Food Hydrocolloids 44 (2015) 309e319

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

Characterization of tara gum edible films incorporated with bulk

chitosan and chitosan nanoparticles: A comparative study
John Antoniou, Fei Liu, Hamid Majeed, Fang Zhong*
Key Laboratory of Food Colloids and Biotechnology, Ministry of Education, School of Food Science and Technology, Jiangnan University, Wuxi 214122,
PR China

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

Article history: Tara gum films were successfully produced with the inclusion of bulk chitosan or chitosan nanoparticles
Received 29 April 2014 at various concentrations. The composites films were compared in terms of antimicrobial activity,
Accepted 9 September 2014 thermomechanical, physicochemical and barrier properties. The thermal stability of the films was
Available online 28 September 2014
studied using thermogravimetric analysis (TGA). Fourier-transform infrared spectroscopy (FTIR) and X-
ray diffraction (XRD) measurements were used to study the interactions and compatibility between the
Keywords:
polysaccharides with in the films. The microstructure of the films was analyzed using atomic force
Antimicrobial edible films
microscopy (AFM) and scanning electron microscopy (SEM). Incorporation of chitosan nanoparticles
Galactomannan
Tara gum
improved mechanical, physicochemical and barrier properties. Tensile strength was increased by
Chitosan nanoparticles 35.73 MPa while the elongation was decreased by 7.21%. Water solubility and water vapor permeability
Characterization (WVP) were reduced by 74.3% and 22.7%, respectively. The compact structure of the chitosan nano-
particles reduced the free volume of the polymer matrix more than bulk chitosan by obstructing the
diffusion of water and thereby decreasing the moisture content of the films. Additionally, the micro-
structure of the films showed that the nanoparticles were distributed homogenously with in the
structure and increased the roughness of the surface. However, tara gum films with bulk chitosan
exhibited better antimicrobial activity. Incorporation of chitosan nanoparticles produced films less
effective against Escherichia coli compared to Staphylococcus aureus, and their antimicrobial activity was
reduced at high concentrations probably due to agglomeration.
© 2014 Published by Elsevier Ltd.

1. Introduction limitation of their industrial use. Over recent years, in order to

Natural galactomannans are an alternative material that can be
used for the production of edible films/coatings based on their
edibility and biodegradability (Cerqueira, Bourbon, et al., 2011).
Galactomannans are reserve polysaccharides composed of linear
chains of (1 / 4)-b-D-mannopyranosyl residues having single side
chain units of (1 / 6)-a-D-galactopyranosyl (Dey, 1978). Among
the most commercial galactomannans is tara gum (TG) which is the
ground endosperm of the seeds of tara tree (Cesalpinia spinosa).
Galactose substitution in TG occurs on approximately every third
mannose unit. TG functions mainly as a good thickener and stabi-
lizer but due to the lower galactose substitution compared to
fenugreek and guar gum it can also produce a stronger film
(Embuscado & Huber, 2009). However, relatively poor mechanical
and water vapor barrier properties of those films result in a major
* Corresponding author. Tel.: þ86 510 85328307.
E-mail address: fzhong@jiangnan.edu.cn (F. Zhong).
overcome these drawbacks of biodegradable films, different types
of nano-fillers have being introduced. By adding appropriate
nanoparticles, it is possible to produce materials with improved
mechanical reinforcement, higher thermal stability and barrier
properties, and lower moisture sensitivity (Chivrac, Pollet, &
Ave
rous, 2009; Sorrentino, Gorrasi, & Vittoria, 2007).
In view of maintaining the edibility of the films much attention
has been focused on polysaccharide nano-fillers. The most common
polysaccharides used for nanoparticle production in edible films
are cellulose, starch and chitosan. Cellulose nanoparticles such as
microcrystalline cellulose, nano-fibrils cellulose and cellulose
whiskers, have been used for reinforcing hydroxypropyl methyl
cellulose based films (Bilbao-Sa
inz, Avena-Bustillos, Wood, Wil-
liams, & McHugh, 2010; Bilbao-Sainz, Bras, Williams, Se
nechal, &
Orts, 2011). Likewise, Azeredo et al. (2010) and Chang, Jian, Zheng,
Yu, and Ma (2010) improved the mechanical and water vapor bar-
rier properties of chitosan and glycerol-plasticized starch films
respectively with the addition of cellulose nanostructures in the
polymer matrix. Similar results have been reported for starch-

http://dx.doi.org/10.1016/j.foodhyd.2014.09.023
0268-005X/© 2014 Published by Elsevier Ltd.
310 J. Antoniou et al. / Food Hydrocolloids 44 (2015) 309e319
based nanoparticles. Angellier, Molina-Boisseau, Dole, and
Dufresne (2006) and Kristo and Biliaderis (2007) have shown the
reinforcing effect of waxy maize starch nano-crystals in glycerol-
plasticized starch and sorbitol-plasticized pullulan films. Spray
dried and vacuum freeze dried starch nanoparticles have also been
successfully incorporated into starch films (Shi, Wang, Li, &
Adhikari, 2013a, 2013b).
Among the polysaccharides that produce nano-fillers with
attractive features is chitosan. Chitosan nanoparticles can be pro-
duced based on ionic gelation as a result of inter- and intra-
molecular cross-linking of chitosan's protonated amino groups by
multivalent polyanions. Sodium tripolyphosphate (TPP) is a very
popular polyanion because it is non-toxic and forms gels with
desirable properties (De Moura et al., 2009). Chitosan nanoparticles
have been successfully used as fillers to improve mechanical and
barrier properties as well as the thermo-stability of films, decrease
solubility and produce more compact and dense materials (Chang,
Jian, Yu, & Ma, 2010a; De Moura, Avena-Bustillos, McHugh, Krochta,
& Mattoso, 2008; De Moura et al., 2009). Additionally, chitosan
compared to other bio-based food packaging materials has the
advantage of antibacterial activity (Dutta, Tripathi, Mehrotra, &
Dutta, 2009; Lei et al., 2014). The antimicrobial activity of chito-
san has been observed against a wide variety of microorganisms
including fungi, algae, and bacteria (Huang et al., 2012; Rabea,
Badawy, Stevens, Smagghe, & Steurbaut, 2003). The broad antimi-
crobial properties of chitosan nanoparticles have also been re-
ported and has been attributed to their high surface area and
charge density (Qi, Xu, Jiang, Hu, & Zou, 2004; Xing, Chen, Liu, Cha,
& Park, 2009). However, a direct comparison of the antimicrobial
activity between bulk chitosan and chitosan nanoparticles within
films has not been reported.
In this study, chitosan nanoparticles (CSNPs) and bulk chitosan
(CS) were incorporated in glycerol-plasticized TG films. The aim of
the study was to compare the effect of bulk CS and CSNPs on TG
films in terms of thermomechanical, physicochemical, morpho-
logical and barrier properties. Additionally, the antimicrobial ac-
tivity of the composite films was examined against Escherichia coli
(Gram-negative) and Staphylococcus aureus (Gram-positive), two of
the most currently observed food-borne bacteria. The structural
changes of TG film were examined by FTIR and XRD analysis.
2. Materials and methods
2.1. Materials
Tara gum was obtained from Dymatic Chemicals Co., Ltd.
(Guangdong, China) with molecular weight ~1000 kDa which was
determined by gel permeation chromatography (GPC) (Tosoh Corp.,
HLC-8320GPC, Japan). Chitosan of molecular mass 100 kDa and DD
of 90% derived from crab shells was obtained from Golden-Shell
Biochemical Co., Ltd. (Hangzhou, China). Sodium tripolyphos-
phate was purchased from Sinopharm Chemical Reagent Co., Ltd
(Shanghai, China). E. coli HB2151 and S. aureus ATCC 25923 were
purchased by Distance Biotechnology Co., Ltd. (Hangzhou, China)
and Haibo Biotechnology Co., Ltd. (Qingdao, China), respectively.
Other reagents were all commercially available and used as
received. Distilled water was used in all experiments.
2.2. Preparation of chitosan nanoparticles (CSNPs)
CSNPs were obtained according to the procedure first reported
by Calvo, Remunan-Lopez, Vila-Jato, and Alonso (1997) with mod-
ifications. The formation is based on the ionic gelation of CS with
TPP anions. In an acidic environment CS is solubilized by proton-
ation of the amino groups, rendering the polymer positively
charged. CS (1.5 mg/mL) was dissolved in aqueous solution con-
taining acetic acid at concentration 3 times that of CS solution. TPP
was obtained in aqueous solution at a concentration of 2.0 mg/mL.
The TPP solution was added drop-wise to the CS solution under
vigorous magnetic stirring at room temperature and the formation
of CSNPs started spontaneously via the TPP-initiated ionic gelation
mechanism. The final solution was ultra-sonicated (VCX 500, Sonics
and Materials Inc., USA) for 0.5 min in order to break any aggre-
gations and reduce particle size.
2.3. Characterization of CSNPs
2.3.1. Measurement of particle size and z-potential of CSNPs
The measurements of particle size, polydispersity index (PDI)
and z-potential of nanoparticles were performed using a Zetasizer
Nano-ZS (Malvern Instruments, Worcestershire, UK) on the basis of
dynamic light scattering (DLS) techniques.
2.3.2. Transmission electron microscopy (TEM)
The morphology of CS and CSNPs was examined by a high-
performance digital imaging TEM (JEOL 2100, Hitachi High-
Technologies Corp., Tokyo, Japan). One drop of the suspended so-
lution was spread onto a carbon-coated copper grid and stained
with 2% (w/v) phosphotungstic acid. After drying at room tem-
perature the samples were placed for TEM analysis using acceler-
ating voltage of 100 kV.
2.4. Preparation of edible films
The TG composite solutions were prepared using the method
described by Pinheiro et al. (2011) and Martins et al. (2012) with
modifications. CSNPs and CS solutions (150 mL) were prepared at
different concentrations 0, 5, 10, 15% w/w on dry basis of the weight
of TG. 1% w/v TG was dissolved in each solution at 45  C for 2 h until
a homogeneous solution was obtained. 20% w/v glycerol was added
as a plasticizer in the solution in order to make the final films less
brittle and easier to handle. The acetic acid concentration was the
same for all solutions (0.42% v/v). Film-forming solutions were then
centrifuged at 3000 g for 10 min to remove un-dissolved matter and
air bubbles. 75 mL of solutions were poured into square plastic petri
dishes (10  10 cm) and dried at 35  C for 24 h. The obtained films
were conditioned at 53 ± 1% RH and 25 ± 1  C for at least 3 days in a
controlled environmental chamber (Shanghai, China).
2.5. Film characterization
2.5.1. Film thickness
Film thickness was measured with a digital micrometer
(Shanghai, China) to the nearest 0.001 mm. Ten thickness mea-
surements were taken on each testing sample in different points
and the mean values were used in mechanical and permeability
calculations.
2.5.2. Mechanical properties
A texture analyzer (Stable Micro Systems, Surrey, UK) was used
in order to measure mechanical properties according to ASTM D882
(ASTM, 2001) with some modifications. The films were initially cut
into strips (2  8 cm) after their equilibration at 53 ± 1% RH for 72 h.
The initial grip separation and crosshead speed used were set at
50 mm and 0.5 mm/s, respectively. Tensile strength (TS) and
elongation (E%) were calculated from the plot of stress (tensile
force/initial cross-sectional area) versus strain (extension as a
fraction of the original length) using the following Equations (1)
and (2):
 J. Antoniou et al. / Food Hydrocolloids 44 (2015) 309e319
TS ¼ F=ðL  XÞ
E% ¼ 100%  ðl  l1 Þ=l1
where F is the tensile force (N), L the width of the film (mm), X the
thickness (mm), l1 is the initial length of the film and l is the length
of the film at breaking point.
2.5.3. Dynamic mechanical analysis (DMA)
DMA (TA Instruments, Q800, New Castle, Del., USA) was per-
formed in order to measure stiffness and damping of the samples
which are reported as storage modulus (E0 ), loss modulus (E00 ) and
tan d (¼E00 /E0 ). Samples with dimensions 1  2 cm were analyzed
using frequency sweep tests. After equilibrating at 25  C for 5 min,
the frequency sweep tests were performed at a frequency range of
0.1e100 Hz at amplitude of 10 mm (within the LVR) and a preload
force of 0.01 N.
2.5.4. Thermogravimetric analysis (TGA)
TGA measurements were carried out in N2 atmosphere using a
Mettler Toledo TGA 1 Thermogravimetric Analyzer (Greifensee,
Switzerland). Samples were weighed at approximately 5e10 mg of
dry matter. Measurements started at 25  C and continued up to
500  C, with a linear increase of 10  C min1. Data analysis was
performed with STARe Thermal Analysis Software Version 12.10.
2.5.5. Moisture content
The moisture content (M%) of the films was determined by
drying 2  2 cm pieces at 105  C for 24 h. The samples were initially
equilibrated at 53 ± 1% RH for 72 h and after drying they were
cooled to room temperature at 0% RH in a desiccator containing
silica gel. The percent moisture content was calculated using the
following Equation (3):
M% ¼ 100%  ðW0  W1 Þ=W0
where W0 and W1 are the weight of initial and dried samples
respectively.
2.5.6. Water solubility
Water solubility (S%) was expressed as percentage of the film
dry matter solubilized after 24 h immersion in distilled water and it
was based on the method described by Cuq, Gontard, Cuq, and
Guilbert (1996) with a few modifications. Pre-weighted (W0)
dried sheet films (2  2 cm) were immersed in 30 mL distilled
water at 25 ± 1  C under constant agitation for 24 h followed by
centrifugation at 150 g for 10 min. The un-dissolved matter was
dried at 105  C for 24 h until constant weight (W1). All tests were
carried out in tetra-plicate and water solubility (S%) was calculated
using the following Equation (4):
S% ¼ 100%  ðW0  W1 Þ=W0
2.5.7. Water vapor permeability (WVP)
The water vapor permeability (WVP) and water vapor trans-
mission rate (WVTR) were determined according to ASTM E96
(ASTM, 1995) with some modifications. Initially the films were cut
in square pieces (4  4 cm) after equilibration at 53 ± 1% RH for
72 h. The square pieces were sealed in cups containing 10 mL of
distilled water and placed in desiccator containing silica gel. The
weight of the cups was monitored every 2 h over 24 h. The slope of
each line was calculated by linear regression (R2 > 0.99) of weight
change vs. time. WVTR and WVP were calculated based on
311
(1) Equations (5) and (6) by Gennadios, Weller, and Gooding (1994)
and McHugh, Avena-Bustillos, and Krochta (1993):
(2)
WVTR ¼ Slope=Film area (5)
WVP ¼ ðWVTR  LÞ=ðPA1  PA2 Þ (6)
where PA1 is the vapor partial pressure at film outer surface in the
cabinet, PA2 the vapor partial pressure at film inner surface in cup
and L the average film thickness.
2.5.8. Fourier-transform infrared (FTIR) spectroscopy
The IR spectra of the films were determined using an infrared
spectrometer (FTIR) (Thermo Fisher Scientific Inc., Nicolet iS10,
USA). The spectra were obtained in an Attenuated Total Reflectance
mode (ATR) between 500 and 4000 cm1, using 16 scans at a res-
olution of 2 cm1. Data analysis of each film was performed with
OMNIC 8.3 Thermo Fisher Scientific Inc.
2.5.9. X-ray diffraction
X-ray diffraction (XRD) pattern of the film samples was analyzed
by X-ray diffractometer (D8 Advance, Bruker AXS, Germany).
Samples were prepared by placing rectangular shape of each film
(3  3 cm) on a glass slide and the spectra were recorded using Cu-
Ka radiation and a nickel monochromator filtering wave at 40 kV
and 30 mA with scanning at 2q ¼ 5e60 and scanning speed of
4 min1.
2.5.10. Atomic force microscopy (AFM)
The surface morphology of the films was analyzed using
atomic force microscopy AFM (Bruker Dimension Icon, Bruker Co.,
Germany). Samples after equilibration at 53 ± 1% RH for 72 h were
cut into thin pieces that fit into AFM imaging, and were stuck on
double-sided tape. Samples were scanned in noncontact mode
(3) using a sharpened cantilever with a spring constant of 25 N/m and
50 mm  50 mm images were obtained. The resulting data for each
sample was transformed into a 3D image (Fabra, Talens, & Chiralt,
2009). All samples were triplicated and analyzed using Bruker
Nanoscope analysis software (Version 1.40) to calculate the
roughness values and cross-section profiles. The obtained pa-
rameters were: Ra, which is the arithmetic average of the absolute
values of the surface height deviations (Z) measured from the
mean plane and Rq, which is the root mean square average of
height deviations taken from the mean image data plane. Ra and
Rq were calculated from the following Equations (7) and (8),
respectively:
1 XN  
Z 
Ra ¼ (7)
N j¼1 j
sffiffiffiffiffiffiffiffiffiffiffi
P 2
(4) Zi
Rq ¼ (8)
N
2.5.11. Measurement of contact angle
Contact angle (CA) was determined in fresh films with sessile
drop method, by depositing a drop of a liquid onto the film surface.
5 mL distilled water was carefully dropped on films through a
2.0 mL mm syringe (KDL Corp., Shanghai, China) and contact angles
were quickly measured before the swelling started. The measure-
ments were carried out in open air and at a room temperature of
25  C. Both left and right contact angles expressed in degrees were
automatically calculated from the digitalized image software
312
belonging to the equipment (DataPhysics Instruments GmbH,
OCA15EC, Germany). Measurements were taken in triplicate for
each type of film.
2.5.12. Scanning electron microscopy (SEM)
Morphology of films was analyzed by a scanning electron mi-
croscope (SEM, S-4800, Hitachi, Japan). Samples were attached to
double-sided adhesive tape and then mounted on the specimen
holder. The samples were sputter coated with 10 mm thickness of
gold under vacuum and scanned with an accelerating beam voltage
of 1 kV.
2.5.13. Antimicrobial tests
The tested organisms E. coli and S. aureus were grown in
lysogeny broth (LB) and tryptic soy broth (TSB) mediums respec-
tively for 24 h at 37  C. The antimicrobial test was carried out ac-
cording to the method developed by Seydim and Sarikus (2006)
with some modifications. The inhibitory zone test on solid medium
was used for determination of the antimicrobial effects of the films.
TG films were cut into discs (diameter ¼ 10 mm) with a punch and
sterilized under UV light before the antimicrobial tests. 3 discs were
placed carefully into each petri dish containing solid medium,
where 0.1 mL seeding culture had been spread. The concentrations
of the E. coli and S. aureus seeding culture were 2  108 CFU/mL and
2  106 CFU/mL, respectively. The petri dishes were then incubated
at 37  C for 24 h in the appropriate incubation chamber. The plates
were examined to find the ‘zone of inhibition’ of the film discs. The
diameter of the zone was measured with a sliding caliper
(Shanghai, China) and the area of the whole zone was calculated.
2.5.14. Color evaluation
The color of the films was determined using a colorimeter
(Hunterlab, UltraScan Pro1166, USA) and values of the Hunter scale
were expressed as L* ¼ 0 (black) to L* ¼ 100 (white), a* (green-
ness) to þa* (redness), b* (blueness) to þb* (yellowness) (Francis
& Clydesdale, 1975). The difference in color (DE*) was calculated
according to the Equation (9) by Gennadios, Weller, Hanna, and
Froning (1996):
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
DE* ¼ ðDL*Þ2 þ ðDa*Þ2 þ ðDb*Þ2
where DL*, Da*, Db* are the differentials between the color
parameter of the samples and of the white standard (L* ¼ 99.38,
a* ¼ 0.24, b* ¼ 0.11). Measurements were taken in triplicate for
each type of film.
Fig. 1. TEM micrographs of (a) bulk chitosan and (b) chitosan nanoparticles.
J. Antoniou et al. / Food Hydrocolloids 44 (2015) 309e319
2.6. Statistical analysis
OriginPro 8 SR4 (Version 8.0, Northampton, USA) and SPSS
Statistics software (Version 17.0, SPSS Inc. Chicago, IL) were used to
analyze the resulting data. Data were initially evaluated by analysis
of variance (ANOVA), and significant results were further analyzed
using Duncan's multiple range tests (P < 0.05) to compare the mean
values of films' properties.
3. Results and discussion
3.1. Chitosan nanoparticle characterization
CSNPs were prepared by the ionic interaction between the
positively charged amino groups (NHþ 3 ) of CS and the negatively
charged phosphate groups (P3 O5 10 ) of TPP. Their average particle
size of CSNPs was measured to be 180.25 ± 2 nm with poly-
dispersity index (PDI) 0.15 ± 0.02 and z-potential 23.7 ± 1.98 which
was lower than that of bulk CS 26.6 ± 0.28. TEM micrographs
revealed the different structure between CS and CSNPs (Fig. 1). It
can be observed that CSNPs had a more compact structure
compared with the diffused CS molecular chains.
3.2. Mechanical properties
The mechanical properties of TG films with incorporation of CS
and CSNPs are shown in Fig. 2. The initial tensile strength of the TG
films was 22.71 ± 2.98 MPa and increased to a maximum value of
58.44 ± 3.23 MPa when 10% w/w of CSNPs was added. However,
further increase in the CSNPs concentration (15% w/w) had no
significant effect on the TS of the films (P > 0.05). Chang et al.
(2010a) reported that this may be ascribed to the agglomeration
of the nanoparticles during the film formation. Incorporation of
CSNPs increased the polymer's stiffness confirming the reinforcing
effect of the nanoparticle in the polymeric matrix. This is consistent
with previous results obtained by De Moura et al. (2009) & Pereira
de Abreu, Paseiro Losada, Angulo, and Cruz (2007). As shown in
Fig. 2b, the percentage elongations of the films decreased slightly
when CSNPs were incorporated. With increasing concentration of
(9) CSNPs, E% presented significant variation (P < 0.05) only when the
concentration was 15% w/w.
Addition of CS had the same trend as CSNPs for both TS and E%
but it was less effective. At corresponding concentrations it
improved the TS less than the nanoparticles and exhibited the same
E% (P > 0.05). Based on these findings, addition of CSNPs up to 10%
 J. Antoniou et al. / Food Hydrocolloids 44 (2015) 309e319 313

a a b
a

bc b a
c ab b b b
c
d d

Fig. 2. Effect of inclusion of ( ) bulk chitosan and ( ) chitosan nanoparticles on (a) tensile strength and (b) elongation of tara gum film. Different letters in the same graph indicate a
statistically significant difference (P < 0.05).

w/w to TG films resulted in significant improvement of the me- broad temperature range (Diab, Biliaderis, Gerasopoulos, &
chanical properties. Sfakiotakis, 2001). The increase in stiffness with increasing con-
centration of CSNPs and CS is also suggested with the increase of
3.3. Dynamic mechanical analysis (DMA) tan d (Fig. 4).

The frequency dependence of storage modulus (E0 ), loss
modulus (E00 ) and tan d for TG films with addition of the two forms
of chitosan was measured with a DMA and results are shown in
Figs. 3 and 4. Based on Perfetti, Jansen, Wildeboer, Van Hee, and
Meesters (2010), the higher E0 values compared to E00 and the low
dependence on the frequency sweep indicate that the films were in
a glassy state for the frequency range tested. Therefore, it can be
assumed that the testing temperature was below the glass transi-
tion temperature Tg of the films (Cespi, Bonacucina, Mencarelli,
Casettari, & Palmieri, 2011). As shown in Fig. 3 with increasing
concentration of CS the E0 increased while E00 had no significant
change. It is known that storage modulus is related to the stiffness
of the material (Yu, Wang, & Ma, 2008). CSNPs compared to CS
increased more the E0 indicating that incorporation of the nano-
particles produced stiffer and stronger films. This is in agreement
with the results from the mechanical testing. It is possible that the
change of E0 is related to the water content of the films which
behaved as a plasticizer increasing the flexibility (Table 1). It has
been reported before that the mechanical behavior of biopolymers
is very sensitive to the hydration of the material and occurs over a
3.4. Thermogravimetric analysis (TGA)
Thermogravimetric analysis was performed in order to study
the decomposition pattern and the thermal stability of the films.
Fig. 5 shows two mass loss events for all samples. The first
occurred near 100  C and resulted in a weight loss of approxi-
mately 10% for TG and reduced slightly with increasing chitosan
content. It may be attributed to the loss of the water content of
each film. The second mass loss event resulted in a weight loss of
approximately 60%, due to polysaccharide thermal decomposition.
The derivative of the weight loss curve (DTG) presented temper-
ature peaks at 287.33, 291.83 and 292.16  C for TG, CS and CSNPs at
15% w/w, respectively. These results are in agreement with
Cerqueira, Souza, et al. (2011), Mothe
and Rao (2000) and
Vendruscolo et al. (2009) who presented values for different gums
ranging from 40 to 60% for the second mass loss at temperature
peaks of 290e315  C. Incorporation of chitosan in both forms
improved slightly the thermal stability of TG films. Chitosan has
been found to be more thermally stable compared to other gums
(Zohuriaan & Shokrolahi, 2004).

a b

Fig. 3. The influence of applied frequency on storage modulus (line) and loss modulus (dots) for (a) bulk chitosan and (b) chitosan nanoparticles of tara gum films ( 0%, 5%,
10%, 15% w/w).
314 J. Antoniou et al. / Food Hydrocolloids 44 (2015) 309e319

Fig. 4. The influence of applied frequency on tan d for (a) bulk chitosan and (b) chitosan nanoparticles of tara gum films ( 0%, 5%, 10%, 15% w/w).

Table 1
Effect of inclusion of bulk chitosan (CS) or chitosan nanoparticles (CSNPs) on solubility and moisture content of tara gum films.a

Film type CS/CSNP content (% w/w) Thickness (mm) Moisture content (%) Solubility (%) WVP (g mm m2 h1 kPa1)

TG 0 0.073 ± 0.005c 14.72 ± 0.09a 24.00 ± 1.30a 0.462 ± 0.013a

TG þ CS 5 0.074 ± 0.010c 14.59 ± 0.86a 21.32 ± 1.54a 0.415 ± 0.026b
10 0.077 ± 0.008c 14.29 ± 0.49ab 16.31 ± 2.61b 0.405 ± 0.011b
15 0.082 ± 0.003bc 12.94 ± 0.15c 10.37 ± 2.31c 0.419 ± 0.027b
TG þ CSNPs 5 0.079 ± 0.010bc 14.50 ± 0.41a 17.94 ± 1.70b 0.410 ± 0.010b
10 0.088 ± 0.005ab 13.05 ± 0.62bc 14.55 ± 2.16b 0.357 ± 0.041c
15 0.094 ± 0.005a 11.26 ± 0.68d 6.16 ± 1.17d 0.365 ± 0.013c
a
Values are given as mean ± standard deviation. Different superscript letters in the same column indicate a statistically significant difference (P < 0.05).

3.5. Moisture content
The moisture content is an important property in edible films.
Changes in the moisture content can affect the physicochemical
and thermomechanical properties of the films (Appelqvist, Cooke,
Gidley, & Lane, 1993; Diab et al., 2001). Addition of glycerol due
to its hydrophilic nature attracted water into the polymer matrix
creating more mobile regions with greater inter-chain distances
into the polymer matrix (Cerqueira, Souza, Teixeira, & Vicente,
2012; Tapia-Bla
cido, Sobral, & Menegalli, 2011; Zhang & Han,
2006). Therefore, because of the high content of glycerol, TG films
were found to have high moisture content 14.72 ± 0.09. However,
as shown in Table 1 with increasing concentration of CS and CSNPs
the moisture content of TG films was found to decrease despite the
increasing thickness of the films. The most profound effect was
shown by CSNPs while CS exerted significant effect (P < 0.05) only
at the highest concentration (15% w/w). The compact structure of
CSNPs compared to bulk CS allowed them to occupy more free
volume in the polymer matrix and therefore reduced more the
moisture content. Additionally, it is possible that the compact
structure of CSNPs allowed less hydroxyl groups of the CS chain
exposed to connect with water molecules.
3.6. Water solubility
The water solubility of the TG composite films is presented in
Table 1. Addition of CS and CSNPs decreased significantly (P < 0.05)
the S% of the films. It was observed that when chitosan in any form

a b

Fig. 5. Thermogravimetric curves of tara gum films incorporated with (a) bulk chitosan and (b) chitosan nanoparticles obtained at a heating rate of 10  C min1 under N2
atmosphere ( 0%, 5%, 10%, 15% w/w).
 J. Antoniou et al. / Food Hydrocolloids 44 (2015) 309e319
was added, TG films maintained their structure after 24 h immer-
sion in water. On the other hand plain TG films dissolved imme-
diately into an amorphous mass. With increasing CS concentration
the amino groups increase accordingly causing decrease of S%
(García, Pinotti, Martino, & Zaritzky, 2004; Wu, Zhong, Li,
Shoemaker, & Xia, 2013). Shen, Wu, Chen, and Zhao (2010) sug-
gested that the reduction of water solubility is a combined result of
the strong hydrogen bonding interaction between chitosan and
polymer and the low water solubility or relative hydrophobicity of
chitosan itself. CSNPs exhibited lower S% compared to CS with the
lowest value at 6.16 ± 1.17% when the concentration was 15% w/w.
De Moura et al. (2008) similarly reported that cellulose films with
bulk CS had higher S% compared to CSNPs and that S% decreased
with increasing particle size. Same as bulk CS, CSNPs were able to
interact strongly with the TG chains but their compact structure
obstructed the diffusion of water and therefore reduced solubility.
3.7. Water vapor permeability (WVP)
As shown in Table 1, water vapor permeated easily the TG film.
The high WVP can be a result of the high glycerol concentration.
Films plasticized with hydrophilic plasticizers such as glycerol favor
the adsorption of water molecules and increase the inter-chain
spacing of the polymer resulting in increasing WVP (Aydinli &
Tutas, 2000; Gontard, Guilbert, & Cuq, 1993; Yang & Paulson,
2000). When 5e15% w/w CS and CSNPs were added to the TG
films the WVP values decreased with the lowest to be at
0.357 g mm m2 h1 kPa1 when 10% w/w of CSNPs was incor-
porated. At the highest concentration (15% w/w) for both CS and
CSNPs the WVP values had no significant change (P > 0.05). Simi-
larly, Chang et al. (2010a) reported that superfluous concentration
could aggregate easily decreasing their effective content and
eventually increase water vapor permeation. Nanoparticles have
more ability in occupying the empty spaces of the porous film
matrix (De Moura et al., 2009). As a result, the more compact
structure of the films containing CSNPs created a more difficult
pathway for water molecules to permeate and therefore decreased
the WVP of TG films (Chang, Jian, Yu, & Ma, 2010b; Kristo and
Biliaderis (2007); Yu et al., 2008).
3.8. FTIR & XRD analysis
The FTIR spectra of TG films with addition of CS and CSNPs is
shown in Fig. 6. The major peaks in the IR spectra appeared in two
regions, 3700e2500 cm1 and 1700e700 cm1. The broad band at
a b
Fig. 6. Effect of inclusion of (a) bulk chitosan and (b) chitosan nanoparticles on FTIR spectrum of tara gum film (
315
3700e3000 cm1 is a result of OeH stretching vibration associated
with free, inter, and intra-molecular bound hydroxyl groups and
the peaks in the region 3000e2800 cm1 is a result of CeH
stretching (Yuen, Choi, Phillips, & Ma, 2009). The peak of 2927 cm1
is typical CeH vibration (Anicuta, Dobre, Stroescu, & Jipa, 2010).
Different concentration of CS and CSNPs resulted in different
magnitudes of absorbance in these regions depending on the
moisture content. For both composites, the CS characteristic band
at 1559 cm1, which is assigned to the stretching vibration of amino
groups of CS, shifted to 1567 cm1 in the films spectra (Anicuta
et al., 2010). With increasing concentration of CS the absorbance
of this peak increased respectively. The broad band between 1132
and 981 cm1 results from the stretching vibrations of alcoholic
CeO in CeOeH bonds (1072 cm1) and in the asymmetric and
symmetric CeOeC bonds (Jamro
z et al., 2007). Cerqueira et al.
(2012) have reported these vibrations at 1018 cm1 in all gal-
actomannans and the small shift (1024 cm1) is probably related to
the high amount of glycerol due to the hydrogen bonds formed by
the hydroxyl groups of both galactomannan and glycerol structures.
Non-significant shifts in the wavenumber of the ether bonds are
present in the region 1100e900 cm1.
The crystalline nature of TG films incorporated with CS and
CSNPs was tested by the X-ray diffraction (XRD) analysis (Fig. 7).
Basically three peaks were observed, a rather broad at 2q ¼ 15.28
and two small at 2q ¼ 6.06 and 26.2 . The very broad peak at
2q ¼ 15.28 indicates that TG was an amorphous material. No
characteristic peaks of CS could be observed probably due to the
low concentration of CS or interaction with the TG polymer matrix.
It is possible that interaction between CS and TG might have caused
destruction of the CS molecules necessary for the formation of
regular crystallites (Wu et al., 2013). However, it was observed that
with incorporation of CS, the peak at 2q ¼ 6.06 disappeared.
Additionally the broad peak at 2q ¼ 15.28 became sharper with
lower intensity. According to Yakimets et al. (2007) it is possible
that the changes in the intensities of the peaks at 6.06 and 15.28
are related to the reduction of water content in the films.
3.9. Film microstructure
The microstructure of the films was studied using SEM for the
cross-section and AFM for the surface morphology. Fig. 8 shows
that incorporation of CS and CSNPs into the films promoted
changes in the film's morphology. Plain TG films presented a
smooth and uniform structure with an undulate appearance which
was probably due to shrinkage during freezing in liquid nitrogen.
0%, 5%, 10%, 15% w/w).
316 J. Antoniou et al. / Food Hydrocolloids 44 (2015) 309e319
Fig. 7. X-ray diffraction (XRD) patterns of tara gum films incorporated with (a) bulk chitosan and (b) chitosan nanoparticles (
Fig. 8. SEM observations of the cross-section of (a) TG (b) TG þ CS and (c) TG þ CSNPs films (CS & CSNPs ¼ 10% w/w of TG).
Addition of CS in the film disrupted the smooth structure and
irregular shapes appeared. On the other hand, the small spherical
nanoparticles appeared evenly distributed over the film's structure.
This even distribution of CSNPs compared to the aggregated chains
of CS allowed them to create a more homogenous structure by
occupying more free volume and thus improve the mechanical
properties.
The surface structure of TG films analyzed by AFM showed a
marked influence very similar to the structure of the cross-section
when CS and CSNPs were incorporated (Fig. 9). The intense peaks
that appear in all the films are attributed to un-dissolved matter in
the TG solution. Addition of CS and CSNPs increased the roughness
of the film's surface and the Ra values were increased from
34.3 ± 2.8 to 45.1 ± 4.2 and 51.4 ± 3.3 nm, respectively (Table 2).
Although the Ra values between films with CS and CSNPs had no
significant difference, the appearance of the two films was
different. Cross-sectional profiles as a function of vertical distance
revealed the difference between the two surfaces (Fig. 10). With
addition of CSNPs the surface became rougher with a lot of small
sharp peaks that are probably attributed to nanoparticles. Rq values
translate also this difference between the surfaces of the films
where addition of CS increased it from 48.7 ± 2.4 to 57.2 ± 5.5 nm
and CSNPs to 75.3 ± 8.5 nm. Increase in the surface root mean
square roughness has also been reported by Azeredo et al. (2010)
with addition of cellulose nano-fibers in glycerol plasticized chi-
tosan films. It is possible that the diffused chains of bulk CS could
accumulate more smoothly on the surface of the film compared
with the more compact CSNPs. This significant increase in rough-
ness of the films with addition of CSNPs changed the water barrier
properties of the films. With increasing roughness the CA of the
surface also increased (Fig. 9) and the CA values correspond to those
of Ra (Table 2). Incorporation of CSNPs made the surface of the films
0%, 10%, 15% w/w).
more hydrophobic which correlates with the results of WVP and
water solubility.
3.10. Antimicrobial tests
The antimicrobial activity of TG edible films incorporated with
CS and CSNPs against E. coli and S. aureus is shown in Table 3. After
24 h of incubation the film disc was diffused through the adjacent
medium creating a clear inhibitory zone. The area of this zone was
calculated by measuring the diameter and then subtracting the
initial film area. For the control TG films the film area was
contaminated by the bacteria and it was assumed that there is no
inhibitory zone thus the diameter was valued as zero. With
increasing CS concentration the inhibitory zone increased almost
linearly for both the bacteria. It was found that CS had slightly
higher antimicrobial activity against S. aureus compared to E. coli at
all concentrations. It is known that chitosan's antimicrobial activity
is higher against Gram-positive (S. aureus) bacteria than the Gram-
negative (E. coli) (Allan & Hadwiger, 1979; Huang et al., 2012; Li,
Kennedy, Peng, Yie, & Xie, 2006; Sudarshan, Hoover, & Knorr,
1992). However, the opposite result has also been reported sug-
gesting that the protonated amino groups of CS can form electro-
static interactions with anionic groups of microbial cell
membranes, leading to the leakage of proteinaceous and other
intracellular constituents of the microorganisms (Chung, Wang,
Chen, & Li, 2003; Pranoto, Rakshit, & Salokhe, 2005; Shahidi,
Arachchi, & Jeon, 1999). Zheng and Zhu (2003) suggested that the
mechanisms of the antimicrobial activity of CS are different against
gram-positive and negative bacteria. For S. aureus, CS can form a
polymer membrane on the surface of the cell preventing nutrients
from entering the cell. For E. coli, chitosan's antimicrobial activity
was attributed to pervasion through the microbial cell disturbing
 J. Antoniou et al. / Food Hydrocolloids 44 (2015) 309e319 317

Fig. 9. 3D images of AFM and contact angles of water sessile drops of (a) TG (b) TG þ CS and (c) TG þ CSNPs films (CS & CSNPs ¼ 10% w/w of TG).

Table 2
Roughness parameters Ra and Rq obtained from AFM images and contacts angles of nanoparticles are able to penetrate into the cells causing a signifi-
water sessile drops. Four samples were analyzed in each case (CS & CSNPs ¼ 10% w/ cant increase in membrane permeability and leading to cell death
w of TG).a (Kanmani & Rhim, 2014; Kim et al., 2007; Ravishankar Rai &
Films Ra (nm) Rq (nm) CA ( ) Jamuna Bai, 2011). In our case CSNPs presented a lower antimi-
crobial activity compared to CS. It is possible that the size of CSNPs
TG 34.3 ± 2.8b 48.7 ± 2.4c 74.1 ± 2.5c
TG þ CS 45.1 ± 4.2a 57.2 ± 5.5b 84.4 ± 1.3b
compared to non-edible nanoparticles wasn't small enough in or-
TG þ CSNPs 51.4 ± 3.3a 75.3 ± 8.5a 88.6 ± 2.2a der to accumulate in the bacterial membrane. It is also estimated
a that when the CSNPs concentration increased, the particle size
Values are given as mean ± standard deviation. Different superscript letters in
the columns indicate significant difference (P < 0.05). increased even further due to aggregation during the film forma-
tion. Moreover, because the formation of CSNPs is based on ionic
the metabolism of the bacteria. Another mechanism has been re- gelation mechanisms the surface charge of CS was reduced making
ported by Sudarshan et al. (1992) which involves the binding of it less effective against Gram-negative bacteria (E. coli). For both CS
chitosan with DNA to inhibit RNA synthesis and therefore inhibition and CSNPs the antimicrobial activity was found to be inversely
of growth. We believe that the antimicrobial activity of CS can be a correlated with the water solubility of the films.
combination of more than one mechanism.
The antimicrobial activity of CSNPs for concentration up to 10%
w/w was found to be the same as chitosan's against S. aureus but 3.11. Color evaluation
decreased significantly (P < 0.05) for E. coli. However, when the
concentration increased from 10% to 15% w/w, no significant Table 4 shows the values for the color parameters (a*, b*, and L*)
improvement (P > 0.05) was observed in the antimicrobial activity of hunter scale and total color difference (DE*) of TG films incor-
of the films against E. coli and it decreased for S. aureus. Similar porated with CS and CSNPs at different concentrations. Because the
result has been obtained by Shen et al. (2010) where 15% w/w of CS used was obtained from crab shells it had a yellow color which
chitosan had no significant improvement in antimicrobial activity affected the color of TG films. Increasing concentration of CS
of sweet potato starch films. According to previous studies, reduced the lightness (L*), increased slightly the greenness (a*)
and of course increased the yellowness (þb*) of the films (P < 0.05).
However, when CS was incorporated in the films in the form of
nanoparticle, the yellowness of the films was reduced significantly
(P < 0.05). At the highest concentration of CSNPs (15% w/w) the
yellowness was reduced from 15.07 ± 0.32 to 12.75 ± 0.65
compared to CS. This change in the þb* component had also a
significant effect (P < 0.05) on the total difference in color DЕ*.
Similar results with respect to the b* component have been ob-
tained before and the different values are probably due to the
different source or processing of chitosan (Casariego et al., 2009;
Rhim, Hong, Park, & Ng, 2006).

Table 3
Antimicrobial activity of tara gum films containing bulk chitosan (CS) and chitosan
nanoparticles (CSNPs) against food pathogenic bacteria of E. coli and S. aureus.a

Film type CS/CSNP content (% w/w) Inhibitory zone (mm2)

E. coli S. aureus
f
TG 0 0.0 ± 0.0 0.0 ± 0.0d
TG þ CS 5 65.43 ± 10.36d 78.45 ± 10.13c
10 112.0 ± 16.06b 117.96 ± 11.08b
15 138.87 ± 12.35a 157.42 ± 10.95a
TG þ CSNPs 5 41.53 ± 7.60e 85.30 ± 12.30c
10 87.32 ± 6.72c 111.71 ± 3.12b
15 85.80 ± 6.17c 93.41 ± 8.59c
a
Fig. 10. Cross-sectional profiles of the analyzed AFM regions as a function of vertical Values are given as mean ± standard deviation. Different superscript letters in
distance of (a) TG (b) TG þ CS and (c) TG þ CSNPs films (CS & CSNPs ¼ 10% w/w of TG). the same column indicate a statistically significant difference (P < 0.05).
318 J. Antoniou et al. / Food Hydrocolloids 44 (2015) 309e319

Table 4
Hunter color values (L*, a*, and b*) and total color difference (DE*) of tara gum films with bulk chitosan (CS) and chitosan nanoparticles (CSNPs) at different concentrations.a

Films CS/CSNP content (% w/w) Hunter scale DE*

L* a* b*

TG 0 92.58 ± 0.11a 1.29 ± 0.09a 2.61 ± 0.33f 7.25 ± 0.20f

TG þ CS 5 91.60 ± 0.15b 1.97 ± 0.08b 8.43 ± 0.47d 11.59 ± 0.97e
10 90.79 ± 0.17c 2.22 ± 0.11c 12.02 ± 0.40b 14.96 ± 0.50c
15 90.01 ± 0.17d 2.35 ± 0.16cd 15.07 ± 0.32a 19.48 ± 0.95a
TG þ CSNPs 5 91.45 ± 0.14b 1.95 ± 0.02b 6.80 ± 0.23e 10.44 ± 0.26e
10 90.65 ± 0.12c 2.28 ± 0.02c 9.96 ± 0.43c 13.19 ± 0.40d
15 89.80 ± 0.25d 2.48 ± 0.05d 12.75 ± 0.65b 16.79 ± 1.14b
a
Values are given as mean ± standard deviation. Different superscript letters in the columns indicate significant difference (P < 0.05).

4. Conclusions
CS and CSNPs were successfully incorporated in the TG films.
CSNPs presented better mechanical, physicochemical and barrier
properties compared to CS. The presence of CSNPs improved
significantly the tensile strength of the films without reducing
notably the elongation. DMA results proved that CSNPs produced a
more stiff and strong material. The improvement in the properties
of the TG films was attributed to the reduction of the free volume in
the polymer matrix by the more compact structure of the CSNPs
containing films compared to those containing bulk CS. As a result
of the occupied empty spaces by CSNPs, the moisture content and
WVP of the TG films was decreased. Furthermore, their compact
structure obstructed the diffusion of water, increased the rough-
ness and hydrophobicity of the film's surface and reduced water
solubility. A slight improvement in the thermal stability of TG films
was observed by TGA with addition of CS and CSNPs although FTIR
showed no significant interactions between the polysaccharides
and XRD analysis revealed the amorphous structure of TG and the
composites. However, CS compared to CSNPs exhibited better
antimicrobial activity. CSNPs were found to be less effective against
E. coli (Gram-negative) compared to S. aureus (Gram-positive) and
their antimicrobial activity was reduced at high concentrations
probably due to agglomeration. This study lays foundation in
developing edible galactomannan-based films containing chitosan
nanoparticles and indicates that use of nanotechnology can
improve their functionality for food applications.
Acknowledgments
This work was financially supported by National 863 Program
2011BAD23B02, 2013AA102207, NSFC 31171686, 30901000, 111
project-B07029 and PCSIRT0627.
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