International Journal of Biological Macromolecules 107 (2018) 17–27

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

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

Preparation of antimicrobial hybrid nano-materials using regenerated cellulose and

metallic nanoparticles
a a,b c,∗
Shiv Shankar , Ahmed A. Oun , Jong-Whan Rhim
a
Department of Food Engineering and Bionanocomposite Research Institute, Mokpo National University, 61 Dorimri, Chungkyemyon, Muangun, Jeonnam 534-729,
Republic of Korea
b
Food Engineering and Packaging Department, Food Technology Research Institute, Agricultural Research Center, Giza, Egypt
c
Center for Humanities and Sciences, and Department of Food and Nutrition, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of
Korea

article info abstract

Article history: In this study, antimicrobial hybrid nano-materials were prepared by one-pot syntheses of silver (Ag), copper oxide (CuO), or
Received 23 December 2016 Received in zinc oxide (ZnO) nanoparticles (NPs) during regeneration of cellulose from cotton linter (CL) and microcrystalline cellulose
revised form 5 July 2017 Accepted 22 (MCC). SEM micrographs indicated that the metallic nanopar-ticles were attached to the surface of the regenerated cellulose.
August 2017 Available online 1
EDX and ICP results showed that more AgNPs were adsorbed on the cellulose than CuONPs or ZnONPs. FTIR results
September 2017
revealed that the metallic nanoparticles were attached to the cellulose through the interaction with the hydroxyl group of
cellulose. XRD results showed the characteristic diffraction peaks of individual metallic nanoparticles. The thermal stability of
Keywords:
the R-CL and R-MCC increased in the hybrids with AgNPs and ZnONPs. The R-cellulose/metallic NPs hybrids showed
Regenerated cellulose
Metallic nanoparticles strong antibacterial activity against E. coli and L. monocytogenes. Thus, the hybrid nano-materials can be used as nanofillers
Hybrid nano-materials for the preparation of antibacterial packaging films.
Antibacterial activity
© 2017 Elsevier B.V. All rights reserved.

1. Introduction advantages of organic polymers such as light weight, flexibility, and

Cellulose is one of the most abundant biopolymers that has been widely
used in reinforcing filler materials for the preparation of biopolymer-based
nanocomposites. Cellulose nanoparticles from natural resources are of various
shapes and sizes, depending on the source of cellulose and preparation
method [1–3]. The excel-lent mechanical properties, nanosize, high aspect
ratio, availability, low cost, and renewability of cellulose make it an excellent
can-didate for a reinforcing material [4–7]. However, monofunctional
cellulose nanoparticles or fibrils can improve only a few proper-ties of a host
polymer, but cannot expect additional functional properties such as
antibacterial and UV-barrier properties [8]. The synthesis of cellulose
nanofibers containing dispersed functional inorganic nanoparticles has
attracted substantial research interest due to their potential applications in
catalysis, electronics, sensors, nanocomposites, drug delivery, and food
packaging [9]. More-over, these composite fibers have been shown to possess
both the
∗
Corresponding author at: Center for Humanities and Sciences, and Department of Food and
Nutrition, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic
of Korea.
E-mail address: jwrhim@khu.ac.kr (J.-W. Rhim).
moldability, and inorganic particles such as specific functionality
(antibacterial and UV barrier), high strength, and thermal stability.
Ionic and nano forms of metals can be applied to the cellulose to impart
the antibacterial property [10–12]. The adsorption pro-cess is the conventional
method to insert metal ions in cellulose through the ion exchange interaction
+
between metal ions and the H of hydroxyl groups of cellulose [13–15]. For
the incorporation of nanometals into cellulose, two different methods are
usually used. First, the metal ions are reduced to nanoparticles then deposited
on cellulose [11,16,17]. In the second, the metal ions are absorbed on
cellulose then reduced in situ [18,19].
Nanocellulose or nanocrystals are usually prepared by remov-ing the
amorphous region of the cellulose using acid hydrolysis methods. However, a
regeneration process coupled with homoge-nization and ultrasonication is a
simple approach to transforming the cellulose to cellulose fibrils, nanofibrils,
nanocrystals, hydro-gels, bioplastics, films, and membranes [20]. Moreover,
using a NaOH/urea aqueous solution is considered a green, non-toxic, and
low-cost method for dissolving cellulose pulps rapidly at a low temperature
[21]. In this one-step green synthesis of metal-lic nanoparticles, the NaOH
solution hydrates the cellulose chains through the formation of new hydrogen-
bond networks and func-tions as a reducing agent for metal ions. Here,
cellulose can be used

https://doi.org/10.1016/j.ijbiomac.2017.08.129 0141-
8130/© 2017 Elsevier B.V. All rights reserved.
18 S. Shankar et al. / International Journal of Biological Macromolecules 107 (2018) 17–27
to prevent the aggregation of metallic nanoparticles and to improve their
distribution along the cellulose chains [22].
Nano-sized silver (Ag), copper (Cu), and zinc (Zn) particles have a strong
antimicrobial activity against a broad range of bacteria [23–26]. Although the
exact mechanism of the action of the NPs has not been established yet,
various probable mechanisms of the action of the metallic NPs are available
in the literature [23,27,28]. Metallic NPs can act on bacteria in a number of
ways: by adsorbing NPs on the cell wall, destruction of the cell membrane by
free radi-cals, by releasing reactive oxygen species, by metal ions interacting
with respiratory enzymes, by interaction with DNA and protein through sulfur
and phosphorus, and by destroying cells [29,30]. In general, the antimicrobial
activity of the metallic NPs depends on the synthesis method of NPs, their
sizes, and shapes, as well as the types and nature of the capping agents. For
instance, when the NPs are deposited on inorganic or organic carriers, the
surface and chemical nature of the carrier influence the release of NPs or
metal ions, the interaction with bacteria, and the tendency to aggregate [31].
Therefore, the present study focussed on the preparation of regenerated
cellulose/metallic nanoparticle composites using the one-step green synthesis
method. The prepared nanocomposites were characterized using SEM, EDX,
FTIR, XRD, ICP-OES, and TGA. Also, the antibacterial activity of R-
cellulose/metallic NPs was eval-uated.
2. Materials and methods
2.1. Materials
Silver nitrate was purchased from Daejung Chemicals & Metals Co., Ltd.
(Siheung, Gyeonggi-do, Korea). Zinc nitrate hexahydrate, brain heart infusion
broth (BHI), and tryptic soy broth (TSB) were obtained from Duksan Pure
Chemicals Co., Ltd. (Ansan, Gyeonggi-do, Korea). Copper nitrate and
microcrystalline cellulose were procured from Sigma-Aldrich (St. Louis, MO,
USA). Cotton linter pulp (CLP-A; -cellulose 99.1%, viscosity 8.4 cP, DPn
465, DPw 1745) was generously donated by COMSCO (Korea Minting and
Secu-rity Printing Corporation, Daejeon, Korea). Escherichia coli ATCC
25922, Listeria monocytogenes ATCC 15313, Staphylococcus aureus, and
Staphylococcus epidermis KCTC 1917 were obtained from the Korean
Collection for Type Cultures (KCTC, Seoul, Korea).
2.2. Preparation of hybrid nano-materials
The hybrid nano-materials of cellulose fibers, cotton linter (CL) and
microcrystalline cellulose (MCC) with metallic nanoparticles (Ag, Cu, and
Zn) were prepared by the method of Shankar & Rhim
[26] with slight modification. For this, 4 g of CL fibers or MCC were added
separately into 400 g of a mixed solution of 7% (w/w) NaOH and 12% (w/w)
urea. The solutions were vigorously stirred for 30 min at room temperature
◦
and kept in a freezer at −20 C for 16 h. Then the solution was again mixed
using a magnetic stirrer at 1000 rpm for 20 min until the CL fibers or MCC
◦ ◦
were dissolved completely. Then the solutions were heated to 80 C while
stirring and 50 mM of silver nitrate, copper nitrate, or zinc nitrate solution
◦
were added dropwise while continuously heating and stirring at 80 C for 4 h.
The solution was cooled to room temperature and an excess amount of double
distilled water was added to produce regenerated cellulose. The regenerated
cellulose/metallic nanopar-ticles (RC-MNPs) were washed with double
distilled water until the pH of the content reached 7–8. The solution was
homoge-nized using a high shear homogenizer (T25 basic, Ika Labotechnik,
Janke & Kunkel Gmbh & Co., KG Staufen, Germany) at 10,000 rpm for 10
min, and sonicated using a high intensity ultrasonic proces-sor (Model VCX
750, Sonic & Materials, Inc., Newtown, CT, USA)
for 5 min using a 2 mm probe at 75% amplitude. The suspended
◦
nanocomposites were dried in an oven at 60 C to get the RC-MNPs hybrid
nano-material powder.
2.3. Characterization of hybrid nano-materials
The morphologies of the RC-MNPs hybrid nano-materials were observed
using a field emission scanning electron microscope (FE-SEM, S-4800,
Hitachi Co., Ltd., Matsuda, Japan) at the accelerated voltage of 5.0 kV. The
shapes and sizes of the RC-MNPs hybrid nano-materials were determined
using a field transmission electron microscope (TEM). The image analysis
was done using a JEM-2100F instrument (JEOL USA, Inc., MA, USA)
operated at an accelerated voltage of 200 kV.
The elemental composition of the RC-MNPs hybrid nano-materials was
analyzed using energy dispersive X-ray spectroscope (EDX) in the SEM
instrument.
Fourier transform infrared (FTIR) spectra of the RC-MNPs hybrid nano-
materials were obtained using an attenuated total reflectance-Fourier
transform infrared (ATR-FTIR) spectropho-tometer (TENSOR 37
Spectrophotometer with OPUS 6.0 software, Billerica, MA, USA) at ambient
temperature using the KBr disk method. The disk was scanned in wavenumber
−1 −1
ranging from 4000 to 600 cm with a resolution of 4 cm .
The crystalline nature of the RC-MNPs hybrid nano-materials was
characterized with powdered X-ray diffraction (XRD) using an XRD
diffractometer (PANalytical Xpert Pro MRD diffractome-ter, Amsterdam,
Netherlands). The spectra were recorded using monochromatic Cu K radiation
◦
at a wavelength of 0.154056 nm in the 2 range of 5–80 at a scanning rate of
◦
0.4 /min at room tem-perature. The X-ray generator tension and current were
40 kV and 30 mA, respectively. The crystallite size (D, nm) of the R-CL, R-
MCC, R-cellulose/AgNPs, R-cellulose/CuONPs, and R-cellulose/ZnONPs
was calculated using Scherrer’s equation with the most intense XRD peaks of
corresponding NPs [26].
D= K / 1/2 cos
where K is the Scherrer constant (0.94), is the X-ray wavelength
( = 0.154056 nm), 1/2 is the full width at the half maximum of the diffraction
peak (FWHM), and is Bragg’s angle.
X-ray photoelectron spectroscopy (XPS) measurements were carried out
with a Thermo K-Alpha XPS instrument (Thermo Sci-entific, NY, USA) at
−9
∼1 × 10 Torr. The core-level spectra from the respective samples were
recorded with monochromated alu-minum K radiation (photon energy =
1486.6 eV) at a pass energy of 20 eV and an electron take-off angle (angle
◦
between the electron emission direction and surface plane) of 90 . The core-
level binding energies were aligned with the C1s (adventitious carbon)
binding energy of 284.6 eV.
The thermal stability of the RC-MNPs hybrid nano-materials was tested
using a thermogravimetric analyzer (Hi-Res TGA 2950, TA Instrument, New
Castle, DE, USA). About 10 mg samples were taken in a standard aluminum
◦
pan and heated from 30 to 600 C at a rate of 10 C/min under a nitrogen
3
flow of 50 cm /min. The derivative form of TGA (DTG) was obtained using
differentials of TGA values, calculated using a central finite difference method
[32]:
DTG = (wt+ t − wt- t )/2 t
where wt+ t and wt- t were the residual weights of the samples at time t + t and t
− t, respectively, and t was the time interval for reading the residual sample
weights. The maximum decompo-sition temperature (Tmax ) of RC-MNPs
hybrid nano-materials was obtained from DTG curves, and the
weight loss (%) and residual char were determined from the
TGA curves.
 S. Shankar et al. / International Journal of Biological Macromolecules 107 (2018) 17–27
The quantity of nanoparticles attached to the cellulose was determined by
using an inductive coupled plasma-optical emission spectrometer (ICP-OES,
Spectro Arcos, AMETEK Inc., Berwyn, PA, USA) equipped with a Babington
nebulizer and a double pass spray chamber. For this, 200 mg of RC-MNPs
hybrid nano-materials were dispersed into 50 mL of deionized water, and 1
mL of concentrated nitric acid was added. The suspension was kept stirring
◦
overnight at room temperature. After, it was heated at 70 C for 1 h then
cooled and filtered through a Whatman No. 541 filter paper. The filtrate was
analyzed using the ICP-OES. A linear calibration curve was obtained using
the standard solutions of each metal (Cu, Ag, and Zn in water) ranging from
0.2 to 10 mg/L. The instrument was oper-ated at a plasma power of 1400 W, a
coolant flow of 12 mL/min, an auxiliary flow of 1 mL/min, and a nebulizer
flow of 1 mL/min with the line selections at 325 nm, 328 nm, and 214 nm for
Cu, Ag, and Zn, respectively. Three replicated samples were analyzed, and the
results were presented as mean ± SD.
2.4. Antibacterial activity
The antibacterial activity of the RC-MNPs hybrid nano-materials was
evaluated using the viable cell colony count method [24]. A single colony of
L. monocytogenes and E. coli was aseptically inocu-lated into 20 mL of BHI
◦ the cellulose fibers as a single nanoparticle or in aggregated form. Yu et al.
and TSB broths, respectively, and incubated at 37 C for 16 h. Each culture
broth was centrifuged at 3000 rpm for 10 min. The cell pellets were
suspended in 100 mL of sterile TSB and BHI broths, respectively, and diluted
5 the ZnONPs were uniformly distributed on the surface of cellulose
ten times with sterile distilled water. 20 mL of diluted broths containing 10 –
6 nanocrystals with an average size of 46.2 nm.
10 CFU/mL bacteria were put into 100 mL conical flasks containing 4 mg of
◦
the hybrid RC-MNPs and subsequently incubated at 37 C for 15 h while
being mildly shaked. The same diluted broth without the hybrid NPs was used
as the control. At 3 h intervals, samples were taken out, seri-ally diluted and
◦
plated on BHI and TSB agar plates. The plates were incubated at 37 C for 20
h, and the cell viability of each pathogen was calculated by counting the
bacterial colonies on the plates.
2.5. Statistical analysis
Statistical analysis was performed by one-way analysis of vari-ance
(ANOVA), and the significance of each mean property value was determined
(p < 0.05) with Duncan’s multiple range test using the SPSS statistical
analysis computer program for Windows (SPSS Inc., Chicago, IL, USA).
3. Results and discussion
3.1. Morphology of RC-MNPs hybrid nano-materials
Hybrid nano-materials composed of regenerated cellulose (R-CL and R-
MCC) and metallic nanoparticles (ZnONPs, CuONPs, and AgNPs) were
prepared using a hydrothermal process. The CL and MCC were dissolved
◦
completely in a solution of NaOH/urea after incubation at −20 C for 16 h.
◦ followed by copper and zinc. The peak intensity indicated the relative
After adding zinc nitrate, copper nitrate, and silver nitrate to the heated (80
C) CL and MCC solu-tions, the colors of the solutions changed to milky
white, dark brown, and brownish yellow, respectively, which indicated the
for-mation of zinc oxide, copper oxide, and silver NPs. The addition of an
excess of water made the RC-MNPs hybrid nano-materials pre-cipitate. Here,
the NaOH in the solution played a crucial role in the formation of ZnONPs,
CuONPs, and AgNPs [24,33–35]. The regen-erated cellulose/ZnONPs,
cellulose/CuONPs, and cellulose/AgNPs hybrid nano-materials were white,
dark brown, and brownish yel-low, respectively. The SEM micrographs of the
regenerated CL, MCC, and RC-MNPs hybrid nano-materials are shown in
Fig. 1. The sur-faces of R-CL and R-MCC were smooth and porous with
small cracks.
19
Table 1
Concentration of metallic NPs the hybrid nano-materials.
Samples Ag (mg/g) Cu (mg/g) Zn (mg/g)
R-CL/AgNPs 102.75 – –
R-MCC/AgNPs 97 – –
R-CL/CuONPs – 29 –
R-MCC/CuONPs – 27 –
R-CL/ZnONPs – – 4
R-MCC/ZnONPs – – 8
The porous structure of the cellulose fibers acted as a microreac-tor for the
nucleation and growth of metallic nanoparticles [36]. The NPs deposits on the
surface of cellulose fibers are shown in the RC-MNPs hybrids (Fig. 1). The
shapes and sizes of the NPs were greatly influenced by the type of cellulose
support. The sizes of AgNPs in R-CL/AgNPs and R-MCC/AgNPs hybrids
were 40–60 nm and 50–150 nm, respectively; however, the shapes were
nearly spherical in both cases. On the contrary, the CuONPs in the R-
CL/CuONPs and the R-MCC/CuONPs hybrids looked like rods and had
irregular shapes with lengths of 50–150 nm and 30–70 nm, respectively. The
ZnONPs were spherical in shape with a diameter of 20–30 nm in both the R-
CL/ZnONPs and R-MCC/ZnONPs hybrids. Ahamed et al. [37] prepared
hybrid nano-materials of regenerated cellulose with chitosan and silver
nanoparticles, they observed that the AgNPs were distributed on the surface of
[38] reported the synthesis of cellulose nanocrystal/zinc oxide nanohybrids
using the Fischer esterification and precipitation method and also found that
The shapes and sizes of the hybrid nano-materials were deter-mined using
TEM (Fig. 2). The sizes of AgNPs in R-CL/AgNPs and R-MCC/AgNPs
hybrids were 60–100 nm and 10–30 nm, respec-tively. The AgNPs of R-
CL/AgNPs were found to be in the form of single NPs as well as in the
aggregated form, but they were well-separated from each other in the R-
MCC/AgNPs. The CuONPs in the R-CL/CuONPs and the R-MCC/CuONPs
hybrids were irregular in shape and 50–150 nm and 20–30 nm, respectively.
The ZnONPs were also irregular in shape and 30–50 nm in both the R-
CL/ZnONPs and R-MCC/ZnONPs hybrids. There was not much difference in
the sizes of NPs when measured by SEM and TEM, except in the case of
AgNPs. The AgNPs in the case of R-MCC/AgNPs were well-separated from
each other as shown in the TEM image. However, the other NPs were in both
single as well as aggregated forms.
3.2. EDX and ICP analyses
The elements in the RC-MNPs hybrid nano-materials were ana-lyzed
using an EDX elemental analyzer equipped with an FE-SEM instrument, and
the results are shown in Fig. 3. The peaks for zinc, copper, and silver in their
respective positions indicated the presence of these elements in the RC-MNPs
hybrids. The peaks for Ag, Cu, and Zn were around 2.95, 0.95, and 1.01 KeV,
respec-tively [23,26,33], with the peak intensities being the highest for silver
concentration of each element in the RC-MNPs hybrid nano-materials and
provided information on the percentage of metallic NPs attached to the
surface of the R-cellulose.
The actual amount of Zn, Cu, and Ag nanoparticles in the RC-MNPs
hybrid nano-materials was determined by performing the ICP element
analysis, and the results are presented in Table 1. The results showed that the
amount of AgNPs was the highest (∼103 mg/g CL and 97 mg/g MCC)
followed by CuONPs (∼29 mg/g CL and 27 mg/g MCC) and ZnONPs (∼4
mg/g CL and 8 mg/g MCC). Among the types of cellulose, the CL exhibited
higher binding capacity with AgNPs and CuONPs than the MCC. The higher
binding
20 S. Shankar et al. / International Journal of Biological Macromolecules 107 (2018) 17–27

Fig. 1. SEM images of R-cellulose/metallic NPs composites.

of AgNPs to the cellulose fibers might be due to the larger surface area of
AgNPs compared with that of CuONPs and ZnONPs. The ICP analysis results
were consistent with the results of the SEM and EDX analyses, which showed
that the binding of AgNPs with R-cellulose was the highest followed by that
of CuONPs and ZnONPs. El-Rafie et al. [11] prepared nanosilver coated
cotton fabrics to test bind-
ing capacity of the AgNPs with the cotton fabrics using an
atomic absorption spectrophotometer, and they found that
the silver con-tents were 109.07, and 97.85 mg/kg of the
fabrics when treated with AgNPs in the presence and absence
of a binder, respectively. However, in the present study, the
silver content is around 1000 times higher in the R-cellulose
compared to the amount reported
 S. Shankar et al. / International Journal of Biological Macromolecules 107 (2018) 17–27 21

Fig. 2. TEM micrographs of R-cellulose/metallic NPs composites.

by El-Rafie et al. [11]. The higher amount of adsorption of NPs in the present 3.3. FTIR and XRD analyses
study might be due to the dispersion of the metal ions over the completely
dissolved CL and MCC solutions by the NaOH/urea solution. The dissolution FTIR analysis was carried out to test the functional groups present in the
of CL and MCC caused an increase in the surface area for the higher RC-MNPs hybrids, and the FTIR spectra of the regen-erated MCC, CL, and
interactions with the metal ions [39]. RC-MNPs hybrids are shown in Fig. 4. The peaks around 3300 and 2902
−1
cm were due to the O H and C H stretch-ing vibrations of cellulose,
−1
respectively [40]. The peak at 1649 cm
22 S. Shankar et al. / International Journal of Biological Macromolecules 107 (2018) 17–27
Fig. 3. EDX of R-cellulose/metallic NPs composites.
could be assigned to the bending of the O H groups of cellulose [41]. The
−1
peaks at 3300 and 1649 cm decreased significantly in the RC-MNPs
hybrids, which indicated the role of the O H group in the formation and
stabilization of the metallic nanoparticles. The R-CL and R-MCC showed the
−1
characteristic peak of C H stretching vibrations at 2902 cm . However, this
peak shifted to the higher frequency when the hybrid was formed with
metallic NPs, indicat-ing a certain interaction between metallic nanoparticles
and the
C H group of cellulose. Other absorption peaks observed in the range of
−1 −1
1800 cm to 600 cm were related to the deformation, wagging and
twisting modes of the anhydro-glucopyranose units [10]. The absorption peak
−1
at 1450 cm corresponded to the CH2 bending vibration and was ascribed to
the ‘crystallinity band’ of the cellulose. The increase in the intensity of the
−1
peak at 1450 cm was observed in the RC-MNPs compared with the R-CL
and R-MCC, which indicated an increase in the crystallinity after the forma-
−1
tion of nanoparticles. The absorption peak at 893 cm , which was attributed
to the C O C stretching vibration of -(1 → 4)-glycosidic linkages of cellulose
(amorphous band), was decreased in the C-MNPs hybrids [42].
The X-ray diffraction patterns of R-CL, R-MCC, and RC-MNPs hybrids
are shown in Fig. 5. The diffractograms of the R-CL and R-MCC showed the
characteristic crystalline peaks of cellulose II structure at 12.03, 20.19, and
◦ respectively. However, the crystallite sizes of ZnONPs in R-
22.5 , corresponding with the (−110), (110), and (200) lattice planes,
respectively [32,43]. The cellulose converted from cellulose I to cellulose II
after treatment with NaOH. The diffractograms of the RC-MNPs hybrids
exhibited the peaks of the metal and metal oxide nanoparticles as well as the
peaks of cellulose. However, the intensity of the cellulose peaks decreased
after the formation of the RC-MNPs hybrids. This might be due to
the presence of metallic nanoparticles (MNPs) on the surface of cellulose
fibers which masked the cellulose to reduce the inten-sity of cellulose peaks.
These results were also consistent with those of EDX and ICP tests. The
masking effect on the cellulose peak was the highest in the R-cellulose/AgNPs
hybrids followed by the R-cellulose/CuONPs and R-cellulose/ZnONPs. Since
the amount of silver on the surface of cellulose fibers was the highest, the R-
cellulose/AgNPs hybrids exhibited the highest masking effect on the cellulose
peaks in XRD diffractograms. Cai et al. [34] also reported the XRD
diffraction peaks of cellulose II with (-110), (110), and (200) lattice planes in
addition to the peaks of silver, gold, and platinum nanoparticles. They also
found that the intensity of cel-lulose peaks decreased after depositing metallic
nanoparticles on the surface of the cellulose fibers.
The crystallite sizes of R-cellulose and R-cellulose/metallic nanoparticles
were estimated by the Scherrer equation. The crys-tallite sizes of R-CL and R-
MCC at the peak positions at 2 = 21.1 and 21.7 ◦ were
2.21 and 2.41 nm,
respectively. The crystallite sizes of AgNPs in R-CL/AgNPs and
R-MCC/AgNPs for peak (111) were 12.64 and 13.63 nm,
respectively. The crystallite sizes of CuONPs in R-CL/CuONPs
and R-MCC/CuONPs for peak (111) were 12.55 and 13.53 nm,
CL/ZnONPs and R-MCC/ZnONPs for peak (002) were 14.56
and 10.92 nm, respectively. The crystallite sizes of the NPs
determined by the XRD peak were smaller than those
determined by the SEM and TEM. The difference in the sizes
of nanoparticles determined by the three different methods
(SEM, TEM, and XRD) was mainly due to the fact that the XRD
peak measures the sizes of a single crystal of nanoparticles,
however, TEM and SEM measure the sizes
 S. Shankar et al. / International Journal of Biological Macromolecules 107 (2018) 17–27
Fig. 4. FTIR spectra of R-cellulose/metallic NPs composites.
of nanoparticles in the form of single particles or bulk/aggregated forms.
3.4. XPS analysis
To determine the oxidation state of metal and metal oxide nanoparticles,
XPS measurement was performed at the Ag3d, Cu2p, and Zn2p core levels of
AgNPs, CuNPs, and ZnNPs, respectively (Fig. 6). The Ag3d5/2 and Ag3d3/2
core level binding energies for AgNPs appeared around 367 and 373 eV,
respectively, which is in good agreement with the bulk metallic silver values
[25,44]. The XPS results of R-cellulose/CuONPs reveal the presence of CuO
2+ 2+
(Cu ) in the nanocrystals, as shown by the Cu2p 3/2 peaks. Cu has one
main peak around 933 eV and shake-up satellites at higher bind-ing energies
around 940 and 942 eV. The shake-up satellites are characteristic of materials
9 2+
having a d configuration in the ground state, i.e., Cu [45]. In the case of R-
cellulose/ZnONPs, the Zn2p3/2 peak was found around 1021 eV. That
corresponded to zinc linked to oxygen in ZnONPs [46]. The XPS results
revealed that the oxida-tion states of silver, copper, and zinc were in the form
0 2+ 2+
of metallic silver (Ag ), copper oxide (Cu ), and zinc oxide (Zn ).
3.5. Thermal stability
The thermal stability of R-CL, R-MCC, and RC-MNPs hybrids was tested
using TGA, and the results are shown in Fig. 7. The
23
Fig. 5. XRD patterns of R-cellulose/metal NPs composites.
TGA thermograms exhibited the weight loss pattern of the sam-ples due to
their thermal degradation, and the DTG curves showed the maximum
decomposition temperature at each stage of degra-dation. The TGA
thermograms showed the two steps of thermal degradation of the samples. The
initial weight loss caused by the moisture evaporation was observed in the
◦
temperature range of 55–100 C. The main step of thermal degradation was
◦
observed in the range of 250–360 C, which was mainly due to the
degradation of cellulosic materials [32,47]. The thermostability of hybrid
nano-materials increased in the case of AgNPs, and ZnONPs incorporated R-
CL and R-MCC. However, it decreased in the CuONPs incorpo-rating R-CL
and R-MCC. Since metallic NPs are more thermostable as compared to
cellulose molecules, the inclusion of metallic NPs increased the
thermostability of the hybrid nanocomposites. In the R-CL/CuONPs and R-
◦
MCC/CuONPs, the main thermal degradation started from 200 C with the
◦
maximum degradation at 321.6 and 315.8 C, respectively. Table 2 shows the
maximum decomposi-tion temperature and char content after final
degradation. After the final thermal decomposition, the char content of the R-
CL, R-CL/AgNPs, R-CL/CuONPs, R-CL/ZnONPs, R-MCC, R-MCC/AgNPs,
R-MCC/CuONPs, and R-MCC/ZnONPs was 0.03, 51.2, 39.8, 25.2, 24.6,
62.2, 48.6, and 31.0%, respectively. The residual char content was higher in
the AgNPs hybrid, followed by the CuONPs and ZnONPs hybrids due to the
higher content of individual nanoparticles in the hybrid nano-materials. The
char residues of TGA results were also consistent with the results of SEM and
ICP tests, which showed that
24 S. Shankar et al. / International Journal of Biological Macromolecules 107 (2018) 17–27

Fig. 6. XPS spectra of R-cellulose/metallic NPs composites.

the cellulose-based nano-materials contained more AgNPs than CuONPs or
ZnONPs.
3.6. Antibacterial activity
The antibacterial activity of the RC-MNPs hybrid nano-materials was
evaluated against two representative foodborne pathogenic
bacteria, E.coli and L. monocytogenes, using a viable colony count method,
and the results are shown in Fig. 8. Hybrid nano-materials with AgNPs
attached to the R-CL or R-MCC showed higher antibac-terial activity against
both E. coli and L. monocytogenes compared with the hybrids of CuONPs and
ZnONPs. The R-CL/AgNPs and R-MCC/AgNPs hybrids exhibited strong
antibacterial activity to completely destroy the Gram-negative bacterium, E.
coli, in 6 h.
 S. Shankar et al. / International Journal of Biological Macromolecules 107 (2018) 17–27 25

Fig. 7. TGA and DTG curves of R-cellulose/metal NPs composites.

Table 2 E. coli and L. monocytogenes. The difference in the antibacterial activity of

Thermogravimetric analysis results of R-cellulose/metallic NPs composites.
the NPs against Gram-negative and Gram-positive bacte-ria might be due to
Samples ◦ ◦
Tmax ( C) Decomposition at Tmax (%) Char at 600 C (%) the differences in their cell wall structures and thicknesses [25,29]. Gram-
R-CL 333.3 44.8 0.03 positive bacteria possess a thick pep-tidoglycan layer just below the cell
R-CL/AgNPs 339.2 30.3 51.2 membrane which protects the bacteria from external stresses and reduces the
R-CL/CuONPs 321.6 34.6 39.8 penetration of toxic substances. However, the outer layer of Gram-negative
R-CL/ZnONPs 333.3 32.3 25.2
bacteria allows a higher permeability of small molecules that make them more
R-MCC 321.6 30.2 24.6
R-MCC/AgNPs 327.5 26.3 62.2 sensitive to toxins [48].
R- MCC/CuONPs 315.8 32.9 48.6
R- MCC/ZnONPs 327.5 30.1 31.0

The R-CL/AgNPs and R-MCC/AgNPs hybrids completely destroyed the
Gram-positive bacterium, L. monocytogenes, in 6 and 9 h, respectively. Also,
the R-cellulose/CuONPs progressively inhib-ited the growth of bacteria and
completely killed E. coli and L. monocytogenes in 9 and 12 h, respectively.
On the contrary, the R-cellulose/ZnONPs showed the bacteriostatic activity
against both
4. Conclusion
Hybrid nano-materials composed of regenerated cellulose (R-CL and R-
MCC) and metallic nanoparticles (AgNPs, CuONPs, and ZnONPs) were
prepared using a hydrothermal method. The results revealed that the metallic
nanoparticles attached to the surface of CL and MCC to form hybrid nano-
materials. The R-CL exhibited a
26 S. Shankar et al. / International Journal of Biological Macromolecules 107 (2018) 17–27

Fig. 8. Antibacterial activity of R-cellulose/metallic NPs composites.

higher binding capacity with AgNPs and CuONPs than the R-MCC. The
metallic nanoparticles formed a hybrid with R-CL and R-MCC, with the free
hydroxyl groups present on the surface of CL and MCC. The hybrid nano-
materials exhibited the crystalline peaks of cor-responding metals. The
deposition of metal nanoparticles masked the peaks of R-CL and R-MCC, and
the intensity of masking was dependent on the concentration of metallic
nanoparticles. The ther-mostability of CL and MCC increased when AgNPs
and ZnONPs were combined. The hybrid nano-materials exhibited strong
antibacte-rial activity against foodborne pathogenic bacteria. Thus, they can
be used as nanofillers for the preparation of antibacterial nanocom-posite
films for active food packaging applications.
Acknowledgements
This research was supported by the Agriculture Research Cen-ter (ARC
710003) program of the Ministry of Agriculture, Food and Rural Affairs,
Korea. Shiv Shankar acknowledges the financial support from the Korea
Research Fellowship Program through the National Research Foundation of
Korea (NRF) funded by the Min-istry of Science, ICT and Future Planning
(2016H1D3A1903910).
References
[1] Y.F. Liu, H.S. Wang, G. Yu, Q.X. Yu, B. Li, X.D. Mu, A novel approach for the
preparation of nanocrystalline cellulose by using phosphotungstic acid, Carbohydr.
Polym. 110 (2014) 415–422.
 S. Shankar et al. / International Journal of Biological Macromolecules 107 (2018) 17–27
[2] M.K.M. Haafiz, A. Hassan, Z. Zakaria, I.M. Inuwa, M.S. Islam, M. Jawaid,
Properties of polylactic acid composites reinforced with oil palm biomass
microcrystalline cellulose, Carbohydr. Polym. 98 (2013) 139–145.
[3] A.A. Oun, J.W. Rhim, Characterization of nanocelluloses isolated from Ushar
(Calotropis procera) seed fiber: effect of isolation method, Mater. Lett. 168 (2015) 146–
150.
[4] B. Braun, J.R. Dorgan, Single-step method for the isolation and surface
functionalization of cellulosic nanowhiskers, Biomacromolecules 10 (2009) 334–341.
[5] B.M. Cherian, L.A. Pothan, T. Nguyen-Chung, G. Mennig, M. Kottaisamy, S.
Thomas, A novel method for the synthesis of cellulose nanofibril whiskers from
banana fibers and characterization, J. Agric. Food Chem. 56 (2008) 5617–5627.
[6] J.H. He, T. Kunitake, A. Nakao, Facile in situ synthesis of noble metal nanoparticles in
porous cellulose fibers, Chem. Mater. 15 (2003) 4401–44063.
[7] P. Lu, Y.L. Hsieh, Preparation and properties of cellulose nanocrystals: rods, spheres,
and network, Carbohydr. Polym. 82 (2010) 329–336.
[8] H. Yu, C. Yan, X. Lei, Z. Quin, J. Yao, Novel approach to extract thermally stable
cellulose nanospheres with high yield, Mater. Lett. 131 (2014) 12–15.
[9] J.W. Rhim, H.M. Park, C.S. Ha, Bio-nanocomposites for food packaging
applications, Prog. Polym. Sci. 38 (2013) 1629–1652.
[10] M. Bajpai, P. Gupta, S.K. Bajpai, Silver(I) ions loaded cyclodextrin-grafted-cotton
fabric with excellent antimicrobial property, Fibers Polym. 11 (2010) 8–13.
[11] M.H. El-Rafie, H.B. Ahmed, M.K. Zahran, Characterization of nanosilver coated cotton
fabrics and evaluation of its antibacterial efficacy, Carbohydr. Polym. 107 (2014) 174–
181.
[12] M.K. Zahran, H.B. Ahmed, M.H. El-Rafie, Surface modification of cotton fabrics for
antibacterial application by coating with AgNPs-alginate composite, Carbohydr. Polym.
108 (2014) 145–152.
[13] H.E. Emam, A.P. Manian, B. Siroka, H. Duelli, B. Redl, A. Pipal, T. Bechtold,
Treatments to impart antimicrobial activity to clothing and household cellulosic-
textiles −why nano-silver? J. Clean. Prod. 39 (2013) 17–23.
[14] S. Ifuku, M. Tsuji, M. Morimoto, H. Saimoto, H. Yano, Synthesis of silver
nanoparticles templated by TEMPO-mediated oxidized bacterial cellulose
nanofibers, Biomacromolecules 10 (2009) 2714–2717.
[15] T. Saito, A. Isogai, Ion-exchange behaviour of carboxylate groups in fibrous cellulose
oxidized by the TEMPO-mediated system, Carbohydr. Polym. 61 (2005) 183–190.
[16] H.J. Lee, S.Y. Yeo, S.H. Jeong, Antibacterial effect of nanosized silver colloidal solution
on textile fabrics, J. Mater. Sci. 38 (2003) 2199–2204.
[17] S. Ravindra, Y. Murali Mohan, N. Narayana Reddy, K. Mohana Raju, Fabrication of
antibacterial cotton fibres loaded with silver nanoparticles via green approach, Coll. Surf.
A 367 (2010) 31–40.
[18] L.C.S. Maria, A.L.C. Santos, P.C. Oliveira, A.S.S. Valle, H.S. Barud, Y. Messaddeq,
S.J.L. Ribeiro, Preparation and antibacterial activity of silver nanoparticles impregnated
in bacterial cellulose, Polimeros 20 (2010) 72–77.
[19] R. Tankhiwale, S.K. Bajpai, Graft copolymerization onto cellulose-based filter paper and
its further development as silver nanoparticles loaded antibacterial food-packaging
material, Colloids Surf. B: Biointerfaces 69 (2009) 164–168.
[20] S. Wang, A. Lu, L. Zhang, Recent advances in regenerated cellulose materials, Prog.
Polym. Sci. 53 (2016) 169–206.
[21] J. Cai, L. Zhang, Rapid dissolution of cellulose in LiOH/urea and NaOH/urea
aqueous solutions, Macromol. Biosci. 5 (2005) 539–548.
[22] S. Azizi, M.B. Ahmad, M.Z. Hussein, N.A. Ibrahim, Synthesis, antibacterial and thermal
studies of cellulose nanocrystal stabilized ZnO-Ag heterostructure nanoparticles,
Molecules 18 (2013) 6269–6280.
[23] S. Shankar, J.W. Rhim, Effect of copper salts and reducing agents on characteristics and
antimicrobial activity of copper nanoparticles, Mater. Lett. 132 (2014) 307–311.
[24] S. Shankar, X. Teng, G. Li, J.W. Rhim, Preparation, characterization, and
antimicrobial activity of gelatin/ZnO nanocomposite films, Food Hydrocolloids
45 (2015) 264–271.
[25] S. Shankar, L. Jaiswal, P.R. Selvakannan, K.S. Ham, J.W. Rhim, Gelatin-based
dissolvable antibacterial films reinforced with metallic nanoparticles, RSC Adv. 6
(2016) 67340–67352.
[26] S. Shankar, J.W. Rhim, Amino acid-mediated synthesis of silver nanoparticles and
preparation of antimicrobial agar/silver nanoparticles composite films, Carbohydr.
Polym. 130 (2015) 353–363.
27
[27] S. Shankar, J. Chorachoo, L. Jaiswal, S.P. Voravuthikunchai, Effect of reducing agent
concentrations and temperature on characteristics and antimicrobial activity of silver
nanoparticles, Mater. Lett. 137 (2014) 160–163.
[28] P. Kanmani, J.W. Rhim, Physical, mechanical and antimicrobial properties of gelatin
based active nanocomposite films containing AgNPs and nanoclay, Food Hydrocolloids
35 (2014) 644–652.
[29] A. Orsuwan, S. Shankar, L.F. Wang, R. Sothornvit, J.W. Rhim, Preparation of
antimicrobial agar/banana powder blend films reinforced with silver nanoparticles,
Food Hydrocolloids 60 (2016) 476–485.
[30] S. Prabhu, E. Poulose, Silver nanoparticles: mechanism of antimicrobial action, synthesis,
medical applications, and toxicity effects, Int. Nano Lett. 2 (2012) 1–10.
[31] A.M. Eremenko, I.S. Petrik, N.P. Smirnova, A.V. Rudenko, Y.S. Marikvas, Antibacterial
and antimycotic activity of cotton fabrics impregnated with silver and binary
silver/copper nanoparticles, Nanoscale Res. Lett. 11 (2016) 1–9.
[32] S. Shankar, J.W. Rhim, Preparation of nanocellulose from micro-crystalline cellulose:
the effect on the performance and properties of agar-based composite films,
Carbohydr. Polym. 135 (2016) 18–26.
[33] S. Shankar, X. Teng, J.W. Rhim, Properties and characterization of agar/CuNP
bionanocomposite films prepared with different copper salts and reducing agents,
Carbohydr. Polym. 114 (2014) 21517–21524.
[34] J. Cai, S. Kimura, M. Wada, S. Kuga, Nanoporous cellulose as metal
nanoparticles support, Biomacromolecules 10 (2009) 87–94.
[35] T. Jiang, L. Liu, J. Yao, In situ deposition of silver nanoparticles on the cotton fabrics,
Fibers Polym. 12 (2011) 620–625.
[36] S. Liu, L. Zhang, J. Zhou, R. Wu, Structure and properties of cellulose/Fe2O3
nanocomposite fibers spun via an effective pathway, J. Phys. Chem. C 112 (2008)
4538–4544.
[37] M.I.N. Ahamed, S. Sankar, P.M. Kashif, S.K.H. Basha, T.P. Sastry, Evaluation of
biomaterial containing regenerated cellulose and chitosan incorporated with silver
nanoparticles, Int. J. Biol. Macromol. 72 (2015) 680–686.
[38] H.Y. Yu, G.Y. Chen, Y.B. Wang, J.M. Yao, A facile one-pot route for preparing
cellulose nanocrystal/zinc oxide nanohybrids with high antibacterial and photocatalytic
activity, Cellulose 22 (2015) 261–273.
[39] M. Gopiraman, H. Bang, G. Yuan, C. Yin, K.H. Song, J.S. Lee, I.M. Chung, R. Karvembu,
I.S. Kim, Noble metal/functionalized cellulose nanofiber composites for catalytic
applications, Carbohydr. Polym. 132 (2015) 554–564.
[40] J.I. Morán, V.A. Alvarez, V.P. Cyras, A. Vázquez, Extraction of cellulose and
preparation of nanocellulose from sisal fibers, Cellulose 15 (2008) 149–159.
[41] L.F. Wang, S. Shankar, J.W. Rhim, Properties of alginate-based films reinforced with
cellulose fibers and cellulose nanowhiskers isolated from mulberry pulp, Food
Hydrocolloids 63 (2017) 201–208.
[42] M. Adsul, S.K. Soni, S.K. Bhargava, V. Bansal, Facile approach for the dispersion of
regenerated cellulose in aqueous system in the form of nanoparticles, Biomacromolecules
13 (2012) 2890–2895.
[43] H.Y. Yu, Z.Y. Qin, B. Sun, J.M. Yao, One-pot green fabrication and antibacterial activity
of thermally stable corn like CNC/Ag nanohybrid, J. Nanopart. Res. 16 (2014) 2202–
2215.
[44] P. Prieto, V. Nistor, K. Nouneh, M. Oyama, M. Abd-Lefdil, R. Diaz, XPS study of silver,
nickel and bimetallic silver-nickel nanoparticles prepared by seed-mediated growth,
Appl. Surf. Sci. 258 (2012) 8807–8813.
[45] C.K. Wu, M. Yin, S. O’Brien, J.T. Koberstein, Quantitative analysis of copper oxide
nanoparticle composition and structure by X-ray photoelectron spectroscopy, Chem.
Mater. 18 (2006) 6054–6058.
[46] S.B. Amor, M. Jacquet, P. Fioux, M. Nardin, XPS characterisation of plasma treated
and zinc oxide coated PET, Appl. Surf. Sci. 255 (2009) 5052–5061.
[47] M. Abdollahi, M. Alboofetileh, M. Rezaei, R. Behrooz, Comparing physico-mechanical
and thermal properties of alginate nanocomposite films reinforced with organic and/or
inorganic nanofillers, Food Hydrocolloids 32 (2013) 416–424.
[48] J. Quirós, J.P. Borges, K. Boltes, I. Rodea-Palomares, R. Rosal, Antimicrobial electrospun
silver-, copper- and zinc-doped polyvinylpyrrolidone nanofibers, J. Hazard. Mater. 299
(2015) 298–305.