Industrial Crops & Products 139 (2019) 111548

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Industrial Crops & Products

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

Unveiling the physicochemical properties of natural Citrus aurantifolia T

crosslinked tapioca starch/nanocellulose bionanocomposites
⁎
Wei Tieng Owia, Hui Lin Onga,b,c, , Sung Ting Samd, Al Rey Villagraciae, Cheng-kuo Tsaif,
Hazizan Md Akilg
a
School of Materials Engineering, Universiti Malaysia Perlis, Kompleks Pusat Pengajian Jejawi 2, Taman Muhibbah, 02600, Arau, Perlis, Malaysia
b
Centre of Excellence for Biomass Utilization, Universiti Malaysia Perlis, Lot 17, Kompleks Pusat Pengajian Jejawi 2, 02600, Arau, Perlis, Malaysia
c
Taiwan-Malaysia Innovation Center for Clean Water and Sustainable Energy (WISE Center), Lot 17, Kompleks Pusat Pengajian Jejawi 2, 02600, Arau, Perlis, Malaysia
d
School of Bioprocess Engineering, Universiti Malaysia Perlis, Kompleks Pusat Pengajian Jejawi 3, 02600, Arau, Perlis, Malaysia
e
Physics Department, De La Salle University, Manila, 0922, Philippines
f
Department of Biomedical Engineering and Environment Sciences, National Tsing Hua University, Hsinchu, 30013, Taiwan
g
School of Materials and Mineral Resources Engineering, Engineering Campus, Universiti Sains Malaysia, 14300, Nibong Tebal, Penang, Malaysia

A R T I C LE I N FO A B S T R A C T

Keywords: Bionanocomposites from low cost and non-scarce resource tapioca starch (TS)-based film were synthesized for
Tapioca starch food packaging applications. Two types of crosslinkers, namely Citrus aurantifolia, also known as lime juice (LJ),
Crosslinking and a commercialized citric acid (CA), were used one at a time with glycerol via solvent casting for comparison
Citrus aurantifolia purposes. Varying amounts of nanocellulose (NC) extracted from oil palm empty fruit bunches (OPEFB) were
Nanocellulose
added as the reinforcing filler via acid hydrolysis. Structural, tensile, physical properties and water vapor per-
Oil palm empty fruit bunch
Bionanocomposite
meability characterizations were carried out. Crosslinking between TS and LJ through esterification were vali-
dated through the Fourier transform infrared spectroscopy, degree of substitution and degree of di-esterification
results. Ten parts per hundred parts of starch (phs) of LJ enhanced the flexibility of TS bionanocomposite as its
break elongation of 106% was the highest, while NC (1 phs) increased its tensile strength with the highest value
of 13.5 MPa. Crosslinkers did not considerably affect the optical property of TS bionanocomposites but the
addition of NC reduced its transparency. TS bionanocomposite films with both LJ and NC have lower water
vapor permeability (WVP). The WVP of LJ-crosslinked TS film with 1 phs NC reduced about 55%, compared to
neat TS film. This outcome showed that LJ is usable in starch crosslinking as the results obtained were com-
parable to those of the commercialized citric acid. The developed LJ-crosslinked TS bionanocomposites with
enhanced tensile properties and water resistance could be used as food packaging, especially for dry food.

1. Introduction
Nowadays, research on using biodegradable starch as alternative
material to replace or minimize the use of petrochemical-based plastic,
particularly for food packaging, is being carried out. Piñeros-Hernandez
et al. (2017) stated that about 39.6% of total plastics needs are taken up
by packaging application and constitute the largest market share.
However, polymers, such as polypropylene and polyethylene used in
plastic packaging application, are often derived from non-biodegrad-
able sources and linked to environmental pollution. Low density poly-
ethylene (LDPE) is one of the most common polymers used as food
packaging found in the market worldwide. According to Gray et al.
(2018), LDPE as a low cost polymer with special properties, such as
good process-ability and high strength, is extensively used in many
applications. Yet, LDPE is a non-degradable polymer that can increase
the accumulation of plastic waste. In addition, production of green
materials from bio-based substances is now very crucial due to issues of
global warming and fossil fuel depletion (Kengkhetkit and
Amornsakchai, 2014). Among the natural polymers, starch proved to be
attractive due to its low price, wide availability, renewability and
compostability without toxic residue (Nakthong et al., 2017; Xie et al.,
2013). Many researchers reported the good quality of films from star-
ches from different crops like corn (Wang et al., 2017; Qin et al., 2016;
Xu et al., 2015; Teacă et al., 2013), potato (Ghosh Dastidar and

⁎
Corresponding author at: School of Materials Engineering, Universiti Malaysia Perlis, Kompleks Pusat Pengajian Jejawi 2, Taman Muhibbah, 02600, Arau, Perlis,
Malaysia.
E-mail addresses: o.w.tieng@gmail.com (W.T. Owi), hlong@unimap.edu.my (H.L. Ong), stsam@unimap.edu.my (S.T. Sam),
al.villagracia@dlsu.edu.ph (A.R. Villagracia), vicenttsai0120@gmail.com (C.-k. Tsai), hazizan@usm.my (H.M. Akil).

https://doi.org/10.1016/j.indcrop.2019.111548
Received 24 October 2018; Received in revised form 5 July 2019; Accepted 6 July 2019
0926-6690/ © 2019 Elsevier B.V. All rights reserved.
W.T. Owi, et al.
Netravali, 2012), wheat (Goudarzi et al., 2017), rice (Colussi et al.,
2015), cassava (Chuang et al., 2017; López-Córdoba et al., 2017; Mei
et al., 2015), etc. Despite the interesting and promising potential uses of
starch to replace conventional polymers such as LDPE, the uses of
starch-based materials are limited to some applications due to its nat-
ural intractability, brittleness, poor mechanical properties, poor water
barrier properties and low water resistance (Al-Hassan and Norziah,
2012). According to Li et al. (2018), the poor resistance to water can be
attributed to the existence of numerous hydroxyl groups in starch.
An approach to enhance the performance of starch for various ap-
plications is through chemical crosslinking. Crosslinking could induce
the formation of networks to improve the properties of starch-based
products. According to Colivet and Carvalho (2017), the characteristics
of modified crosslinked starches are exploited in the preparation of
films as they have higher tensile property in term of break elongation
than native starch, leading to more flexible films. Crosslinkers, such as
epichlorohydrin and sodium trimetaphosphate were traditionally used
to treat starch but they are toxic and expensive (Reddy and Yang,
2010). To overcome these problems, green crosslinkers, such as citric
acid, tartaric acid, malonic acid and succinic acid were introduced
(Shen et al., 2015). Moreover, Zheng et al. (2017) introduced a fully
bio-based crosslinking agent for the purpose of food packaging, such as
dialdehyde carboxymethyl cellulose (DCMC) for soy protein isolate
(SPI) film. They reported that crosslinking improved the tensile
strength and water barrier properties of DCMC-crosslinked SPI films.
Ghanbarzadeh et al. (2011) reported that citric acid (CA) significantly
improved the tensile properties and reduced the water vapor perme-
ability of starch. Citric acid might be the most frequently used chemical
for starch crosslinking because it is nutritionally harmless, however its
manufacture requires more resources and expense. Based on the ex-
isting works of CA on starch, Citrus aurantifolia juice or lime juice (LJ)
could be a new option as a potential crosslinker. To the best of our
knowledge there is no report on using LJ to crosslink starch. Lime juice
is found to have the most concentrated CA compared to other citrus
products (Brima and Abbas, 2014), and is expected to be able to
crosslink starch, as well as improve its properties.
Another approach to overcome the limitations of starch is by de-
veloping bionanocomposites materials where the starch matrix is re-
inforced with nano-size natural filler. Nanocellulose-based fiber is
commonly used as a biodegrable filler. Starch and nanocellulose have
similar chemical structures which lead to strong adhesion via hydrogen
bond (Soykeabkaew et al., 2012). Nanocellulose with huge surface area
could improve tensile strength (Shankar and Rhim, 2016) and at the
same time create a tortuosity of the pathway in starch matrix to hinder
water molecules passing through (Versino and García, 2014). Teixeira
et al. (2009) utilized cellulose nanofibrils to reinforce thermoplastic
cassava starch. Nanocellulose (NC) can be extracted from many sources
including cotton, wood, bamboo, fungi, as well as industrial and crop
wastes (Rol et al., 2018). At present, our NC was extracted from oil
palm empty fruit bunch (OPEFB), a type of oil palm waste. Oil palm
waste is abundant in Malaysia as it is one of the vital crops that con-
tribute to Malaysia’s economy.
The objective of this study was to develop NC-reinforced bionano-
composite tapioca starch (TS) films crosslinked with lime juice (LJ) as a
new potential natural crosslinker. Nanocellulose was extracted from
OPEFB through acid hydrolysis. Combined effects of LJ and NC on
structural, tensile properties, transparency, color and water vapor
permeability of TS bionanocomposite films were investigated.
Commercialized CA was added into TS bionanocomposite films in order
to compare and justify the acceptability of using LJ for starch cross-
linking.
2
Industrial Crops & Products 139 (2019) 111548
2. Experimental
2.1. Materials and reagents
Oil palm empty fruit bunch, OPEFB, was collected from United Oil
Palm Industries Sdn. Bhd., Nibong Tebal, Pulau Pinang (Malaysia).
Tapioca starch, TS (10% of moisture), was purchased from Thye Huat
Chan Sdn. Bhd. (Malaysia). Fresh green Citrus aurantifolia, commonly
called lime, was obtained from a local hypermarket, Giant Superstore
(Malaysia). Glycerol, C3H8O3 (≥ 99%), sulphuric acid, H2SO4
(95–98%), acetic acid glacial, CH3COOH (99.85%), ethanol, C2H6O
(95%) and sodium hydroxide, NaOH (≥ 98%) were provided by HmbG
Chemicals (Germany). Sodium chlorite, NaClO2 (80%), was supplied by
Acros Organics (Belgium). Potassium bromide, KBr (FTIR grade ≥
99%), sodium tetraborate decahydrate, Na2B4O7‧10H2O (≥ 99.5%) and
sodium hydrogen sulfate, NaHSO4 (approximately 100%), were pro-
cured from Sigma Aldrich (US). Copper (II) sulfate, CuSO4 (≥ 99%) and
potassium hydroxide, KOH (≥ 85%) were purchased from Merck
(Germany). Murexide, C8H8N6O6 (approximately 100%) was obtained
from Riedel-de Haën (Germany). Phenolphthalein, C20H14O4 (≥ 95%),
was acquired from Fisher Scientific (US) and silica gel was obtained
from Bendosen (Malaysia). All chemicals were used as received without
supplementary treatment.
2.2. Nanocellulose preparation
Microcellulose (MC) was first extracted from OPEFB according to
the procedure in our previous work (Owi et al., 2016). In brief, ground
OPEFB was treated with 4 wt% NaOH solution to remove the hemi-
celluloses and then subsequently bleached using 1.7 wt% NaClO2 in an
acetic acid buffer solution for delignification. Both treatments were
carried out at 80 °C for 2 h with constant stirring. Following treatment,
the MC produced was cleaned using distilled water until pH neutral and
dried. In order to produce nanocellulose (NC), 10 g of previously ob-
tained dried MC was acid-hydrolyzed in a preheated glass bottle con-
taining 200 ml of H2SO4 solution (64 wt%). The acid hydrolysis was set-
up as a water bath system performed at 45 °C for 45 min with constant
stirring. Iced distilled water (100 ml) was added into the reaction so-
lution to quench the acid hydrolysis process. Excess concentrated
H2SO4 was cleared out in a centrifuge at a speed of 10,000 rpm for
10 min. Centrifugation was repeated by adding distilled water onto the
NC until its solution became turbid to ensure that the NC suspended
well in distilled water, and the pH of the suspension reached a pH of 5
(Owi et al., 2017). The zeta potential value of NC obtained is -35.8 mV,
and the average particle size of NC is 58.4 nm. The NC suspension was
freeze dried at a temperature of -90 °C and vacuum pressure of 11.1 Pa
using an EYELA FDS-1000 freeze dryer for 72 h to obtain its dry weight
prior to TS film preparation.
2.3. Solution casting and crosslinking of tapioca starch and its
bionanocomposite films
Neat tapioca starch (TS) film was prepared by mixing 4 g of TS,
100 ml of distilled water and 0.8 g of glycerol (20% based on dry weight
of TS) in a beaker. The TS mixture was then heated up to the required
gelatinization temperature, GTemp (70, 80 and 90 °C), held at that
temperature for 30 min and stirred continuosly. Following heating, the
gelatinized solution was cast onto a Teflon-coated mold (15 cm internal
diameter), and then dried at 50 °C for 18 h in an air circulating oven.
For crosslinked film, a desired amount of LJ (pH 3.4), 3, 5, 10, 15, 20
and 25 parts per hundred parts of starch (phs) was mixed together with
the TS mixture before being heated. The TS film with LJ was subse-
quently crosslinked at a variety of temperatures, CTemp (105, 120 and
150 °C) for different durations, CT (10, 15, 20, 30, 35 and 40 min). The
specimen codes and formulations of crosslinked TS films with variations
of LJ content are tabulated in Table S1 (supplementary data).
W.T. Owi, et al.
Non-crosslinked TS bionanocomposite film was prepared by dis-
persing the appropriate amount of NC, 1, 2, 3, 4 and 5 phs into 100 ml
of distilled water containing 0.8 g of glycerol using IKA T25 digital
Ultra-Turrax homogenizer at a speed of 10,000 rpm for 15 min. Next,
4 g of TS were added into the NC mixture prior to heating it to 80 °C for
30 min. The gelatinized solution was homogenized at 10,000 rpm for
another 10 min and then poured onto a Teflon-coated mold (15 cm
internal diameter), and dried at 50 °C for 18 h. For crosslinked TS
bionanocomposite film, LJ (10 phs) was mixed with the non-crosslinked
TS-NC mixture before the heating process. A similar procedure to that
of LJ, commercial CA was added into the non-crosslinked TS-NC mix-
ture. The crosslinking temperature and time were fixed at 105 °C and
15 min, respectively for crosslinked TS bionanocomposite film. The
specimen codes and formulations of non-crosslinked and crosslinked TS
bionanocomposite films with variations of NC content are tabulated in
Table S2 (supplementary data). The pH values of all starch solutions are
listed in Table S1 and Table S2. Prior to characterization, all TS films
were conditioned in a humidity chamber at 50% relative humidity (RH)
for at least 48 h.
2.4. Determination of citric acid content in lime juice
Acid-base titration was applied based on Brima and Abbas (2014) to
determine the concentration of CA (C6H8O7) in LJ. Three drops of
phenolphthalein indicator solution were added in every titration
throughout the experiment. In order to determine the molarity of NaOH
solution (1 M), standardization was carried out using sodium hydrogen
sulfate (NaHSO4). The molarity of the NaOH solution could be calcu-
lated based on chemical Eq. (1).
NaOH + NaHSO4 → NaSO4 + H2O (1)
For specimen preparation, the juice was obtained by hand squeezing
the fresh green limes (size of around 3 to 4 cm in diameter), and then
strained through a cloth filter to eliminate any pulp. Twenty five (25)
ml of lime juice was utilized to carry out each titration with five (5)
replicates againts 1 M of NaOH solution, and phenolphthalein was
dropped in LJ as indicator. The volume of NaOH used was recorded
when the color of the mixture changed from colorless to pink. The
concentration of C6H8O7 could be calculated based on chemical Eq. (2),
while other acids might present at negligible amounts (Scherer et al.,
2012).
3 NaOH + C6H8O7 → Na3C6H5O7 + H2O (2)
2.5. Functional groups identification of tapioca starch and its
bionanocomposite films
The functional groups of chosen non-crosslinked and crosslinked TS
bionanocomposite films were identified with a Perkin Elmer Spectrum
RX Fourier transform infrared (FTIR) spectrometer by applying the at-
tenuated total reflection (ATR) method. The specimens were cut into
squares of 5 mm × 5 mm dimension. The FTIR spectra were recorded
from 4000 to 650 cm−1 for 32 scans and at a resolution of 4 cm−1.
2.6. Degree of substitution of tapioca starch and its bionanocomposite films
Acid-base titration was carried out in order to obtain the degree of
substitution (DS) of TS and its bionanocomposite films according to
Zain et al. (2018). One half (0.5) g of specimen was placed into a 250 ml
conical flask containing 50 ml of distilled water and dispersed by stir-
ring continuously. The mixture was then saponified with 0.5 N of NaOH
solution. The dissolution of specimens in NaOH solution was carried out
at a temperature of 25 °C for 24 h with an agitation speed of 500 rpm. A
phenolphthalein solution was dropped into the mixture as indicator.
Excess NaOH was back-titrated with 0.5 N of H2SO4 solution until the
3
Industrial Crops & Products 139 (2019) 111548
color changed from pink to colorless. The volume of H2SO4 solution was
recorded. Neat TS film was used as control specimen. Tests were per-
formed in triplicate for each specimen. DS was calculated using the
following equations:
MW
(Vblank − Va) × Na × × 100
% substitution = 1000
Ws (3)
162 × % substitution
Degree of Substitution, DS =
100MW − [(MW − 1) × % substitution]
(4)
where, Vblank (ml) is the volume of H2SO4 solution used during titration
of non-crosslinked TS film, Va (ml) is the volume of H2SO4 solution used
during titration of crosslinked TS films, Na (mol/L) is the normality of
H2SO4 solution, MW (g/mol) is the molecular weight of ester group and
Ws (g) is the weight of specimen.
2.7. Complexometric titration of citric acid with copper (II) sulfate
In order to determine the degree of di-esterification (DDE), the
complexometric titration of citric acid (CA) with copper (II) sulfate
(CuSO4) was carried out following the methodology proposed by
Menzel et al. (2013). Based on the stable complex formation reaction of
copper (II)-ions with free and asymmetrically mono-esterified CA, two
independent titrations were performed on all crosslinked TS films. In
the first titration, the crosslinked TS specimen (0.3 g) was hydrolyzed
with 50 ml 0.1 M KOH for 20 min in a boiling water bath. After cooling
the solution to 25 °C, the pH of the solutions was adjusted to 8.5 with
5 N acetic acid and a borax/boric acid buffer. The solution was then
made up to 250 ml with distilled water and a spatula tip of murexide
was added as indicator. The specimen solution was titrated with a
0.02 M of CuSO4 solution until the color changed from purple to orange.
The objective of the first titration was to measure the total amount of
CA, including free and esterified CA. In the second titration, the
crosslinked TS specimen was not hydrolyzed but only swollen in 2 ml of
distilled water for 20 min, and its pH was adjusted to 8.5 with borax/
boric acid buffer solution. Murexide was added into the specimen so-
lution, and the specimen was titrated with a 0.02 M of CuSO4 solution.
This titration was used to measure free plus mono-esterified CA. Hence,
the difference between the titrations of the hydrolyzed and non-hy-
drolyzed crosslinked TS specimens enabled the DDE to be calculated
(Eq. 5).
2 × mCA × wdiester × MAGU
Degree of Di‐esterification, DDE =
MCA × mTS (5)
where, mCA (g) is the amount of CA added, wdiester is the weight fraction
of CA taking part in a diester linkage given as a percentage, 2 is a factor
to reflect that two anhydroglucose units are esterified by one CA mo-
lecule, MAGU (162 g/mol) is the molar mass of one anhydroglucose unit,
MCA (192 g/mol) is the molar mass of CA and mTS (g) is the amount of
TS in the CA-containing film. The measurements of DDE were per-
formed in triplicate.
2.8. Tensile properties and fractured morphology of tapioca starch
bionanocomposite films
The tensile strength (MPa) and break elongation (%) of the TS and
its bionanocomposite films were evaluated according to ASTM-D882-12
(ASTM, 2012) using an Instron Universal 5569 tensile testing machine
with load of 50 kN. The film specimens were cut into rectangular strips
with dimensions of 100 mm in length and 10 mm in width. The thick-
ness of the film specimens were measured using a Mitutoyo IP65 digi-
matic micrometer with accuracy of ± 0.001 mm and the average
thickness values of five specimens for each formulation were recorded.
The initial gauge length was fixed at 50 mm and the test speed was set
for 10 mm/min at 25 °C.
W.T. Owi, et al.
The tensile fractured surfaces of all TS bionanocomposite film spe-
cimens were viewed under a JEOL JSM-6460 LA scanning electron
microscope (SEM) with an accelerated voltage of 10 kV. Small strips of
film specimens of 5 mm × 10 mm dimension were placed on the alu-
minium stubs using double-sided tapes and then coated with platinum
by a JEOL JFC-1600 auto fine coater.
2.9. Optical properties of tapioca starch and its bionanocomposite films
The ultraviolet-visible (UV–vis) spectra of the non-crosslinked and
crosslinked TS bionanocomposite films were recorded from 200 to
800 nm by using a Hitachi U-4100 UV–vis spectrophotometer in
transmittance mode. Specimens were cut into a circular shape of 2.5 cm
diameter and placed directly on the solid holder of the spectro-
photometer. Measurements were performed using air as reference. The
UV–vis spectra were recorded as single measurement without repeti-
tion. The transparency value (T value) of each film was calculated using
Eq. (6) in accordance to Wang et al. (2017).
T value = A600 / X (6)
where A600 is the absorbance value at 600 nm and X is the thickness
(mm) of the TS films. According to Eq. (6), a higher T value represents a
lower degree of transparency. Absorbance ( A ) values were obtained
from transmittance values by applying the Lambert-Beer equation as
shown in Eq. (7) (Oleyaei et al., 2016).
A = 2 − log10 %Transmittance (7)
2.10. Color measurement for tapioca starch and its bionanocomposite films
Surface colors of the non-crosslinked and crosslinked TS bionano-
composite films were evaluated using a Premier Colorscan Instruments
Pvt. Ltd. model SS5100A fluoro spectrophotometer. Square-shaped film
specimens of 4 cm × 4 cm dimension were placed on a black standard
plate (L* = 1, a* = 0 and b* = 0). The luminosity (L*), chromaticity
parameters a* (red-green) and b* (yellow-blue) of TS and its bionano-
composite films were measured. The color measurement was recorded
as single measurement without repetition.
2.11. Water vapor permeability of tapioca starch and its bionanocomposite
films
Determination of TS and its bionanocomposite films’ water vapor
permeability (WVP) were carried out in accordance with the ASTM-
E96/E96M-10 standard methodology (ASTM, 2010) with minor mod-
ification, using the desiccant method. A circular film specimen was
attached and sealed on top of the glass test cup with an internal dia-
meter of approximately 20 mm and depth of 45 mm. Twenty (20) g of
silica gel were filled into the glass test cup and placed below the film
specimen to obtain a 0% relative humidity (RH) environment. The film
specimen and silica gel were separated with sufficient space to provide
an air gap of approximately 6 mm, such that the glass test cup could be
shaken during each weight measurement. The set-up of equipment used
for water vapor permeability test is illustrated in Fig. S1 (supplemen-
tary data). The weights of specimens with glass test cup were measured
using a Sartorius electronic balance with accuracy of ± 0.0001 g. Fol-
lowing the recording of the initial weights of the specimens with the
glass test cup, they were stored in the humidity chamber at 50% RH and
temperature of 25 °C. At designated periods of time, the specimens with
glass test cup were taken out and weighed. Twenty weight measure-
ments were made within 48 h.
A graph of weight gained (g) against time (h) for each glass test cup
was plotted. The slope of the linear portion of the plot (representing
weight change) indicates the amount of water vapor diffusion across
the film per unit of time, expressed as grams per hour (g/h), as shown in
Fig. 1. The result shown in Fig. 1 is for a WVP test on neat TS film.
4
Industrial Crops & Products 139 (2019) 111548
Fig. 1. Weight gain of non-crosslinked neat TS film (80 °C GTemp) as a function
of time.
From Fig. 1, the weight change for neat TS film is 3.25 × 10−3 g/h.
The water vapor transmission rate (WVT) (g/mm2.h) and water vapor
permeability (WVP) (g/mm.h.kPa) were calculated using the following
equations:
(W / t )
WVT =
A (8)
WVT
WVP = X
S (R1 − R2) (9)
where (W / t ) is the weight change at the time it occurred (g/h), A is the
test area (mm2), X is the thickness of the film (mm), S is the saturation
vapor pressure at the test temperature (kPa), R1 and R2 are the RH in
the humidity chamber and in the glass test cup, respectively. All TS and
its bionanocomposite films were examined in triplicate.
2.12. Solubility of tapioca starch and its bionanocomposite films in water
The solubility of non-crosslinked and crosslinked TS bionano-
composite films in water was measured according to the method de-
scribed by Ghanbarzadeh et al. (2011) and Merino et al. (2018). Ap-
proximately 0.5 g of each specimen was dried in an air-circulating oven
at 50 °C until it reached a constant weight. Then, its initial weight was
recorded. The specimens were immersed in 50 ml of distilled water at
25 °C for 24 h with agitation speed of 500 rpm. After 24 h, the speci-
mens were removed from the distilled water and dried at 105 °C for
24 h. The final weight of specimens was recorded and the percentage of
water solubility (WS) was calculated using Eq. (10).
Wi − Wf
WS (%) = × 100
Wi (10)
where Wi (g) is the initial weight of the specimen, and Wf (g) is the final
weight of the specimen after immersion in distilled water. The mea-
surement of WS was performed in triplicate.
2.13. Statistical analysis
Minitab 17 was used for statistical analysis of the data. Data was
subjected to one-way analysis of variance (ANOVA), and Tukey’s test
was used to detect the differences among mean values of the TS bio-
nanocomposite films properties at a confidence level of p ≤ 0.05. The
mean values of DS, DDE, tensile test, WVP test and WS for TS and its
bionanocomposite films were tested for statistically significant differ-
ences. Statistically significant differences between groups were denoted
by different English alphabets on the graphs.
W.T. Owi, et al.
3. Results and discussion
3.1. Citric acid content in lime juice
In order to use the same amount of commercial citric acid (CA) for
the preparation of crosslinked TS bionanocomposite films, it is neces-
sary to determine the amount of CA in lime juice (LJ). According to
Scherer et al. (2012), CA is the main acid present in C. aurantifolia while
other acids such as tartaric acid, malic acid and ascorbic acid are pre-
sent at negligible amounts. This means that the total amount of acid in
LJ can approximate the amount of CA in LJ. From the titration result,
the average CA concentration in LJ from Malaysia was found to be
8,530 mg/100 ml. This value is higher than the values of 6,797, 6,581,
and 7,023 mg/100 ml for all 3 batches of Citrus aurantifolia obtained
from Brazil, which were determined by Scherer et al. (2012). The ef-
fectiveness of using LJ on the crosslinked TS bionanocomposite films
would be proven by comparing the TS bionanocomposite films’ prop-
erties prepared using commercial CA.
3.2. Structural properties of tapioca starch and its bionanocomposite films
The FTIR spectra of non-crosslinked TS, crosslinked TS using 10 phs
LJ, and TS bionanocomposite films with 1 phs NC are shown in Fig. 2.
There is no difference between the FTIR spectra of the non-crosslinked
neat TS (Fig. 2a) and non-crosslinked TS bionanocomposite (Fig. 2b).
This means that the addition of NC does not lead to significant chemical
changes in terms of their functional group due to the structure simi-
larity between starch and NC. The broad peak at 3,334 cm−1 is assigned
to the OeH stretching being related to the hydrogen-bonded hydroxyl
group in TS. This peak became broader for TS bionanocomposite films,
which might be due to the hydrophilic characteristic of NC, while the
sharp peak at 2,928 cm−1 pertains to the aliphatic C–H stretching. The
peak at 1,643 cm−1, also known as characteristic peak, of starch (Ma
et al., 2009) is assigned to the OeH bending which is believed to be a
Fig. 2. FTIR spectra of (a) non-crosslinked Neat TS (80 °C GTemp), (b) non-crosslinked NC1 (80 °C GTemp, 1 phs NC), (c) crosslinked LJ10 (80 °C GTemp, 105 °C
CTemp, 15 min CT, 10 phs LJ), (d) crosslinked LJ-NC1 (80 °C GTemp, 105 °C CTemp, 15 min CT, 10 phs LJ, 1phs NC), (e) crosslinked CA (80 °C GTemp, 105 °C
CTemp, 15 min CT), and (f) crosslinked CA-NC1 (80 °C GTemp, 105 °C CTemp, 15 min CT, 1phs NC).
5
Industrial Crops & Products 139 (2019) 111548
property of tightly-bound water existing in the starch. The peak at
1,353 cm−1 is attributed to the bending modes of O–C–H, C–C–H and
C–O–H (Mei et al., 2015). The peak at 1,152 cm−1 could be assigned to
C–O stretching. The two peaks at 1,080 and 1,021 cm−1 represent the
glucopyranose ring OeC stretching vibrations (Ghosh Dastidar and
Netravali, 2012). The peaks at 928 and 859 cm−1 are both ascribed to
C–H bending mode.
All films have similar peaks, except for the extra peak at 1,728 cm−1
for LJ-crosslinked TS (Fig. 2c), 1,726 cm−1 for LJ-crosslinked TS bio-
nanocomposite (Fig. 2d), 1,722 cm−1 for CA-crosslinked TS (Fig. 2e),
and 1,721 cm−1 for CA-crosslinked TS bionanocomposite (Fig. 2f). The
peaks ranging from 1,721 to 1,728 cm−1 in all crosslinked TS and its
bionanocomposite films are ascribed to C]O stretching vibration
which related to the ester carbonyl (C]O) and carboxyl (C(=O)OH)
functional group (Reddy and Yang, 2010). Similar results were obtained
by Shen et al. (2015) for their crosslinked starch film using CA; they
reported that the peak at 1,735 cm−1 indicating the carbonyl groups. In
addition, Ghosh Dastidar and Netravali (2012) uncovered a new peak at
1,725 cm−1 that confirmed the presence of ester bond in crosslinked
potato starch using malonic acid. Teacă et al. (2013) obtained a new
peak occurred at 1,742 cm−1 for carbonyl peak in the FTIR of corn
starch crosslinked using tartaric acid. However, the broad extra peaks
ranging from 1,721 to 1,728 cm−1 found in this study might be a
coalescence peak caused by the ester bond and carboxyl groups in CA.
The reason is because it is unlikely that all the carboxyl groups are
esterified (Shi et al., 2007). Although the ester bond found on FTIR
spectra demonstrates that the esterification occurred between TS and
CA, the same reaction between glycerol and CA could have occurred
(Garcia et al., 2014). Furthermore, the new peaks could be associated to
the overlapping bands from carboxyl groups of residual CA as Demitri
et al. (2008) reported a peak at 1,715 cm−1 for pure CA. These events
are not captured in the FTIR spectra by the presence of a new peak.
Therefore, the crosslinking are further supported by the measurements
of degree of substitution and degree of di-esterification.
W.T. Owi, et al.
Fig. 3. Proposed reaction mechanism for crosslinked TS bionanocomposite films.
Overall, the new extra peaks found on the FTIR spectra of cross-
linked TS and its bionanocomposite films could be attributed to ester
carbonyl group which indicates the crosslinking of TS and LJ through
esterification. The proposed crosslinking mechanism of TS bionano-
composite film through esterification is demonstrated in Fig. 3. TS
could react with the crosslinker by esterification and form ester bonds.
Subsequently, the NC could form hydrogen bonds with TS.
3.3. Degree of substitution of tapioca starch and its bionanocomposite films
The degree of substitution (DS) for starch derivative is defined as
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Industrial Crops & Products 139 (2019) 111548
the average number of hydroxyl groups (OH) substituted per D-gluco-
pyranosyl ring (Ren et al., 2016). Since every ring possesses three OH
groups, the maximum possible value of DS is 3. Nevertheless, the pri-
mary OH group (C-6) is much more reactive than the two secondary OH
groups (C-2 and C-3) due to steric hindrance (Ghosh Dastidar and
Netravali, 2012). The DS value obtained for crosslinked neat TS film
using 10 phs LJ is 0.30, while the DS value for crosslinked neat TS film
using commercial CA is 0.29. This indicates that the crosslinking of TS
film using LJ occurred, and this is in agreement with the FTIR results in
the previous section. The DS values for crosslinked TS (obtained from
Malysia) films in this study are higher than the DS values of 0.178 for
W.T. Owi, et al.
Fig. 4. DS of crosslinked TS bionanocomposite films using LJ and CA as a
function of NC content (80 °C GTemp, 105 °C CTemp, 15 min CT, 10 phs LJ/
CA).
crosslinked TS (obtained from China) film using CA, which was re-
ported by Mei et al. (2015). The variation of DS could be affected by
numerous factors, such as botanical origin, type of reagent, reagent
concentration and the properties of the size and structure of starch
granules (Colussi et al., 2015). Similarly, the DS values obtained by
Ghosh Dastidar and Netravali (2012) are different for potato and corn
starch cured with the same amount of malonic acid.
Fig. 4 presents the DS of crosslinked TS bionanocomposite films
using 10 phs LJ and CA as a function of NC content. In general, the
crosslinked TS bionanocomposite films with LJ as crosslinker show
higher DS values compared with the crosslinked TS bionanocomposite
films using commercialized CA. The DS values for TS bionanocomposite
films crosslinked with 10 phs LJ decreased significantly from 0.30 to
0.21, with the increasing amount of NC from 1 to 5 phs. This trend is
consistent with the DS values of TS bionanocomposite films crosslinked
with commercialized CA which decreased from 0.29 to 0.19, with the
same increasing amount of NC. This result revealed that the addition of
NC would interrupt the esterification process between starch and
crosslinker. This might be due to the chemical match between TS and
NC as shown in Fig. 3, and a strong hydrogen (H) bonding resulting
from OH groups of both TS and NC (Ghosh Dastidar and Netravali,
2013). Overall, DS results of LJ-crosslinked and CA-crosslinked TS
bionanocomposite films are comparable, and so justified the use of LJ as
natural crosslinker for starch.
3.4. Degree of di-esterification according to complexometric titration of
citric acid with copper (II) sulfate
The degree of substitution (DS) discussed earlier indicates the
crosslinking of TS bionanocomposites using LJ and commercial CA.
However, the DS values do not specifically identifies the esterification
of TS by CA in LJ or commercial CA alone. Therefore, to further prove
that LJ could be used to crosslinked TS films, the degree of di-ester-
ification (DDE) of all crosslinked TS bionanocomposite films were
computed from the complexometric titration of CA with copper (II)
sulfate as reported by Menzel et al. (2013). The DDE value obtained for
crosslinked neat TS film using 10 phs LJ is 0.0648 whereas the DDE
value for crosslinked neat TS film using commercial CA is 0.0594. This
confirmed the formation of di-ester through crosslinking of TS using LJ
and commercial CA. However, the DDE values obtained for both LJ-
crosslinked and CA-crosslinked TS were lower than the DS value. This
was expected as the measurement of DS might include mono-ester, di-
7
Industrial Crops & Products 139 (2019) 111548
Fig. 5. DDE of crosslinked TS bionanocomposite films using LJ and CA as a
function of NC content (80 °C GTemp, 105 °C CTemp, 15 min CT, 10 phs LJ/
CA).
ester as well as unreacted CA. Moreover, the results of DS may include
the crosslinking between glycerol and CA. Fig. 5 displays the DDE of
crosslinked TS bionanocomposite films using 10 phs LJ and CA as a
function of NC content. Similar to the trends of DS values for both LJ-
and CA-crosslinked TS bionanocomposites, the DDE values decreased
significantly when NC content increased from 1 to 5 phs. Thus, the
addition of NC affected the value of DDE. This is consistent with the
results of DS for crosslinked TS bionanocomposite films.
3.5. Tensile properties and fractured morphology of tapioca starch
bionanocomposite films
Tensile properties of neat TS films are influenced by various factors,
such as processing condition and additive content, so it is necessary to
optimize the tensile properties against these factors. In addition, tensile
results were used as a benchmark to determine the optimum processing
parameters for making TS and its bionanocomposite films throughout
this study. Fig. 6 exhibits the tensile properties resulting from the
varying of gelatinization temperature (GTemp), crosslinking tempera-
ture (CTemp), crosslinking time (CT) and LJ content. Rising gelatini-
zation temperature increased the tensile strength up to the maximum,
8.18 MPa at 80 °C, but then it decreased beyond that temperature
(Fig. 6a). High gelatinization temperature caused the reduction of the
break elongation of neat TS films. Thus, 80 °C is chosen as optimum
gelatinization temperature for the remainder of the study.
Different crosslinking temperatures resulted in an identical trend for
both tensile strength and break elongation (Fig. 6b). The optimum
crosslinking temperature was 105 °C. In Fig. 6c, an opposing trend was
found between tensile strength and break elongation when the cross-
linking time was varied. The most suitable crosslinking time was 15 min
to obtain a crosslinked neat TS film with a good balance of tensile
strength and break elongation. Prolonging the crosslinking time would
damage the starch backbones, and resulting in degrading tensile
properties (Xu et al., 2015). The trend between tensile strength and
break elongation of TS film obtained was also opposing when the LJ
content is increased (Fig. 6d). Generally, the tensile strength increased
and the break elongation decreased with the addition of LJ. This shows
that crosslinking of TS using LJ provides better intermolecular inter-
action between molecules, leading to better strength (Reddy and Yang,
2010). Similarly to the results in FTIR, glycerol-CA esters may have
formed and affected the tensile properties of crosslinked TS films. Shen
et al. (2018) reported that the tensile strength of glycerol-CA ester is
W.T. Owi, et al. Industrial Crops & Products 139 (2019) 111548

Fig. 6. Effects of (a) gelatinization temperature (non-crosslinked), (b) crosslinking temperature (80 °C GTemp, 15 min CT, 15 phs LJ), (c) crosslinking time (80 °C
GTemp, 105 °C CTemp, 15 phs LJ) and (d) LJ content (80 °C GTemp, 105 °C CTemp, 15 min CT) on tensile strength and break elongation of non-crosslinked and LJ-
crosslinked neat TS films. Fixed values of the parameters are given and the LJ content is based on the dry weight of starch (4 g).

difficult to measure due to high brittleness. Thus, ten (10) phs of LJ is
chosen as the optimum amount for TS films crosslinking to obtain a
balanced tensile strength and break elongation.
The effect of NC added into non-crosslinked and crosslinked TS
bionanocomposite films on tensile properties is shown in Fig. 7. With
sufficient content of NC added into the TS matrix, a significantly
improved tensile strength can be obtained in expense to the reduction
of break elongation. Fig. 7a shows the tensile strength increasing from
8.18 MPa for neat TS to the highest value of 13.46 MPa for non-cross-
linked TS bionanocomposite film with 1 phs NC, but this highest value
was reduced for higher NC content. A similar tensile strength trend was
found for LJ and CA crosslinked TS bionanocomposite films when

Fig. 7. (a) Tensile strength and (b) break elongation of non-crosslinked (80 °C GTemp), LJ-crosslinked (80 °C GTemp, 105 °C CTemp, 15 min CT, 10 phs LJ) and CA-
crosslinked (80 °C GTemp, 105 °C CTemp, 15 min CT) of TS and its bionanocomposite films.

8
W.T. Owi, et al. Industrial Crops & Products 139 (2019) 111548

varying the NC content. In Fig. 7b, the addition of NC reduces break starch-based films. However, the fractured surface of all crosslinked TS
elongation of non-crosslinked TS bionanocomposite films. Break elon- bionanocomposite films had fewer pores and cracks, compared to non-
gation remarkably increased from 9.1% for neat TS to 105.5% for LJ- crosslinked TS bionanocomposite films, except for the film with 5 phs
NC1 when crosslinked with LJ. This result is comparable to 96.6% (CA- NC. The cracks and pores found on the fractured surface of non-cross-
NC1) break elongation for CA-crosslinked TS bionanocomposite film. linked TS bionanocomposite films provide poor tensile properties in
Excessive NC content lowers the break elongation too. High tensile terms of break elongation. The fractured surface of crosslinked TS
strength could be driven by the similarities in chemical structure be- bionanocomposite films with fewer defects provides a higher value of
tween NC and TS that promote a good interaction between them. A break elongation. This result is in good agreement with earlier tensile
large surface area of nano-size NC induces a strong interfacial interac- results.
tion via hydrogen bond among NC and TS (Shankar and Rhim, 2016),
while the lowering of break elongation when increasing the NC content
3.6. Optical properties and color measurement of TS and its
might be due to the rigid nature of the NC (Slavutsky and Bertuzzi,
bionanocomposites
2014). Break elongation improvement of crosslinked TS bionano-
composite film is due to the role of LJ or CA as plasticizer, which in-
Selected TS bionanocomposite films for non-crosslinked, LJ-cross-
creases the interstitial volume of substance or macromolecular mobility
linked and CA-crosslinked are shown in Fig. 9. Generally, the TS films
of the TS, hence causing the polymeric network to become less dense by
are colorless and translucent. However, as shown in Fig. 9, the TS films’
lowering intermolecular forces (Wang et al., 2014). Consequently, the
transparency against visible light decreases with increasing NC content.
extensibility and flexibility of TS films are enhanced. Overall, the in-
Crosslinking of TS films using LJ and CA does not affect the transpar-
clusion of 1 phs of NC provided the best tensile strength for both non-
ency.
crosslinked and crosslinked TS bionanocomposite films. However, LJ-
The UV–vis transmittance spectra of TS bionanocomposite films
crosslinked TS bionanocomposite film with 1 phs NC provided the best
shown in Fig. 10 were acquired to study the optical transparency. The
tensile properties as its tensile strength and break elongation improved.
assessment of TS films’ optical properties is relevant as this is a key
Fractured surfaces of the non-crosslinked and crosslinked TS bio-
property in some applications, mainly for food packaging. In Fig. 10a,
nanocomposite films following the tensile test are shown in Fig. 8. This
there are drastic differences between the UV transmittance of non-
figure presents a good evidence for the strong interfacial adhesion be-
crosslinked neat TS, TS with 1 phs NC, coded as NC1, and TS with 5 phs
tween TS and NC due to its rough surface. The fractured surface of non-
NC, coded as NC5. In the visible zone at 600 nm, the neat TS film ex-
crosslinked TS bionanocomposite films with cracking lines and pores
hibits the highest transparency with a UV transmittance value of
can be seen in the figure. This can be attributed to the brittleness of the
35.57%. However, this value is considerably low as the starch granules,

Fig. 8. Morphology of non-crosslinked (80 °C GTemp), LJ-crosslinked (80 °C GTemp, 105 °C CTemp, 15 min CT, 10 phs LJ) and CA-crosslinked (80 °C GTemp, 105 °C
CTemp, 15 min CT) TS bionanocomposite films’ fractured surface at magnification of X1,000.

9
W.T. Owi, et al. Industrial Crops & Products 139 (2019) 111548

Fig. 9. Digital images of TS bionanocomposite films for non-crosslinked (80 °C GTemp), LJ-crosslinked (80 °C GTemp, 105 °C CTemp, 15 min CT, 10 phs LJ) and CA-
crosslinked (80 °C GTemp, 105 °C CTemp, 15 min CT) with variation of NC content.

Fig. 10. UV–vis transmittance spectra of (a) non-crosslinked (80 °C GTemp) and (b) crosslinked (80 °C GTemp, 105 °C CTemp, 15 min CT, 10 phs LJ/CA) TS
bionanocomposite films.

which consist of semi-crystalline amylose, scattered a portion of light
causing a lower UV transmittance value at the wavelength of 600 nm
(Wang et al., 2017). On the other hand, in Fig. 10b, there were slight
differences between the UV transmittance of non-crosslinked neat TS,
LJ-crosslinked (10 phs) TS, coded as LJ10, CA-crosslinked TS (same
amount to 10 phs LJ), coded as CA, LJ-crosslinked TS with 1 phs NC,
coded as LJ-NC1, and CA-crosslinked TS with 1 phs NC, coded as CA-
NC1.
Furthermore, the transparency value (T value) could be calculated
from the absorbance obtained in the UV–vis region (600 nm) over TS

10
W.T. Owi, et al. Industrial Crops & Products 139 (2019) 111548

Table 1
Transmittance (%T), T value and color parameters for non-crosslinked (80 °C GTemp), LJ-crosslinked (80 °C GTemp, 105 °C CTemp, 15 min CT, 10 phs LJ) and CA-
crosslinked (80 °C GTemp, 105 °C CTemp, 15 min CT) TS and its bionanocomposite films.
Property Non-crosslinked LJ-crosslinked CA-crosslinked

TS NC1 NC5 LJ10 LJ-NC1 LJ-NC5 CA CA-NC1 CA-NC5

UV-C (256 nm), %T 25.31 21.83 18.65 24.03 21.03 19.47 25.83 23.69 21.60
UV-B (298 nm), %T 24.46 21.80 19.07 23.31 20.39 18.88 24.40 24.12 21.41
UV-visible (600 nm), %T 35.57 33.53 31.39 33.85 33.89 31.99 34.49 34.41 33.08
T value 2.99 3.16 3.35 3.14 3.15 3.30 3.08 3.09 3.20
L* 39.78 40.31 41.14 40.10 40.27 40.78 39.88 39.91 40.51
a* 0.11 0.11 0.08 0.06 0.17 0.15 0.13 0.09 0.08
b* −0.24 0.04 0.24 −0.18 −0.42 −0.41 −0.48 −0.32 −0.31

Fig. 11. WVP of (a) crosslinked TS films as a function of LJ content (80 °C GTemp, 105 °C CTemp, 15 min CT), and (b) non-crosslinked (80 °C GTemp) and crosslinked
TS bionanocomposite films using LJ (80 °C GTemp, 105 °C CTemp, 15 min CT, 10 phs LJ) and CA (80 °C GTemp, 105 °C CTemp, 15 min CT) as a function of NC
content.

film thickness. The average of TS bionanocomposite film thickness is
approximately 0.15 mm. The addition of NC did not affect the film’s
thickness. The calculated T values are listed in Table 1. The lowest T
value recorded is 2.99 for neat TS, then it increased slowly to 3.35 for
NC5. This means that the neat TS provided the highest degree of
transparency among all TS films. Qin et al. (2016) documented a similar
trend in transparency for maize starch films when the concentration of
chitin nano-whiskers was increased from 0 to 5%. The trend of trans-
parency for LJ-crosslinked and CA-crosslinked TS bionanocomposite
films matched the trend for non-crosslinked TS bionanocomposite films.
UV regions are divided into three; they are UV-A at the wavelength
of 320–400 nm, UV-B at the wavelength of 280–320 nm and UV-C at the
wavelength of 200–280 nm. For all specimens, only two peaks are
found in the UV-B (298 nm) and UV-C (256 nm) regions, as listed in
Table 1. The UV transmittance values (%) in both UV regions are re-
duced with increasing NC content, which indicates that UV-light is
blocked by NC. Shankar and Rhim (2016) also observed that increasing
the amount of NC and micro-crystalline cellulose in agar-based film led
to a lower UV transmittance at 280 nm. The UV filtering effect is ben-
eficial in food packaging applications since UV radiation may cause the
formation of free radicals in food products through a variety of organic
photochemical reactions. Nanocellulose dominated the results of
transparency and UV-shielding effect of TS bionanocomposite films.
The factors that suggest that NC could hinder the light passage across
the TS bionanocomposite films are its nano-size dimension with large
specific surface area (Oleyaei et al., 2016), the rigidity of cellulose
nanocrystals (Oun and Rhim, 2015) and the amount of NC in TS films
(Kim et al., 2014), causing transparency loss. The transmittance of light
might be prevented by the NC embedded in the interspaces of TS bio-
nanocomposite films (Qin et al., 2016). Overall, the results revealed
that all TS bionanocomposite films are translucent and NC reduces the
transparency of the TS bionanocomposite films. These results justified
the digital images of TS bionanocomposite films depicted in Fig. 9.
Color determination is crucial as it is related to the appearance of a
packaged product, which might affect the product’s demand as well as
consumers’ preference. The results of color parameters (L*, a* and b*)
measurement of non-crosslinked, LJ-crosslinked and CA-crosslinked TS
bionanocomposite films are shown in Table 1. Luminosity, L*, of the TS
bionanocomposite films increases with increasing NC content in-
dicating that the specimens become lighter in color. This might be in-
duced by the white color of NC introduced into the TS bionano-
composite films. The chromaticity parameter a* decreased, while
parameter b* increased. This means the color of TS bionanocomposite
films incorporating NC tends towards green and faintly increases yel-
lowness. The values of L* and a* for crosslinked TS bionanocomposite
films changed slightly. However, the values of b* for crosslinked TS
bionanocomposite films decreased. This indicates that the films’ color
tends towards blue. Overall, the changes in color were not obvious and
would minimally affect the apparent acceptability of TS bionano-
composite films.
3.7. Water vapor permeability of tapioca starch and its bionanocomposite
films
The TS bionanocomposite films’ water vapor permeability (WVP)
was significantly influenced by LJ and NC addition. Fig. 11a shows a
significant decrease of WVP values of LJ-crosslinked TS films, however
increasing the LJ amount further had an insignificant effect. Fig. 11b
shows a significant reduction of TS bionanocomposite films’ WVP for
varying NC content and, similarly to LJ, the addition of commercialized

11
W.T. Owi, et al.
CA also lowers the WVP values. This result improved the TS bionano-
composite films’ WVP beyond that of Piñeros-Hernandez et al. (2017)
who reported an increased WVP with rosemary extracts added into
starch films. To use TS bionanocomposite films in food packaging, films
should at least reduce the moisture formation between food and the
surrounding environment. WVP reduction for crosslinked TS bionano-
composite films could be attributed to the hydrophilic OH groups
substitution with hydrophobic ester groups (Ghanbarzadeh et al.,
2011), while the addition of NC into TS also reduced the WVP of TS
bionanocomposite films. This is due to the high surface area of NC
which increases the tortuosity of the pathway for water molecules to
pass through the TS bionanocomposite films (Versino and García,
2014). Similarly, Espino-Pérez et al. (2018) reported that cellulose
nanocrystals had a tortuos effect on the polylactide nanocomposites,
which improved its water barrier properties. However, the addition of
NC in LJ-crosslinked and CA-crosslinked TS bionanocomposite films
had higher WVP compared to LJ-crosslinked and CA-crosslinked TS
without NC films. This may be due to the nature of NC which is also
hydrophilic, making it absorb moisture from its surroundings. This af-
fected the results of WVP, but further increment of NC amount had an
insignificant effect. The addition of 1 phs of NC on LJ-crosslinked TS
bionanocomposite film is sufficient enough to provide a good WVP as
55% reduction had been achieved.
3.8. Solubility of tapioca starch and its bionanocomposite films in water
The results of water solubility (WS) measurements are shown in
Fig. 12. The addition of both LJ and NC into TS significantly affected
the solubility of TS bionanocomposite films in water. Fig. 12a shows
that the inclusion of LJ in TS (until 10 phs LJ) reduced the solubility of
crosslinked TS film in water, but it increased beyond 10 phs LJ. The %
WS was 36.49% for non-crosslinked neat TS film and decreased sig-
nificantly to 31.59% for crosslinked TS film containing 10 phs of LJ.
The crosslinking reaction can reinforce the starch network both che-
mically and physically, thus resulting in lower WS (Menzel et al., 2013),
whereas the increase of WS for crosslinked TS films beyond 10 phs LJ
was probably due to the excess LJ which could not interact with TS.
Excessive LJ might interact with water and interrupt the network
through hydrogen bonds, thus increasing its solubility in water
(Ghanbarzadeh et al., 2011).
Fig. 12b shows that the addition of NC (1 to 5 phs) into TS decreased
the %WS from 36.49% to 22.45% for non-crosslinked TS bionano-
composites, 31.59% to 21.92% for LJ-crosslinked TS bionanocompo-
sites and 33.12% to 22.10% for CA-crosslinked TS bionanocomposites.
Fig. 12. Water solubility of (a) crosslinked TS films as a function of LJ content (80 °C GTemp, 105 °C CTemp, 15 min CT), and (b) non-crosslinked (80 °C GTemp), LJ-
crosslinked (80 °C GTemp, 105 °C CTemp, 15 min CT, 10 phs LJ) and CA-crosslinked (80 °C GTemp, 105 °C CTemp, 15 min CT) of TS films as a function of NC content.
12
Industrial Crops & Products 139 (2019) 111548
This is an indication of strong interactions between individual NC-na-
nofiller and TS chains within the TS film matrix. According to Ilyas
et al. (2018), the capability of NC in interacting with TS chains was due
to the interaction of starch hydrogen bonds with the hydroxyl groups of
NC. This interaction enhanced water stability of TS bionanocomposite
films. A similar WS result was reported by Aila-Suárez et al. (2013);
they stated that the addition of cellulose and cellulose nanoparticle in
starch film promoted the interactions among hydroxyl groups of both
polysaccharides, resulting in reduced WS. The trends of WS for either
non-crosslinked, LJ-crosslinked or CA-crosslinked TS bionanocompo-
sites were similar to WVP when NC amount is increased, indicating the
enhancement of TS films’ water resistance.
4. Conclusion
The achievement of starch crosslinking using LJ was revealed
through these three outcomes: 1) new peak found in FTIR that corre-
sponded to the ester carbonyl group, 2) results from DS that confirmed
there was substitution on the hydroxyl group of starch, and 3) results
from DDE that confirmed the crosslinking between TS and LJ or CA. In
general, the tensile strength of TS bionanocomposite films increased
with the addition of NC, while the addition of LJ made the TS biona-
nocomposite films more flexible with a higher break elongation per-
centage. Acceptable transparency and color on TS bionanocomposite
films were observed. The WVP of crosslinked TS bionanocomposite
films was sharply reduced from 11.97 to 5.35 (x10−7 g/mm.h.kPa).
Similar to the results of WVP, the addition of both LJ and NC into TS
decreased the %WS, indicating good TS films’ water resistance. All re-
sults suggest that LJ is a suitable natural crosslinker, and it can replace
the commercialized CA. The advantage of using starch instead of fossil
fuel based packaging material is that starch is a natural resource.
Further improvement of TS bionanocomposite films should be focused
on the mechanical properties, in order to compete and replace the ex-
isting material usually used for food packaging such as LDPE which has
a higher mechanical strength.
Acknowledgements
The authors wish to acknowledge the Ministry of Education,
Malaysia, for providing financial support through the MyBrain15-
MyPhD Scholarship. In addition, the authors are grateful to Prof. Ruey-
an Doong from Department of Biomedical Engineering and
Environmental Sciences, National Tsing Hua University, for providing
some laboratory facilities.
W.T. Owi, et al.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.indcrop.2019.111548.
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