International Journal of Biological Macromolecules 237 (2023) 124187

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International Journal of Biological Macromolecules

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

Preparation of the starch-lipid complexes by ultrasound treatment:

Exploring the interactions using molecular docking
Zongwei Hao , Huajian Xu , Yiyang Yu , Shengjun Han , Zongyan Gu , Yu Wang , Chao Li ,
Qiang Zhang , Changyue Deng , Yaqing Xiao , Yingnan Liu , Kang Liu , Mingming Zheng ,
Yibin Zhou *, Zhenyu Yu *
Food Processing Research Institute, China
Anhui Engineering Laboratory for Agroproducts Processing, China
Key Laboratory of Agricultural Product Fine Processing and Resource Utilization, Ministry of Agriculture and Rural Affairs, China
School of Tea and Food Science & Technology, Anhui Agricultural University, Hefei 230036, China

A R T I C L E I N F O A B S T R A C T

Keywords: In this work, Corn Starch (CS)-Lauric acid (LA) complexes prepared by different ultrasound times were explored
Ultrasound for multi-scale structure and digestibility. The results showed that the average molecular weight of the CS
Molecular interaction decreased from 380.478 to 323.989 kDa and the transparency increased to 38.55 % after 30 min of ultrasound
Starch-lipid complexes
treatment. The scanning electron microscope (SEM) results revealed a rough surface and agglomeration of the
Multi-scale structure
prepared complexes. The complexing index of the CS-LA complexes increased by 14.03 % compared to the non-
ultrasound group. The prepared CS-LA complexes formed a more ordered helical structure and a more dense V-
shaped crystal structure through hydrophobic interactions and hydrogen bonding. In addition, fourier transforms
infrared spectroscopy and the molecular docking revealed that the hydrogen bonds formed by CS and LA pro­
moted the formation of an ordered structure of the polymer, retarding the diffusion of the enzyme and thus
reducing the digestibility of the starch. With correlation analysis, we provided insight into the multi-scale
structure-digestibility relationship in the CS-LA complexes, which provided a basis for the relationship be­
tween structure and digestibility of lipid-containing starchy foods.

1. Introduction
Starch is an abundant carbohydrate polymer consisting mainly of
amylose and amylopectin [1]. It is widely used in many industrial ap­
plications with its abundant resources, low cost, renewable and biode­
gradable nature. According to the research of Englyst [2], starch can be
divided into rapidly digested starch (RDS), slowly digested starch (SDS)
and resistant starch (RS) at different hydrolysis times. As one of the most
consumed polysaccharides in the human diet, starch plays an important
role in human nutrition, however the intake of RDS immediately in­
creases the body's blood glucose levels, which can lead to diet-related
metabolic chronic diseases such as type II diabetes [3]. RS is not
digested in the digestive tract but can be degraded by the microbial
community in the colon through anaerobic fermentation, which helps to
regulate the intestinal microbiota and prevent metabolic diseases [4].
Therefore, the modification of starch by various means for enhancing
the RS content in starchy foods is receiving more and more attention
from researchers.
There are five types of RS (RS1 to RS5) and many studies classify the
starch-lipid complex as a new type of resistant starch, which is RS5. The
complex formation, physicochemical properties, structural character­
ization and digestibility of RS5 are currently being extensively investi­
gated [5]. It has a strong resistance to starch hydrolases which
effectively prevents diet-induced obesity. Kang et al. [6] showed that
microwave treatment promoted the formation of corn starch-lauric acid
complexes. In this way, the content of resistant starch was increased,
which provided a new idea for the preparation of low-glycaemic foods.
In the preparation of starch-lipid complexes, intramolecular and inter­
molecular forces played a key role in stabilising their structure [7]. Of
these, intramolecular interactions include van der Waals forces and
hydrogen bonding, which help to stabilise the single helix conformation.
In contrast, the intermolecular interactions are mainly hydrophobic

* Corresponding authors.
E-mail addresses: zhouyibin@ahau.edu.cn (Y. Zhou), yuzhenyuHF@163.com (Z. Yu).

https://doi.org/10.1016/j.ijbiomac.2023.124187
Received 1 February 2023; Received in revised form 2 March 2023; Accepted 22 March 2023
Available online 27 March 2023
0141-8130/© 2023 Published by Elsevier B.V.
Z. Hao et al.
between the starch helix cavity and the guest molecule. In addition,
some guest molecules formed hydrogen bonds with the amylose at the
entrance to the helix [8]. There are many factors (amylose chain length,
molecular weight, type and saturation of lipids, etc.) that affect the
formation of starch-lipid complexes. Liu et al. [9]. showed that moderate
concentrations of ionic liquids can promote the formation of V-shaped
inclusion complexes, improving thermal stability and increasing the
short-range ordered structure of the complexes.
In recent years, there are many works showing that many chemical,
physical and enzymatic techniques have been used to accelerate the
formation of V-complexes [10,11]. Compared to chemical and enzy­
matic technologies, ultrasonic technology is a green and novel non-
thermal processing technology with the advantages of short action
time, high efficiency, no pollution, easy operation and low energy
consumption, which is widely used in food processing. Ultrasound
treatment can cause disintegration of the expanded starch granules
through cavitation effects, mechanical forces and shock waves,
degrading preferentially the amorphous regions of the starch granules
and releasing more linear amylose molecules to participate in the for­
mation of the complexes [12]. In addition, the application of ultrasound
promoted complexation reactions between starch and guest molecules
and improved intermolecular interactions [13]. Despite the wide
application of ultrasound in starch modification, the application of ul­
trasound for the preparation of starch-lipid complexes is relatively little
studied, and there is a lack of coherent studies on the conditions of
starch-lipid complex formation and its correlation with structural and
digestive properties, where intermolecular interactions are still unclear.
Therefore, RS5 was prepared in this study by using ultrasound-
assisted lauric acid (LA) and corn starch (CS) complexes. The main ob­
jectives of this study were to systematically understand the effects of
ultrasonic treatment on the multiscale structure of the complexes and
influence of these interactions on digestibility. The mechanism of the
interaction between CS and LA was described by molecular perspective.
It is expected to be used to provide a theoretical basis for the preparation
of starch-lipid complexes with hypoglycaemic effects and promote in the
development of healthy starchy foods.
2. Materials and methods
2.1. Materials
The native corn starch (containing about 23 % amylose) and Porcine
pancreas α-amylase (EC3.2.1.1, 12 U/mg) were purchased from Yuanye
Bio-Technology Co., Ltd. (Shanghai, China). Lauric acid was obtained
from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Amy­
loglucosidase (EC3.2.1.3, 260 U/mL) from Aspergillus niger was bought
from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). The other
chemicals used in this study were all analytical grade.
2.2. Preparation of starch-lipid complex by ultrasonic treatment
2.2.1. Ultrasonic treatment of starch paste
5 g native CS was dispersed in 100 mL distilled water and pasted in a
water bath with constant stirring at 90 ◦ C for 30 min to prepare a certain
concentration (5 wt%) of starch paste. The starch paste was ultra­
sonicated for 10, 20 and 30 min by using the ultrasonic cell crusher
(SCIENTZ-IID; Ningbo Scientz Biotechnology Co., Ningbo, China). The
ultrasound amplitude was selected as 50 % (400 W/cm2), and the pulse
duration was 5 s on and 5 s off, and they were named as CS-10, CS-20
and CS-30 respectively. The CS paste without ultrasonic treatment was
prepared as a control.
2.2.2. Preparation of CS-LA complexes
LA (0.5 g) was dissolved in ethanol and added to the starch paste
with sonication for 10, 20 and 30 min. The mixture was stirred at 90 ◦ C
for 60 min. After that, the precipitate was obtained by centrifugation at
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International Journal of Biological Macromolecules 237 (2023) 124187
5000 rpm for 10 min and washed three times with 50 % ethanol/water.
The precipitates were dried at an oven at 40 ◦ C for 24 h. The obtained
samples were named as CS-LA-10, CS-LA-20 and CS-LA-30. The CS-LA
complex without ultrasound treatment was prepared as a control.
2.3. The viscosity of starch paste by ultrasonic treatment
We used a rheometer equipped (DHR-1, TA Instruments, New Castle,
USA) with a 40 mm diameter parallel plate to measure the apparent
viscosity of the starch paste (obtained in 2.2.1). Test conditions: the gap
value was chosen to 1000 μm, the temperature was 25 ◦ C, and the shear
rate was in the range of 0.1 to 100 l/s for steady-state shear rheology
experiments, which were acquired and recorded automatically by a
computer.
2.4. The transparency of starch paste by ultrasonic treatment
We measured the translucency of the starch paste at 650 nm by using
a UV–Vis spectrophotometer (DU-730, Beckman Coulter Corporation,
USA) with distilled water as a blank.
2.5. Gel permeation chromatography (GPC)
The average molecular weight of CS was determined by gel perme­
ation chromatography (GPC) combined with multi-angle laser scattering
(MALLS) (Waters, USA) and an oscillometric detector. 5 mg of CS was
dissolved in 10 mL of DMSO with 50 mmol/L LiBr and heated at 60 ◦ C
for 12 h [14]. The suspension was passed through a 4.5 μm filter
membrane. The chromatographic system used for the tests was a GPC
combined with a multi-angle laser scattering system equipped with a
Styragel HMW 6E column. The flow rate was 0.5 mL/min.
2.6. Complexing index (CI) value determination
We determined the complexing index (CI) of the complexes accord­
ing to the method of Tang et al. [15]. In short, 0.4 g of the sample (dry
base) was accurately weighed and dispersed into distilled water (5 mL).
Then the mixture was heated at 95 ◦ C for 15 min with shaking. After
cooling to 25 ◦ C, adding 25 mL of distilled water and vortexing for a few
minutes. 0.5 mL of sample was taken and mixed with 15 mL of distilled
water and 2 mL of iodine solution (2.0 % KI and 1.3 % I2). The CI was the
absorbance value at 690 nm (UV–vis, Shimadzu UV-2600i, Japan),
calculated from the following equation.
CI(%) = [(AO − AS ) × 100 ]/AO
where A0 indicates the absorbance value of the starch paste; As indicates
the absorbance value of the CS-LA complex with different ultrasound
treatment times.
2.7. Scanning electron microscopy (SEM) of CS-LA complex
The samples were dispersed evenly on double-sided adhesive,
sprayed with gold for 30 s and imaged scanning electron microscope (S-
4800, Hitachi, Tokyo, Japan) with an accelerating voltage of 3 kV at
1000–4000× resolution.
2.8. X-ray diffraction test of CS–LA complex
We used XRD (Rigaku D/max-TTR-III, Rigaku Corporation, Tokyo,
Japan) to analyze the crystalline structure of the samples and deter­
mined the crystalline shape. Diffraction conditions: the generator
voltage and current set at 40 kV and 40 mA, scan rate 2◦ /min, diffraction
angle 2θ in the range 5◦ to 40◦ for the data. The obtained XRD patterns
were used to calculate the relative crystallinity by using MDI Jade 6
software version (Materials Data, Inc., California, USA).
Z. Hao et al.
2.9. Fourier-transformed infrared (FT-IR) spectroscopy of CS–LA
complex
FTIR spectrum of the sample was observed by a FTIR spectrometer
(Nicolet 6700, Thermo Fisher Scientific, USA). Samples (1 mg) were
milled with that of the KBr pellets (100 mg) and FTIR spectra were
observed in the spectral range of 4000 to 400 cm− 1 by scan accumula­
tion rate of 32 scans at 4 cm− 1 resolution. The obtained spectra were
deconvoluted (1200–800 cm− 1) and the absorbance ratios 1047 cm− 1/
1022 cm− 1 and 1022 cm− 1/995 cm− 1 were used to study the short-range
ordered structure of the complexes.
2.10. Laser confocal micro-Raman (LCM-Raman) spectroscopy of CS–LA
complex
The samples were placed on slides, flattened and tested under a
Raman spectrometer (Horiba scientific, France). Test conditions: The
laser light source was 785 nm and the scan range was 3200–100 cm− 1.
Analysis of the data was conducted by using Peakfit 4.12 (Jandel Scien-
tific Software, San Rafael, USA) to obtain half-peak widths at 480 cm− 1
which were used to characterize the short-range molecular ordering of
the complexes.
13
2.11. C CP/MAS nuclear magnetic resonance (NMR) spectroscopy
We used NMR spectroscopyss (AVANCE III HD 400 MHz,Bruker,
Karlsruhe, Germany) to study the structure of the complexes. According
to the method of Tan et al. [16], 40 mg of the complex was dissolved in
0.6 mL of DMSO and heated to form a clear solution, which was scanned
1000 times to obtain a 13C NMR spectrum. The analysis of helical con­
formations in starch was carried out by using MestReNova software
(Mestrelab Research Corporation., Spain).
2.12. Differential scanning calorimetry (DSC)
The thermal properties of the samples were obtained by using a DSC
8000 (Perkin Elme Corporation., USA). 2 mg of starch was weighed into
an aluminium crucible and 6 μL of deionised water was added. The
crucible was sealed and equilibrated at room temperature for 24 h. An
empty sealed crucible was used as a blank and scanned at a rate of 10 ◦ C/
min over a temperature range of 30–140 ◦ C. The onset temperature (To),
peak temperature (Tp), conclusion temperature (Tc) and enthalpy
change (ΔH) were recorded and analyzed.
2.13. In vitro digestion characteristics of CS–LA complex
According to the method reported by Englyst [2] et al., with minor
modifications. 200 mg of sample was weighed into a centrifuge tube, 15
mL of pH 5.2 sodium acetate buffer was added in a water bath at 95 ◦ C
for 30 min. After cooling to room temperature, 5 mL of mixed enzyme
solution (α-amylase (12 U/mg) and glucosidase (260 U/mL) was added.
At 20 and 120 min, 0.5 mL of digest was removed and 4.5 mL of
anhydrous ethanol was added to deactivate the enzyme, then the
glucose content was determined by the glucose oxidase-peroxidase
(GOPOD) (JianCheng Bioengineering Institute, Nanjing, China) assay
kit. Each test was analyzed in triplicate. The RDS, SDS and RS contents
were calculated according to the following equations.
RDS(%) = [(G20 − G0 ) × 0.9 ]/TS × 100
SDS(%) = [(G120 − G20 ) × 0.9 ]/TS × 100
RS(%) = (1 − RDS − SDS) × 100
where G0 indicates the amount of glucose when unhydrolysed; G20 in­
dicates the amount of glucose released within 20 min of hydrolysis; G120
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International Journal of Biological Macromolecules 237 (2023) 124187
indicates the amount of glucose released within 120 min of hydrolysis;
and TS is the mass of total starch dry base (mg).
2.14. First-order kinetic analysis of in vitro digestibility
According to Goni et al. [17], who simulated human intestinal
digestion, the starch hydrolysis curve follows the first order reaction
equation as follows.
( )
C = C∞ 1 − e− kt
where C is the rate of hydrolysis of the sample at time t; C∞ is the rate of
hydrolysis of the sample at equilibrium; and k is the kinetic constant of
digestion.
The logarithmic slope (LOS) graphical method was used to measure
the in vitro digestibility of samples [18,19]. We calculated the logarithm
of slope (LOS) graph by representing the first order derivative of the first
order equation in logarithmic form.
( )
ln
dC
= − kt + ln(C∞ k)
dt
where ln (dC/dt) represents the logarithm of the slope, and k and C∞ are
used to construct the model-fitted starch digestion curve from the
equation.
2.15. Computational methods
In order to better investigate the possible sites of action of the non-
covalent intermolecular interactions of the CS-LA complex, we down­
loaded the structural formula of LA from the PubChem database and
converted it to 3D format by using the Open Babel program. Due to the
huge molecular weight of starch and the multi-scale structure inside the
granule, the whole starch structure system is difficult to be simulated by
the DFT theory. Therefore, we chose the starch model (DP = 10) as the
computational model for the starch molecule. Firstly, the geometric
structure optimization of the starch (DP = 10) and LA molecular
monomer structure was performed using the B3LYP/6-31G(d) method.
Then, the electrostatic potential (ESP) distribution was plotted using the
Multiwfn program. Randomized images of 100 CS-LA complexes were
generated with the Genmer program and optimized with the semi-
empirical algorithm of the Molclus program. In the case of ZPE and
BSSE corrections, the same DFT basis set was used to further optimize
the initial composite structure with minimal energy. Low density
gradient RDG assays were performed in Multiwfn to analyze and map
molecular interactions to evaluate and map non-covalent interactions
between molecules [20,21].
2.16. Statistical analysis
All experiments were operated in triplicate to calculate the means ±
standard deviation. PyMol software (Version 2.4.0a Open-Source,
Schrödinger, LLC, U.S.A.) and Discovery Studio Visualizer 2021 soft­
ware (BIOVIA, U.S.A.) were used to capture conformation images. Sta­
tistical significance was assessed by analysis of variance (ANOVA) in the
Statistix 8.0 program.
3. Results and discussion
3.1. Starch paste viscosity analysis
As can be seen from Fig. 1A, the apparent viscosity of the CS paste
decreased with the increase in shear rate and stabilized after a shear rate
of 10 s− 1, which was typical of shear thinning, a characteristic feature of
pseudoplastic fluids. The apparent viscosity of the CS paste decreased
sharply with the ultrasonic treatment. This was due to the fact that the
original starch was dextrinised with intertwined macromolecular chains
Z. Hao et al. International Journal of Biological Macromolecules 237 (2023) 124187

Fig. 1. Apparent viscosity(A), Visual photograph of starch paste (B) and Transparency of starch paste (C) for CS with different ultrasonic treatment times. Different
letters in the same chart represent significant differences between different treatments (P < 0.05).

and had a high viscous resistance to flow, however, treatment with ul­
trasound caused some of the macro-molecular chains of the starch to
break and the crystalline structure to change, reducing the resistance to
flow [22,23] and thus presenting a lower viscosity.
3.2. Transparency analysis
The transparency of the CS paste is reflected by its transmittance. The
effect of sonication on the transparency of the CS paste is shown in
Fig. 1B-C. When the sonication time reached 30 min, the light trans­
mission increased to 38.55 %. This was mainly due to the destruction of
the amorphous and small crystalline regions of the starch granules at
this stage, which also allowed the starch chains to be more fully
dispersed in the paste, reducing the reflection of light and thus
increasing the light transmission. On the other hand, it may be due to the
fact that some of the amylopectin molecules in the starch paste were
broken at this stage and more starch was dissolved, resulting in easier
diffusion of the starch chains in the water.
3.3. Average molecular weight analysis
GPC is a method for separating starch molecules and measuring their

Fig. 2. RI response and Molecular weight distribution profiles of CS (A) and CS treated with sonication for 10 (B), 20 (C) and 30 (D) min.

4
Z. Hao et al.
average molecular weight according to their molecular size. It has the
advantages of fast detection, high separation and good reproducibility,
which has been widely used to determine the relative molecular mass of
starch and polysaccharides. Fig. 2 shows that the molecular weight of CS
paste decreased from 380.478 kDa to 323.989 kDa as the sonication time
was increased to 30 min. This was mainly due to the fact the effects of
ultrasound on CS were mainly from its mechanical and cavitation effects
[24]. During the ultrasonic treatment, the cross-linked spatial mesh
structure formed by the CS was disrupted and the rate of disruption of
the entanglement points increased, which led to its faster dispersion in
water. On the other hand, ultrasound degraded amylose and amylo­
pectin, severed α-1,4 glycosidic bonds and α-1,6 glycosidic bonds, and
formed new short amylose. It was the breaking of bonds and the
weakening of intermolecular interactions in the starch molecule that led
to the reduction in its molecular weight. This was consistent with the
results of increased transparency of starch paste [25].
3.4. CI values of the starch-lipid complexes
The complexing index can characterize the amount of starch-fatty
acid complexes. The ability of starch to bind to iodine decreases when
starch is bound to lipids, and the degree of starch-lipid binding can be
reflected by measuring the content of starch‑iodine complexes [26].
According to Fig. 4A, the CI values of CS-LA complexes were signifi­
cantly increased with the increase of ultrasound treatment time. The CI
values increased from 3.78 % to 17.81 % at a treatment time of 30 min.
This was mainly attributed to the cavitation effect of ultrasound
breaking down the swollen starch granules and releasing more amylose
from its internal matrix, which increased the availability of amylose
bound to lipids [27]. In addition, the above transparency analysis
confirmed that sonication improved the ligand dispersion in the dex­
trinized starch suspension and increased the contact between CS and LA,
resulting in a better V-shaped inclusion, which was consistent with the
work of Kang et al. [28].
3.5. Microstructure analysis
Fig. 3 shows scanning electron microscopy images of native CS and
starch complexes at different magnifications. The native CS granules had
a more regular morphology, with a flat surface and exhibited an
approximate spherical shape, which was consistent with previous re­
ports [29]. The surface of the CS-LA complexes became rough. This was
due to the fact that temperature rising caused by ultrasonic treatment
moved amylose out of starch granules, thus giving LA the opportunity to
Fig. 3. SEM images of Native CS and CS-LA complexes with different ultrasonic treatment times. (A:Native CS, B:Control, C:CS-LA-10, D:CS-LA-20, E:CS-LA-30. The
magnification of A-E and A1-E1 is 1000 x and 4000 x respectively).
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International Journal of Biological Macromolecules 237 (2023) 124187
bridge the mobile amylose chains and form some loosely connected
complexes through hydrogen bonds on the granule surface, showing a
“rough, uniform net” [30]. On closer inspection, it can be seen that the
large granules are made up of small “flakes” of granules. This further
suggested that the amylopectin was degraded by sonication into more
amylose [31], and pushed LA into the amylose helical cavity to form
inclusions that showed spherical crystals to further aggregate. On the
other hand, it may be related to the hot air drying during the preparation
process, where ultrasonication led to aggregation between small parti­
cles [32]. This may be attributed to the depolymerisation, degradation
and rearrangement effects in the CS-LA complexes induced by the son­
ication treatment.
3.6. XRD analysis
The X-ray diffraction patterns of native CS, LA and CS-LA complexes
are shown in Fig. 4B. It revealed that native CS has strong diffraction
peaks at 15.0◦ , 17.0◦ , 18.0◦ and 23.0◦ , which was typical of A-type
starch [33]. When the CS was pasted at 90 ◦ C, the double helix of the
swollen starch was opened, the crystalline structure was completely
disrupted and the internal lamellar structure was exposed where its
crystalline shape was changed by interaction with LA molecules under
ultrasonic treatment [12]. The CS-LA complexes showed strong
diffraction peaks around 7.5◦ , 13◦ and 20◦ , which were typical of a V-
shaped crystal structure, indicating the formation of a single helical
complex between the starch and the guest molecule [34]. Noticeably,
the relative crystallinity of the complexes increased significantly with
the increase in ultrasonic time, increasing from 16.40 % to 24.47 %
(Table 1) as compared to the un-sonicated complex. This was mainly due
to the fact that pasted starch granules were disrupted by ultrasonic
treatment, releasing more linear starch chains. In addition, the change in
peak intensity with increasing sonication time was attributed to increase
in amylose content and improved LA dispersion by using sonication
treatments, where LA molecules were forced to entrap into hydrophobic
cavities within the V-type amylose helices to facilitate V-type CS-LA
inclusion complexes. This was consistent with the work of Raza et al.
[35], who studied the induction of starch-linoleic acid complexes by
multi-frequency power sonication, which significantly increased their
relative crystallinity with increasing sonication power. These findings
indicated that the appropriate ultrasonic treatment time may be more
favourable for the formation of starch-lipid complexes.
Z. Hao et al. International Journal of Biological Macromolecules 237 (2023) 124187

Fig. 4. The complexing index (A), X-ray diffraction patterns (B), FT-IR spectra (C) and Infrared deconvolution map (D) for Native CS and CS-LA complexes with
different ultrasonic treatment times. Different letters in the same chart represent significant differences between different treatments (P < 0.05).

Table 1
XRD, FT-IR, FWHM and thermal propertie of Native CS and CS-LA complexes with different ultrasonic treatment times.
1
Sample Crystallinity (%) R1047/1022 R1022/995 FWHM at 480 cm− T0 (◦ C) Tp (◦ C) Tc (◦ C) ΔH (J.g− 1)
a b d a e d e
Native CS 28.21 ± 0.61 0.785 ± 0.009 0.850 ± 0.008 18.483 ± 0.444 70.48 ± 0.32 74.49 ± 0.28 78.36 ± 0.25 10.35 ± 0.50a
Control 16.40 ± 0.29e 0.706 ± 0.005d 1.316 ± 0.006a 19.498 ± 0.709a 92.64 ± 0.23c 95.66 ± 0.08c 100.40 ± 0.96d 1.19 ± 0.15d
CS-LA-10 18.87 ± 0.83d 0.739 ± 0.023c 1.224 ± 0.004b 17.019 ± 0.405b 91.83 ± 0.04d 95.68 ± 0.21c 101.96 ± 0.23c 2.57 ± 0.25c
CS-LA-20 21.71 ± 1.16c 0.813 ± 0.003b 1.143 ± 0.038bc 15.544 ± 0.405c 104.83 ± 0.05b 106.98 ± 0.01b 109.58 ± 0.04b 3.18 ± 0.12c
CS-LA-30 24.47 ± 1.18b 0.851 ± 0.002a 1.108 ± 0.058c 13.477 ± 0.490d 110.00 ± 0.26a 115.39 ± 0.31a 119.30 ± 0.34a 4.62 ± 0.28b

To, Tp, Tc and ΔH indicate gelatinization parameters onset, peak, conclusion temperature and gelatinization enthalpy, respectively.
All the values are the mean ± standard deviations. The mean values with different superscripts in a column indicate significant differences (p < 0.05).

3.7. FT-IR analysis to understand the changes in short-range ordered
structure
FT-IR spectroscopy is an effective method for analysing the compo­
sition and structure changes of samples. Fig. 4C-D shows the FTIR
spectrum of native CS and the CS-LA complexes obtained by ultrasonic
treatment at different times. Compared to native CS, the CS-LA com­
plexes resulted in two additional absorption peaks at 1740 cm− 1 and
2850 cm− 1, which represented to the stretching vibrations of the
carbonyl and methylene groups of the LA molecule, respectively
[36,37]. The creation of the carbonyl band here was probably due to the
breakage of the hydrogen bond between the carboxyl groups in the
crystalline state of LA and the incorporation of LA in the amylose double
helix, where a new hydrogen bond was formed with the starch molecule
[38]. It confirmed the V-shaped complex formation between the CS and
LA by the ultrasonic treatment. In addition, the peak position of the

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Z. Hao et al.
vibrational stretching of the hydroxyl group (-OH) of the native CS was
3433.73 cm− 1, but the characteristic peak of the CS-LA complex treated
by different ultrasonic times was shifted towards a lower wavelength.
The lower the wavenumber of the vibration peak, the more certain
groups appear or the stronger their interaction [39]. The lower wave­
lengths of CS-LA complexes showed that the stability of hydroxyl group
increased, which may be caused by the cross-linking of hydrogen bonds
between the carbonyl group of LA and the hydroxyl group of amylose
and superposition of the hydroxyl groups of starch and LA molecule
[10]. As such, we speculated that hydrogen bonding was the main non-
covalent bond during the formation of the CS-LA complexes. Van Soest
et al. [40]. has shown that the intensity ratios of 1047 cm− 1/1022 cm− 1
and 1022 cm− 1/995 cm− 1 indicate the degree of order in the crystalline
region and the sensitivity of the amorphous structure, respectively.
Therefore, their ratios can be used to characterize the short-range or­
dered structure of their starches. The values of R1047/1022 are shown in
Table 1, and it could be found that R1047/1022 increased significantly
with the increase of ultrasonic time, indicating that during sonication
and the presence of LA could improve the short-range order of starch
granules [41].
3.8. LC-Raman analysis
The FWHM values of the characteristic peaks at 480 cm− 1 for native
CS and CS-LA complexes are determined by Raman spectroscopy to
investigate the effect of different times of ultrasonic treatment on the
short-range molecular ordering of the complexes. It has been shown that
the FWHM values decrease with increasing short-range molecular order
of the starch structure [42]. As shown in Fig. 5A, there were no new
peaks in the spectra of native CS and the addition of LA, indicating that
13
Fig. 5. The LCM-Raman spectra (A), C CP/MAS NMR spectra (B), Digestibility curves (C) and associated LoS plots (D) for Native CS and CS-LA complexes with
different ultrasonic treatment times.
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International Journal of Biological Macromolecules 237 (2023) 124187
no chemical changes and new bond formation occurred during the
sonication preparation of the complexes. Moreover, the Table 1 showed
a significant decrease in the FWHM of the complexes after different
times of ultrasonic treatment. The FWHM decreased from 19.498 for the
control to 13.477 for CS-LA-30, which indicated a further increase in the
short-range ordering of the complexes. As well as expected, appropriate
ultrasonic treatment caused the CS-LA samples to contain more short-
range structure than the control. We can infer that the formation of
the complexes were promoted by ultrasound but also that LA accelerated
the self-assembly of amylose [7,43], which was in accordance with the
findings of FT-IR spectroscopy.
3.9. Helical structures analysis
The solid-state 13C CP/MAS NMR spectra of native CS and CS-LA
complexes were shown in Fig. 5B. With the incorporation of LA mole­
cules, all complexes exhibited a different spectrum from the native CS.
The 13C NMR spectra of the treated and control samples had an addi­
tional resonance peak at approximately 31.7 ppm and the intensity of
the peak here became stronger with the increasing treatment time which
indicated better binding and stronger interaction between them. This is
a typical sign of the V-shaped structure of the starch-lipid complex [44].
According to relevant studies [45], the intensity of the peak in the C1
region has reflected the change in the double helix structure of starch. As
can be seen in Fig. 5B, sonication can promote the formation of helical
structures of the complexes. At the same time, the hydrogen bonds
formed between LA and starch influence the rearrangement of the starch
structure to form a double helix. At longer ultrasonic treatment times
(≥20 min), the hydrogen bonding between LA and starch increased,
forcing the starch chains to align more closely to form more double helix
Z. Hao et al. International Journal of Biological Macromolecules 237 (2023) 124187

structures [46]. It was notable that the typical peaks C2, 3, 5 (70–79 the formation of a single helix inclusion complex of amylose and LA by
ppm) of starch in the treated group were not significantly changed by the ultrasound treatment, which had a more dense crystal structure. The
comparing with CS. However, its vibrational intensity was significantly more dense structure resulted in the inhibition of the swelling of the
reduced, which was mainly due to the fact that it represented amor­ starch granules and reduced the binding site of starch to the enzyme,
phous amylose, and it was influenced by the cluster structure of which was consistent with the SEM observation. In other words, the
amylose, which was inversely proportional to the intensity of the ultrasound treatment promoted the interaction of amylose and ligand
vibrational peaks [47]. During the ultrasonic treatment, LA was com­ molecules, forcing the starch chains to align closely to form more
bined with more amylose molecules to form the CS-LA complex, causing double-helix structures through hydrogen bonding. [6]. In addition, the
the cluster structure of weaker binding capacity. ligand molecules could alter the amylose structure and change the
amylose-amylose and amylose-amylopectin stacking arrangement in the
3.10. DSC analysis amorphous region of the starch granules, which reduced the starch di­
gestibility [49]. These results were consistent with the previously re­
The changes in the crystalline structure of starch could be reflected ported finding [12] that sonication facilitated the formation of more
by measuring the thermal properties of starch and calculating and starch-lipid complexes.
analysing the changes in T0, Tp, Tc and ΔH. The thermal properties of the
native CS and V-complexes are shown in Table 1. Only one peak 3.12. First-order kinetics analysis
appeared in the detection temperature range for both the native CS and
V-complexes. In the case of starch, when heated with water, the Fig. 5D shows the hydrolysis curves and LOS plots of CS and ultra­
branched starch double helix would be unheliced by the hydrogen sonically treated CS-LA complexes. All samples were rapidly digested
bonding of the water molecules, so that the heat absorption peak within 60 min and stabilized after 120 min. At the same time, the LOS
detected by DSC could reflect the change in the starch helix. Kiatpon­ plot exhibited a linear relationship with a constant k value, suggesting
glarp et al. [48] has shown that ΔH is positively correlated with the that the digestion of CS and CS-LA complexes was a single-phase process
number of long and short-range ordered structures of starch. After the [50]. The k value is determined by the catalytic nature of the amylase
transformation of the original starch into a V-complex, the pasting itself, which was higher for the natural CS compared to the un-sonicated
temperature of the complex shifted to the right, while the correspondent complex. The k value decreased significantly with increasing sonication
ΔH increased gradually with the increasing ultrasonic time. This was time, which indicated that the diffusion of amylase into the starch
due to the fact that the CS-LA complex was a single helix structure granules became slower [51]. This was mainly attributed to the fact that
formed by amylose and LA molecules, and the ultrasonic treatment LA promoted the internal helical structure of starch through hydro­
released more amylose, which facilitated the formation of more amylose phobic interactions and formed dense V-shaped crystal structures
single helices, making it require more energy to unhelix. The shift in the through non-covalent interactions. On the other hand, the hydrogen
heat absorption peak of the V-complex may be caused by the presence of bonds formed between LA and starch promoted the formation of an
LA which can increase the arrangement of starch molecules becoming ordered structure of aggregates, which retarded the diffusion of the
more ordered during sonication [41]. enzyme. The difference in digestion rate is mainly dependent on C∞.
With increasing treatment time, it resulted in a significant decrease in
3.11. In vitro starch digestibility C∞ of the complexes, which was consistent with the digestibility data in
the above table (Table 2).
The in vitro digestibility (RS, SDS and RDS) of CS and CS-LA com­
plexes with different ultrasound time treatments are shown in Table 2. It 3.13. Molecular docking and quantum chemistry
was shown that the RDS content of CS was 76.09 % and the RS content
was 10.45 %. Compared with the non-sonication group, the hydrolysis The analysis of the molecular surface electrostatic potential (ESP) of
rate of the samples decreased significantly after the formation of com­ free monomers is one of the common tools used in quantum chemistry to
plexes between CS and LA, with RDS decreasing from 74.31 % to 61.21 study non-covalent interactions [52]. Fig. 6A-B shows the ESP distri­
% and RS increasing from 18.95 % to 24.88 %. This suggested that the V- bution of starch (DP = 10) and LA. The color of the scale in the figure
complex exhibited a higher resistance to digestibility. This may be due to ranges from red to blue, where the darker the red represents the lower
electron cloud density and the more positive charge here, and the darker
Table 2
the blue represents the higher electron cloud density and the more
In vitro digestibility of Native CS and CS-LA complexes with different ultrasonic negative charge here. The ESP value has a good correlation with
treatment times. whether the site has the ability to act as a hydrogen bond acceptor or
donor. Fig. 6C shows the RDG scatter plot, which can visualize the non-
Sample RDS(%) SDS(%) RS(%) Hydrolysis kinetics
covalent interactions in the system such as hydrogen bonding, van der
k C∞ (%) R2 Waals forces and strong spatial site resistance effects by visualizing the
(min− 1)
images [53]. The distribution of non-covalent interactions in real space
CS 76.09 13.46 10.45 0.138 ± 89.269 0.999468 in the starch (DP = 10)-LA complex can be seen from Fig. 6D. Among
± 1.34a ± 1.07c 0.011a ± 0.608a
them, the hydrogen bonding interaction represented by the blue pattern
±
0.93ab
Control 74.31 6.74 ± 18.95 0.118 ± 82.242 0.998375 was mainly distributed near the regions with higher electron cloud
± 0.27a 0.22c ± 0.45b 0.012b ± 0.978b density such as the hydroxyl group of the starch molecule and LA, which
CS-LA- 68.20 8.01 ± 23.79 0.107 ± 77.074 0.999471 was consistent with the results of ESP analysis above. The van der Waals
10 ± 0.27b 0.27c ± 0.51a 0.006c ± 0.523c force interaction represented by the green pattern was mainly distrib­
CS-LA- 63.52 12.00 24.48 0.093 ± 75.404 0.999418
20 ± 0.31c ± 0.89b ± 1.20a 0.004d ± 0.539d
uted in the spatial region where the starch molecules and LA molecules
CS-LA- 61.21 13.92 24.88 0.087 ± 74.978 0.999679 were in contact with each other, while the strong spatial site resistance
30 ± 1.08d ± 0.60a ± 0.75a 0.003e ± 0.400e effect represented by the red pattern was mainly distributed in the
Data were expressed as mean ± standard deviation of triplicate. Different su­ glucose ring of the starch molecules [54]. The above results suggested
perscript letters in the same column indicating significantly difference (p < that the addition of LA in the sonication environment, they induced
0.05). RDS, rapidly digestible starch (%); SDS, slowly digestible starch (%); RS, starch to form locally ordered structural domains at different scales
resistance starch (%); C∞, equilibrium hydrolysis percentage (%); k, digestion including single and double helices, V-shaped crystals and nano­
rate coefficient (min− 1); R2, correlation coefficient. aggregates through non-covalent interactions (hydrophobic and

8
Z. Hao et al. International Journal of Biological Macromolecules 237 (2023) 124187

Fig. 6. The distribution of electrostatic potential (ESP) on the molecular surface of starch (DP = 10) and lauric acid monomers, starch (DP = 10) (A) and lauric acid
(B); The red spheres represent oxygen atoms, cyan spheres represent carbon atoms and white spheres represent hydrogen atoms in the molecular structure. The RDG
scatter (C) and the distribution of non-covalent interactions in real space (D) for the starch (DP = 10) -LA complex system; Where the red spheres represent oxygen
atoms, the blue spheres represent carbon atoms, and the white spheres represent hydrogen atoms.

Fig. 7. Pearson correlation analysis between the in vitro digestibility parameters and complexing index (CI), relative crystallinity (RC), R1047/1022, R1022/995, and full
width at half-maximum (FWHM).

9
Z. Hao et al.
hydrogen bonding) to resist the degradation by amylase.
3.14. Correlation analysis
During the ultrasonication process, the synergistic effects of water
molecules, mechanical forces and LA molecules can lead to different
degrees of alteration in the multiscale structure of CS, affecting its
digestive performance. The Pearson correlation coefficients between the
digestive performance of the sonicated CS-LA complex and its multiscale
structure are displayed in Fig. 7. The CS-LA complexes showed a sig­
nificant negative correlation (P < 0.001) with the complexing index and
relative crystallinity for the RDS content, and a positive significant
correlation (P < 0.001) with R1022/995 and FWHM. The structures
associated with the SDS and RS contents were opposite to the RDS
contents, where RS showed a significant negative correlation with R1022/
995 and FWHM (P < 0.001) and a positive correlation with complexing
index, relative crystallinity and R1047/1022 (P < 0.01). In summary, the
multi-scale structure of CS determined the digestibility of the lipid
complexes during the ultrasonic co-preparation process. The greater the
degree of lipid complexation in the system, the more dense and orderly
the structure, which improved the slow digestibility of the CS-LA com­
plex to some extent.
4. Conclusion
This work investigated the changes in physicochemical properties
and multiscale structure of CS-LA complexes under ultrasonic treatment
to better understanding how ultrasonication influenced the digestive
properties of their complexes. The findings indicated that the apparent
viscosity of the starch paste decreased significantly with the average
molecular weight decreasing from 380.478 to 323.989 kDa and the
transparency increasing to 38.55 % in comparison to the CS without
ultrasound treatment. As the ultrasonic time increased, the complexing
index of the CS-LA complexes increased by 14.03 %. The SEM results
revealed that the surface of the prepared complexes was rough and
agglomerated. The CS-LA complexes prepared by ultrasound assistance
promoted a single helix structure inside the starch through hydrophobic
interactions. They formed dense V-shaped crystal structures through
non-covalent interactions. In addition, FTIR, LCM-Raman and molecular
docking showed that CS and LA promoted the formation of an ordered
structure of the complex by forming hydrogen bonds, retarding the
diffusion of the enzyme and thus reducing the digestibility of the starch.
Through correlation analysis, we initially established the multi-scale
structure-digestive performance interactions, which can provide basic
data for the creation of starch-based nutritional health foods that meet
the needs of different populations.
CRediT authorship contribution statement
Zongwei Hao: Writing – original draft, Conceptualization, Software,
Methodology, Data curation. Huajian Xu: Software, Writing – review &
editing. Yiyang Yu: Methodology, Writing – review & editing. Sheng­
jun Han: Methodology, Software, Writing – review & editing. Zongyan
Gu: Software. Yu Wang: Software. Chao Li: Software. Qiang Zhang:
Validation. Changyue Deng: Visualization. Yaqing Xiao: Formal
analysis. Yingnan Liu: Formal analysis. Kang Liu: Formal analysis.
Mingming Zheng: Formal analysis. Yibin Zhou: Funding acquisition,
Writing – review & editing. Zhenyu Yu: Funding acquisition, Writing –
review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
10
International Journal of Biological Macromolecules 237 (2023) 124187
Data availability
Data will be made available on request.
Acknowledgements
This work was supported by the National Natural Science Foundation
of China (32172162, 32201972), Anhui Agricultural University Foun­
dation for Stability and Introduction of Talent (rc352008), Natural
Science Youth Fund Project of Anhui Agricultural University
(K2135002), the Key Research and Development Program of Anhui
Province (202204c06020077) and Scientific Research Projects for Uni­
versities in Anhui Province (KJ2020A0135). The authors would like to
thank the Shiyanjia Lab (www.shiyanjia.com) for the assistance of GPC
analysis.
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