Carbohydrate Polymers 223 (2019) 115020

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Preparation and properties of potato amylose-based fat replacer using super- T

heated quenching
⁎
Yuying Hua, Chunmei Lia, Joe M. Regensteinb, Lufeng Wanga,c,
a
College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, 430070, China
b
Department of Food Science, Cornell University, Ithaca, NY, 14853, USA
c
Key Laboratory of Environment Correlative Dietology, Ministry of Education, Huazhong Agricultural University, Wuhan, Hubei, 430070, China

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

Keywords: A potato amylose-based fat replacer (PAFR) was prepared using super-heated quenching at different amylose
Potato amylose ratios. The effects of amylose ratio on texture attributes, rheological properties, water distribution, and mi-
Fat replacer crostructure of PAFR were investigated. The results showed amylose contributed to the gel structure of PAFR,
Spherulites with a positive correlation of G′ and G′′ with amylose ratio. PAFR with 85% amylose ratio generated better
Super-heated quenching
texture attributes and gel properties. The low-field 1H nuclear magnetic resonance (LF-NMR) indicated amylose
was advantageous for the tight interaction with water molecules through hydrogen bonds, which then developed
a creamy texture of PAFR. In addition, the microstructure changed from a three-dimensional network structure
to a lamellar structure, while some spherulites of 2–10 μm diameter, similar to fat globules in size, started to
appear with the increasing amylose ratio. This study on amylose impact would provide great insights for future
application of fat replacer.

1. Introduction
As a major ingredient in many foods, fat plays an important role in
the texture, mouthfeel and flavor of foods. Fat is also a critical nutrient
for animals and human due to its many physiological functions.
However, excessive intake of fat would not only lead to obesity, but also
increase the incidence of cardiovascular disease, diabetes, etc. Even
though the potential cause of harm to human by excessive consumption
of fat has been well understood, it is still often challenging for con-
sumers to keep the intake of dietary fat below the recommended level
(Whybrow, Macdiarmid, Craig, Clark, & Mcneill, 2016). This is partly
due to the creamy taste provided by fat, which is among consumers’
favorites. Therefore, more attention needs to be paid to fat-free or low-
fat foods where calorie density can be reduced while fat-like mouthfeel
can be maintained (Sandrou & Arvanitoyannis, 2000). Fat replacers are
food ingredients that can provide a fat-like mouthfeel, reduced calorie
density, and desirable textural attributes in food matrices. They have
been developed to reduce the calorie density of foods so as to tackle the
rise in overweight and obesity in many countries.
Carbohydrate-based fat replacers are generally safe with a low
calorie density (0–4 cal/g) compared to fat (Akoh, 1998). As compared
with protein-based fat replacers, they are generally more cost-effective
(Peng & Yao, 2017) and recognized as GRAS (Generally Recognized as
Safe). Among the carbohydrate-based fat replacers, starch is one of the
most frequently used ingredients as it’s relatively inexpensive and
readily available (O’Connor & O’Brien, 2011). Starch-based fat re-
placers have been studied and applied in a wide variety of low-fat food
products including cheeses, sausages, yogurt, mayonnaise, and frozen
desserts (Peng & Yao, 2017). In regard to the simulation mechanism,
there are two hypotheses for starch-based fat replacer to mimic the
mouthfeel and texture characteristics of fat. The first explanation is that
starch would form a gel three-dimensional network structure at a sui-
table condition (Alexander, 1995; Yackel & Cox, 1992), where the
water trapped in the gel could provide a fat-like sensation. In addition,
some reports have showed that the linear amylose played an important
role in the three-dimensional network structure formation of starch gel
(Chronakis, 1998; Wang & Wang, 2015; Yang & Xu, 2007). Previous
study (Lii, Tsai, & Tseng, 1996) had found that amylose molecules
would leach out of the starch granules during gelatinization, then the
three-dimensional network structure would form when cooling down.
Other researchers further suggested that the gel matrix of fat replacer
can also be formed with the linear chains of amylopectin interacted
with amylose molecules (Chronakis, 1998; Yang & Xu, 2007).
The second explanation is that the starch could form some spher-
ulites similar in size to fat globules (2–10 μm) during super-heating and
quenching treatment, which would provide the lubricating mouthfeel of

⁎
Corresponding author at: College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, 430070, China.
E-mail address: pipilu001@foxmail.com (L. Wang).

https://doi.org/10.1016/j.carbpol.2019.115020
Received 4 February 2019; Received in revised form 20 June 2019; Accepted 21 June 2019
Available online 22 June 2019
0144-8617/ © 2019 Elsevier Ltd. All rights reserved.
Y. Hu, et al.
fat. Spherulites are semi-crystalline entities with some degree of radial
symmetry which would show the ‘Maltese cross’ using polarized light. It
could be formed by heating a starch suspension and then re-
crystallization with slow cooling without any disturbances (Singh,
Lelane, Stewart, & Singh, 2010; Steeneken & Woortman, 2009; Ziegler,
Nordmark, & Woodling, 2003). Moreover, it had been observed that the
spherulites made from high amylose maize starch had a radially or-
iented structure, a central cavity and small holes away from the center
(Nordmark & Ziegler, 2002). Creek (2007) suggested that a radially
oriented layered structure of spherulites can be made from corn amy-
lose with varying degrees of polymerization (DP). These authors re-
ported that the spherulite formation was favored by amylose. In addi-
tion, Creek, Ziegler, and Runt (2006) confirmed that amylopectin from
potato starch did not form spherulite.
There are numerous researches focused on the properties and ap-
plications of fat replacers. Among these studies, several preparation
methods and physicochemical properties of starch-based fat replacer
have been reported (Babu, Parimalavalli, & Mohan, 2018; Brennan &
Tudorica, 2008; Peng & Yao, 2017). Furthermore, amylose plays an
important role in the simulation mechanism of starch-based fat re-
placer. However, there are a few studies describing the role of amylose
in fat replacer in detail. Moreover, the effects of amylose on the prop-
erties, microstructure and simulation mechanism of starch-based fat
replacer were still unclear. The objective of this study is to investigate
the effects of amylose ratio on macro-properties and microstructures of
PAFR treated by super-heated quenching, which would contribute to
the development of simulation mechanism of starch-based fat replacer.
The textural attributes, rheological properties, water distribution, mi-
crostructure and spherulite formation of PAFR were investigated. It
may contribute to further understand the formation of fat replacer and
develop a new starch-based fat replacer for food industry.
2. Materials and methods
2.1. Materials
Potato starch (apparent amylose ratio, 22.8%) was obtained from
Dacheng Co., Ltd. (Changchun, Jilin, China). High amylose potato
starch (apparent amylose ratio, 97.6%) was obtained from Hengdailao
Biotechnology Co., Ltd. (Shanghai, China). An amylose standard was
purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All
the other chemicals were of analytical grade and obtained from
Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
2.2. Sample preparation
PAFR was prepared using the super-heated quenching method
(Steeneken & Woortman, 2009) with some modifications. The mixtures
of potato starch and high amylose potato starch (4.5:0.0, 4.0:0.5,
3.0:1.5, 2.0:2.5, 1.0:3.5, and 0.0:4.5) were dispersed with distilled
water (starch: water = 10%, w/w) and the pH was adjusted to 5.5 with
5% citric acid (Babu et al., 2018; Steeneken & Woortman, 2009).
Samples with different amylose ratios were determined by double-wa-
velength spectroscopy method and calculated for an average of three
repetitions. All samples were held in sealed polytetrafluoroethylene
containers (length 8.0 cm, internal width 3.2 cm). Then, all samples
were subsequently heated in a water bath (98 °C, 11 min) while shaking
by hand to gelatinize the starch and prevent granule settling. After full
gelatinization, the samples were quickly placed in an autoclave and
transferred to an oil bath pot preheated to 175 °C for 7.5 min. The ex-
periment condition in this research was based on our previous tests by
the orthogonal experimental design for optimization process. Finally,
the container was quickly quenched with liquid nitrogen. After the
sample was rapidly cooled down by liquid nitrogen for 20 min (at -
40 °C), the PFAR was obtained.
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Carbohydrate Polymers 223 (2019) 115020
2.3. Textural profile analysis (TPA)
The final fat replacers, after stored at 4 °C for 12 h, were then
sheeted into a 2 × 2 x 2 cm cube for analysis, and all experiments were
triplicated. The texture profile analysis (TPA) was performed using a
TA.XT Plus Texture Analyzer (Stable Micro Systems, Surrey, UK) fitted
with a P / 0.5 R (5 mm diameter) probe. Samples were compressed
twice up to 5 mm (25% compression) of the original height at a test
speed of 1 mm/s to obtain TPA parameters (hardness, cohesiveness and
gumminess) (Huang, Kennedy, Li, Xu, & Xie, 2007). The Texture Ex-
ponent TEE32 V 3.0.4.0 Software (Stable Micro Systems) was used to
collect the data and prepare the force-time curves. TPA results were
given in terms of hardness, cohesiveness and gumminess (Bourne,
2010). Hardness is defined as the maximum peak force during the first
compression cycle. Cohesiveness is usually characterized as deforma-
tion resistance of ingredients (Seetapan, Limparyoon, Gamonpilas,
Methacanon, & Fuongfuchat, 2015) and determined as the ratio of the
positive force area during the second compression to which during the
first compression. Gumminess is used to describe the viscosity property
of semi-solid ingredients (Correa, Anon, Perez, & Ferrero, 2010), and it
shows the amount of required energy used for disintegration and
mastication of semi-solid food (Farahnaky, Azizi, Majzoobi, Mesbahi, &
Maftoonazad, 2013; Phimolsiripol, Siripatrawan, & Cjk, 2011). The
analysis was performed in triplicate.
2.4. Color measurements
The color profile was measured by High-quality Colorimeter (NH
310, 3NH Technology Co., Ltd., China) for six times. A standard white
plate provided with the meter was used to calibrate the colorimeter.
The color was expressed as L* (lightness), a* (redness ± greenness),
and b* (yellowness ± blueness). The average value of six replicates
was recorded.
2.5. Rheological measurements
Rheological measurements were performed using a DHR2
Rheometer (TA Instruments, New Castle, DE, USA). For the rheological
tests, the PAFR samples prepared from Section 2.2 were transferred to
the rheometer plate, and then the upper parallel plate was moved down
to give a 1 mm gap. The excess sample was carefully trimmed, and silica
oil was spreaded around the edge of the plate to prevent moisture
evaporation. Samples were equilibrated at 25 °C for 2 min for equili-
brium. The linear viscoelastic range for each sample was determined
with a strain sweep from 0.01% to 100% at the frequency of 1 Hz.
The steady shear experiments were conducted over the shear rate
range of 0.1–100 s−1. Frequency sweep was performed from 0.1 to
100 Hz at 25 °C with a constant strain of 1.0%, which was within the
linear viscoelastic range (Chen, Tong, Ren, & Zhu, 2014). Temperature
sweep was performed from 25 to 100 °C in tension mode working at
1 Hz, 1.0% of amplitude and 5 °C/min heating rate. To avoid moisture
loss during temperature sweep measurements, a protective cover was
applied. Storage modulus (G') and loss modulus (G”) were obtained in
frequency sweep and temperature sweep using the software provided
with the instrument. All the measurements were performed in tripli-
cate.
2.6. Low field 1H nuclear magnetic resonance (LF-NMR)
Distribution and migration of the water in PAFRs were recorded as
the transverse relaxation time (T2) using a low-field 1H nuclear mag-
netic resonance (NMI20-015V-1, Shanghai Niumag Co. Ltd., China).
The instrument was equipped with a 15 mm probe. The relaxation
curves were obtained using a Carr-Purcell-Meiboom-Gill (CPMG) pulse
sequence. Data from 2000 echoes were obtained as eight-scan repeti-
tions. The repetition time between two successive scans was set at 1 s
Y. Hu, et al.
(Bosmans, Lagrain, Ooms, Fierens, & Delcour, 2014; Curti, Carini,
Tribuzio, & Vittadini, 2014). Approximately 1.0 g PAFR samples were
placed in the probe, and all the results need to be normalized in order to
obtain the signal amplitude of protons corresponding to the unit mass
sample for data analysis.
2.7. X-ray diffraction (XRD)
The samples were lyophilized in a freeze dryer (LGJ-10, Beijing
Songyuan Huaxing Technology Development Co., Ltd., Beijing, China)
for 48 h for XRD test. The XRD patterns of PAFR were obtained using a
Rigaku MiniFlex II desktop X-ray diffractometer (D8 Advance, Bruker,
Germany), operating at a current of 40 mA and voltage of 40 kV. Dry
powdered samples were exposed to Cu Kα radiation (0.1512 nm) and
scanned between 2θ = 5 and 45° at a scanning rate of 10°/min with a
step size of 0.02°.
2.8. Scanning electron microscopy analysis (SEM)
A scanning electron microscope (JSM-6390, Hitachi, City, Japan)
was used to observe the microstructure of PAFRs. Samples were rapidly
freeze-dried and mounted on an aluminum stub using double-sided
adhesive tape, coated with gold palladium using a sputter coater (JFC-
1600), then examined at an accelerating voltage of 15 kV.
Representative digital images of samples were obtained at 1000 × and
3000 × magnifications.
2.9. Statistical analysis
All the measurements were performed at least three times with data
expressed as mean ± standard deviations. One-way analysis of var-
iance (ANOVA) was carried out to assess for any significant differences
between the means. ANOVA was analyzed using the SPSS 24.0
Statistical Software Program (SPSS, Inc., Chicago, IL, USA). Significant
differences (p < 0.05) were determined using Duncan’s multiple range
tests.
3. Results and discussion
3.1. Texture profile analysis (TPA) of PAFR
The effects of amylose ratio on TPA of PAFR were presented in
Fig. 1. Generally, hardness increased initially and then decreased with
the increasing amylose ratio (Fig. 1A), and the peak values were ob-
served at 69.58% and 85.17% amylose ratio of PAFRs (with the cor-
responding values of 501.7 ± 2.3 and 506.9 ± 3.2 g). The textural
properties during storage were predominantly affected by short-term
retrogradation of amylose and intensive long-term retrogradation of
amylopectin (Dobosz et al., 2019; Rahmati, Mazaheri Tehrani,
Daneshvar, & Koocheki, 2014; Vamadevan & Bertoft, 2018). After ge-
latinization, the retrogradation of amylose molecules and straight
chains of amylopectin occurred, which followed by the formation of
bundle structure. Generally, as compared to amylopectin, amylose with
regular molecular arrangement is always more prone to retrogradation,
where starch molecules assembled together and then formed a starch-
gel matrix. While it is difficult for branched amylopectin to aggregate
with greater space steric resistance (Liu, 2008). Therefore, the higher
the amylose ratio and the smaller the branch structure were, the easier
it was for starch molecules to coagulate and form a regular structure,
which is related to the hardness increase of PAFR. Moreover, the pos-
sible reason for the poor textural properties at 22.82% was attributed to
the amylopectin matrix gel, which had not been formed yet. The initial
increase of hardness was associated with the stronger interaction be-
tween amylose and water molecules, while the amylopectin gel matrix
had not been formed. Similar result had also been reported where the
gel strength of potato starch was positively correlated with the amylose
3
Carbohydrate Polymers 223 (2019) 115020
content (Yuan et al., 2017). By contrast, amylopectin has a relatively
low degree of ordering during retrogradation, and is more prone to
develop a three-dimensional network gel with lower hardness and
higher springiness, when compared with amylose (Zhang, 2012). The
reason for the low hardness at 97.64% may be that the substantial
quantities of amylose and tiny amounts of amylopectin may influence
the formation of the three-dimensional network structure of PAFR
during retrogradation. Dobosz et al. (2019) had indicated that the
creation of a three-dimensional network of starch gel was resulted from
the interactions between amylose and amylopectin. A similar result has
also been demonstrated in the study of Mua and Jackson (1997), which
indicated that the starches exhibiting harder gels tend to have higher
amylose content and longer amylopectin chains.
As showed in Fig. 1B, the cohesiveness of PAFR decreased with the
increasing amylose ratio. The reason may be that the texture properties
were mainly related to the retrogradation of starch. And in the process
of starch retrogradation, the ordering degree of amylopectin was rela-
tively low. There were more entanglements between amylopectin mo-
lecules. As compared to the bundle structure formed by amylose mo-
lecule, they were more likely to form the associated network structure,
which resulted in the larger cohesiveness of PAFR (Zhang, 2012). The
reassociation of amylopectin was proved to be a critical part in the
formation of cohesive starch gel (Hedayati & Niakousari, 2018). Liu,
Liu, Li, Li, and Su (2011)) had also found that amylopectin molecule
produced a certain degree of cross-linking, and it was easier to form a
network structure, resulting in the reinforcement of cohesiveness.
Fig. 1C recorded the gumminess changes at different amylose ratios
of PAFR. It was exhibited that with the increasing amylose ratio of
PAFR, more energy was needed to masticate the PAFR on the whole.
The highest value of gumminess was observed at 60.23%, indicating
that the three-dimensional network structure of PAFR was strongest
and stable to mimic the structure of fat at this amylose ratio. The fol-
lowing decrease in gumminess with the increasing amylose ratio may
be caused by the weak entanglement between linear chain molecules,
and the poor ability to form the three-dimensional network gel when
this system was at high level of amylose. According to López and García
(2012), amylopectin has a certain reinforcement effect on the network
structure of starch gel; also, the lower swelling ratio of starch with
higher amylose content may be another possible factor contributing to
the low value of gumminess (Romero-Bastida et al., 2018).
3.2. Color analysis of PAFR
Color analysis was conducted to depict the color changes of PAFR at
different amylose ratios (Table 1). The L* value is an estimation of food
lightness and goes from 0 to 100 (darkest black to brightest white),
higher L* value represents a whiter sample (Zare, Boye, Orsat,
Champagne, & Simpson, 2011). The high L* value denotes a colorless,
luminous and homogenous sample, whereas low L* value denotes
otherwise (Nandi & Guha, 2018). A positive correlation (0.965) was
observed between L* value and amylose ratio of PAFR. All samples
depicted high values of L* (from 65.72 to 76.06), which provided a
colorless appearance of PAFR. Meanwhile, this was in accordance with
the result of sensory evaluation. With the increasing amylose ratio, the
values of a* and b* both presented the trend of increasing at first and
decreasing afterwards. In addition, it was worth noting that a* value
changed from positive to negative when the amylose ratio rose from
22.82% to 44.50%, indicating the changes from greenish to reddish
characteristics. While b* values of all samples were negative, indicating
the blueish characteristic of PAFRs. According to the color analysis of
PAFR, it was colorless and easy to be accepted by consumers, which did
not influence the appearance and color of the food products.
3.3. Rheological properties of PAFR
As exhibited in Fig. 2A, all samples presented typical shear thinning
Y. Hu, et al. Carbohydrate Polymers 223 (2019) 115020

Fig. 1. Effect of amylose ratio on hardness (A), cohesiveness (B) and gumminess (C) of PAFRs. Error bars represent the standard errors of the means of replicates.
Values with different small letters indicate significant differences (P < 0.05).

Table 1 reduction of winding point and inter-laminar shear stress (Zhang,

Effect of amylose ratio on the changes in L* (lightness), a* (redness ± green- 2012). Amylose molecules have short molecular chains, low molecular
ness), and b* (yellowness ± blueness) of PAFRs. weights and a few branches, so as to make them equipped with higher
Amylose ratio Color orientation, thus improving the shear thinning behavior. Many studies
have also demonstrated that the higher amylose ratio was, the more
a* b* L* pronounced the shear thinning phenomenon presented (Valle, Colonna,
Patria, & Vergnes, 1996; Xie et al., 2009). Also, it was found that higher
22.82% 1.41 ± 0.03a −2.73 ± 0.07c 65.72 ± 0.25a
44.50% −1.38 ± 0.06b −6.03 ± 0.18a 68.76 ± 0.90 b amylose ratio resulted in a higher apparent viscosity over the same
30.23% −1.58 ± 0.13bc −3.76 ± 0.13b 72.02 ± 0.87c shear rate range, which was the same with the study reported by Xie
69.58% −1.88 ± 0.22c −3.33 ± 0.23b 73.18 ± 0.63c et al. (2009). The higher viscosity with the increasing amount of
85.17% −1.91 ± 0.26c −2.02 ± 0.36d 72.71 ± 0.54c amylose was resulted from high-ordered shorter chains of amylose
97.64% −1.66 ± 0.03bc −1.19 ± 0.17e 76.06 ± 0.78d
molecules (Romero-Bastida et al., 2018).
Mean ± standard deviation values (n = 6) in the same column for each sample Frequency sweep was usually performed to evaluate the changes of
followed by different letters are significantly different (p < 0.05). structure and viscoelasticity of starch gel (Chen et al., 2018). G' and G”
were quantified with frequency sweep from 0.1 to 100 Hz, and damping
behaviors of decreasing viscosity (defined as non-Newtonian flow) or factor (tanδ) was calculated by the ratio of G” and G'. In regard to G' and
pseudoplastic fluid characteristics with the increasing shear rate. This G”, the values in all samples increased significantly with the increasing
reason could be that the increasing shear rate promoted the thermal frequency (Fig. 2B and C) and showed a certain dependency in the
motion of starch molecules, contributing to the molecular expansion range of frequency acquired (0.1–100 Hz), indicating that the rheolo-
and chain segment activity, also reduced the interaction force among gical properties of PAFR samples were gradually enhanced with the
starch molecules. Furthermore, the increased flexibility and fluidity of increasing external force in the linear viscoelastic region. Meanwhile, it
starch molecules led to the decrease in apparent viscosity of PAFR was well known that G' and G” are the most important parameters in
(Islam, Dos Mohd, & Noor, 2001). And the higher the amylose ratio was rheological analysis, which are associated with solid and liquid prop-
in PAFR, the more pronounced the shear thinning phenomenon was. erties in semi-solid sample (Peng, Li, Ding, & Yang, 2017). If G' pre-
This phenomenon happened when starch molecules gradually straigh- dominates G”, it indicates a gel-like behavior of sample. It was mon-
tened and oriented under shear stress, which contributed to the itored that the curve of G' was always higher than that of G”, suggesting
that it exhibited a gel-like behavior and the elastic property dominated

4
Y. Hu, et al.
Fig. 2. Steady shear flow curves of PAFRs (A); Storage modulus (G') (B) and loss modulus (G”) (C), as well as damping factor (tanδ) (D) of PAFRs at different amylose
ratios in frequency sweep；Storage modulus (G') (E) and loss modulus (G”) (F) of PAFRs at different amylose ratios in temperature sweep.
viscous component throughout the frequency sweep (Liu et al., 2016;
Okechukwu & Rao, 1997). Both of the G' and G” curves showed an
apparent rise as the amylose ratio increased, particularly from the in-
itial stage of 22.82%. Moreover, the G' values of 85.17% and 97.64%
samples were very close. This might be caused by that amylose and
water molecules could combine easily through hydrogen bonds, so that
amylose had a positive reinforcement effect on the structure of PAFR,
thus stabilizing the system. Lii et al. (1996) had also summarized si-
milar results on the rheological properties of rice starch with different
amylose content. Furthermore, the reinforcement effect of amylose on
rheological properties of PAFR was more pronounced at lower amylose
ratio, while it was less remarkable at high amylose ratio. The tanδ is
usually characterizing the changes in the viscoelastic property of a
polymer structure. Normally, a low tanδ means that the material be-
haves more like a solid than a liquid (Su et al., 2018). As the tanδ < 1
(Fig. 2D), all samples presented typical weak gel structures and semi-
solid properties (Wu, 2014), which was in accordance with the textural
analysis in this study. The continuously increasing tanδ with frequency
indicated that inner structure of PAFR became much stronger and dis-
played more elastic structure due to the intensified molecular en-
tanglement, which was beneficial to the stability of PAFR structure.
However, the rheological properties of PAFR exhibited a limited rise
5
Carbohydrate Polymers 223 (2019) 115020
through all analysis above, where the PAFR system became relatively
stable and only displayed a minor change when the amylose ratio
reached a certain degree (around 85%). The reason could be that the
starch gel is formed by an interpenetrating amylose gel with the swollen
gelatinized starch granules (Ring, 2010), and the numerous amylose
would hinder the swelling of amylopection. The leaching amylose
molecules would tend to accumulate towards the surface of the starch
granules (Chen et al., 2018), which would lead to the restricted for-
mation of viscoelastic gel. Although it is difficult for branched amylo-
pection molecules to aggregate with each other, it could provide a rigid
structure support for the three-dimensional network structure of PAFR.
Therefore, the proper ratio of amylose was conducive to form a strong
and stable network structure of PAFR, and to mimic the fat.
In temperature sweep of PAFR, Fig. 2E and F depicted that the
curves of G' and G′′ rose dramatically with the increasing amylose ratio,
in addition, the trend of rise was more significant at lower amylose
stage. Previous researches had elucidated that the amylose concentra-
tion was fundamental to the elastic characteristic of starch gel and the
gel strength was reinforced with short-chain amylose (Chen et al.,
2018). Also the gel structure formed with linear amylose is more stable
than that of amylopectin especially at high temperature. The reason
might be that amylose molecules could easily combine with water
Y. Hu, et al.
molecules by hydrogen bonds, and it was relatively different for amy-
lopectin with spatial steric resistance (Liu, 2008). Both of G' and G”
decreased significantly as the temperature rose from 25 to 95 °C, in-
dicating that higher temperature would weaken the three-dimensional
network structure of PAFR, which was resulted from the fracture of
hydrogen bonds. Overall, starch-based fat replacer is less stable and
always destroyed during high temperature process. The thermal in-
stability characteristic of PAFR at high temperature may be due to the
fact that the high temperature would destroy the intermolecular in-
teraction between starch and water molecules and lead to the loss of
water. Then, the three-dimensional network structure of starch gel
would be destroyed. For that the starch-based fat replacer have been
used to achieve fat mimetic properties by retaining substantial quantity
of water into weak gel structures (Luo & Xu, 2011). This result further
suggested that PAFR was not suitable for the application with high-
temperature process. But it was worth noting that the structure of PAFR
equipped with much amylose molecules was relatively stronger, and the
corresponding ability to withstand high temperature was more out-
standing, as presented in Fig. 2E and F.
3.4. Water distribution and migration in PAFR
The fat-like mouthfeel of starch-based fat replacer mainly originates
from the water trapped in the gel (Luo & Xu, 2011). Low-field 1H nu-
clear magnetic resonance (LF-NMR) has been widely applied to ex-
amine the water distribution and migration in food, in which was es-
timated by transverse relaxation time (T2) (Huang, Guo, Wang, Ding, &
Cui, 2016). Inversion spectra of transverse relaxation time (T2) of
PAFRs were recorded in Fig. 3. In this study, T2 was used to confirm the
water distribution and migration in PAFR and explore the interaction
between water and starch molecules by molecule freedom. For the in-
version spectra of T2, the x-axis is associated with water activity and a
longer T2 indicates a higher degree of molecule freedom. The y-axis
represents the signal amplitude of protons and the peak area expresses a
relative content of water in each part. T2 was often divided into three
regions, including the bonded water T21, weakly bonded water T22 and
mobile water T23 (Resing & Neihof, 1970). As showed in Fig. 3, there
were three peaks of T21 (0.01–1 ms), T22 (1–100 ms) and T23
(100–1000 ms), which represented the bonded water, the weakly
bonded water and mobile water, accounting for about 0.002%, 3–5%
and 95–97% of signal amplitude, respectively (Table. 2). Apparently,
Fig. 3. Representative 1H T2 distributions of transverse relaxation times at different amylose ratios in PAFRs.
6
Carbohydrate Polymers 223 (2019) 115020
the mobile water accounted for the most fraction of the total water
content in PAFR. While the bonded water only appeared in the PAFR
with least amylose ratio (22.82%), this part of water was tightly bound
to starch molecules. The weakly bonded water in PAFR was combined
with starch molecules by hydrogen bonds as main force. And the mobile
water was mainly combined with starch molecules by van der Waals
forces in PAFR, which was only subjected to minimal binding force and
can easily become removable water fraction (Raun et al., 1999), ac-
counting for the most fraction of total water content in PAFR.
The effects of amylose ratio on the transverse relaxation time (T2)
and the fractions of signal amplitude of protons in each part of water
were presented in Table 2. The values of T2 represented the binding
force of water molecules combined with starch molecules, which was
used to describe the distribution of water molecules. And the signal
amplitude of protons and peak area represented the content of the
corresponding water molecules, which was used to characterize the
migration of water molecules (Peng et al., 2017). As showed in Table 2,
the fractions of water reflected by T22 and T23 respectively presented
decreasing and increasing trends with the increase of amylose ratio,
which represented the corresponding changes of weakly bonded water
and mobile water. As calculated, the T22 fractions of PAFR decreased
from 5.11 to 3.68%, while the T23 fractions increased from 94.88 to
96.32% (Table 2). On further analysis, some weakly bonded water was
migrated from three-dimensional network structure of PAFR and
transformed to mobile water as amylose ratio continuously increased. It
might result from the stronger trend of linear amylose molecules to
aggregate by hydrogen bonds, subsequently it tightly enwrapped the
branched amylopectin molecules. Ordinarily, the increase in molecular
associations between the starch chains would lead to the exudation of
water from the network structure of gel (Karim, Norziah, & Seow,
2009). As demonstrated in previous study, starch-based fat replacers
have been used to achieve fat mimetic properties by retaining sub-
stantial quantity of water into weak gel structures (Luo & Xu, 2011).
Hence, increasing fluidity of water in PAFR was beneficial to mimic the
lubricating mouthfeel of fat.
As described in Table 2, the T22 and T23 were negatively correlated
with amylose ratio, with correlation coefficients of - 0.995 and - 0.960,
respectively. A negative correlation (- 0.922) was observed between the
total peak area and amylose ratio of PAFR. With the increase of amy-
lose, the bonded water had disappeared in PAFR, which only appeared
in 22.82% sample as analyzed above. The small quantity of bonded
Y. Hu, et al. Carbohydrate Polymers 223 (2019) 115020

Note: a The signal amplitude of protons in one gram. N.D. = not detected. T21, the transverse relaxation time of the bonded water; T22, the transverse relaxation time of the weakly bonded water; T23, the transverse
water was proposed to have transformed to weakly bonded water. For

127.07ab
111.87ab
392.32b
38.93ab
60.10a
77.41a
Total area of Peak
more specific observation showed in Fig. 3, inversion spectra of T2
gradually shifted toward the left (shorter transverse relaxation time),

±
±
±
±
±
±
and the peak time of T22 and T23 respectively reduced from 12.35 to

9208.21
9300.63
8912.31
9084.38
9112.30
8700.15
5.78 ms and from 305.39 to 126.45 ms, indicating that the binding
force (hydrogen bonds) of the weakly bonded water and mobile water
were significantly enhanced. The strong hydrogen bonds promoted
water molecules to trap in three-dimensional network structure of
amplitude of protons in

PAFR. So that this system would be equipped with strong water holding
force and stable structure, which would further provide a steady
Fractions of signal

0.28bc
0.07bc
0.33b
0.09a

mouthfeel of PAFR. This was in accordance with the rheological ana-

0.08c
0.17c
each part (%)a

lysis, where both of G′ and G′′ in PAFR presented positive correlations

±
±
±
±
±
±

with amylose ratio. It was proved that a great amount of amylose

94.88
95.70
95.89
95.94
96.23
96.32

promoted the interaction force of starch molecules with water mole-

cules, especially for weakly bonded water and mobile water, which is
117.16ab
102.66ab
365.96a

the key for starch-based fat replacer to mimic the lubricating mouthfeel
54.65ab

46.34ab
88.04b

of fat.
Area of Peak

±
±
±
±
±
±
8736.67
8900.27
8546.04
8715.25
8769.07
8479.15

3.5. X-ray diffractograms of PAFR

X-ray diffractogram (XRD) is an important analytical technique used

11.31d
12.62a

10.88c
7.98b

9.33e
8.12e
Peak time (ms)

to characterize starch structures and detect any changes in the starch

Mean ± standard deviation values in the same column for each sample followed by different letters are significantly different (p < 0.05).

crystallinity pattern due to different processing techniques (Dankar,

±
±
±
±
±
±

Haddarah, Omar, Pujola, & Sepulcre, 2018). The crystalline pattern of

305.39
200.92
180.57
162.11
138.79
126.45

starch was divided into three types as reported by Hizukuri (1969),

T23
Transverse relaxation time (T2), area of peak and fractions of signal amplitude of protons of PAFRs detected by low-field 1H NMR.

including A and B crystalline patterns with individual composition and

C crystalline pattern composed of a mixture of A and B crystalline
amplitude of protons in

patterns. XRD patterns of three significant samples at different amylose

ratios were depicted in Fig. 4. PAFR showed a typical B-type diffraction
Fractions of signal

pattern with the characteristic reflection at 5.6°, 17.0°, 22.0° and 24.0°
each part (%)a

0.28bc
0.07bc
0.33b
0.07a

0.08c
0.17c

as marked in Fig. 4, which was the same as the native crystalline pat-
±
±
±
±
±
±

tern of potato starch. Besides, crystallinity and spherulite number have

5.11
4.30
4.11
4.06
3.77
3.68

increased with the rising amylose ratio of PAFR treated by super-heated

quenching, which was in accordance with the results of scanning
10.51cd
24.54bc
11.04bc

27.71d
29.91b

electron microscopy (SEM) in 3.6. The reason may be that shorter chain
8.31a

length and less interaction force of amylose molecules would lead to an

Area of Peak

±
±
±
±
±
±

easier migration of amylose molecules and favor the formation of

470.38
400.36
366.27
369.13
343.24
321.00

2–10 μm diameter spherulites during super-heated quenching treat-

ment. This result corresponded with the previous study which revealed
that amylose was more easily prone to form crystallites and entangle-
Peak time (ms)

12.35 ± 0.92a

6.75 ± 0.43d
9.79 ± 0.66b

5.78 ± 0.40e
8.65 ± 0.60c
8.11 ± 0.20c

ments (Koch, Gillgren, Stading, & Andersson, 2010). Furthermore, some

studies have confirmed that the small particles of spherulites were
beneficial to mimic the fat globules in fat replacers (Singh et al., 2010;
T22

Steeneken & Woortman, 2009; Ziegler et al., 2003).

Fractions of signal amplitude
of protons in each part (%)a

0.0026 ± 0.0010
N.D.
N.D.
N.D.
N.D.
N.D.
0.24 ± 0.09
Area of Peak

relaxation time of the mobile water.

N.D.
N.D.
N.D.
N.D.
N.D.
Peak time (ms)

0.49 ± 0.02
N.D.
N.D.
N.D.
N.D.
N.D.
T21
Amylose

22.82%
44.50%
60.23%
69.58%
85.17%
97.64%
Table 2

ratio

Fig. 4. X-ray diffractograms of PAFRs at three typical amylose ratios of 22.82%,

60.23% and 97.64%.

7
Y. Hu, et al.
Fig. 5. Scanning electron microscopy (SEM) of PAFRs at the amylose ratios of 22.82%, 60.23% and 97.64% (a: × 1000, b: × 3000).
3.6. SEM analysis of PAFR
Fig. 5 exhibited representative SEM micrographs of PAFRs in mag-
nifications of × 1000 and × 3000. It showed that PAFR samples had a
continuous three-dimensional network structure with some uniform
holes after super-heated quenching treatment, which was formed re-
sulting from the swelling of starch granules and the leaching of starch
chains during heat treatment (Lee & Rhee, 2007). Meanwhile, the
continuous three-dimensional network structure was responsible for the
elasticity and the water holding capacity of starch gel, which corro-
borated the textural attributes, rheological properties and water dis-
tribution above. Several studies have reported that sufficient amylose
chains between the starch granule were very necessary to form a well-
developed starch network structure (Xie, Liu, & Cui, 2006). Further-
more, many short chains with a larger void structure and a shrub-like
structure appeared in the system when the amylose ratio reached a
certain level (nearly 60.00%). After many micropores appeared, the
three-dimensional network structure began to transform into a lamellar
structure, which presented advantages for simulating the fat-like
mouthfeel. This was consistent with the result reported by Fanta,
Felker, Shogren, and Salch (2006)). As showed in Fig. 5b, many mi-
cropores were observed in the three-dimensional network structure
with increasing amylose ratio. With a great amount of amylose mole-
cules, more spherulites of 2–10 μm diameter began to appear in the
8
Carbohydrate Polymers 223 (2019) 115020
three-dimensional network structure of PAFR. Previous studies have
confirmed that high amylose content of corn starch, mung bean starch
and so on would form starch spherulites after high temperature treat-
ment (> 170 °C) then followed by quenching treatment (Creek, Ziegler,
& Runt, 2006; Nordmark & Ziegler, 2002; Ziegler et al., 2003). And this
result was also proved by the crystallinity of PAFR in XRD analysis. The
phenomenon of spherulite formation favored by linear chains of amy-
lose might be related to the folding of amorphous amylose to crystalline
lamellae (Nordmark & Ziegler, 2002). Meanwhile, crystallization of
spherulite also has been reported to be hindered by the presence of high
proportions of amylopectin (Ziegler, Creek, & Runt, 2005). So that a
large number of amylose molecules favored the formation of spherulite.
This size of spherulite was below the threshold value (10 μm) for human
tongue to touch the particles, which would result in a smooth grainy
mouthfeel and further produce a fat-like smooth grainy feel (Singh
et al., 2010; Yi, Huang, Pan, & Wang, 2014). Steeneken and Woortman
(2009) had also indicated that the ice cream-like creamy taste of fat
replacer partially came from the synthesis of starch spherulite and the
layered three-dimensional network structure.
4. Conclusion
This study showed the significant effects of amylose ratio on the
structure and properties of PAFR. Higher hardness and gumminess, as
Y. Hu, et al.
well as lower cohesiveness were observed in the samples with in-
creasing amylose ratio, and PAFR with 85% amylose ratio presented
better texture properties. Both non-Newtonian fluids properties and a
gel-like behavior of PAFR were investigated, while G′ and G′′ were
positively correlated with amylose ratio. Moreover, PAFR presented the
characteristic of thermal instability by temperature sweep. LF-NMR and
SEM analysis suggested that PAFR with high amylose ratio had stronger
water holding force and was prone to form the lamellar structure. Some
spherulites at the size of 2–10 μm diameter appeared in the three-di-
mensional network with increasing amylose ratio, which was similar to
fat globules in size and contributed to the simulation of PAFR. These
results suggested that amylose contributed to the gel structure of starch-
based fat replacer, and the PAFR with suggested 85% amylose ratio
presented the desirable properties. The insights provided in this study
will be beneficial for the further understanding in the simulation me-
chanism of starch-based fat replacer, as well as the potential application
of this technology in food industry.
Acknowledgments
This study was financially supported by National Natural Science
Foundation of China (Grant No.31601504)
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