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=
Preparation
and Characterization of Chitosan Nanocomposite Based on Nanoscale Silver an=
d Nanomontmorillonite=
=
=
Fatemeh-Sadat Ebnerasool and Negar <=
span
class=3DSpellE>Motakef-Kazemi,
<=
span
style=3D'mso-bookmark:OLE_LINK8'>The chitosan nanocomposites were rapidly prepared by simple solution
method. This biopolymer matrix was modified by prepared nanoscale silver (A=
g)
using in situ synthesis from precursor and <=
span
style=3D'mso-bookmark:OLE_LINK8'>=
nanomontmorillonite<=
span
style=3D'mso-bookmark:OLE_LINK8'> (NMMT). Moreover, the samples were characterized by fourier transform infrared (FTIR) spectroscopy,
thermogravimetric analysis (TGA), field emission scanning electron microsco=
py
(FESEM), and energy dispersive x-ray spectroscopy (EDX). Moreover, the water
vapor properties (WVP) of nanocomposites were investigated using gravimetric
standard. In addition, the antibacterial activity of nanocomposite was meas=
ured
by the well diffusion method on Muller–Hinton Agar against Escherichia coli<=
/span><=
span
style=3D'mso-bookmark:OLE_LINK8'> (E. col=
i)
=
by zone inhibition. Finally, based on the
obtained results, the nanocomposite can have a good candidate for different
applications and food packaging industry.
Keywords:=
span> Nanocomposite,
Chitosan, Nanoscale silver, Nanomontmorillonite,
Packaging.
1. Introduc=
tion
Today, nanotechnology has expanded in various fields duo to the potenti=
al of
application benefits in the world [1]. Nanomaterials have size of 1-100 nm =
that
showed unique properties because of small size and high surface area [2]. In
recent years, population growth was resulted in the extension of packaging =
for food
source protection [3]. Since, nanotechnology can be affected because of the
wide application in production, processing, storage, packaging, and transpo=
rt
of food products with environment protection and earth resources [4]. The main factor is included a lot of effort to pr=
oduce functional
materials, food processing, product development, methods, and tools design<=
/span>
[5]. The application of nanocomposite is expanded
because of its unique properties in food
packaging [6,7]. The nanocomposites can control the permeability of polymer=
and
increase shelf life to improve the efficiency of
packaging materials [8]. In packaging industry, the use of biodegra=
dable
material can reduce the waste and results to co=
nsume the
less material precursor [9].
=
The nanomaterials have been developed the
research work in food products [10]. Recently, biodegradable nanocomposite
has been used to investigate barrier materials and antimicrobials in packaging [11]. Moreover,
nanocomposite based on biopolymer =
=
can be named such as starch [12,13], cellulose [14], and chitosan (CS) [15,16] for food
packaging. Moreover, chitosan is derived fr=
om
chitin, and its use has expanded as biomaterials [17]. The ch=
itosan
has several excellent properties such as biodegradability, non-toxicity, and
antimicrobial properties which investigated in food packaging widely [18-20=
]. The polymer nanocomposite included the polymer as matrix phase an=
d nanomaterial as filler phase to improvement of physical and
mechanical properties [21]. In addition, the use of nanocomposites c=
an
enhance the applications of different properties for nanomaterials in food
packaging technology [22]. Moreover, silver nanoparticles are well kn=
own
as antimicrobial agents in curative and preventive health care with low
toxicity for humans [23,24]. Clay
filler has received significant attention because of suitable dispersion,
thermal stability, and barrier properties in polymer nanocomposites =
[25].
In the present study, chitosan nanocomposite
based on nanoscale silver and =
nanomontmorillonite has been prepared by a simple method using in situ<=
/i> synthesis <=
/span>of nanoscale silver for food packaging. The aim of this work is to study antibac=
terial
activity against Escherichia coli and barrier for water vapor permeability<=
/span> of this nanocomposite.
2. Experime=
ntal
procedure
2.1. Materi=
als
All chemica=
ls
used were analytical grade. Chitosan was obtained from Sigma–Aldrich with
medium molecular weight. Glacial acetic acid, trisodium citrate and silver nitrate=
(AgNO3)
were obtained from Merck. Ultra-pure water was used for the preparation of =
all
reagents solutions. Moreover, the modified montmorillonite clay was obtained
from Nano Pasargad Novin=
span> Company. The test strains, Escherichia coli A=
TCC 1399,
were procured from Islamic Azad University.
2.2.1. Prep=
aration
of nanoscale Ag
The =
nanoscale silver was prepared by reducing silver nitrate using trisodium citrate. AgNO3 solution (2 M) was
added to the 1% acetic acid solution. Then trisodium=
span>
citrate solution (4 M) was added to the resulting solution and stirred for =
2 h.
The nanoscale silver was centrifuged at 10,000=
rpm
for 10 min and dried at room temperature for 48 h [26]. 2.2.2. Prep=
aration
of chitosan–=
nanoscale Ag na=
nocomposite
Chitosan
aqueous solution of 2 wt.% was prepared by
dissolving chitosan powder
(2 g) in 100 mL of 1% (v/v) acetic acid. Then AgNO3<=
/sub> solution (2 M) and trisodium
citrate (4 M) were added to the chitosan solution and stirred for 2 h at room
temperature. The preparation of chitosan–nanoscale Ag nanocomposite was don=
e by
in
situ synthesis of nanoscale silver<=
span
style=3D'font-family:"Times New Roman","serif";mso-ascii-theme-font:major-b=
idi;
mso-hansi-theme-font:major-bidi;mso-bidi-theme-font:major-bidi'>. The mixtu=
re
solution
2.2.3. Prep=
aration
of chitosan–NMMT nanocomposite
Chitosan
aqueous solution of 2 wt.% was prepared in acetic acid solution (1%=
v/v).
The nanomontmorillonite was prepared by dispers=
ing of
montmorillonite clay into 10 mL of 1% =
acetic
acid solution and vigorously stirring for 24 h. The
nanomontmorillonite solution (1% and 3 wt.%) was added slowly to chitosan solution and stirred
continuously for 2 h. Then the mixture was cast =
onto
glass plates and dried at room temperature for 72 h [26-27].
2.2.4. Prep=
aration
of chitosan–nanoscale Ag–NMMT nanocomposite
Chitosan
aqueous solution of 2 wt.% was prepared by 1% (v=
/v)
acetic acid. Then AgNO3 solution (2 M), tris=
odium
citrate=
(4 M), and nanomontmorillonite solution (=
1%
and 3% wt) were added into the chitosan solutio=
n and
stirred for 2 h at room temperature. Then the mixture was cast onto glass
plates and dried at room temperature for 72 h [26].
2.3. Charac=
terization
of samples
The samples were characterized by Fourier transform infrared spectroscopy, thermogravimetric
analysis, field emission scanning electron microscop=
y, and
energy-dispersive x-ray spectroscopy. FTIR <=
/a>spectra
were obtained using a FTIR spectrophotometer (SHIMADZU Co) in the range of
400–4000 cm−1 at a resolution of 4 cm−1 i=
n a KBr matrix. The thermogravimetric analysis was measur=
ed by
a TGA-SF1 (Mettler Co) that carried out to 800 =
◦C
at the heating rate of 20 ◦C/min under
nitrogen atmosphere. The morphologies of the samples were analyzed on a MIR=
A3 TeScan LMU (TeScan Co) FESEM
at 5 kV. Afterwards, the specimens were coated with a thin
conductive gold layer before observation. The EDX analysis was measured by =
usingASK SEM-CL View VIS (OXFORD INSTRUMENTS Co. The
antimicrobial activity was investigated for pure chitosan, montmorillonite,=
and
chitosan-based nanocomposites by measurement of the minimum inhibition
concentration (MIC) against E. coli as gram-negative bacteria. The c=
ells
of E. coli were cultivated on Mueller-Hinton Agar and incubated at 3=
7ºC
for 1 day. The samples were systematically diluted from 0.02% (w/v) to
0.000625% (w/v) to determine of the MIC values.
The WVP of
films was determined using gravimetric standard ASTM E96-=
05 [28].
Glass bottles were used to perform the test with a diameter of 20 mm and de=
pth
of 45 mm. The bottles were filled with 3 g of CaCl2 for maintaining
a relative humidity (0% RH) and covered with the film specimen. =
The
result bottles were placed in a container containing K=
2SO4
super saturated solution (97% RH) at 25 ◦C. Then the bottl=
es
were weighed ten times at 3 h intervals. Afterwards, water vapor transmissi=
on
rate (WVTR) was determined from slope of mass change of bottle versus time
curve divided by area of glass bottle mouth (m2). Then the WVP of
film was calculated using Eq. 1:
WVP =3D WVT=
R × L
/ ΔP (1)
=
where WVTR is wa=
ter
vapor transmission rate (g/m2s) through film, L is the mean
thickness of film (m), and ∆P is partial water vapor pressure differe=
nce
(Pa) across film [21].
3. Results =
and
Discussion
3.1. FTIR <= o:p>
The FTIR spectra
are shown for CS, nanoscale Ag, NMMT, CS-nanoscale Ag nanocompos=
ite,
CS-NMMT nanocomposite and CS-nanoscale Ag-NMMT nanocomposite in Fig. 1. The
spectrum of chitosan (Fig. 1 a) shows the band at 1647 cm−1 that
corresponds to the amide function due to acetylated amine, where=
as
the band at 1600 cm−1 is corresponded to the free amine I
function due to deacetylated amine. The broad b=
and at
3400-3500 cm−1 is assigned with the overlapping=
O-H stretching bands. The N-H bending, C-H bendin=
g, and
C-O stretching are shown at 1600cm−1, 1382 cm−1=
and 1087 cm−1 respectively. The results corresponded to the
results [27, 29]. The FTIR spectrum of NMMT (Fig.1 b) is shown the vibration
bands at 3624 cm−1 for =
O-H,
at 3425 cm−1 due to interlayered O-H, at 1641 cmͨ=
2;1
for H-O-H bending, at 1035 and 914 cm
Fig 1. Transmissi=
on
FTIR spectra of (a) pure CS, (b) NMMT, (c) CS-NMMT nanocomposite, (d)
CS-nanoscale Ag-NMMT nanocomposite, (e) CS-nanoscale Ag nanocomposite and (=
f)
nanoscale Ag.
The weight-=
loss
curve was obtained by heating of samples and determined thermal stability
analysis as the function of temperature. The TGA analysis is shown for CS-N=
MMT nanocomposite
that two weight loss regions were observed=
(Fig. 2. a), about 100°C (related to loss =
of
water), and about 300°C (related to degradation of chitosan), at
the result the remained weight =
perent
was related to NMMT.<=
/span>
The TGA analysis is shown for CS-nanoscale Ag nanocomposite that two weight loss regions were observed
(Fig. 2. b), about 100°C (related to loss =
of
water), and about 300 °C (related to degradation of chitosan), at the result
the remained weight perent was rela=
ted to
silver. The TGA analysis is shown for CS-nanoscale =
Ag-NMMT nanocomposite
that two weight loss regions we=
re
observed (Fig. 2. c), about 100°C
(related to loss of water), and about 300°C (related to degradation of
chitosan), at the result the remained weight perent was related to NMMT and
silver. Based on TGA results, thermal stability of nanocomposite
which are as follows: CS-NMMT > CS-nanoscale
Ag- NMMT > CS-nanoscale Ag that the presence of NMMT and nanoscale Ag have
been caused an increase and decrease in this property respectively.
CS-nanoscale Ag- NMMT was average thermal stability because of the opposite
effects of two fillers. The TGA results are comparable to
those of previous researchers [32].
Fig 2. The TGA of =
(a)
CS-NMMT nanocomposite, (b) CS-nanoscale Ag nanocomposite, and (c) CS-nanosc=
ale
Ag-NMMT nanocomposite.
3.3. FESEM =
The size and
morphology structures of samples were studied using FESEM that shown nanoscale
Ag, NMMT, CS-nanoscale Ag nanocomposite, CS-NMMT nanocomposite, =
CS-nanoscale
Ag-NMMT nanocomposite in Fig. 3. Moreover, the NMMT has shaped sheet-like layer silicates and that included a two-dimension=
al
layer with thickness of about 1 nm and length and width of about 300 nm to
several microns (Fig.3 a). The morphology of nanoscale Ag has been na=
norod
arrays with the mean diameter of about 40 nm<=
span
lang=3DEN style=3D'font-family:"Times New Roman","serif";mso-ascii-theme-fo=
nt:major-bidi;
mso-hansi-theme-font:major-bidi;mso-bidi-theme-font:major-bidi;mso-ansi-lan=
guage:
EN'> and the length of about 200 nm (Fig.3 b).
Fig 3. The SEM of =
(a)
NMMT, (b) nanoscale Ag, (c) CS-nanoscale Ag nanocomposite, (d) CS-NMMT
nanocomposite and (e) CS-nanoscale Ag-NMMT nanocomposite.
The EDX
analysis is shown for nanoscale Ag, Cs-nanoscale Ag nanocomposit=
e,
(c) CS-NMMT nanocomposite and CS-nanoscale Ag-NMMT nanocomposite in figure =
4. The
intense signal has been at 3 keV that confirmed=
the
presence of nanoscale silver (Fig.4 a). The EDX analysis of CS-nanoscale Ag
nanocomposite has been the signal of nanoscale silver at 3 keV
and appeared the other signal of C, O, and N due to ch=
itosan
(Fig.4 b). Based on the result is confirmed the presence of CS <=
/a>and nanoscale silver.
The EDX analysis of CS-NMMT has been the signal of clay for Al and Si and
appeared the other signal of C, O, and N due to chitosan (Fig.4 c). The EDX
analysis of CS-nanoscale Ag-NMMT nanocomposite has indicated the presence of silver, chitosan and clay in the nanocomposite (Fig.4 d).
<=
span
style=3D'font-family:"Times New Roman","serif";mso-ascii-theme-font:major-b=
idi;
mso-hansi-theme-font:major-bidi;mso-bidi-theme-font:major-bidi'>Fig 4. EDX analys=
is
of (a) nanoscale Ag, (b) CS-nanoscale Ag nanocomposite, (c) CS-NMMT
nanocomposite and (d) CS-nanoscale Ag-NMMT nanocomposite.
=
3.5. WVP tests
WVP test is used to study water vapor barrier properties of packag=
ing
films and investigated the inhibition of the humidity exchange
between food and environment. The WVP of samples was determined by the gravimetric method usi=
ng
WVP (Table 1). The chitosan film was showed the reduction in WVP permeability. The increase and decrease in WVP permeability were observed because of=
the =
addition
of nanoscale Ag and NMMT respectively into chitosan nanocomposite films. The
nanoscale Ag can form the hydrogen bond and resulted to the increase passage
while NMMT with high aspect ratio can dispers=
e uniformly in polymer matrix and lead to prevent permeability.
Table 1. =
span>WVP of samp=
les.
Sample |
Sample |
WVP (g/(m s Pa)) |
1 |
CS soluti=
on
2% wt |
1.92 × 10=
-10 |
2 |
CS-Silver nancomposite |
4.31 × 10-10 |
3 |
CS-MMT |
1.63 × 10=
-10 |
4 |
CS-MMT na=
ncomposite
5% |
1.17 × 10-10 |
5 |
CS- Silver
-MMT nancomposite 3% |
4.78 × 10=
-10 |
6 |
CS- Silver -MMT nancomposite 5% |
3.25 × 10-10 |
The
antibacterial activity of samples was tested against E. coli bacteria using=
the =
well
diffusion method on Muller–Hinton Agar. All tests were
done in triplicate. The antibacterial tests were summarized in Table 2 that observed
the effect of chitosan and nanoscale Ag while NMMT has not exhib=
ited
any effect. The inhibition zone was the same =
span>for =
nanoscale
Ag and chitosan–nanoscale Ag and the effect of nanoscale Ag has been evident
apparent in comparison with chitosan. The minimum inhibitory concentration =
was
0.005 mol/L for chitosan–nanoscale Ag composite=
. Antibacterial activity results are comparable to those of previous researchers [33].
<=
span
style=3D'font-family:"Times New Roman","serif";mso-ascii-theme-font:major-b=
idi;
mso-hansi-theme-font:major-bidi;mso-bidi-theme-font:major-bidi'>Table 2. Inhibition
zone of samples.
Sample |
Sample |
Inhibition zone (mm) |
1 |
|
|
2 |
|
|
3 |
|
|
4 |
|
|
5 |
|
|
4. Conclusi=
ons
The chitosan
nanocomposites were successfully prepared from NMMT and in situ synthesis of nanoscale silver
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