ad-
dition, both Hunter a- and b-values of AgNP included films increased
significantly, which indicates the increase in redness and yellowness
of the nanocomposite films. Consequently, the total color difference
(ΔE) of AgNP included nanocomposite films increased dramatically.
This is mainly due to the development of brown color caused by the
plasmonic effect of AgNPs (Chhatre et al., 2012; Rhim et al., 2013). The
plasmonic effect of AgNPs was clearly shown in the absorption spectra
of the film samples as shown in Fig. 1. Though the neat carrageenan
and carrageenan/clay mineral films did not show any absorption peak
at visible light region, the AgNPs included films exhibited a characteris-
tic absorption peak at 420 nm. It is the evidence of formation of silver
nanoparticles, since AgNPs are known to have surface plasmon reso-
nance transition peaks at visible light region of 400–450 nm (Chhatre
et al., 2012; Pandey et al., 2012).
UV barrier or transparency properties of the films were evaluated by
measuring the transmittance of the films at 280 and 660 nm, and their
ð4Þ results were also shown in Table 1. As observed by the naked eye, the
neat carrageenan film was highly transparent, and showed high transmit-
tance values at both visible and UV ranges (91 and 74.7%, respectively).
Both transmittance values of the films decreased significantly (p b 0.05)
after the formation of nanocomposite with clay mineral and AgNPs,
however, the degree of decrease in transmittance was dependent on
the type of nanofillers. Compared with the neat carrageenan film, the
transmittance of visible (T660 nm) and UV (T280 nm) light of the clay
mineral included composite film decreased by 38% and 61%, respectively,
however, those of the AgNPs included composite film decreased by 93%
and 98%, respectively. This is mainly due to the prevention of light
transmission by the nanoparticles dispersed in the polymer matrix
(Kanmani and Rhim, 2014b). The UV barrier property of packaging film
is desirable to prevent UV light driven reactions such as lipid oxidation,
discoloration, formation of off-flavor, and loss of nutrients of the pack-
aged food (Ramos et al., 2013).
 J.-W. Rhim, L.-F. Wang / Applied Clay Science 97–98 (2014) 174–181 177

Table 1
Apparent surface color and transmittance of carrageenan and carrageenan-based nanocomposite films.

Films L a b ΔE T280 nm (%) T660 nm (%)

Carrageenan 93.3 ± 0.1b −0.52 ± 0.05a 3.91 ± 0.13a 1.48 ± 0.14a 74.7 ± 1.1d 91.0 ± 0.6d
Car/AgNPs 45.3 ± 1.2a 1.35 ± 0.24b 11.91 ± 0.55d 50.05 ± 1.22b 1.5 ± 0.1a 6.0 ± 1.6a
Car/clay 93.6 ± 0.2b −0.70 ± 0.02a 4.75 ± 0.16b 1.96 ± 0.21a 29.0 ± 1.4c 56.5 ± 3.0c
Car/AgNP/clay 43.9 ± 1.6a 3.48 ± 0.80c 10.81 ± 1.04c 51.42 ± 2.52b 2.9 ± 0.1b 9.4 ± 2.4b

Each value is the mean of three replicates with the standard deviation. Any two means in the same column followed by the same letter are not significantly (p N 0.05) different by Duncan's
multiple range tests.

3.2. Characterization
Surface morphology of the carrageenan and carrageenan-based
nanocomposite films was evaluated using SEM, and the results are
shown in Fig. 2. All the films showed homogenous and smooth surface
structures with compact structural integrity. The SEM images did not
show any noticeable differences in microstructure between the neat
carrageenan and nanocomposite films except that the AgNP-included
nanocomposite films showed distinctive AgNP images on the surface.
The SEM images also show that AgNPs are uniformly dispersed in the
nanocomposite films, however, aggregation of AgNPs is observed in
the carrageenan/AgNP/clay mineral composite film. Similar results of
microstructure were observed with gelatin-based nanocomposite
films (Kanmani and Rhim, 2014b).
The crystalline nature of the film samples was evaluated using XRD
analysis and the resulting XRD patterns of the carrageenan and
carrageenan-based nanocomposite films are shown in Fig. 3. As shown
in Fig. 3a, the first broad band and the second minor peak observed
commonly at 2θ around 20° and 28° may be attributed to the carrageen-
an base film (Rhim et al., 2013). Except that, no characteristic diffraction
peaks were observed in the neat carrageenan and carrageenan/clay
mineral films. However, carrageenan/AgNP and carrageenan/AgNP/
clay mineral films exhibited similar diffraction profiles and diffraction
peaks at 2θ of 38.2° and 44.4°, which can be attributed to the (111)
and (200) crystallographic planes of face-centered cubic (fcc) silver
crystals, respectively (Pandey et al., 2012). Unexpectedly, both AgNP-
included films showed unexpected intense peak at 2θ of 32.31°, which
corresponds to the diffraction peak of Na2O2 (Hill et al., 1997). Presum-
ably the Na2O2 originates from sodium citrate used for the reduction of
AgNO3. The carrageenan/AgNP film exhibited more intense peaks than
the carrageenan/AgNP/clay mineral film. The approximate mean crys-
tallite size (D) of the AgNPs in the composite films, calculated using
the Scherrer equation, ranged from 23.7 to 35.6 nm, which was compa-
rable to that of 21.3 to 23.8 nm of agar/AgNP composite films (Rhim
et al., 2013). However, the peak positions of the carrageenan/clay min-
eral composite and carrageenan/AgNP/clay mineral composite films
2.0
1.5
Absorbance (A.U.)
were not changed compared with that of the neat clay mineral powder
as observed in the XRD patterns (Fig. 3b), which indicates that the orig-
inal interlayer spacing (d-spacing) of the clay mineral (1.86 nm) has not
been changed by compositing with carrageenan and AgNPs. This seems
to be contradictory to the finding of Ahmad et al. (2009a) who prepared
nanocomposite of natural clay mineral, Mt, and AgNPs formed by a
chemical reduction method and found that the d-spacing of Mt in-
creased from 1.24 nm to 1.30 nm due to the intercalation of AgNPs.
The difference may be caused by the different type of clay mineral
used in the present study. The organically modified Mt (Cloisite® 30B)
has already been intercalated with quaternary ammonium salt, and
the d-spacing of the organically modified clay mineral has already
been increased from 1.24 nm to 1.86 nm (Rhim et al., 2011). Therefore
simple mixing with AgNPs and Cloisite® 30B did not further increase
the d-spacing of the clay mineral interlayer.
The FT-IR spectra of carrageenan and carrageenan-based nanocom-
posite films were shown in Fig. 4. All the films exhibited characteristic
peaks of carrageenan in the range of 3200–3600 cm− 1, which were
due to the O–H stretching of carrageenan (Hezaveh and Muhamad,
2013). The peaks that appeared at 2935 and 2885 cm−1 correspond to
C–H stretching vibrations of alkane groups in the carrageenan polymer
chain (Hezaveh and Muhamad, 2013). A typical amide I peak at
1644 cm − 1 appeared in the spectra of all nanocomposite films
(Hezaveh and Muhamad, 2013). A peak observed at 1218 cm − 1
corresponded to sulfate ester groups of the carrageenan (Pereira
et al., 2009). A peak observed at 920 cm − 1 corresponded to the 3,
6-anhydro-D-galactose (Distantina et al., 2013a) and a characteristic
peak observed at 844 cm−1 was due to galactose-4-sulfate (Distantina
et al., 2013b). In general, there was no significant changes of functional
groups observed in all of the nanocomposite films including the
nanofillers (AgNPs and clay mineral), which indicates that the structure
of the carrageenan has not been changed in the carbohydrate region
(700–1700 cm−1) by blending with the nanoparticles. However, the
peak intensity increased in all the peaks of AgNP included films,
which is presumably due to the van der Waals interaction between
AgNPs and the carrageenan polymer (Shameli et al., 2010). On the con-
trary, there was no change in intensity of peaks of clay mineral included
films, which may be due to the formation of weak intermolecular hy-
drogen bond between carrageenan and clay mineral particles. Similar
patterns of FT-IR result were observed in the nanocomposite films
with combined use of AgNPs and clay mineral.

3.3. Mechanical properties

1.0
Carrageenan Tensile test was performed using Instron universal testing machine
Carrageenan/Clay to measure the mechanical properties of the film samples, and the re-
Carrageenan/AgNP
sults of the tensile strength (TS), elongation at break (EB), and elastic
Carrageenan/AgNP/Clay
0.5 modulus (EM) of carrageenan and carrageenan-based nanocomposite
films are shown in Table 2. The TS, EB, and EM of the neat carrageenan
film were 56.5 MPa, 4.0%, and 3.6 GPa, respectively, which indicates
that the carrageenan film is mechanically stronger than most of com-
0.0
300 400 500 600 700 modity plastic films or PLA film (Rhim et al., 2013), but the flexibility
Wavelength (nm) of the carrageenan film is very low. The TS of carrageenan nanocompos-
ite films increased significantly (p b 0.05) with an increase in the range
Fig. 1. UV–vis absorption spectra of carrageenan and carrageenan-based nanocomposite of 14.3 to 26.2% compared to that of the neat carrageenan film. The clay
films. mineral was more effective in increasing the TS of carrageenan film than
178 J.-W. Rhim, L.-F. Wang / Applied Clay Science 97–98 (2014) 174–181

Carrageenan Carrageenan/AgNPs

Carrageenan/Clay Carrageenan/AgNPs/Clay

Fig. 2. SEM images of carrageenan and carrageenan-based nanocomposite films.

AgNP. Such increase in TS of nanocomposite films has been frequently
observed with other biopolymer-based nanocomposite films such as
agar/clay mineral (Rhim, 2011) and agar/AgNP nanocomposite films
(Rhim et al., 2013). The increase in mechanical strength of the nano-
composite films is mainly attributed to the physical attraction between
the filler and polymer matrix. The higher TS of the carrageenan-based
nanocomposite films may be explained by the increase of contact area
between carrageenan and nanoparticles. Shameli et al. (2010) found
through FT-IR test that the major force between biopolymer and
AgNPs of their nanocomposite film was due to the presence of van der
Waals interactions between the hydroxyl groups of biopolymer and
the partial positive charge on the surface of the AgNPs. However, the
EM of all nanocomposite films, namely, the stiffness of the films,
decreased significantly by blending with clay mineral or AgNPs.
3.4. Water vapor permeability (WVP) and water contact angle (CA)
The WVP of the carrageenan and carrageenan-based nanocomposite
films are also shown in Table 2. The WVP of the neat carrageenan
film was 1.29 × 10 − 9 g·m/m 2·Pa·s, but it decreased significantly
(p b 0.05) after blending with nanofillers. The WVP of carrageenan/
clay mineral nanocomposite film was lower than that of carrageenan/
AgNP nanocomposite film, and the WVP of nanocomposite film blend-
ing with both nanofillers was in-between. This is presumably because

a b
Intensity (a.u.)

Clay (Cloisite 30B)

Carrageenan/Clay
Carrageenan/AgNPs/Clay

0 2 4 6 8 10
2 degree)

Fig. 3. X-ray diffractograms of carrageenan and carrageenan-based nanocomposite films observed at higher angles (2θ = 10–90°) for silver nanoparticles and observed at lower
angles (2θ = 1–10°) for clay nanoparticles.
 J.-W. Rhim, L.-F. Wang / Applied Clay Science 97–98 (2014) 174–181 179

Fig. 4. FT-IR spectra of κ-carrageenan and κ-carrageenan-based nanocomposite films.

of the difference in the shape or structure of nanofillers, i.e., layered

plate-like structure of the clay mineral and spherical shape of the
AgNPs. The clay mineral dispersed in the polymer matrix might develop
more efficient tortuous path for moisture vapor diffusion then the
spherical AgNPs. A similar result on the effect of nanofillers on the
WVP that has been observed with gelatin-based films has been reported
(Kanmani and Rhim, 2014a, 2014b). The CA of the carrageenan and
carrageenan-based nanocomposite films is also shown in Table 2. The
CA is usually used as a measure of the film surface hydrophobicity or hy-
drophilicity (Phan et al., 2009; Su et al., 2010). It is generally used as a
measure of wettability of surface or an estimation of the resistance of
the film against liquid water. The CA of the neat carrageenan film was
48.0 ± 0.8°, which was consistent with previously reported value of
48.09 ± 1.69° (Rhim, 2012). The CA of nanocomposite films has been
increased significantly (p b 0.05) compared with that of the neat carra-
geenan film. Not like the WVP, the CA of AgNP-included nanocomposite Fig. 5. TGA and DTG thermograms of carrageenan and carrageenan-based nanocomposite
films increased more than that of clay mineral-include nanocomposite films.

film. This is probably because the AgNPs are more hydrophobic than
the clay mineral. According to the manufacturer's report, the organoclay
mineral used in the present study (Cloisite® 30B) is the least hydropho-
bic Mt clay mineral among their organically modified clay mineral pro-
duction lines. However, the CA of all the nanocomposite films was less
than 65°, so the film surface of nanocomposite films is still considered
as hydrophilic (Vogler, 1998).
3.5. Thermal stability
Thermal stability of the carrageenan and carrageenan-based
nanocomposite films was tested by TGA analysis, and the results
of TGA thermograms and their DTG curves are shown in Fig. 5. The TGA
thermograms exhibit the weight decreasing pattern of the films during
thermal decompositions and the DTG curves indicate clearly the inflec-
tion point of thermal decomposition. As shown in the TGA thermograms,
thermal decomposition of the carrageenan and carrageenan-based nano-
composite films followed multistep decomposition. Each step of thermal
destruction is attributed to evaporation of moisture, volatilization of
glycerol used as a plasticizer, and decomposition of polymer matrix,
which were observed around 100 °C, 290 °C, and 400 °C, respectively
(Rhim, 2013). After the final thermal destruction, residuals of the neat
carrageenan, carrageenan/AgNP, carrageenan/clay mineral, and carra-
geenan/AgNP/clay mineral films were 40.1%, 42.7%, 41.3%, and 44.5%,
respectively. TGA results revealed that thermal stability of carrageenan
film increased after the formation of nanocomposite with clay and
AgNPs, in which the effect was more pronounced in the AgNP-included
films.
3.6. Antimicrobial activity
The antimicrobial activity of carrageenan and carrageenan-based
nanocomposite films was investigated using agar well diffusion method
using food-borne pathogens such as E. coli and L. monocytogenes. The
results of the antimicrobial activities determined by the diameter of in-
hibition zone (DI) of the film samples are shown in Fig. 6. As expected,

Table 2
Tensile properties, water vapor permeability (WVP), and water contact angle (CA) of the carrageenan and carrageenan-based nanocomposite films.

Films TS EB EM WVP CA
(MPa) (%) (GPa) (×10−9 g·m/m2·Pa·s) (°)

Carrageenan 56.5 ± 20.1a 4.0 ± 0.7a 3.6 ± 0.3b 1.29 ± 0.12c 48.0 ± 0.8a
Car/AgNPs 64.6 ± 20.3ab 6.2 ± 4.6b 3.1 ± 0.4a 1.14 ± 0.07bc 62.5 ± 0.9d
Car/clay 71.3 ± 8.4b 4.9 ± 1.0ab 3.1 ± 0.3a 0.94 ± 0.11a 50.5 ± 1.3b
Car/AgNPs/clay 68.9 ± 11.7b 5.2 ± 1.1ab 3.2 ± 0.4a 1.09 ± 0.05ab 56.3 ± 2.0c

Each value is the mean of three replicates with the standard deviation. Any two means in the same column followed by the same letter are not significantly (p N 0.05) different by Duncan's
multiple range tests.
180 J.-W. Rhim, L.-F. Wang / Applied Clay Science 97–98 (2014) 174–181
E. coli
Car/AgNP Carrageenan
Car/AgNPs/Clay
Car/Clay
Fig. 6. Antimicrobial activity of carrageenan and carrageenan-based nanocomposite films against E. coli and L. monocytogenes.
the neat carrageenan film didn't show any inhibitory activity against
both Gram-positive (L. monocytogenes) (DI = 5 mm) and Gram-
negative (E. coli) bacteria (DI = 5 mm), however, the composite films
included with AgNPs or clay mineral exhibited clear antibacterial activ-
ity against both pathogens. The AgNP-included nanocomposite films
showed pronounced antimicrobial activity against Gram-negative bac-
teria (DI = 7.96 mm) with slight antimicrobial activity against Gram-
positive bacteria (DI = 5.76 mm). On the contrary, the clay-included
nanocomposite film showed distinctive antimicrobial activity against
Gram-positive bacteria (DI = 6.26 mm) with slight antimicrobial activ-
ity against Gram-negative bacteria (DI = 5.21 mm). However, the com-
bined use of clay mineral and AgNPs exhibited more pronounced
antimicrobial activity against both Gram-positive (DI = 6.34 mm) and
Gram-negative bacteria (DI = 7.43 mm). Similar results on antimicrobi-
al activity of the organoclay mineral and AgNPs have been observed
with gelatin-based nanocomposites (Kanmani and Rhim, 2014b).
The difference in antimicrobial activity of both nano-fillers may be
attributed to their structure and mode of antimicrobial action as well
as difference in the cell wall structure of indicator microorganisms.
Gram-positive bacteria are composed of a three-dimensional thick
peptidoglycan layer consisting of linear polysaccharide chains cross-
linked with short peptides to form a complex cell wall structure,
which might hinder the AgNPs to penetrate into Gram-positive bacteria
(Priyadarshini et al., 2013). On the contrary, Gram-negative bacteria are
coated with negatively charged outer membrane with thin peptidogly-
can layer which might facilitate AgNPs to penetrate more easily into the
Gram-negative bacteria (Priyadarshini et al., 2013). The antimicrobial
activity of the organoclay mineral is mainly due to the strong antimicro-
bial activity of alkyl quaternary ammonium salt group (methyl tallow
bis-2-hydroxyethyl quaternary ammonium) in the organoclay mineral,
Cloisite® 30B (Hong and Rhim, 2008). The quaternary ammonium salt
is expected to bind irreversibly to the phospholipids and proteins of
the bacterial cell membranes to disrupt the lipid bilayers of the cell
membrane, and to cause cell lysis. Although the clear mechanism of
antimicrobial activity of AgNPs has not been established yet, several
mechanistic actions for antimicrobial activity of AgNPs have been
reported. One is cell membrane disruption due to the interaction of
AgNPs with phosphorous and sulfur containing compounds of proteins
and DNA, preventing DNA replication, and leading to cell death (Morones
et al., 2005). The second is binding of the positively charged AgNPs
with negatively charged bacterial cell membranes, which disrupt cell
walls and surface proteins leading to cell death (Sui et al., 2006). A third
is the penetration of AgNPs into the bacteria, which inactivates the
enzymes and produces H2O2 leading to cell death (Raffi et al., 2008).
Though the carrageenan-based nanocomposite films that each
L. monocytogenes
Carra/AgNPs Carrageenan
Car/AgNPs/Clay
Car/Clay
composed of nanofiller have not exhibited antimicrobial activity
comprehensively against both Gram-positive and Gram-negative
bacteria, the antimicrobial activity of the carrageenan-based nanocom-
posite film has been greatly improved by the combined use of both
AgNPs and the organoclay mineral. The carrageenan-based nanocom-
posite films with strong antimicrobial activity against both Gram-
positive and Gram-negative pathogenic bacteria are expected to have
high potential for the application as an active antimicrobial food pack-
aging material.
4. Conclusion
Antimicrobial nanocomposites films were prepared with carrageen-
an and antimicrobial nanofillers such as AgNPs and organically modified
clay mineral. Both nanofillers influenced film properties such as surface
color, mechanical, water vapor barrier, thermal, and antimicrobial prop-
erties. The AgNP-included nanocomposite films exhibited characteristic
plasmonic effect of AgNPs with the maximum light absorption at
420 nm and all the nanocomposite films, especially composed of
AgNPs, showed UV barrier property. The improved UV barrier property
of the composite film can be used as a UV screening food packaging film.
Mechanical strength (determined by the TS), water vapor barrier prop-
erty (determined by the WVP), and thermal stability (determined by
TGA analysis) of all the nanocomposite films increased significantly. In
addition, the carrageenan-based nanocomposite films exhibited charac-
teristic antimicrobial activity against both Gram-positive and Gram-
negative food borne pathogenic bacteria depending on the type of
nanofiller. While the AgNP-included nanocomposite film exhibited
strong antimicrobial activity against Gram-negative bacteria, the organ-
ically modified clay-included nanocomposite films exhibited strong
antimicrobial activity against Gram-positive bacteria. The combined
use of nanofillers showed strong antimicrobial activity against both
Gram-positive and Gram-negative bacteria. These results suggests that
the carrageenan-based nanocomposite films with promising antimicro-
bial activity with improved film properties have a high potential for the
application as an active packaging to secure food safety and to prolong
the shelf-life of packaged foods.
Acknowledgments
This research was supported by R&D Convergence Support Program
(ARC 710003) of the Ministry for Agriculture, Food and Rural affairs,
Republic of Korea.
 J.-W. Rhim, L.-F. Wang / Applied Clay Science 97–98 (2014) 174–181
References
Ahmad, M.B., Shameli, K., Darroudi, M., Yunus, W.M.Z.W., Ibrahim, N.A., 2009a. Synthesis
and characterization of silver/clay nanocomposites by chemical reduction method.
Am. J. Appl. Sci. 6, 1909–1914.
Ahmad, M.B., Shameli, K., Darroudi, M., Yunus, W.M.Z.W., Ibrahim, N.A., Hamid, A.A.,
Zagar, M., 2009b. Antibacterial activity of silver/clay/chitosan bionanocomposites.
Res. J. Biol. Sci. 4, 1156–1161.
Appendini, P., Hotchkiss, J.H., 2002. Review of antimicrobial food packaging. Innov. Food
Sci. Emerg. Technol. 3, 113–126.
Armentano, I., Bitinis, N., Fortunati, E., Mattioli, S., Rescignano, N., Verdejo, R., Lopez-
Manchado, M.A., Kenny, J.M., 2013. Multifunctional nanostructured PLA materials
for packaging and tissue engineering. Prog. Polym. Sci. 38, 1720–1747.
Borchert, H., Shevchenko, E.V., Robert, A., Mekis, I., Kornowski, A., Grübel, G., et al., 2005.
Determination of nanocrystal sizes: a comparison of TEM, SAXS, and XRD studies of
highly monodisperse CoPt3 aprticles. Langmuir 21, 1931–1936.
Carja, G., Kameshima, Y., Nakajima, A., Dranca, C., Okada, K., 2009. Nanosized silver-
anionic clay matrix as nanostructured ensembles with antimicrobial activity. Int. J.
Antimicrob. Agents 34, 534–539.
Cha, D.S., Choi, J.H., Chinnan, M.S., Park, H.J., 2002. Antimicrobial films based on Na-
alginate and κ-carrageenan. LWT Food Sci. Technol. 35, 715–719.
Chhatre, A., Solasa, P., Sakle, S., Thaokar, R., Mehra, A., 2012. Color and surface plasmon
effects in nanoparticles systems: case of silver nanoparticles prepared by micro-
emulsion route. Colloids Surf. A Physicochem. Eng. Aspects 404, 83–92.
De Azeredo, H.M.C., 2009. Nanocomposite for food packaging applications. Food Res. Int.
42, 1240–1253.
Distantina, S., Rochmadi, Fahrurrozi, M., Wiratni, 2013a. Synthesis of hydrogel film based
on carrageenan extracted from Kappaphycus alvarezii. Mod. Appl. Sci. 7 (8), 22–30.
Distantina, S., Rochmadi, Fahrurrozi, M., Wiratni, 2013b. Preparation and characterization
of glutaraldehyde-crosslinked kappa carrageenan hydrogel. Eng. J. 17 (3), 57–66.
Duncan, T.V., 2011. Applications of nanotechnology in food packaging and food safety:
barrier materials, antimicrobials and sensors. J. Colloid Interface Sci. 363, 1–24.
Falguera, V., Quintero, J.P., Jiménez, A., Muñoz, J.A., Ibarz, A., 2011. Edible films and
coatings: structures, active functions and trends in their use. Trends Food Sci.
Technol. 22, 292–303.
Hezaveh, H., Muhamad, I.I., 2013. Modification and swelling kinetic study of kappa-
carrageenan-based hydrogel for controlled release study. J. Taiwan Inst. Chem. Eng.
44, 182–191.
Hill, G.S., Holah, D.G., Kinrade, S.D., Magnuson, V.R., Polyakov, V., Sloan, T.A., 1997. The X-
ray structure of a sodium peroxide hydrate, Na2O2·H2O, and its reactions with carbon
dioxide: relevance to the brightening of mechanical pulps. Can. J. Chem. 75, 46–51.
Hong, S.I., Rhim, J.W., 2008. Antimicrobial activity of organically modified nanoclay. J.
Nanosci. Nanotechnol. 8, 5818–5824.
Incoronato, A.L., Buonocore, G.C., Conte, A., Lavorgna, M., Del-Nobile, M.A., 2010. Active
systems based on silver-montmorillonite nanoparticles embedded into bio-based
polymer matrices for packaging applications. J. Food Prot. 73, 2256–2262.
Kanmani, P., Rhim, J.M., 2014a. Properties and characterization of biocomposite films
prepared with various biopolymers and ZnO nanoparticles. Carbohydr. Polym. 106,
190–199.
Kanmani, P., Rhim, J.M., 2014b. Physical, mechanical and antimicrobial properties of
gelatin based active nanocomposite films containing AgNPs and nanoclay. Food
Hydrocoll. 35, 644–652.
Kim, Y.H., Lee, D.K., Cha, H.G., Kim, C.W., Kang, Y.S., 2007. Synthesis and characterization
of antimicrobial Ag–SiO2 nanocomposite. J. Phys. Chem. C 111, 3629–3635.
Llorens, A., Lloret, E., Picouet, P.A., Trbojevich, R., Fernandez, A., 2012. Metallic-based
micro and nanocomposites in food contact materials and active food packaging.
Trends Food Sci. Technol. 24, 19–29.
Malchová, K., Praus, P., Pavlíčkvá, Z., Turicová, M., 2009. Activity of antimicrobial
compounds immobilized on montmorillonite. Appl. Clay Sci. 42, 364–368.
Martins, J.T., Bourbon, A.I., Pinheiro, A.C., Souza, B.W.S., Cerqueira, M.A., Vicente, A.A.,
2013. Biocomposite films based on κ-carrageenan/locust bean gum blends and
clays: physical and antimicrobial properties. Food Bioprocess Technol. 6, 2081–2092.
Morones, J.R., Elechiguerra, J.L., Camacho, a., Holt, K., Kouri, J.B., Ramirez, J.T., 2005. The
bactericidal effect of silver nanoparticles. Nanotechnology 16, 2346–2352.
Ojijo, V., Ray, S.S., 2013. Processing strategies in bionanocomposites. Prog. Polym. Sci. 38,
1543–1589.
181
Pandey, S., Goswami, G.K., Nanda, K.K., 2012. Green synthesis of biopolymer-silver nano-
particle nanocomposite: an optical sensor for ammonia detection. Int. J. Biol.
Macromol. 51, 583–589.
Pereira, L., Amado, A.M., Critchley, A.T., Velde, F.V.D., Ribeiro-Claro, P.J.A., 2009. Identifica-
tion of selected seaweed polysaccharides (phycocolloids) by vibrational spectroscopy
(FTIR-ATR and FT-Raman). Food Hydrocoll. 23, 1903–1909.
Phan, T.D., Debeafort, F., Kuu, D., Voilley, A., 2009. Biopolymer interactions affect the func-
tional properties of edible film based on agar, cassava and arabinoxylan blends. J.
Food Eng. 90, 548–558.
Priyadarshini, S., Gopinath, V., Meera Priyadharsshini, N., MubarakAli, D., Velusamy, P.,
2013. Synthesis of anisotropic silver nanoparticles using novel strain, Bacillus flexus
and its biomedical application. Colloids Surf. B Biointerfaces 102, 232–237.
Raffi, M., Hussain, F., Bhatti, T.M., Akhter, J.I., Hameed, A., Hasan, M.M., 2008. Antibacterial
characterization of silver nanoparticles against E. coli ATCC 15224. J. Mater. Sci.
Technol. 24, 192–196.
Rai, M., Yadav, A., Gade, A., 2009. Silver nanoparticles as a new generation of antimicro-
bials. Biotechnol. Adv. 27, 76–83.
Ramos, O.L., Reinas, I., Silva, S.I., Fernandes, J.C., Cerqueira, M.A., Pereira, R.N., Vicente, A.A.,
Fatima Poças, M., Pintado, M.E., Malcata, F.X., 2013. Effect of whey protein purity and
glycerol content upon physical properties of edible films manufactured therefrom.
Food Hydrocoll. 30, 110–122.
Raquez, J.M., Habibi, Y., Muraiu, M., Dubois, P., 2013. Polylactide (PLA)-based nanocom-
posites. Prog. Polym. Sci. 38, 1504–1542.
Reddy, M.M., Vivekanandhan, S., Misra, M., Bhatia, S.K., Mohanty, A.K., 2013. Biobased
plastics and bionanocomposites: current status and future opportunities. Prog.
Polym. Sci. 38, 1653–1689.
Rhim, J.W., 2011. Effect of clay contents on the mechanical and water vapor barrier prop-
erties of agar-based nanocomposite films. Carbohydr. Polym. 86, 691–699.
Rhim, J.W., 2012. Physical–mechanical properties of agar/κ-carrageenan blend film and
derived clay nanocomposite film. J. Food Sci. 77 (12), N66–N73.
Rhim, J.W., 2013. Effect of PLA lamination on performance characteristics of agar/κ-
carrageenan/clay bio-nanocomposite film. Food Res. Int. 51, 714–722.
Rhim, J.W., Wang, L.F., 2013. Mechanical and water barrier properties of agar/κ-
carrageenan/konjac glucomannan ternary blend hydrogel film. Carbohydr. Polym.
96, 71–81.
Rhim, J.W., Hong, S.I., Ha, C.S., 2009. Tensile, water vapor barrier and antimicrobial
properties of PLA/nanoclay composite films. LWT Food Sci. Technol. 42, 612–617.
Rhim, J.W., Lee, S.B., Hong, S.I., 2011. Preparation and characterization of agar/clay
nanocomposite films: the effect of clay type. J. Food Sci. 76 (3), N40–N48.
Rhim, J.W., Wang, L.F., Hong, S.I., 2013. Preparation and characterization of agar/silver
nanoparticles composite films with antimicrobial activity. Food Hydrocoll. 33,
327–335.
Shameli, K., Ahmad, M.B., Yunus, W.M.Z.W., Ibrahim, N.A., Rahman, R.A., Kokar, M.,
Darroudi, M., 2010. Silver/poly(lactic acid) nanocomposites: preparation, characteri-
zation, and antibacterial activity. Int. J. Nanomed. 5, 573–579.
Silvestre, C., Duraccio, D., Cimmino, S., 2011. Food Packaging based on polymer
nanomaterials. Prog. Polym. Sci. 36, 1766–1782.
Sorrentino, A., Gorrasi, G., Vittoria, V., 2007. Potential perspectives of bionanocomposites
for food packaging applications. Trends Food Sci. Technol. 18 (2), 84–95.
Sozer, N., Kokini, J.L., 2008. Nanotechnology and its applications in the food sector. Trends
Biotechnol. 27 (2), 82–89.
Su, J.F., Huang, Z., Zhao, Y.H., Yuan, X.Y., Wang, X.Y., Li, M., 2010. Structure and properties
of carboxymethyl cellulose/soy protein isolate blend edible films cross linked by
Maillard reactions. Carbohydr. Polym. 79, 145–153.
Sui, Z.M., Chen, X., Wang, L.Y., Xu, L.M., Zhuang, W.C., Chai, Y.C., Yang, C.J., 2006. Capping
effect of CTAB on positively charged Ag nanoparticles. Phys. E. 33, 308–314.
Vogler, E.A., 1998. Structure and reactivity of water at biomaterial surfaces. Adv. Colloid
Interface Sci. 74, 69–117.
Xie, F., Pollet, E., Halley, P.J., Avérous, L., 2013. Starch-based nano-biocomposites. Prog.
Polym. Sci. 38, 1590–1628.
Yoksan, R., Chirachanchai, S., 2010. Silver nanoparticle-loaded chitosan-starch based
films: fabrication and evaluation of tensile, barrier and antimicrobial properties.
Mater. Sci. Eng. C 30, 891–897.