Food Hydrocolloids 57 (2016) 132e138

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

A fast and efcient approach to prepare starch nanocrystals from

normal corn starch
Asad Mohammad Amini, Seyed Mohammad Ali Razavi*
Food Hydrocolloids Research Centre, Department of Food Science and Technology, Ferdowsi University of Mashhad (FUM), POBox: 91775-1163, Mashhad,
Iran

a r t i c l e i n f o a b s t r a c t

Article history: In present study, simultaneous treatment of corn starch by ultrasound and sulphuric acid hydrolysis was
Received 22 October 2015 investigated focussing a reduction in starch nanocrystal preparation time while increasing the yield.
Received in revised form Different acid/starch concentrations, temperatures, and time durations were assessed through degree of
24 January 2016
hydrolysis, yield, particle size, and surface charge. It was observed that the method is capable of pro-
Accepted 27 January 2016
Available online 30 January 2016
ducing nanocrystals within 45 min with particle size smaller than 100 nm and up to 21.6% yield. The
surface charge of nanocrystals was observed to be increased at elevated levels of independent param-
eters. Round edge nanocrystals with diameters smaller than 50 nm were observed under TEM. The re-
Keywords:
Crystallinity
sults revealed that sonication during nanocrystal preparation not only preserved the crystalline
Hydrolysis structure, but also reasonably increased the efciency of hydrolysis to produce starch nanocrystals. The
Nanotechnology results of the present study would be of great importance to adopt large scale production of nanocrystals
Polydispersity index from starch.
Starch 2016 Elsevier Ltd. All rights reserved.
Ultrasound
Zeta potential

1. Introduction hinder the industrial production and applicability of SNCs, there-

During the last decade, starch nanocrystals (SNCs) have been the
subject of invaluable studies due to their abundance, biodegrad-
ability and biocompatibility. Over the years, the preparation pro-
cess of SNCs has been evolved considerably, most evidently by
Angellier, Choisnard, Molina-Boisseau, Ozil, and Dufresne (2004).
The preparation methods and applications of starch nanocrystals
have been reviewed extensively (Dufresne, 2014; Hu, Fu, Huang,
Anderson, & Chang, 2015; Kim, Park, & Lim, 2015; Le Corre &
Angellier-Coussy, 2014; Le Corre, Bras, & Dufresne, 2010; Lin,
Huang, Chang, Anderson, & Yu, 2011; Lin, Huang, & Dufresne, 2012;
Mary, Pothan, & Thomas, 2013). From the literature, it was evident
that the most commonly used protocols in preparing nanocrystals
from starch are mainly based on acid hydrolysis because of its
simplicity and controllability, but there are some issues associated
with the method; acid hydrolysis is time consuming and the yield
or recovery of SNCs are low (Kim et al., 2015; LeCorre, Bras, &
Dufresne, 2011a; Le Corre, Bras, Choisnard, & Dufresne, 2012;
Romdhane, Aurousseau, Guillet, & Mauret, 2015a). These issues
* Corresponding author.
E-mail address: s.razavi@um.ac.ir (S.M.A. Razavi).
fore, considerable efforts have been conducted in recent years to
overcome the mentioned issues (Jin et al., 2015; Kim, Park, & Lim,
2008; LeCorre et al., 2011a, 2012a; LeCorre, Vahanian, Dufresne, &
Bras, 2012b; Romdhane et al., 2015a).
Recently, the application of ultrasound treatment to disperse or
break the aggregates of SNC and/or to produce nanoparticles from
starch has been the subject of some studies. In 2008, Kim et al.
hydrolysed waxy rice starch using a-amylase enzyme followed by
sonication in ethanol. The authors reported that sonication
increased the particle size and decreased the X-ray diffraction in-
tensity. Kim, Han, Kweon, Park, and Lim (2013) applied ultrasound
on waxy maize starch hydrolysates in two different ways and
concluded that sonication increased the yield of starch nano-
particles, but with loss of crystallinity. In another study, Kim, Park,
Kim, and Lim (2013) prepared SNCs from waxy maize starch by
performing hydrolysis at 4
C followed by sonication. Bel Haaj,
Magnin, Petrier, and Bou (2013) prepared nanoparticles with
low crystallinity or amorphous nature from normal and waxy
maize starch using ultrasound treatment. Using ultrasound-
assisted oxidation method, starch nanoparticles have been pre-
pared from waxy maize starch with low crystallinity (Sun, Fan, &
Xiong, 2014). Goncalves, Norena, da Silveira, and Brandelli (2014)

http://dx.doi.org/10.1016/j.foodhyd.2016.01.022
0268-005X/ 2016 Elsevier Ltd. All rights reserved.
 A. Mohammad Amini, S.M.A. Razavi / Food Hydrocolloids 57 (2016) 132e138
treated pinhao seeds' starch by sonication and reported that
nanoparticles have been achieved without destructing the crys-
talline structure. More recently, Jin et al. (2015) reported that real-
time sonication increased the performance of cross-ow ultral-
tration system for waxy maize starch SNCs while the structure of
SNCs has been preserved.
To the best of our knowledge, most of the published works
focused on dispersing the previously prepared SNCs and/or fabri-
cating starch nanoparticles which barely were crystalline and there
is no published work regarding the production of nanocrystals by
simultaneous acid hydrolysis and ultrasound treatment. Therefore,
the objectives of the present study were: i) to investigate the in-
uence of coupling sonication and sulphuric acid hydrolysis on
yield, particle size and homogeneity, crystallinity, and surface
charge of nanocrystals from native corn starch, and ii) to shorten
the preparation time of SNCs to less than an hour. In order to
compare the results, the two optimal acid and starch concentration
described in the literature were used; i.e. 15% starch slurry in 3.16 M
acid solution (Angellier et al., 2004), and 40% starch in acid solution
with 4.5 M concentration (Le Corre, Bras et al., 2012). Other studied
variables were sonication temperature (10, 20, and 40
C) and
exposure time (15, 30, and 45 min).
2. Materials and methods
2.1. Materials
Normal corn starch with 27.4 1.1% amylose content and sul-
phuric acid were purchased from Sigma (St. Louis, USA) and Merck
(Darmstadt, Germany), respectively. The ltration of starch hy-
drolysates were performed using MN-619de lter paper (Macher-
eyeNagel GmbH & Co., Germany) with an average retention
capacity of 1e2 mm. At all preparations deionised water was used,
unless otherwise specied.
2.2. Nanocrystals preparation procedure
The acid hydrolysis procedure to produce SNC was simulta-
neously performed during continuous sonication using a 40 kHz
QuickSonic bath-type ultrasound apparatus (Schder-Schall Co.,
Germany) with 350 W nominal power. Initially, the temperature
inside ultrasound bath was adjusted to appropriate values (10, 20,
and 40
C), then, dispersions of starch with 15 and 40% (w/w)
concentration were prepared in 3.16 and 4.5 M sulphuric acid so-
lutions, respectively. The dispersions were placed immediately at a
height of 20 cm from transducer and submitted to sonication at
100% amplitude for 15, 30, and 45 min. The temperature inside the
bath was traced real-time using digital thermometer (0.01
C) and
adjusted to specied value by replacing with appropriate amount of
ice water to prevent temperature rise during operation. Treated
samples were washed successively until neutrality, ltered by lter
paper (1e2 mm), lyophilised and placed in air-tight containers
before performing the experiments. The degree of hydrolysis and
SNC yield were calculated as the percent ratio of dissolved solids
and hydrolysis residues (after ltration) based on the initial starch
weight, respectively.
2.3. Particle size analysis
Particle size measurements on nanocrystals were performed at
25
C using a Vasco-3 particle size analyser (Cordouan Technolo-
gies, France) after 30 s homogenisation with ultrasound bath. A
value of 1.35 was used as refractive index and particle size distri-
bution of samples were characterised based on cumulants and
Pade-Laplace methods. Also, the polydispersity index (PDI) was
133
measured as the ratio of the square of particle size standard devi-
ation to mean particle size. At least three independent measure-
ments were performed and the data were averaged.
2.4. Zeta potential measurement
Zeta potential of nanocrystals, z, was measured based on micro-
electrophoresis technique using a ZetaCompact zetameter (CAD
Instruments, France) at pH 7.0 0.1 and 25
C. The data were the
mean of at least three replicates.
2.5. Degree of crystallinity
X-ray diffraction (XRD) pattern of different samples treated for
45 min were recorded using a Bruker D8 Advance spectrometer
(Bruker AXS GmbH, Germany) operating at Cu-Ka wavelength,
target voltage and current of 1.541874 , 40 kV and 40 mA,
respectively. The scans were performed in diffraction angle (2q)
range of 4e40
at a step size of 0.04
. Relative crystallinity was
calculated based on the method described by Hermans and
Weidinger (1948).
2.6. Morphology
Scanning electron microscopy (SEM) micrographs of starch hy-
drolysates before ltration were recorded using LEO 1450VP mi-
croscope (LEO, Germany). The samples were sprinkled onto double-
sided adhesive tapes attached to aluminium stubs, coated for 60 s
with gold-palladium using SC7620 sputter coater (Quorum Tech-
nologies, UK) and photographed in microscope at an accelerating
voltage of 20 kV.
Transmission electron microscopy (TEM) was performed using a
Philips CM120 microscope (Philips, Netherlands) with an acceler-
ating voltage of 80 kV. A drop of nanocrystal suspension was
deposited onto a carbon coated copper grid, negatively stained
with a 2% aqueous solution of uranyl acetate and the image was
recorded on a Kodak SO-163 lm.
2.7. Statistical analyses
The experimental data were analysed through GLM procedure
of SAS software version 9.1 (SAS Inc., USA). Statistical signicance of
samples through mean comparisons were conducted using Dun-
can's multiple range test at a condence level of 95%.
3. Results and discussion
3.1. Degree of hydrolysis
The degree of hydrolysis (DOH) of corn starch samples in rela-
tion to different levels of temperature, exposure time, and acid/
starch concentrations are illustrated as Fig. 1a & b. As it can be seen,
by an increase in treatment time from 15 to 45 min at 10
C, the
DOH increased signicantly (p < 0.05) from 6.9 to 18.7% and from
4.7 to 16.3% for samples treated in 3.16 (Fig. 1a) and 4.5 M acid
(Fig. 1b), respectively. At treatment temperature of 20
C, the DOH
of samples treated in 3.16 M acid was increased signicantly from
10.5 to 21.1% by an increase in exposure time from 15 to 45 min
(Fig. 1a), while it was increased to a lower extent (8.1e18.6%) in
4.5 M acid solution (Fig. 1b). By elongation of treatment, the DOH of
samples treated at 40
C in 3.16 and 4.5 M concentrations raised
prominently from 21.6 to 53.9% and 15.4e39.8%, respectively
(Fig. 1a & b).
The results revealed that the efciency of hydrolysis was
strongly temperature dependent, so that the DOH prominently
134 A. Mohammad Amini, S.M.A. Razavi / Food Hydrocolloids 57 (2016) 132e138
Fig. 1. The variations in degree of hydrolysis as a response to different ultrasound exposure time and temperatures; (a) 15% starch in 3.16 M acid solution, (b) 40% starch in 4.5 M
acid solution. The error bars are standard deviation and different letters represent statistical signicance (p < 0.05).
increased by raising temperature from 10 to 40
C at similar time
and concentrations. It is generally accepted that the hydrolysis of
starch by acid is temperature dependent, in which higher rates of
granule degradation will be achieved at higher temperatures (Kim,
Park et al., 2013; Le Corre, Bras et al., 2012). By contrast, the ef-
ciency of ultrasound treatment decreases at higher temperatures
because it is believed that the increased vapour pressure of solvent
at elevated temperatures diminish the magnitude of shear forces
generated upon the collapse of cavitation bubbles (Caruso et al.,
2009; Mohammad Amini, Razavi, & Mortazavi, 2015). Therefore,
the increased DOH by raising temperature will be expected by
considering the sole effect of acid. In the situation that there is an
opposite temperature dependence for ultrasound and acid, the
direct trend of DOH with temperature could be explained by the
inuence of temperature on solution properties so that probably,
higher treatment temperatures reduced the viscosity facilitating
cavitation in solution, and on the other hand, the dissociation of
acid increased by the combined effect of ultrasound and tempera-
ture rise because the mobility and activity of H strongly depends
on temperature (Hoover, 2000; Utrilla-Coello et al., 2014).
Generally, it was observed that the DOH of samples treated in
3.16 M acid concentration were signicantly higher compared with
those hydrolysed by 4.5 M acid (Fig. 1). This could be attributed to
the lower starch concentration in samples treated by 3.16 M acid
solution compared with 40% starch concentration for samples
hydrolysed in 4.5 M acid. Furthermore, the efciency of ultrasound
treatment is believed to be dependent on concentration, so that the
higher concentration reduces the free solvent available to form
cavitation, resulting in suppression of shear forces induced by the
explosion of cavitation bubbles (Caruso et al., 2009; Czechowska-
Biskup, Rokita, Lotfy, Ulanski, & Rosiak, 2005).
The extent of hydrolysis obtained after treatment beyond
30 min at 40
C at the present study were close to the values re-
ported by Wang, Blazek, Gilbert, and Copeland (2012) after 6 days
of hydrolysis but lower than those obtained by Goncalves et al.
(2014) after 5 days of starch hydrolysis.
3.2. SNC yield
Based on the results, by raising temperature from 10 to 40
C,
the yield of SNC increased signicantly (p < 0.05) at all time
durations and acid/solution concentrations, except samples treated
for 15 min in 4.5 M acid at 10
C and 20
C (Fig. 2a & b). Considering
the results of DOH (Section 3.1), a higher portion of amorphous
lamellae of starch granules has been degraded at elevated tem-
peratures facilitating the extraction of crystallites observed as an
increased SNC yield. The results are in accordance with the
increasing trend of SNC extraction by elevating temperature as
reported by Le Corre, Bras et al. (2012), whereas, Kim, Park et al.
(2013) observed opposite trend.
Also, elongation of exposure time from 15 to 45 min enhanced
the extraction of SNC so that at similar conditions, samples treated
in 3.16 M acid were more evidently (p < 0.05) of higher SNC yield
(Fig. 2a) compared with those obtained by 4.5 M sulphuric acid
solution (Fig. 2b). In analogy, Le Corre, Bras et al. (2012) concluded
that the inuence of starch concentration on SNC yield has been
dispensable so that at temperatures below 30
C the yield has been
decreased by an increase in starch concentration, whereas, it had
no inuence at higher temperatures. The authors attributed the
observed trend to the fact that the hydrolysis has not been cata-
lysed at ambient temperatures, but at higher temperatures, the acid
existed in excess.
The values of SNC yield obtained after 30 min treatment and
beyond at 40
C in both acid concentrations in the present study
were generally higher than the values in the literature (Angellier
et al., 2004; Kim et al., 2008; Le Corre, Bras et al., 2012; LeCorre,
Vahanian, Dufresne, & Bras, 2012; Putaux, Molina-Boisseau,
Momaur, & Dufresne, 2003), but lower than the value (78%) re-
ported by Kim, Park et al. (2013) after 6 day of hydrolysis at 40
C.
It is believed that at early stages of acid hydrolysis, both amylose
and amylopectin in amorphous lamellae of granules will be
attacked by hydronium ion (H3O) of acid resulting in a random
cleavage of amylose and depolymerisation of amylopectin
(Ashogbon & Akintayo, 2014; Gao, Vasanthan, Hoover, & Li, 2012;
Hoover, 2000; Kim, Lee, Kim, Lim, & Lim, 2012; LeCorre, Bras, &
Dufresne, 2011b; Putaux et al., 2003; Utrilla-Coello et al., 2014).
Due to the similarities in the mode of action of ultrasound and acid
hydrolysis, i.e. solubilisation of amorphous lamellae of granules and
chain scission, it could be expected that simultaneous sonication
and acid hydrolysis will synergistically accelerate the inuence of
acid on starch resulting in more efcient extraction of crystallites
observed as an increase in SNC yield. Also, this may be due to the
 A. Mohammad Amini, S.M.A. Razavi / Food Hydrocolloids 57 (2016) 132e138 135

Fig. 2. The yield of corn starch nanocrystals as a function of varying sonication temperature and time for samples with (a) 15% starch and 3.16 M acid solution and (b) 40% starch and
4.5 M acid concentrations. Different letters on error bars (standard deviation) represent statistical signicance.

mechanical damage of granule surface by ultrasound which pre- hydrolysed fragments rather than aggregates of blocklets.
sumably facilitated the penetration of acid into granule interior. The samples obtained after treatment at 10
C in 3.16 M acid
showed two distinct particle size peaks which they came closer by
elongation of exposure time from 15 to 45 min. The same trend was
3.3. Particle size and homogeneity
observed for samples treated by 4.5 M acid at 10
C but particle size
values were signicantly higher compared with samples prepared
The results of particle size analysis for corn starch nanocrystals,
by acid solution with lower concentration. Treatment of samples at
characterised based on cumulants and Pade-Laplace methods, are
higher temperatures for 15 and 30 min in 3.16 M acid solution
presented in Table 1. Based on the results of Pade-Laplace method,
decreased the mean particle size and the size distributions were
it was observed that most of samples comprise a bimodal distri-
still bimodal but by further treatment to 45 min, a monomodal
bution with two distinct particle size peaks. Similar observations
distribution with particle size lower than 125 nm was observed
have been reported regarding the bimodal particle size distribution
(Table 1). In case of samples treated by 4.5 M acid, the particle size
(LeCorre et al., 2011a,b). The second peak has been attributed to the
distributions were still bimodal at 20
C at all exposure durations
aggregates (LeCorre et al., 2011a), while in the present study it may
but with reasonably smaller particle size values compared to their
be related to the fragments that were not fully hydrolysed. The
counterparts at 10
C. At 40
C, the same bimodal distribution of
mean particle size corresponding to both peaks decreased upon
particle size was observed, except samples hydrolysed for 45 min.
elongation of exposure time and elevation of temperature; this
Based on the results of cumulants particle size distribution
conrms that the second peak in present work represents non-

Table 1
The results of particle size analysis of corn starch nanocrystals.

Concentration Temperature Time Mean particle size (nm) Polydispersity index

Pade-Laplace method Cumulants method

First peak Second peak

3.16 M acid 15% w/v starch 10
C 15 776.5 18.4 (7.5%) 295.2 10.0 (92.5%) 294.2 7.7b 0.277 0.002a
30 662.4 24.6 (7.0%) 234.7 15.3 (93.0%) 223.6 6.0 de 0.209 0.018d
45 351.3 19.4 (2.5%) 173.9 5.7 (97.5%) 91.8 2.2 hi 0.175 0.023e
20
C 15 631.3 20.6 (5.5%) 229.2 7.5 (94.5%) 260.5 2.0c 0.261 0.037 ab
30 400.9 15.0 (3.5%) 174.2 17.0 (96.5%) 218.2 11.0e 0.190 0.003 de
45 e 125.0 5.9 (100%) 86.2 3.1i 0.170 0.076e
40
C 15 472.3 21.7 (6.0%) 138.1 4.5 (94.0%) 199.5 16.6f 0.147 0.012f
30 324.0 11.1 (8.0%) 131.1 3.8 (92.0%) 95.0 4.0 hi 0.104 0.023g
45 e 109.9 1.7 (100%) 82.9 2.0i 0.078 0.004h
4.5 M acid 40% w/v starch 10
C 15 1050.8 36.3 (9.5%) 472.3 11.7 (90.5%) 360.0 6.2a 0.280 0.047a
30 708.1 9.5 (6.0%) 248.1 15.1 (94.0%) 240.0 10.2d 0.242 0.013 bc
45 495.0 20.9 (4.0%) 210.4 14.1 (96.0%) 176.2 5.0g 0.241 0.015 bc
20
C 15 706.9 24.3 (6.0%) 251.9 14.6 (94.0%) 270.7 11.2c 0.262 0.153 ab
30 517.9 10.6 (4.5%) 195.2 7.7 (95.5%) 235.7 3.9d 0.207 0.013d
45 381.2 7.2 (1.0%) 135.1 8.8 (99.0%) 108.5 1.0h 0.183 0.013e
40
C 15 479.9 16.8 (6.0%) 162.2 9.0 (94.0%) 239.7 13.6d 0.231 0.001c
30 380.4 12.4 (6.0%) 137.2 5.3 (94.0%) 98.4 6.5 hi 0.133 0.021f
45 e 125.2 2.2 (100%) 82.2 16.9i 0.101 0.031g

The values are mean standard deviation and different letters in columns represent signicant statistical differences (p < 0.05). The values in parenthesis stand for the
percentage share of each peak from total size distribution.
136 A. Mohammad Amini, S.M.A. Razavi / Food Hydrocolloids 57 (2016) 132e138
(Table 1), it was observed that increasing temperature and expo-
sure time reduced mean particle size of SNCs. The differences be-
tween samples were signicant at exposure time of 15 min at all
temperatures and concentrations, while treatment of samples for
45 min barely changed particle size (82e92 nm) so that the dif-
ferences were not signicant except those prepared at 10 and 20
C
by 4.5 M acid. Again, smaller particle size was observed for SNCs
prepared by acid/starch solution of lower concentration.
Concluding, by an increase in temperature and time of hydrolysis,
mean particle size decreased for both types of solutions, more
efciently at lower acid/starch concentration.
Based on the results of particle size analysis, the size of samples
treated for 45 min in 3.16 M solution at all temperatures and for
samples obtained from 4.5 M solution at 40
C for 45 min were
more or less close to the values reported by others ranging from 40
to 150 nm (Angellier et al., 2004; Chen et al., 2008; Kim, Park et al.,
2013; Putaux et al., 2003; Zheng, Ai, Chang, Huang, & Dufresne,
2009).
The results of plydispersity index (PDI) for nanocrystals are
summarised in Table 1. Based on the results, it was revealed that PDI
decreased as temperature and treatment time increased, showing
that more homogenous SNC samples have been obtained, espe-
cially those prepared in 3.16 M acid at 40
C (p < 0.05). The results
conrmed that the dilute acid/starch solution enhanced the pro-
duction of a more homogenous SNC compared with treatment by
4.5 M acid/40% starch, so that the differences were signicant
except those prepared at 20
C.
It could be concluded that by elongation of treatment, the
produced fragments have been broken by the ultrasound to smaller
size particles, as evidenced by particle size distribution that was
changed from bimodal to monomodal. A similar conclusion on the
effect of ultrasound has been made by Kim, Park et al. (2013). On
the other hand, further treatment by ultrasound at 40
C beyond
30 min had no signicant inuence on particle size while increased
the homogeneity (lower PDI). This may conrm that sonication had
higher efciency on breaking the larger particles rather than
degradation of smaller size species, because the yield of SNCs were
observed to increase upon treatment progress (refer to Section 3.2)
while reducing the mean particle size.
3.4. Morphology
Based on SEM observations, it was revealed that simultaneous
treatment of corn starch by ultrasound and acid hydrolysis caused
granule disruption to more or less submicron fragments through
mechanical damage to granule surface and synergistically elimi-
nation of amorphous regions. The fragments resulting from the
action of acid and sonication are evident in Fig. 3a & b. Also, it is
clear that mechanical effect of microjets and/or hot-spots of ul-
trasound accelerated the inuence of acid by providing open
channels for acid to eliminate the amorphous growth rings and
granule hylum (Fig. 3c).
From TEM image of hydrolysates before ltration (Fig. 3d), it can
be inferred that the main effect of ultrasound and acid hydrolysis at
early stages is to liberate large fragments of crystalline lamellae of
granules through degradation of amorphous regions. This may
conrm the results of particle size analysis (see Section 3.3). After
progressive treatment and ltration of hydrolysates, TEM obser-
vations showed that SNCs with particle size lower than 50 nm can
be achieved in the shape of grape-like aggregates (Fig. 3e) or iso-
lated round edge particles (Fig. 3f). In contrast to the platelet-like
parallelepipedal morphology observed in case of waxy maize
SNCs (Angellier et al., 2004; Angellier, Molina-Boisseau, Belgacem,
& Dufresne, 2005; Putaux et al., 2003; Thielemans, Belgacem, &
Dufresne, 2006), some researchers reported round edge, polygon,
or irregular shape platelets (LeCorre, Bras, & Dufresne, 2011c; Le
Corre et al., 2010; Kim et al., 2012, 2015).
The observed particles in TEM images could be corresponded to
the amylopectin blocklets which are the building blocks of crys-
talline lamellae of granule because it has been reported that the
diameter of blocklets vary in the range of 20e500 nm based on the
botanical origin of starch; specically in the range of 10e30 nm for
normal corn starch (Baker, Miles, & Helbert, 2001; LeCorre et al.
2011a; Kim et al., 2012).
3.5. Zeta potential
It is widely accepted that the surface properties of SNCs are
dependent on the method utilised for fabrication, so that it has
been postulated that SNCs prepared by sulphuric acid hydrolysis
contain sulphate esters along with carboxyl groups, while sulphate
groups are absent in SNCs prepared by hydrochloric acid
(Romdhane, Aurousseau, Guillet, & Mauret, 2015b; Wei, Hu et al.,
2014; Wei, Xu, Jin, & Tian, 2014). In addition, it has been reported
that the majority charged groups on the surface of SNCs prepared
by sulphuric acid hydrolysis were the weak acidic, i.e. carboxylic
acid, groups which are independent of the hydrolysis time
compared with strong acidic groups of sulphate esters (Romdhane
et al., 2015b).
In present study, the surface charge of SNCs were characterised
by determining zeta potential (z) rather than measuring sulphate
content by conductometric method. The results revealed that
raising temperature of treatment signicantly increase the z of
SNCs produced by both acid concentrations (Fig. 4). Also, elonga-
tion of sonication exposure time caused an abrupt (p < 0.05) in-
crease in z of SNC samples, regardless of acid concentration. The
differences between acid concentrations were only signicant for
samples treated at 10 and 20
C for 45 min, while all levels of
exposure time at 40
C caused signicant differences (Fig. 4a & b).
The highest values of z were observed for samples prepared at
40
C, especially for samples with lower acid/starch concentration
(Fig. 4a).
It was observed that by increasing the extent of hydrolysis in
relation to an increase in independent parameters, the charged
groups exist on the surface of nanocrystals increased, as charac-
terised by higher absolute z values. Similar results have been re-
ported by Romdhane et al. (2015b). Therefore, the SNCs prepared
from 15% starch concentration by 3.16 M acid at 40
C could yield
more stable suspensions because of the higher surface charge
compared with other samples. The values of z in present study for
SNCs prepared at 40
C for 30 min and beyond varied from 18.4
to 31.9 mV which were comparable to the reported values (12
to 27 mV) measured at similar pH value for waxy maize starch
nanocrystals (Wei, Hu et al., 2014,Wei, Xu et al., 2014; Romdhane
et al., 2015a,b).
3.6. Crystallinity
Unlike starch nanoparticles, the most important characteristic
of SNCs is their crystallinity which is essential in applied point of
view, therefore, any degradation of crystalline structure during the
preparation process should be avoided. There are some evidence
that sonication disrupt the crystalline structure of SNCs (Bel Haaj
et al., 2013; Kim et al. 2008, Kim, Han et al., 2013,Kim, Park et al.,
2013), thus, to ensure that the crystallinity of SNCs has not been
diminished by sonication treatment, XRD analysis was performed
on samples with the highest level of ultrasound duration (45 min).
The results of crystallinity of SNC samples prepared at different
temperature and concentrations are presented in Fig. 5.
As it can be seen, elevating temperature from 10 to 40
C caused
 A. Mohammad Amini, S.M.A. Razavi / Food Hydrocolloids 57 (2016) 132e138 137

Fig. 3. SEM micrographs of corn starch granules sonicated in acidic medium (a, 10000X; b, 20000X; c, 30000X); TEM images of corn starch nanocrystals before (d, scale bar 100 nm)
and after ltration (e & f, scale bar: 50 nm).

Fig. 4. The zeta potential of corn starch nanocrystals as affected by sonication temperature and exposure time for samples prepared in: a) 3.16 M acid and 15% starch concentration,
and b) 4.5 M acid and 40% starch suspension. Different letters on error bars (standard deviation) represent signicant differences (p < 0.05).

an increase in crystallinity of SNC samples ranging from 28.1 to
36.6%. Also, the results revealed that samples prepared by lower
acid and starch concentration were more crystalline than those
prepared by 4.5 M sulphuric acid at the same conditions. The re-
sults clearly indicated that ultrasound treatment for 45 min had no
inuence on crystalline nature of SNC, unlike other reports (Bel Haj
et al., 2013; Kim et al. 2008, Kim, Han et al., 2013,Kim, Park et al.,
2013). In accordance with the present results, an increase in de-
gree of crystallinity after acid hydrolysis has been reported by
others (LeCorre et al., 2011c; Kim et al., 2012; Wang et al., 2012).
4. Conclusions
The present study focused on adopting a fast and efcient
method to prepare starch nanocrystals from normal corn starch
through simultaneous coupling of sulphuric acid hydrolysis and
ultrasound. Different parameters including starch and acid con-
centrations, temperature, and time duration of sonication were
assessed in relation to the yield, particle size, surface charge, and
crystallinity of nanocrystals. The results proved that by applying
ultrasound at higher temperatures it could be possible to prepare
nanocrystals in reasonable yield and short time comparable to the
available protocols in the literature. Through different character-
isations, it was conrmed that the prepared samples were in
nanometric size range with preserved crystalline structure. The
results of present study provide the opportunity to prepare starch
nanocrystals in appropriate time scale and yield for industrial
scales, especially when it has the applicability to be coupled with
other isolation methods such as membrane ltration to adopt
continuous operations needed by the industry.
138 A. Mohammad Amini, S.M.A. Razavi / Food Hydrocolloids 57 (2016) 132e138
Fig. 5. The variations in crystallinity of corn starch nanocrystals as affected by tem-
perature and concentration at sonication exposure time of 45 min.
References
Angellier, H., Choisnard, L., Molina-Boisseau, S., Ozil, P., & Dufresne, A. (2004).
Optimization of the preparation of aqueous suspensions of waxy maize starch
nanocrystals using a response surface methodology. Biomacromolecules, 5,
1545e1551.
Angellier, H., Molina-Boisseau, S., Belgacem, M. N., & Dufresne, A. (2005). Surface
chemical modication of waxy maize starch nanocrystals. Langmuir, 21,
2425e2433.
Ashogbon, A. O., & Akintayo, E. T. (2014). Recent trend in the physical and chemical
modication of starches from different botanical sources: a review. Starch/
rke, 66, 41e57.
Sta
Baker, A. A., Miles, M. J., & Helbert, W. (2001). Internal structure of the starch
granule revealed by AFM. Carbohydrate Research, 330, 249e256.
Bel Haaj, S., Magnin, A., Petrier, C., & Bou, S. (2013). Starch nanoparticles formation
via high power ultrasonication. Carbohydrate Polymers, 92, 1625e1632.
Caruso, M. M., Davis, D. A., Shen, Q., Odom, S. A., Sottos, N. R., White, S. R., et al.
(2009). Mechanically-induced chemical changes in polymeric materials.
Chemical Reviews, 109(11), 5755e5798.
Chen, G., Wei, M., Chen, J., Huang, J., Dufresne, A., & Chang, P. R. (2008). Simulta-
neous reinforcing and toughening: new nanocomposites of waterborne poly-
urethane lled with low loading level of starch nanocrystals. Polymer, 49(7),
1860e1870.
Czechowska-Biskup, R., Rokita, B., Lotfy, S., Ulanski, P., & Rosiak, J. M. (2005).
Degradation of chitosan and starch by 360-kHz ultrasound. Carbohydrate
Polymers, 60(2), 175e184.
Dufresne, A. (2014). Crystalline starch based nanoparticles. Current Opinion in
Colloid & Interface Science, 19(5), 397e408.
Gao, J., Vasanthan, T., Hoover, R., & Li, J. (2012). Structural modication of waxy,
regular, and high amylose-maize and hulless barley starches on partial acid
hydrolysis and their impact on physicochemical properties and chemical
modication. Starch/Sta rke, 64, 313e325.
Goncalves, P. M., Norena, C. P. Z., da Silveira, N. P., & Brandelli, A. (2014). Charac-
terization of starch nanoparticles obtained from Araucaria angustifolia seeds by
acid hydrolysis and ultrasound. LWT - Food Science and Technology, 58, 21e27.
Hermans, P. H., & Weidinger, A. (1948). Quantitative X-ray investigations on the
crystallinity of cellulose bers. A background analysis. Journal of Applied Physics,
19, 491e506.
Hoover, R. (2000). Acid-treated starches. Food Reviews International, 16, 369e392.
Hu, F., Fu, S., Huang, J., Anderson, D. P., & Chang, P. R. (2015). Structure and prop-
erties of polysaccharide nanocrystals. In J. Huang, P. R. Chang, N. Lin, &
A. Dufresne (Eds.), Polysaccharide-based nanocrystals: Chemistry and applications
(pp. 15e62). Weinheim: Wiley-VCH Verlag GmbH & Co. KGaA.
Jin, Y., Hengl, N., Baup, S., Pignon, F., Gondrexon, N., Sztucki, M., et al. (2015). Ul-
trasonic assisted cross-ow ultraltration of starch and cellulose nanocrystals
suspensions: characterization at multi-scales. Carbohydrate Polymers, 124,
66e76.
Kim, H.-Y., Han, J.-A., Kweon, D.-K., Park, J.-D., & Lim, S.-T. (2013). Effect of ultrasonic
treatments on nanoparticle preparation of acid-hydrolyzed waxy maize starch.
Carbohydrate Polymers, 93, 582e588.
Kim, H.-Y., Lee, J. H., Kim, J.-Y., Lim, W.-J., & Lim, S.-T. (2012). Characterization of
nanoparticles prepared by acid hydrolysis of various starches. Starch/Sta rke, 64,
367e373.
Kim, H.-Y., Park, D. J., Kim, J.-Y., & Lim, S.-T. (2013). Preparation of crystalline starch
nanoparticles using cold acid hydrolysis and ultrasonication. Carbohydrate
Polymers, 98, 295e301.
Kim, J.-Y., Park, D.-J., & Lim, S.-T. (2008). Fragmentation of waxy rice starch granules
by enzymatic hydrolysis. Cereal Chemistry, 85(2), 182e187.
Kim, H.-Y., Park, S. S., & Lim, S.-T. (2015). Preparation, characterization and utiliza-
tion of starch nanoparticles. Colloids and Surfaces B: Biointerfaces, 126, 607e620.
Le Corre, D., & Angellier-Coussy, H. (2014). Preparation and application of starch
nanoparticles for nanocomposites: a review. Reactive and Functional Polymers,
85, 97e120.
Le Corre, D., Bras, J., Choisnard, L., & Dufresne, A. (2012). Optimization of the batch
preparation of starch nanocrystals to reach daily time-scale. Starch/Sta rke, 64,
489e496.
Le Corre, D., Bras, J., & Dufresne, A. (2010). Starch nanoparticles: a review. Bio-
macromolecules, 11, 1139e1153.
LeCorre, D., Bras, J., & Dufresne, A. (2011a). Ceramic membrane ltration for
isolating starch nanocrystals. Carbohydrate Polymers, 86, 1565e1572.
LeCorre, D., Bras, J., & Dufresne, A. (2011b). Evidence of micro- and nanoscaled
particles during starch nanocrystals preparation and their isolation. Bio-
macromolecules, 12(8), 3039e3046.
LeCorre, D., Bras, J., & Dufresne, A. (2011c). Inuence of botanic origin and amylose
content on the morphology of starch nanocrystals. Journal of Nanoparticle
Research, 13(12), 7193e7208.
LeCorre, D., Vahanian, E., Dufresne, A., & Bras, J. (2012). Enzymatic pretreatment for
preparing starch nanocrystals. Biomacromolecules, 13(1), 132e137.
Lin, N., Huang, J., Chang, P. R., Anderson, D. P., & Yu, J. (2011). Preparation, modi-
cation, and application of starch nanocrystals in nanomaterials: a review.
Journal of Nanomaterials, 1e14. Article ID: 573687.
Lin, N., Huang, J., & Dufresne, A. (2012). Preparation, properties and applications of
polysaccharide nanocrystals in advanced functional nanomaterials: a review.
Nanoscale, 4, 3274e3294.
Mary, S. K., Pothan, L. A., & Thomas, S. (2013). Applications of starch nanoparticles
and starch-based bionanocomposites. In A. Dufresne, S. Thomas, & L. A. Pothen
(Eds.), Biopolymer nanocomposites: Processing, properties, and applications (pp.
293e307). New Jersey: John Wiley & Sons Inc.
Mohammad Amini, A., Razavi, S. M. A., & Mortazavi, S. A. (2015). Morphological,
physicochemical, and viscoelastic properties of sonicated corn starch. Carbo-
hydrate Polymers, 122, 282e292.
Putaux, J.-L., Molina-Boisseau, S., Momaur, T., & Dufresne, A. (2003). Platelet
nanocrystals resulting from the disruption of waxy maize starch granules by
acid hydrolysis. Biomacromolecules, 4(5), 1198e1202.
Romdhane, A., Aurousseau, M., Guillet, A., & Mauret, E. (2015a). Cross ow micro-
ltration of starch nanocrystals suspensions. The Canadian Journal of Chemical
Engineering, 93(2), 412e418.
Romdhane, A., Aurousseau, M., Guillet, A., & Mauret, E. (2015b). Effect of pH and
ionic strength on the electrical charge and particle size distribution of starch
nanocrystal suspensions. Starch/Sta rke, 67, 319e327.
Sun, Q., Fan, H., & Xiong, L. (2014). Preparation and characterization of starch
nanoparticles through ultrasonic-assisted oxidation methods. Carbohydrate
Polymers, 106, 359e364.
Thielemans, W., Belgacem, M. N., & Dufresne, A. (2006). Starch nanocrystals with
large chain surface modications. Langmuir, 22, 4804e4810.
Utrilla-Coello, R. G., Hernandez-Jaimes, C., Carrillo-Navas, H., Gonzalez, F.,
Rodriguez, E., Bello-Perez, L. A., et al. (2014). Acid hydrolysis of native corn
starch: morphology, crystallinity, rheological and thermal properties. Carbo-
hydrate Polymers, 103, 596e602.
Wang, S., Blazek, J., Gilbert, E., & Copeland, L. (2012). New insights on the mecha-
nism of acid degradation of pea starch. Carbohydrate Polymers, 87, 1941e1949.
Wei, B., Hu, X., Li, H., Wu, C., Xu, X., Jin, Z., et al. (2014). Effect of pHs on dispersity of
maize starch nanocrystals in aqueous medium. Food Hydrocolloids, 36, 369e373.
Wei, B., Xu, X., Jin, Z., & Tian, Y. (2014). Surface chemical compositions and dis-
persity of starch nanocrystals formed by sulfuric and hydrochloric acid hy-
drolysis. PLoS One, 9(2), e86024. http://dx.doi.org/10.1371/
journal.pone.0086024.
Zheng, H., Ai, F., Chang, P. R., Huang, J., & Dufresne, A. (2009). Structure and prop-
erties of starch nanocrystal-reinforced soy protein plastics. Polymer Composites,
30(4), 474e480.