RESEARCH ARTICLE
Cassava Starch
High-Efficient Preparation of Cross-Linked Cassava
Starch by Microwave-Ultrasound-Assisted and its
Physicochemical Properties
Jin Sun, Ri-Mei He, Feng-Yuan Gao, Zong-Liang Kou, Li-Hong Lan, Ping Lan,*
and An-Ping Liao
Cross-linked cassava starch (CCS) is prepared by a simple method assisted
by microwave-ultrasound using cassava starch as raw material. Several
factors affecting cross-linking reaction such as starch concentration,
microwave–ultrasound power ratio, pH, reaction time, and amount of
(NaPO3)6 are investigated by orthogonal experiment in terms of sedimen-
tation volume for the CCS and the optimum conditions are established.
Under these conditions, the sedimentation volume of the prepared CCS is
3.50. After cross-linking, the crystallinity measured by X-ray diffraction
(XRD) decreases which is also confirmed by Fourier transform infrared
(FTIR) spectroscopy. Atomic force microscopy (AFM) and scanning
electron microscopy (SEM) observations indicate that the obtained CCS is
in rough spherical with the same average sizes as native starch.
Thermogravimetric analysis (TGA) reports CCS exhibit higher thermal
stability. In addition, the physicochemical properties of CCS are also
studied. It is found that the freeze-thaw stability, acid resistance, and
thermal stability are highly improved, indicating that CCS has high
application value in the food industry.
1. Introduction
Starch is one of the major nutrients for human beings. It is
non-toxic, biocompatible, and biodegradable. However, its
application in many industries, including the food industry, is
limited owing to its shortcomings, such as low thermal stability
and susceptibility to extreme pH conditions.[1–4] Thus,
researchers are currently working on modifying raw starch
J. Sun, F.-Y. Gao, Z.-L. Kou, Prof. L.-H. Lan, Prof. P. Lan, Prof. A.-P. Liao even days to complete.[35] To the best of our knowledge, the use of
Guangxi Key Laboratory for Polysaccharide Materials and Modifications
Key Laboratory of Chemical and Biological Transformation Process of
Guangxi Higher Education Institutes
School of Chemistry and Chemical Engineering of Guangxi University for
Nationalities
Nanning 530006, China
E-mail: gxlanping@163.com
R.-M. He
Guangxi Zhuang Autonomous Institute of Metrology and Test
Nanning, China
DOI: 10.1002/star.201800273
Starch - Stärke 2019, 71, 1800273 1800273 (1 of 9)
www.starch-journal.com
so as to expand its application range.[5–10]
Chemical reagents are usually used to
modify the performance of starch; how-
ever, homogeneously mixing these
reagents with starch to permeate its
internal structure is difficult. Therefore,
more highly efficient methods by which
starch can be modified have recently been
the subject of attention and are showing
great potential.[11,12] To that end, micro-
waves, a type of electromagnetic wave, can
heat reactants without conducting that
heat, accelerate the reaction rate of the
thermal effect from internal expan-
sion,[13,14] and improve the contact be-
tween the starch and the reagent.
Ultrasound can provide hotspots and
strong aforementioned mechanical
effects, while heat transfer can be im-
proved with the microwave’s selective
heating.[15,16] The combination of these
technologies is a new technology by which
to promote chemical reaction.[17]
Cross-linked starch is a type of modified starch that attracted
considerable attention because of its high resistance to acids,
alkalis, shears, and enzymes.[18–21] This type of starch has been
widely used in food,[22,23] biomedicine,[24,25] papermaking,[26,27]
and other[28] industries. The cross-linking reaction that forms
distarch phosphate could result from a reaction between C2 and
C2/C3, between C6 and C6 or between C6 and C2/C3 of the sugar
ring. Figure 1(a) provides one possible structure of cross-linked
cassava starch (CCS). The preparation of cross-linked starch
comprises mainly wet cross-linking technology,[29,30] microwave-
assisted technology,[31,32] and ultrasonic wave technology.[33,34]
However, traditional cross-linking reaction take several hours or
microwave–ultrasound technology to prepare cross-linked starch
has not been reported, and the reactions using this technology
can be brought to completion within minutes rather than within
hours or days.
The aim of our study was to develop a new method by which CCS
can be prepared using microwave–ultrasound irradiation and
sodium hexametaphosphate [(NaPO3)6] as the cross-linking reagent.
Our results not only helped develop a simple, facile, efficient, and
environmentally friendly method, but also provide the experimental
basis for the application of CCS in the food industry.
© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
Figure 1. a) A possible structure of CCS. b) Schematic diagram of UWave-1000
multifunctional microwave chemistry reaction workstation.
2. Experimental Section
2.1. Materials
Cassava starch (water content of 13%) was purchased from
Guangxi Wuming Anning Starch Co. Ltd. (Wuming, China).
Sodium hydroxide (NaOH), (NaPO3)6, absolute ethanol
(C2H5OH), and hydrochloric acid (HCl) were supplied by
Tianjin Bodi Chemical Co. Ltd. (Tianjin, China) and used
without modification. All chemicals and reagents used in the
study were of analytical grade.
2.2. Preparation of CCS
Cassava starch was dispersed in 50 mL of a NaOH solution at
pH ¼ 9.0, and then placed in a UWave-1000 multifunctional
microwave chemistry reaction workstation [see Figure 1(b)], in
Starch - Stärke 2019, 71, 1800273 1800273 (2 of 9)
www.starch-journal.com
which the temperature, reaction time, and
microwave–ultrasound power were set.
(NaPO3)6 was added dropwise during the
reaction. After the reaction was complete, the
obtained mixture was centrifuged with etha-
nol several times in order to remove any
excess NaOH and other impurities. Then
starch granules were then dried in a vacuum
oven at 40  C for 3 h, yielding a CCS sample,
which was then kept in a desiccator at room
temperature until further analysis.
2.3. Characterizations and Measurements
2.3.1. Determination of the Sedimentation
Volume
The degree of substitution (DS) of cross-
linked starch is generally too small to measure
directly, and the sedimentation volume (S) is
negatively correlated with the DS; the smaller
the S, the larger the DS.[36] The measurement
volume was determined according to the
method described by Tang[37] and Chen,[57]
and the procedure was as follows: 0.5 g of CCS
was dispersed into 25 mL of distilled water and
heated to 82–85  C for 2 min until the sample
completely gelatinized, after which it was
cooled to room temperature. The resulting
paste (10 mL) was then transferred to a
centrifuge tube and centrifuged at 4000 rpm
for 2 min. The difference of supernatant (V)
and the paste volume (10 mL) were calculated
as the degree of sedimentation.
S ¼ 10  V ð1Þ
2.3.2. Single-Factor Experiment
The effects of starch concentration (A), microwave–ultrasound
power ratio (B), reaction time (C), pH (D), and amount of sodium
hexametaphosphate [(NaPO3)6] (E) on the S of CCS were
investigated. Based on the pre-experimental results, the
concentration of starch was set to 35%, the microwave–
ultrasound power ratio to 24:500 W, the reaction time to
9 min, the pH to 9.0, and the (NaPO3)6 volume to 9 mL. For
the single-factor experiment, only one of the above settings was
changed.
2.3.3. Orthogonal Experimental Design for CCS
An orthogonal test of L16 (45) was designed (see Table 1) in order
to further optimize the significant factors affecting the cross-
linking.
© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
Table 1. Design orthogonal experiments.
Level A starch concentration (%) B power ratio of microwave-ultrasound (W:W)
1 25 24:300
2 30 24:400
3 35 24:500
4 40 24:600
2.4. Structural Characterization of Cross-Linked Starch
The morphology of the native starch and CCS-1, CCS-2, CCS-3
were examined using a SUPRA 55 sapphire scanning electron
microscopy (SEM). The starch samples were sputter-coated with
gold.
Atomic force microscopy (AFM) images were taken at
ambient temperature and controlled humidity using a Bruker
Multimode 8 Scanning Probe Microscope. Starch samples were
spread directly onto an AFM sample holder. The images were
recorded using the instrument’s ScanAsyst mode. Images of
the native starch and CCS topographics and amplitude were
taken simultaneously. Silicon probes were used with a nominal
spring constant of 20–100 N m1 and a scan frequency of
0.5 Hz. The scan areas were of 2.2  2.2, 3  3, 4.1  4.1, and
5  5 mm2.
The X-ray diffraction (XRD) patterns of the obtained
products were measured using an X’Pert PRO X-ray
diffractometer (Rigaku Co, Tokyo, Japan) at 40 mA and
40 kV with Cu Kα radiation. The samples were scanned from
5 to 70 with a scanning speed of 8 min1 and a step size of
0.026 . The crystallinity (Rc) of the samples was calculated
using MDI Jade 6.0 software (Materials Data, Inc., Livermore,
CA, USA). According to the method described by Nara,[38,39]
the area above the smooth curve was taken to correspond to
the crystalline portion (Ac), and the lower area between the
smooth curve and a linear baseline was taken as the
amorphous section (Aa). The ratio of the crystalline area to
total diffraction area was taken as the degree of crystallinity.
The Rc values were using Equation (2)
Ac
Rc ¼
Aa þ Ac
Fourier transform infrared (FTIR) spectroscopy was con-
ducted using a MAGNA-1R550 FTIR Spectrum Scanner. The
spectra were recorded in transmission mode during 32 scans at a
resolution of 2 cm1 within a range of 4000–500 cm1. The
specimens were prepared by grinding the samples in potassium
bromide (KBr) and pressing them into a mold.
Thermogravimetric analysis (TGA) measurements were
conducted via a synchronous thermal analysis (STA449C/4/G,
Netzsch, Germany). Approximately 3 mg of each sample was
loaded into a platinum pan and heated from 25 to 700  C at a
heating rate of 10  C min1. All the measurements were done
under a nitrogen atmosphere with a gas flow of 20 mL min1.
Starch - Stärke 2019, 71, 1800273
www.starch-journal.com
Factors
C reaction time (min) D pH E amount of (NaPO3)6 (mL)
7 7 5
8 8 7
9 9 9
10 10 11
2.5. Physicochemical Properties of CCS
The freeze-thaw stability, acid resistance, and thermal stability of
the four starches (native starch, CCS-1, CCS-2, CCS-3) were
determined.
2.5.1. Analysis of Freeze-Thaw Stabilities
The freeze-thaw stability of the native starch and CCS was
determined using freeze-thaw cycle treatment (FTC). Starch
samples were heated to 95  C under constantly mild agitation for
30 min, followed by cooling in a centrifuge tube (m0). The
gelatinized paste was frozen at 40  C for 24 h, and then thawed
at 25  C for 2 h. This process was repeated up to six cycles. The
samples was weighed before and after centrifugation at
3000 rpm for 10 min, and the weights were recorded as m1
and m2. The percent water separation (%WS) was calculated as
follows
m1  m2
%WS ¼  100% ð3Þ
m1  m0
2.5.2. Analysis of Acid Resistance
A 3% starch slurry suspension was heated to gelatinization at
90  C, and the viscosity was measured after cooling to room
temperature. The pH of the starch paste was then adjusted to 3.0
using HCl and the viscosity was measured again.
ð2Þ
2.5.3. Thermal Stability Analysis of CCS Paste
A 4% starch slurry suspension based on dry starch was
prepared and gelatinized. The viscosity at different temper-
atures were determined, and a viscosity–temperature curve was
created.
2.5.4. Rotational Viscosity Measurement
Using a DV-3T viscometer, the apparent viscosities of completely
gelatinized native starch and CCS aqueous solutions based on
dry mass was determined.
1800273 (3 of 9) © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
3. Results and Discussion
3.1. Influence of Process Variables on the Sedimentation
Volume
As shown in Figure 2(a), the CCS sedimentation volume were
different under different conditions as follows: microwave–
ultrasound power ratio ¼ 24:500 W, pH ¼ 9.0, (NaPO3)6 volume
¼ 9 mL, and reaction time ¼ 9 min. The sedimentation volume
obviously decreased as the starch concentration rose from 15 to
35%, which means that the DS gradually increased. However, no
further remarkable decreases in the sedimentation volume were
observed as the starch concentration continued to increase. This
might be because as the concentration of starch increased from
15 to 35%, the concentration of the reactant, the effective
collision of the starch with the cross-linking agent, and the
degree of cross-linking increased. When the concentration of
starch continued increasing to 40%, it became more viscous and
stirring became more difficult, which resulted in a reaction that
was more inhibited from effectively proceeding. Therefore,
when the concentration of starch is 35%, the reaction proceeded
more fully.
The influence of the microwave–ultrasound power ratio on
the sedimentation volume is shown in Figure 2(b), when the
starch concentration was 35%, pH ¼ 9.0, (NaPO3)6 volume ¼
9 mL, and reaction time ¼ 9 min. Because of the thermal and
mechanical effects of microwaves, a high-power setting would
gelatinize the starch. Therefore, the microwave was kept at 24 W,
which provided a heat source to the reaction system without
causing gelatinization of the starch. As seen from Figure 3(b), we
found that as the microwave–ultrasound power ratio increased
from 24:200 W to 24:500 W, the CCS sedimentation volume
Figure 2. Effects of starch concentration (a), power ratio of microwave-ultrasound (b), reaction time (c), pH (d), and amount of sodium
hexametaphosphate (e) on the sedimentation volume of CCS.
Starch - Stärke 2019, 71, 1800273 1800273 (4 of 9)
www.starch-journal.com
decreased from 6.7 to 2.6 mL, following by a sharp increase of the
power ratio equal to 24:600 W. The reaction was most likely
caused by ultrasound increasing the collision probability of the
starch and the cross-linking agent to increase the DS. However,
as the ultrasound power increases, starch might be gelatinized
partially, resulting in a decrease in the DS. Therefore, the
microwave–ultrasound power ratio chosen for the experiment is
24:500 W.
As seen from Figure 2(c), the effect of the reaction time on the
sedimentation volume was studied under the experimental
conditions: starch concentration ¼ 35%, microwave–ultrasound
power ratio ¼ 24:500 W, pH ¼ 9.0, and (NaPO3)6 volume ¼ 9 mL.
When the reaction time increased from 5 to 9 min, a noticeable
decrease in the CCS sedimentation volume was observed.
However, with a further increase in the reaction time from 9 to
10 min, the CCS sedimentation volume increased. The reason
for this might be that as the reaction time increased, the contact
between the starch and the (NaPO3)6 was more efficient, and
under the thermal and mechanical actions of the microwave, the
temperature of the reaction system gradually rose, which
exacerbated the irregular movement of the molecules and
increased the probability of collision. Coupled with the cavitation
of the ultrasound wave, the molecular structure in the emulsion
system was exposed to more active molecules because of the
prolonged ultrasonic action time, thus promoting the progress of
the reaction and increasing the degree of starch cross-linking.
When the reaction time was increased to 10 min, the degree of
cross-linking decreased, which might be due to CCS degradation
under longer microwave–ultrasound exposure; therefore, the
optimum reaction time is 9 min.
Figure 2(d) shows the effect of pH on the sedimentation
volume of CCS under the following cross-linking conditions:
© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
Figure 3. SEM images of (A) native starch and (B) CCS-1, (C) CCS-2, (D) CCS-3 and two-dimensional AFM images taken from different scanning areas
randomly selected 2.2  2.2, 3  32, 4.1  4.1, 5  5 mm2 areas for (E1–E4, respectively) native starch and (F1–F4, respectively) CCS prepared under the
optimal conditions (starch concentration ¼ 25%, microwave-ultrasound power ratio ¼ 24:400 W, reaction time ¼ 7 min, pH ¼ 9, (NaPO3)6
volume ¼ 5 mL).
starch concentration ¼ 35%, microwave–ultrasound power ratio
¼ 24:500 W, (NaPO3)6 volume ¼ 9 mL, and reaction time ¼ 9
min. Within the pH range of 7.0–12.0, the sedimentation
volume first slightly decreased and then significantly decreased;
that is, the degree of cross-linking first increased and then
decreased, with the highest degree at pH ¼ 9.0. As the pH value
increased, the OH content increased, which prompted the
release of the starch’s active center and increased the active
group of the reaction, which, in turn, increased the DS of CCS.
When the pH was more than 9, a noticeable increase in the
sedimentation volume was observed, which can be attributed to
gelatinization becoming more obvious as the pH increased,
resulting in the reaction of starch with (NaPO3)6 becoming more
difficult. Therefore, CCS prepared at pH ¼ 9.0 resulted in the
highest DS.
The effect of the amount of (NaPO3)6 on the sedimentation
volume is shown in Figure 2(e). When the starch concentration
was 35%, the microwave–ultrasound power ratio 24:500 W,
the pH 9.0, and reaction time = 9 min, the sedimentation
volume decreased at (NaPO3)6 ranged from 5 to 9 mL, which
means the DS of CCS increased. As the amount of (NaPO3)6
increased, the probability of collision with the reactants
increased, subsequently promoting the reaction; however,
when the volume of (NaPO3)6 was >9 mL, the movement of
the starch molecules was hindered, resulting in a decrease in
the DS of CCS. Therefore, the cross-linking effect is ideal when
the volume of (NaPO3)6 is 9 mL.
Starch - Stärke 2019, 71, 1800273
www.starch-journal.com
Based on the results of the single-factor experiment, the best
technological conditions was determined. According to Table 2,
the preferred experiment protocol was A1B2C1D3E1, in which
the starch concentration was 25%, the power ratio of
microwave-ultrasound 24:400 W, the reaction time 7 min,
the pH 9, and the volume of (NaPO3)6 5 mL. In addition, the
R-value of Factor B was 0.91, which was the biggest factor
affecting the DS of CCS. The concentration of starch was a
secondary factor related to the system viscosity and the
concentration of the reactants. The higher the pH, the higher
the OH content in the system, leading to better starch
swelling and exposure of its active centers. In summary, the
order of factor influence was as follows: microwave–ultrasound
power ratio > starch concentration > pH > (NaPO3)6 volume >
reaction time. The results of the verification experiments are
presented in Table 3, and the experimental scheme of the
orthogonal test was verified to be optimal. It is worth
mentioning that the cross-linking reaction can be completed
within 5 min, which is much faster than that reported in other
literatures.[33,40]
3.2. CCS Characterizations
Figure 3 shows the morphology of native starch and CCS-1,
CCS-2, CCS-3. The morphology of the native starch granules
were sphere or hemisphere, with an average size of 15 mm. After
1800273 (5 of 9) © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com www.starch-journal.com

Table 2. The orthogonal layout and the results of experiments. roughness of CCS could be attributed to the reaction between
starch and (NaPO3)6. In addition, the AFM analysis verified the
Number A B C D E S results of SEM.
1 25 24:300 7 7 5 5.13 XRD is an effective means by which to study the crystalline
properties of starch. The XRD diffraction patterns of the native
2 25 24:400 8 8 7 5.30
starch and cross-linked starches are shown in Figure 4(a).
3 25 24:500 9 9 9 5.20 Diffractogram of native starch and cross-linked starches had
4 25 24:600 10 10 11 5.27 peaks at angles 15 , 17 , 18 , and 23 (2θ), which indicated that
5 30 24:500 8 10 5 4.73 the starches might be the A-type structure.[42,43] The peak
intensity decreased, and the dispersion diffraction peak
6 30 24:600 7 9 7 5.13
increased, which indicated that both the native starch and the
7 30 24:300 10 8 9 5.33
CCS are composed of a crystalline region and an amorphous
8 30 24:400 9 7 11 5.03 region. According to our results, the crystallinity of native starch,
9 35 24:600 9 8 5 5.07 CCS-3, CCS-2, CCS-1 were 25.44, 24.54, 24.18, 22.18%,
10 35 24:500 10 7 7 3.67
respectively, as calculated using MDI jade 6.0. This might be
because the cross-linking reaction mainly happened in the
11 35 24:400 7 10 9 5.67
amorphous region and thus the crystal structure changed little.
12 35 24:300 8 9 11 5.13 Therefore, we supposed that the cross-linking reaction firstly
13 40 24:400 10 9 5 5.07 happened in amorphous and then in crystalline region.[44] All of
14 40 24:300 9 10 7 3.87 these observations were consistent with the results obtained
from the SEM assay mentioned above and in agreement with the
15 40 24:600 8 7 9 4.07
study by Liu.[51]
16 40 24:500 7 8 11 4.33 As seen from Figure 4(b), there is no significant difference
K1 4.89 5.14 4.94 5.53 5.00 between native starch and CCS except for the bands at 1013
K2 4.95 4.73 5.19 5.00 5.51 and 3400 cm1. We found an asymmetry at 3400 cm1 in the
K3 5.13 5.64 5.33 4.99 5.05
CCS spectra, which might indicate the substitution of the
OH groups. The band at 2931 cm1 was strong and sharp,
K4 5.67 5.12 5.17 5.12 5.06
representing the antisymmetry stretching vibration of the
R 0.78 0.91 0.39 0.54 0.51 carbon-hydrogen bond (–CH2). In general, the telescopic
vibration of P5 5O appears at 1300–1150 cm1, and the
chemical bond of COP exhibits strong absorption at
cross-linking, it can be seen that there are tiny cracks on the 1050–970 cm1.[45] In our study, the absorbance of P5 5O
surface of the cross-linked starches (Figure 3B–D) and as the (1300–1150 cm1) was much weaker than POC
degree of cross-linking increases, surface of CCS particles (1013 cm1). It has been shown in previous studies that
become rough. These results were consistent with those of some bands are highly sensitive to polymer conformations
Majzoobi.[41] within 950–1200 cm1.[46,47] The band at 1013 cm1 is
The AFM technique is a powerful tool for studying surfaces characteristic of the amorphous structure in starch, which
that are often inaccessible using other experimental technique. is characteristic of amorphous structure in starch, which
Figure 3(E1–F4) show two-dimensional AFM images of the increases with decreasing crystallinity and is commonly used
derivative surfaces of the native starch (Figure 3E1–E4) and CCS to judge the mutual transformation between the crystalline
(Figure 3F1–F4) prepared under the optimal conditions taken in and the amorphous state in the starch molecules.[48] The
the ScanAsyst mode. Table 4 lists the average roughness (Ra) of absorbance ratio of 1050/1013 cm1 could be used to quantify
the native starch and CCS. The surface of the native starch was the degree of order in the starch samples.[49] In our study, the
smooth, and the values of Ra under different scanning areas CCS band at 1013 cm1 was somewhat greater than that of
were 3.37, 4.32, 6.47, 10.8 nm. CCS was much rougher native starch, which indicated a decrease in the crystallinity.
(Ra ¼ 7.64, 9.39, 20.3, and 75.5 nm) than native starch. The These results showed that the cross-linking reaction was
height difference of CCS under 2.2  2.2, 3  3, 4.1  4.1, successful, which was in agreement with the results of the
5  5 mm2 areas were 66.8, 87.9, 199.5, 1131.1 nm, respectively, XRD analysis and consistent with the results obtained by Gao
which were larger than those of native starch. The greater et al.[50] and Liu et al.[51]
A TGA chart of native starch and CCS prepared under the
optimum conditions is shown in Figure 4(c). In the case of native
starch, degradation started at a temperature of 275  C. But for
Table 3. Orthogonal verification experiment. CCS, the degradation temperature started at a higher tempera-
ture of 299  C. Temperature of native starch and CCS with 5%
Number A B C D E S Relative deviation (%) weight loss (T5) was almost the same 85  C which could be
1 3.52 0.19 attributed to the evaporation of free-water in the same
molecules,[52] and the temperature corresponding to 10% weight
2 25 24:400 7 9 5 3.50 0.19
loss (T10) of native starch and CCS were 254 and 272  C,
3 3.52 0.19
respectively. The maximum thermal degradation temperature

Starch - Stärke 2019, 71, 1800273 1800273 (6 of 9) © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com www.starch-journal.com

Table 4. Average roughness (Ra) and height difference were measured by atomic force microscopy (AFM) of the native starch and CCS.

Native starch CCS

Scanning area Ra (nm) Height difference (nm) Ra (nm) Height difference (nm)

2.2  2.2 μm 2
3.37 30.1 7.64 66.8
3  3 μm2 4.32 41.6 9.39 87.9
4.1  4.1 μm2 6.47 66.1 20.3 199.5
5  5 μm2 10.8 109.4 75.5 1131.1

(Tmax) for native starch and CCS were 315 and 335  C.
Moreover, the remaining weight of the CCS was 16.98 wt.% at
700  C, which was higher than that of native starch. Therefore,
the thermal stability of CCS enhanced which makes it
appropriate to be used in different industries like food and
pharmaceutical industry in various ways. Akhilesh[53] also
reported that cross-linked starch exhibited higher thermal
stability.
3.3. Analysis of the Freeze-Thaw Stability (Syneresis)
Freeze-thaw stability is an important indicator that affects the
quality of starch-based frozen foods,[54] and the development
of starch-based food processing industry.[55] During the
storage process of starch paste, water is separated as the
starch ages. Water separation is an important indicator by
which to judge the freeze-thaw stability of starch products.
The less the water separation, the higher the freeze-thaw
stability.[56] As shown in Figure 5(a), water separation
from gelatinized native starch paste increased from 24.72
to 67.85% after one to six freeze-thaw cycles, but for CCS-1,
CCS-2, and CCS-3, the water separation decreased. In
particular, CCS-3, which had a lower DS, showed a higher
freeze thaw stability than those of CCS-1 and CCS-2. This
indicates that CCS has a good freeze-thaw stability, which
was also attributed to the introduction of phosphate groups
that were positively charged and repellent to each other.
Therefore, the CCS paste could form a stable three-
dimensional gel after freezing. These results were consistent
with those of Chen.[57]
3.4. Analysis of Acid Resistance
Figure 5(b) shows the results of acid resistance of native starch
and CCS at different DS. These results showed that the viscosity
of the native starch significantly decreased after acid treatment,
whereas that of CCS remained basically unchanged. In addition,
the native starch was sensitive to acids, which accelerated particle
breakage and caused a significant drop in viscosity. On the other
hand, CCS formed an inorganic, highly stable ester bond against
the actions of the acid; therefore, it maintained a certain
structural stability in the acidic environment, and the acid
resistance enhanced as the values of DS increased, which
indicated that CCS was more suitable than the native starch for
use in developing acidic drinks.
3.5. Analysis of the Thermal Stability of CCS Paste
Whether the cross-linked starch can maintain a good paste
viscosity at different temperatures is directly related to the
quality of the product.[58] Therefore, it is valuable to study the
thermal stability of cross-linked starch paste. We see from
Figure 5(c) that the viscosity of the native starch paste
significantly changed with temperature, whereas that of CCS
paste was relatively stable with temperature and its viscosity
was positively correlated with the DS values. This character-
istic could be caused by the strong chemical bond resulting
from the cross-linking reaction, which inhibits expansion,
cracking and viscosity reduction in the particles. In addition,
the inhibition is more pronounced as the CCS DS values
increase.

Figure 4. a) X-ray diffraction of native starch and CCS-1, CCS-2, CCS-3; (b) FTIR of native starch and CCS prepared under the optimal conditions (starch
concentration ¼ 25%, microwave-ultrasound power ratio ¼ 24:400 W, reaction time ¼ 7 min, pH ¼ 9, (NaPO3)6 volume ¼ 5 mL); (c) TGA curves of
native starch and CCS prepared under the optimal conditions (starch concentration ¼ 25%, microwave-ultrasound power ratio ¼ 24:400 W, reaction
time ¼ 7 min, pH ¼ 9, (NaPO3)6 volume ¼ 5 mL).

Starch - Stärke 2019, 71, 1800273 1800273 (7 of 9) © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
Figure 5. a–c) Freeze-thaw stabilities, acid resistance, and viscosity thermal stabilities of native starch and CCS-1, CCS-2, CCS-3.
4. Conclusion
In this research, we developed a rapid and facile microwave–
ultrasound method that can assist in the preparation of CCS.
The optimal experimental scheme and the influence of
various factors on the results were obtained through
orthogonal testing. The optimum conditions for the prepared
CCS were as follows: starch concentration ¼ 25%, microwave–
ultrasound power ratio ¼ 24:400 W, pH ¼ 9.0, (NaPO3)6 ¼ 5
mL, and reaction time ¼ 7 min. The order of influence on the
DS of each factor was as follows: microwave–ultrasound
power ratio > starch concentration > pH > (NaPO3)6 volume
> reaction time.
The FTIR analysis revealed that the amount of CCS crystalline
starch changed. The XRD results showed that, compared with
the native starch, the crystal form of CCS did not change, but the
relative crystallinity decreased. The SEM and AFM analysis
revealed that CCS was spherical with rough surface. The TGA
results confirmed the thermal stability of cross-linked starch. In
addition, its freeze-thaw stability, acid resistance, and thermal
stability were greatly improved, These characteristics indicate
that CCS has great potential for application in the food industry
and that it can be used for processing quick-frozen foods and
improving the quality of the products. Cross-linking reaction
could be completed within 5 min using the microwave–
ultrasound technology. In summary, using the microwave–
ultrasound technology is a highly effective method by which to
prepare CCS.
Abbreviations
AFM, atomic force microscopy; CCS, cross-linked cassava starch; CCS-1,
CCS-2 and CCS-3 represent samples with a sedimentation volume of 3.5,
4.07, and 5.07 mL, respectively; DS, degree of substitution; FTIR, Fourier
transform infrared spectroscopy; SEM, scanning electron microscopy;
TGA, thermogravimetric analysis; XRD, X-ray diffraction.
Acknowledgments
The author would like to thank the Guangxi Key Laboratory for
Polysaccharide Materials and Modifications, Key Laboratory of Chemical
and Biological Transformation Process of Guangxi Higher Education
Institutes, Guangxi University for Nationalities. This work was supported
financially by the Guangxi Biological Polysaccharide Separation,
Starch - Stärke 2019, 71, 1800273 1800273 (8 of 9)
www.starch-journal.com
Purification and Modification Research Platform (GKZY18076005), Key
Project of Graduate Education Innovation Program of Guangxi University
for Nationalities (gxun-chxzs2017127; gxun-chxzs2018049).
Conflict of Interest
The authors declare no conflict of interest.
Keywords
characterization, cross-linked cassava starch, microwave-ultrasound,
physicochemical properties
Received: September 30, 2018
Revised: December 14, 2018
Published online: April 9, 2019
[1] S. N. Moorthy, Starch/Stärke 2015, 54, 559.
[2] D. d. Souza, A. F. Sbardelotto, D. R. Ziegler, L. D. F. Marczak,
I. C. Tessaro, Food Chem. 2016, 191, 36.
[3] H. F. Zobel, Starch/Stärke 2010, 40, 44.
[4] D. Hwang, B. Kim, M. Bail, Starch/Stärke 2010, 61, 438.
[5] M. Witczak, L. Juszczak, R. Ziobro, J. Korus, Food Hydrocolloid. 2012,
28, 353.
[6] H. X. Xiao, Q. L. Lin, G. Q. Liu, F. X. Yu, Molecules 2012, 17, 10946.
[7] F. W. Xie, L. Yu, H. S. Liu, L. Chen, Starch/Stärke 2010, 58, 131.
[8] Y. J. Zhang, Z. Q. Huang, C. Yang, A. Huang, H. Y. Hu, Z. Q. Gong,
G. S. Sun, K. Huang, Starch/Stärke 2013, 65, 461.
[9] X. Qi, R. F. Tester, Starch/Stärke 2016, 68, 807.
[10] N. R. d. Oliveira, B. Fornaciari, S. Mali, G. M. Carvalho, Starch/Stärke
2018, 70, 1700170.
[11] M. Zhou, L. Shi, F. Cheng, Y. Lin, P. X. Zhu, Starch/Stärke 2018, 70,
1700250.
[12] X. X. Yan, Y. J. Chang, Q. Wang, Y. J. Fu, L. L. Ren, J. Zhou, Starch/
Stärke 2018, 70, 1700213.
[13] M. Saha, C. Eskicioglu, J. Marin, Bioresour. Technol. 2011, 102, 7815.
[14] G. X. Xing, S. F. Zhang, B. Z. Ju, J. Z. Yang, Starch/Stärke 2010, 58,
464.
[15] Y. Wu, X. F. Du, H. H. Ge, Z. Lv, Starch/Stärke 2011, 63, 217.
[16] M Sujka, J. Jamroz, Food Hydrocolloid. 2013, 31, 413.
[17] C. Leonelli, T. J. Mason, Chem. Eng. Process. 2010, 49, 885.
[18] Y. J. Wang, L. F. Wang, Starch/Stärke 2015, 52, 406.
[19] N. S. Yussof, U. Utra, A. K. Alias, Starch/Stärke 2013, 65, 285.
[20] B. L. Kuniak, R. H. Marchessault, M. Que, Starch/Stärke 2010, 24,
110.
© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com www.starch-journal.com

[21] P. V. Hung, N. Morita, Carbohydr. Polym. 2005, 59, 239. [42] Y. R. Zhang, X. L. Wang, G. M. Zhao, Y. Z. Wang, Carbohydr. Polym.
[22] N. Reddy, Y. Yang, Food Chem. 2015, 118, 702. 2012, 87, 2554.
[23] S. J. Jun, K. Y. Lee, H. G. Lee, Starch/Stärke 2014, 66, 468. [43] S. Dhital, A. K. Shrestha, J. Hasjim, M. J. Gidley, J. Agr. Food Chem.
[24] S. Matsumoto, R. J. Chirstie, N. Nishiyama, K. Miyata, A. Ishii, 2010, 60, 634.
M. Oba, H. Koyama, Y. Yamasaki, K. Kataoka, Biomacromolecules [44] H. Fredriksson, J. Silverio, R. Andersson, A. C. Eliasson, P. Aman,
2009, 10, 119. Carbohydr. Polym. 1998, 35, 119.
[25] C. Saikia, A. Hussain, A. Ramteke, H. K. Sharma, T. K. Maji, Starch/ [45] D. M. Suflet, G. C. Chitanu, V. I. Popa, React. Funct. Polym. 2006, 66,
Stärke 2014, 66, 760. 1240.
[26] S. L. Wang, F. F. Zhang, F. S. Chen, Z. Q. Pang, BioResources 2013, 8, [46] R. H. Wilson, P. S. Belton, Carbohydr. Res. 1988, 180, 339.
3574. [47] R. H. Wilson, M. T. Kalichevsky, S. G. Ring, P. S. Belton, Carbohydr.
[27] H. J. Liu, L. Ramsden, H. Corke, Starch/Stärke 1999, 51, 249. Res. 1987, 166, 162.
[28] K. Feng, G. Wen, Int. J. Polym. Sci. 2017, 58, 275. [48] B. Z. Li, L. J. Wang, D. Li, Y. L. Chiu, Z. J. Zhang, J. Shi, X. D. Chen,
[29] M. M. Shi, F. Gu, J. C. Wu, S. J. Yu, Q. Y. Gao, Starch/Stärke 2013, 65, Z. H. Mao, J. Food Eng. 2009, 92, 255.
947. [49] Z. Z. Wang, B. Chen, X. Zhang, Y. Li, W. Z. Fang, X. T. Yu, L. P. Dang,
[30] T. T. Kou, Q. Y. Gao, Carbohyd. Res. 2018, 458, 13. Starch/Stärke 2016, 68, 1.
[31] A. V. Singh, L. K. Nath, M. Guha, R. Kumar, Pharmacol. Pharm. 2011, [50] F. Gao, D. Li, C. H. Bi, Z. H. Mao, B. Adhikari, Carbohydr. Polym.
2, 42. 2014, 103, 310.
[32] Q. Lin, J. X. Pan, Q. L. Lin, Q. J. Liu, J. Hazard. Mater. 2017, 2, 150. [51] J. Liu, B. Wang, L. Lin, J. Y. Zhang, W. L. Liu, J. H. Xie, Food
[33] M. J. Jivan, A. Madadlou, M. Yarmand, Food Chem. 2013, 141, 1661. Hydrocolloid. 2014, 36, 45.
[34] M. Majzoobi, B. Sabery, A. Farahnaky, T. T. Karrila, Iran. Polym. J. [52] Q. Sun, G. Min, Y. Li, L. Xiong, Carbohydr. Polym. 2014, 111, 133.
2012, 21, 513. [53] A. V. Singh, L. K. Nath, Int. J. Biol. Macromol. 2011, 50, 14.
[35] Q. Chang, X. Hao, L. L. Duan, J. Hazard. Mater. 2008, 159, 548. [54] S. Puncha-Arnon, W. Jiranuntakul, D. Uttapap, Carbohydr. Polym.
[36] W. Wang, L. Guan, P. Seib, Y. Shi, Carbohydr. Polym. 2018, 2015, 134, 344.
196, 18. [55] J. P. Ye, R. Yang, C. M. Liu, S. J. Luo, J. Chen, X. T. Hu, J. Y. Wu, Food
[37] H. Tang, M. Sun, Y. Li, S. Dong, Polym. Bull. 2013, 70, 3331. Chem. 2018, 268, 324.
[38] N. H. Cheetham, L. Tao, Carbohydr. Polym. 1998, 36, 277. [56] P. J. White, I. R. Abbas, L. A. Johnson, Starch/Stärke 2010, 41,
[39] S. Nara, T. Komiya, Starch/Stärke 1983, 35, 407. 176.
[40] A. N. Jyothi, S. N. Moorthy, K. N. Rajasekharan, Starch/Stärke 2010, [57] B. Chen, L. Dang, X. Zhang, W. Fang, M. Hou, T. K. Liu, Z. Z. Wang,
58, 292. Food Chem. 2017, 219, 93.
[41] M. Majzoobi, N. Seifzadeh, A. Farahnaky, G. Mesbahi, J. Texture [58] X. Fu, Q. Huang, F. X. Luo, J. South China Univ. Technol. 2007, 35,
Stud. 2015, 46, 105. 91.

Starch - Stärke 2019, 71, 1800273 1800273 (9 of 9) © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim