Study of chitosan with different degrees of acetylation as cardboard paper coating

Carbohydrate Polymers 210 (2019) 56–63

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Study of chitosan with different degrees of acetylation as cardboard paper
coating

T

⁎

Mariane Gatto, Deise Ochi, Cristiana Maria Pedroso Yoshida , Classius Ferreira da Silva
UNIFESP - Federal São Paulo University, Institute of Environmental, Chemical and Pharmaceutical Sciences, SP, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords:
Coating
Chitosan
Chemical reacetylation
Acetylation degree

The biodegradability of chitosan is significant for packaging systems. Another relevant property of chitosan is its
degree of acetylation (DA), which affects other properties, such as crystallinity and hydrophobicity. The DA can
be modulated by chitin deacetylation or even chitosan reacetylation. The novelty of this paper is the application
of reacetylated chitosan as a coating for cardboard paper surfaces to improve the barrier and mechanical

properties of the paper. Chitosan with 2% DA was reacetylated to yield chitosan with 48% DA. Both samples
were applied as cardboard paper coating, and the coated materials were characterized. The paper-film system of
chitosan with 2% DA had better water barrier and mechanical resistance. Heterogeneous deacetylation of chitin
reduced the solubility of chitosan because molecular groups were distributed in blocks, increasing the hydrophobicity of the polymer.

1. Introduction
In the 20th century, petrochemical-based materials were extensively
used as packaging materials. In general, the barrier properties of the
paper packaging material can be controlled by coating with petroleumbased derivatives polymers (e.g., polyolefins and ethylene vinyl alcohol) and waxes (Rastogi & Samyn, 2015). However, there is a demand
for product manufacturers to focus on ecofriendly packaging solutions,
as sustainability concerns are increasing as well as the replacement of
the nonrenewable sources (mainly from petroleum) with renewable
sources (Robertson, 2012). Examples of such renewable materials include biopolymers such as starch, cellulose, animal or plant-based
proteins, lipids, and chitin/chitosan (Cutter, 2006).
Paper is a biodegradable material widely applied as packaging
material in many industries such as electronics, food, and pharmaceuticals because of its low cost, biodegradability, good resistance, light
weight, and recyclability. However, hydrophilicity and porosity limit
their packaging applications (Rastogi & Samyn, 2015). Cellulose chains
are linked by hydrogen bonds, which directly affect the physicochemical properties (i.e., solubility and crystallinity) and mechanical properties of paper (Kondo, Koschella, Heublein, Klemm, & Heinze, 2008).
The hydrogen bonds favor adsorption and transport of water molecules,
gases, and oils through the cellulose network, resulting in poor barrier
properties. Moreover, water adsorption causes fiber swelling, spoiling
material shape and reducing mechanical properties, which is not

⁎

desirable in packaging applications (Rastogi & Samyn, 2015). Therefore, despite the versatile advantages of paper, it is still important to
improve its mechanical properties; water absorption; and barrier to gas,
moisture, and fat (Reis, Yoshida, Reis, & Franco, 2011). The required
properties of paper depend on its application; for example, some food

packaging applications require high permeability of oxygen or even low
water permeability; on the other hand, paper for fried potatoes package
has to be oil-impermeable. The barrier and mechanical properties of
paper can be modified by coating using a nonrenewable polymer;
however, this transforms an ecofriendly material in a more polluting
material. On the other hand, biopolymers coatings offer environmental
advantages such as biodegradability, better recyclability, nontoxicity,
and biocompatibility, compared to conventional synthetic polymers
(Tang, Kumar, Alavi, & Sandeep, 2012). Therefore, coating paper surface with renewable biopolymers is an interesting method to improve
the barrier and wettability of paper as packaging materials (Khwaldia,
Basta, Aloui, & El-Saied, 2014).
For packaging applications, chitosan films are characterized by selective permeability to gases (CO2 and O2), good mechanical properties,
and biodegradability. However, high permeability to water vapor limits
their use, as this is not desirable for packaging applications. Promising
results have been obtained by the addition of lipids, waxes, and clays to
improve hydrophobicity, although this often reduces chemical and
mechanical properties (Elsabee & Abdou, 2013; MacIel, Yoshida, &
Franco, 2012). Composite coatings or multilayer coatings on paper

Corresponding author at: UNIFESP Federal University of São Paulo, Laboratory of Biotechnology and Natural Products, Rua São Nicolau, 210, Diadema, SP, Brazil.
E-mail address: (C.M.P. Yoshida).

/>Received 15 October 2018; Received in revised form 11 January 2019; Accepted 16 January 2019
Available online 17 January 2019
0144-8617/ © 2019 Elsevier Ltd. All rights reserved.


Carbohydrate Polymers 210 (2019) 56–63

M. Gatto et al.

distribution can be obtained by chitosan reacetylation under homogeneous conditions. Chitosan reacetylation in homogeneous conditions
produces more uniform structures (Chitosan 3, Eq. (3)). If the starting
chitosan is extensively deacetylated, the obtained copolymer will have
randomly distributed GlcN and GlcNAc units (Aiba, 1991).
This aim of this work is to evaluate the coating application of
deacetylated chitosan with 2% DA and reacetylated chitosan with 48%
DA on the cardboard paper surface and test the effects of these different
chitosan coatings on the final material properties.

surface have been prepared to combine the good mechanical resistance
of paper material with the barrier properties of coatings materials.
Khwaldia et al. (2014) observed that caseinate and caseinate/chitosan
bilayer coatings improved the water vapor barrier and mechanical
properties of paper packaging. Atkinson et al. (2017) applied chitosan
as paperboard coating and obtained better water wicking properties
and thus less moisture absorption, compared to using paper coated with
phenol formaldehyde resin. Kuusipalo, Kaunisto, Laine, and Kellomäki
(2005) applied chitosan as a wet-end additive in paperboard to improve
the mechanical and barrier properties and bending strength. Chitosan
with low molar mass (0.768 × 105g/mol) coating yielded paper with
lower water absorption, more smoothness, and higher dry strength than
chitosan with medium molar mass (2.375 × 105 g/mol) (Habibie,
Hamzah, Anggaravidya, & Kalembang, 2016).
Chitosan is the main chitin derivative and consists of 2-amino-2deoxy-D-glucopyranose (GlcN) units and 2-acetamido-2-deoxy-D-glucopyranose (GlcNAc), the latter in proportions below 50% of the biopolymer chain. The GlcNAc proportions in the macromolecule are represented by the degree of acetylation (DA) (Chatelet et al., 2001).
Chitin is insoluble in most organic acids, while chitosan is soluble in
dilute acidic solutions. Chitosan is a strong base because of primary
amino group presents in a GlcN unit. At low pH, these amino groups
(pKa 6.3) become protonated and positively charged. Chitosan becomes
a water-soluble cationic polyelectrolyte, increasing the pH (above 6),

which causes loss of charge, reducing solubility. This soluble-insoluble
transition occurs at a pKa value of 6.0–6.3. Chitosan solubility is
strongly dependent on chitosan DA (presence of GlcN and GlcNAc
units). Increasing the acetyl groups promotes changes in chitosan
structure; it transforms into a more crystalline chain. Therefore, chitosan solubility can be controlled by manipulating the DA (Pillai, Paul, &
Sharma, 2009).
A sample of chitin with 100% acetylation (i.e., 100% of the residues
are GlcNAc) can be deacetylated to obtain “Chitosan 1″ with 60%
acetylation degree; that is, 60% of the residues are GlcNAc (Eq. 1). In
parallel, another partial deacetylation can be performed to obtain
“Chitosan 2″ with 3% acetylation degree; that is, 3% of the residues are
GlcNAc (Eq. 2). However, if “Chitosan 2″ is subjected to a reacetylation
reaction, “Chitosan 3″, also with 60% acetylation degree, can be obtained (Eq. 3). Although Chitosan samples 1 and 3 have the same
amount of GLcNAc residues, the properties of these two samples can be
extremely different since the distributions of these residues are different. Block-like residues of GlcNAc and GlcN are present in Sample 1;
however, such residues are statistically distributed in Sample 3 (nonblock residues), promoting different properties in both samples. When
the GlcN residues are in blocks (Chitosan 1), they are less accessible to
protonation, and therefore, the solubilization of chitosan is more difficult.

Chitin (DA = 100%)

Chitin (DA = 100%)

Deacetylation 1

→

Deacetylation 2

→

Chitosan 1 (DA = 60%)

(1)

Chitosan 2 (DA = 3%)

(2)

Chitosan 2 (DA = 3%) + Acetic Anhydride
= 60%)

Reacetylation

→

2. Materials and methods
2.1. Materials
Original chitosan with 2% DA, provided by Mahtani Chitosan
(Veraval, India).
2.2. Homogeneous reacetylation
Reacetylation was carried out as described by Vachoud, Zydowicz,
and Domard (1997). Chitosan with 2% DA (C-2) was dissolved (1% w/
w) in aqueous acetic acid (AcA) solution in stoichiometric proportions
([AcA] = [-NH2]) and was agitated overnight. Afterward, 1,2-propanediol was added in two portions to the chitosan/AcA solution. The first
portion was added in its pure form (in an amount corresponding to 90%
v/v of the chitosan solution), and the solution was agitated for 2 h.
A solution was prepared by mixing the second portion of 1,2-propanediol (in an amount corresponding to 10% v/v of initial chitosan
solution) and acetic anhydride (AAn). The AAn amount, mAAn(g), was
determined by Eq. (4), where mc(g) is the amount of chitosan, DA1 and

DA2 are initial and desired DAs, respectively (DA2 = 0.48), WH2O is
water content determined through thermogravimetric analysis, MAAn is
the molar mass of acetic anhydride (102.1 g/mol), and M0 average mass
of the repetitive unit of chitosan.

mAAn =

mc (DA2 − DA1 )(1 − w H2 O ) MAAn
MO

(4)

The AAn solution was homogenized for 45 min under agitation and
was added dropwise to the chitosan solution. The mixture was kept
under agitation overnight. The resulting mixture was precipitated using
concentrated NH4OH, washed 10 times with distilled water, frozen, and
then freeze-dried. The resulting reacetylated chitosan with 48% DA is
referred as C-48 in this paper.
2.3. Chitosan characterization
2.3.1. Proton nuclear magnetic resonance spectroscopy
Proton nuclear magnetic resonance (H NMR) spectroscopy was
performed before and after reacetylation to determine the DA. Here,
8 mg of chitosan was dissolved in 1 mL of D2O acidified with 5 μL of HCl
(37%). Spectra were recorded on an ALS300 300 MHz (Bruker) spectrometer at 300 K. The DA (%) was calculated using (5):

Chitosan 3 (DA

1

⎛ ICH 3 ⎞

DA (%) = ⎜ 1 3
⎟ × 100
I
⎝ 6 (H 2 − H 6) ⎠

(3)

Furthermore, the distribution of N-acetyl groups is related to chitosan solubility (Franca, Freitas, & Lins, 2011). Chitosan is a linear copolymer obtained from chitin deacetylation or partial chitosan reacetylation to obtain chitosan with higher DA. These reactions can
occur in homogeneous (one phase reaction) or heterogeneous (more
than one phase, i.e., solid and liquid) conditions, resulting in copolymers with a different distribution of GlcN and GlcNAc units. Chitosan
produced from chitin deacetylation under heterogeneous conditions
occurs preferentially in amorphous regions of the molecule, forming
block copolymers and resulting in a more hydrophobic polymer (Chitosan 1, Eq. (1)). Chitosan with a higher DA and different N-acetyl group

(5)

where ICH3 represents the signal of methyl hydrogen in the acetyl group,
and I(H2-H6) is the signal of hydrogen connected to carbon 2 and 6 of the
glucopyranosyl ring.
2.3.2. Thermogravimetric analysis
Samples of chitosan powder (3–10 mg) were placed in open platinum capsules in a thermogravimetric analysis equipment (DTG-60,
Shimadzu, Japan). The temperature range was from 30 to 200 °C
(10 °C/min) under N2 flow at 100 mL/min. The water content was
calculated according to the mass loss up to 105 °C.
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M. Gatto et al.

1994a). Ten samples of dimensions 0.125 m × 0.125 m were preconditioned in a desiccator containing silica for 72 h at room temperature (25 ± 2 °C). Samples were individually weighed on a semianalytical scale with a precision of 0.01 g and attached to the Cobb test
equipment (Regmed, Brazil). Moreover, 100 mL of water was added to
contact with the surface delimited by the apparatus ring (internal diameter of 11.28 ± 0.02 cm) for 120 s. Soon after the specimen was removed, it was placed between two sheets of absorbent paper and
pressed by a conditioning roller (Regmed, Brazil) to remove excess
water. Lastly, the samples were weighed. Water absorption capacity
(Abs, g/m²) was determined using (7), where Mf and Mi(g) are final and
initial sample mass, respectively.

2.3.3. Size exclusion chromatography
Average molar mass (Mw) was determined by a size exclusion
chromatograph attached to a refraction index and multi-angle scattering measuring equipment (Wyatt Dawn ES multi-angles, United
States). The used column was TSKgel G6000PW + G2500PW (30 cm
length and 7.8 mm diameter). The mobile phase was an acid acetic
buffer solution (0.15 mol.L−1 ammonium acetate/0.2 mol.L−1) flowing
through the column at 0.5 mL/min.
2.3.4. Fourier transform infrared spectroscopy
Fourier transform infrared (FTIR) spectroscopy was performed at
room temperature using a universal attenuated total reflectance accessory (ATR), Nicolet iN10MXDA (Thermo Fisher, United States). Each
sample was evaluated by taking 64 scans with spectral range of
650–4000 cm−1 and 4 cm−1 resolution.

Abs =

(Mf − Mi )
A

× 100

(7)

2.6.5. Moisture content
Based on ASTM D644-99 (ASTM, 2007), the moisture content evaluation experiment was executed in triplicate. The samples were cut
into squares of 1 cm × 1 cm and dried in a TE-393 forced air circulation
oven (Tecnal, Brazil) at 105 ± 2 °C for 24 h. Moisture content (g H2O/
100 g paper) was calculated according to (8), where Mw and Md are wet
and dried mass, respectively.

2.3.5. Differential scanning calorimetry
Powdered chitosan samples (2.8–3 mg) were placed in aluminum
capsules hermetically closed in a differential scanning calorimeter DSC60 (Shimadzu, Japan). The capsules were exposed to a temperature of
20 °C–200 °C at 10 °C/min.
2.4. Chitosan suspension

M − Md ⎞
Moisture Content = ⎛ w
× 100
Md ⎠
⎝

Chitosan suspension was prepared as described by Yoshida (2009).
First, 1.0% (w/w) of chitosan was dispersed in the aqueous acid solution. Acetic acid was then added in stoichiometric proportions, according to the DA (DA = 2% or DA = 48%) and chitosan weight. The
system was kept under agitation overnight.

⎜

⎟

(8)

2.6.6. Water vapor transmission rate
The procedure was based on ASTM E96-00 (ASTM, 2000). Five discshaped samples were cut from each sample and fixed to the top of
permeation cells containing silica gel. The cells were conditioned in
desiccators containing saturated saline (sodium chloride) at relative
humidity of 75 ± 5% and kept in a temperature-controlled chamber
(25 ± 0.2 °C). Using an analytical scale, the mass gain of the system
(cell and sample) was determined at 24 h intervals over 120 h. The
water vapor transmission rate (WVTR) (gH2O/m2day) was calculated
using (9), where G (g) is the weight gained by the system, A (m²) is the
area exposed to vapor transmission, and t (day) is the number of days
the sample spent in the chamber.

2.5. Chitosan coating application on paper
First, 3 mL of chitosan suspension was dispersed on a cardboard
surface (Triplex TP 250 g/m2 Suzano Papel e Celulose Ltda., Brazil)
using a 60 μm bar (TKB Ericken, Brazil). Coated sheets were dried in a
forced air circulation oven (MA035/1000, Marconi Equipamentos,
Brazil) at 100 °C for 90 s. Each sheet sample was coated three times
(MacIel et al., 2012). The studied systems were paper coated with
chitosan of 2% DA (CP-2) and 48% DA (CP-48) and paper without
coating (P).

WVTR = G /(A. t )

(9)

2.6. Chitosan-coated cardboard paper characterization
2.6.7. Taber stiffness
Stiffness was determined according to T489 om-92 (TAPPI, 1994b).
Ten specimens of each system were cut in both directions of cellulosic

fibers (machine direction MD and cross-machine direction CD) in dimensions of 38.1 mm × 70.0 mm using a pneumatic guillotine (Regmed, Brazil). Samples were evaluated using an RI-5000 stiffness meter
(Regmed, Brazil) at an angle of 15°.

2.6.1. Coating evaluation: colored solution penetration
The evaluation procedure was adapted from Marcy (1995) and
executed in triplicate. Erythrosine in isopropanol solution (0.5% w/w)
was applied to uncoated (matte side) and coated paper samples (coated
side) covering the entire surface. Samples were held upright for 60 min
and dried in a TE-393 forced air circulation oven (Tecnal, Brazil) at
50 °C for 30 min.

2.6.8. Fat barrier
The methodology was based on Ham-Pichavant, Sèbe, Pardon, and
Coma, (2005). Different test solutions containing castor oil, toluene,
and n-heptane were prepared. One drop of test solution was applied to
the sample paper surface, and the sample was kept for 15 s. Excess
solution was removed, and the appearance of the opposite surface was
observed. The highest kit number for the test solutions that did not
cause blemishes was the adopted as the kit rating value of fat repellency. Kit nº1 and Kit nº12 were respectively the least and the most
aggressive solutions for producing a stain on the opposite surface.

2.6.2. Grammage
Following ASTM D646-96 (ASTM, 1996), 10 samples of each system
were cut into squares of 0.016 m² (0.125 m × 0.125 m) and weighed on
an analytical scale. Grammage G (g/m²) was calculated using (6),
where M (g) is the paper weight, and A (m²) is the area.

G = M /A

(6)

2.6.3. Thickness
Thickness was measured using a digital micrometer (0.001 mm)
(Mitutoyo, Japan) in quintuplicate. For each sample, six measurements
were carried out at different points.

2.6.9. Scanning electron microscopy
Samples were cut in squares of 1 cm × 1 cm and kept in a silicacontaining desiccator for 48 h at 25 °C. The metallic coating was conducted using a K450 sputter coater (Emitech, France), gold layer
thickness estimated at 200 Å. Micrographs of the surface (1000×

2.6.4. Cobb test
Cobb test was executed as described in T-441 om-90 (TAPPI,
58


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M. Gatto et al.

band at 1313 cm−1; and a CeCH3 deformation band at 1380 cm−1. The
C-2 spectra presented angular deformation bands of NH (amide II) at
1560 cm−1 (Focher, Naggi, & Torri, 1992). A less intensity of C]O
(1630 cm−1), amide III (1313 cm−1) and CeCH3 (1380 cm−1) peaks
were observed in the C-2 spectra, compared to C-48; this was associated
with the lower content of GlcNAc units. Focher et al. (1992) observed
that the bands at 1420–1435 cm−1were related to CH2. Modifications
in the CH2OH group were observed in the C-2 and C-48 samples.
Duarte, Ferreira, Marvão, and Rocha (2002) reported CO stretching at
1159, 1074, and 1025 cm−1. The asymmetric absorbance of CO at
1159 cm−1 could indicate changes promoted by depolymerization

during the deacetylation process.
In the differential scanning calorimetry (DSC) thermograms (Fig. 3),
an endothermic peak was identified between 20 °C and 100 °C, with an
enthalpy of 242.60 J/g at 58.37 °C for C-2 and 251.39 J/g at 58.03 °C
for C-48. These peaks are attributed to the evaporation of water
bounded to chitosan at hydroxyl groups and free amine in the amorphous region of chitosan (Ghosh, Azam Ali, & Walls, 2010). The C-48
endothermic peak was slightly higher than that of C-2, despite that C48 had a significantly higher WC, as shown in Table 1. Although the
water evaporation enthalpies for both samples were practically equal,
their WCs were quite distinct. It is essential to consider that the analytical conditions were very different for both techniques. In DSC
analysis, the evaporation of water took place in a few minutes and at
temperatures below that used to determine the WC. Moreover, in the
WC determination tests, the sample was subjected to 24 h drying at
105 °C. Thus, water strongly bonded to the chitosan chain could be
removed, whereas in the DSC, not all the water molecules were removed, but only those weakly bonded to the chitosan chain. Kittur,
Harish Prashanth, Udaya Sankar, and Tharanathan (2002) observed
that a higher number of amine groups in chitosans with lower DA allowed more bondings with water molecules, and therefore, lower DA
was related to higher endothermic peaks. They prepared chitosan with
different DAs using sodium hydroxide and observed that the water
evaporation enthalpy was inversely proportional to the DA when the
chitosan samples were obtained directly from chitin; for example, the
sample with 52% DA presented enthalpy 20% lower than the sample
with 11% DA. Our results showed that the difference between the enthalpies of the sample with 2% DA, obtained directly from chitin, was
only 3% higher that of the reacetylated sample with 48% DA. Therefore, the distribution of the chitosan residues, in blocks or not, affects
the water evaporation enthalpy probably due to the accessibility of the
water to the residues.
The exothermic peaks observed between 280 °C and 340 °C were
associated with chitosan degradation (Ng, Cheung, & McKay, 2002).
Guinesi and Cavalheiro (2006) evaluated different chitosan samples
with different DAs and observed a similar exothermic peak related to
GlcN group degradation in a temperature range of 296 °C–299 °C. In

their study, GlcNAc group degradation occurred near 404 °C, but this
was not observed in our samples. Moreover, they assessed the heterogeneous N-deacetylation of α-chitin to obtain chitosan with different
DAs, where the produced blocks of GlcNAc and GlcN along the chitosan
chain were probably due to the deacetylation. The block-like chitosan
could present different thermal events, compared to the chitosan with
statistical distribution of acetylated and non-acetylated residues. In our
study the GlcNAc content in C-2 was shallow; thus, an exothermic peak
at 404 °C was not expected. Nam, Park, Ihm, and Hudson, (2010) observed two separate peaks in DSC thermograms, which showed that a
block-like structure was obtained in the chitosan backbone. Our results
did not show the separate peaks probably due to the random backbone
structure, as seen in C-48.
The enthalpy for C-2 was 223.67 J/g at 311.00 °C and for C-48 was
134.15 J/g at 302.53 °C. Since high energy and high temperature are
required to degrade 2% DA chitosan, C-2 had higher thermal stability
than C-48. Hamdi et al. (2019) also observed high thermal stability for
lower DA. However, they did not evaluate GlcN and GlcNAc

magnification) were obtained using a scanning electron microscope
with energy dispersive X-ray detector (Leo 440i, LEO Electron
Microscopy/Oxford).
2.6.10. Differential scanning calorimetry
Samples of the paper systems (2.8–3.0 mg) were packed in hermetically sealed aluminum capsules. The capsules were exposed to a
heating rate of 10 °C/min under a temperature of 20–550 °C and N2
flow of 100 mL/min in a DSC-60 equipment (Shimadzu, Japan).
2.7. Statistical analysis
The statistical analysis was ANOVA for one criterion using
Assistat7.7 version developed by Universidade Federal de Campina
Grande-UFCG.
3. Results and discussion
3.1. Chitosan characterization

The initial chitosan sample had a DA of 2%. After the reacetylation
process, a sample with high DA (48%) and higher content of GlcNAc
groups was obtained. Chitosan with 48% DA was expected to exhibit
hydrophobic properties similar to chitin while maintaining partial solubility in acidic conditions to enable posterior paper coating.
Nonetheless, random distribution of GlcN and GlcNAc related to
homogeneous acetylation affected the hydrophilic/hydrophobic property of chitosan to a greater extent than the quantity of these groups.
The increase in water content in C-48 illustrates this effect.
Reacetylation also caused an expected increase in molar mass, as seen
in Table 1, since acetyl groups were added, converting GlcN units into
GlcNAc.
As stated by Kasaai (2009), chemical modification such as acetylation of chitosan results in a change in H NMR spectra. Patterns observed
in the H NMR spectra of samples were also identified by Hirai, Odani,
and Nakajima (1991). The signal at 2.1 ppm indicates the presence of
hydrogen of the methyl group from N-acetyl, and it is more intense in
the reacetylated chitosan C-48 sample spectrum (Fig. 1b) than in the C2 sample (Fig. 1a). This may be associated with the higher quantity of
GlcNAc units obtained from the reacetylation reaction. The signals at
3.3 and 4.8 ppm represent the hydrogen atoms attached to carbon 2 and
carbon 1 of D-glucosamine ring, respectively. A higher signal was observed in this interval (Fig. 1); this could indicate a higher quantity of
GlcNAc units in C-48 chitosan than in C-2 chitosan. According to
Vårum, Antohonsen, Grasdalen, and Smidsrød (1991), peaks between
3.5 and 4.5 ppm are related to hydrogen atoms attached to carbon 3
and carbon 6 of GlcNAc and GlcN units and hydrogen atom attached to
carbon 2 of GlcNAc unit; the signal between 4.5 and 5.0 ppm was related to hydrogen atoms attached to carbon 1 of GlcN unit and carbon 1
of GlcNAc.
The FTIR spectra of C-2 and C-48 shown in Fig. 2 present the typical
bands for chitosan samples.
The C-48 spectra presented specific bands of chitosan: NH (amide II)
at 1560 cm−1; CO band at 1660 cm−1, which is related to C]O of intermolecular hydrogen bond with NH groups; C]O band at 1623
cm−1, which is related to hydrogen bonds of NH group and OH attached to carbon at position 6 of the glucopyranoside ring; amide III
Table 1

Degree of acetylation (DA), water content (WC), and molar mass (Mw) of
chitosan with 2% DA (C-2) and reacetylated chitosan (C-48).
Chitosan Samples

DA (%)

WC (%)

Mw (105g/mol)

C-2
C-48

2
48

5.05
16.37

5.238
6.186

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Carbohydrate Polymers 210 (2019) 56–63

M. Gatto et al.

Fig. 1. H-RMN spectra of (a) C-2 and (b) C-48.

Table 2
Properties of cardboard paper samples coated with three layers of C-2 and C-48
suspensions and uncoated cardboard paper (P).
Properties

Cardboard Paper Samples
P

Average thickness (μm)
Grammage (g/m²)
Moisture content
(gH2O/100 g
paper)
Water absorption Cobb
test (g/m²)
WVTR (g H2O/m²day)
Taber stiffness MD
(mN.m)
Taber stiffness CD
(mN.m)

CP-2
a

CP-48
b

3.78 ± 0.01
252.23 ± 1.79a
8.20 ± 0.01b

3.90 ± 0.01
252.40 ± 1.93a
8.80 ± 0.10b

4.08 ± 0.01c
259.93 ± 2.86b
9.86 ± 0.06a

45.21 ± 2.75a

39.74 ± 1.44b

52.16 ± 1.51c

298.17 ± 3.34a
18.10 ± 0.61a

280.69 ± 2.92b
18.91 ± 0.71b

291.44 ± 8.81ab
17.26 ± 0.60c

7.05 ± 0.22a

8.34 ± 0.49b

7.66 ± 0.27c

Fig. 2. FTIR spectra of C-2 and C-48 samples.
Note: In the same row, superscript with different letters indicates that the mean
values are statistically different. Criteria: p < 0.01 for G, E, MD, and CD. For
moisture content, Cobb test, and WVPR, p < 0.05.

Average thickness increased by about 3% and 8% for CP-2 and CP48, respectively, compared with uncoated cardboard paper (P)
(Table 2). The slight increase of coated cardboard paper thickness may
be related to the penetration of chitosan solution into the cellulose
network. Khwaldia et al. (2014) found that the thickness of caseinate
and caseinate/chitosan bilayer coatings on paper ranged between 3.70
and 16.88 μm, applying coatings from 5 to 16 g/m2. Thickness is directly related to the physical and optical characteristics of the paper
(tensile strength, transparency, color, whiteness, and electrical resistance).
Furthermore, the grammage of CP-48 was statistically higher than
those of P and CP-2. This is because C-48 had the highest WC and molar
mass (Table 1). However, the difference between the grammages of P
and CP-2 was not statistically significant, despite the application of
three chitosan layers. This result suggests that the water affinity property has an essential effect on the grammage of coated cardpaper. The
moisture content of CP-2 was not statistically different from that of the
uncoated sample. The moisture content of CP-48 was about 12% and
20% higher than those of CP-2 and P, respectively. Moreover, CP-48
was observed to be more hydrophilic than the CP-2. Despite having a
high content of GlcNAc (hydrophobic) groups, the random distribution
of molecules in the reacetylated chitosan caused the predominance of
hydrophilic property, favoring the hydrogen bonds with water molecules. These hydrogen bonds reduce the water vapor barrier property,
increasing the interaction between paper and environmental humidity.
The water absorption capacity (Cobb test) of CP-2 was 12% less
than that of P, which is favorable to packaging material application. In
cellulosic materials, water absorption is related to the water resistance

Fig. 3. DSC thermograms of C-2 and C-48.

distribution. On the other hand, according to Wanjun, Cunxin, and
Donghua (2005), the random distribution of acetyl group in chitosan
with 85% deacetylation degree stabilized the main chain, slowing
scissions caused by thermal treatment.

3.2. Chitosan-coated paper characterization
Application of three layers of C-2 and C-48 chitosan coatings (estimating 2.3 g/m2 of weight coating) resulted in a smooth, uniform, and
yellowish cardboard paper surface. The systems did not exhibit delamination after rigorous handling, indicating good compatibility between chitosan and cellulose fibers. MacIel et al. (2012) applied chitosan coating containing anthocyanin and observed similar
characteristics.
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and depends on the type of cellulose and the coating material. Reis et al.
(2011) obtained similar results for chitosan-coated Kraft paper. Cellulosic fibers are highly hydrophilic because of the hydrogen bonding of
cellulose molecules with water molecules, and this reduces the mechanical properties of paper sheets. However, the Cobb test showed that
the water absorption of CP-48 was 15% and 31% higher than those of P
and CP-2, respectively. This also confirmed that CP-48 presented higher
hydrophilicity than CP-2. As previously mentioned, the random distribution of the GlcN and GlcNAc groups in the chain favored the solubility of chitosan, which may have increased the hydrophilicity of the
reacetylated chitosan.
Habibie et al. (2016) observed that the grammage values of the
control sample and paper sheet coated with 1% (w/w) of chitosan were
similar, but the coated paper sheet had lower water absorption. However, the sample treated with 1% of chitosan and 20% of filler presented
higher grammage but a slightly lower water absorption.
Furthermore, the transport properties of coated packaging materials
are influenced by the type of coating material, composition, and weight

(Aloui, Khwaldia, Slama, & Hamdi, 2011). Chitosan coating significantly reduced WVTR to 6% in CP-2; however, the WVTR difference
between CP-48 and P was not statistically significant. Considering that
paperboard has poor water barrier property, the reduction of WVTR
should be due to the chitosan coating. The pores between the cellulose
fibers network were probably filled with chitosan suspension, which
reduced the permeation of the water vapor molecules through the
cellulose fibers network. Bordenave, Grelier, Pichavant, and Coma
(2007) observed a reduction of WVTR of chitosan-coated paper, which
was associated with the decrease of the preferential pathway for water
across the cellulose network filled with chitosan suspension. In the
present study, the diffusion rate of water molecules was higher in reacetylated chitosan coating. The hydrophilic character of the reacetylated chitosan CP-48 was again reflected by the non-reduction of
the WVTR, as its value was similar to that of the uncoated cardboard
paper.
Stiffness tests were performed in the machine direction, MD (that is,
the fibers were aligned along the direction of travel in a papermaking
machine) and in the cross-machine direction, CD (fibers were transversely aligned) (Brodnjak, 2017). For all samples, Taber stiffness for
MD test exceeded the double of that for CD. The resistance of fibers in
MD was always high due to fibers alignment (Reis et al., 2011).
Moreover, CP-2 presented the highest Taber stiffness in MD and CD.
The resistance and flexibility of chitosan film may have improved the
bonds of cellulose fibers in the coated papers, compared to the uncoated
paper (MacIel et al., 2012). In addition, CP-48 showed lower Taber
stiffness than P. Since CP-48 had the highest moisture content, water
molecules might have weakened the bonds between the cellulose fibers,
reducing the mechanical properties of paper.
As for coating process evaluation, red-colored aqueous solution
penetrated through the sample, which presented colored spots on the
opposite surface (Fig. 4). In CP-2 and CP-48, the opposite surfaces were
clean, indicating that chitosan suspensions formed a uniform and
homogeneous coating on the cardboard paper surface. Improvement of

barrier properties may be related to the solids content in the coated
paper, which fills the fibrous structure of the paper. Prevention of
aqueous solution penetration through the material matrix is a desired
property for packaging (Reis et al., 2011).

Fig. 4. Samples P, CP-2, and CP-48 paper systems during visual analysis.

3.2.2. Morphology
Application of three layers of chitosan resulted in a more homogeneous paper surface (Fig. 5b and c), compared to the uncoated paper
(Fig. 5a). Chitosan suspension penetrated the cellulose fibers network,
filling the paper pores.
Furthermore, a continuous and uniform film was not observed on
the surfaces of CP-2 and CP-48. Although three coating layers were
used, chitosan suspension was still absorbed by cellulose fibers, filling
empty spaces, and thus, a continuous film was not formed on the surface. Anyway, there was some difference between Fig. 5(a) – (c), which
is probably due to the rugosity. Fernandes et al. (2009) concluded that
the first layers of chitosan solution penetrate the cardboard paper
sheets progressively, and at least three chitosan layers are required for
film formation due to saturation of the cellulose fibers matrix.
3.2.3. DSC analysis
In the DSC thermogram shown in Fig. 6, the endothermic peaks
between 20 °C and 100 °C can be attributed to the evaporation of water
attached to paper cellulose chain in P and also that attached to chitosan
and cellulosic chains in CP-2 and CP-48 samples. The exothermic peak
between 330 °C and 420 °C was associated with the degradation of the
paper-film system of the samples.
Chitosan as coating affects the thermal properties of cardboard
paper samples. The degradation enthalpy of CP-2 was 91.67 J/g at
375.1 °C, while that of CP-48 was 81.18 J/g at 375.0 °C, and that of P
was 78.62 J/g at 371.5 °C. Acetylated chitosan with 2% DA had better

thermal stability, reinforcing the results regarding the higher resistance
of CP-2. Habibie et al. (2016) attributed the thermal property variation
to the greater interaction between the amino groups in chitosan and the
hydroxyl groups in cellulose.
4. Conclusion

3.2.1. Fat barrier
Storage of high-fat content foodstuffs requires packaging materials
with a fat barrier. In the uncoated cardboard paper (P), the fat barrier
was represented by kit test number 8. Papers CP-2 and CP-48 achieved
absolute fat barrier, indicating that chitosan is resistant to fat diffusion.
Cationic groups (NH3+) in chitosan interacted electrostatically with
anionic groups of lipid, retaining fat and preventing the appearance of
spots on the opposite surface (Ham-Pichavant et al., 2005).

Cardboard paper coated with chitosan of 2% DA is a potential
packaging material because of the following advantages: reduced water
absorption capacity due to absolute fat barrier, decreased WVTR, and
higher resistance (higher Taber stiffness in CD and MD), compared to
uncoated cardboard paper. Moreover, C-48 coating presented a higher
hydrophilicity than the original chitosan C-2 coating. Block distribution
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M. Gatto et al.

Fig. 6. DSC thermogram for P, CP-2, and CP-48.

of molecules unit in C-2 decreased solubility because of the unavailability of the NH3+ groups to interact with water. The high availability
of free NH3+ in C-48, attributed to the random distribution of units, is
related to its higher solubility. Although C-48 is expected to exhibit
hydrophobicity because of a higher amount of acetyl groups compared
to C-2, the results of this work indicate the opposite. In addition, GlcN
and GlcNAc distribution in chitosan influenced hydrophilicity/hydrophobicity more than DA. For coating cardboard papers, chitosan of 2%
DA obtained from heterogeneous chitin deacetylation can be considered
an advantageous alternative to films from synthetic polymers. The
paper-chitosan packaging system is environmentally friendly due to its
high recyclability and biodegradability.
Acknowledgments
This work was financially supported by Brazilian entities FAPESP
(grant #2016/25120-7, São Paulo Research Foundation - FAPESP),
CAPES (Coordination for the Improvement of Higher Education
Personnel) and CNPq (grant #249270/2013-7, National Council for
Scientific and Technological Development - CNPq). We want to thank
Prof. Dr. Laurent David and Prof. Dr. Thierry Delair from the
Département Matériaux et Ingénierie des Surfaces - Ingénierie des
Matériaux Polymère - Université Claude Bernard Lyon 1.
References
Aiba, S. I. (1991). Studies on chitosan: 3. Evidence for the presence of random and block
copolymer structures in partially N-acetylated chitosans. International Journal of
Biological Macromolecules, 13(1), 40–44. />90008-I.
Aloui, H., Khwaldia, K., Slama, M. B., & Hamdi, M. (2011). Effect of glycerol and coating
weight on functional properties of biopolymer-coated paper. Carbohydrate Polymers,
86(2), 1063–1072. />ASTM D646-96 (1996). Standard test method for grammage of paper and paperboard (mass
per unit area). West Conshohocken, PA: ASTM International />D0646-96www.astm.org.
ASTM D644-99 (2007). Standard test method for moisture content of paper and paperboard by
oven drying (withdrawn 2010). West Conshohocken, PA: ASTM Internationalhttps://
doi.org/10.1520/D0644-99R07www.astm.org.

ASTM E96-00 (2000). Standard test methods for water vapor transmission of materials. West
Conshohocken, PA: ASTM International />astm.org.
Atkinson, J., Mondala, A., Senger, Y. S., Al-Mubarak, R., Young, B., Pekarovic, J., et al.
(2017). Chitosan as paperboard coating additive for use in HVAC (heating, ventilation and air conditioning) applications. Cellulose Chemistry and Technology, 51(5–6),
477–481.
Bordenave, N., Grelier, S., Pichavant, F., & Coma, V. (2007). Water and moisture susceptibility of chitosan and paper-based materials: Structure-property relationships.
Journal of Agricultural and Food Chemistry, 55(23), 9479–9488. />1021/jf070595i.
Brodnjak, U. V. (2017). Experimental investigation of novel curdlan/chitosan coatings on
packaging paper. Progress in Organic Coatings, 112, 86–92. />porgcoat.2017.06.030.
Cutter, C. N. (2006). Opportunities for bio-based packaging technologies to improve the
quality and safety of fresh and further processed muscle foods. Meat Science, 74(1),
131–142. />
Fig. 5. SEM 1000× magnification of paper-film surface of (a) P (b) CP-2, and
(c) CP-48.

62


Carbohydrate Polymers 210 (2019) 56–63

M. Gatto et al.

formation in regioselectively functionalized 3-mono-O-methyl cellulose. Carbohydrate
Research, 343(15), 2600–2604. />Kuusipalo, J., Kaunisto, M., Laine, A., & Kellomäki, M. (2005). Chitosan as a coating
additive in paper and paperboard. TAPPI Journal.
MacIel, V. B. V., Yoshida, C. M. P., & Franco, T. T. (2012). Development of a prototype of
a colourimetric temperature indicator for monitoring food quality. Journal of Food
Engineering, 111(1), 21–27. />Marcy, J. E. (1995). Integrity testing and biotest procedures for heat-sealed containers. Plastic
package integrity testing-assuring seal quality. Herndon, VA: Institute of Packaging
Professionals35–47.

Nam, Y. S., Park, W. H., Ihm, D., & Hudson, S. M. (2010). Effect of the degree of deacetylation on the thermal decomposition of chitin and chitosan nanofibers.
Carbohydrate Polymers, 80(1), 291–295. />030.
Ng, J. C. Y., Cheung, W. H., & McKay, G. (2002). Equilibrium studies of the sorption of Cu
(II) ions onto chitosan. Journal of Colloid and Interface Science, 255(1), 64–74. https://
doi.org/10.1006/jcis.2002.8664.
Pillai, C. K. S., Paul, W., & Sharma, C. P. (2009). Chitin and chitosan polymers: Chemistry,
solubility and fiber formation. Progress in Polymer Science (Oxford), 34(7), 641–678.
/>Rastogi, V., & Samyn, P. (2015). Bio-Based Coatings for Paper Applications. Coatings,
5(4), 887–930. />Reis, A. B., Yoshida, C. M. P., Reis, A. P. C., & Franco, T. T. (2011). Application of chitosan
emulsion as a coating on Kraft paper. Polymer International, 60(6), 963–969. https://
doi.org/10.1002/pi.3023.
Robertson, G. L. (2012). Food packaging: Principles and practice (3rd ed.). Boca Raton, FL,
USA: CRC Press.
Tang, X. Z., Kumar, P., Alavi, S., & Sandeep, K. P. (2012). Recent advances in biopolymers
and biopolymer-based nanocomposites for food packaging materials. Critical reviews in
food science and nutrition. />TAPPI Test Methods T 441 om-90 (1994a). Water absorptiveness of sized (non-bibulous)
paper and paperboard (Cobb test). Atlanta: TAPPI – Technical Association of The Pulp
And Paper Industry.
TAPPI Test Methods T489 om-92 (1994b). Stiffness of paperboard. Atlanta: TAPPI
-Technical Association of the Pulp and Paper Industry.
Vachoud, L., Zydowicz, N., & Domard, A. (1997). Formation and characterisation of a
physical chitin gel. Carbohydrate Research, 302(3–4), 169–177. />1016/S0008-6215(97)00126-2.
Vårum, K. M., Antohonsen, M. W., Grasdalen, H., & Smidsrød, O. (1991). Determination
of the degree of N-acetylation and the distribution of N-acetyl groups in partially Ndeacetylated chitins (chitosans) by high-field n.m.r. spectroscopy. Carbohydrate
Research, 211(1), 17–23. />Wanjun, T., Cunxin, W., & Donghua, C. (2005). Kinetic studies on the pyrolysis of chitin
and chitosan. Polymer Degradation and Stability, 87(3), 389–394. />1016/j.polymdegradstab.2004.08.006.
Yoshida (2009). Chitosan tailor-made films: The effects of additives on bamer and mechanical properties. Packaging Technology and Science, 22(November), 161–170.

Duarte, M. L., Ferreira, M. C., Marvão, M. R., & Rocha, J. (2002). An optimized method to
determine the degree of acetylation of chitin and chitosan by FTIR spectroscopy.

International Journal of Biological Macromolecules, 31, 1–8. />S0141-8130(02)00039-9.
Elsabee, M. Z., & Abdou, E. S. (2013). Chitosan based edible films and coatings: A review.
Materials Science and Engineering C, 33(4), 1819–1841. />msec.2013.01.010.
Fernandes, S. C. M., Freire, C. S. R., Silvestre, A. J. D., Neto, C. P., Gandini, A., Desbriéres,
J., et al. (2009). A study of the distribution of chitosan onto and within a paper sheet
using a fluorescent chitosan derivative. Carbohydrate Polymers, 78(4), 760–766.
/>Focher, B., Naggi, A., & Torri, G. (1992). Chitosans from Euphausia superba. 2:
Characterization of solid state structure. Carbohydrate Polymers, 18, 43–49. https://
doi.org/10.1016/0144-8617(92)90186-T.
Franca, E. F., Freitas, L. C. G., & Lins, R. D. (2011). Chitosan molecular structure as a
function of N-acetylation. Biopolymers, 95(7), 448–460. />21602.
Ghosh, A., Azam Ali, M., & Walls, R. (2010). Modification of microstructural morphology
and physical performance of chitosan films. International Journal of Biological
Macromolecules, 46(2), 179–186. />Guinesi, L. S., & Cavalheiro, É. T. G. (2006). The use of DSC curves to determine the
acetylation degree of chitin/chitosan samples. Thermochimica Acta, 444(2), 128–133.
/>Habibie, S., Hamzah, M., Anggaravidya, M., & Kalembang, E. (2016). The effect of
chitosan on physical and mechanical properties of paper. Journal of Chemical
Engineering and Materials Science, 7(1), 1–10. />0235.
Hamdi, M., Nasri, R., Hajji, S., Nigen, M., Li, S., & Nasri, M. (2019). Acetylation degree, a
key parameter modulating chitosan rheological, thermal and film-forming properties.
Food Hydrocolloids, 87(May), 48–60. />027.
Ham-Pichavant, F., Sèbe, G., Pardon, P., & Coma, V. (2005). Fat resistance properties of
chitosan-based paper packaging for food applications. Carbohydrate Polymers, 61(3),
259–265. />Hirai, A., Odani, H., & Nakajima, A. (1991). Determination of degree of deacetylation of
chitosan by H NMR spectroscopy. Polymer Bulletin, 26(1), 87–94. />1007/bf00299352.
Kasaai, M. R. (2009). Various methods for determination of the degree of N-acetylation of
chitin and chitosan: A review. Journal of Agricultural and Food Chemistry, 57,
1667–1676. />Khwaldia, K., Basta, A. H., Aloui, H., & El-Saied, H. (2014). Chitosan-caseinate bilayer
coatings for paper packaging materials. Carbohydrate Polymers, 99, 508–516. https://
doi.org/10.1016/j.carbpol.2013.08.086.

Kittur, F. S., Harish Prashanth, K. V., Udaya Sankar, K., & Tharanathan, R. N. (2002).
Characterization of chitin, chitosan and their carboxymethyl derivatives by differential scanning calorimetry. Carbohydrate Polymers, 49(2), 185–193. />10.1016/S0144-8617(01)00320-4.
Kondo, T., Koschella, A., Heublein, B., Klemm, D., & Heinze, T. (2008). Hydrogen bond

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