Carbohydrate Polymers 213 (2019) 199–207

Contents lists available at ScienceDirect

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

Multi-scale structure and pasting/digestion features of yam bean tuber T

starches
⁎
Dongling Qiaoa, Wenyao Tua, Anping Liaob, Nannan Lic, Binjia Zhangc,f, , Fatang Jianga,
Lei Zhongb, Siming Zhaoc, Liang Zhangd, Qinlu Line
a
Glyn O. Phillips Hydrocolloid Research Centre at HBUT, School of Food and Biological Engineering, Hubei University of Technology, Wuhan, 430068, China
b
Department of Chemical Engineering, Guangxi Key Laboratory Cultivation Base for Polysaccharide Materials and Modifications, Guangxi University for Nationalities,
Nanning, 530008, China
c
Group for Cereals and Oils Processing, College of Food Science and Technology, Key Laboratory of Environment Correlative Dietology (Ministry of Education), Huazhong
Agricultural University, Wuhan, 430070, China
d
School of Food Science and Engineering, Yangzhou University, Yangzhou, 225127, China
e
National Engineering Laboratory for Rice and By-product Deep Processing, College of Food Science and Engineering, Central South University of Forestry and Technology,
Changsha, 410004, China
f
Jinjian Cereals Industry Co., Ltd., Changde, 415000, China

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

Chemical compounds studied in this article: This work discloses the multi-scale structure and pasting/digestion behaviors of yam bean tuber starches (YB-GD
Starch (PubChem CID: 24836924) and YB-SC). Compared to potato starch, YB starches exhibited varied pasting/digestion features that were un-
Water (PubChem CID: 962) derstood from a multi-scale structural view. Especially, YB starches showed relatively polydisperse lamellae, less
Keywords: perfect crystallites, and fewer amylose molecules. These features should reduce the bulk density of starch chain
Yam bean tuber starch packing, and could facilitate the water or enzyme diffusion in starch matrixes. Consistently, not only was starch’s
Structure resistance to hydrothermal effects weakened (shown by reduced pasting temperature), but also the enzyme
Multi-scale absorption to starch chains and the subsequent hydrolysis events were accelerated. Furthermore, YB starch
Physicochemical features
molecules had more short chains, which played roles in reducing the paste viscosity along with the reduced
granule size and in enhancing the paste stabilities during heating and cooling. Also, those molecular features
tended to speed up the enzyme diffusion and digestion events.

1. Introduction starch closely related to the thickening and/or gelling performance

Yam bean (Pachyrhizus erosus L. Urban), originated from the
Amazon region and Mexico semiarid region, belongs to the family
Leguminosae, subfamily Papilionoidea. It is easy to cultivate, growing
well in tropical and subtropical regions and in acid and basic soils, and
has high potentials in nitrogen fixation (Stamford, Freitas, Ferraz, &
Santos, 2002). Yam bean produces tuberous roots with high yields
(Forsyth et al., 2002), which contain relatively high amounts of car-
bohydrate and vitamin C, adequate protein, as well as some potassium,
sodium, phosphorus, calcium and magnesium (Noman, Hoque, Haque,
Pervin, & Karim, 2007). Due to the advantages of easy-cultivation, high-
yield and high nutritional value, this crop is feasible to be included in
dietary for humans, especially in the areas where malnutrition is pre-
valent (Noman et al., 2007).
Starch, as a component of yam bean tuber, can affect the practical
performance of yam bean tuber related foods. Especially, the pasting of
(Iida, Tuziuti, Yasui, Towata, & Kozuka, 2008). The digestion of starch
can release glucose and thus is correlated to the metabolic diseases
(Type II diabetes, cardiovascular diseases, etc.) (Robertson, Currie,
Morgan, Jewell, & Frayn, 2003; Zou, Sissons, Gidley, Gilbert, & Warren,
2015). The physicochemical properties, including pasting and digest-
ibility, of starch are governed by its hierarchical (multi-scale) structure
resulting from the assembly of amylose and amylopectin chains on
multiple length scales, and this multi-scale structural system involves
the whole granule, the growth rings, the semicrystalline lamellae, the
crystallites, and the helices (Blazek & Gilbert, 2011; Doutch & Gilbert,
2013; Flanagan, Gidley, & Warren, 2015; Luengwilai & Beckles, 2009;
Perez & Bertoft, 2010). Thus, to rationally use yam bean tuber starch
resources, it is indispensable to understand the properties of the starch
based on the multi-scale structural features.
Previous investigations of starches from yam bean tuber primarily
concerned the composition (Forsyth et al., 2002; López et al., 2010), the

⁎
Corresponding author.
E-mail address: zhangbj@mail.hzau.edu.cn (B. Zhang).

https://doi.org/10.1016/j.carbpol.2019.02.082
Received 12 November 2018; Received in revised form 26 January 2019; Accepted 24 February 2019
Available online 25 February 2019
0144-8617/ © 2019 Elsevier Ltd. All rights reserved.
D. Qiao, et al.
physicochemical properties (De, Mélo, Krieger, & Stamford, 1994;
Forsyth et al., 2002), the functional behaviors (López et al., 2010; Mélo,
Stamford, Silva, Krieger, & Stamford, 2003) and the nutritional values
(Noman et al., 2007). An investigation had also been conducted to
explore the impact of high energy milling (Martínez-Bustos, López-Soto,
San Martín-Martínez, Zazueta-Morales, & Velez-Medina, 2007) on the
physicochemical and thermal behaviors of yam bean tuber starches.
However, there is still limited understanding of the practical properties
(such as pasting and digestion features) of yam bean tuber starch from a
hierarchical structural viewpoint. The lack of this understanding pre-
vents us from rationally linking the multi-scale structure of yam bean
tuber starch to its properties and functions, and thus is undesired for the
development of yam bean tuber products with regulated performance.
Hence, two starches (YB-GD and YB-SC) from yam bean tubers
cultivated in different provinces in China were used as the materials,
along with a potato starch (a typical tuber starch) as the comparison.
Combined analytical methods covering multiple length-scale orders
were used to inspect the multi-scale structural features and the pasting
and digestion properties of yam bean tuber starches. Then, the struc-
ture-property relationship for yam bean tuber starch was discussed
from the viewpoint of multi-scale structure. This allowed a profound
understanding of how the pasting and digestion behaviors of yam bean
tuber starch differ from the potato starch. The present results could be
helpful in promoting the application of yam bean tuber starch in food
products.
2. Materials and methods
2.1. Materials
Two starches were isolated from yam bean tubers cultivated in
Guangdong (YB-GD) and Sichuan (YB-SC) province in China (detailed
in Section 2.2), and a potato starch was purchased from Wuhan LinHe
Food Co., Ltd. (China). As measured using a SEC method (Cave,
Seabrook, Gidley, & Gilbert, 2009), the amylose content for YB-GD, YB-
SC and potato starch was 16.89%, 15.78% and 21.03%, respectively. α-
Amylase from porcine pancreas (A-3176; activity 25 unit/mg), and
amyloglucosidase from Aspergillus niger (10115, activity 65 unit/mg)
were purchased from Sigma-Aldrich.
2.2. Isolation of YB-GD and YB-SC starches
YB-SD and YB-SC starches were isolated from yam bean tubers using
an earlier method (Stevenson, Jane, & Inglett, 2007) with modifica-
tions. Peeled and cut tuberous roots (about 1 cm3) were immersed into
0.3% (w/v) aqueous sodium metabisulphite solution, and blended using
a blender (Joyoung JYL-C022, Shandong, China). The obtained yam
bean puree was filtered through a 106 μm mesh screen, and the filtrate
was kept at 4 °C for 12 h to acquire the starch deposit. Then, the su-
pernatant was discarded, and the rest was centrifuged at 8000 rpm for
30 min. The recovered starch was washed three times with 0.1 M NaCl,
three times with ultrapure water and two time with ethanol. The re-
sulted starches were dried in a convection oven at 35 °C for 48 h. Note
that the reference potato starch was used as purchased; this starch had
different isolation procedures with the YB starches. The isolation
method may affect starch structural features. Here, the purchased po-
tato starch and the two isolated YB starches were used directly; then,
with the potato starch reference, the multi-scale structure and pasting/
digestion features of YB starches were understood.
2.3. Scanning electron microscopy (SEM)
SEM observations were performed on a scanning electron micro-
scope (JEOL-Model 6390, Japan) operating at 15 kV. The samples were
mounted on a metal stage and then coated with gold. Magnifications of
1000× and 4000× were used for all samples.
200
Carbohydrate Polymers 213 (2019) 199–207
2.4. Laser diffraction analysis
A laser-diffraction analyzer (Mastersizer 2000, Malvern, UK) was
used to measure the starch granule size distribution. Starch was added
to the reservoir and fully dispersed in distilled water until an ob-
scuration value above 10% was achieved. Mastersizer 2000 software
(Ver. 5.60) was adopted to analyze the related parameters (D[4, 3], D
[3, 2], d0.5 and d0.9), and the volume size distribution in the specific
range was calculated using the Microsoft excel 2010 (Redmond, WA,
USA). All the results were reported as the averages of three replicates.
2.5. Small angle X-ray scattering (SAXS)
SAXS measurements were conducted on a NanoSTAR system
(Bruker, Germany) operated at 30 W. The Cu Kα radiation (wavelength,
λ = 0.1542 nm) was used as the X-ray source. A VÅnTeC-2000 detector
(active area 140 × 140 mm2 and pixel size 68 × 68 μm2) was used to
collect the scattering data. The starch slurries (40% starch concentra-
tion) were kept under ambient conditions for 4 h to achieve equili-
brium, and the slurries were used as the samples. The empty sample cell
with water was used as the background. All data were background
subtracted and normalized, and the data in the region of
0.008 < q < 0.20 Å−1 were used as the SAXS results. The scattering
vector, q (Å−1), was defined as q = 4πsinθ/λ (2θ, scattering angle)
(Suzuki, Chiba, & Yano, 1997).
2.6. X-ray diffraction (XRD)
An X-ray powder diffractometer (D8 Advance, Bruker, USA) was
used to investigate the crystalline structure of the starches, operated at
40 kV and 30 mA. The XRD patterns were acquired for a 2θ range of 4-
40°, with a step size of 0.02° and a step rate of 0.5 s per step. The re-
lative crystallinity (Xc, %) was calculated using the PeakFit software
(Ver. 4.12) according to Eq. (1).
n
∑i = 1 Aci
Xc =
At (1)
Where Aci is the area under each crystalline peak with index i, and At is
the total area of the diffraction pattern.
2.7. Size exclusion chromatography (SEC)
The molecular structure of fully and debranched starches were
characterized using an Agilent 1100 Series SEC system (Agilent
Technologies, Waldbronn, Germany) with a differential refractive index
(RI) detector (Shimadzu RID-10 A, Shimadzu Corporation, Kyoto,
Japan) (Liu, Halley, & Gilbert, 2010). As shown previously (K. Wang
et al., 2015), the GRAM precolumn, GRAM 100 and GRAM 3000 col-
umns (PSS, Germany) were used for the size separation of fully bran-
ched starch molecules at 80 °C, and the starch molecules were eluted
using DMSO/LiBr solution at 0.3 mL/min. Then, a series of pullulan
standards were used for calibration to convert elution volume to hy-
drodynamic volume (Vh, or the corresponding radius Rh) with the
Mark–Houwink equation (Cave et al., 2009). The SEC weight size dis-
tributions w(logVh) for fully branched starch molecules acquired from
the RI signal was plotted as a function of Rh (Huo et al., 2018; Vilaplana
& Gilbert, 2010).
To evaluate the SEC size distributions and chain length distributions
(CLDs) of debranched starch molecules, the starches were debranched
by isoamylases following a previous method (Liu et al., 2010). Each
debranched starch was freeze-dried and then dissolved in DMSO/LiBr
solution for SEC analysis. The same Agilent 1100 SEC system, with
GRAM precolumn, GRAM 100 and GRAM 1000 columns, was applied to
analyze the debranched starch molecules at a flow rate of 0.6 mL/min.
Here, for debranched starch molecules containing linear glucan chains,
D. Qiao, et al.
Fig. 1. SEM images of YB-GD, YB-SC and potato starch granules.
the hydrodynamic volume Vh of linear chains was converted to the
degree of polymerization (DP) by Mark-Houwink equation (Cave et al.,
2009).
2.8. Rapid visco analyzer (RVA) analysis
A rapid visco analyzer (RVA) (RVA4500, Perten, Sweden) was used
to explore the viscosity of each starch in water. About 3 g of the starch
was placed into a sample test canister containing 25 g of distilled water.
The RVA impeller was jogged up and down and rotated in the canister
to suspend the starch in the liquid. The RVA canister and impeller were
then positioned in the RVA, and the trial was started. The test profile
involved stirring at 960 rpm for 10 s at 50 °C, stirring at 160 rpm for 50 s
at 50 °C (this rotation rate was maintained in all subsequent stages of
the test), heating from 50 °C to 95 °C within 7.5 min, holding at 95 °C
for 5 min, cooling from 95 °C to 50 °C within 7.5 min, and holding at
50 °C for 2 min. The total test time was 23 min.
2.9. Digestion behaviors
Starch digestion in vitro was carried out in duplicated according to a
reported method (Qiao et al., 2017; Zou et al., 2015) with modifica-
tions. A centrifuge tube with 90.0 mg of starch and 6.0 mL of deionized
water was incubated at ambient temperature for 12 h, followed by
adding 10.0 mL of pH 6.0 sodium acetate buffer solution and incubating
at 37 °C in water bath for 10 min. Then, 5 mL of freshly prepared
201
Carbohydrate Polymers 213 (2019) 199–207
enzyme buffer solution containing 24 unit/mL α-amylase and 42 unit/
mL amyloglucosidase was pipetted into the tube containing starch to be
digested. Afterwards, 100 μL of the digested solution was collected at
each time point and mixed with 900 μL of ethanol to terminate the
digestion. The glucose concentration of the digestion solution was
measured using a glucose oxidase/peroxidase reagent (GOPOD Re-
agent, Megazyme). The glucose solution (1 mg/mL) was used as the
standard. The percentage of digested starch was calculated according to
Eq. (2).
100μL× 1.0mg/mL 100% 162
SD (%) = A sample × × 10 × 210 × ×
A glucose 90mg 180 (2)
Where SD is the percentage of starch digested; Asample and Aglucose are
the absorbance values for the starch digestion solution and glucose
standard, respectively; the value of 10 × 210 is the computational
multiple from 100 μL aliquots to 21.0 mL reaction solution; 162/180 is
the transformation coefficient from glucose to starch in weight.
Based on the first-order kinetic model (Eq. (3)), the logarithm of the
slope (LOS) plot (Eq. (4)) combined with the non-linear curve fitting
method was adopted to analyze the digestion rate of starch
(Butterworth, Warren, Grassby, Patel, & Ellis, 2012; Qiao et al., 2017).
The LOS plot can distinguish the number of specific digestions stages
with specific digestion rates throughout the whole digestion period
based on the changes in the slope of digestion pattern (ln(dCt/dt))
against time (t). But the LOS plot uses the numerical derivative of
discrete rate data points and thus is inherent inaccurate regarding the
D. Qiao, et al.
rate coefficient (i.e., kLOS). Hence, the LOS plot was mainly used to
reveal the number of digestion stages, and the non-linear curve fitting
based on first-order kinetic in Eq. (1) is employed to obtain the rate
coefficient for each starch digestion (i.e., kfitting) stage with increased
accuracy.
Ct = C∞ (1 − e−k × t )
dCt
ln = −k × t + ln(C∞ × k )
dt
Where Ct (%) is the amount of starch digested at a given time (t (min));
C∞ (%) is the estimated percentage of starch digested at the end point
of a digestion stage; k (min−1) is the coefficient of starch digestion rate.
The calculated digestion data (ln[(Ci+2−Ci)/(ti+2− ti)]) at each time
point ((ti+2+ ti)/2), except the last two points, was used to obtain the
LOS pattern and the related fit curve.
2.10. Statistical analysis
Data were expressed as means ± standard deviations (SD). A sta-
tistical difference of P < 0.05 was considered to be significant.
ANOVA analysis was carried out in Microsoft excel 2010 (Redmond,
WA, USA).
3. Results and discussion
3.1. Granule features
The SEM micrographs of YB-GD, YB-SC and potato starches are
presented in Fig. 1. YB-GD and YB-SC displayed spherical, polygonal,
and irregular shapes with a relatively smooth surface. No visible dif-
ference in morphology was observed for the two yam bean tuber star-
ches. The potato starch showed ellipsoidal shape along with a smooth
surface, consistent with earlier findings (Binjia Zhang, Dhital, Flanagan,
& Gidley, 2014, 2014b).
The granule size distribution analyzed by laser diffraction for the
three tuber starches are included in Fig. 2, and the related parameters
are listed in Table 1. YB-GD and YB-SC displayed a dominant granule
size distribution peak I in the range of ca. 2–50 μm, and a smaller
shoulder peak II between ca. 50–300 μm. Relative to YB-SC, YB-GD had
a smaller peak I but a slightly stronger peak II, and thus showed larger
size values (D[4, 3] and d(0.9)). For the potato starch, the granule size
distribution peak shifted to significantly higher size values (ca.
10–200 μm) than those for YB-GD and YB-SC. Consistently, the potato
starch displayed evidently larger size parameters such as d(0.5), and
d(0.9) than did YB starches (see Table 1).
3.2. Lamellar structure
The semicrystalline lamellae of starch granules, i.e., alternating
Fig. 2. Granule size distribution profiles of YB-GD, YB-SC and potato starches.
Carbohydrate Polymers 213 (2019) 199–207
crystalline and amorphous lamellae, formed by the assembly of starch
chains on the nanoscale (9–10 nm), could be well evaluated by the
SAXS technique (Qiao et al., 2017; Binjia Zhang, Xie, Shamshina et al.,
2017; Binjia Zhang, Xie, Wang et al., 2017). The logarithmic SAXS
patterns of three tuber starches are shown in Fig. 3. Due to the alter-
nating arrangement of crystalline and amorphous lamellae, a scattering
(3)
peak centred at a q value of ca. 0.065 Å−1 is presented on the SAXS
patterns. The position (q) of this peak could be used to calculate the
(4) interlamellar repeat distance (d) of the semicrystalline lamellae with
the Woolf-Bragg's equation (d = 2π/q). The interlamellar repeat dis-
tance (10.5 nm) of YB-SC was larger than that (9.8 nm) of YB-GD, with
the smallest value (9.4 nm) for potato starch. Also, the half width at
half-maximum of the scattering peak is associated with the poly-
dispersity of the crystalline − amorphous lamellae (Witt, Doutch,
Gilbert, & Gilbert, 2012). Compared with the potato starch, the YB
starches displayed a broader peak, suggesting the existence of relatively
polydisperse semicrystalline lamellae in YB starches.
3.3. Crystalline structure
The crystalline structure of starch can be distinguished by XRD
(Buléon, Colonna, Planchot, & Ball, 1998; Perez & Bertoft, 2010). The
double helices of starch can be organized in monoclinic or hexagonal
crystalline unit cells, constructing the A- and B-type polymorphs, re-
spectively (Gérard, Planchot, Colonna, & Bertoft, 2000). The C-type
structure is in fact a superposition of A- and B-type structures. The
starches from different botanical origins may show varied crystalline
types (Gérard et al., 2000; S. Wang & Copeland, 2015). Fig. 4 shows the
XRD patterns of YB-GD, YB-SC and potato starches. Potato starch dis-
played a typical B-type polymorphic structure with the strongest dif-
fraction peak at around 17°, several smaller peaks at 15°, 22° and 24°,
and a characteristic peak at ca. 5.6°. Except for the B-type diffraction
peaks, YB-GD and YB-SC showed additional A-type diffraction peaks at
ca. 15° and 23°. This indicates that YB-GD and YB-SC starches had a C-
type crystalline structure. Also, YB-GD and YB-SC starches displayed a
crystallinity degree (Xc) at around 44% which is similar to that of po-
tato starch (about 45%).
3.4. Size distributions of whole starch molecules
Typical SEC weight size distributions of fully branched YB-GD, YB-
SC and potato starches are shown in Fig. 5a, normalized to yield the
same height of the highest peak for comparisons. All starches displayed
a bimodal size distribution corresponding to amylose at smaller Rh
values and amylopectin at larger Rh values. Note that the SEC separa-
tion of whole amylopectin molecules suffers from unavoidable shear
scission, calibration limitation, separation limit of SEC columns and low
recovery (Cave et al., 2009; Syahariza, Sar, Hasjim, Tizzotti, & Gilbert,
2013). Thus, the SEC size distribution of amylopectin component
cannot be used to make structural inferences, and only the distribution
of smaller molecule (amylose) was considered. Table 2 presents the
results for the average Rh of amylose (denoted as R̄h, amylose ). The results
indicated that YB-GD, YB-SC and potato starch showed a similar size for
amylose molecules.
3.5. Size distributions and chain-length distributions of debranched starch
The SEC size distributions and chain-length distributions (CLDs) of
debranched starches are presented as weigh distributions (w (log Vh)) in
Fig. 5b. All weight distributions were normalized to yield the same
global maximum to enable relative comparisons. All samples showed
commonly seen features, i.e., multiple smaller bumps for amylose
branches (approximately Rh ≥ 4 nm or DP ≥ 100) and two large peaks
for amylopectin branches (about Rh < 4 nm or DP < 100). The two
amylopectin peaks corresponded to the amylopectin short chains (viz.,
Ap1) (DP ≤ 30) confined to a single lamella and the amylopectin long
202
D. Qiao, et al.
Table 1
Granule size distribution parameters of YB-GD, YB-SC and potato starchesA.
Sample D[4, 3] D[3, 2]
bB b b
YB-GD 19.2 ± 0.5 10.8 ± 0.2
YB-SC 14.0 ± 0.1c 10.7 ± 0.1b
Potato starch 47.5 ± 0.0a 34.3 ± 0.1a
A
D[4, 3], volume weight mean; D[3, 2], surface weight mean; d(0.5), 50% of the overall particles showed a size less than this value (μm); d(0.9), 90% of the overall
particles showed a size less than this value (μm).
B
Values followed by the different uppercase letter within a column differ significantly (P < 0.05).
Fig. 3. SAXS patterns of YB-GD, YB-SC and potato starches.
Fig. 4. XRD patterns of YB-GD, YB-SC and potato starches.
chains (viz., Ap2) (31 ≤ DP < 100) spanning more than a single la-
mella. The distribution patterns of amylopectin chains from YB-GD and
YB-SC were similar; the potato starch showed a stronger peak of Ap2
chains, suggesting a larger proportion of amylopectin long chains in the
potato starch than in the YB starches.
Apparent differences were observed in the weight size distributions
and CLDs of amylose chains among those starches (enlarged in Fig. 5c).
For the starches, the amylose weight distributions had two unresolved
bumps. This indicates the existence of two groups of amylose chains,
including shorter chains (denoted by Am1) (DP 200–2600) and longer
chains (denoted by Am2) (DP 2600–20000). The area under the re-
spective peak of Am1 chains (AAm1) or Am2 chains (AAm2) was used to
indicate relative amount of corresponding amylose chains in starch. The
amylose content was obtained from the weight size distributions by
calculating the ratio of the area under the curve of whole amylose range
(about Rh ≥ 4 nm) to the area under the curve of whole starch dis-
tribution. All parameters (AAm1, AAm2 and amylose content) are shown
in Table 2. There were no visible differences in the AAm1 and AAm2
values among YBeGD and YBeSC; the YB starches had a larger AAm1
Carbohydrate Polymers 213 (2019) 199–207
d(0.5) d(0.9) Volume size distribution
0-10 μm 0-50 μm
b a
12.1 ± 0.2 30.4 ± 0.3 37.4 ± 1.2 94.2 ± 0.2b
12.0 ± 0.1b 22.6 ± 0.2c 35.9 ± 0.4a 99.3 ± 0.1a
40.7 ± 0.1a 86.8 ± 0.4a 0.3 ± 0.0b 65.9 ± 0.0c
but a smaller AAm2 than did the potato starch. In addition, the order of
amylose content was GBeSC < YBeGD < potato starch with sig-
nificant difference from 15.8% to 21.0%.
3.6. Pasting properties
Fig. 6 shows the pasting profiles of starches, and the related para-
meters are listed in Table 2. The pasting temperature (Tps) of the star-
ches had an order of YB-SC < YB-GD < potato starch. This reveals
that the starch granules of the YB starches (rather than the potato
starch) were easier to swell and rupture in aqueous medium (Li, Shu,
Zhang, & Shen, 2011). The YB starches displayed a lower peak viscosity
(ηpk) than did the potato starch, but their ηp values were still higher
than that for commonly used regular maize starch (Qiu et al., 2015).
Also, the YB starches exhibited a reduced breakdown viscosity
(Δηbreakdown) as compared to potato starch, suggesting a higher paste
stability under shearing with heating. In addition, the YB-SC sample
had a lower setback viscosity (Δηsetback) than did the potato starch. That
is, YB-SC showed weakened retrogradation tendency and enhanced
paste stability during cooling with shearing. These pasting features
indicate that YB starch may serve as food agents with reduced pasting
temperature and increased paste stabilities under heating and cooling
with shearing.
3.7. Digestion behaviors
Fig. 7 includes the digestion curves and LOS plots, with their fit
curves, for the three starches. All of the starches only showed one linear
range in the LOS plot curve as identified by rate constant kLOS. This
revealed a monophasic digestion behavior under the first-order ki-
netics. Since the digestion rate can be affected by the concentration of
enzymes used in the experiment, the digestion course of the starches is
in fact pseudo-first-order (Butterworth et al., 2012). The rate coefficient
(kLOS) acquired from the LOS plot has inherent inaccuracy. Thus, this
LOS method was just used to show the number of digestion steps, and
the non-linear curve fitting based on first-order kinetics was used to
obtain the digestion rate coefficient (kFitting) (Yu, Tao, & Gilbert, 2018).
Table 2 lists the digestion parameters of the starches. The kFitting value
for three tuber starches was in an order of YB-GD ≈ YB-SC > potato.
This reflects that YB starches had no significant variations in kFitting that
is somewhat higher than that of potato starch.
3.8. Discussion on the structure-property relationship
3.8.1. Structure-pasting relationship
Following the multi-scale structure and the pasting characteristics,
we suggest a schematic representation for the structure-pasting re-
lationship of yam bean tuber starch (shown in Fig. 8). With potato
starch as a comparison, the YB starch showed a reduced granule size (an
increased specific surface area), relatively polydisperse semicrystalline
lamellae, and less perfect crystallites with weak points. These structural
features on multiple scales could facilitate the permeation of water
molecules into the starch granules. Consequently, the starch structures
203
D. Qiao, et al. Carbohydrate Polymers 213 (2019) 199–207

Fig. 5. SEC weight size distributions of whole starch molecules (a), and SEC weight size distributions and chain-length distributions of debranched starch molecules
(b and c) for YB-GD, YB-SC and potato samples.

displayed a reduced resistance to heat-induced swelling and rupture in
water medium, as reflected by the lowered pasting temperature Tps.
Consistent with this, previous investigations show that the pasting
temperature of starch could be decreased, when the perfection of starch
structures are certainly reduced by treatments such as ball-milling (Tan
et al., 2015). In addition, the YB starch contained a reduced content of
amylose molecules, accompanied with fewer amylose long chains
(Am2) and amylopectin long chains (Ap2). The glucan chains should
less effectively (densely) assemble within the multi-level structures,
also playing some roles in lowering the pasting temperature.
With the temperature increasing from Tps, the paste viscosity con-
tinuously increased and could reach a peak value (i.e., peak viscosity
ηpk). The smaller granule size of YB starch allowed the formation of
fully swollen granules with a decreased size. As a result, the swelled
granules of YB starch were more closely packed within a relatively
small volume than did those of potato starch. Also, the YB starch mo-
lecules displayed less amylose and amylopectin long chains, thus
having a weaker capacity to hold water molecules. All these facts
contributed to the reduction in the peak paste viscosity. Then, with the
starch pasting continued, the fully swelled granules at the peak visc-
osity could be gradually disrupted, allowing the emergence of break-
down viscosity Δηbreakdown. In fact, the maximumly swollen starch
granules are called as granule “ghosts” having a nonordered shell
formed by randomly entangled glucan chains (Miao et al., 2018; B.

Table 2
Molecular, pasting and digestion parameters of YB-GD, YB-SC and potato starches A.
YB-GD YB-SC Potato starch

aB a
R̄h, amylose (nm) 20.2 ± 0.5 19.9 ± 0.5 18.1 ± 0.1a
a a
The area under the peak of Am1 and Am2 AAm1 30.3 ± 2.3 36.2 ± 4.9 17.0 ± 0.0b
AAm2 69.7 ± 2.3b 63.8 ± 4.9b 82.9 ± 0.0a
Amylose content (%) 16.9 ± 0.1b 15.8 ± 0.2c 21.0 ± 0.4a
Tps (°C) 62.3 ± 0.0b 61.3 ± 0.2c 63.7 ± 0.3a
ηpk (cP) 6783.8 ± 74.5b 6783.5 ± 42.5b 8120.0 ± 48.0a
Δηbreakdown (cP) 5082.3 ± 89.6b 5122.5 ± 29.5b 5535.5 ± 37.5a
Δηsetback (cP) 1190.7 ± 22.9a 912.0 ± 43.0b 1243.5 ± 43.5a
LOS plot kLOS (min−1) 0.0031 ± 0.0001a 0.0030 ± 0.0000a 0.0010 ± 0.0000b
Non-linear curve fitting kFitting (min−1) 0.0035 ± 0.0001a 0.0033 ± 0.0001a 0.0013 ± 0.0001b

A
Parameters from SEC: R̄h, amylose , average Rh of amylose; AAm1 or AAm2, amounts of shorter or longer amylose chains in starch. Parameters from RVA: Tps, pasting
temperature; ηpk, peak viscosity; Δηbreakdown, breakdown viscosity; Δηsetback, setback viscosity. Parameters of starch digestion: kLOS (min−1), digestion rate coefficient
derived from LOS plot; kFitting (min−1), digestion rate coefficient derived from non-linear curve fitting.
B
The different inline letters within a row indicate significant difference P < 0.05.

204
D. Qiao, et al.
Fig. 6. Pasting profiles for YB-GD, YB-SC and potato starches.
Zhang, Dhital et al., 2014, 2014b). The chain features of YB starch, such
as the reduced content for amylose and amylopectin long chains,
probably allowed the more robust entanglement of glucan chains in the
shell of granule ghosts. This resulted in the enhanced paste stability of
YB starch under heating with shearing (shown by the reduced
Δηbreakdown).
Finally, accompanying the paste cooling, the adjacent chains of
starch molecules reassembled to induce an increase in the paste visc-
osity (as reflected by the setback viscosity Δηsetback). Other than the
lower amylose content, the amylose and amylopectin molecules in YB
starch contained elevated proportions of relatively short chain fractions
(denoted as Am1 and Ap1 above). These chain characteristics tended to
suppress the interactions and/or the reorganization of starch chains
during cooling. In this way, the YB starch such as YB-SC could show the
enhanced paste stability (the weakened retrogradation tendency) under
Fig. 7. Typical digestion curves, LOS plots and nonlinear fitting curves for YB-GD, YB-SC and potato starches. , experimental data; , LOS plot data;
curve for LOS plot data; , fit curve based on first-order kinetic model.
205
Carbohydrate Polymers 213 (2019) 199–207
cooling with shearing (confirmed by the reduced Δηsetback).
3.8.2. Structure-digestion relationship
Typically, there are two main enzymes, α-amylase and amyloglu-
cosidase, in the digestion system of starch. α-amylase molecules ran-
domly hydrolyze the α-1,4 linkages of starch glucan chains, and amy-
loglucosidase molecules cleave the terminal (or next-to-terminal)
linkage at the non-reducing ends of starch chains. The digestion process
of granule starch is heterogeneous, which involves the diffusion of
enzyme molecules to starch substrate as well as the absorption (glucan
chain-enzyme complex formation) and subsequent catalytic events
(glucoside linkage hydrolysis) (Colonna, Leloup, & Buléon, 1992; Bin
Zhang, Dhital, & Gidley, 2013). Thus, the rate of starch digestion is
correlated with the rate of the enzyme diffusion in the substrate and the
enzyme-substrate complex formation. It is found that the digestion rate
of starch can be altered by a wide range of features such as the granule
surface structure, the polymorphic type, and the molecule chain fea-
tures (Blazek & Copeland, 2010; Syahariza et al., 2013), since such
features probably change the enzyme diffusion in the substrate and
subsequently the enzyme absorption and hydrolysis events.
Based on this theoretical basis, the structural and digestion features
mentioned above enable an understanding of the structure-digestion
relationship of yam bean tuber starch (Fig. 8). More specifically, re-
lative to the potato starch, the YB starch displayed more polydisperse
amorphous-crystalline lamellae, less perfect A-type polymorphs in C-
type crystallites, and fewer amylose molecules. These multi-scale
structural features could reduce the bulk density of molecule chain
packing in starch substrate. This reduced bulk density, with the reduced
granule size (the elevated specific surface area), tended to facilitate the
diffusion of enzyme molecules in the substrate matrixes. Then, the
absorption of enzyme molecules to the glucan chains, i.e., the formation
of enzyme-glucan complexes at the molecular scale, could be
, linear fit
D. Qiao, et al.
Fig. 8. Schematic representation for the variations in pasting and digestion features among YB (YB-GD and YB-SC) and potato starches.
accelerated, which led to a proper increase in the enzyme hydrolysis
rate of starch glucan chains. This is certainly consistent with earlier
findings that the present of micro pores on starch granule surface (the
reduced bulk density of granules) promote the migration of enzyme into
the granule and eventually speed up the starch digestion (Shrestha
et al., 2012). Furthermore, YB starches contained increased ratios of
short glucan chains (Am1 and Ap1), which might lead to a less flexible
structure and allow less effective interactions between amylose and/or
amylopectin chains. The events here also contributed to the migration
of enzyme molecules in the substrate matrixes towards the glucan
chains. This allowed a faster absorption of enzyme molecules on the
glucan chains to form starch-enzyme complexes and then certainly in-
creased the rate of starch chain hydrolysis resulting from the enzyme
catalytic events.
4. Conclusions
Using combined techniques, this work reveals the multi-scale
structure and the pasting and digestion behaviors of yam bean tuber
starches (YB-GD and YB-SC). Relative to the potato starch comparison,
YB starches showed varied pasting/digestion features such as reduced
pasting temperature and paste viscosity, enhanced paste stabilities and
a properly increased digestion rate. Then, the variations in starch
properties were rationalized from a multi-scale structural view.
Specifically, YB starches had relatively polydisperse lamellae, less
perfect crystallites, and fewer amylose molecules, which probably re-
duced the bulk density of starch chain packing in the structures and
could promote the diffusion of water or enzyme molecules in starch
matrixes. Then, not only was starch structures’ resistance to hydro-
thermal effects weakened (indicated by reduced pasting temperature),
but also the enzyme absorption and hydrolysis events could be ac-
celerated. Furthermore, the YB starch molecules had more short chains.
These features tended to reduce the paste viscosity along with the re-
duced granule size and enhance the paste stabilities during heating and
cooling related to the robustness of swollen granules and the re-
assembly of starch chains. In addition, the molecular features should
also speed up the digestion of starch, presumably due to less effective
interactions between substrate glucan chains. Hence, the present results
provide insights into the pasting and digestion behaviors of YB starch,
which is of value for the rational usage of YB starch resources.
206
Carbohydrate Polymers 213 (2019) 199–207
Acknowledgments
The authors would like to acknowledge the National Natural
Science Foundation of China (31801582, and 31701637), the Project
funded by China Postdoctoral Science Foundation (2018M642865), the
Scientific Research Project from Hubei Province Department of
Education (Q20181407), and the Fundamental Research Funds for the
Central Universities (2662016QD008). This work is also supported by
the Open Project Program (GXPSMM18ZD-03) from Guangxi Key
Laboratory Cultivation Base for Polysaccharide Materials and
Modification, Guangxi University for Nationalities, and the Guangxi
Biological Polysaccharide Separation, Purification and Modification
Research Platform (GKZY18076005). The authors would like to thank
Dr. Cheng Li, Dr. Enpeng Li and Mr. Shiqing Zhou from Prof. Robert
Gilbert’s lab at Yangzhou University for their assistance on SEC ana-
lysis. Also, B. Zhang thank the Young Elite Scientists Sponsorship
Program by China Association for Science and Technology, the Chutian
Sholars Program of Hubei Province, and the Shishan Sholars Program of
Huazhong Agricultural University.
References
Blazek, J., & Copeland, L. (2010). Amylolysis of wheat starches. II. Degradation patterns
of native starch granules with varying functional properties. Journal of Cereal Science,
52(2), 295–302.
Blazek, J., & Gilbert, E. P. (2011). Application of small-angle X-ray and neutron scattering
techniques to the characterisation of starch structure: A review. Carbohydrate
Polymers, 85(2), 281–293.
Buléon, A., Colonna, P., Planchot, V., & Ball, S. (1998). Starch granules: Structure and
biosynthesis. International Journal of Biological Macromolecules, 23(2), 85–112.
Butterworth, P. J., Warren, F. J., Grassby, T., Patel, H., & Ellis, P. R. (2012). Analysis of
starch amylolysis using plots for first-order kinetics. Carbohydrate Polymers, 87(3),
2189–2197.
Cave, R. A., Seabrook, S. A., Gidley, M. J., & Gilbert, R. G. (2009). Characterization of
starch by size-exclusion chromatography: The limitations imposed by shear scission.
Biomacromolecules, 10(8), 2245–2253.
Colonna, P., Leloup, V., & Buléon, A. (1992). Limiting factors of starch hydrolysis.
European Journal of Clinical Nutrition, 46(Suppl 2), S17–32.
De, A., Mélo, E., Krieger, N., & Stamford, T. L. M. (1994). Physicochemical properties of
jacatupé (Pachyrhizus erosus L. urban) starch. Starch - Stärke, 46(7), 245–247.
Doutch, J., & Gilbert, E. P. (2013). Characterisation of large scale structures in starch
granules via small-angle neutron and X-ray scattering. Carbohydrate Polymers, 91(1),
444–451.
Flanagan, B. M., Gidley, M. J., & Warren, F. J. (2015). Rapid quantification of starch
molecular order through multivariate modelling of 13C CP/MAS NMR spectra.
Chemical Communications, 51(80), 14856–14858.
Forsyth, J. L., Ring, S. G., Noel, T. R., Parker, R., Cairns, P., Findlay, K., et al. (2002).
D. Qiao, et al.
Characterization of starch from tubers of Yam Bean (Pachyrhizus ahipa). Journal of
Agricultural and Food Chemistry, 50(2), 361–367.
Gérard, C., Planchot, V., Colonna, P., & Bertoft, E. (2000). Relationship between
branching density and crystalline structure of A- and B-type maize mutant starches.
Carbohydrate Research, 326(2), 130–144.
Huo, Y., Zhang, B., Niu, M., Jia, C., Zhao, S., Huang, Q., et al. (2018). An insight into the
multi-scale structures and pasting behaviors of starch following citric acid treatment.
International Journal of Biological Macromolecules, 116, 793–800.
Iida, Y., Tuziuti, T., Yasui, K., Towata, A., & Kozuka, T. (2008). Control of viscosity in
starch and polysaccharide solutions with ultrasound after gelatinization. Innovative
Food Science & Emerging Technologies, 9(2), 140–146.
Li, W. H., Shu, C., Zhang, P. L., & Shen, Q. (2011). Properties of starch separated from ten
mung bean varieties and seeds processing characteristics. Food and Bioprocess
Technology, 4(5), 814–821.
Liu, W.-C., Halley, P. J., & Gilbert, R. G. (2010). Mechanism of degradation of starch, a
highly branched polymer, during extrusion. Macromolecules, 43(6), 2855–2864.
López, O. V., Viña, S. Z., Pachas, A. N. A., Sisterna, M. N., Rohatsch, P. H., Mugridge, A.,
et al. (2010). Composition and food properties of Pachyrhizus ahipa roots and starch.
International Journal of Food Science & Technology, 45(2), 223–233.
Luengwilai, K., & Beckles, D. M. (2009). Structural investigations and morphology of
tomato fruit starch. Journal of Agricultural and Food Chemistry, 57(1), 282–291.
Martínez-Bustos, F., López-Soto, M., San Martín-Martínez, E., Zazueta-Morales, J. J., &
Velez-Medina, J. J. (2007). Effects of high energy milling on some functional prop-
erties of jicama starch (Pachyrrhizus erosus L. Urban) and cassava starch (Manihot
esculenta Crantz). Journal of Food Engineering, 78(4), 1212–1220.
Mélo, E. A., Stamford, T. L. M., Silva, M. P. C., Krieger, N., & Stamford, N. P. (2003).
Functional properties of yam bean (Pachyrhizus erosus) starch. Bioresource
Technology, 89(1), 103–106.
Miao, L., Zhao, S., Zhang, B., Tan, M., Niu, M., Jia, C., et al. (2018). Understanding the
supramolecular structures and pasting features of adlay seed starches. Food
Hydrocolloids, 83, 411–418.
Noman, A. S. M., Hoque, M. A., Haque, M. M., Pervin, F., & Karim, M. R. (2007).
Nutritional and anti-nutritional components in Pachyrhizus erosus L. Tuber. Food
Chemistry, 102(4), 1112–1118.
Perez, S., & Bertoft, E. (2010). The molecular structures of starch components and their
contribution to the architecture of starch granules: A comprehensive review. Starch/
Stärke, 62(8), 389–420.
Qiao, D., Xie, F., Zhang, B., Zou, W., Zhao, S., Niu, M., et al. (2017). A further under-
standing of the multi-scale supramolecular structure and digestion rate of waxy
starch. Food Hydrocolloids, 65, 24–34.
Qiu, S., Yadav, M. P., Chen, H., Liu, Y., Tatsumi, E., & Yin, L. (2015). Effects of corn fiber
gum (CFG) on the pasting and thermal behaviors of maize starch. Carbohydrate
Polymers, 115, 246–252.
Robertson, M., Currie, J., Morgan, L., Jewell, D., & Frayn, K. (2003). Prior short-term
consumption of resistant starch enhances postprandial insulin sensitivity in healthy
subjects. Diabetologia, 46(5), 659–665.
Shrestha, A. K., Blazek, J., Flanagan, B. M., Dhital, S., Larroque, O., Morell, M. K., et al.
(2012). Molecular, mesoscopic and microscopic structure evolution during amylase
207
Carbohydrate Polymers 213 (2019) 199–207
digestion of maize starch granules. Carbohydrate Polymers, 90(1), 23–33.
Stamford, N., Freitas, A., Ferraz, D., & Santos, C. (2002). Effect of sulphur inoculated with
Thiobacillus on saline soils amendment and growth of cowpea and yam bean le-
gumes. The Journal of Agricultural Science, 139(3), 275–281.
Stevenson, D. G., Jane, J.-l., & Inglett, G. E. (2007). Characterisation of Jícama (Mexican
potato) (Pachyrhizus erosus L. urban) starch from taproots grown in USA and Mexico.
Starch - Stärke, 59(3-4), 132–140.
Suzuki, T., Chiba, A., & Yano, T. (1997). Interpretation of small angle X-ray scattering
from starch on the basis of fractals. Carbohydrate Polymers, 34(4), 357–363.
Syahariza, Z. A., Sar, S., Hasjim, J., Tizzotti, M. J., & Gilbert, R. G. (2013). The importance
of amylose and amylopectin fine structures for starch digestibility in cooked rice
grains. Food Chemistry, 136(2), 742–749.
Tan, X., Zhang, B., Chen, L., Li, X., Li, L., & Xie, F. (2015). Effect of planetary ball-milling
on multi-scale structures and pasting properties of waxy and high-amylose corn-
starches. Innovative Food Science & Emerging Technologies, 30, 198–207.
Vilaplana, F., & Gilbert, R. G. (2010). Two-dimensional Size/Branch length distributions
of a branched polymer. Macromolecules, 43(17), 7321–7329.
Wang, S., & Copeland, L. (2015). Effect of acid hydrolysis on starch structure and func-
tionality: A review. Critical Reviews in Food Science and Nutrition, 55(8), 1081–1097.
Wang, K., Wambugu, P. W., Zhang, B., Wu, A. C., Henry, R. J., & Gilbert, R. G. (2015). The
biosynthesis, structure and gelatinization properties of starches from wild and cul-
tivated African rice species (Oryza barthii and Oryza glaberrima). Carbohydrate
Polymers, 129, 92–100.
Witt, T., Doutch, J., Gilbert, E. P., & Gilbert, R. G. (2012). Relations between molecular,
crystalline, and lamellar structures of amylopectin. Biomacromolecules, 13(12),
4273–4282.
Yu, W., Tao, K., & Gilbert, R. G. (2018). Improved methodology for analyzing relations
between starch digestion kinetics and molecular structure. Food Chemistry, 264,
284–292.
Zhang, B., Dhital, S., & Gidley, M. J. (2013). Synergistic and antagonistic effects of α-
amylase and amyloglucosidase on starch digestion. Biomacromolecules, 14(6),
1945–1954.
Zhang, B., Dhital, S., Flanagan, B. M., & Gidley, M. J. (2014). Mechanism for starch
granule ghost formation deduced from structural and enzyme digestion properties.
Journal of Agricultural and Food Chemistry.
Zhang, B., Xie, F., Shamshina, J. L., Rogers, R. D., McNally, T., Halley, P. J., et al. (2017).
Dissolution of starch with aqueous ionic liquid under ambient conditions. ACS
Sustainable Chemistry & Engineering, 5(5), 3737–3741.
Zhang, B., Xie, F., Wang, D. K., Zhao, S., Niu, M., Qiao, D., et al. (2017). An improved
approach for evaluating the semicrystalline lamellae of starch granules by synchro-
tron SAXS. Carbohydrate Polymers, 158, 29–36.
Zhang, B., Zhao, Y., Li, X., Zhang, P., Li, L., Xie, F., et al. (2014). Effects of amylose and
phosphate monoester on aggregation structures of heat-moisture treated potato
starches. Carbohydrate Polymers, 103, 228–233.
Zou, W., Sissons, M., Gidley, M. J., Gilbert, R. G., & Warren, F. J. (2015). Combined
techniques for characterising pasta structure reveals how the gluten network slows
enzymic digestion rate. Food Chemistry, 188, 559–568.