Antimicrobial Edible Film Prepared from Bacterial Cellulose Nanofibers/Starch/Chitosan for a Food Packaging Alternative

Abstract

As a contribution to the growing demand for environmentally friendly food packaging films, this work produced and characterized a biocomposite of disintegrated bacterial cellulose (BC) nanofibers and tapioca starch/chitosan-based films. Ultrasonication dispersed all fillers throughout the film homogeneously. The highest fraction of dried BC nanofibers (0.136 g) in the film resulted in the maximum tensile strength of 4.7 MPa. 0.136 g BC nanofiber addition to the tapioca starch/chitosan matrix increased the thermal resistance (the temperature of maximum decomposition rate from 307 to 317°C), moisture resistance (after 8 h) by 8.9%, and water vapor barrier (24 h) by 27%. All chitosan-based films displayed antibacterial activity. This characterization suggests that this environmentally friendly edible biocomposite film is a potential candidate for applications in food packaging.

Full text

Research Article
Antimicrobial Edible Film Prepared from Bacterial Cellulose
Nanofibers/Starch/Chitosan for a Food Packaging Alternative
Hairul Abral ,
1
Angga Bahri Pratama ,
2
Dian Handayani ,
3
Melbi Mahardika ,
4
Ibtisamatul Aminah ,
3
Neny Sandrawati ,
3
Eni Sugiarti,
5
Ahmad Novi Muslimin ,
5
S. M. Sapuan ,
6
and R. A. Ilyas
7
1
Department of Mechanical Engineering, Andalas University, 25163 Padang Sumatera Barat, Indonesia
2
Program Studi Teknik Mesin, Universitas Dharma Andalas, 25000 Padang Sumatera Barat, Indonesia
3
Laboratory of Sumatran Biota/Faculty of Pharmacy, Andalas University, 25163 Padang, Sumatera Barat, Indonesia
4
Department of Biosystems Engineering, Institut Teknologi Sumatera, 35365 South Lampung, Indonesia
5
Laboratory of High-Temperature Coating, Research Center for Physics Indonesian Institute of Sciences (LIPI), Serpong, Indonesia
6
Department of Mechanical and Manufacturing Engineering, Faculty of Engineering, Universiti Putra Malaysia,
43400 UPM Serdang, Selangor, Malaysia
7
School of Chemical and Energy Engineering, Faculty of Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor Bahru,
Johor, Malaysia
Correspondence should be addressed to Hairul Abral; habral@yahoo.com
Received 2 January 2021; Revised 9 March 2021; Accepted 15 March 2021; Published 1 April 2021
Academic Editor: Victor H. Perez
Copyright © 2021 Hairul Abral et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
As a contribution to the growing demand for environmentally friendly food packaging films, this work produced and
characterized a biocomposite of disintegrated bacterial cellulose (BC) nanofibers and tapioca starch/chitosan-based films.
Ultrasonication dispersed all fillers throughout the film homogeneously. The highest fraction of dried BC nanofibers
(0.136 g) in the film resulted in the maximum tensile strength of 4.7 MPa. 0.136 g BC nanofiber addition to the tapioca
starch/chitosan matrix increased the thermal resistance (the temperature of maximum decomposition rate from 307 to
317
°
C), moisture resistance (after 8 h) by 8.9%, and water vapor barrier (24 h) by 27%. All chitosan-based films displayed
antibacterial activity. This characterization suggests that this environmentally friendly edible biocomposite film is a
potential candidate for applications in food packaging.
1. Introduction
The demand for affordable environmentally friendly plastic
substitutes made from renewable sources continues to
increase resulting in a growing interest in the research
community in plant-based replacements for nondegradable
plastics [1–3]. For the food packaging industry, promising
cheap and abundant eco-friendly edible film-forming mate-
rials include starches, pure bacterial cellulose nanofibers,
and chitosan [4]. However, films made of starch alone have
low mechanical and thermal properties, high moisture
absorption, and poor antimicrobial resistance [5, 6]. Many
attempts have been conducted previously to reduce these
weaknesses by adding environmentally friendly fillers to the
starch film [7–10]. Of these edible fillers, cellulose fiber and
chitosan have significant potential, with one being one of
the most abundant in nature[10, 11]. Cellulose fiber has good
mechanical properties and flexibility but no antimicrobial
activity [12]. The tensile and thermal properties of the starch
film were improved after reinforcement with randomly ori-
ented cellulose fibers from kenaf [13], water hyacinth [14],
and softwood [15]. However, these short cellulose nanofiber
preparation methods tend to use potentially harmful chemi-
cals and result in a high residue of hemicellulose, lignin, or
other impurities. Nanofiber from bacterial cellulose pellicle
has none of these disadvantages because it consists of large
Hindawi
International Journal of Polymer Science
Volume 2021, Article ID 6641284, 11 pages
https://doi.org/10.1155/2021/6641284

amounts of pure cellulose fibers with nanosized diameters
[16]. Short BC nanofibers can be prepared from this pellicle
using a high-shear homogenizer with a rotating blade to
disintegrate the long fibers into nanosized lengths [17].
Recently, previous work has reported the interesting results
that the microbial activity of the disintegrated BC/chitosan
film was less than that of the BC sheet/chitosan one [1].
Chitosan has several advantages including antimicrobial
activity, controlled release of food ingredients and drugs, rel-
atively low cost, and widespread availability from a stable
renewable source [5, 18]. Numerous studies have been per-
formed on the development of edible biocomposite films
made from chitosan, cellulose, and starch [19, 20]. Of course,
as a food packaging material, this polysaccharide-based edi-
ble film should protect foods against deterioration due to
microorganisms, moisture, dust, odors, and mechanical
forces [21, 22]. There have been many previous studies
reporting on the improved properties of the biocomposite
film. However, characterizations of chitosan and disinte-
grated BC nanofiber content on tapioca starch-based bio-
composites’properties have yet to be reported. This study
understands the effect of the addition of both chitosan and
disintegrated nanofiber from BC pellicle on the properties
of this edible biocomposite film more completely. All sam-
ples were characterized by field emission scanning electron
microscopy (FESEM), X-ray diffraction (XRD), Fourier
transform infrared (FTIR), and thermogravimetric analysis
(TGA). Opacity, moisture absorption (MA), water vapor per-
meability (WVP), and tensile properties were also measured.
2. Materials and Experiment
2.1. Materials. Local (Padang, Indonesia) commercially avail-
able tapioca starch (Cap Pak Tani brand) was washed once
with distilled water to obtain pure tapioca starch. The pure
wet starch was dried using a drying oven (Universal Oven
Memmert UN-55) for 20 h at 50
°
C. The dried starch was fil-
tered through a 63 μm mesh cloth. The chemical composi-
tion of dry starch granules was analyzed according to a
previous amylopectin method [23]. The pure dried granular
starch for this present work contained 14.5% of amylose
and 85.5% of glycerol (Brataco brand) purchased from
Brataco (Padang, Indonesia) and chitosan (degree of deacety-
lation: 94%) from CV. Chi Multiguna (Indramayu, Indone-
sia). Acetic acid (CH
3
COOH, 5%) was used as the solvent
for chitosan.
2.2. Preparation of Single BC Nanofiber and Films. Single BC
nanofiber was isolated and characterized as in our previous
work [24]. Briefly, a wet pellicle with a dimension
(248 × 151 × 22 mm) was purchased from a local small-
scale industry in Padang, Indonesia. This pellicle, which is a
common addition to drinks or desserts in the form of nata
de coco, is the result of a week-long fermentation of coconut
water, glucose, and acetic acid with Acetobacter xylinum in a
static closed container. Integration of the pellicle was carried
out with a homogenizer and ultrasonication probe. The crys-
tallinity index of the disintegrated BC nanofiber was about
71%. Figure 1 displays the steps of sample preparation from
raw BC until biocomposite. The film sample was prepared
as follows:
Starch film. About 10 g purified tapioca granules, 100 mL
distilled water, and 2 mL glycerol were mixed in a glass bea-
ker (250 mL) using a hot plate stirrer (Daihan Scientific
MSH-200) at 500 rpm and 65
°
C until completely gelatinized.
The gel suspension was sonicated using an ultrasonic cell
crusher (SJIA-1200W) at 600 W for 1 min, then poured onto
a petri dish (d= 145 mm) and dried in a drying oven (Mem-
mert Germany, Model 55 UN) at 50
°
C for 20 h.
Chitosan film. Simultaneously, a mixture of 2.5 g chitosan
and 100 mL acetic acid was prepared in a glass beaker
(250 mL) and heated using a hot plate stirrer at 80
°
C for 2
hours until gelatinization. The chitosan gel was filtered using
74 μm cheesecloth. The gel was poured onto a petri dish
which was dried using the drying oven at 50
°
C for 20 h.
Starch/chitosan film. The two gels, starch and chitosan,
were blended in the ratio of 80 : 20 (70 g total weight) using
an ultrasonic cell crusher at 600 W for 1 min keeping the
temperature below 65
°
C. The gel was poured onto a petri dish
for drying as described in the starch sample preparation.
Biocomposite film. The blended starch/chitosan gels were
mixed with the appropriate BC suspension (10, 15, or 20 mL).
Each gel suspension was sonicated at 600 W for 1 min, then
cast onto a petri dish and dried in the drying oven at 50
°
C
for 20 h.
Abbreviations used for the studied samples with their
compositions are shown in Table 1.
2.3. Characterization
2.3.1. FESEM Morphology of the Fracture Surface. The tensile
sample’s fracture surface morphology after the tensile test
was observed using JIB-4610F FESEM from JEOL (Tokyo,
Japan). At about 25 mm from its fracture surface, the tensile
specimen was cut using a steel scissor (in perpendicular ten-
sile direction) and placed on a specimen holder. All samples
were coated with gold (Au). An accelerati ng voltage of 10 kV
with 8 mA was set up for testing.
2.3.2. X-Ray Diffraction. The XRD pattern was recorded
using an X’Pert PRO PANalytical instrument (Philips Ana-
lytical, Netherlands) with CuKαradiation (λ=0:154)at
40 kV and 30 mA. The scanning range was 5
°
to 50
°
. The crys-
tallinity index (CI) of the biocomposites was calculated using
CI = I002 −Iam
ðÞ
I002

× 100, ð1Þ
where I002 and Iam are the peak intensities of crystalline and
amorphous regions, respectively [25].
2.3.3. Opacity Measurement. The opacity of the film was mea-
sured using a UV-Vis spectrophotometer (Shimadzu UV
1800, Japan) in the range 400-800 nm according to ASTM
D 1003-00 (Standard Test Method for Haze and Luminous
Transmittance of Transparent Plastics). Films of 0.38 mm
thickness were cut into 10 mm × 25 mm rectangles. The
opacity measurement was repeated 3 times.
2 International Journal of Polymer Science

2.3.4. Fourier Transform Infrared. FTIR spectra of films were
recorded using a PerkinElmer FTIR spectrometer (Frontier
Instrument, USA), equipped with deuterated triglycine sul-
fate, DTGS, detector, and extended range KBr beam splitter.
This spectrometer was used in the frequency range in the
wavenumber range of 4000-600 cm
-1
, at resolution 4 cm
-1
with 32 scans per sample. Samples (10 mm × 10 mm) were
dried in the oven at 50
°
C until constant weight before
characterization. The samples were made in powder and
mixed with KBr as well as followed by the pressure within
the pellet ultrathin layer [26].
2.3.5. Moisture Absorption and Water Vapor Permeability.
Moisture absorption (MA). MA was determined using the
method described in a previous study [27]. All biocomposite
samples were dried in a drying oven (Memmert Germany,
Model 55 UN) at 50
°
C until a constant weight was achieved.
The dried sample was stored in a closed chamber with 75%
RH at 25
°
C. The samples were we ighed every 30 min for 7 h
with a precision balance (Kenko) with a 0.1 mg accuracy.
MA was calculated using
MA = wh−wo
ðÞ
wo
,ð2Þ
where whis the final weight and wois the initial weight of the
sample. MA determination was repeated 5 times for each
film.
Water vapor permeability (WVP). WVP was measured
according to the method described by previous work [28].
WVP determination was repeated 3 times for each film.
2.3.6. Thermogravimetric Analysis (TGA) and Derivative
Thermogravimetry (DTG). TGA and DTG of all samples
were characterized using a differential scanning calorimeter
(Linseis TA type PT 1600, Germany). About 25 mg of the
film was positioned on the microbalance located inside the
Table 1: Composition of the starch film, chitosan film, and biocomposite films used in the study.
Sample code Tapioca starch
(g)
Nanofiber suspension
(mL)
Dried nanofibers
(g)
Aquades
(mL)
Glycerol
(mL)
Chitosan
(g)
Acetic acid
(mL)
GU 10 ——100 2 ——
CH —— ———2.5 100
GU/CH 10 ——100 2 2.5 100
GU/CH/10BC 10 10 0.068 100 2 2.5 100
GU/CH/15BC 10 15 0.102 100 2 2.5 100
GU/CH/20BC 10 20 0.136 100 2 2.5 100
10% NaOH, 12 h Electrical blender
12000 rpm, 1 h
High-shear homogenizer
60 min,
10000 rpm
e suspension was filtered
200T mesh cheesecloth (74 𝜇m)
BC suspensionsGelatinized starchChitosan gel
Bionanocomposite
film
Dried in a drying
Poured onto a
petri dish
(d = 145 mm)
Blended using
ultrasonic cell
crusher at 600 W
Suspension (starch/BC) and chitosan gel
with composition 80:20
(70 g total weight)
Figure 1: Steps of sample preparations for biocomposite.
3International Journal of Polymer Science

furnace. The test was carried out from 35
°
C up to 550
°
C with
a heating rate of 10
°
C/min in a nitrogen atmosphere.
2.3.7. Tensile Properties. Tensile properties of the biocompo-
sites, including tensile strength and elongation at break, were
measured using COM-TEN 95T Series 5K (Pinellas Park,
USA) and were performed according to the ASTM D 638
type V standard. Before the test, all samples were stored in
a desiccator with 50 ± 5%relative humidity at 25
°
C for 48 h.
Samples were then tested at room temperature and RH 75%
using a tensile test speed of 5 mm/min. The testing of the film
was repeated at least three times for each fiber content.
2.3.8. Antimicrobial Activity. The antibacterial activity of the
starch/chitosan-based biocomposite films was assayed using
the agar diffusion method (Bauer, Kirby, Sherris, and Turck,
1966). Four microbe strains were used: Gram-positive Staph-
ylococcus aureus and Bacillus subtilis bacteria and Gram-
negative Escherichia coli and Pseudomonas aeruginosa. The
microbial suspensions in saline solution (NaCl 0.85% sterile)
were standardized using the McFarland scale to inoculate
petri dishes containing nutrient agar for bacter ia. 6 mm film
diameter disks were placed on the inoculated agar then incu-
bated at 30
°
C for 24 h. The diameter of the growth inhibition
zones around the film disks was gauged visually. All tests
10.0 kV 10 𝜇m8.3 3 SEM_SEI
(a)
10.0 kV 10 𝜇m8.9 3 SEM_SEI
(b)
10.0 kV 100 𝜇m8.9 3 SEM_SEI 10.0 kV 10 𝜇m8.8 3 SEM_SEI
(c)
10.0 kV 1 𝜇m9.5 3 SEM_SEI
(d)
10.0 kV 1 𝜇m9.0 3 SEM_SEI
(e)
Figure 2: FESEM images of the fracture surface for the pure starch film (a), pure chitosan film (b), starch/chitosan film (c), GU/CH/15BC
film (d), and GU/CH/20BC film (e).
4 International Journal of Polymer Science

were carried out in triplicate, and the antibacterial activity
was expressed as the mean of the inhibition diameters (mm).
2.3.9. Statistical Analysis. Experimental data were analyzed
using IBM SPSS Statistics 25.0 (IBM Corporation, Chicago,
USA). One-way analysis of variance (ANOVA) and a ptest
were used to identify the significance of any effects of varying
nanofiber content on properties of the biocomposites. Dun-
can’s multiple range tests were used on the MA and WVP
results using a 95% (p≤0:05) confidence level.
3. Results and Discussion
3.1. Morphological Biocomposites. Figure 2 displays FESEM
fracture surface micrographs for the pure starch film (a), pure
chitosan film (b), starch/chitosan film (c), GU/CH/15BC film
(d), and GU/CH/20BC film (e). The surface of the starch film
was rough (Figure 2(a)) probably as a result of a long tortu-
ous way of the polymer chains looking for the weak section
of the chain structure. Meanwhile, the CH film had a smooth
fracture surface (Figure 2(b)) attributed to unimpeded crack
propagation and corresponding to its brittle properties.
10 20 30 40 50 60
0
200
400
600
800
1000
1200
1400
Intensity (count)
Shi
GU
CH
GU/CH
GU/CH/10BC
GU/CH/15BC
GU/CH/20BC
Figure 3: The XRD patterns of all samples.
Table 2: Crystallinity index, opacity value, and thermal properties of all samples.
Films Crystallinity index (%) Opacity (AUnm)∗Maximum decomposition temperature (
°
C)
GU 11 280:5±0:16
d
268
CH 17 450:8±0:04
f
311
GU/CH 14 202:3±0:98
a
307
GU/CH/10BC 35 208:1±1:09
b
313
GU/CH/15BC 36 237:9±0:63
c
314
GU/CH/20BC 37 328:3±0:76
e
317
∗Different letters a, b, c, d, e, and f in the same column indicate significant differences in means (p≤0:05).
100
80
60
40
20
0
400 500 600 700 800
Wavelength (nm)
Transmittance (%)
GU
CH
GU/CH
GU/CH/10BC
GU/CH/15BC
GU/CH/20BC
Figure 4: The transmittance of all samples.
5International Journal of Polymer Science

Figure 2(c) displays the GU/CH fracture surface, which was
rougher than the CH sample. The different chemical struc-
tures of both these substances produce a weaker structural
section in which the crack propagates with a longer tortuous
way resulting in microscopic features known as a beach mark
as shown by the yellow arrow in the inset of Figure 2(c) which
marks an interruption of the cracking progress. Adding BC
into the blends continuously increases the surface roughness
of the biocomposite (yellow arrow in Figures 2(d) and 2(e)).
In these figures, disintegrated BC nanofibers were dispersed
homogeneously after ultrasonication. A similar result also is
supported by previous studies [14, 17, 24, 29, 30].
3.2. X-Ray Diffraction. The X-ray diffraction curves for all
studied films are shown in Figure 3. All films show a similar
semicrystalline pattern with prominent peaks at about 2θ=
20°and 23
°
. The crystallinity index (CI) of each sample is
shown in Table 2. The GU/CH blend film has a CI value of
14% between the CI of chitosan (17%) and the CI of the
starch film (11%). The addition of any fraction of nanofibers
to the starch-based matrix improves the CI value of the
biocomposite films (around 164% increase compared to
GU/CH). This increased value indicates better filler disper-
sion in the starch matrix thanks to ultrasonication [2]. Sam-
ples before mixing with nanofibers display the main peak
position at 2θ=23
°. The addition of the nanofibers shifted
the position toward the left side (2θ=20
°). According to pre-
vious work, shifting the peak position to the left side can be
associated with an increase in tensile residual stress resulting
from increases in the polymer chains’interlayer spacing [31].
3.3. Transparency. In food packaging applications, high
transparency can be an essential property [32]. The transpar-
ency values for all samples are displayed in Figure 4. The GU
film displays low transmittance at all wavelengths. The high-
est transparency at 800 nm belongs to the CH film. There-
fore, after mixing starch with chitosan, the GU/CH film
became more transparent. However, the BC nanofiber addi-
tion to this GU/CH film significantly decreased (p≤0:05)
the transparency of the biocomposite film. This phenomenon
is because increasing amounts of the nanofiber increase the
amount of reflected light in the film. The lowest transparency
was consequently measured on the film with the highest fiber
loading, the GU/CH/20BC film (28.9% less than the GU/CH
film). This value still agrees with previous work [33]. Despite
the decreasing transparency, this film was still clear enough
to see through easily. The consistency of transparency read-
ings in each of the repeats for each fiber loading, as shown
by the small standard deviation values, confirms that the
cellulose fibers are homogeneously dispersed in the starch-
based matrix. This result is consistent with Figure 2(e) which
shows beach marks spread evenly on all fracture surfaces of
the biocomposite film.
3.4. FTIR Spectra. Structural changes in the starch-based film
after mixing with chitosan or/and nanofibers can be observed
using an FTIR curve. Figure 5 displays an FTIR curve of the
mean transmittance values for three samples of each biocom-
posite. Shifts of peak intensity, broadening of absorption
peaks, and appearance of new bands in the FTIR spectra cor-
respond to structural changes [34]. There are absorption
peaks in the GU film at about 3247 cm
−1
(–OH stretching)
and 2917 cm
-1
due to CH stretching. The band at 1650 cm
-1
is present due to the deformation vibration of the absorbed
water molecules. The characteristic absorption peak of chito-
san is the band at 1559 cm
-1
, which is assigned to the stretch-
ing vibration of the amino group of chitosan. Another band
at 3367 cm
-1
is due to amine NH symmetric vibration. The
wavenumber and Tvalue of O-H stretching shift from
100
80
60
40
20
0
4000 3500 3000 2500 2000 1500 1000
Wavenumber (cm–1)
Transmittance (%)
GU
CH
GU/CH
GU/CH/10BC
GU/CH/15BC
GU/CH/20BC
(a)
GU
CH
GU/CH
GU/CH/10BC
GU/CH/15BC
GU/CH/20BC
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
3500 3450 3400 3350 3300 3250 3200 3150 3100 3050
Absorbance
Wavenumber (cm–1)
(b)
Figure 5: FTIR spectrum resulting from triplicate measurements of each film. The full spectrum from 4000 to 250 cm
-1
(a). Sections of the
spectrum for O-H stretching vibration (b).
6 International Journal of Polymer Science

3290 cm
−1
and 17.9% for the GU film to 3288 cm
−1
and
19.6% for the GU/CH blend film. Similar shifting was also
observed on the GU/CH-based biocomposite film due to
the presence of nanofibers. For example, Tof the GU/CH
film at about 3290 cm
−1
is 19.6% but 24.5% for the
GU/CH/20BC film. As expected, increasing concentrations
of BC increased the peak Tand shifted the wavenumber of
O-H functional groups. This case is probably a result of
increasing hydrogen bonds between starch and/or nanofiber
polymer chains and amino functional groups [35].
3.5. Moisture Absorption and Water Vapor Permeability.
Figure 6(a) shows the moisture absorption (MA) of the pure
starch film, chitosan film, and starch/chitosan-based biocom-
posite film. The pure chitosan film has the lowest MA (17.7%
after 8 h in the humid chamber). The presence of nanofibers
25
20
15
10
5
0
021 3456789
7.0 7.5 8.0 8.5 9.0
16
18
20
22
A
B
C
C
D
E
–1
Time (h)
Moisture absorption (%)
7.0
7.5
8.0
16
18
20
22
A
B
C
C
D
E
GU
CH
GU/CH
GU/CH/10BC
GU/CH/15BC
GU/CH/20BC
(a)
2.50E–010
2.00E–010
1.50E–010
1.00E–010
5.00E–011
5.00E–011
0.00E+000
0.00E+000
8 12162024
Time (h)
20 24
A
A,B
B,C
C,D
D
C,D
GU
CH
GU/CH
GU/CH/10BC
GU/CH/15BC
GU/CH/20BC
(b)
Figure 6: Average value of moisture absorption (a) and WVP (b) of each studied film. Different letters a, b, c, d, and e in the inset indicate
significant differences (p≤0:05).
7International Journal of Polymer Science

in the starch/chitosan-based film results in a decrease in MA
of the biocomposites. Higher nanofiber loading leads to
lower average MA values. This result is because nanofiber
and chitosan are more hydrophobic than the neat starch film.
Dispersion of fillers in the starch film homogeneously results
in decreasing MA of the biocomposite film. Better intermo-
lecular hydrogen bonding between the starch matrix and
the chitosan and fiber improves the moisture resistance of
the biocomposite film due to reducing the number of free
hydroxyl groups. This result is consistent with the FTIR pat-
tern (Figure 5) showing the weakest intensity of O-H stretch-
ing and O-H of absorbed water peaks in the films with the
highest nanofiber content due to the reduction in free
hydroxyl groups. Similar findings of reducing moisture
absorption with increased fiber loadings have also been
reported previously [30]. Figure 5(b) shows water vapor per-
meability (WVP) of both starch and chitosan films and bio-
composite films. As expected, the pattern of WVP with the
addition of nanofibers is similar to that of MA. There is a
decrease in the WVP value in films that contain more nano-
fibers. WVP of the GU/CH/20BC sample is 27% lower than
that of the GU/CH film after 24 h. The decrease in WVP is
because moisture is absorbed less readily into the biocompo-
site for the reasons described above.
Also, well-dispersed nanofiber hinders the path for water
molecule diffusion through the film due to the more com-
pact, homogeneous polymer structures [29]. As shown in
Figure 6, the WVP value of the GU/CH/20BC film is 3:7×
10−11 gm
−1s−1Pa−1(24 h), similar to that found in a previous
study on the improvement of the shelf life of yam starch/chi-
tosan-coated apples [36]. Therefore, this film has a good
potential for the shelf life of various food types.
3.6. Thermal Properties. Figures 7(a) and 7(b) show TGA and
DTG curves of each tested film as a function of temperature.
There are three stages of weight loss of the film shown in a
TGA graph. The first stage at 100-150
°
C is related to weight
loss in the film due to the evaporation of the absorbed mois-
ture. This small amount of dehydration is evident in the DTG
curve (Figure 7(b)). The weight loss for the second stage at
250-350
°
C is attributed to the decomposition of starch, chito-
san, and nanofibers. In the temperature range of 360–570
°
C,
a third weight loss was observed due to a final decomposition
to ash. The temperature of the maximum decomposition rate
(Tm) at the second stage was higher (311
°
C) for starch than
for chitosan (268
°
C). As expected, the addition of chitosan
to starch decreased the Tmvalue slightly (307
°
C). However,
the thermal resistance of the starch/chitosan-based film
became higher with the addition of nanofibers (Figures 7(a)
and 7(b)). For example, the Tmof the GU/CH film increased
from 10
°
C to 317
°
C after adding dried nanofibers of 0.136 g.
This increased value is probably because of the higher crys-
tallinity in the sample [30]. Also, the higher thermal resis-
tance resulted from better interfacial hydrogen bonding
between starch and nanofiber dispersed homogeneously
[17, 30]. This result is consistent with the high CI value of
films with high nanofiber content, as shown in the XRD
curve (Table 2).
3.7. Tensile Properties. Figure 8 shows the tensile properties
of all tested samples. TS for the GU/CH film was 2.6 MPa, a
value between pure GU (1.4 MPa) and CH (3.2 MPa). As
expected, the nanofiber addition to the GU/CH film led to
an increase in its TS value. The maximum TS was 4.7 MPa,
measured on the GU/CH/20BC film, reinforced with the
highest fiber loading (0.136 g). This increased TS value prob-
ably results from the increased crystallinity index (Table 2),
better nanofiber dispersion (Figure 2), and better interfacial
hydrogen bonding between the nanofibers with the GU/CH
matrix [20]. The pure chitosan film is the least brittle of all
the films with an EB of only 0.98%. After mixing the chitosan
with starch, the EB of the GU/CH film became higher (11%).
GU
CH
GU/CH
GU/CH/10BC
GU/CH/15BC
GU/CH/20BC
100 200 300 400 500
0
20
40
60
80
100
Weight (%)
(a)
100 200 300 400 500
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
–0.2
–0.4
DTG (%/min)
GU
CH
GU/CH
GU/CH/10BC
GU/CH/15BC
GU/CH/20BC
(b)
Figure 7: TGA (a) and DTG (b) charts of all samples.
8 International Journal of Polymer Science

Even the addition of the BC nanofibers to the GU/CH film
improved its EB. This tendency is attributable to longer tor-
tuous pathways of the crack propagation through the matrix
due to the BC nanofibers. Further nanofiber loading did not
result in statistically significant changes in EB of the biocom-
posite film.
3.8. Antibacterial Activity. Table 3 displays the diameters of
antibacterial activity inhibition zones against all microorgan-
isms tested in studied samples. BC nanofibers did not inhibit
any microorganisms. This phenomenon is in good agree-
ment with previous work [37]. A similar appearance was also
displayed by the GU film without antibacterial activity. How-
ever, all chitosan-contained films were effective against
Gram-positive bacteria and Gram-negative bacteria. This
result could be due to the numerous active ingredients pres-
ent in chitosan. Chitosan could adsorb the electronegative
substance in the cell, and it disturbs the physiological activi-
ties of the bacteria and kills them [38].
4. Conclusion
This work characterized a tapioca starch/chitosan-based film
reinforced by bacterial cellulose nanofiber. All chitosan-
based films had antibacterial activity. 0.136 g nanofiber addi-
tion to this film led to the highest tensile strength and the
highest thermal resistance. The presence of nanofibers
increased moisture resistance and water barrier properties.
The addition of the nanofibers led to a decrease in transpar-
ency. However, the resulting translucent biocomposite film
could still be seen through clearly. Overall, this biocomposite
film could become a food packaging alternative for replacing
hydrocarbon-based plastics.
Table 3: Antibacterial activity of the films.
Films Diameter of inhibition zones (mm) against microorganisms∗
SA BC EC PA
CH 20:9±1:913:9±7:212:8±0:215:1±0:1
GU 0000
GU/CH 18 ± 1:612:3±6:412:3±6:912:0±3:5
GU/CH/10BC 14:7±6 11:9±6:111:4±5:511:3±6:5
GU/CH/15BC 12:1±7:210:5±4:212:5±2:510:3±3:9
GU/CH/20BC 14:5±5:810:6±4:910:3±1:513:4±3:3
BC nanofibers 0000
∗SA = Staphylococcus aureus;BC=Bacillus subtilis;EC=Escherichia coli;PA=Pseudomonas aeruginosa.
1.4
3.2
2.6
3.6
4.1 4.7
E
D
C
B
C
A
5
4
3
2
1
0
GU
CH
GU/CH
GU/CH/10BC
GU/CH/15BC
GU/CH/20BC
Samples
Tensile strength (MPa)
(a)
C
24.7%
0.98%
A
B
11%
D
43.7%
CC
20.9%
21.5%
GU
CH
GU/CH
GU/CH/10BC
GU/CH/15BC
GU/CH/20BC
Samples
50
40
30
20
10
0
Elongation at break (%)
(b)
Figure 8: Tensile strength (a) and elongation at break (b) of each sample. Different letters a, b, c, d, and e in the vertical bar chart indicate
significant differences (p≤0:05).
9International Journal of Polymer Science

Data Availability
We have data supporting the results of this work. The Micro-
soft Word document data used to support the findings of this
study are available from the corresponding author upon
request. Please email habral@yahoo.com.
Conflicts of Interest
We affirm that there is no conflict of interest.
Acknowledgments
Acknowledgment is addressed to Universitas Andalas for
supporting research funding with project name PDU
KRP1GB UNAND (number T/5/UN.16.17/PT.01.03/IS-
PDU-KRP1GB/2020).
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