International Journal of Biological Macromolecules 103 (2017) 656662

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International Journal of Biological Macromolecules

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

Preparation and characterization of non-crystalline granular starch

and corresponding carboxymethyl starch
Bao Zhang a , Xiaomin Li a , Qiutao Xie c , Han Tao a,b, , Wu Wang a , Han-Qing Chen a,
a
School of Food Science and Engineering, Hefei University of Technology, 193 Tunxi Road, Hefei, Anhui 230009, PR China
b
School of Biological and Medical Engineering, Hefei University of Technology, 193 Tunxi Road, Hefei, Anhui 230009, PR China
c
Hunan Agricultural Product Processing Institute, Hunan Academy of Agricultural Sciences, Changsha, PR China

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

Article history: Native corn starch slurried in 50% ethanol solution was treated at 60 C, 70 C, 80 C, and 85 C, respec-
Received 15 February 2017 tively. The resultant starches were investigated by polarized microscopy, scanning electron microscopy
Received in revised form 7 May 2017 (SEM), differential scanning calorimetry (DSC), and X-ray diffractometry (XRD). The Maltese cross of
Accepted 22 May 2017
ethanol-heating treated starch gradually weaken with increasing temperature and completely disap-
Available online 22 May 2017
peared at 85 C. SEM data indicated the treated granular exhibited a rougher surface with more pores
and grooves than native starch granular, but the shape of the treated starch was still intact. DSC and
Keywords:
XRD data conrmed ethanol-heating treated starch changed from crystalline to non-crystalline struc-
Non-crystalline granular starch
Ethanol-heating treatment
ture at 85 C. The highest degree of substitution (DS) and corresponding reaction efciency (RE) for the
Carboxymethyl starch carboxymethylation of native starch were 0.66 and 36.7%, respectively, while the corresponding DS and
RE for non-crystalline granular starches increased by 0.29 and 16.1% at the same reaction condition,
respectively.
2017 Elsevier B.V. All rights reserved.

1. Introduction
Starch is the most abundant storage carbohydrate in the bio-
sphere and the main source of energy in human diets [13]. It is
also a kind of low-costly renewable and biodegradable raw mate-
rial with distinct physiochemical properties [4,5]. These qualities
account for increasing applications of native starch on various
industries for food and non-food utilizations. However, the appli-
cation of native starches has been limited in the food industry for
several factors, such as retrogradation, instability of paste under
shearing, acid or freezing, and poor paste clarity et al. [68]. There-
fore, most native starches are required to be modied chemically
and/or physically to enhance their functional properties and/or
to minimize their defects [9]. Starch derivatives are used in food
products as thickeners, gelling agents, encapsulating agents, in
papermaking as wet-end additives for dry strength, surface sizes,
and coating binders et al. [1013].
Among these starch derivatives, carboxymethyl starch (CMS)
as a kind of etheried starch has attracted a lot of attention in
both research and industry [14]. The presence of functional group
Corresponding authors.
E-mail addresses: nancyfoodscience@hotmail.com (H. Tao),
hanqchen@hfut.edu.cn (H.-Q. Chen).
(CH2 COO ) yields starch with many unique properties, such as low
gelatinization temperature, excellent exibility, improved paste
storage stability and clarity [15]. Therefore, the CMS is applied
to wide industry such as thickening of food, drug release, tex-
tile sizing, paper coating, and for water-based adhesives [14,16].
However, the degree of substitution of CMS was limited due to
crystallinity of starch, which decreased reaction efciency of starch
derivatives. Non-crystalline granular starch, an intermediate state
between native semi-crystal structure and paste, is a peculiar form
of starch, which shows a granular shape but without crystalline
structure [17]. This new type of starch has many merits, such as, its
physical and chemical reaction might be accelerated. We suppose
that the non-crystalline granular starch may increase the reactiv-
ity of the starch granules and make the sodium monochloroacetate
easier to access to the starch granules.
Ethanol can be able to complete with starch granular to
absorb water, so the granular presents limited swelling and can
obtain granular shape when heating above gelatinization tem-
perature. Therefore, the objective of the present works was to
prepare non-crystalline granular starch using ethanol-heating
treatment, and resultant starches were investigated by polar-
ized microscopy, scanning electron microscopy (SEM), differential
scanning calorimetry (DSC), and X-ray diffractometry (XRD). Fur-

http://dx.doi.org/10.1016/j.ijbiomac.2017.05.131
0141-8130/ 2017 Elsevier B.V. All rights reserved.
 B. Zhang et al. / International Journal of Biological Macromolecules 103 (2017) 656662
thermore, the CMS was synthesized compared non-crystalline
granular starch with native starch.
2. Materials and methods
2.1. Materials
Corn starch was purchased from Puluoxing Starch Co., Ltd.
(Hangzhou, China). All other chemicals and reagents were of ana-
lytical grade and purchased from Sinopharm Chemical Reagent Co.,
Ltd. (Suzhou, P.R. China).
2.2. Preparation of non-crystalline granular starch
Non-crystalline granular starch was prepared according to pre-
vious method with slight modication [18]. Native corn starches
(100 g, dry basis) were added to a three-necked round-bottom ask
with 400 mL 50% ethanol solution equipped with motor-driven stir-
rer, which was heated in a water bath set at 60 C, 70 C, 80 C,
and 85 C for 2 h, respectively. The treatment was quenched by the
addition of 500 mL ethanol. After cooling to room temperature, the
starch slurry was ltered with lter paper. The residue was fur-
ther washed 3 times with 2 L of ethanol. The residual starch was
dried in a vacuum oven for 24 h at 40 C, and then passed through
a 100-mesh screen.
2.3. Light microscopy
The morphological structure of the starch samples was observed
using an Olympus BX41TF light microscope (Olympus Co., Tokyo,
Japan). The native and different temperature treated starches were
dispersed in deionized water. A drop of starch dispersion was
placed in the middle of the microscope slide with a cover glass
thereon, which was observed with normal light and polarized light,
and the images were recorded at 400 magnication.
2.4. Particle size distribution of starches
The size distribution of native and different temperature treated
starches in suspension was determined using a Malvern Master
Sizer 2000 (Malvern, UK). The sizes of the samples in suspension
were measured in deionized water at 25 C. The weight concen-
tration of the measured particles was 0.05 mg/mL. Before the
measurement, the suspensions were sonicated for 15 min to obtain
nely dispersed starch particles. The measurements were done in
triplicate.
2.5. Field emission scanning electron microscopy (FE-SEM)
The samples of native and different temperature treated
starches were xed on aluminum specimen stubs with double-
sided adhesive tape, coated with a thin lm of gold (10 nm), and
placed in a vacuum evaporator. The samples were observed in a
SEM (Hitachi S-4800; Hitachi Company, Japan) at an accelerating
voltage of 5 kV.
2.6. Differential scanning calorimetry (DSC)
A differential scanning calorimeter (Seiko Instruments, Inc.,
Chiba, Japan) was used to measure thermal properties of native
and different temperature treated starches according to previ-
ous reported method with a slight modication [19]. The samples
(about 2 mg) and deionized water (two times weight of the starch)
were mixed and hermetically sealed in an aluminum pan. All sam-
ples were allowed to equilibrate for 24 h at 4 C. The gelatinization
properties of starches were determined by heating the pans at a
657
constant rate of 10 C/min from 30 C to 100 C under a continuous
ow of dry N2 gas with an empty pan sealed as a reference.
2.7. X-ray patterns
XRD analyses of native and different temperature treated
starches were carried out on a Bruker D8 Focus X-ray power diffrac-
tometer (Bruker AXS, Inc., Karlsruhe, Germany) with Nickel ltered
Cu-Ka radiation at a voltage of 40 kV and current of 30 mA as
described previously [19]. All samples were stored in a dessicator,
wherein a saturated solution of NaCl maintained the atmosphere at
constant humidity. Samples were stored in the dessicator for sev-
eral days at 25 C before measurements. The relative crystallinity of
the starches and diffraction angle of X-ray pattern were analyzed
by Jade 5.0 software (Materials Data, Inc., CA, USA).
2.8. Preparation of carboxymethyl starch
Carboxymethylation of native and non-crystalline granular
starches was carried out in 90% isopropanol solution according
to previous reports with slight modication [14]. Starch sam-
ple (50 g dry weight) was added to three-necked round-bottom
ask with 450 mL 90% isopropanol solution, equipped with motor-
driven stirrer. The temperature was maintained at 40 C, then
sodium hydroxide (22 g) was added to isopropanol solution in the
ask and the mixture was stirred at 250 rpm for 30 min. Sodium
monochloroacetate was added to the mixture and reaction time
was 4 h. At the end of reaction, the starch slurry was ltered, sus-
pended in methanol and neutralized with acetic acid. Following
ltration, the slurry was dispersed again in 80% methanol and it was
washed several times with 80% methanol until the silver nitrate
test for chloride of the ltrate was negative. The slurry obtained
was suspended in acetone, stirred for 20 min, and dried in an oven
at 40 C for 24 h. The degree of substitution (DS) and reaction ef-
ciency (RE) of resultant starch samples were determined according
to the methods described in previous reports [20].
2.9. Statistical analysis
All experiments described above were performed at least in trip-
licates, and the results presented were the means and standard
deviations of each experiment. The signicant differences between
the results were determined by analysis of variance (ANOVA).
3. Results and discussion
3.1. Light microscopy
Fig. 1 showed the polarized light (Left) and normal light (Right)
of native and different temperature treated starches. Birefringence
was a symbol of the average radial orientation of helical struc-
tures [21]. The native starch exhibited a typical birefringence cross
under polarized light, since the ordered starch (crystallites, etc.) and
the disordered starch generated the differed density and refractive
index in the starch granular [22]. Starch granular partially swelled
at 60 C and 70 C, and the Maltese cross still existed. Maltese cross
completely disappeared at 85 C, which indicated the ordered semi-
crystalline nature of starch was destroyed [23]. Although crystalline
structure dramatically changed, the integrity of starch granular still
was observed due to that ethanol could limit the starch granular
swelling.
3.2. Size distribution of starches
Water molecules formed hydrogen bonds with the exposed
hydroxyl groups of amylose and amylopectin in starch, causing
658 B. Zhang et al. / International Journal of Biological Macromolecules 103 (2017) 656662

Fig. 1. The polarized light (Left) and normal light (Right) of native and different temperature treated starches: (A, a) Native starch; (B, b) 60 C; (C, c) 70 C; (D, d) 80 C; (E,
e) 85 C.
 B. Zhang et al. / International Journal of Biological Macromolecules 103 (2017) 656662 659

Fig. 2. Size distribution of native and different temperature treated starches.

swelling of the starch granular [24]. Size distribution of native
and different temperature treated starches was investigated by
a Malvern Master Sizer 2000 (Fig. 2). The curves of particle size
distributions became larger with increasing temperature because
hydration of starch granular (swelling) could be induced by heating.
3.3. Field emission scanning electron microscopy (FE-SEM)
SEM micrographs of native and different temperature treated
starches were shown in Fig. 3. Some morphological modications
were observed on the surface of the starch granular treated with
different temperature, but these changes did not signicantly affect
the shape of the granular. The granular exhibited a rougher surface
with more pores and grooves than the native starch granular. The
surface layer was peeled off after ethanol-heating treatment, indi-
cating that surface lipids were removed [25]. Swelling of granular
was positively correlated with ethanol-heating treatment temper-
ature, but the integrity of the starch granular was still observed
[23]. This phenomenon could be attributed to that ethanol was able
to protect starch granular from swelling while thermal treatment
could destroy crystalline structure of the granular, which was in
agreement with the results of light microscopy.
3.4. Differential scanning calorimetry (DSC)
The DSC curves of native and different temperature treated
starches were presented in Fig. 4. The onset (To ) temperature of
gelatinization gradually increased with increasing ethanol-heating
660 B. Zhang et al. / International Journal of Biological Macromolecules 103 (2017) 656662

Fig. 3. SEM micrographs of native and different temperature treated starches: (A) native starch; (B) 60 C; (C) 70 C; (D) 80 C; (E) 85 C.

treated temperature, which indicated that thermal treatment change could not be determined, which indicated crystalline region
rstly destroyed the weaker crystalline structure of starch gran- was completely destroyed and had been transformed into amor-
ular. The conclusion (Tc ) temperature of gelatinization maintained phous region.
unchanged, suggesting that the strongest crystalline structure of
the starch granular was not inuenced. When the temperature was
3.5. X-ray patterns
85 C, To and Tc were disappeared, which claimed that there was
no crystalline structure in the starch granular. The gelatinization
X-ray diffraction patterns of native and different temperature
enthalpy change also decreased with the increase of tempera-
treated starches were shown in Fig. 5. Native corn starch with a
ture. When the temperature was 85 C, the gelatinization enthalpy
typical A-type crystalline polymorphic structure was characterized
 B. Zhang et al. / International Journal of Biological Macromolecules 103 (2017) 656662 661

Native starch

o
60 C

o
70 C

o
80 C

o
85 C

40 60 80 100
Fig. 5. X-ray diffraction patterns of native and different temperature treated
o
Temperature ( C) starches.

Fig. 4. DSC curves of native and different temperature treated starches.

by clear diffraction peaks around 2 = 15 , 17 , 18 , and 23 [26].
The intensity of the diffraction peak decreased with the increase of
temperature because thermal treatment gradually melt the double-
helical crystalline structure. When the treated temperature was
85 C, X-ray diffraction patterns of starch transferred from typical
A-type diffraction peak to dispersion type diffraction peak, sug-
gesting there was no the crystalline area inside the granular. These
results were in agreement with the results of DSC which indicated
the gelatinization enthalpy change was zero at 85 C. The results
also suggested that the loss of crystallinity could be due to the rup-
ture of starch granular, which in the presence of water together
with heating treatment caused the breakage of chemical bonds in
starch molecules [27]. Furthermore, the peaks at 20 for different
temperature treated samples disappeared, which could be ascribed
to the loss of starch lipids after treating, since the peaks at 20 was
a V-characteristic of amylose-lipid complex [28,29].
3.6. Preparation of carboxymethyl starch
The effects of the molar ratio of sodium monochloroacetate
to anhydroglucose unit (nSMCA /nAGU ) on the DS and the RE for
the carboxymethylation of native and non-crystalline granular
starches were presented in Fig. 6. The DS increased with increas-
ing nSMCA /nAGU while a decline in the RE was observed as the
nSMCA /nAGU ratio increased before 1.8, which was attributed to
that the increasing concentration of SMCA resulted more contact
between starch and etherifying agent [27]. The highest DS and
corresponding RE for the carboxymethylation of non-crystalline
granular starches were 0.95 and 52.8%, respectively, however,
the corresponding DS and RE for native starch were 0.66 and
36.7% at the same molar ratio of sodium monochloroacetate to
anhydroglucose unit, respectively. These results indicated that the
non-crystallization process of native starch was benecial to syn-
thesize CMS.
4. Conclusions
Native corn starch was slurried in 50% ethanol solution treated
with different temperature treatment. The resultant starches were
investigated by polarized microscopy, SEM, DSC, and XRD. It could
be included that ethanol-heating treatment method could lead
to our objective non-crystalline granular starches: with integrity
granular shape, while without the crystalline area inside the gran-
ular. Change from crystalline to non-crystalline structure was
conrmed by DSC and XRD. The highest DS and corresponding
RE for the carboxymethylation of non-crystalline granular starch

100
1.0 Nonocrystalline granular starch
Native starch Nonocrystalline granular starch
Native starch
0.8 80
RE (%)

0.6
60
DS

0.4
40
0.2

0.0 20
0 1 2 0 1 2

nSMCA/nAGU nSMCA/nAGU

Fig. 6. The effect of the molar ratio of sodium monochloroacetate to anhydroglucose unit on the degree of substitution (DS) and the reaction efciency (RE) for the
carboxymethylation of native and non-crystalline granular starches.
662 B. Zhang et al. / International Journal of Biological Macromolecules 103 (2017) 656662
were 0.95 and 52.8%, respectively. However, the corresponding
DS and RE for native starch were 0.66 and 36.7% at the same
molar ratio of sodium monochloroacetate to anhydroglucose unit,
respectively. These results indicated that the non-crystallization
process of native starch was benecial to synthesize CMS. This
non-crystalline granular starch was seen as a new raw material
to prepare various modied starch and products.
Acknowledgements
This study was nancially supported by the Natural Science
Foundation of Anhui Province (No. 1708085QC78) and National
Natural Science Foundation of China (No. 31601429).
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