Food Chemistry 268 (2018) 324–333

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Improvement in freeze-thaw stability of rice starch gel by inulin and its T

mechanism
⁎
Jiangping Ye, Rong Yang, Chengmei Liu, Shunjing Luo, Jun Chen, Xiuting Hu, Jianyong Wu
State Key Laboratory of Food Science and Technology, Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, China

A R T I C LE I N FO A B S T R A C T

Keywords: Three types of inulin with different degree of polymerization (average DP < 10, DP ≥ 10, and DP > 23) were
Freeze-thaw stability used to improve the freeze-thaw stability of rice starch gel. The gels with or without addition of inulin were
Rice starch subjected to seven freeze-thaw cycles (FTC). Inulin enhanced the water holding capacity and reduced the
Inulin amount of freezable water of the gels, thereby decreasing the syneresis of the gels during seven FTC. In addition,
Syneresis
the amylose and amylopectin retrogradation of the gels were retarded. By adding inulin, the microstructure of
Retrogradation
gel network was stabilized, and the deterioration in viscoelastic properties of the gels during seven FTC was
reversed. Therefore, inulin was an effective additive for preserving the quality of freeze-thawed rice starch gels.
Furthermore, low DP inulin had higher water holding capacity than high DP one, as a result the inulin with lower
DP was more effective.

1. Introduction tapioca starch gel (Pongsawatmanit & Srijunthongsiri, 2008), and

Starch-based frozen foods such as rice noodles, dumplings and
steamed bread are continually being marketed worldwide due to the
fast-paced lifestyle nowadays. When starch-containing foods are sub-
jected to repeated freezing and thawing from factory to table, their
sensory and physicochemical properties are extensively deteriorated
(Charoenrein & Preechathammawong, 2012; Wang et al., 2013). During
freezing, water in these frozen foods turns into ice, resulting in phase
separation. Upon thawing, the resultant water can be easily released
from the food matrix, which is known as syneresis (Arunyanart &
Charoenrein, 2008). The phase separation leads to the formation of a
starch-rich regions where amylose and amylopectin chains tend to re-
associate and form crystallites (retrogradation) (Ferrero, Martino, &
Zaritzky, 1994; Lee, Baek, Cha, Park, & Lim, 2002; Muadklay &
Charoenrein, 2008). Repeated freeze–thaw cycle (FTC) promotes the
phase separation and ice growth, therefore aggravates the problems of
syneresis and retrogradation.
Hydrocolloids are commonly used to improve the freeze-thaw sta-
bility of starch gels. By adding a small amount of hydrocolloids, it is
possible to prevent syneresis and retrogradation of the gels, and thus
improving the sensory and physicochemical properties of corn starch
gel (Ferrero et al., 1994), sweet potato starch gel (Lee et al., 2002),
starch-based frozen foods (Arocas, Sanz, & Fiszman, 2009).
Charoenrein, Tatirat, Rengsutthi, and Thongngam (2011) reported that
the adverse effects of FTC on the syneresis and microstructure of rice
starch gels were minimized by konjac glucomannan. Although the in-
fluences of conventional hydrocolloids have been investigated, appli-
cation of novel hydrocolloids is still needed to satisfy the growing de-
mands for maintaining higher quality and nutrition value of food
products by consumers.
Inulin is a heterogeneous collection of fructose polymers. It consists
of chain-terminating glucosyl moieties and a repetitive fructosyl
moiety, which are linked by β(2,1) bonds. As a food component, inulin
offers a unique combination of technological and nutritional ad-
vantages (Franck, 2002; Schaafsma & Slavin, 2015). Typically, inulin
was used as a prebiotic, sugar replacer, fat replacer, quality improver
and fiber supplement in dairy products, frozen desserts and baked foods
(Franck, 2002). Currently, some of successful applications of inulin are
made in starchy food system, such as bakery food (bread, cakes) and
cereal breakfast (Shoaib et al., 2016), gluten-free bread and pasta
(Drabińska, Zieliński, & Krupa-Kozak, 2016), spaghetti (Padalino et al.,
2017). Particularly, inulin had shown a modification effect on the
rheological, gelatinization and retrogradation properties of starch gels
(Luo et al., 2017; Witczak, Witczak, & Ziobro, 2014) and starch-based

Abbreviations: RS, rice starch gel; DP, degree of polymerization; LPI, low DP inulin (DP < 1 0); MPI, medium DP inulin (DP ≥ 10); HPI, high DP inulin (DP > 23); FTC, freeze-thaw
cycle; SEM, scanning electron microscopy; NMR, nuclear magnetic resonance; DSC, differential scanning calorimeter; WXRD, wide angle X-ray diffraction; FTIR, fourier transform
infrared spectroscopy; d.b., dry weight basis
⁎
Corresponding author.
E-mail address: jianyongwu@ncu.edu.cn (J. Wu).

https://doi.org/10.1016/j.foodchem.2018.06.086
Received 21 March 2018; Received in revised form 15 June 2018; Accepted 18 June 2018
Available online 19 June 2018
0308-8146/ © 2018 Elsevier Ltd. All rights reserved.
J. Ye et al.
foods (Juszczak et al., 2012; Ziobro, Korus, Juszczak, & Witczak, 2013),
and the effect varied with the degree of polymerization (DP) of inulin.
Therefore, we speculated that inulin may also affect the freeze-thaw
stability of starch gels. However, to the best of our knowledge, the ef-
fect of inulin on the freeze-thaw stability of rice starch gel is still un-
known.
The aim of this study was to explore whether three types of inulin
(average DP < 10, DP ≥ 10, and DP > 23) could improve the freeze-
thaw stability of rice starch gel. Syneresis, microstructure, water mo-
bility as well as retrogradation properties of inulin-added rice starch
gels were investigated during FTC and compared with those of native
rice starch gels. A particular emphasis was given to elucidate the po-
tential physicochemical mechanism by which inulin improved starch
freeze-thaw stability.
2. Materials and methods
2.1. Materials
Three types of commercially available inulin with different average
DP: low DP inulin (LPI) (DP < 10), medium DP inulin (MPI) (DP ≥ 10)
and high DP inulin (HPI) (DP > 23) were purchased from BENEO-
Orafti Inc. (Belgium). It was preliminarily confirmed that the average
molecular weight of LPI, MPI, and HPI was 1.1, 2.7, and 7.5 KDa, re-
spectively, by gel permeation chromatography (Agilent 1200, Agilent
Inc., California, USA). Rice starch, containing 22.90% amylose, 0.34%
protein and 0.20% fat, was kindly provided by Golden Agriculture
Biotech Co., Ltd. (Shanggao, Jiangxi, China). All other chemicals and
reagents were of analytical grade unless otherwise stated.
2.2. Sample preparation
Rice starch or rice starch containing inulin slurry (5% inulin on
basis of RS, 8% total solids, d.b.), where inulin amounts were selected
according to Witczak et al. (2014), was gelatinized by heating in a
water bath at 95 °C for 25 min. All the obtained pastes were cooled
down to form gel in an incubator at 25 °C for 4 h. The resultant rice
starch-inulin gels containing LPI, MPI and HPI were referred to as RS-
LPI, RS-MPI and RS-HPI, respectively. A pure rice starch gel, which was
prepared in the same way except the substitution with inulin, was re-
ferred to as RS and used as control.
2.3. Freeze-thaw cycle (FTC)
All the gels (RS, RS-LPI, RS-MPI, RS-HPI) prepared in Section 2.2
were recognized as freeze-thaw cycle 0 (FTC-0). Subsequently, all the
samples were frozen in a freezer (BCD-610 W, Siemens, China) at
−20 °C for 20 h and then thawed in an incubator at 25 °C for 4 h. This
freeze-thaw cycle (FTC) was repeated 1, 3, 5 and 7 times, respectively,
and the corresponding samples were labeled as FTC-1, FTC-3, FTC-5,
and FTC-7, respectively.
2.4. Syneresis measurement
Syneresis of the freeze-thawed gels was measured according to the
centrifugation-filtration method that improved in our previous work
(Ye et al., 2016). The key to this method is an experimental device
assembled by a syringe and a centrifuge tube. The liquid in starch gel
can be effectively separated by using the device after centrifuging at
1000g for 15 min. The liquid separated from the freeze-thawed gels was
collected and the percentage of syneresis was calculated according to
Eq. (1).
Weight of separated liquid from gel (g )
Syneresis = × 100%
Total weight of gel before centrifuging (g ) (1)
325
Food Chemistry 268 (2018) 324–333
2.5. Water mobility determination
Water mobility of the freeze-thawed gels was determined on a nu-
clear magnetic resonance (NMR) (EDUMR, Niumag Co., Ltd., Shanghai,
China) according to the method described previously (Ritota, Gianferri,
Bucci, & Brosio, 2008) with minor modifications. The samples were
loaded in NMR tubes which were then sealed to prevent the evapora-
tion of water. The spin-spin relaxation time (T2) was measured using
the Carr–Purcell–Meiboom–Gill (CPMG) sequences. The sequence had a
pulse separation of 200 μs between the 90° and 180° pulse. A total of
1024 data points were collected by 32 scans with a recycle delay of
1.0 s. The transverse relaxation curves were fitted to an exponential
model (Eq. (2)) using the Multi Exp Inv Analysis software (Niumag Co.,
Ltd., Suzhou, China), as follows:
− t
A = A 0e T2 (2)
where A is the echo amplitude at time t, A0 is the spin-spin relaxation
amplitude, T2 is the spin-spin relaxation time.
2.6. Microstructure characteristics
Microstructure of the freeze-thawed gels was observed by scanning
electron microscopy (SEM). In brief, the samples were freeze-dried and
cut with a razor blade. With the cut surface upward, the samples were
adhered to specimen holder and then sputter coated with a thin film of
gold (10 nm) using a vacuum evaporator. The microstructure of the
samples was observed on a SEM (Quanta-200, FEI Inc., Netherlands) at
130 × magnifications with an accelerating voltage of 20 kV.
2.7. Thermal properties
The freeze-thawed gels were air-dried and their thermal properties
were determined using a differential scanning calorimeter (DSC) (7000
Series, Hitachi Corp., Japan) according to Zhang, Liu, et al. (2014). In
brief, the dried sample (2–4 mg, d.b.) was loaded into an aluminum pan
and then a calculated amount of water was added to obtain a sample/
water ratio of 1:2 (w/v). Samples were hermetically sealed and allowed
to equilibrate for 24 h at ambient temperature. The instrument was
calibrated using indium and an empty aluminum pan was used as re-
ference. Subsequently, the sample pan was heated from 20 to 100 °C at
a rate of 10 °C/min. The onset, peak and conclusion temperatures (To,
Tp and Tc) and the enthalpy (ΔH) were analyzed to characterize the
thermal properties of the samples by using the instrument software
(Universal Analysis Program, Hitachi Corp., Japan).
2.8. X-ray diffractogram
The freeze-thawed gels were air-dried and their X-ray diffractogram
was analyzed by wide angle X-ray diffraction (WXRD). WXRD was
carried out on an X-ray powder diffractometer (Bede D1 HR, Bede
Scientific Inc., Durham, UK) equipped with a copper tube operating at
40 kV and 40 mA, producing CuKα radiation of 0.1546 nm wavelength.
The spectra scanned over a diffraction angle (2θ) ranged from 5° to 40°
at a step size of 0.02° (2θ) per second.
2.9. FTIR spectroscopy analysis
FTIR spectra of samples were obtained in KBr pellets using an FT-IR
a Nicolet 5700 (Thermo Nicolet Co., Waltham, USA) spectrometer over
a wavelength range of 400–4000 cm−1. Data analysis was carried out
using the OMNIC 6.2 software (Thermo Electron Corporation, Madison,
WI, USA). Spectra were baseline-corrected at 1200 and 800 cm−1 by
drawing a straight line and the spectra were deconvoluted. A half-band
width of 26 cm−1 and an enhancement factor of 2.4 with triangular
apodization were employed. Intensity measurements were performed
J. Ye et al. Food Chemistry 268 (2018) 324–333

on the deconvoluted spectra by recording the height of the absorbance 65

bands from the baseline. The ratios of absorbance height 1047 cm−1 (a)
and 1022 cm−1 were obtained for the deconvoluted spectra (Wu,
McClements, Chen, Hu, & Liu, 2016). 60

2.10. Dynamic rheological analysis

55

Syneresis (%)
The FTC was simulated according to the method of (Yamazaki et al.,
2013) on a rheometer (DCR-302, Anton Paar, Austria). A parallel-plate
geometry with a diameter of 40 mm was used. The measuring gap be- 50
tween the plates was fixed at 1000 μm. Samples of FTC-0 prepared in RS
Section 2.2 were loaded on the base plate and excessive sample was RS-LPI
wiped off with a spatula. Water evaporation was minimized by coating 45 RS-MPI
the periphery of geometry with low viscosity (0.02 Pa·s) silicon oil. The RS-HPI
run temperature was precisely controlled by a Peltier system in the base
plate. The loaded samples were allowed to relax and equilibrate at 25 °C 40
1 3 5 7
for 1 min. Afterwards, a frequency sweep was conducted over the range FTC
of 0.1–10 Hz at 2% strain (within the linear viscoelastic region) and ten
points per decade, to characterize the viscoelastic properties of the (b)
180
freeze-thawed gels. The mechanical spectra were obtained by recording
storage modulus (G′), and loss modulus (G″), and then the loss tangent RS
(tan δ = G″/G′) as a function of angular frequency (Hz) was graphed. RS-LPI
160 RS-MPI
Immediately after the frequency sweep, the samples were frozen and
RS-HPI
thawed on the rheometer as follows: cooled to −20 °C in 8 min and held
for 1 min, and then heated to 25 °C at 20 °C/min and held for 1 min (as T2 (ms) 140
FTC-1). The FTC was repeated up to 7 times and the frequency sweep
described above was conducted after each FTC.
120
2.11. Statistical analysis

Results are expressed as mean ± standard deviations of triplicate 100

analyses for each sample unless otherwise stated. The statistical ana-
lyses were performed using SPSS for Windows (Version 17.0, SPSS Inc.,
USA). A comparison of the means was ascertained by Ducan's test at 5%
level of significance using one-way analysis of variance (ANOVA).
3. Results and discussions
3.1. Syneresis
Syneresis is a phenomenon that water separates from starch gels
upon repeated freezing and thawing (Karim, Norziah, & Seow, 2000).
The syneresis is undesirable because it usually causes quality dete-
rioration of starch-based frozen foods. In order to improve the freeze-
thaw stability of rice starch-based foods, three types of inulin with
different average DP (LPI, MPI and HPI) were used as additives to
minimize the syneresis of rice starch gel during seven freeze–thaw cy-
cles (FTC) in this study (Fig. 1a). Syneresis was not observed in any of
the freshly prepared gels (FTC-0). Overall, the syneresis values of all
gels (RS, RS-LPI, RS-MPI and RS-HPI) increased with increasing number
of FTC, and the syneresis occurred rapidly in the first three cycles then
slowed down in the following cycles. For example, the syneresis values
of RS (control) reached 49.29% after FTC-1, and rapidly increased to
57.09% after FTC-3, and then slowly increased from 57.09% to 60.50%
through subsequent FTC. More importantly, all the syneresis values of
RS-LPI, RS-MPI and RS-HPI were significantly (p < 0.05) lower than
those of RS (control) during seven FTC, suggesting that all three types
of inulin could significantly reduce the syneresis of rice starch gel.
Furthermore, the average values of syneresis during seven FTC were RS-
LPI < RS-MPI < RS-HPI < RS, and they were significantly different
from FTC-1 to FTC-5 (p < 0.05), indicating that inulin with lower DP
had a better effect on reducing the syneresis of rice starch gel.
When a starch gel is frozen, the free water forms ice crystals, which
results in the concentration of starch in the unfrozen phase. Upon
thawing, the water is easily released from the gel network, giving rise to
the phenomenon known as syneresis. It has been reported that in starch
80
0 1 3 5 7
FTC
Fig. 1. Syneresis (a) and water mobility (b) of rice starch gel after freeze-thaw
cycles (0, 1, 3, 5 and 7) as affected by addition of inulin with different DP. DP,
degree of polymerization; LPI, low DP inulin (DP < 10); MPI, medium DP
inulin (DP ≥ 10); HPI, high DP inulin (DP > 23); RS, rice starch gel; FTC,
freeze-thaw cycle; T2, spin–spin relaxation time.
gel containing ingredients which can bind to water molecules such as
hydrocolloids or sugars, syneresis is reduced (Arunyanart &
Charoenrein, 2008; Lee et al., 2002; Muadklay & Charoenrein, 2008).
As a kind of soluble dietary fiber in nature, inulin has high hydro-
philicity and could form a tri-dimensional gel network after heating
with starch (Franck, 2002). Therefore, we speculated that after adding
small amount of inulin (5%), parts of the free water in rice starch gel
was immobilized and converted to bound water. The bound water was
not easy to form ice crystals upon freezing (verified by SEM in Section
3.3) and hard to release from the gel network upon thawing, by which
the syneresis of the rice starch gel was significantly reduced. Moreover,
low DP inulin was more effective than high DP one in reducing sy-
neresis of rice starch gel, probably because low DP inulin had higher
water holding capacity (Luo et al., 2017), which was verified by NMR in
Section 3.2. Therefore, inulin could effectively improve the freeze-thaw
stability of rice starch gels by reducing their syneresis, and the inulin
with lower DP was more effective.
3.2. Water mobility
In Section 3.1, we speculated that low DP inulin had higher water
holding capacity than high DP one, and as a result low DP inulin is more
effective than high DP one in reducing syneresis of rice starch gel. To
verify the speculation, the water mobility of rice starch gels with or

326
J. Ye et al.
without addition of inulin during seven FTC was determined by using
NMR (Fig. 1b). The spin-spin relaxation time (T2) measured by NMR is a
good indicator of water mobility for foodstuffs (Zhu, 2017). However,
to the best of our knowledge, the T2 of freeze-thawed starch gel has
never been investigated. In this study, the T2 values of all rice starch
gels were decreased rapidly in the first cycle and slowed down in the
following cycles. Although the T2 values of all rice starch gels decreased
with the increasing number of FTC, the order (RS-LPI > RS-MPI >
RS-HPI > RS) was maintained during seven FTC. These results showed
that RS-LPI had the highest water mobility, followed by RS-MPI, RS-HPI
and RS.
In foods, water is typically described as free water or bound water.
The concept of free and bound water is key to understanding water
mobility of food systems, but it is inaccurate to describe water only as
such. Water does not exist as two discrete states. There is a continuum
of free and bound water in food systems where many layers of water
molecules form around macromolecules (Khan, Wellard, Nagy,
Joardder, & Karim, 2016). The water layers furthest from the macro-
molecule have the highest mobility while the layers directly partici-
pating in hydrogen bonding with hydrophilic sites on the macro-
molecule have the lowest mobility (Au, Acevedo, Horner, & Wang,
2015). Therefore, inulin-bound water can be divided into firmly bound
water and loosely bound water, based on the bonding strength of the
water molecules with the inulin, and the latter is relatively lower and
more likely to transform into free water (Chen, Wu, Li, & Wang, 2017).
That is to say, rice starch gels with high amount of inulin-bound water
probably also have high water mobility. Our result showed that RS-LPI
had the highest water mobility followed by RS-MPI and RS-HPI, in-
dicating that the amount of inulin-bound water was highest in RS-LPI
followed by RS-MPI and RS-HPI. In other words, LPI had the highest
water holding capacity, followed by MPI and HPI, which validated the
speculation we proposed at the beginning of this section. Therefore,
inulin could effectively improve the freeze-thaw stability of rice starch
gels by enhancing their water holding capacity, and the inulin with
lower DP was more effective.
3.3. Microstructure
In Section 3.1, we speculated that the water bound to inulin was less
freezable, and as a result the syneresis of the rice starch gels with ad-
dition of inulin was significantly reduced. To verify the speculation, the
microstructure of rice starch gels with or without addition of inulin
during seven FTC was examined using SEM (Fig. 2). The SEM specimens
were prepared by freeze-drying in this study. Therefore, the freezable
water in gel specimens would be first frozen into ice crystals and then
sublimated into vapor. In the places where the freezable water was
sublimated, ice cells (solid arrow in Fig. 2) or micropores were formed
(Wu, Chen, et al., 2016). Thus, the more micropores mean the more
freezable water in gels. Overall, the micropores in all gels (RS, RS-LPI,
RS-MPI and RS-HPI) became larger and more well-defined with in-
creasing number of FTC, suggesting that freezable water was con-
tinuously released from the gel network upon FTC. Moreover, the mi-
crostructural changes occurred rapidly in the first three cycles and
slowed down in the following cycles. The above microstructural
changes correlated closely with the results of syneresis. After FTC-1
(Fig. 2a1–d1), all gels developed a spongy structure which consists of
ice cells and fibrillar network (hollow arrow in Fig. 2). However, clear
differences were observed between the microstructure of rice starch
gels with inulin added (RS-LPI, RS-MPI and RS-HPI) and the one
without inulin added (RS). RS-LPI (Fig. 2b1), RS-MPI (Fig. 2c1) and RS-
HPI (Fig. 2d1) showed much smaller ice cells and thinner fibrillar
network than RS (Fig. 2a1). In addition, the ice cells and fibrillar net-
work of RS-LPI appeared to be the smallest and thinnest among all the
gels. These microstructural differences maintained from FTC-1 to FTC-
7, indicating that the water holding capacity during FTC was increased
by addition of inulin, because the inulin-bound water was less
327
Food Chemistry 268 (2018) 324–333
freezable. In accordance with our results, previous studies had stated
that the addition of hydrocolloids (such as xanthan, konjac gluco-
mannan) and sucrose could improve the microstructure by decreasing
the size of ice cells in starch gels (Charoenrein et al., 2011; Sae-kang &
Suphantharika, 2006). Therefore, inulin could effectively improve the
freeze-thaw stability of rice starch gels by stabilizing their micro-
structure, and the inulin with lower DP was more effective.
As far as is known, at least two phenomena are responsible for the
quality deterioration of starch-based frozen foods, one is syneresis and
the other is retrogradation of starch gel. To better understand the effect
of inulin on the freeze-thaw stability of rice starch gels, the retro-
gradation behavior of the gels was further analyzed by DSC, WXRD, and
FTIR.
3.4. Thermal properties
It is well recognized that the retrogradation of starch gel can be
considered as a two-step processes: (i) a short-term one related to the
rearrangement and recrystallization of leached amylose chains, and (ii)
a long-term one that involves the reorganization and recrystallization of
amylopectin chains (Fu, Chen, Luo, Liu, & Liu, 2015). Thermal prop-
erties including transition temperatures (To, Tp, and Tc) and enthalpies
(ΔH) measured by DSC correspond to the heat involved in melting
down the double helices and crystallites of amylose and/or amylo-
pectin. However, only amylopectin retrogradation could be quantified
by DSC in the temperature range of 10–120 °C (Karim et al., 2000). In
order to specifically investigate the effect of inulin on the long-term
amylopectin retrogradation, thermal properties of rice starch gels with
or without addition of inulin during seven FTC were determined using
DSC in the temperature range of 10–100 °C in this study (Table 1). No
endothermic peak was observed in any of the freshly prepared gels
(FTC-0), because the amylose crystallites formed in short-term retro-
gradation was unable to detect under the experimental conditions.
Overall, the transition temperatures of all rice starch gels increased
with increasing number of FTC, which might be an indication of more
perfect double helices and crystallites of amylopectin (Witczak et al.,
2014). However, the increase of the transition temperatures was re-
tarded by the addition of inulin, and the inulin with lower DP was more
effective.
On the other hand, the enthalpy (ΔH) provides an overall measure
of double helices and crystallites (quality and quantity) (Zhang, Chen,
et al., 2014). It has been reported that the ΔH values of corn (Zhang,
Chen, et al., 2014), and potato, chestnut, and yam starch gels gradually
decreased with increasing number of FTC (Jaime Vernon-Carter et al.,
2016). The authors attributed the decline in ΔH values to the physical
stress induced by repeated ice-water transformations. The double he-
lices and crystallites of retrogradated might be partially disrupted by
the stress. On the contrary, our results showed that the ΔH values of all
rice starch gels gradually increased with the number of FTC, indicating
that amylopectin retrogradation was progressive enhanced during
seven FTC. Similar trend was found in a study of Chinese water chestnut
starch gels (Wang, Yin, Wu, Sun, & Xie, 2008). It is well known that
when a starch gel is frozen, the concentration of starch phase is in-
creased. High solid concentration facilitated the re-association of
amylopectin chains to form double helices and crystallites. Most im-
portantly, almost all the ΔH values of RS-LPI, RS-MPI and RS-HPI were
significantly (p < 0.05) lower than those of RS (control) during seven
FTC, suggesting that all three types of inulin could significantly inhibit
the amylopectin retrogradation of rice starch gel. Furthermore, the
order of the ΔH values during seven FTC was RS-LPI < RS-MPI < RS-
HPI < RS, indicating that inulin with lower DP had a better inhibitory
effect on the amylopectin retrogradation. For example, the ΔH values of
RS-LPI, RS-MPI, RS-HPI and RS were 1.35, 1.81, 2.13 and 2.23, re-
spectively after FTC-7. The entanglements of inulin with amylopectin
may compete with amylopectin-amylopectin interactions, resulting in
the reduction of amylopectin retrogradation and thereby lower ΔH
J. Ye et al. Food Chemistry 268 (2018) 324–333

Fig. 2. Microstructure of rice starch gel after freeze-thaw cycles (0, 1, 3, 5 and 7) as affected by addition of inulin with different DP. DP, degree of polymerization;
LPI, low DP inulin (DP < 10); MPI, medium DP inulin (DP ≥ 10); HPI, high DP inulin (DP > 23); RS, rice starch gel; FTC, freeze-thaw cycle. The lower case letters
a, b, c and d on the SEM images represented the samples RS, RS-LPI, RS-MPI and RS-HPI, respectively. The Arabic numbers 0, 1, 3, 5 and 7 on the SEM images
represented the treatments FTC-0, FTC-1, FTC-3, FTC-5 and FTC-7, respectively. Solid arrow, ice cells; hollow arrow, fibrillar network. The scale bar on the SEM
images equals to 500 μm.

values. In addition, inulin with lower DP was easier to align and as-
sociate with amylopectin chains owing to the greater flexibility of
smaller molecules. Therefore, inulin could effectively improve the
freeze-thaw stability of rice starch gels by inhibiting the amylopectin
retrogradation, and the inulin with lower DP was more effective.
3.5. X-ray diffractogram
WXRD analysis was applied to further prove the inhibitory effect of
inulin on both amylose and amylopectin retrogradation of rice starch
gels (Fig. 3). All freeze-thawed gels displayed a clear B-type with a
superimposed V-type diffraction pattern. The B-type diffraction pattern
was characterized by the peaks at about 5.6°, 15°, 17°, 22°, and 24° (2θ),
indicating the formation of retrograded amylose and/or amylopectin
crystallites (Derycke et al., 2005). The V-type diffraction pattern was
identified by the peaks near 2θ = 13° and 20° (Derycke et al., 2005). All
the diffraction peaks were clearly observed in RS (control), while two
peaks (2θ = 5.6° and 13°) were disappeared or relatively small in the
gels with inulin added (RS-LPI, RS-MPI and RS-HPI), suggesting that all
three types of inulin could effectively inhibit the amylose and
amylopectin retrogradation of rice starch gels. Moreover, the lowest
peak intensities of 2θ = 22° and 24° were found in RS-LPI during seven
FTC, and the overall peak intensity from FTC-0 to FTC-1 was also lowest
in RS-LPI, indicating that inulin with lower DP exhibited higher in-
hibition on the retrogradation of rice starch gels. WXRD results further
confirmed that inulin not only entangled with amylopectin chains but
also may entangle with amylose chains, resulting in less interactions of
both amylose-amylose and amylopectin-amylopectin, and thereby
lower peak intensity. Therefore, inulin could effectively improve the
freeze-thaw stability of rice starch gels by inhibiting both amylose and
amylopectin retrogradation, and the inulin with lower DP was more
effective.
3.6. FTIR spectroscopy analysis
The FTIR spectra (4000–400 cm−1) of RS, RS-LPI, RS-MPI, and RS-
HPI at FTC-0 are shown in Fig. 4. The characteristic bands at wave-
numbers around 3400 and 2930 cm−1 are attributed to the hydrogen
bonded –OH and –CH groups of the starch molecule, respectively, while
peaks at wavenumbers 1160, 1080, and 1022 cm−1 are related to C–O

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Table 1
Thermal properties and ratio of FTIR of rice starch gel after freeze-thaw cycles (0, 1, 3, 5 and 7) as affected by addition of inulin with different DP.
Samples FTC Thermal properties A1047 cm−1/A1022 cm−1

To (°C) Tp (°C) Tc (°C) ΔH (J/g)

RS 0 – – – – 1.05 ± 0.01g
1 53.39 ± 0.04ef 59.71 ± 0.01fg 64.26 ± 0.07h 0.51 ± 0.01h 1.13 ± 0.03f
3 54.08 ± 0.18d 60.52 ± 0.01d 66.27 ± 0.09c 1.07 ± 0.03e 1.28 ± 0.00bc
5 55.88 ± 0.08b 61.87 ± 0.03bc 67.33 ± 0.03b 1.83 ± 0.01c 1.29 ± 0.02ab
7 56.78 ± 0.01a 62.66 ± 0.34a 68.37 ± 0.21a 2.23 ± 0.05a 1.35 ± 0.04a
RS-LPI 0 – – – – 0.79 ± 0.07j
1 47.91 ± 0.03j 54.59 ± 0.25k 60.12 ± 0.31k 0.29 ± 0.06j 0.97 ± 0.04h
3 50.37 ± 0.06i 57.67 ± 0.36i 64.12 ± 0.20h 0.47 ± 0.04hi 1.13 ± 0.02f
5 50.98 ± 0.20h 59.35 ± 0.16g 64.67 ± 0.22g 0.97 ± 0.01f 1.16 ± 0.03ef
7 53.04 ± 0.44f 60.15 ± 0.17def 65.11 ± 0.28ef 1.35 ± 0.01d 1.22 ± 0.02cde
RS-MPI 0 – – – – 0.89 ± 0.00i
1 50.54 ± 0.15i 56.52 ± 0.29j 62.53 ± 0.12j 0.40 ± 0.01i 1.03 ± 0.04gh
3 51.61 ± 0.12g 58.62 ± 0.08h 64.98 ± 0.09fg 0.74 ± 0.03g 1.14 ± 0.01f
5 53.66 ± 0.06e 60.25 ± 0.05de 65.43 ± 0.11e 1.13 ± 0.04e 1.20 ± 0.00def
7 54.38 ± 0.08d 61.52 ± 0.39c 66.49 ± 0.02c 1.81 ± 0.05c 1.25 ± 0.02bcd
RS-HPI 0 – – – – 1.00 ± 0.07gh
1 51.46 ± 0.35g 58.43 ± 0.26h 63.67 ± 0.36i 0.44 ± 0.02hi 1.06 ± 0.01g
3 53.54 ± 0.03e 59.88 ± 0.06ef 65.83 ± 0.04d 0.92 ± 0.01f 1.17 ± 0.00ef
5 55.19 ± 0.08c 61.59 ± 0.06c 66.59 ± 0.16c 1.40 ± 0.03d 1.24 ± 0.01bcd
7 56.11 ± 0.27b 62.14 ± 0.03b 68.04 ± 0.15a 2.13 ± 0.07b 1.28 ± 0.01bc

DP, degree of polymerization, LPI, low DP inulin (DP < 10); MPI, medium DP inulin (DP ≥ 10); HPI, high DP inulin (DP > 23); RS, rice starch gel; FTC, freeze-thaw
cycle; “–”, undetectable; To, onset temperature of gelatinization; Tp, peak temperature of gelatinization; Tc, conclusion temperature of gelatinization; ΔH, enthalpy of
gelatinization. Values were mean ± standard deviation of three replicates. Mean values in each column with different lower case letters were significantly different
by Ducan’s test (p < 0.05).

bond stretching. Absorbance at wavelengths at 930, 853, 762, and
575 cm−1 are due to the entire anhydroglucose ring stretching vibra-
tions (Kizil, Irudayaraj, & Seetharaman, 2002). For the starch, the band
at 1639 cm−1 is related to moisture content of the sample, corre-
sponding to bending of water overlapped with the asymmetric
stretching of carboxylated anion (–COO–) (Gamonpilas et al., 2011;
Xiong, Li, Shi, & Ye, 2017). Further, this characteristic band shift to low
wavenumber (1600 cm−1) which is much closer to –COO– antisym-
metric stretching vibration with the addition of inulin. It can be as-
sumed that inulin is involved in interacting with water molecules in the

Fig. 3. X-ray diffractogram of rice starch gel after freeze-thaw cycles (0, 1, 3, 5 and 7) as affected by addition of inulin with different DP. DP, degree of poly-
merization; LPI, low DP inulin (DP < 10); MPI, medium DP inulin (DP ≥ 10); HPI, high DP inulin (DP > 23); RS, rice starch gel; FTC, freeze-thaw cycle.

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J. Ye et al. Food Chemistry 268 (2018) 324–333

(a)
RS-HPI

RS-MPI

RS-LPI

RS
1600
853
762
575
1160
2930 1633 1383 1022

3449

4000 3500 3000 2500 2000 1500 1000 500

-1
Wavenumber (cm )
1047 1022
1047 1022
(b) (c)

RS RS-LPI

1200 1100 1000 -1

900 800 1200 1100 1000 900 800
Wavenumber (cm ) -1
Wavenumber (cm )
1047 1022
1047 1022
(d) (e)

FTC-7

FTC-5

FTC-3

FTC-1

RS-MPI RS-HPI FTC-0

1200 1100 1000 900 800 1200 1100 1000 900 800
-1 -1
Wavenumber (cm ) Wavenumber (cm )

Fig. 4. FTIR spectra (a) of rice starch gel with inulin at FTC-0 and deconvoluted FTIR spectra (b, c, d, e) of rice starch gel after freeze-thaw cycles (0, 1, 3, 5 and 7) as
affected by addition of inulin with different DP. DP, degree of polymerization; LPI, low DP inulin (DP < 10); MPI, medium DP inulin (DP ≥ 10); HPI, high DP inulin
(DP > 23); RS, rice starch gel; FTC, freeze-thaw cycle.

330
J. Ye et al.
amorphous region of RS (Kizil et al., 2002; Xiong et al., 2017).
The deconvoluted FTIR spectra (1200–800 cm−1) of rice starch gels
during FTC are illustrated in Fig. 4 and the corresponding absorbance
ratios of 1047 cm−1/1022 cm−1 are summarized in Table 1. The ratio
of 1047 cm−1/1022 cm−1 has been used to determine the amount of
ordered crystalline domains to the amount of amorphous domains in
starch (Wu, McClements, et al., 2016). According to Zhang, Liu, et al.
(2014), as retrogradation proceeded, the system became more ordered
and the ratio of 1047 cm−1/1022 cm−1 would be increased. In this
study, the ratios of all the samples increased with increasing of FTC,
suggesting that all the gel samples were progressively retrograded
during FTC. More importantly, at the same FTC, the ratios of RS-LPI,
RS-MPI and RS-HPI were lower than those of RS (control) and the ratio
of RS-LPI was the lowest. Therefore, all three types of inulin could ef-
fectively inhibit the retrogradation of rice starch gel and the inulin with
lower DP exhibited better inhibiting effect.
3.7. Rheological properties
Insight in the viscoelastic properties (tan δ) of rice starch gels were
obtained by frequency sweep tests with a small deformation amplitude
for predicting their processability and quality (Fig. 5). As shown in
Fig. 5, all the gels behaved more solid-like (lower tan δ) at lower fre-
quencies and more liquid-like (higher tan δ) at higher frequencies. In
addition, the values of tan δ decreased gradually with the increasing
number of FTC, indicating that repeated FTC resulted in a firmer and
more solid-like structure of rice starch gels. Similar trend was found in
the gel systems of corn starch, guar gum and xanthan gum (Yamazaki
et al., 2013). Most importantly, a clear difference in the values of tan δ
was observed between the gels with inulin added (RS-LPI, RS-MPI and
RS-HPI) (Fig. 5b–d) and the one without inulin added (RS) (Fig. 5a).
Without FTC (FTC-0), the values of tan δ were below 0.1 in RS
throughout the frequencies accessed (0.1–10 Hz). Unlike RS (control),
the values of tan δ in RS-LPI, RS-MPI and RS-HPI were found to exceed
Fig. 5. Rheological properties (tan δ = G″/G′) of rice starch gel after freeze-thaw cycles (0, 1, 3, 5 and 7) as affected by addition of inulin with different DP. DP,
degree of polymerization; LPI, low DP inulin (DP < 10); MPI, medium DP inulin (DP ≥ 10); HPI, high DP inulin (DP > 23); RS, rice starch gel; FTC, freeze-thaw
cycle.
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Food Chemistry 268 (2018) 324–333
0.1 at 0.5 Hz, 3 Hz, and 4 Hz, respectively. These result suggested that
the addition of inulin resulted in a softer and more liquid-like structure
(higher tan δ) of rice starch gels, and the inulin with lower DP was more
effective. There are considerable literatures on the phenomenon that
the presence of a hydrocolloid makes a starch gel more liquid-like
(Kaur, Singh, Singh, & McCarthy, 2008). Such more liquid-like structure
of rice starch gels could be attributed to the water absorption capability
of inulin, especially the inulin with lower DP, as evidenced by the re-
sults of syneresis, SEM and NMR. Another reason was that the retro-
gradation of rice starch gels was inhibited by inulin, especially the in-
ulin with lower DP, as confirmed by DSC, WXRD, and FTIR results.
3.8. Potential mechanism
Based on the above results, the underlying mechanism of how inulin
improved the freeze–thaw stability of rice starch gels is outlined in
Fig. 6. Typical pasting (Fig. 6b), retrogradation (Fig. 6c) and syneresis
(Fig. 6d) phenomena of rice starch are shown. If inulin was pre-added in
the rice starch-water suspension (Fig. 6a) before heating, the syneresis
and retrogradation of rice starch gel could be inhibited by the following
mechanisms: (i) inulin had a strong water holding capacity (as evi-
denced by NMR) (Fig. 6e), and the water bound to inulin was less
freezable (as evidenced by SEM). Thus, less ice crystals would form in
the gel upon freezing and as a result less water would release upon
thawing. In this way, syneresis of the gel was inhibited; (ii) inulin
molecules entangled with the leached amylose and amylopectin chains
(Fig. 6f). This entanglements may compete with the interactions be-
tween amylose-amylose and amylopectin–amylopectin, whereby the
retrogradation of the gel was inhibited (as evidenced by DSC, WXRD,
and FTIR).
4. Conclusions
Inulin enhanced the water holding capacity and reduced the amount
J. Ye et al.
Fig. 6. A schematic diagram depicting the potential mechanism of the improving effect of inulin on the freeze-thaw stability of rice starch gel. (a) rice starch-water
suspension; (b) rice starch paste; (c) rice starch gel; (d) the water released from gel matrix due to syneresis; (e) rice starch paste with inulin added; (f) rice starch gel
with inulin added.
of freezable water of rice starch gel, thereby effectively decreasing the
syneresis of the gels during seven freeze-thaw cycles. In addition, ret-
rogradation of rice starch gel was retarded after addition of inulin,
probably because the molecular entanglements of inulin-starch com-
peted with the interactions of amylose-amylose and amylopectin–a-
mylopectin. By adding inulin, microstructure of rice starch gel network
was stabilized, and the deterioration in viscoelastic properties of the gel
during freeze-thaw cycles was reversed. Therefore, inulin could effec-
tively improve the freeze-thaw stability of rice starch gel. In addition,
low DP inulin had higher water holding capacity than high DP one, as a
result the inulin with lower DP was more effective. Thus, inulin may be
a promising component for improving the freeze-thaw stability of
starch-based frozen foods such as frozen dumplings, steamed bread and
noodles.
Acknowledgements
This study was supported financially by the National Natural
Science Foundation of China, China (31701641 and 31571875),
Graduate Innovative Special Funds Project of Nanchang University
(cx2016006).
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