International Journal of Biological Macromolecules 160 (2020) 437–445

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International Journal of Biological Macromolecules

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

Preparation and characterization of broken-rice starch nanoparticles

with different sizes
Huaxi Xiao a, Fan Yang b, Qinlu Lin a,⁎, Qian Zhang a, Lin Zhang a, Shuguo Sun a,
Wenfang Han a, Gao-Qiang Liu a,c,⁎
a
National Engineering Laboratory for Deep Processing of Rice and Byproducts, Central South University of Forestry & Technology, Changsha 410004, China
b
Shanxi Technology and Business College, Taiyuan 030006, China
c
Hunan Provincial Key Laboratory of Forestry Biotechnology & International Cooperation Base of Science and Technology Innovation on Forest Resource Biotechnology, Central South University of
Forestry & Technology, Changsha 410004, China

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

Article history: Broken-rice starch nanoparticles with different mean particle diameters for 100, 200, 400 and 800 nm were pre-
Received 23 March 2020 pared by nanoprecipitation, alkali freezing, cross-linking and H2SO4 hydrolysis methods respectively, and their
Received in revised form 12 May 2020 structural, morphological and physicochemical properties were systematically characterized. The results showed
Accepted 22 May 2020
that broken-rice starch nanoparticles had higher water absorption rate, and the maximum water absorption rate
Available online 27 May 2020
was obtained from the 100 nm starch granules being 91.53%, which means an increase about 2.07-fold in water
Keywords:
absorption rate as compared with native rice starch. The stability of native rice starch is the worst, but the viscos-
Broken rice ity characteristic value is always higher than that of starch nanoparticles in the whole gelatinization process. The
Starch nanoparticles FT-IR spectrum showed that only starch nanoparticles prepared by cross-linking method showed the character-
Particle diameter istic peak of secondary amide structure at 1714 cm−1, but the structure of was basically the same as native starch.
Water absorption The X-ray diffraction pattern revealed that there were obvious characteristic diffraction peaks near 2θ for 15°, 17°,
Crystalline structure 19° and 23° for the 800 nm starch nanoparticles and native rice starch, while the characteristic diffraction peaks
of other starch nanoparticles disappeared in varying degrees due to the changed crystal structure.
© 2020 Elsevier B.V. All rights reserved.

1. Introduction
In recent years, nanoparticle has become one of the hot spots in the
field of research because of its high specific surface area and quantum
size effect. Among them, biodegradable nanoparticles are widely used
in food, medicine, biotechnology and other fields because of their
good biodegradability. Starch is one of the three major nutrients, and
it is closely related to human life. As a typical biodegradable natural
polysaccharide, starch is a renewable substance produced by many
plants as a source of stored energy. Starch nanoparticles produced via
nano-technological processes have drawn more attention in recent
years because they provide greater opportunity for mass production
and are non-hazardous to human health [1]. These starch nanoparticles
have been widely used in food packaging [2], plastic fillers [3], diagnosis
and the treatment of cardiovascular diseases, and drug delivery [4,5]
due to their mechanical properties and renewable nature.
Starch nanoparticles have been prepared using a variety of methods
[6], including high pressure homogenization and emulsification [7], acid
⁎ Corresponding authors at: 498 Southern Shaoshan Road, Changsha 410004, China.
E-mail addresses: linedu2018@163.com (Q. Lin), gaoliuedu@csuft.edu.cn (G.-Q. Liu).
hydrolysis [8], extrusion [9], and nanoprecipitation [10]. The anti-
solvent nanoprecipitation is a simple, fast and reproducible method to
fabricate synthetic and natural polymer nanoparticles [11]. It typically
allows the preparation of very fine particles with an improved control
over particle properties, such as size, morphology and physical state,
and is suitable for producing nanoparticles from a range of different
food ingredients. Chin et al. [12] prepared starch nanoparticles by
anti-solvent precipitation from sago starch with the diameters in the
range of 300–400 nm. Tan et al. [13] also obtained nanosphere using
acetylated waxy maize starch by a nanoprecipitation process, and
found the mean diameter increased from 249 to 720 nm as the concen-
tration of the starch in the acetone raised from 1 to 20 mg/mL. Hebeish
et al. [14] produced SNPs by nanoprecipitation from the starch solution,
and found the diameters raised from 132 to 220 nm with the concentra-
tions of maize starch increased from 2.5% to 10%. Additionally, Juna et al.
[15] studied the influence of temperature (75, 100, and 130 °C) on the
shapes and sizes of waxy corn starch nanoparticles via
nanoprecipitation, and observed the sizes of starch nanoparticles de-
creased from 600 to 238 nm with increasing temperature. However,
up to now, the physical and chemical properties of starch nanoparticles
with different sizes prepared by different methods are still unclear,
which limits the development and application of starch nanoparticles.

https://doi.org/10.1016/j.ijbiomac.2020.05.182
0141-8130/© 2020 Elsevier B.V. All rights reserved.
438 H. Xiao et al. / International Journal of Biological Macromolecules 160 (2020) 437–445
China is a large rice producing country. It is inevitable that a large
amount of broken-rice will be produced in rice processing, with a con-
servative estimate of 30 million tons per year [16]. The added value of
the broken-rice is low, and the price is less than half of the ordinary
rice, so it will bring huge economic losses to farmers and rice processing
enterprises. In addition, the taste of broken-rice is not good, and it
should not be used as food directly. However, the ingredients of broken
rice are no different from the normal rice, and they have similar physical
and chemical properties and the integrity of starch particles [16], sug-
gesting it is a good resource for starch production.
Currently, regarding starch nanoparticles, most of the reported work
was done using normal maize or waxy maize [8,10,13,14] and waxy
corn or normal corn [6,10,15], but data on broken-rice starch nanopar-
ticles are relatively scarce. Recently we have developed a new
nanoparticulate system for acetylated starch nanocrystals (ASN) with
low DS (DS for 0.14) using broken-rice [16], and also have developed
hydrophobic films based on ASN with high DS using broken-rice for
controlled drug delivery. [17]. In this paper, we report on the synthesis
of broken-rice starch nanoparticles with different particle diameters
using nanoprecipitation, alkali freezing, cross-linking and H2SO4 hydro-
lysis methods, respectively. The structural, morphological and physico-
chemical properties of the rice starch nanoparticles were systematically
investigated, and the effects of different prepared methods on the phys-
icochemical properties of nanoparticles were analyzed and compared.
2. Materials and methods
2.1. Materials
Rice starch (amylose content was 23.5%) was extracted from
broken-rices. Other chemicals were purchased from NongHe Co. Ltd.
(Hunan province, China). All of the chemicals are of analytical grade
and used without further purification, and deionized water was used
throughout.
2.2. Preparation of starch nanoparticles
Starch nanoparticles for mean diameter 100 nm were prepared
using nanoprecipitation method. Starch solutions were prepared by dis-
solving 0.1 g of starch powder in 10 mL of dimethylsulfoxide (DMSO) at
100 °C for 1 h. An aliquot of starch solution (1 mL) at room temperature
was added dropwise into a fixed quantity of absolute ethanol (20 mL)
including 1 mL 4% span80 under moderate magnetic stirring for
30 min. The resulting mixture was then centrifuged with 3000 rpm
and rinsed three times with absolute ethanol to remove DMSO and
free surfactant. Then, the obtained starch nanoparticles were
lyophilized.
Starch nanoparticles for mean diameter 200 nm were prepared by
the alkali freezing treatment method [18]. Briefly, 10 g of dry starch
were dispersed into the prepared sodium hydroxide–urea aqueous so-
lutions with concentration 6:4:90 (NaOH:Urea:H2O), stirred for
10 min, stored in a refrigerator (−15 °C) for 24 h, and then thawed
with continuous stirring at 1000 rpm for 30 min at 0–4 °C to obtain ho-
mogeneous and viscous dispersions. After then, the viscous dispersions
were dialyzed against distilled water (Scheme 1). The final product,
starch nanoparticles were obtained by freeze drying.
Starch nanoparticles for mean diameter 400 nm were prepared
using cross-linking method. 1 g rice starch was dispersed in 50 mL dis-
tilled water, 1 drop of 2 mol/L NaOH was added, which were gelatinized
in boiling water bath for 10 min, and then ultrasonic vibration was con-
tinued for 30 min under the condition of 600 W ultrasonic power, and
cooled to room temperature. The mixed solution of 200 mL soybean
oil and 0.5% Span80 was stired in a constant temperature water bath
at 45 °C until Span80 is completely dissolved. Under the condition of
800 R/min mechanical agitation, 25 mL gelatinized starch solution was
added to the mixed solution of 400 mL soybean oil and Span80 drop
by drop, and 0.1 g crosslinker (N, N-methylenebisacrylamide) was
added, 30 min later, the initiator (0.4 g ammonium persulfate and
0.2 g sodium bisulfite) was added for another 30 min later, continue
the reaction for 2 h, centrifugation at 4000 R/min for 20 min, discard
the supernatant, precipitate was washed several times with absolute
ethanol, freeze-drying and seal storage.
Starch nanoparticles for mean diameter 800 nm were prepared
using H2SO4 hydrolysis method according to the procedure described
in the literature [8]. The typical preparation method is as follows:
36.73 g of starch were mixed with 250 mL of 3.16 M H2SO4 at 40 °C
and were poured into a flask with a continuous horizontal and circular
stirring (100 rpm) for 6 days. The suspension was washed by successive
centrifugations with distilled water until it reached neutrality. Then the
solvent exchange was done from distilled water to acetone by succes-
sive centrifugations at 8000 rpm and 10 °C for 15 min.
2.3. Particle size and distribution analysis
The volume mean diameter and particle size distribution of native
starch, starch nanoparticles were measured by laser light diffraction
particle size analyzer (Mastersizer 2000, Malven Instruments, UK).
The measurement procedure and principle were as follows: 1 g or so
of starch sample were immersed into 50 mL of anhydrous ethanol for ul-
trasonic dipersion so as to prevent agglomeration of sample. 500 mL of
anhydrous ethanol was added into a beaker as dispersing medium for
measuring the background concentration and then 30 mL of sample
was poured into another beaker and measured until the obscuration
seemed to be in the selected range, in the process of measurement,
the sample was circulated in company with liquid flow. The particle
size distributions were calculated from the intensity of light diffracted
at each angle using Mie theory. Refractive indices of ethanol and sample
used were 1.320 and 1.200 at room temperature, respectively. The ab-
sorbance of sample was taken as 0 [19]. Particle size characteristics
were indicated with Mastersizer 2000 Software v. 5.22. Results are
expressed as D10, D50 and D90, in which D10, D50 and D90 are partical
sizes (D) in microns at which accumulated particle size distribution per-
centages of the sample are 10%, 50% and 90%, respectively.
2.4. Scanning electron microscopy (SEM)
The surface morphologies of the starches and the starches films
without drug and BSA-loaded films after drug release were further eval-
uated by scanning electron microscopy (SEM). The samples were sprin-
kled onto a double-backed cellophane tape attached to a stub before
coating with gold palladium (thick 100 A). The specimens were exam-
ined with a JOEL–JEM 6380 LV (JOEL Ltd., Tokyo, Japan) scanning elec-
tron microscope at an accelerating voltage of 25 kV.
2.5. Determination of absorptive capacity for liquids
Starch (3 g) and liquid (10 mL, water) were mixed and stirred for
30 min at room temperature, and then the mixture was filtrated by vac-
uum filtration, placed on a filter. After dripping of the liquids ceased, the
weight of the liquid-impregnated sample was measured, from which
the adsorptive capacity of the starch for the liquids was calculated as
the weight of liquid-impregnated sample divided by the dry weigh of
the sample.
2.6. Determination of the stability and dispersion of the starches
Native rice starch and rice starch nanoparticles were dispersed into
PBS buffer solution in test tube respectively at a concentration of
4 mg/mL at 25 °C room temperature, the tube mouth was sealed and
tubes were put in the ultrasonic power of 200 W for 10 min, then
tubes were put on the test tube rack for 20d and 40d at room
 H. Xiao et al. / International Journal of Biological Macromolecules 160 (2020) 437–445 439

Scheme 1. Preparation of starch nanoparticles for mean diameter 200 nm

temperature, pictures were taken to illustrate the stability and disper- 2.9. Fourier transform infrared spectroscopy (FT-IR)
sion of starches.
FT-IR spectra of native and acetylated starch nanocrystals were mea-
sured using an FTIR spectrometer (NEXUS870，Nicolet, USA). The
2.7. Amylose contents determination
starch samples were collected using the KBr pellet method, the pow-
dered samples mixed with dry KBr in the ratio of 1:100. The scanning
Amylose contents were determined by fully automated flow injec-
wavenumber region was from 4000–400 cm−1 at a resolution of
tion system (FIAStar 5000, FOSS, Ltd., Sweden). This is a new type of
4 cm−1, the total scans were 64. The starch samples and KBr were
continuous flow analysis technique. This technology is that a certain
dried at 60 °C in a vacuum oven before measurement.
volume of sample solution is injected into a flowing and non-air spacing
reagent solution (or water) containing the stream according to com-
2.10. X-ray diffraction (XRD) analysis
pared method and the work curve drawn by standard solution mea-
sured the concentration of a substance in the sample solution. After
X-ray diffraction (XRD) analyses for the starch polymer fifilms and
defatting, spread the rice starch in a thin layer on a dish or watch glass
BSA powder were carried out on an X-ray diffractometer (D-Max-
or place them in paper bags and leave for 2 days in the same room to
2500, Rigaku, Japan) equipped with a copper tube operating at 40 kV
allow evaporation of residual methanol and for moisture content equi-
and 250 mA. Diffractograms were obtained, the scanning region of the
librium to be reached. Weigh in duplicate 100 ± 0.5 mg of the test sam-
diffraction angle (2θ) was from 4° to 50° at a rate of 4°/min, a step size
ple into 100 mL volumetric flasks. To the test portion, carefully add 1 mL
of 0.02°, 1°/1° divergence slit/scattering slit, 0.30 mm receiving slit.
of ethanol 95% (V/V) using a pipette, washing down any of the test por-
MDI Jade 5.0 was used to analyze the diffractograms.
tion adhering to the side of the flask. Add 9.0 mL of 1 mol/L sodium hy-
droxide solution using a pipette and mix. Then heat the mixture in a
2.11. Statistical analysis
boiling water bath for 10 min to disperse the amylose. Allow to cool to
room temperature without shaking for at least 2 h (or overnight).
Each measurement was carried out using at least three fresh, inde-
Make up to the metered volume with water and mix vigorously.
pendently prepared samples. The results were reported as the mean
value and standard deviation. The data were subjected to analysis of
2.8. Pasting properties variance (ANOVA) using the SPSS v.17.0 statistical software package
(SPSS Inc., Chicago, IL). Duncan's multiple range test was also used to
The pasting properties of rice starch were determined with the rapid determine the difference of the means from the ANOVA using a signifi-
visco analyzer (RVA super 4, Newport Scientific, Australia). Viscosities cance test level of 5% (p b 0.05).
of starches were recorded with starch suspensions (Moisture Content
12.0%, sample 3.00 g, water 25.00 mL). Underwent a controlled heating 3. Results and discussion
and cooling cycle under constant shear where the sample was held at
50 °C for 1 min, heated from 50 to 95 °C at 5 °C min−1 and held at 3.1. Morphologies of native starch granule and starch nanoparticles
95 °C for 2.7 min, cooled from 95 to 50 °C at 5 °C min−1 and held at
50 °C for 2 min. The initial speed of blender in 10s was 960 r/min, The morphologies of native starch granules and starch nanoparticles
after that it was maintained to 160 r/min. Pasting parameters such as prepared by nanoprecipitation (100 nm), alkali freezing (200 nm),
pasting temperature, peak viscosity, breakdown (peak viscosity-hot cross-linking (400 nm) and H2SO4 hydrolysis (800 nm) method were
paste viscosity), final viscosity, and setback (cold paste viscosity-peak examined by SEM (Fig. 1). SEM micrographs obviously demonstrate
viscosity) were recorded. that native starch granules were much larger than the starch
440 H. Xiao et al. / International Journal of Biological Macromolecules 160 (2020) 437–445
Fig. 1. SEM micrograph of starch and starch nanoparticles: (1) native starch, (2) 100 nm, (3) 200 nm, (4) 400 nm, (5) 800 nm.
nanoparticles. Native rice starch granules were observed to be mostly
oval and irregular granular shaped with a smooth surface when evalu-
ated using SEM, and a range in diameter of about 2–6 μm, the morphol-
ogy of starch nanoparticles prepared by nanoprecipitation and Alkali
freezing were mainly of irregular spherical and unclear shape with the
particle sizes ranging from 100 to 200 nm with some aggregation. It
was observed that the starch nanoparticles consisted of aggregates
properties, a result to hydrogen bond interactions via the surface hy-
droxyl groups [16]. The starch nanoparticle prepared by cross-linking
was emulsified from small microspheres to nanoparticles with rough
surface and irregular shape in oil phase. The morphology of starch nano-
particles prepared by acid hydrolysis showed cracks appear on the sur-
face with clear edges and corners due to the fact that the amorphous
area is destroyed after acid hydrolysis.
3.2. Particle size analysis
SEM images can provide visual comparison of starch particles at dif-
ferent size levels. In order to obtain a statistical result, the number inten-
sity distribution of the particle size was determined using the light
scattering method. The particle size distribution diagrams have been
presented in Fig. 2. In the diagrams, as indicated, the size distribution
changed with different preparing methods for starch nanoparticles.
The average size of native rice starch was around 2.3 μm, while the av-
erage sizes of starch nanoparticles prepared by nanoprecipitation, alkali
freezing, cross-linking and H2SO4 hydrolysis method were much
smaller than that of native starch, they were about 100 nm, 200 nm,
400 nm and 800 nm, respectively. These results suggested that
nanoprecipitation, alkali freezing, cross-linking and H2SO4 hydrolysis
can result in and facilitate the reduction of particle size, which agreed
with the SEM images (Fig. 1).
In acid hydrolysis, the starch was treated with sulfuric acid, and only
the amorphous area was hydrolyzed. Although the molecular size
reached the nanometer level, the particle size was still large. In the
cross-linking method, starch solution is separated from the liquid
phase in the form of small microspheres under the action of rapid
agitation and cross-linking agent, and relatively small starch nanoparti-
cles are obtained. For alkali freezing method, NaOH and water mole-
cules are separated due to low temperature and high mechanical
agitation, which destroys the hydrogen bond between starch, and urea
prevents the re-association of hydrogen bond, so the starch nanoparti-
cles with smaller particle size are obtained. In the process of preparing
starch nanoparticles by alcohol precipitation, ultrasonic cavitation effec-
tively destroys the internal structure of starch molecules. Starch chains
contract and gather synchronously in ethanol solution, and precipitate
out of the system under the action of gravity to obtain starch nanopar-
ticles with the smallest average particle size.
3.3. Determination of absorptive capacity for water
Fig. 3 shows the absorptive capacity of starch and starch nanoparti-
cles. Water absorption value decreased with the increasing of starch
mean particle diameter, the water absorption value of native starch
was 29.84%, the water absorption value of starch nanoparticle with
the smallest particle size for 100 nm was 91.53%. After being treated
by external physical and chemical factors, the internal α-1,4 and α-1,6
glycoside bonds of rice starch were destroyed, the hydrophilic groups
OH groups were exposed, and the reactivity of rice starch was improved.
Moreover, when the starch particles changed from small microspheres
to nanoparticles, the specific surface area of starch particles was in-
creased, so the adsorption capacity was enhanced.
3.4. Determination of the stability and dispersion of the starches
The stability and dispersion of native starch granules and starch
nanoparticles prepared by nanoprecipitation (100 nm), alkali freezing
(200 nm), cross-linking (400 nm) and H2SO4 hydrolysis (800 nm)
methods were showed in Fig. 4 after placing them in tube for 0 d (A),
10 d (B), 20 d (C) respectively. Fig. 4A showed that all starches were
in a uniform and stable state. The native rice starch and starch nanopar-
ticle for 800 nm showed a milky white suspension due to their relatively
large particle size, while the starch nanoparticles for 100, 200 and
 H. Xiao et al. / International Journal of Biological Macromolecules 160 (2020) 437–445 441

Fig. 2. The particle size distribution of starch and starch nanoparticles: (1) native starch, (2) 100 nm, (3) 200 nm, (4) 400 nm, (5) 800 nm.

Fig. 3. The water absorption of native starch and starch nanoparticles with different mean particle diameters for 100 nm by nanoprecipitation, 200 nm by alkali freezing treatment, 400 nm
by cross-linking and 800 nm by H2SO4 hydrolysis.

Fig. 4. The stability and dispersion of starch and starch nanoparticles: (1) native starch, (2) 100 nm, (3) 200 nm, (4) 400 nm, (5) 800 nm. A: 0 d, B: 10 d, C: 20 d.
442 H. Xiao et al. / International Journal of Biological Macromolecules 160 (2020) 437–445
400 nm were in a transparent state due to their small particles. After
placing starches in tube for 10 days, Fig. 4B showed that native rice
starch particles were all immersed in the bottom of the test tube, and
starch nanoparticle for 800 nm was dispersed in PBS buffer solution
with a small amount of precipitation observed on 10 d standing, while
the starch nanoparticles for 100, 200 and 400 nm were well dispersed
in PBS buffer solution without precipitation observed. After 20 days,
Fig. 4C showed that native rice starch particles and starch nanoparticle
for 800 nm all sink into the bottom of the tube under the action of grav-
ity, the starch nanoparticles for 100, 200 and 400 nm have no obviously
change, and the solution is basically transparent. These results indicated
that the stability and dispersion of starch nanoparticles were better than
that of native starch.
3.5. Pasting properties
The pasting properties of native starch and starch nanoparticles have
been investigated by using the rapid visco analyzer (RVA). The pasting
properties reflected the molecular events occurring in starch granules.
Therefore, the integrity of starch granule and hydration properties
resulting from the starch native properties or from the inter- or intra-
molecular interactions during hydrothermal treatment could be easily
investigated by measuring the pasting curves of starches before and
after modification [20]. The pasting curves and property values of native
starch and starch nanoparticles have been shown in Fig. 5 and Table 1.
Native rice starch exhibited a characteristic pasting curve of native
starch with pasting temperature (77.6 °C), peak viscosity (3141 cP),
trough viscosity (2275 cP), breakdown (866 cP), final viscosity
(3877 cP) and setback value (1602 cP). Granular starch was insoluble
into cold water. When heated in excess water, the starch granules
swelled and the ordered structures were disrupted at the pasting tem-
perature range, resulting in an increase in viscosity [21]. However,
starch nanoparticles with different mean particle diameters for 100,
200, 400 and 800 nm revealed different pasting curves as compared
with native rice starch. The viscosity of starch nanoparticles with differ-
ent mean particle diameters for 100, 200 and 400 nm aqueous solution
basically kept unchanged during the heating cycle, the viscosity of
starch nanoparticle for 800 nm aqueous solution increased compared
to starch nanoparticles for 100, 200 and 400 nm, but evidently de-
creased compared to native rice starch. As shown in Fig. 5 and Table 1,
Fig. 5. RVA pasting profiles of starch and starch nanoparticles: (1) native starch, (2) 100 nm, (3) 200 nm, (4) 400 nm, (5) 800 nm.
the pasting temperatures and amylose contents of starch nanoparticles
for 100, 200, 400 and 800 nm are higher than that of native rice starch.
In the process of preparing nanoparticle, most of rice starch is trans-
formed into amylose with single orientation, which makes the amylose
content more than the native rice starch, and the amylose content is
more higher, the particles are less likely to break during the heating
pasting process, which results in the rising of the pasting temperature
[22]. Due to the size effect, the chain length of amylose is limited to a
certain extent. The smaller the particle size is, the higher the content
of short amylose is [23]. Therefore, the pasting temperature of starch
nanoparticle for 100 nm and 200 nm is 92.2 °C and 91.4 °C respectively,
that is significantly higher than other starches.
As shown in Fig. 5, the viscosity of native rice starch increased faster
than that of starch nanoparticles. This is due to the fact that the intra-
molecular and inter-molecular hydrogen bonds of native starch parti-
cles break with the increasing of temperature, and the crystal structure
disappears, which result in the sharp rise of viscosity. Starch nanoparti-
cles for 100, 200 and 400 nm have the similar gelatinization curves, the
viscosity value of starch nanoparticle for 800 nm is higher than that of
other nanoparticle. This is because during the acidolysis process, only
the noncrystalline area of starch is destroyed, and the crystalline area
is preserved, which basically keeps the same crystal structure as the na-
tive rice starch. Moreover, the content of long amylose in nanoparticle
for 800 nm is high. Compared with the short amylose in other starch
nanoparticle, the hydrogen bond is easier to break, which makes the ge-
latinization characteristic value higher than that of nanoparticle for 100,
200 and 400 nm.
The bonding force between amylose molecules are strong due to the
hydrogen bonds, and the pasting of starch with high amylose content
was difficult than that of starch with low amylose content [24], there-
fore, the pasting temperature of rice starch with high amylose content
was higher than that of rice starch with low amylose content. The
peak viscosity and breakdown viscosity of starch were negatively corre-
lated with the amylose content, this is consistent with previous reports
[25]. The starch granules swell with the increasing of temperature and
the amylose in starch granules obstruct the swelling, thus, the peak vis-
cosity, trough visco and breakdown viscosity decrease with the increas-
ing of amylose content. Setback and final viscosity essentially measures
the extent of retrogradation of the starch paste, and the setback and
final viscosity are higher, the starch is more easy to retrogradate
 H. Xiao et al. / International Journal of Biological Macromolecules 160 (2020) 437–445 443

Table 1
Pasting properties and amylose contents of starch and starch nanoparticles.

Sample Amylose content (%) Peak visco (cP) Trough visco (cP) Breakdown (cP) Final visco (cP) Setback (cP) Peak time (min) Pasting temp (°C)

Native starch 23.5 3141 2275 866 3877 1602 6.07 77.6
100 nm 27.3 246 174 72 315 141 5.53 92.2
200 nm 26.2 305 234 71 375 141 5.47 91.4
400 nm 25.1 381 314 67 453 139 5.93 80.9
800 nm 24.3 1005 842 163 1289 447 5.93 79.9

through arrangement of amylose molecules. Low degree of polymeriza-
tion of starch molecules obstruct amylose molecules to arrange
parallelly and draw close to each other to combine through hydrogen
bonds so that the starch with low degree of polymerization is uneasy
to retrogradate. Research by Angellier et al. [26] showed that the degree
of polymerization of starch nanoparticle decreased, which resulted in
the lower the setback viscosity and final viscosity of rice starch nanopar-
ticle compared with that of native rice starch.
3.6. Fourier transformation infrared spectroscopy (FT-IR) analysis
The FT-IR spectra of native rice starch and rice starch nanoparticles
with different mean particle diameters for 100, 200, 400 and 800 nm
are shown in Fig. 6. Native starch and starch nanoparticles have the sim-
ilar FT-IR spectra, a broad band in starch due to hydrogen-bonded hy-
droxyl groups appeared at 3600–3200 cm−1 [27] and 1650 cm−1, the
absorption peak intensity of stretching and bending vibration of hydro-
gen bonding appeared at 3402 cm−1, 3352 cm−1, 3375 cm−1,
3390 cm−1, 3388 cm−1 and 1650 cm−1 respectively, but, the vibration
peak of starch nanoparticles hydrogen bonding OH group becomes
wider and stronger, it is because after different treatment, the ɑ-1,6 gly-
coside bond and ɑ-1,4 glycoside bond in starch are destroyed, which
makes the starch chain break and the hydrogen bond association is
weakened. The absorption peaks at 2931 cm−1, 2920 cm−1,
2926 cm−1, 2927 cm−1 and 2924 cm−1 were the stretching vibration
of C\\H in the starch glucose unit. The absorption peaks at
1641 cm−1, 1635 cm−1, 1637 cm−1, 1631 cm−1 and 1634 cm−1 belong
to the C_O stretching vibration in\\CHO. The C\\H bending vibration
at 1273 cm−1, 1274 cm−1, 1276 cm−1, 1273 cm−1 and 1282 cm−1
slightly increased the peak strength, which was caused by the asymmet-
ric bending vibration of C\\H.
In the FT-IR spectrum of starch nanoparticle for 100 nm prepared by
nanoprecipitation method, there is no characteristic absorption peak of
\\CH3 and \\S_O groups in DMSO, which indicates that DMSO is
only used as an organic solvent, does not interact with the groups on
starch molecules, and effectively prevents the recombination of starch
molecules in the form of hydrogen bond. In the post-treatment process,
it can be well miscible with ethanol and can be completely removed. In
the FT-IR spectrum of starch nanoparticle for 200 nm prepared by alkali
freezing method, the characteristic absorption peak of amide group in
urea molecule is not observed, which shows that urea has the same ef-
fect with DMSO, urea not only prevents the adsorption agglomeration
between starch granules, but also does not react with the hydroxyl
group of starch granules to generate starch carbamate. In the FT-IR spec-
trum of starch nanoparticle for 800 nm prepared by H2SO4 hydrolysis
method, sulfuric acid mainly hydrolyzes α-1,4 and α-1,6 glycoside
bonds in starch chain, and does not react with starch groups. Compared
with native rice starch, the molecular chain structure of starch nanopar-
ticles prepared by the above three methods did not change. In the FT-IR
spectrum of starch nanoparticle for 400 nm prepared by cross-linking
method, the absorption peak at 1714 cm−1 was the characteristic
peak of secondary amide structure, which indicated that starch was ob-
viously cross-linked, but the structure of starch nanoparticle was basi-
cally the same as native starch. In conclusion, there is no significant
difference in the structure between the different mean particle diame-
ters of starch nanoparticles and native rice starch, which also indicate
that the structure of starch glucose unit is relatively stable.
3.7. X-ray diffraction (XRD) analysis
The X-ray diffraction patterns of native rice starch and rice starch
nanoparticles with different mean particle diameters for 100, 200, 400
and 800 nm are shown in Fig. 7.
Starch is a biosynthesized polymer-containing semi-crystalline
granule with varying polymorphic types and degree of crystallinity.
Polymorphism of the a-glucans is one of the main characteristics of

Fig. 6. FT-IR spectra of starch and starch nanoparticles: (1) native starch, (2) 100 nm, (3) 200 nm, (4) 400 nm, (5) 800 nm.
444 H. Xiao et al. / International Journal of Biological Macromolecules 160 (2020) 437–445
Fig. 7. X-ray diffractograms of starch and starch nanoparticles: (1) native starch, (2) 100 nm, (3) 200 nm, (4) 400 nm, (5) 800 nm.
the crystalline parts in starch granules. Native starch granules contain
crystalline regions as shown by their unique X-ray diffraction patterns
(XRD) [28]. As shown in Fig. 7, the diffraction pattern of native rice
starch displays typical peaks of A-type amylose allomorph, which was
characterized by peaks at 2θ value at 11.2° and a strong peak at 15°, a
double strong peak at 17.2° and 17.9°, and a strong peak at 23° [29].
Rice starch nanoparticle which was prepared by H2SO4 hydrolysis
with mean particle diameter for 800 nm also displays a characteristic
typical A-type crystalline pattern, this indicates that the acid hydrolysis
does not change or transform the native crystalline state of starch. How-
ever, the diffraction peaks of starch nanoparticle for 800 nm were
sharper than those of native starch. A sharper diffraction peak is an indi-
cation of a higher crystallinity value in the structure of starch. During
the acid hydrolysis process, acid attacked amorphous areas more rap-
idly than the crystalline ones [30]. This process became very sharp,
which is associated with an increase in crystalline regions and with
losses in amorphous fractions [31]. The X-ray diffraction patterns of
starch nanoparticles for 100 and 200 nm exhibited the V-type diffrac-
tion peaks, which included new, observed diffraction peaks at
13.6°and 19.8°; meanwhile, the diffraction peaks at 15°, 17.2°, 17.9°
and 23° completely vanished [32]. During preparing starch nanoparticle
for 100 nm, with the continuous swelling of starch molecules in DMSO
solvent, some hydrogen bonds were destroyed, the internal spiral struc-
ture of starch stretched, and the crystal structure were destroyed. The
most diffraction peak of starch nanoparticle for 200 nm prepared by al-
kali freezing method disappeared, and the crystal structure was
destroyed under the action of sodium hydroxide and urea. For starch
nanoparticles for 400 nm, the diffraction peaks at 15°, 17.2° and 17.9°
completely vanished. Only the diffraction peaks near 23° were retained,
and the diffraction curve of 2θ was basically amorphous. The
crosslinking agent breaks the hydrogen bond in and between the starch
molecules, and the crosslinking network structure destroys the ability of
starch molecules to change from amorphous to crystalline.
4. Conclusions
In this study, broken-rice starch nanoparticles with different mean
particle diameters for 100, 200, 400 and 800 nm were synthesized by
nanoprecipitation, Alkali freezing, cross-linking and H2SO4 hydrolysis
method respectively under controlled conditions using rice starch
with medium amylose content. The synthesis methods used are simple,
fast and easy to perform, and we further reported the differential struc-
tural and morphological properties of native starch and starch nanopar-
ticles. The results demonstrated that the scale of starch granule
synthesized by nanoprecipitation method is the smallest, while the
scale of starch granule synthesized by H2SO4 hydrolysis method is the
largest. Native starch and starch nanoparticles by H2SO4 hydrolysis for
800 nm showed a typical A-type crystalline structure, starch nanoparti-
cles for 100, 200 and 400 nm displayed a typical V-type crystalline struc-
ture, suggesting that the crystalline patterns of nanoparticles except the
granule by H2SO4 hydrolysis were not related to that of native starch.
Native starch and starch nanoparticles have the similar FT-IR spectra.
The stability and dispersion of starch nanoparticles were better than
that of native starch. Moreover, it also was observed that water absorp-
tion value decreased with the increasing of starch mean particle diame-
ter. In addition, the starch nanoparticles showed a low pasting property,
the pasting temperatures and amylose content of starch nanoparticles
are higher than those of native rice starch, while peak viscosity, break-
down, final viscosity, and setback are lower than those of native rice
starch, which may serve as an effective nanocarrier by coating and
entrapping, protecting, and releasing hydrophobic bioactive com-
pounds for functional food, pharmaceutical, and cosmetic industries.
Credit author statement
Huaxi Xiao: Investigation, Data curation, Writing - Original draft
preparation. Fan Yang: Software, Investigation. Qinlu Lin: Conceptuali-
zation, Reviewing and Editing. Qian Zhang: Validation. Lin Zhang: Inves-
tigation, Resources. Shuguo Sun: Methodology, Project administration.
Wenfang Han: Formal analysis. Gao-Qiang Liu: Conceptualization,
Editing, Supervision.
Acknowledgements
This study was financially supported by the Changsha Science and
Technology Project (kq1907098 and kq1907096), Intergovernmental
Key Project of the International Science and Technology Cooperation
Research of China (No. 2017YFE0108100), Project of International
 H. Xiao et al. / International Journal of Biological Macromolecules 160 (2020) 437–445
Cooperation Base of Science and Technology Innovation on Forest Re-
source Biotechnology of Hunan Province (2018WK4008), the Project
of Chang-Zhu-Tan National Independent Innovation Demonstration
Zone (2018XK2007), Changsha Engineering Research Center of Food
Storage and Preservation (kq1901146) and National Natural Science
Foundation of China (31801497).
Compliance with ethical standards
This article does not contain any studies with human participants or
animals performed by any of the authors.
Declaration of competing interest
The authors declare that they have no conflict of interest.
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