Mango kernel starch films as affected by starch nanocrystals and cellulose nanocrystals

Carbohydrate Polymers 211 (2019) 209–216

Contents lists available at ScienceDirect

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

Mango kernel starch films as affected by starch nanocrystals and cellulose
nanocrystals

T

Ana Priscila M. Silvaa, Ana Vitória Oliveiraa, Sheyliane M.A. Pontesb, André L.S. Pereirab,
⁎
Men de sá M. Souza Filhoc, Morsyleide F. Rosac, Henriette M.C. Azeredoc,d,
a

Federal University of Ceara, Department of Chemical Engineering, Campus Pici, Bl. 709, 60455-760, Fortaleza, CE, Brazil
Federal University of Ceara, Department of Organic and Inorganic Chemistry, Campus Pici, Bl. 940, 60451-970, Fortaleza, CE, Brazil
c
Embrapa Agroindústria Tropical, R. Dra. Sara Mesquita, 2270, Fortaleza, CE, 60511-110, Brazil
d
Embrapa Instrumentaỗóo, R. 15 de Novembro, 1452, Caixa Postal 741, São Carlos, SP, CEP 13560-970, Brazil
b

A R T I C LE I N FO

A B S T R A C T

Keywords:
Biopolymers

Polysaccharides
Food packaging
Nanoparticles
Reinforcement

Mango seeds have been used to obtain components for nanocomposite films, namely, starch and starch nanocrystals (SNC) from seed kernels, and cellulose nanocrystals (CNC) from seed shells. Lignin was also recovered
from shells. Starch-based films were prepared with different contents and combinations of SNC and CNC. SNC
exhibited round-like rather than platelet-like morphology, and their effect on water vapor barrier was not as high
as that of the needle-like CNC. Also, CNC were more effective than SNC to increase elastic modulus. On the other
hand, CNC impaired more the elongation. The optimized conditions (1.5 wt% CNC and 8.5 wt% SNC on a starch
basis) resulted in a film with enhanced strength, modulus, and barrier to water vapor when compared to the
unfilled film, although the elongation has been impaired.

1. Introduction
In the last few decades, there has been an increasing focus on process and product development as part of a biorefinery approach, as well
as a transition from petrochemistry to bioeconomy. Those are aspects of
global trends aiming to reduce wastes (particularly non-biodegradable
wastes, but also food wastes) and to improve the economics of food
processing. Food processing byproducts can be converted into a variety
of chemicals, including biopolymers that represent alternatives for
fossil based polymers for some applications, including food packaging.
Mango is an important tropical fruit, very appreciated for its peculiar flavor. The world mango production in 2016 has been about 46.5
million metric tons (Statista, 2018),‘Tommy Atkins’ being the most
produced mango variety (UNCTAD, 2016). Mangoes have been recently
suggested as potential feedstocks for integrated biorefineries (Arora,
Banerjee, Vijayaraghavan, MacFarlane, & Patti, 2018; Banerjee et al.,
2018; Matharu, Houghton, Lucas-Torres, & Moreno, 2016). The mango
seed (stone) is a major by-product of mango processing, representing
about 22% of the weight of Tommy Atkins mangoes (Plant-O-Gram,
2015); it contains a kernel containing more than 50% starch by weight

(Kaur, Singh, Sandhu, & Guraya, 2004), and a shell whose major
component (more than 50%) is cellulose (Henrique, Silvério, Flauzino
Neto, & Pasquini, 2013).
⁎

Starch has a variety of food and non-food applications.
Conventional edible starch sources such as corn, potato, and cassava are
usually of choice for food applications, whereas non-edible sources such
as food byproducts have been frequently preferred for non-food applications, in order to avoid issues around food versus non-food competition, as well as to allow sustainable use of resources (Persin et al.,
2011). Starch has been frequently used for production of biodegradable
films for food packaging purposes. However, since the tensile and
barrier properties of starch films are usually poor, many studies have
focused on the use of nano-reinforcements to improve their properties.
Cellulose nanocrystals (CNC) have been widely studied and proven to
be effective to enhance the performance of starch films (Chen, Liu,
Chang, Cao, & Anderson, 2009; Slavutsky & Bertuzzi, 2014; TerrazasHernandez et al., 2015). The effects of starch nanocrystals (SNC) on
starch films have also been studied (Angellier, Molina-Boisseau, Dole, &
Dufresne, 2006; García, Ribba, Dufresne, Aranguren, & Goyanes, 2011;
Piyada, Waranyou, & Thawien, 2013), which are favored by the high
affinity provided by the identical nature of matrix and filler. A previous
study has aimed to compare SNC and CNC effects on thermoplastic
starch (González, Retegi, González, Eceiza, & Gabilondo, 2015), but
only at the 1 wt% reinforcement level.
Some recent studies have proposed mango kernel starch as a matrix
for films or coatings, usually without nanofillers (Nawab, Alam, Haq, &

Corresponding author at: Embrapa Instrumentaỗóo, R. 15 de Novembro, 1452, Caixa Postal 741, São Carlos, SP, CEP 13560-970, Brazil.
E-mail address: (H.M.C. Azeredo).

/>Received 7 November 2018; Received in revised form 2 February 2019; Accepted 3 February 2019

Available online 05 February 2019
0144-8617/ © 2019 Elsevier Ltd. All rights reserved.


Carbohydrate Polymers 211 (2019) 209–216

A.P.M. Silva et al.

2.3. Isolation of starch nanocrystals (SNC)

Hasnain, 2016, 2018). Our group have studied the properties of mango
kernel starch films as affected by SNC from mango kernel starch
(Oliveira et al., 2018), demonstrating that those nanocrystals have
improved tensile and barrier properties of the films. In this study, SNC
(from mango kernels) and CNC (from mango kernel shells) have been
combined as an attempt to further improve the physical properties of
mango kernel starch films, as well as to compare the effects of SNC and
CNC (both from mango seeds) on those films.

The production of starch nanocrystals (SNC) was based on an acid
hydrolysis procedure adapted from the method described by Angellier,
Choisnard, Molina-Boisseau, Ozil, and Dufresne (2004), as described by
Oliveira et al. (2018). 73.5 g of mango kernel starch were added to
500 mL of a mixture (volume ratio, 1:1) of HCl 3.16 M and H3PO4
3.16 M at 40 °C in a silicone bath under stirring (150 rpm) for 5 days.
The suspensions were then submitted to six centrifugation-sonication
cycles consisting of centrifugation (25,384 g, 20 min, 4 °C), homogenization of the precipitate in a Walita Billy mixer (Walita, São Paulo,
Brazil) for 1 min, sonication on a Unique Cell Disruptor (Unique,
Campinas, SP, Brazil) for 20 min at 450 W. The suspension was then
dialysed until pH 6.5, then homogenized with an Ultra-Turrax T50

(5000 rpm, 5 min), added with 0.01 vol% chloroform, and stored at
4 °C. The SNC yield was taken as the average yield from the three
batches, based on the initial dry weight of starch. The SNC suspension
was the mixture of the suspensions from the three batches, and its solid
content was determined by gravimetry after drying at 105 °C until
constant weight.

2. Materials and methods
2.1. Materials and reagents
Mangoes (Mangifera indica L. cv. ‘Tommy Atkins’) at ripening stage
3 (GTZ, 1992) were purchased at a local market (CEASA, Maracanaú,
CE, Brazil). Sodium hydroxide (NaOH), hydrochloric acid (HCl),
phosphoric acid (H3PO4), sulfuric acid (H2SO4), acetic acid (C2H4O2),
and hydrogen peroxide solution (H2O2 30 vol%) were purchased from
Sigma-Aldrich Ltd. (St. Louis, USA), and absolute ethanol
(C2H5O, > 99.9%), from Merck (Germany).

2.4. Processing of mango seed shells and isolation of cellulose nanocrystals
(CNC)
2.2. Isolation and characterization of mango kernel starch (MKS)
The processing of mango seed shells was carried out in three batches, and the yield of CNC and lignin were calculated as averages of the
values from those batches.

Mangoes were manually depulped. The starch isolation was adapted
from the method described in the previous study by our group (Oliveira
et al., 2018). The mango stones were decorticated, and the kernels were
immersed in distilled water. After all stones were decorticated, the
kernels were removed from water, cut into small pieces (about 1 cm3),
and immersed into sodium metabisulfite 0.2 wt% (kernel: solution
ratio, 1:2, wt/vol) for 48 h at 4 °C, again to avoid browning.

The kernels were then divided into three batches for further processing. The kernels were drained and ground in distilled water (kernel:water weight ratio, 1:1) with a cutter (R 502 VV., Roubot-Coupe,
Vincennes, France) for 3 min at 1750 rpm, the resulting suspension was
filtered in a 60 mesh sieve stirred (200 rpm) for 10 min. The retentate
was again ground in water in the cutter and filtered, and the procedure
was repeated until there was no retentate. The starch slurry was allowed to settle for 40 min, separated, added with a 0.05 M NaOH solution (at a 1:2 slurry/solution weight ratio) in order to remove soluble
fiber, and stirred at 200 rpm for 2 h at 25 °C. The suspension was then
centrifuged (20,000 g, 4 °C, 20 min) in a High-Speed Refrigerated
Centrifuge CR22GIII (Hitachi, Tokyo, Japan). The starch precipitate
was collected, repeatedly washed in distilled water (at a 1:2 precipitate/water weight ratio), stirred (200 rpm, 5 min), and centrifuged,
until the starch suspension reached pH 7. Absolute ethanol was added
to the suspension (2:1 ethanol/suspension volume ratio), which was
stirred (200 rpm, 1 h), then allowed to settle for 2 h at 25 °C. The decanted starch was washed with distilled water, vacuum filtered through
28 μm filter paper, allowed to dry in an oven (40 °C, 24 h), ground with
an analytical mill (Ika A11, Staufen, Germany), and stored in sealed
glass flasks. The starch yield was expressed as extracted starch to initial
raw kernels (wt%, on a dry basis), as an average value of the three
batches.
The starch was analyzed for ashes (ASTM E1755-01, 2015ASTM
E1755-01, 2015) and proteins (Bradford, 1976) in triplicate. The
amylose content was measured according to the standard method ISO
(6647)-1 (2015), also in triplicate. A scanning electron micrograph
(SEM) was obtained from a starch powder placed on a stub and coated
with a 20 nm layer of gold by using a Quorum QT150ES metallizer
(Quorum, Laughton, UK). The micrograph was taken with a Quanta 450
FEG-FEI (Quanta, Hillsboro, USA), with an acceleration voltage of
15 kV, viewed at 2000× magnification. The starch granule sizes were
taken as averages of 50 measurements taken by using the software
ImageJ.

2.4.1. Isolation of CNC

The CNCs were obtained from previously dried mango seed shells
(in an oven at 50 °C until constant weight). The dried shells were
ground in a Willye knife mill (STAR FT-80, Fortinox, Piracicaba, Brazil)
with a 10 mesh (2.0 mm) sieve, forming the mango shell powder (MSP).
The MSP was then submitted to acetosolv pulping. The acetosolv
solution (93 wt% acetic acid and 0.3 wt% hydrochloric acid in distilled
water) was added to MSP at a 10:1 wt-to-volume ratio, and the mixture
was cooked under reflux at 120 °C with stirring (150 rpm) for 90 min in
a flat-bottom flask in a mineral oil bath. The mixture was filtered (in a
28 μm mesh) to separate the black liquor from the mango shell acetosolv pulp (MSAP). The MSAP was washed with acetic acid (99.7%) at
80 °C until the washing liquid was colorless and dried (60 °C, 24 h).
Bleaching was then carried out. For each 10 g of MSAP, 200 mL
NaOH 4% (w/v) were heated to 40 °C, when the AP was added under
stirring. When the temperature of the mixture reached 65 °C, 60 mL
H2O2 30 vol% were added. After 60 min at this same temperature, more
60 mL H2O2 30 vol% were added, and the reaction was kept for more
90 min. The material was then vacuum-filtered, washed with distilled
water until its pH was equal to that of the washing water, and ovendried at 50 °C until constant weight, producing the bleached acetosolv
pulp (MSBAP).
The acid hydrolysis method used to obtain the CNC was based on a
mixture of sulfuric acid 11.3 M with hydrochloric acid 8 M at a volume
ratio of 3:1, which was defined from a series of preliminary tests. The
acid solution (200 mL) was stirred in a round-bottom flask immersed in
a 45 °C mineral oil bath. When the solution temperature reached 45 °C,
10 g of MSBAP were added, and the hydrolysis was carried out for
60 min under stirring. The reaction was quenched by adding cold (4 °C)
deionized water at a 1:3 volume ratio (suspension/water). The suspension was then centrifuged (26,400 g, 15 min, 20 °C), and the supernatant was discarded. Deionized water (500 mL) was added again,
and two more centrifugation cycles were conducted. The final precipitate was added again with deionized water, and the suspension was
sonicated with a Unique Cell Disruptor (60 W, in three 2 min cycles
interspersed with 1 min intervals). The suspension was then dialysed

against deionized water until neutrality, and the CNC suspension was
stored at 4 °C. The CNC yield was based on the initial dry weight of
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A.P.M. Silva et al.

MSBAP. The CNC suspension was the mixture of the suspensions from
the three batches, and its solid content was determined by gravimetry
after drying at 105 °C until constant weight.

samples and conditioned for 48 h under controlled temperature and
relative humidity (23 ± 1 °C, 50 ± 3% RH) before analyses.

2.4.2. Lignin recovery
The lignin-rich black liquor (obtained as a byproduct of the CNC, as
described in 2.4.1) was concentrated in a rotary evaporator (R-210/
215, Buchi, Flawil, Switzerland) at 65 °C for lignin recovery. The concentrated black liquor was diluted 10 times in hot (∼80 °C) distilled
water, and kept for 24 h. The mixture was then vacuum filtered (with
8 μm filter paper), washed with distilled water until neutrality, and
dried at 50 °C in air-circulating oven until constant weight. The resulting material was then ground to a dark powder (lignin) with an
analytical mill (Ika A11, Staufen, Germany). The lignin content (purity)
of the powder was determined according to TAPPI T222 om-22 (2000),
and the lignin yield was calculated on an MSP basis.

2.7. Film characterization
2.7.1. Moisture content
The moisture contents of the different films were measured by using

a Marte ID50 infrared scale (Marte, São Paulo, SP, Brazil).

2.7.2. Tensile tests
Tensile properties of 125 mm × 12.5 mm film strips (with at least
five replicates) were measured according to D882-12 (ASTM, 2012),
using a Universal Testing Machine (Emic DL-3000, São José dos Pinhais, Brazil) with a load cell of 100 N, initial grip separation of 100 mm,
and crosshead speed of 12.5 mm/min. Before the test, the sample
thicknesses were measured with an Akrom KR1250 coating thickness
tester (Akrom, São Leopoldo, RS, Brazil) to the nearest 1 μm.

2.5. Characterization of SNC and CNC
SEM micrographs were obtained from starch granules and 2 wt%
SNC and CNC suspensions previously sonicated (Unique Cell Disruptor,
50 W, 1 min), placed onto Formvar coated grids, and stained with 1%
phosphotungstic acid. The nanostructures were examined using a
Tescan Vega 3 microscope (Tescan, Brno, Czech Republic) equipped
with a STEM detector at an acceleration voltage of 30 kV. The SNC and
CNC average dimensions were taken as an average of 100 measurements taken by using the software Gimp 2.8.
The zeta potentials of SNC and CNC were measured in triplicate
using a Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire,
UK), for suspensions diluted to 0.05 wt% with deionized water.
X-ray diffraction (XRD) analyses were carried out on MKS, SNC, and
CNC by using a DMAXB diffractometer (Rigaku, Japan), using CuKα
radiation, operated at 40 kV and 40 mA, at the 2ϴ range of 3–40°, with
a step size of 0.05°, and scanning speed of 1° min−1. The diffractograms
were deconvoluted by using Origin 9.0 software, and the crystallinity
indexes (CrI) were calculated as the ratio between the sum of areas
under the curves corresponding to the crystalline peaks and the sum of
the areas of all deconvoluted peaks.
Fourier-transform infrared spectroscopy (FTIR) spectra of MKS,

SNC, and CNC were recorded in a Schimatzu Prestige-21 spectrophotometer, using KBr pellets, in the wavenumber range from 4000 to
400 cm−1, with a resolution of 4 cm−1 over 64 scans.

2.7.3. Water vapor permeability (WVP)
The water vapor permeability (WVP) determination, with at least
five replicates, was based on the method E96/E96M-16 (ASTM, 2016)
at 24 °C, using 2 mL distilled water inside permeation cells (inner diameter, 24 mm; height, 10 mm) and silica gel as the desiccant material,
in an Arsec DCV040 desiccator with air circulation (Arsec, Vargem
Grande Paulista, SP, Brazil). Eight measurements were made within
24 h. Before the test, the sample thicknesses were measured with an
Akrom KR1250 coating thickness tester (Akrom, São Leopoldo, RS,
Brazil) to the nearest 1 μm.
2.7.4. Fourier transform infrared (FTIR)
Fourier-transform infrared spectroscopy (FTIR) spectra were recorded in a Schimatzu Prestige-21 spectrophotometer, using KBr pellets, in the wavenumber range from 4000 to 400 cm−1, with a resolution of 4 cm−1 over 64 scans.

2.7.5. Scanning electron microscopy (SEM)
The scanning electronic microscopy (SEM) micrographs of the films
were taken using a QUANTA FEG-FEI 450 microscope. The samples
were mounted on the aluminum stub using carbon-coated double sided
adhesive tape, coated with a 20 nm-thick gold layer by using a Quorum
QT150ES metallizer, and examined using an accelerating voltage of
5 kV and a magnification of 2000 times.

2.6. Formation of bionanocomposite films
Twelve films were produced, according to a central composite design (CCD), with two variables: total nanofiller content (SNC + CNC,
from 0 to 6 wt% on the starch matrix) and degree of replacement of SNC
with CNC (from 0 to 100 wt%), as presented in Table 2 with the corresponding responses. Each film was made from 12.5 g mango kernel
starch previously gelatinized in 125 mL distilled water (95 °C, for
30 min, under stirring). Glycerol (3.13 g) was then added to the gelatinized starch, which was kept at 60 °C for 15 min under stirring, then
stabilized to 40 °C min (also under stirring). The SNC and/or CNC suspensions (in volumes according to the set concentrations for each

treatment), previously mixed and sonicated with a Unique USC-1400
bath sonicator (40 kHz, 135 W) in five 1-min sonication cycles interspersed with 30 s intervals, were then added to the gelatinized starch
dispersion, and the mixture was stirred for more 30 min at 40 °C. The
final film forming dispersion was then homogenized with an Ultra
Turrax T25 (Ika, Staufen, Germany) at 10,000 rpm for 30 min. Air
bubbles were removed under vacuum (using an MA 057/1 vacuum
pump, Marconi, Piracicaba, SP, Brazil), and the films were cast on
Mylar® substrates on leveled glass plates for a thickness of 1.5 mm. The
films were allowed to dry at room conditions (23 °C, 54% RH, 20 h).
After being detached from the Mylar surfaces, films were cut into test

2.7.6. Statistical analyses
Results of tensile tests and WVP were analyzed using the software
Minitab® (Minitab Inc., State College, PA, USA). Full quadratic models
were fit to the experimental responses by using the DOE (Design of
Experiments)/Response Surface analysis, and the regressions were
evaluated in terms of their determination coefficients (R2 values) and
the significance of their F values.
The optimized conditions were defined by using the Response
Optimizer function of the Minitab® software, which aims to identify a
combination of conditions that jointly optimizes a set of responses
(using maximizing, minimizing, or targeting goals for each response),
as required by the user, who establishes lower or upper as well as target
values. The optimization was based on the following combination:
maximizing all tensile properties (i.e., finding a proper balance among
them), while minimizing WVP. The definition of lower/upper and
target values for each response were based on the ranges obtained for
each one of them. The conditions set as optimum were experimentally
verified (with three repetitions), and the mean relative deviation
modulus (G) was calculated for each response.

211


Carbohydrate Polymers 211 (2019) 209–216

A.P.M. Silva et al.

granules presented oval or spherical shapes (Fig. 1), and the SNCs were
round like those reported for waxy maize SNCs (García et al., 2011).
The major X-ray diffraction peaks of both MKS and SNC (Fig. 1)
were observed at 2θ around 12°, 15°, a doublet one at 17–18°, and
another near 23°, a typical A-type starch pattern (Angellier et al., 2006;
Mukurumbira, Mariano et al., 2017). Although Le Corre et al. (2011)
has described that A-type starches typically produce square-like nanocrystals, the A-type mango kernel starch of this study produced roundlike nanocrystals, corroborating our previous study (Oliveira et al.,
2018). This may be due to the sonication applied on SNC isolation.
Indeed, the morphology of starch nanostructures was demonstrated to
depend on the method used to produce them, ultrasound generating
ellipsoidal nanoparticles instead of the platelet-like nanocrystals produced by acid hydrolysis (Haaj, Thielemans, Magnin, & Boufi, 2016).
The combination of acid hydrolysis and ultrasound used in this study
may explain the production of round-like nanocrystals. Some peaks of
SNC were sharpened when compared to those of MKS, which probably
results from the selective acid hydrolysis of the starch (Mukurumbira,
Mellem, & Amonsou, 2017). The CrI of the SNC was about 50% higher
than that of MKS, confirming the effectiveness of the hydrolysis in
partially removing the amorphous parts of starch.
The FTIR spectra of the MKS and SNC are almost identical (Fig. 2A),
except that the height ratio of the bands at 1045 and 1018 cm−1 increased in SNC, indicating that the ratio of crystalline to amorphous
starch has increased (Warren, Gidley, & Flanagan, 2016), as expected.
Also, the band at 2930 cm−1 (CeH region), which varies with the
amylose/amylopectin ratio (Kizil, Irudayaraj, & Seetharaman, 2002),

was lower in SNC, which is ascribed to decreased amylose content,
which was also expected, since amylopectin is the crystalline fraction.
Some bands are present in MKS and both kinds of nanocrystals, such as
those ascribed to antisymmetric CeOeC stretching of glycosidic links at
around 1160 cm−1 (Robert, Marquis, Barron, Guillon, & Saulnier,
2005), CeO stretch at 1034 cm−1 (Mohebby, 2008; Peng et al., 2012),
CeH and OeH bending at 1420–1430 cm−1 and 1317–1337 cm−1,
respectively (Peng et al., 2012).
The CNC yield (Table 1) was similar to the one reported from sugarcane bagasse pith (Oliveira, Bras, Pimenta, Curvelo, & Belgacem,
2016). The CNCs were needle-like (Fig. 1), with an average aspect ratio
(L/d) slightly higher than those reported for CNCs from cotton fibers
(Sapkota, Natterodt, Shirole, Foster, & Weder, 2017; Teixeira et al.,
2010), and good dispersability, as represented by their high zeta potential (Table 1). The XRD pattern of CNC (Fig. 1) exhibited two main
crystalline domains at 2Ɵ about 12° (plane 110) and 22° (plane 020)
(French, 2014; Santmartí & Lee, 2018).
The yield of lignin powder (on MSP) was 15%, while its purity was
89%, indicating that about 88% of the lignin in MSP (Table 1) was
recovered.

Table 1
Determinations on components from mango kernels and mango seed shell.
Determination

Average ( ± standard deviation)

MKS

38.8 ± 3.5
0.12 ± 0.02
0.02 ± 0.001

25.1 ± 1.95
10.2 ± 2.1
39.5
24.4 ± 2.7
67.1 ± 21.7
−24 ± 3
62.6
15.8 ± 2.1
270 ± 71
18 ± 5
15
−54 ± 4
80.3

Yield (wt%, on dry kernels)
Ashes (wt%)
Proteins (wt%)
Amylose (wt%)
Granule size (μm)
Crystallinity index (%)
Yield (wt% on starch)
Particle size (nm)
Zeta potential (mV)
Crystallinity index (%)
Yield (wt% on MSP)
Length (nm)
Diameter (nm)
Aspect ratio (L/d)
Zeta potential (mV)
Crystallinity index (%)

SNC

CNC

MKS: mango kernel starch; SNC: starch nanocrystals (from MKS); CNC: cellulose
nanocrystals (from MSP).

3. Results and discussion
3.1. Characterization of film components
The amylose content of MKS (Table 1) may be considered as
medium, when compared to those classified by Colussi et al. (2017).
Medium- to high-amylose starches are more suitable to form films, since
the mainly linear chains of amylose allow it to form more hydrogen
bonds when compared to amylopectin (Colussi et al., 2017). Also,
amylose tends to re-associate easier than amylopectin by forming
double-helix on retrogradation due to its linear structure (Zhu et al.,
2017). Starches with higher amylose contents result in films with better
tensile strength and barrier properties (Fu et al., 2018; Wang et al.,
2017), although more rigid and brittle (Biduski et al., 2018; Wang et al.,
2017). On the other hand, high-amylose starches should be avoided to
obtain starch nanocrystals, since nanocrystals are mostly derived from
amylopectin (Le Corre, Bras, & Dufresne, 2011). Thus, a medium
amylose may be regarded as suitable to obtain both starch and starch
nanocrystals for films.
The SNC yield from MKS was similar to those reported from amadumbe starch (Mukurumbira, Mariano, Dufresne, Mellem, & Amonsou,
2017) and waxy maize (Sanchez de la Concha et al., 2018). The MKS
Table 2
Experimental conditions and responses for film properties.
Film

Control
1
2
3
4
5
6
7
8
9
10
11

Replacement of SNC
with CNC (wt%)

Total nanofiller
content (wt%)

C

U

C

U

−1.41
−1

1
−1
1
−1.41
1.41
0
0
0
0
0

0
14.5
85.5
14.5
85.5
0
100
50
50
50
50
50

−1.41
−1
−1
1
1
0

0
−1.41
1.41
0
0
0

0
3.16
3.16
8.84
8.84
6
6
2
10
6
6
6

σ

ε

E

WVP

3.2. Characterization of films

13.05
14.37
16.25
16.72
19.77
15.50
18.04
13.47
25.10
25.41
23.50
25.66

17.72
10.71
8.78
10.66
5.43
11.92
7.35
15.31
2.53
3.50
3.44
3.28

1158
1181
1420
1311

1751
1247
1577
1214
1967
1902
1848
1898

The tensile properties and water vapor permeability of the films are
shown in Table 2. When compared to an MKS film containing just 5 wt
% SNC (from our previous study, Oliveira et al., 2018), the film #8 from
this study (containing 5 wt% SNC and 5 wt% CNC) presented higher
tensile strength and modulus, corroborating the higher effectiveness of
combined SNC and CNC on tensile properties when compared to SNC
alone.
Fig. 3 presents the contour plots of the models, while Table 3 presents the regression coefficients and statistical evaluation, indicating
that all the regressions were significant.
The replacement of SNC with CNC resulted in decreasing WVP
(reflected by a significant and negative regression coefficient, Table 3)
and increasing modulus, indicating that CNC were more effective than
SNC to increase stiffness and water vapor barrier of the films.
The higher effect of CNC on modulus was also observed by González
et al. (2015), and is ascribed to the higher aspect ratio of CNCs, which

1.579
1.483
1.298
1.341
1.248

1.386
1.303
1.334
1.133
1.311
1.263
1.269

C: coded values, according to the central composite design, varying from −1.41
to 1.41 (corresponding to 0 to 100 wt% replacement of SNC with CNC, and from
2 to 10 wt% total nanofiller (SNC + CNC) content on films. U: uncoded (experimental) values; σ: tensile strength (MPa); ε: elongation at break (%); E:
elastic modulus (MPa); WVP: water vapor permeability (g mm h−1 kPa−1 m-2).
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A.P.M. Silva et al.

Fig. 1. SEM micrographs and XRD diffractograms of mango kernel starch (MKS), starch nanocrystals (SNC), and cellulose nanocrystals (CNC). Magnification of
micrographs: MKS: 2000×; SNC: 28,000×; CNC: 41,000×.

elongation has been impaired, as observed in previous studies with SNC
alone (Kristo & Biliaderis, 2007; Oliveira et al., 2018), CNC alone (El
Achaby et al., 2017; Pereira et al., 2017; Sukyai et al., 2018), and
combined CNC and SNC (González et al., 2015).
The FTIR spectrum of the film containing 5 wt% of each kind of
nanocrystals (film #8) exhibited some changes when compared to that
of the control film (Fig. 4). The 1043 cm−1 to 1022 cm−1 height ratio
increased with the addition of nanofillers, which is ascribed to the increasing crystalline to amorphous starch ratio (Htoon et al., 2009;

Warren et al., 2016). The band at 1155 cm−1 (antisymmetric CeOeC
stretching of glycosidic links, according to Robert et al., 2005) had its
intensity increased with the addition of nanofillers, as previously reported from CNC addition to alginate films (Huq et al., 2012); the
presence of charged sulfate ester bonds on CNC resulting from sulfuric
acid hydrolysis may have contributed to this band (Zhao, Zhang,
Lindström, & Li, 2015). Other bands were increased with the addition of
nanocrystals, such as those at 1462 cm−1 (CH2 bending), 1423 cm−1
(CH2 scissoring), 1202 cm−1 (OH bending, probably increased by the
S]O vibration on CNC), 1111 cm−1 (ring breathing of CeOeC), and
931 cm−1 (out-of-plane bending of OeH).
The unfilled MKS film (Fig. 5, A1 and A2) presented a relatively
smooth surface, with a good transparency. On the other hand, the film
with 5 wt% CNC and 5 wt% SNC (Fig. 5, B1 and B2) was rougher and
more opaque, which is ascribed to aggregations of nanocrystals, which
was also noticed previously on starch films added with SNC (Jiang, Liu,
Wang, Xiong, & Sun, 2016; Li et al., 2015; Oliveira et al., 2018) or CNC
(Johar & Ahmad, 2012). None of the films exhibited cracks or discontinuities, and the signs of nanocrystal aggregation were not reflected
in impaired tensile and barrier properties; on the contrary, those
properties were improved by those nanocrystals (Table 2, Fig. 3).

Fig. 2. FTIR spectra of: MKS: mango kernel starch; SNC: starch nanocrystals;
CNC: cellulose nanocrystals.

results in higher mechanical reinforcement (González et al., 2015;
Wang, Tian, & Zhang, 2010). Although the effects of replacing SNC with
CNC on strength and elongation were not significant, their values may
be interpreted as a tendency of CNC to be more effective than SNC on
enhancing film strength, but also less favorable to elongation.
The better effectiveness of CNC (when compared to SNC) on barrier
properties would be surprising if the SNC had presented platelet-like

morphology, such as those from waxy maize starch (González et al.,
2015; Putaux, Molina-Boisseau, Momaur, & Dufresne, 2003), which
would be expected to create a more tortuous diffusion path for the
permeants (González et al., 2015; Le Corre, Bras, & Dufresne, 2010; Le
Corre, Bras, & Dufresne, 2012). On the other hand, the round-like
morphology presented by the SNC in this study (Fig. 1) partly explains
why this was not the case. Moreover, the barrier properties do not
depend only on the diffusion, but also on polarity and structural features of the polymer (González et al., 2015), and the lower crystallinity
of SNC when compared to CNC (Table 1) could be a major reason for
their lower effectiveness on decreasing WVP.
Increasing the total nanofiller (SNC + CNC) content resulted in increased strength and modulus and decreased WVP, although the

3.3. Optimization
The optimization was based on the following criteria: (a) tensile
strength: goal, maximizing; lower, 14 MPa; target, 16 MPa; (b) elastic
modulus: goal, maximizing; lower, 1200 MPa; target, 1400 MPa; (c)
elongation at break: goal, maximizing; lower, 9%; target, 12%; (d)
WVP: goal, minimizing; upper, 1.5 g mm kPa−1 h−1 m−2; target,
1.3 g mm kPa−1 h−1 m−2. The optimized conditions (with a composite
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A.P.M. Silva et al.

Fig. 3. Contour plots for tensile properties and water vapor permeability of films.

desirability of 0.96) were thus established as 10 wt% total nanocrystals,
with 15% replacement of SNC with CNC, that is to say, 1.5 wt% CNC

and 8.5 wt% SNC. The predicted responses at those conditions, as well
as the corresponding experimental responses and mean relative deviation modulus (G), are presented at Table 4. The strength of the film
obtained at those conditions was well above the lowest acceptable
value for food packaging (4 MPa, according to Tajeddin, Rahman, &
Abdulah, 2010), and its elongation was about 10%, which has been
considered by Khwaldia, Banon, Desobry, and Hardy (2004) as the
minimum value required for most industrial applications of edible
films. The mean relative deviation modulus (G) values of all models
(Table 4) were between 5 and 10, indicating reasonably good predictions (Roy, Gennadios, Weller, & Testin, 2000).

Table 3
Regression coefficients (in coded values) for film properties.
Term

WVP

σ

ε

E

Constant
x
y
x2
y2
x.y
R2 (%)
F

p

1.281
−0.050
−0.059
0.045
−0.010
0.023
85.19
5.75
0.04

24.85
1.066
2.789
−4.352
−3.098
0.292
90.82
9.90
0.01

3.408
−1.703
−2.684
3.019
2.661
−0.825
84.99
5.66

0.04

1882
143.3
190.6
−256.8
−167.6
50.37
92.93
13.14
< 0.01

WVP: water vapor permeability; σ: tensile strength; ε: elongation at break; E:
elastic modulus. x: replacement of SNC with CNC; y: total nanofiller
(SNC + CNC) content on films. Regression terms in bold are significant
(p < 0.05).

4. Conclusions
Starch and starch nanocrystals (SNC) were obtained from mango
kernels, and cellulose nanocrystals (CNC), from mango seed shells.
Mango kernel starch films were prepared with different combinations of
SNC and CNC. When compared to SNC, CNC were more effective to
enhance overall tensile properties and water vapor barrier of the films,
whereas SNC tended to exhibit a lower effect on decreasing elongation.
The optimized conditions (1.5 wt% CNC and 8.5 wt% SNC on a starch
basis) resulted in a film with enhanced strength and modulus (30% and
17% higher respectively) and water vapor barrier (22% lower permeability) than the unfilled film, although the elongation has been reduced by 40% (but still with a value suitable for edible film applications). The nanocomposite mango kernel starch films may be used for a
variety of food applications as renewable and biodegradable, even edible, films.
Acknowledgements

Fig. 4. FTIR spectra of mango kernel starch films: unfilled (control) and with
5 wt% CNC and 5 wt% SNC (film #8).

The authors gratefully acknowledge the financial support of the
Brazilian
Agricultural
Research
Corporation
(Embrapa,
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A.P.M. Silva et al.

Fig. 5. SEM micrographs and pictures of mango kernel starch films. (A1) and (A2): unfilled (control) film; (B1) and (B2): film #8 (with 5 wt% CNC and 5 wt% SNC).
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Table 4
Predicted and experimental responses obtained from the optimized conditions

(1.5 wt% CNC and 8.5 wt% SNC).
Responses

Predicted

Experimentala

G

σ (MPa)
ε (%)
E (MPa)
WVP (g mm kPa−1 h−1 m−2)

16.87
10.73
1353
1.238

16.68 ± 1.400
10.35 ± 0.593
1299 ± 57.55
1.265 ± 0.091

8.52
7.18
6.33
7.16

G: relative deviation modulus.

a
Averages from at least four replicates of 3 repetitions.

02.14.04.002.00.00). They also thank the Central Analítica-UFC/CTINFRA/MCTI-SIS-NANO/Pró-Equipamentos CAPES for the use of their
facilities for the SEM analyses, the X-Ray Laboratory at Federal
University of Ceara for the XRD analyses (National Council for
Scientific and Technological Development, CNPq, 402561/2007-4), as
well as Celli R. Muniz (Embrapa Agroindústria Tropical) for the SEM
micrographs of starch nanocrystals. We thank the Coordination for the
Improvement of Higher Education Personnel (CAPES, 1376677 and
2017SLR-17925) and Fundaỗóo Cearense de Apoio ao Desenvolvimento
Cientớco e Tecnolúgico (FUNCAP, BMD-0008-00640.01.11/15) for the
scholarships granted to A.V. Oliveira, A.L.S. Pereira, and A.P.M. Silva
respectively. The authors also acknowledge CNPq for the Research
Productivity Fellowships granted to M.F. Rosa and H.M.C. Azeredo
(305504/2016-9 and 302381/2016-3 respectively).
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