Research Article

Received: 11 July 2017 Revised: 29 October 2017 Accepted article published: 6 November 2017 Published online in Wiley Online Library: 22 November 2017

(wileyonlinelibrary.com) DOI 10.1002/jsfa.8771

Structural, thermal, and morphological

characteristics of cassava amylodextrins
Mariana Souza Costa,a Diogo Paschoalini Volanti,b
Maria Victória Eiras Grossmannc and Célia Maria Landi Francoa*

Abstract
BACKGROUND: Amylodextrins from cassava starch were obtained by acid hydrolysis, and their structural, thermal and morpho-
logical characteristics were evaluated and compared to those from potato and corn amylodextrins.

RESULTS: Cassava starch was the most susceptible to hydrolysis due to imperfections in its crystalline structure. The crystalline
patterns of amylodextrins remained unchanged, and crystallinity and peak temperature increased with hydrolysis time, whereas
thermal degradation temperature decreased, independent of treatment time and starch source. Cassava amylodextrins had sim-
ilar structural and morphological characteristics to those from corn amylodextrins due to their A-type crystalline arrangements.
A-amylodextrins were structurally and thermally more stable than potato amylodextrins (B-type). Starch nanocrystals (SNC)
were observed by transmission electron microscopy from the third day of hydrolysis in cassava amylodextrins, whereas potato
and corn amylodextrins displayed SNC only on the fifth day. A-SNC displayed platelet shapes, whereas B-SNC were rounded. The
SNC shape was related to the packing form and geometry of unit cells of allomorphs A and B.

CONCLUSION: Microstructures (agglomerated crystalline particles) and nanostructures (double helix organization) were
observed for amylodextrins. Cassava starch was shown to be a promising material for SNC production, since it requires less
hydrolysis time to obtaining more stable crystals.
© 2017 Society of Chemical Industry

Supporting information may be found in the online version of this article.

Keywords: starch; acid hydrolysis; crystallinity; thermal properties; nanostructure

INTRODUCTION organized areas of the granules (amorphous regions) are pref-

Starch is constituted of amylose and amylopectin, which are syn-
thesized in a semi-crystalline granular structure. The starch crys-
talline lamellae are arranged into spherical blocklets of different
sizes (20–500 nm), depending on the starch source.1 The crys-
talline concentric layers are formed into lamellae by amylopectin
double helices packed in an ordered form, whereas the amorphous
regions are composed of amylopectin branching points and amy-
lose in a disordered configuration.2
Starch granules have different polymorphisms exhibiting differ-
ent X-ray diffraction patterns (A-, B- and C-types), which depend
on the branch chain length of their amylopectins. Starches with
amylopectin longer branch chains have a B-type pattern, whereas
those with shorter branch chains display an A-type pattern.3,4 A-
and B-polymorphs differ from one another in packing form and
geometry of the unit cell. The A-type crystallite displays a mono-
clinic unit cell, which is closely packed, whereas the B-type exhibits
a hexagonal arrangement, which is more open, containing 36
water molecules attached to the double helices.5,6 The C-type pat-
tern consists of the combination of both A and B structures.5
Crystalline fractions from starch can be obtained by extensive
hydrolysis with hydrochloric (Lintner amylodextrins) or sulfuric
(Näegeli amylodextrins) acid at a temperature lower than the
erentially degraded, but the main crystalline structure remains
intact.7 These acid-resistant fractions can have different sizes and
shapes depending on the amylose content, amylopectin chain
length, crystalline pattern and blocklet size of the parent starch.
In the early stages of acid hydrolysis the amylose and the long
amylopectin chains are attacked. The amylodextrins are made up
of two main chain populations, which correspond to the linear
dextrins (DP 11–15) and the singly branched dextrins (DP 21–27).
Beyond these chains, small amounts of dextrins with multiple
branches (DP > 35) can also be observed.4–9
Some authors use the term ‘amylodextrins’ whereas others use
‘starch nanocrystals’ (SNC) to refer to the same particles. This
∗ Correspondence to: CML Franco, Department of Food Engineering and Technol-
ogy, UNESP – São Paulo State University, 15054-000 São José do Rio Preto, SP,
Brazil. E-mail: celia@ibilce.unesp.br
a Department of Food Engineering and Technology, UNESP – São Paulo State
University, São José do Rio Preto, SP, Brazil
b Department of Chemistry and Environmental Sciences, UNESP – São Paulo
State University, São José do Rio Preto, SP, Brazil
c Department of Food Science and Technology UEL, Londrina State University,
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gelatinization temperature of starch. During this process, the less Londrina, PR, Brazil

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 www.soci.org
misunderstanding arises from the divergence between the
nomenclature due to variation in size and crystallinity of the
particles, which alter according to the botanical source. In this
study, the term ‘SNC’ will be used to refer to particles with sizes
smaller than 100 nm. SNC are present in the insoluble residues
from acid hydrolysis and result from the complete degradation
of amorphous regions of the granule.10 Thus they can be con-
tinuously obtained during acid hydrolysis. The size of the SNC is
associated with the size of the blocklets of the parent starch and
resistance of the granule to the acid.11 Putaux et al.10 reported
that waxy corn SNC are composed of nanoplatelets, which form
parallelepipedal crystalline blocks that measure 20–40 nm long,
15–30 nm wide and 5–7 nm thick, resembling the monoclinic
structural arrangement of A-type starch. On the other hand,
high-amylose maize SNC were thicker than those from normal
and waxy maize starches.9,11 The format of the SNC has been con-
troversial. Despite some authors having reported that the shape
of the SNC is related to the geometry and form of packaging of
the allomorph of the original starch,10,11 others have shown that,
regardless of crystalline pattern, the SNC have a rounded format.9
Acid-resistant fractions have high thermal stability due to degra-
dation of the granule amorphous region and, consequently, an
increase in the molecular order of the crystals.9,12,13 Studies have
reported that A-crystallites have higher thermal stability than
B-crystallites due to the dense packaging of the double helices
in the crystalline lamellae.9 However, opposite behavior was
reported in the studies of LeCorre et al.12 These authors reported
that B-crystallites are thermally more stable due to the longer
length of their double helices.
SNC have recently received much attention due to their
nanoscale. These nanoparticles can be used in industries and
food applications: for example, to reinforce the matrix of syn-
thetic polymers or biopolymers and fat replacers. Most of the
studies on amylodextrins or SNC have reported the acid action on
waxy maize starch9,10 and starches from conventional botanical
sources such as potato, normal maize, high-amylose maize and
wheat.9,11,12 In this work we have considered the amylodextrins
and SNC from cassava starch.
Cassava (Manihot esculenta) is one of the world’s main carbohy-
drate sources, mainly in tropical areas, and has a great cultural and
economic importance in Brazil. In 2014, world production of cas-
sava was estimated to be 268.28 million tons, Brazil being respon-
sible for 23.25 million tons.14 Besides the consumption of this root
in its natural form, there is a great potential for the industrial use of
cassava in Brazil, especially the starch, because its extraction pro-
cess is simple and cheap. The production of cassava starch in Brazil
is currently around 2.55 million tons.15 Thus, for a better under-
standing of the micro- and nanostructures of cassava starch, the
acid-resistant crystalline fractions, obtained at different hydroly-
sis times, were evaluated. The aim of this study was to systemati-
cally evaluate the structural, thermal and morphological features
of the cassava amylodextrins/SNC and compare them to those
from potato and corn starches.
MATERIALS AND METHODS
Materials
Cassava (Manihot esculenta) roots of the Cascudinha variety were
provided by Promafa – Produtos de mandioca Fadel Ltda (Palmi-
tal, Brazil); corn (Zea mays) grains of the IPR 114 line were kindly
provided by the Agronomic Institute of Paraná (Londrina, Brazil);
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and potato (Solanum tuberosum) tubers of the IAC Ibituaçu line
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MS Costa et al.
were provided by the Agency for Agribusiness Technology of
São Paulo (Campinas, Brazil). The enzyme isoamylase from Pseu-
domonas sp. (EC 3.2.1.68) (Megazyme International, Wicklow, Ire-
land) was used without further processing. All other chemicals
used were of analytical grade.
Isolation of starch
The starches were isolated from cassava roots and potato tubers
following the procedure described by Peroni et al.16 The regular
corn starch was isolated using wet milling as described by Rocha
et al.17
Branch chain length distribution of amylopectin
The branch chain length distributions of amylopectins were ana-
lyzed by high-performance anion-exchange chromatography with
pulsed amperometric detection (HPAEC-PAD) using a ICS 3000
system (Dionex Corporation, Sunnyvale, CA, USA) equipped with
an AS40 automatic sampler. The starches were debranched using
isoamylase enzyme (Megazyme, 3 U) as described by Wong and
Jane,18 filtered (0.22 𝜇m membrane), and 20 𝜇L was automatically
injected into the HPAEC-PAD system. The flow rate was 0.5 mL
min−1 at 30 ∘ C. Eluent A was made up of 200 mmol L−1 NaOH and
eluent B was made up of 100 mmol L−1 NaOH and 1000 mmol L−1
sodium acetate. All eluents were prepared using ultrapure water
(18 MΩ cm−1 ) with N2 sparging. The debranched chains of amy-
lopectin were separated using a Dionex CarboPac™ PA-200 guard
column (3 mm × 50 mm) and a Dionex CarboPac™ PA-200 column
(3 mm × 250 mm).The eluent gradients were: 0–10 min, linear gra-
dient from 0 to 70 mmol L−1 sodium acetate; 10–80 min, linear gra-
dient ending with 350 mmol L−1 sodium acetate; 80–100 min, the
concentration of 350 mmol L−1 sodium acetate was maintained.
Sodium hydroxide concentration was maintained at 100 mmol
L−1 during the whole analysis. The data were analyzed using
Chromeleon software, version 6.8 (Dionex Corporation, Sunnyvale,
CA, USA).
Acid hydrolysis of starch
Acid hydrolysis was performed in duplicate according to the
methodology described by Angellier-Coussy et al.19 The starches
of potato, cassava and corn (37 g, dry weight basis) were dispersed
in 250 mL of 3.16 mol L−1 H2 SO4 and were incubated at 40 ∘ C with
continuous stirring (120 g) for 1, 3, 5 and 7 days. After each day,
the suspensions were centrifuged at 12 000 × g for 10 min. The
solubilized carbohydrates in the supernatant were determined by
the phenol–sulfuric method.20 The extent of hydrolysis was deter-
mined by the ratio between the amount of solubilized total sugars
and the total weight of starch on a dry basis. The acid-resistant
fraction (amylodextrin) was washed with deionized water until it
reached neutrality (pH ∼6.8) and freeze-dried.
Apparent amylose content
Starches and amylodextrins were defatted by dispersing the sam-
ples in 11.5 mol L−1 dimethyl sulfoxide (DMSO) solution in a bath
of boiling water under stirring for 1 h and then stirred for another
16 h at room temperature. The samples were precipitated from
DMSO solution, washed with anhydrous ethanol, recovered by
filtration and dried at 35 ∘ C for 24 h. The iodine affinities were
determined using a potentiometric autotitrator (716 SM Titrino,
Metrohm, Switzerland) as described by Kasemsuwan et al.21 The
apparent amylose content was calculated by dividing the iodine
affinity of the defatted samples by 20%.22
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Characteristics of cassava amylodextrins
X-ray diffraction pattern and crystallinity
The starches and amylodextrins were previously stored in a des-
iccator containing saturated BaCl2 solution (25 ∘ C, Aw = 0.9) for
10 days. Glass sample ports were used for analysis. The X-ray
diffraction pattern of the samples was determined using a bench-
top X-ray diffractometer (MiniFlex 300, Rigaku, Tokyo, Japan)
equipped with Cu–K 𝛼 monochromatic radiation (𝜆 = 0.1542 nm).
The scan was performed at 30 kV and 10 mA for a 2𝜃 range of 3–40∘
with a step size of 0.01. The relative crystallinity was quantitatively
estimated based on the relationship between the peak area and
the total area of the diffractogram.23
Thermal properties
Thermal properties of the starches and amylodextrins were
determined using a differential scanning calorimeter (Pyris 1,
PerkinElmer, Waltham, MA, USA). The samples (4 mg, dry basis)
and deionized water (12 𝜇L) were hermetically sealed in aluminum
pans and equilibrated at room temperature for 12 h. The samples
were scanned from 25 to 125 ∘ C at a rate of 10 ∘ C min−1 . The
calorimeter was calibrated with indium, and an empty aluminum
pan was used as the reference. Pyris 1 software (PerkinElmer) was
used for processing the data.
Thermogravimetric analysis
Thermogravimetric analysis of the starches and amylodextrins
was performed using a thermogravimetric analyzer (TGA 4000,
PerkinElmer). The samples (∼10 mg, dry basis) were placed in the
sample ports and scanned from 30 to 600 ∘ C at 10 ∘ C min−1 under a
nitrogen atmosphere (20 mL min−1 ). The mass loss of the samples
was calculated from the derivative of each curve using Pyris 1
software.
Chain length distributions of amylodextrins
The amylodextrins obtained on days 1, 3, 5 and 7 (1 mg) were dis-
persed in 1 mL ultra-pure water (18 MΩ cm−1 ), placed in a bath of
boiling water for 15 min, cooled, filtered (0.22 mm membrane) and
analyzed using HPAEC-PAD. The instrumental parameters were the
same as those described above (‘Branch chain length distribution
of amylopectin’), but the flow rate was 1.0 mL min−1 at 40 ∘ and the
samples were separated using a Dionex CarboPac™ PA-100 guard
column (4 mm × 50 mm) and a Dionex CarboPac™ PA-100 column
(4 mm × 250 mm). The optimal gradient was based on the method-
ology of Koch et al.24 As the samples were not debranched with
isoamylase, the chains were classified according to their ‘apparent
degree of polymerization’ (DPap ).8
Morphology and size distribution of the starch granules
and amylodextrins
Starches and amylodextrins, previously rinsed with ethanol, were
mounted on a metal plate covered with a carbon double-sided
adhesive tape, submitted to a 14 nm gold layer application using
a sputter coater (SCD 004, Bal-Tec AG, Balzers, Liechtenstein), and
observed in a scanning electron microscope (Philips XL-30 FEG,
Eindhoven, Netherlands) operating at an acceleration tension of
5 kV.
The granule size distribution of the starches was determined
using an image analysis system (Axio Vision Rel 4.8.2.-SP2, Zeiss,
Göttingen, Germany) attached to a light microscope (Axioskop 2
Plus, Zeiss). The starch sample was sprinkled on a glass slide, and
one to two drops of water–glycerin solution (1:1, v/v) were added
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and mixed with the starch. The slide was then covered with a glass
cover slip and observed under the microscope. Three slides for
each sample were made, and 500 granules were measured for each
slide.
The morphology and particle size of the amylodextrins were ana-
lyzed using a transmission electron microscope (TEM JEM-100CX
II, JEOL, Tokyo, Japan) operated at 80 kV. An aqueous suspen-
sion of the amylodextrins (200 g kg−1 ) was homogenized for 180 s
using a bath-type sonicating system (Branson model 3510, Emer-
son, Dubai, UAE) for dispersing the particles in water. A drop of
the suspension (150 𝜇L) was deposited on a support covered with
parafilm, and a carbon-coated microscopy grid was placed over
the drop for 30 s. The carbon-coated microscopy grid was then
dried at room temperature. For each sample, 20 particles were ran-
domly selected9 and their sizes were measured using the software
GIMP 2.8.18.
Statistical analysis
Hydrolysis was performed in duplicate, and all the other experi-
ments were performed at least in triplicate, except for the branch
chain length distribution of amylopectin analyses, which were per-
formed in duplicate. The data were evaluated using Statistica soft-
ware (version 7.0, Statsoft, Tulsa, OK, USA), including analysis of
variance (ANOVA). Differences were assessed using Tukey’s test.
The significance level was set at a P-value <0.05.
RESULTS AND DISCUSSION
Amylopectin branch chain length distribution of the starches
The cassava starch had the highest proportion of short chains (DP
6-12) and also a large number of long branch chains (DP ≥ 37).
This starch displayed a shoulder at DP 19–21 on the amylopectin
branch chain length distribution (Fig. 1). Shoulders on the amy-
lopectin branch chain length distribution indicate imperfections
in the starch crystalline structure.6,25 In turn, the potato starch had
the highest proportion of long branch chains and an average DP
of 23.8, whereas the corn starch contained a large proportion of
short chains and the smallest proportion of long chains and aver-
age DP (Table 1). The potato and corn starches did not display any
shoulder on their branch chain length distributions. Similar results
were observed by Jane et al.25 and Rocha et al.17,26
Acid hydrolysis
The cassava, potato and corn starch granules were degraded by
acid in two stages: the first one, from day 1 to day 3, corresponded
to a quick degradation of the amorphous regions of the gran-
ules, whereas the second one, from day 4 to day 7, at a lower
hydrolysis rate, corresponded to the disruption of the crystalline
areas (Fig. 2a). Acid hydrolysis of starch involves a unilateral elec-
trochemical attack of protons on glycosidic linkages. Therefore,
the less organized packing of amylose and amylopectin branch
points in amorphous areas are more susceptible to acid action,
whereas the dense packing of amylopectin double helices in crys-
talline regions does not allow H3 O+ ions to quickly penetrate the
granule.13
The potato and corn starches displayed similar hydrolysis rates
in the first stage, while in the second one the potato starch was
more resistant to the acid. Potato and corn starches display B-
and A-type crystalline patterns, respectively. B-starch contains
amylopectin long branch chains (B2, B3 and B4) that extend
2753
through multiple clusters.3 The B-starch, therefore, has thicker
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Figure 1. Amylopectin branch chain length distributions of cassava, corn and potato starches determined using a HPAEC–PAD system.

Table 1. Branch chain length distributions of amylopectins of cassava, corn and potato starches

Branch chain length distribution (%)

Starches DP 6-12 DP 13-24 DP 25-36 DP ≥ 37 DP Highest DP

Cassava 26.1 ± 0.1a 42.4 ± 0.1b 13.9 ± 0.0a 17.6 ± 0.0b 23.1b 92
Corn 23.0 ± 0.2b 47.5 ± 0.2a 14.4 ± 0.1a 15.2 ± 0.1c 22.5c 92
Potato 22.1 ± 0.1c 45.9 ± 0.9a 12.9 ± 0.9a 19.2 ± 0.1a 23.8a 97

Values with the same letter in the same column for each starch are not significantly different (P > 0.05). DP, degree of polymerization.

lamellae, which have been described as more resistant to acid.3
Similar results were found by Srichuwong et al.7 and Campanha
and Franco13 when studying the effect of acid hydrolysis on
different starches. Regardless of A-pattern, imperfections in the
crystalline structure of the cassava starch, as discussed earlier, may
have contributed to a lower structural stability of the granule,
making it the most susceptible to acid attack.
Amylose content of the starches and amylodextrins
The cassava, potato and corn starches contained 238, 262 and
284 g kg−1 , respectively, of amylose (Fig. 2b). Similar values were
found by Rocha et al.17,26 The longer branch chains of amylopectin
in potato starch can also form complexes with iodine, as amylose
does, and then the amylose content may be overestimated.25
There was a substantial decrease in the amylose content of all
the samples on the first day of hydrolysis (>70%) because this
polymer forms part of the amorphous areas of the granule that are
more susceptible to the acid (Fig. 2b). The cassava starch, the most
susceptible to hydrolysis, had the lowest amylose content after
the first day of treatment (17 g kg−1 ). On the last day, the cassava,
potato and corn amylodextrins contained, respectively, 6.1, 12.1
and 49.1 g kg−1 amylose. During hydrolysis, the starch granules
swell and allow amylose to exit from the amorphous region and
migrate to the aqueous phase, where it is easily hydrolyzed by
H3 O+ .27 The amylose–lipid complex, naturally present in the corn
starch,5 may have contributed to the slower decrease in the
amylose content of this starch during hydrolysis.
X-ray diffraction and relative crystallinity of the starches
and amylodextrins
As extensively described in the literature, the cassava and corn
starches displayed A-type pattern, while the potato starch dis-
played B-type pattern (Fig. 3a–c), which agrees with the data
from the amylopectin branch chain length distribution shown
in Table 1. The crystalline patterns of the starches remained
unchanged during the whole acid hydrolysis period. However,
during hydrolysis there was a gradual decrease in the intensity
2754
the potato starch, whereas the crystallites of cassava and corn
starches practically remained intact. These two peaks, 22∘ and
24∘ in 2𝜃, appear to merge in a single peak at 23∘ in the potato
starch on the last day of hydrolysis. Hydrolysis of amylopectin
chains causes changes in the organization of double helices2
without altering the crystalline pattern. In this study it was found
that the arrangement of B-type crystallites was more easily inter-
rupted by acid than the arrangement of A-type crystallites, which
is in agreement with data observed by Kim et al.9 The relative
crystallinity (RC) of the starches varied from 33.3% to 36.1%
(Table 2). Similar results were found by Rocha et al.17,26 for cassava,
potato and corn starches. The amylodextrins, independently of
the starch source, had their RC increased up to the fifth day of
hydrolysis, reaching ∼57% (Table 2) as a result of the gradual
degradation of the granule amorphous areas. RC values ranging
between 45% and 50% for acid-resistant fractions from different
starches were found by different researchers.11,27 On the seventh
day of hydrolysis, the RC of the amylodextrins decreased due
to probable disruption of part of the crystalline area. However,
the variation in RC of the amylodextrins during hydrolysis was
different, depending on the starch crystalline pattern. The cassava
and corn amylodextrins had an increase of ∼43% of RC until
the third day of hydrolysis, whereas in the potato amylodextrins
there was a less expressive increase (∼24%) in this same period.
On the other hand, the potato amylodextrins had the highest
decrease in RC (14%) on the last day of treatment, while cassava
and corn amylodextrins had a decrease of ∼5%, suggesting that
the B-type crystalline arrangement was more easily disrupted by
the acid.
Thermal properties of the starches and amylodextrins
The onset gelatinization temperature (T o ) varied from 59.17 ∘ C in
potato starch to 64.66 ∘ C in corn starch, while the gelatinization
enthalpy change (ΔH) ranged from 14.42 to 18.76 J g−1 in corn
and potato starches, respectively (Table 2 and supporting infor-
mation Fig. 7). The cassava starch had the highest gelatinization
temperature range (T c –T o ) and the potato starch the lowest, sug-
gesting a greater homogeneity of crystals in potato starch. Simi-

of the peak at 5.6∘ and of the peaks at 22∘ and 24∘ in 2𝜃 in lar results were found for potato,28 cassava29 and corn17 starches.

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Figure 2. (a) Percentage of hydrolysis of starches as a function of time. (b) Amylose content of starches and amylodextrins obtained on different hydrolysis
days.

Table 2. Thermal properties, relative crystallinity and ratio between single-branch chains and linear chains of starches and their amylodextrins

Hydrolysis time (d) T o (∘ C) T p (∘ C) T c (∘ C) ΔT (∘ C) ΔH (J g−1 ) RC (%) P2 /P1

Cassava
Raw starch 63.83 ± 0.17a 68.77 ± 0.24c 74.63 ± 0.30c 10.80 15.08 ± 0.29bcd 36.06 –
1 50.58 ± 0.09def 61.17 ± 0.24 g 98.53 ± 0.68a 47.96 11.69 ± 0.08 g 48.49 0.58
3 50.00 ± 1.35ef 68.81 ± 0.20c 99.76 ± 0.28a 49.76 16.48 ± 0.78b 51.63 0.45
5 49.20 ± 0.01f 72.47 ± 0.12b 99.47 ± 0.45a 50.27 15.77 ± 0.20bc 56.99 0.35
7 50.54 ± 1.32def 73.05 ± 0.00b 98.67 ± 0.74a 48.13 14.80 ± 0.28bcde 53.99 0.32
Corn
Raw starch 64.66 ± 0.28a 69.91 ± 0.08c 74.70 ± 0.12c 10.04 14.42 ± 0.29cde 34.70 -
1 50.99 ± 0.37def 64.01 ± 1.04de 102.12 ± 2.32a 51.13 14.98 ± 0.84bccd 45.07 0.69
3 51.81 ± 1.22de 72.94 ± 0.09b 100.14 ± 1.79a 48.33 15.57 ± 0.72bc 49.32 0.50
5 52.74 ± 0.51d 76.15 ± 0.83a 99.84 ± 0.21a 47.10 14.31 ± 0.52cdef 57.28 0.35
7 55.41 ± 0.19c 77.24 ± 0.23a 99.26 ± 0.61a 43.86 13.07 ± 0.31efg 54.30 0.35
Potato
Raw starch 59.17 ± 0.07b 62.08 ± 0.00 fg 66.01 ± 0.01d 6.83 18.76 ± 0.52a 33.27 -
1 50.09 ± 0.34ef 60.60 ± 0.34 g 74.09 ± 0.61c 24.00 13.94 ± 0.57def 39.19 0.45
3 50.51 ± 0.72def 62.91 ± 0.25ef 80.49 ± 0.80b 29.99 14.64 ± 0.45cde 41.10 0.40
5 50.29 ± 0.09ef 64.08 ± 0.10de 83.08 ± 0.18b 32.80 12.56 ± 0.30 fg 56.48 0.22
7 50.22 ± 0.37ef 64.25 ± 0.20d 82.94 ± 1.88b 32.72 9.88 ± 0.46 h 48.54 0.17

Values with the same letter in the same column for each starch are not significantly different (P > 0.05). T o , T p , T c : onset, peak and conclusion
temperatures; ΔT = (T c − T o ); ΔH, enthalpy change; RC, relative crystallinity % = [(total area − amorphous area)/total area] × 100, from X-ray diffraction;
P2 /P1 , ratio between areas of peak 2 (DPap ≥ 18, singly branched chains) and peak 1 (DPap ≤ 17, linear chains of amylodextrins).

The amylose–lipid endothermic peak (T o = 87.13 ∘ C, T p = 97.94 ∘ C,
T c = 103.15 ∘ C and ΔH = 1.43 J g−1 ) displayed for the corn starch
practically disappeared in the amylodextrins from the first day
of treatment due to the significant reduction of amylose during
hydrolysis (Fig. 2b).
Independently of the amylodextrins source, there was a decrease
in T o and an increase in the conclusion temperature (T c ) for all
the samples due to the degradation of the amorphous region and
destabilization of the crystalline region during hydrolysis.9,27,30 The
peak temperature (T p ) also decreased on the first day of treatment
due to destabilization of the starch granule structure and then
increased from the third day of hydrolysis because the structures
stabilize the crystalline regions are degraded last.6,7 This increase
was greater for the cassava and corn amylodextrins.
The amylodextrins had a broad gelatinization temperature range
(T c –T o ) since the first day of hydrolysis due to the heterogeneity
of the crystals. The differential scanning calorimeter is a sensitive
instrument and measures the T o when the first particles melt,
so the low T o observed refers to the crystals that have been
destabilized by acid attack. The hydrolysis of the amylose chains
(Fig. 2b) and amylopectin long chains (DP > 37) (Fig. 4) at the first
stage of hydrolysis resulted in the increase of the proportion of
very short chains (DP 1–10), which do not form stable double
helices and cause fragility in the crystalline structure.7 The acid also
2755

become more organized and the double helices (DP 11–15) that attacks the outermost turns of the double helices, causing small

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Figure 3. X-ray diffractograms (a–c) and TGA curves (d–f ) of starches and amylodextrins on different hydrolysis days.

defects because the ends of the double helices become frayed.6 All
this evidence on the first stage of hydrolysis influenced the melting
temperature of amylodextrins, as also observed by Utrilla-Coello
et al.,27 Kim et al.9 and Kong et al.30
The amylodextrins also displayed an increase of 17–27 ∘ C in T c
when comparing with T c of the parent starches. This is because
during the acid action on the granules a reorganization of the
crystalline structure that makes the crystals thermally more stable
2756
There was a greater decrease in the ΔH of the potato amylodex-
trins with hydrolysis, and this was also observed for the RC of
B-type amylodextrins (Table 2). These results suggest that the crys-
tals of A-type amylodextrins have greater thermal and structural
stability than those of B-type amylodextrins.
Thermogravimetric analysis
Independent of their source, the starches and amylodextrins

occurs.9,12,13,27 had similar thermogravimetric behavior with two mass losses

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Characteristics of cassava amylodextrins
Figure 4. Chain length distributions of cassava, corn and potato amylodextrins obtained at 3, 5 and 7 days of hydrolysis determined using an HPAEC-PAD
system.
(Fig. 3d–f ). The first corresponded to the process of dehydra-
tion/evaporation of water (65–130 ∘ C) and the second was
attributed to decomposition of the starch glycosidic units
(265–404 ∘ C) and amylodextrin glycosidic units (270–440 ∘ C).
Independent of the hydrolysis time, the decomposition temper-
ature range of the glycosidic units of amylodextrins was slightly
greater than that of the starches due to the greater energy required
to decompose the remaining crystalline fraction. Besides, a third
mass loss at temperatures between 150 and 250 ∘ C was observed
in the amylodextrins. The probable presence of sulfate groups,
which are formed on the surface of particles due to sulfate esteri-
fication of hydroxyl linkages and sulfuric acid during hydrolysis,31
can cause initial depolymerization of the material. The yield of
the residue (final weight) was greater for amylodextrins than for
starches probably because of the sulfate groups present on the
crystalline particles, which increase the amount of water released
and decrease combustion.32
Chain length distribution of amylodextrins
Anion-exchange chromatograms of the amylodextrins of cassava,
potato and corn obtained on days 1, 3, 5 and 7 of hydrolysis are
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shown in Fig. 4. In general, the amylodextrins had chains with
DPap 2–51. Two main populations, with peak maxima at DPap
11–12 and DPap 22, which corresponded to the linear and singly
branched chains,4,8,9 respectively, were observed, except for the
potato amylodextrins, which had much smaller proportions of
singly branched chains, represented by a shoulder near to DPap 25.
The increase in intensity of the peaks (DPap 11–12 and
DPap 22) in cassava and corn amylodextrins and in the
shoulder in potato amylodextrins during hydrolysis indi-
cates that the longer branch chains of starch were initially
degraded.
Cassava amylodextrin chromatograms exhibited a small peak at
DPap 37, which appeared on the fifth day of hydrolysis, whereas
in those of the corn amylodextrins, small additional peaks near
to DPap 35, 44 and 47 (Fig. 4) were observed from the third
day of treatment. These small peaks corresponded to the chains
linked to branching points, which were resistant to the action of
acid.8 Part of the branching linkages of A-type starch (cassava
and corn starches) are scattered in the crystalline lamellae of the
granule, while in B-type (potato starch) they are mainly clustered
2757
in the amorphous region. In the crystalline area, these linkages
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Figure 5. Scanning electron micrographs of starches and amylodextrins: (a–e) cassava; (f–j) corn; (k–o) potato; (a, f, k) native starches; (b, g, l) day 1; (c, h,
m) day 3; (d, i, n) day 5; (e, j, o) day 7.
are protected from acid action.4 These chains contributed to the
greater increase of crystallinity in A-type amylodextrins (Table 2).
The ratio between the areas of populations of singly branched
chains and linear chains (Table 2) was higher in corn amylodex-
trins, followed by cassava and potato amylodextrins. These results
confirm the larger number of branching points in A-type amy-
lodextrins.
Morphology and average size of starch granules
and amylodextrins
The shape and size of granules were quite variable among the
different starch sources. Cassava starch granules had a round
shape with truncated ends (Fig. 5a), whereas corn starch gran-
ules displayed round and polyhedral shapes (Fig. 5f ). Some corn
starch granules exhibited pores and depressions on their surfaces
because of the high amylase activity in the initial stage of grain
germination.33 Both the corn and cassava starch granules had a
similar size distribution, ranging from 5 to 30 𝜇m, with 14.8 𝜇m
average diameter. Potato starch granules displayed oval and ellip-
tical shapes, smooth surface (Fig. 5k) and size ranging from 10 to
60 𝜇m, with 32.7 𝜇m average diameter.
On the first day of hydrolysis, the granular structure of all the
starches was maintained (Fig. 5b, g, l). Cassava and potato starch
granules kept their original shape, while corn starch granules
exhibited small pieces as if they had been broken. Faceted struc-
tures, characteristic of the crystalline material, appeared on the
third day of hydrolysis for cassava and corn amylodextrins (Fig. 5c,
h) and only on the fifth day of treatment for potato amylodex-
trins (Fig. 5n). These results confirm the greater resistance of B-type
starch to acid action in the second hydrolysis stage (Fig. 2a). Gran-
ules of cassava and potato starches were degraded by acid erosion
2758
of their surface, whereas corn starch granules were degraded by
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MS Costa et al.
acid diffusion into the granule due to its porous structure. Simi-
lar results were observed by Campanha and Franco13 and Fannon
et al.33 There was a great agglomeration of the particles for all the
samples mainly in the last days of hydrolysis (Fig. 5d, e, i, j, n, o).
This particle agglomeration may be related to the higher number
of free hydroxyl groups in the samples with the increase in hydrol-
ysis time. These groups are capable of forming hydrogen linkages
with each other and favor agglomeration.31
The greater extent of hydrolysis may result in a decrease in
nanoparticle production. When acid hydrolysis reaches the sec-
ond stage (crystalline regions) it must be controlled so as not to
degrade the crystalline region. In this study, it was found that
after 7 days of hydrolysis the acid begins to slowly degrade the
crystalline regions, causing loss of nanomaterial. The action of
acid on the starch does not occur uniformly and repetitively along
the amorphous and crystalline structures.27,31 In addition, crys-
talline nanoparticles form agglomerates due to hydrogen linkages
between the hydroxyl groups present on their surface.31 These
facts make it difficult to visualize and quantify crystalline nanopar-
ticles of starch in precipitates of the samples. In order to visualize
these nanoparticles the amylodextrins obtained on days 3, 5
and 7 were also studied using transmission electron microscopy
(Fig. 6). On the third day, nanoparticles smaller than 100 nm,
namely starch nanocrystals (SNC), were observed only in cassava
amylodextrins. These cassava SNC had a square shape and a size of
91.1 nm (Fig. 6a). In the same period, corn amylodextrins displayed
starch particles with a platelet shape and size of 505.14 nm length
and 141.68 nm width (Fig. 6d), whereas potato amylodextrins dis-
played agglomerated particles with micrometric and nanometric
sizes (Fig. 6g). As the hydrolysis time increased, all the amylodex-
trins displayed SNC with a well-defined format. The cassava and
corn SNC (Fig. 6b, e) displayed platelet shapes, whereas potato
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Characteristics of cassava amylodextrins
Figure 6. Transmission electronic micrographs of amylodextrins obtained at 3, 5, and 7 days of hydrolysis: (a, b, c) cassava; (d, e, f ) corn; (g, h, i) potato.
SNC were round (Fig. 6h). The shape of the SNC was related to the
A- and B-type crystalline patterns of their amylodextrins. In the
A-type pattern, the crystallite displays an orthorhombic arrange-
ment, whereas, in the B-type pattern, the arrangement is hexago-
nal. These results are close to those observed in other studies.10,11,19
On the other hand, Kim et al.9 found rounded and oval shapes for
SNC, independent of the crystalline pattern of starch.
The size of the SNC also decreased on the fifth day of treat-
ment. Cassava SNC measured 59.32 nm long and 10.74 nm wide,
whereas corn SNC were 67.32 nm long and 14.32 nm wide. Potato
SNC were 67.25 nm in diameter. The morphology and size of the
SNC can define their use in industrial applications, for example as
an emulsion stabilizer,34 fat replacer35,36 or mechanical reinforcer
in any polymeric matrix,36–38 making the crystalline pattern and
hydrolysis time-critical parameters to be considered for SNC pro-
duction. Although the particle shape did not change, an excessive
agglomeration of particles occurred on the last day of hydroly-
sis, resulting in SNC with increased sizes (Fig. 6c, f, i), regardless of
the botanical source. This behavior probably occurred due to the
greater amount of free hydroxyl groups on the amylodextrin sur-
face, which rendered them more reactive, as discussed before.
Thus, from results of both scanning and transmission electron
microscopy, two structures, micro- and nano-, were seen in all the
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amylodextrins. The microstructures come from the agglomerated
crystalline particles of amylodextrins, which have faceted shapes,
whereas the nanostructures are related to the organization of
double helices in the crystalline lamellae.
Although cassava and corn starches have an A-type crystalline
pattern, their amylodextrins showed subtle differences in peak
temperature and relative crystallinity in the first 3 days of hydrol-
ysis due to the molecular organization of the amorphous and
crystalline regions and morphology of the granules of these
starches. However, in the last days of hydrolysis, cassava and corn
amylodextrins showed similar molecular, thermal and morpho-
logical characteristics, indicating that the organization of the
double helices in A-type or B-type crystalline patterns defines the
nanostructure of starch.
CONCLUSION
Cassava, corn and potato starches displayed different behaviors
during acid hydrolysis due to their structural characteristics. Cas-
sava amylodextrins displayed SNC from the third day of hydrolysis,
whereas potato and corn amylodextrins displayed SNC only on the
fifth day. The arrangement of double helices of A- and B-type crys-
2759
talline patterns influenced the rates of hydrolysis and morphology
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of the SNC. This study showed that the A-type SNC (cassava and
corn starches) had a platelet form and were structurally and ther-
mally more stable than the B-type, which was round. Cassava
starch was shown to be a promising material for the production
of SNC, since 3 days of hydrolysis were sufficient to obtain stable
crystals at the nanometric scale.
ACKNOWLEDGEMENTS
This study was financially supported by the Fundação de Amparo
á Pesquisa do Estado de São Paulo – FAPESP (grant Nos. 2013/
01500-7 and 2014/04686-7) and Conselho Nacional de Desenvolvi-
mento Científico e Tecnológico – CNPq (grant No. 307942/2015-5)
from Brazil.
SUPPORTING INFORMATION
Supporting information may be found in the online version of this
article.
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