International Journal of Food Properties

ISSN: 1094-2912 (Print) 1532-2386 (Online) Journal homepage: http://www.tandfonline.com/loi/ljfp20

Structure and digestibility properties of resistant

rice starch cross-linked with citric acid

Ji Yeon Kim, Yun-Kyung Lee & Yoon Hyuk Chang

To cite this article: Ji Yeon Kim, Yun-Kyung Lee & Yoon Hyuk Chang (2017): Structure and
digestibility properties of resistant rice starch cross-linked with citric acid, International Journal of
Food Properties, DOI: 10.1080/10942912.2017.1368551

To link to this article: https://doi.org/10.1080/10942912.2017.1368551

Published online: 29 Dec 2017.

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 INTERNATIONAL JOURNAL OF FOOD PROPERTIES
https://doi.org/10.1080/10942912.2017.1368551

Structure and digestibility properties of resistant rice starch

cross-linked with citric acid
Ji Yeon Kim, Yun-Kyung Lee, and Yoon Hyuk Chang
Department of Food and Nutrition, Kyung Hee University, Seoul, Korea

ABSTRACT ARTICLE HISTORY

The objective of this present study was to determine the structural, physi- Received 30 March 2017
cochemical, and in-vitro digestibility properties of cross-linked rice starch Accepted 14 August 2017
with citric acid (CARS). Native rice starch (NRS) was cross-linked with differ- KEYWORDS
ent concentrations of citric acid (0, 1, 10, and 30%, w/w) of starch dry basis.
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Rice starch; Cross-linking;

The SEM results showed that cross-linking with citric acid induced changes Citric acid; In-vitro
in the morphology of starch granules. Compared with NRS, a new peak at digestibility
1734 cm−1 appeared in FT-IR spectra of CARS samples, except CARS1. In the
X-ray diffraction patterns, crystalline peaks of all CARS samples became
much smaller or even disappeared, as compared to that of NRS. 13C NMR
spectra of CARS10 and CARS30 showed new peaks at about 45.55 ppm; in
particular, an additional peak at 178.94 ppm occurred in the spectrum of
CARS30. The swelling factor, light transmittance, thermal properties, and
pasting properties of CARS were significantly lower than those of NRS.
Furthermore, resistant starch contents of NRS were significantly increased,
while the rapidly digestible starch and slowly digestible starch contents of
NRS were significantly decreased after cross-linking with citric acid.
Therefore, cross-linked rice starch with citric acid showed potential for
application as a dietary fibre source in the food industry.

Introduction
Rice (Oryza sativa L.) is composed of a significant amount of starch, accounting for approximately
90% of the milled rice by dry weight. The cooking properties of rice depend on starch and its
interactions with other components of the rice endosperm such as lipids and proteins. Rice is
regarded as non-allergenic because of the hypoallergenicity of its residual protein; hence, rice starch
is widely used in infant foods, processed meats, puddings, and sauces as binder, thickening agent,
and inflating agent. However, rice starch has some disadvantages that limit its applications in food
products. Rice starch has a high glycaemic index similar to glucose and shows instability under
various temperature, shear, and pH conditions.[1] To overcome these limitations, native rice starch
(NRS) is often used for chemical or physical modification.
From a nutritional point of view, starch is classified into rapidly digestible starch (RDS, the starch
fractions digested within 20 min after ingestion), slowly digestible starch (SDS, the starch fractions
digested between 20 and 120 min after ingestion), and resistant starch (RS, the starch fractions that
cannot be digested in the small intestine after ingestion) based on the rate and extent of its
digestion.[2] RS yields short-chain fatty acids in the large intestine through fermentation, which is
beneficial to human health similar to the functional properties of dietary fibre.[3]
Of the various chemical modifications of starch, cross-linking has been generally used to modify
native starches using various cross-linking agents such as phosphoryl chloride (POCl3), epichlor-
ohydrin (EPI), and polycarboxylic acids.[4] In the food industry, cross-linked starch is commonly

CONTACT Yoon Hyuk Chang yhchang@khu.ac.kr Department of Food and Nutrition, Kyung Hee University, Seoul Korea
© 2017 Taylor & Francis Group, LLC
 2 J. Y. KIM ET AL.

used to provide stabilized granular structure and restricted swelling as well as nutritionally beneficial
effects.[4] Citric acid is considered as one of the least toxic and inexpensive cross-linking agents, as
compared to many other cross-linking agents. The conventional dry cross-linking method is gen-
erally used for cross-linking between starch molecules and citric acid. For example, when citric acid
is heated, citric acid is dehydrated to form an anhydride, which reacts with starch molecules to form
a starch citrate adduct. Further heating can result in additional dehydration of citric acid and cause
cross-linking between starch molecules and citric acid.[5] Xie and Liu[5] reported that 78.8% RS was
obtained when citric acid (40% of starch dry weight) was reacted with normal corn starch at 140°C
for 7 h. Jyothi et al.[6] showed that citrate cassava starches synthesized by microwave technique were
less susceptible to enzyme hydrolysis than native starch.
Although the functional and structural properties of corn and cassava starches cross-linked with
citric acid have been investigated previously,[5,7] the structural and physicochemical of cross-linked
rice starch with different concentrations of citric acid have not been reported. In addition, the in-
vitro digestibility properties of cross-linked rice starch with citric acid including RDS, SDS, and RS
have not been discussed. Therefore, the specific objectives of the present study were to (1) produce
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cross-linked rice starch with different concentrations of citric acid and (2) determine structural,
physicochemical, and in-vitro digestibility properties of cross-linked rice starch with different
concentrations of citric acid.

Materials and methods

Materials
Rice flour was purchased from Nongshim Co., Ltd. (Seoul, Korea). Citric acid and pancreatin from
porcine pancreas (P7545, 8×USP) were purchased from Junsei Chemical Co. Inc. (Tokyo, Japan),
and Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), respectively. Amyloglucosidase (AMG
300 L, activity 300 AGU/ml) and α-amylase were purchased from Novozyme (Bagsvaerd,
Denmark) and Megazyme International Ireland Ltd. Co. (Wicklow, Ireland), respectively. Other
chemicals were analytical grade reagents.

Starch preparations
The alkaline steeping method,[8] with slight modification, was used for the isolation of rice starch.
Rice flour (500 g) was dispersed in 1500 mL of 0.2% NaOH with constant stirring at 30 °C for 1 h.
Subsequently, the suspension was centrifuged at 1800 × g for 10 min (Combi 408, Hanil Science
Industrial Co., Ltd., Incheon, Korea). The supernatant was discarded and the protein extract was
removed from the precipitate, and the alkaline extraction was repeated twice. The starch precipitates
were then mixed with 1000 mL of distilled water and neutralized at pH 7.0 with 0.1 M HCl. The
starch dispersion was centrifuged and washed twice with distilled water followed by washing 95%
ethanol. The purified starch was dried at 40°C for 24 h, ground, sieved (150 μm), and stored in a
sealed container at 4°C before use.

Chemical composition
Standard AACCI-approved methods were used to determine the chemical composition of isolated
NRS, including the contents of moisture (44-15A), ash (08–01), protein (46-12A), and lipid (30–
10).[9] Total starch content was analysed using the Total Starch Assay Kit (Megazyme International
Ireland Ltd. Co., Wicklow, Ireland). Amylose content of NRS was determined by the method of
Williams et al.[10] The amylose content was measured from a standard curve prepared with mixtures
of potato amylose and maize amylopectin (Sigma-Aldrich, St. Louis, MO, USA).
 INTERNATIONAL JOURNAL OF FOOD PROPERTIES 3

Preparation of cross-linked rice starch with citric acid

Cross-linked rice starch with citric acid was produced based on the method of Klaushofer et al. [11]
with some modifications. Citric acid (1, 10, and 30% of starch dry basis) was dissolved in distilled
water; subsequently, the pH of the solution was adjusted to 3.5 with 10 M NaOH, and finally diluted
to a final volume of 50 mL with distilled water. The citric acid solution (50 mL) was mixed with 50 g
of NRS and the dispersion was conditioned at ambient temperature for 16 h. The mixture was dried
in an air oven at 60°C for 6 h to a moisture level of 5–10% (w/w), ground, and dried in a dry oven
for 4 h at 140°C. The dry mixture was rinsed three times with distilled water to remove unreacted
citric acid. The washed starch was air-dried at 40°C for 24 h, ground into powder, and passed
through a 150 μm sieve. The NRS cross-linked with the three concentrations of citric acid (1, 10, and
30%) were designated to as: CARS1, CARS10, and CARS30, respectively. The degree of substitution
(DS) was determined based on the method of Kweon et al.[12]

Scanning electron microscopy (SEM)

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The morphology of NRS and CARS was determined using Scanning Electron Microscope (S-4700,
Hitachi Co., Tokyo, Japan) according to the method of Atrous et al.[13] with some modifications.
Starch samples were placed on double-sided carbon tape attached to a specimen holder and then
coated with platinum powder. The samples were examined at an accelerating voltage of 10 kV and
magnification of ×10,000.

Fourier-transform infrared (FT-IR) spectroscopy

Fourier-Transform Infrared (FT-IR) spectroscopy of NRS and CARS was performed by the method
of Chung et al. [14] (2011) with some modifications. FT-IR spectra were measured using a Fourier-
transform infrared spectrophotometer (Spectrum One System, Perkin-Elmer, Waltham, MA, USA).
NRS and CARS were ground with potassium bromide (KBr) and pressed into a disc. The spectral
scanning range was 4000 to 500 cm−1.

X-ray (XRD) diffraction

To determine the X-ray diffraction of CARS and NRS, the method of Won et al. [15] was used with
some modifications. X-ray diffraction patterns were obtained by an X-ray diffractometer (X′Pert
PRO, PANalytical, Almelo, Netherlands). X-ray generator tension and current were 40 kV and
40 mA, respectively. The samples were scanned through the range of 2θ from 6 ° to 30°.

13
C nuclear magnetic resonance (NMR) spectroscopy
The 13C NMR spectra were recorded on a JMM-ECZ 500R spectrometer (JEOL Co., Tokyo, Japan).
The samples were prepared for NMR experiments based on the method of Xu and Seib [16] with
some modification. Each sample (2 g) with α-amylase (10 ul) was dispersed in 30 mL of distilled
water; afterwards, sodium acetate (0.3 g) was added. The mixture was heated in a water bath at 85°C
for 2 h under constant agitation to hydrolyse the starch, and then moved to a boiling water bath for
30 min to inactivate the enzyme. After cooling to room temperature, the mixture was centrifuged at
3000 rpm for 10 min and the supernatant obtained was freeze-dried (FD8508, Ilshin BioBase Co.,
Ltd., Yangju, Korea). The freeze-dried starch hydrolysate (0.2 g) was dissolved in 3 mL of D2O for
2 h at room temperature followed by freeze-drying, and the process was repeated once. The D2O-
exchanged starch (0.05 g) was solubilized in 0.5 mL of D2O. 13C NMR spectra were recorded at
400.13 MHz. The 13C spectra were measured at 25°C for 128 scans with an acquisition time of 0.82 s
and a delay time of 1 s.
 4 J. Y. KIM ET AL.

Swelling factor and light transmittance

Swelling factor of NRS and CARS was determined by the method of Tester and Morrison.[17] Light
transmittance of NRS and CARS was measured based on the procedure of Reddy and Seib.[18]

Thermal properties
Thermal properties of NRS and CARS were investigated by the method of Chung and Liu [19]
with some modifications. The thermal properties of NRS and CARS were analysed using a
differential scanning calorimeter (DSC; A200, TA Instruments, New Castle, DE, USA). Starch
(20 mg) and distilled water (1 mL) were added to an aluminium DSC pan. The pan was
immediately sealed and equilibrated at room temperature for 1 h before heating in the DSC.
The starch sample was heated from 30 to 130°C at a heating rate of 10°C/min. An empty
aluminium pan was used as reference. The onset, peak, and final temperature (To, Tp, and Tc,
respectively) and gelatinization enthalpy (ΔH) were calculated using Universal Analysis software
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provided by the manufacturer.

Pasting properties
The pasting properties of NRS and CARS were conducted using a Rapid Visco Analyser (RVA model
4, Newport Scientific Inc., Warriewood, Australia) with Standard Method 1 profile according to
AACC Approved Method 76–21 (American Association of Cereal Chemists International, 2000). [9]
The starch suspensions (14%, wet basis) were held at 50°C, heated at 12°C/min from 50 to 95°C, held
at 95°C for 2.5 min, cooled from 95 to 50°C at the same rate, and held at 50°C for 2 min. The RVA
instrument provided the following parameters: pasting temperature, peak viscosity, breakdown,
setback and final viscosity. The parameters from the RVA were processed by Thermocline version
1.2 software (Newport Scientific Inc.).

In-vitro digestibility
The in-vitro digestibility of NRS and CARS was determined based on the method described by
Englyst et al.[2] with some modifications. Pancreatin (1 g) was dispersed in 12 mL of distilled water
for 10 min. The dispersion was then centrifuged at 1500 × g for 10 min. The supernatant (10 mL)
was mixed with 0.2 mL of amyloglucosidase and 1.8 mL of distilled water. This enzyme solution was
prepared freshly and equilibrated at 37°C for 10 min before each test.
The NRS and CARS (30 mg, db) with 2 glass balls (5 mm diameter) were added to each micro-
tube (2 mL), followed by adding to 0.75 mL of sodium acetate buffer (0.1 M, pH 5.2). The micro-
tubes were equilibrated in a water bath (37°C) for 10 min. The enzyme solution (0.75 mL) was then
added to the sample tube and incubated in a water bath at 37°C with shaking (240 rpm). After
hydrolysis for 20 or 120 min, the tubes were boiled for 10 min to deactivate the enzyme and
terminate the reaction and centrifuged at 5000 × g for 10 min. The glucose content in the super-
natant was measured by the dinitrosalicylic acid (DNS) method. The percentage of hydrolysed starch
was calculated by multiplying a factor of 0.9 to change values for glucose to starch. The rapidly
digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) contents in NRS and
CARS were calculated using the equations (1, 2), and (3).
RDS ð%Þ ¼ ðG20  0:9=WÞ  100 (1)

SDS ð%Þ ¼ ½ðG120  G20 Þ  0:9=W  100 (2)

RS ð%Þ ¼ 100  RDS  SDS (3)
 INTERNATIONAL JOURNAL OF FOOD PROPERTIES 5

where G20 and G120 are glucose content released after 20 and 120 min, respectively; and W is the
starch weight (mg) used for each test.

Statistical analysis
All statistical analyses were performed using SAS version 9.3 (SAS Institute Inc., Cary, NC, USA).
Analysis of variance (ANOVA) was performed using the general linear models (GLM) procedure to
determine significant differences among the samples. Means were compared by using Fisher’s least
significant difference (LSD) procedure. Significance was defined at the 5% level.

Results and discussion

Chemical composition
The moisture, ash, protein, lipid, and total starch contents of NRS were 3.60%, 0.17%, 0.33%, 0.90%,
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and 93.32% (data are not shown), respectively, suggesting that protein, lipid, and ash in rice flour
were removed almost entirely from the isolated starch. The amylose and amylopectin contents of
NRS were 22.11 and 77.78%, respectively (data not shown). These findings were consistent with
previous literatures. [20,21] You et al. [21] reported that the amylose contents of various rice species
ranged 22.4 to 25.5%.

Determination of DS
The DS of CARS varied from 0.01 to 0.27 and showed increase with increasing concentrations of
citric acid to starch (Table 1). Mei et al.[7] reported that the DS values of cassava starch cross-linked
with citric acid ranged from 0.058 to 0.178 when the concentrations of citric acid were increased
from 10 to 30%, respectively. In the case of hydroxylpropylated and oxidized normal potato starch
films cross-linked with citric acid, the degree of di-esterification was increased from 0.008 to 0.054
with the increase of citric acid contents from 5 to 30%, respectively.[22]

Scanning electron microscopy (SEM)

The scanning electron microphotographs of NRS and CARS samples are presented in Figure 1. NRS
had polygonal, and irregular shape with a smooth surface, which was consistent with the previous
publication.[23] Besides, NRS showed some pores on the surface of the granules (depicted by arrows).
However, the surface of CARS appeared rougher than that of NRS. As the concentrations of citric
acid increased, this phenomenon was more pronounced, subsequently, causing the erosion on the
core part of the starch granules. The morphological changes possibly indicate that the citric acid

Table 1. Degree of substitution (DS), swelling factor, light transmittance and thermal properties of native and cross-linked rice
starches prepared with different concentrations of citric acid (1–30%).a
Thermal properties
Sample DS Swelling factor Light transmittance (%T) To (°C)b Tp (°C) Tc (°C) ΔH (J/g)
a a
NRS - 16.29 ± 0.41 23.80 ± 0.35 59.56 ± 0.14a 64.89 ± 0.06a 71.53 ± 0.23a 1.59 ± 0.01a
CARS1 0.01 ± 0.00c 11.30 ± 0.16b 13.46 ± 0.35b 58.26 ± 0.02b 63.10 ± 0.10b 69.05 ± 0.31b 1.31 ± 0.09b
CARS10 0.15 ± 0.00b 6.95 ± 0.00c 2.82 ± 0.00c N.D.c N.D. N.D. N.D.
CARS30 0.27 ± 0.00a 4.07 ± 0.07d 1.95 ± 0.03d N.D. N.D. N.D. N.D.
a
Values with different letters within the same column differ significantly (p < 0.05).
NRS = Native rice starch, CARS = cross-linked rice starch prepared with citric acid.
b
To, onset temperature; Tp, peak temperature; Tc, conclusion temperature; Tc-To, conclusion temperature minus onset temperature
(temperature range); Δ H, enthalpy of gelatinization.
c
N.D.: not detected.
 6 J. Y. KIM ET AL.
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Figure 1. Scanning electron microphotographs of native and cross-linked rice starches prepared with different concentrations of
citric acid (1–30%). The bold arrows indicate the pores on the surface of native starch granules.

penetrated the pores on the surface of starch granules and approached the hilum through the
channels that connect the pores to the hilum.[3] This could result in the erosion on the central
part of starch granules. This result indicated that the cross-linking reaction with citric acid could
affect the granule morphology.

Fourier-transform infrared (FT-IR) spectroscopy

The FT-IR spectra of NRS, CARS, and pure citric acid are presented in Figure 2. Both NRS and
CARS showed a broad peak in the range of 3000–3600 cm−1 and 2932 cm−1, which correspond to
OH and CH stretching, respectively.[24,25] The peaks around 1650 cm−1 and 1420 cm−1 were
attributed to OH and CH bending, respectively.[25] Three peaks observed near 1018–1155 cm−1
are the distinctive peaks of the pyranose form of glucose residues.[26]
In particular, a new peak at 1734 cm−1 appeared in the FT-IR spectra of CARS samples except
CARS1. The absorbance at 1734 cm−1 may be associated with the new characteristic ester group
between the hydroxyl groups of cassava starch molecules and carboxyl groups of citric acids during
cross-linking.[27] Thus, a hydroxyl group of the starch can be reacted with a carboxyl group of the
citric acid during cross-linking, consequently leading to the formation of the ester bond in the CARS
structure.
In the FT-IR spectrum of pure citric acid (Figure 2), the peak observed at 1704 cm−1 was due to
the C = O stretching vibration in the carboxyl groups; and the peak found at 1755 cm−1 was the
interference peak that occur when the carboxyl groups are very close to each other in diacid.[28]
However, these two peaks disappeared in the FT-IR spectra of CARS samples. According to Zhou
et al.,[29] the change of the peak’s position from 1709 cm−1 in pure citric acid to 1743 cm−1 in the
modified starch nanocrystals confirmed that the crosslinking between starch and citric acid
occurred during heating. These authors also noted that the unbound citric acid was thoroughly
washed from the modified starch nanocrystal, leading to disappearance of the 1709 cm−1 peak.
Thus, these results revealed successful cross-linking of NRS with citric acids during the cross-
linking process.
 INTERNATIONAL JOURNAL OF FOOD PROPERTIES 7
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Figure 2. FT-IR spectra of citric acid, native, and cross-linked rice starches prepared with different concentrations of citric acid (1–30%).

X-ray (XRD) diffraction

The X-ray diffraction patterns of NRS and CARS samples are shown in Figure 3. NRS exhibited the
typical A-type crystalline pattern with 2θ reflection intensities at 15, 17, 18 and 23°.[1,30] After cross-
linking with citric acid, the crystalline patterns of CARS1 rarely change in comparison to NRS.
However, higher DS of CARS samples is associated with lower intensity of these peaks; moreover,
the crystalline peaks of CARS30 were not detected. This suggested that cross-linking of citric acid

Figure 3. XRD diffractogram of native and cross-linked rice starches prepared with different concentrations of citric acid (1–30%).
 8 J. Y. KIM ET AL.

should occur both in the amorphous regions and crystalline regions. According to Xie et al.,[3] when
citric acid migrates into the channels and cavities of the starch granules, it could destroy the
crystalline structures due to the concentrated solution of citric acid. Therefore, esterification of the
citric acid group on the amorphous regions and crystalline regions of starch could form a highly
cross-linked starch and restrict the mobility of the starch chain.

13
C NMR spectroscopy
As shown by 13C NMR spectra of NRS and CARS (Figure 4), the signals at around 100 ppm
corresponded to the internal anomeric C-1.[31] The multiplets between 68 and 78 ppm were assigned
to C-2, C-3, C-4, and C-5.[32] The signal at 60.44 ppm was from C-6.[32] There were no significant
differences between NRS and CARS1, probably because DS was too low to change the structure of
NRS. However, 13C NMR spectra of CARS10 and CARS30 had an additional peak near 45.55 ppm,
which might be attributed to the methylene group of citric acid.[33] In case of the spectrum of
CARS30, a new peak appeared at 178.94 ppm, as compared to other samples prepared in this study.
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This peak might be assigned to the carbonyl group of CARS for an ester bond, which confirmed the
reaction between carboxylic groups from citric acid and hydroxyl groups from starch molecules.[31]
This result was confirmed by FT-IR spectra (Figure 2). Therefore, in the present study, the results of
the 13C NMR spectra confirmed the change of structure of NRS after cross-linking.

Swelling factor and light transmittance

The swelling factors of the CARS samples were significantly lower than that of the NRS samples
(Table 1). In addition, the swelling factor of CARS samples significantly decreased with increasing
concentrations of citric acid, in agreement with the results of Mei et al.,[7] who reported that the
swelling power of cross-linked cassava starch significantly decreased with the increase of citric acid.
The findings indicated that cross-linking between starch molecules and citric acid could strengthen
the starch network both chemically and physically, thus resulting in lower swelling factor.[22]
Dastidar et al.[27] noted that the cross-linking of corn starch with malonic acid hindered the
penetration of water into the starch network because of the compact structure of the cross-linked
starch. Additionally, cross-linked tapioca starch with citric acid decreased the absorption of water.[34]

Figure 4. 13
C NMR spectra of native and cross-linked rice starches prepared with different concentrations of citric acid (1–30%).
 INTERNATIONAL JOURNAL OF FOOD PROPERTIES 9

In the present study, we confirmed that the cross-linked structure between starch granules and citric
acid inhibited the absorption of water, subsequently resulting in the reduction of swelling factor. The
light transmittance of NRS and CARS pastes is shown in Table 1. The light transmittance values of
CARS pastes were significantly lower than that of NRS. The light transmittance values of CARS were
significantly decreased with increasing the concentrations of citric acid, which could be associated
with new cross-linking between the starch granules. Jyothi et al.[4] reported that cross-linked starch
had denser granules than native starch and reflected rather than transmitted light.

Thermal properties
The gelatinization parameters of NRS and CARS are shown in Table 1. The onset temperature (To),
peak temperature (Tp), conclusion temperature (Tc), and enthalpy of gelatinization (ΔH) of NRS
were 59.56°C, 64.89°C, 71.53°C, and 1.59 J/g, respectively. The To, Tp, Tc, and ΔH values of CARS
samples were significantly decreased for CARS 1 and not determined for CARS 10 and CARS 30, as
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compared with those of NRS. The new crosslinking between starch granules in CARS can prevent
the water absorption and consequently decrease the swelling and gelatinization of starch granules.
Kartha and Srivastas[35] reported that highly cross-linked starches had lower gelatinization enthalpy
than native starch due to less portion of starch granules that could be gelatinized by heat.
Accordingly, in the present study, cross-linking with citric acid could restrict the hydration, swelling
and gelatinization of CARS molecules, subsequently resulting in the reduction of gelatinization
parameters for CARS samples.

Pasting properties
The pasting properties of NRS and CARS are presented in Table 2. The pasting properties of
CARS30 were not detected due to too high degree of cross-linking. CARS1 and CARS10 showed a
significant decrease in the pasting temperature and peak viscosity, as compared to NRS. The
reinforcement of bonding between starch chains by cross-linking with citric acid can restrict water
absorption and swelling of the CARS granules, leading to a decrease in the peak viscosity of
CARS.[36]
CARS1 and CARS10 exhibited lower breakdown viscosity (2.38–113.73 RVU) than NRS (227.75
RVU). Yussof et al.[37] reported that cross-linked starches showed lower breakdown than native
starch because of their compact granule structure. In the present study, we confirmed that cross-
linking with citric acid could form the compact granule structures of CARS during crosslinking,
consequently improving the resistance against the high heat and shear.

Table 2. Rapidly digestible starch (RDS), slowly digestible starch (SDS), resistant starch (RS), and pasting properties of native and
cross-linked rice starches prepared with different concentrations of citric acid (1–30%).a
Starch fractions (%) Pasting properties
Pasting Peak viscosity Breakdown Setback
Sample RDS SDS RS temperature (°C) (RVU) (RVU) (RVU)
NRS 51.00 ± 0.41a 37.71 ± 0.23a 11.29 ± 0.64d 70.63 ± 0.60a 419.17 ± 2.11a 227.75 ± 0.74a 152.76 ± 1.65a
CARS1 47.52 ± 0.14b 35.13 ± 0.23b 17.35 ± 0.09c 67.80 ± 0.14b 405.02 ± 1.15b 113.73 ± 0.28b 134.46 ± 0.60b
CARS10 39.19 ± 0.78c 25.39 ± 0.05c 35.42 ± 0.74b 64.58 ± 0.04c 13.31 ± 0.12c 9.07 ± 0.06c 0.41 ± 0.35c
CARS30 16.87 ± 0.69d 19.85 ± 0.06d 63.77 ± 0.05a N.D.b 3.83 ± 0.06d 2.38 ± 0.17d 0.08 ± 0.00c
a
Values with different letters within the same column differ significantly (p < 0.05).
NRS = Native rice starch, CARS = cross-linked rice starch prepared with citric acid.
b
N.D.: not detected
 10 J. Y. KIM ET AL.

In-vitro digestibility
The starch fractions including rapidly digestible starch (RDS), slowly digestible starch (SDS), and
resistant starch (RS) of NRS and all CARS are presented in Table 2. The RDS and SDS contents
of NRS were significantly higher than those of CARS. The RDS contents of CARS were
significantly decreased from 47.52 to 16.87%, when the citric acid concentrations were increased
from 1 to 30%, respectively. However, the RS contents of all CARS were significantly higher than
that of NRS. In particular, the RS content of CARS30 was about 5.6 times higher than that of
NRS. These findings could be due to the stearic hindrance by the newly cross-linked structure in
CARS. The presence of citric acid in CARS could sterically inhibit the proper positioning of the
substrate into the active site of digestive enzymes such as α-amylase and amyloglucosidase, and
consequently, restrict enzyme hydrolysis.[38] These results corroborate the results of previous
studies.[6,25]
Another possible explanation for the decrease in the digestibility of starch could be the decrease in
the swelling factor of CARS (Table 1). Chung et al.[39] reported that the swelling of starch granules
could facilitate the access of digestive enzymes to the granule interior, leading to an increase in the
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RDS contents. In the present study, the swelling factor of CARS was decreased with increasing the
concentrations of citric acid, which caused difficulty in access of the digestive enzymes to the starch
granules and subsequently, increased the RS contents of CARS. Therefore, the cross-linking with
citric acid could cause stearic hindrance and restrict the swelling in the CARS granules, thereby
increasing the RS contents of CARS.

Conclusion
The structural, physicochemical, and in-vitro digestibility properties of CARS with different con-
centrations of citric acid were evaluated in the present study. Based on FT-IR, X-ray, SEM, and 13C
NMR results, esterification of the citric acid group on the amorphous regions and crystalline regions
of starch could cause formation of the compact structure of cross-linked starch. When the DS of
CARS were increased, the swelling factor, light transmittance, thermal properties, and pasting
properties of CARS were significantly decreased. These results confirmed that the structural changes
induced by cross-linking with citric acid affected the physicochemical properties of NRS.
Furthermore, the compact granule structure of CARS could sterically hinder the access of digestive
enzymes to the surface of starch, leading to a decrease in digestibility of NRS. Cross-linking with
citric acid improved the RS contents of rice starch. Thus, CARS can be used as an ingredient of
functional food in the food industry.

Funding
This project was conducted by the generous financial support of the Youlchon Foundation (Nongshim Corporation
and its affiliated companies) in Korea.

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