Food Hydrocolloids 82 (2018) 370e378

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Mechanisms of starch gelatinization during heating of wheat flour and

its effect on in vitro starch digestibility
Peng Guo a, b, c, Jinglin Yu a, Les Copeland d, Shuo Wang e, **, Shujun Wang a, b, c, *
a
State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science & Technology, Tianjin 300457, China
b
Key Laboratory of Food Nutrition and Safety, Ministry of Education, Tianjin University of Science & Technology, Tianjin 300457, China
c
School of Food Engineering and Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China
d
The University of Sydney, Sydney Institute of Agriculture, School of Life and Environmental Sciences, 2006, Australia
e
Tianjin Key Laboratory of Food Science and Human Health, School of Medicine, Nankai University, Tianjin 30071, China

a r t i c l e i n f o a b s t r a c t

Article history: This study aimed to understand mechanisms of starch gelatinization during heating of wheat (Triticum
Received 19 December 2017 aestivum L.) flour, and its effects on in vitro starch amylolysis of cooked wheat flour. At water content of
Received in revised form 20%, cooking did not disrupt greatly the crystalline structures of starch in wheat flour. Considerable
11 March 2018
disruption of ordered structures of starch occurred with cooking at a water content of 30%, and starch
Accepted 6 April 2018
Available online 8 April 2018
was fully gelatinized at a water content of 40%. The transition of the X-ray diffraction pattern from A to V
types with increasing water content suggested the formation of starch-lipid complexes during cooking of
flour. Typical starch pasting profiles of cooked wheat flour were observed at water contents of 40 and
Keywords:
Triticum aestivum
50%, whereas longer cooking time decreased the pasting viscosities of the flour. Cooking the flour for
Wheat flour only 5 min with 20% water greatly increased the rate and extent of in vitro enzymatic digestion of starch,
Cooking which were not increased further by longer cooking or higher water content. From this study, we
Structural order conclude that the progression of starch gelatinization during cooking was not greatly affected by non-
In vitro starch digestibility starch components in wheat flour, and that some structural order in amorphous regions of starch
contributed to the development of pasting viscosities.
© 2018 Elsevier Ltd. All rights reserved.

1. Introduction Copeland, 2013). The increasing incidence of type 2 diabetes and

Wheat (Triticum aestivum L.) is one of the most cultivated cereal
grains and supplies the daily calories as an important staple food in
many countries. Wheat is the most important cereal used in
breadmaking and other cereal-based products (cookies, pasta,
cakes, etc.) (Veraverbeke & Delcour, 2002). Wheat flour consists
mainly of starch (about 70e75%) and proteins (about 8e14%). Some
minor components such as lipids (about 2%), non-starch poly-
saccharides (about 2e3%), minerals, vitamins, antioxidants and
other nutrients are also present in the whole wheat flour (Hemery,
Rouau, Lullien-Pellerin, Barron, & Abecassis, 2007). Starch is the
main source of glycemic carbohydrates in the human diet and also a
major determinant of quality of wheat-based products (Wang &
* Corresponding author. No 29, 13th Avenue, Tianjin Economic and Develop-
mental Area (TEDA), Tianjin 300457, China.
** Corresponding author.No 29, 13th Avenue, Tianjin Economic and Develop-
mental Area (TEDA), Tianjin 300457, China.
E-mail addresses: s.wang@tust.edu.cn (S. Wang), sjwang@tust.edu.cn (S. Wang).
cardiovascular diseases associated with consumption of excess
starchy foods is of considerable nutritional concern (Taylor,
Emmambux, & Kruger, 2014; Tian, Chen, Ye, & Chen, 2016;
Willett, Manson, & Liu, 2002). This has led to much research on
how the structural and functional properties of starch, interactions
between starch and other food components, and properties of the
food matrix affect the rate and extent of starch digestion (Singh,
Dartois, & Kaur, 2010; Wang & Copeland, 2013). Moreover, there
is growing interest in using wholemeal flours or adding bran
fractions to refined flours in foods to help moderate post-prandial
blood glucose response (Hemdane et al., 2016; Petitot, Barron,
Morel, & Micard, 2010; Rosa-Sibakov, Poutanen, & Micard, 2015).
Starch granules have a complex hierarchical structure, which is
studied with multiple techniques such as DSC, XRD, SAXS and SEM
over a scale ranging from nano-to micrometers (Pe
rez & Bertoft,
2010; Wang, Li, Copeland, Niu, & Wang, 2015). Upon heating,
starch granules are gelatinized causing disruption of multi-scale
structures, with the extent of disruption depending on tempera-
ture, water content, and length of heating time and rate (Mariotti,

https://doi.org/10.1016/j.foodhyd.2018.04.012
0268-005X/© 2018 Elsevier Ltd. All rights reserved.
 P. Guo et al. / Food Hydrocolloids 82 (2018) 370e378
Zardi, Lucisano, & Pagani, 2005; Wang & Copeland, 2013). At a fixed
heating temperature, water content plays a more important role in
disruption of starch structures and increasing starch digestibility
than length of heating time (Wang, Wang, Guo, Liu, & Wang,
2017b), whereas at a fixed water content, higher temperature re-
sults in greater disruption of starch structure (Pan et al., 2017;
Wang, Sun, Wang, Wang, & Copeland, 2016a; Zhang et al., 2014).
Use of purified starch as a model material has led to an under-
standing of the disassembly mechanisms of starch granules during
thermal food processing. However, the presence of proteins and
lipids in cereal-based food systems may affect the gelatinization
behavior of starch during processing through the potential for
complex formation with starch (Wang, Zheng, Yu, Wang, &
Copeland, 2017d) or by changing water availability (Kim et al.,
2008; Zou, Sissons, Gidley, Gilbert, & Warren, 2015). Interactions
between starch, proteins and lipids during thermal processing have
been studied extensively in model systems (Wang et al., 2017d;
Zhang, Maladen, & Hamaker, 2003; Zhang, Maladen, Campanella, &
Hamaker, 2010). Although the presence of other components in
flour is assumed to affect the functionality of starch for food pro-
cessing and human nutrition, there is little actual information on
this. For example, how proteins in might influence starch gelati-
nization during cooking of doughs and batters is not understood.
Studying the mechanism of starch gelatinization in wheat flour is
an important step towards predicting and, in turn, controlling the
processing and functional properties of more complex cereal-based
food systems. Hence, in the present study, standard wheat flour
was used as a model to study gelatinization properties of starch
cooked with food components that might be present during
processing.
2. Material and methods
2.1. Materials
The standard wheat flour was purchased from local market
(Tianjin, China). Total Starch kit, Glucose Oxidase/peroxidase
(GOPOD) kit, Total Dietary Fiber kit, amyloglucosidase (AMG, 3260
U/ml) were purchased from Megazyme International Ireland Ltd.
(Bray Co., Wicklow, Ireland). Porcine pancreatic a-amylase (PPA,
A3176, EC 3.2.1.1, type VI-B, 16 units/mg), was purchased from
Sigma Chemical Co. (St. Louis, Mo., USA). Other chemical reagents
were of analytical grade.
2.2. Flour analysis
Total starch content of wheat flour was determined using a
Megazyme Total Starch kit according to a modified assay procedure
(DMSO format) (Edwards, Warren, Milligan, Butterworth, & Ellis,
2014). Total dietary fiber content was quantified according to
AOAC Official Methods 991.43 (AOAC, 2012) using Megazyme K-
TDFR assay kit. The protein content (N
5.7) was determined ac-
cording to the Kjeldahl method (AACC method 11e15.02) and the
moisture content was measured by drying the samples in an oven
at 105  C to a constant moisture content (AACC method 46e12.01).
The crude fat content was determined by Soxhlet extraction uti-
lizing petroleum ether as solvent (AACC method 30e25.01). The
total starch, crude protein, crude lipid, total dietary fiber and
moisture contents of wheat flour were 60.7, 12.4, 2.0, 9.4 and 9.9%,
respectively.
2.3. Heat treatment of wheat flour-water mixtures
The heat treatment of wheat flour-water mixtures was per-
formed by a method described elsewhere (Wang et al., 2017b).
371
Briefly, wheat flour (25 g) was weighed accurately into a poly-
propylene bag, and the requisite amount of distilled water was
added to obtain water contents of 20, 30, 40, 50, 60 and 70% (w/w,
dry flour basis). The components were mixed thoroughly during
the addition of water. The bags were sealed by vacuum and allowed
to stand for 12 h at room temperature before heating in a boiling
water bath for 5, 10 and 20 min, respectively. After heating, the
samples were frozen immediately in liquid nitrogen, freeze-dried,
ground into powder and passed through a 60 mesh (250 mm)
sieve for further structural and functional analysis.
2.4. Attenuated total reflectance-fourier transform infrared
spectroscopy
The attenuated total reflectance-Fourier transform infrared
(ATR-FTIR) spectra of raw and heated samples were determined
using a Thermo Scientific Nicolet IS50 FTIR spectrometer (Thermo
Fisher Scientific, USA). Raw and heated samples (150 mg) were
pressed into round tablets and spectra were collected in the range
of 4000e400 cm1. Each spectrum was collected at a resolution of
4 cm1 with an accumulation of 64 scans against air as background.
A minimum of six spectra were collected for each sample. The ratio
of absorbances at 1022/995 cm1 were obtained and used to
determine the short-range molecular order of starch in samples
(Wang, Zhang, Wang, & Copeland, 2016b).
2.5. Laser confocal micro-raman spectroscopy
Laser confocal micro-Raman (LCM-Raman) spectra were ob-
tained using a Renishaw Invia Raman microscope system
(Renishaw, Gloucestershire, United Kingdom) equipped with a
Leica microscope (Leica Biosystems, Wetzlar, Germany); the laser
source was a 785 nm green diode. The spectra from 3200 to
100 cm1 were collected from at least six different positions of each
sample. The full width at half maximum (FWHM) of the band at
480 cm1, which represents the short-range ordered structures in
starch (Wang et al., 2015, 2016b), was obtained using the WIRE 2.0
software.
2.6. X-ray diffraction analysis (XRD)
The X-ray diffractometer (D8 Advance, Bruker, Germany) was
operated at 40 kV and 40 mA with Cu Ka radiation (l ¼ 0.154 nm).
Raw and heated samples were equilibrated in a chamber with a
saturated NaCl solution at 25  C for 7 days prior to analysis. The
samples were scanned from 4 to 40 (2q) at a speed of 2 /min and
a step size of 0.02 . The diffractograms were analyzed and the
relative crystallinity was calculated using the software of TOPAS 5.0
(Bruker, Germany).
2.7. Differential scanning calorimetry (DSC)
Thermal properties of samples were measured using a Differ-
ential Scanning Calorimeter (200F3, Netzsch, Germany) equipped
with a thermal analysis data station. Raw and heated samples were
weighed accurately (approximately 3.0 mg, dry basis) into a 40 ml
aluminum sample pan. Distilled water was added with a pipette to
obtain a starch: water ratio (w/v) of 1:4 in the DSC pans. The pans
were sealed and allowed to stand for 12 h at room temperature
before analysis. The pans were heated from 20  C to 130  C at a rate
of 10  C/min. An empty aluminum pan was used as the reference.
The gelatinization enthalpy (DH), onset temperature (To), peak
temperature (Tp), and conclusion temperature (Tc) were obtained
using data recording software.
372 P. Guo et al. / Food Hydrocolloids 82 (2018) 370e378

2.8. Scanning electron microscopy (SEM) Table 1

Gelatinization transition properties of raw and heated samples.

The morphology of flour before and after hydrothermal treat- Water content To (oC) Tp (oC) Tc (oC) DH (J/g flour)
ment was observed using a scanning electron microscope (JSM- 5 min
IT300LV, JEOL, Japan). The lyophilized powders were mounted on Raw 59.2 ± 0.3c'a 65.1 ± 0.5c'a 71.6 ± 0.3c'a 6.3 ± 0.1a'a
the stubs with double-sided adhesive tape, and coated with gold in 20% 60.8 ± 0.2b'c 66.8 ± 0.3b'c 72.5 ± 0.2b'b 5.8 ± 0.3b'a
a sputter coater (JEC-3000FC, Tokyo, Japan). Images were taken at 30% 64.1 ± 0.5a'b 71.3 ± 0.4a'c 78.4 ± 0.2a'b 2.1 ± 0.2c'a
40% N.D. N.D. N.D. N.D.
an accelerating voltage of 10 kV.
50% N.D. N.D. N.D. N.D.
60% N.D. N.D. N.D. N.D.
2.9. Rapid viscosity analysis (RVA) 70% N.D. N.D. N.D. N.D.
10 min
Raw 59.2 ± 0.3c'a 65.1 ± 0.5c'a 71.6 ± 0.3c'a 6.3 ± 0.1a'a
The pasting profiles of raw and heated wheat flour were
20% 61.9 ± 0.3b'b 67.5 ± 0.3b'b 73.4 ± 0.3b'a 5.8 ± 0.3b'a
determined using a Rapid Viscosity Analyser-Tecmaster (Perten 30% 65.7 ± 0.5a'a 72.7 ± 0.3a'b 78.7 ± 0.9a'b 2.0 ± 0.1c'a
Instruments, Australia). Raw and heated samples (3.0 g, dry basis) 40% N.D. N.D. N.D. N.D.
were weighed exactly into a RVA canister, and distilled water was 50% N.D. N.D. N.D. N.D.
added to obtain a total weight of 28.0 g. The standard profile, in 60% N.D. N.D. N.D. N.D.
70% N.D. N.D. N.D. N.D.
which the sample is equilibrated at 50  C for 1 min, heated at 12  C/
20 min
min to 95  C, held at 95  C for 2.5 min, cooled at 12  C/min to 50  C, Raw 59.2 ± 0.3c'a 65.1 ± 0.5c'a 71.6 ± 0.3c'a 6.3 ± 0.1a'a
and held at 50  C for 2 min was used. The paddle speed was 20% 62.6 ± 0.1b'a 68.3 ± 0.2b'a 74.1 ± 0.7b'a 5.5 ± 0.3b'a
960 rpm for the first 10 s, then 160 rpm for the remainder of the 30% 66.4 ± 0.2a'a 76.1 ± 0.1a'a 81.1 ± 0.2a'a 1.7 ± 0.1c'b
experiment. The peak, trough, breakdown, final and setback vis- 40% N.D. N.D. N.D. N.D.
50% N.D. N.D. N.D. N.D.
cosities and pasting temperature were obtained using the Ther- 60% N.D. N.D. N.D. N.D.
mocline 3.0 software. 70% N.D. N.D. N.D. N.D.

2.10. In vitro starch digestion
In vitro starch digestion was determined according to the
method described elsewhere (Wang et al., 2016a). At specific time
points (10, 20, 40, 60, 80, 100, 120 min) during the digestion, ali-
quots (50 mL) were taken and mixed with 950 mL 95% (v/v) ethanol
to terminate the enzymatic reaction; the content of glucose
released in the digestion solution was measured using the Mea-
gazyme GOPOD kit. The percentage of starch hydrolysed was
calculated by multiplying the glucose content with a factor of 0.9.
The starch digestograms were plotted and fitted to the first-order
rate equation (Gon~ i, Garcia-Alonso, & Saura-Calixto, 1997).
Ct ¼ C∞ (1-e-kt)
where Ct is the amount of starch digested at time t (min), C∞ is the
estimated amount of starch digested at the reaction end point, and
k (min1) is the first order rate coefficient. For ease of interpreta-
tion, Ct was expressed as the percentage of starch hydrolysed. The
value of k can be calculated from the slope of a linear-least-squares
fit of a plot of ln (1-Ct/C∞) against t.
Values are means ± SD. The lowercase letters a0 , b0 and c0 represent significant dif-
ferences between the data for each heating length sample (p < 0.05); The lowercase
letters a, b and c represent significant differences between data for each same water
content sample (p < 0.05).
To, onset temperature; Tp, peak temperature; Tc, conclusion temperature; DH,
gelatinization enthalpy.
N.D., not detected.
samples was observed, indicative of considerable disruption of
starch structure during heating. Longer duration of heating
(20 min) resulted in a further small decrease in enthalpy change
(from 2.1 to 1.7 J/g). Similar results were also observed for pure
wheat starch after heating for different lengths of time (Wang et al.,
2017b). No gelatinization peaks were detected for samples that
were heated with 40e70% water content for only 5 min, indicating
complete gelatinization of starch had occurred under these con-
ditions. To, Tp and Tc of starch in the pre-heated wheat flours
increased with increasing water content or heating time. These
increases in the thermal transition temperatures are likely to
represent more stable starch crystallites that remain after less
stable crystallites melted during pre-heating (Wang et al., 2016b).

2.11. Statistical analysis 3.2. Short-range molecular order of starch

Results are reported as the mean values and standard de-
viations. Statistical analysis was performed by one-way analysis of
variance using the SPSS Statistical Software Program (version 19.0,
IBM Corp., USA) and significant differences were detected by the
Duncan's test (p < 0.05).
3. Results
3.1. Thermal properties of raw and heated samples
Thermal properties of raw and heated samples are summarized
in Table 1. The gelatinization enthalpy of raw flour was 6.3 J/g. At
water content of 20%, there were small decreases in enthalpy
change of samples that were heated for 5, 10 and 20 min (5.5e5.8 J/
g), but differences in enthalpy change between the different heat-
ing times were not significant. When the water content increased
to 30%, a large decrease in gelatinization enthalpies of heated
The ratio of absorbances at 1022/995 cm1 in ATR-FTIR spectra
of raw and heated samples is summarized in Table 2. These ratios
can be used to characterize the short-range molecular order of
starch. Gelatinization disrupts crystalline structure and increases
the proportion of amorphous regions in starch, leading to a
decreased intensity of the band at 995 cm1 ascribed to crystalline
regions and an increased intensity at 1022 cm1 associated with
amorphous regions (Wang, Wang, Liu, Wang, & Copeland, 2017c).
After heating the flour for 5, 10 and 20 min, the ratios of absor-
bances at 1022/995 cm1 increased gradually with increasing water
content to 40%, above which no further significant increases in the
ratio occurred (Table 2). This observation is consistent with the DSC
results, in that long-range crystallinity was completely disrupted at
water content of 40%. At each water content, length of heating time
had little effect on the ratio of absorbances at 1022/995 cm1
(Table 2).
The FWHM of the band at 480 cm1 is sensitive to the changes in
 P. Guo et al. / Food Hydrocolloids 82 (2018) 370e378 373

Table 2 weak. As similar patterns were observed for all the heated sam-
Short-range molecular orders of starch in raw and heated samples determined by ples, only the 2D view of samples that were heated for 5 min are
ATR-FTIR and Raman spectra.
shown (Fig. 1g). With increasing water content, the typical A-type
Water content 5 min Length of heating time 20 min crystalline peaks (denoted by arrows in Fig. 1g) gradually weak-
10 min ened and disappeared at water content of 40%. For raw flour and
IR ratios of absorbances at 1022/995 cm1 flour that was heated with 20% water, no diffraction peak was
Raw 1.104 ± 0.016c'a 1.104 ± 0.016c'a 1.104 ± 0.016d'a observed at 13 (2q). After heating at 30% water, the samples
20% 1.215 ± 0.027b'a 1.217 ± 0.027b'a 1.210 ± 0.043c'a
presented a weak peak at 13 (2q), which became progressively
30% 1.301 ± 0.043a'a 1.322 ± 0.035a'a 1.269 ± 0.021bc'a
40% 1.358 ± 0.056a'a 1.347 ± 0.042a'a 1.350 ± 0.01a'a stronger with increasing water content. In contrast, the peak at
50% 1.324 ± 0.059a'a 1.324 ± 0.059a'a 1.324 ± 0.011ab'a 20 (2q), which was weak for raw flour, became increasingly
60% 1.346 ± 0.054a'a 1.328 ± 0.013a'a 1.332 ± 0.056ab'a obvious with increasing water content to 40e50%. These obser-
70% 1.314 ± 0.011a'a 1.338 ± 0.034a'a 1.343 ± 0.035a'a
vations confirm that the V-type starch-lipid complexes were
FWHM of the band at 480 cm1
Raw 15.5 ± 0.6c'a 15.5 ± 0.6c'a 15.5 ± 0.6c'a
formed during heating of wheat flour.
20% 15.7 ± 0.6c'a 16.0 ± 0.4bc'a 16.0 ± 0.6c'a
30% 16.8 ± 0.7bc'a 16.7 ± 0.8b'a 16.8 ± 0.7bc'a 3.4. Morphology of starch
40% 17.4 ± 0.7ab'a 17.8 ± 0.7a'a 17.8 ± 0.8ab'a
50% 17.6 ± 1.1ab'a 17.9 ± 0.5a'a 18.3 ± 0.9a'a
The microstructure of raw and heated samples is shown in
60% 18.0 ± 0.7a'a 18.2 ± 1.1a'a 18.4 ± 1.1a'a
70% 18.2 ± 1.5a'a 18.5 ± 0.3a'a 18.5 ± 1.8a'a Fig. S1. The raw wheat flour showed the typical large A-type and
small B-type starch granules (Fig. S1a). After heating with 20%
Values are means ± SD. The different lowercase letters a', b' and c' represent sig-
nificant differences between the data in the same column (p < 0.05); and the
water, mostly starch granules were intact, although some starch
lowercase letters a, b and c represent significant differences between the data in the granules seemed to adhere to each other (Fig. S1b-d). After heating
same row (p < 0.05). with 30% water, swelling and deformation of the starch granules
FWHM: full width at half maximum. was clearly apparent (Fig. S1e-g), which increased considerably as
the water content increased to 40e50% (Fig. S1h-m). It is inter-
esting to note that while the starch granule contours could still be
the short-range molecular order of starch during gelatinization or
identified, the crystalline structure of starch was hardly detected by
retrogradation (Wang et al., 2016b). After heating for 5, 10 and
X-ray diffraction (Fig. 1c and d). Similarly, complete disruption of
20 min, the FWHM of the band at 480 cm1 increased gradually
crystalline structures during gelatinization while the contours of
with increasing water content up to 40%, with only small increases
swollen starch granules are still evident has been reported in pre-
being noted at higher water content (Table 2), consistent with the
vious studies (Wang & Copeland, 2012; Wang et al., 2016b). As the
FTIR results. No significant changes were noted between the sam-
water content increased further (60e70%), the starch granules were
ples after heating for different lengths of time at the same water
disrupted completely. Increasing heating times from 5 to 10 or
content.
20 min seemed to bring about few additional changes in granular
morphology of starch in cooked flours.
3.3. X-ray diffraction analysis
3.5. Pasting properties
The XRD patterns of raw and the heated samples are presented
in Fig. 1. The starch in raw wheat flour exhibited A-type diffraction The pasting profiles of raw and heated samples are given in
pattern with strong diffraction peaks at 15.3 , 17.2 , 18.2 and Fig. 2 and the corresponding pasting parameters are summarized in
23.3 (2q) (Fig. 1a). The obvious diffraction peak at around 20.0 (2q) Table 3. The samples heated with water contents of 20e50%
was assigned to endogenous starch-lipid complex. No significant exhibited typical viscosity profiles, although there were gradual
differences in diffraction patterns were observed between raw flour decreases in peak, trough, setback and final viscosities and in-
and samples heated with 20% water for 5, 10 and 20 min, although creases in pasting temperature compared with the raw flour. While
the relative crystallinity of the heated samples was slightly lower the overall crystallinity was completely disrupted after heating at
than that of raw flour (Fig. 1a). After heating with 30% water, the water contents of 40 and 50%, the heated samples still developed
diffraction peaks became weaker and the relative crystallinity the typical viscosity pattern, as was similarly observed for cooked
decreased substantially (19.1, 19.3 and 17.9% after 5, 10 and 20 min, rice flours (Wang et al., 2017a). Clearly, some ordered structures
respectively) (Fig. 1b). With further increases in water content remain, which can swell and develop paste viscosity but are no
(from 40% to 70%), the typical A-type diffraction peaks disappeared, longer detectable by XRD and DSC. Moreover, with water contents
indicating complete disruption of starch long-range crystallinity of 20e50%, the peak, trough, setback and final viscosities decreased
(Fig. 1cef). Two prominent peaks at around 13 and 20 (2q) as length of heating time increased, indicating structural order
emerged, especially at water contents of 30 and 40%, indicating the other than crystalline structures continued to be disrupted pro-
formation of starch-lipid or starch-protein-lipid complexes in gressively. In contrast, the samples that were heated with 60 or 70%
heated samples (Wang et al., 2017d). Similar observations have water displayed different pasting profiles that indicated the pres-
already been reported on heat-moisture treated wheat flour (Chen, ence of cold water-swelling starch. Similar results were also re-
He, Fu, & Huang, 2015) and sorghum flour extrudates (Jafari, ported for heated rice flours (Chung, Liu, Huang, Yin, & Li, 2010;
Koocheki, & Milani, 2017). A small peak at around 17 (2q) was Wang et al., 2017a).
observed for heated samples at water contents of 60 and 70%,
suggesting the retrogradation of starch in heated samples. At each 3.6. In vitro starch digestibility
water content, heating duration had little effect on crystallinity of
samples. Digestograms of starch in raw and heated flour samples are
To further illustrate the effect of water content on the crys- shown in Fig. 3aec and the corresponding fit of the data to the
tallinity of heated samples, the 2D views of XRD diffraction pat- equation for first order kinetics are also presented. Starch in raw
terns are shown in Fig. 1g, which represents a stack of XRD scans wheat flour was hydrolyzed very slowly, with only about 20%
with a colored ruler indicating peak intensities from strong to broken down to glucose after 2 h incubation. Similar digestion
374 P. Guo et al. / Food Hydrocolloids 82 (2018) 370e378

Fig. 1. XRD patterns of raw and heated samples with 20e70% water content (RC, relative crystallinity). a: raw and 20% water content; b: 30% water content; c: 40% water content; d:
50% water content; e: 60% water content; f: 70% water content; g: 2D views of XRD diffraction patterns of raw and heated flour samples for 5 min at various water contents.
 P. Guo et al. / Food Hydrocolloids 82 (2018) 370e378
Fig. 2. Pasting profiles of raw and heated flour samples for 5min (a), 10 min (b) and
20 min (c).
patterns were observed for the samples heated with 20e70% water.
After heating for only 5 min with water content of 20%, the rate and
extent of starch digestibility were increased greatly, with a final
digestion percentage after 2 h of over 85%. Under these heating
conditions, long-range structural order, as measured by XRD and
DSC was almost unaffected. Further increases in water content or
heating time resulted in only minor increases in digestion rate. The
small differences in extent and rate of starch digestion in all heated
samples were consistent with our previous findings that structural
order in gelatinized starch is not the determinant for in vitro starch
digestibility (Wang et al., 2017c).
375
4. Discussion
In the present study, gelatinization properties of starch in wheat
flour during cooking with water were monitored by a combination
of analytical methods. An advantage of using wheat flour as model
system to study starch gelatinization rather than isolated starch
during cooking or simulated food processing is that the effect of
other components on starch gelatinization can be revealed. Thus,
the present study has relevance to foods made from doughs (e.g.,
bakery products, noodles, pasta) and batters (e.g., pancakes, coat-
ings for fried foods). The results showed the short- and long-range
molecular orders of starch in wheat flour were disrupted progres-
sively with increasing water content during cooking. The DSC
transition temperatures increased and enthalpy change decreased
with increasing water content. At a water content of 40% or higher,
the overall loss of structural order in wheat flour indicated the
starch was fully gelatinized during cooking. Nevertheless, remnant
structures in the starch were still able to exhibit some swelling and
pasting properties in samples heated with water content up to 50%.
Length of heating duration had much less effect compared to water
content on the disruption of long- and short-range molecular or-
ders of starch in wheat flour, consistent with previous studies (Sui
et al., 2015; Wang et al., 2017b). Heating for only 5 min at water
content as low as 20% greatly increased the in vitro enzymatic di-
gestibility of starch in wheat flour, as observed for isolated starch
(Wang et al., 2017b). These results confirmed that loss of crystalline
structure in gelatinized starch is not the rate-determining factor for
starch digestion (Wang et al., 2017c). Moreover, our results are
consistent with the proposal that molecular density of amorphous
regions (Zhang, Dhital, & Gidley, 2015) or quantity of flexible a-
glucan chains protruding from the granule surface (Baldwin et al.,
2015) are key structural features that determine the rate of starch
digestion, mostly by influencing the access/binding of alpha-
amylase to starch. The presence of proteins and other constitu-
ents in the flour did not seem to block this access.
The present study of heated wheat flour-water systems showed
that there was no impediment to starch gelatinization or any lesser
degree of disruption of starch structures, compared with isolated
starch in similar experimental systems (Wang et al., 2017b). For
example, the degree of gelatinization after heating with 30% water
was 72e73% for wheat starch (Wang et al., 2017b) and starch in
wheat flour, whereas at a water content of 40% or higher, starch in
both systems was almost completely gelatinized. These results
indicated that non-starch components, either in a free state or after
complexing with starch during heating, do not influence the pro-
gression of starch gelatinization in wheat flour. In previous studies,
proteins and non-starch polysaccharides have been shown to
retard the progression of starch gelatinization in model systems by
reducing water availability for starch gelatinization or by com-
plexing with starch (Jekle, Mühlberger, & Becker, 2016; Mohamed
& Rayas-Duarte, 2003; Tester & Sommerville, 2003). The conclu-
sion drawn from these previous studies was based mainly on the
increased gelatinization temperatures in the presence of added
proteins and non-starch polysaccharides. Our study provided solid
evidences that starch gelatinization in wheat flour was not affected
obviously by the presence of other components during cooking.
A key difference between the flour and isolated starch systems
demonstrated by the present study was the formation of starch-
lipid or starch-lipid-protein complexes during heating of wheat
flour-water mixtures. The clearly evident transformation of the A to
A þ V, and finally to V type XRD trace, and accompanying DSC
analyses, confirmed that starch-lipid or starch-lipid-protein com-
plexes are formed during heating of wheat flour-water mixtures.
The formation of amylose-lipid complexes has been studied mostly
in model mixtures, but relatively little is known about complex
376 P. Guo et al. / Food Hydrocolloids 82 (2018) 370e378

Table 3
Pasting parameters of raw and heated samples.

Water content PV (cP) TV (cP) BV (cP) FV (cP) SV (cP) PT ( C)

5 min
Raw 2380.5 ± 12.4a'a 1567.3 ± 90.0a'a 813.3 ± 4.0a'a 3354.5 ± 12.1a'a 1787.5 ± 4.5a'a 70.2 ± 0.2d'a
20% 1934.5 ± 34.6b'a 1493.0 ± 25.5b'a 441.5 ± 9.2b'a 3065.0 ± 36.8b'a 1572.0 ± 11.3b'a 89.7 ± 0.1c'c
30% 1452.0 ± 5.7c'a 1264.5 ± 4.9c'a 187.5 ± 10.6c'a 2554.5 ± 24.7c'a 1290.0 ± 29.7c'a 93.3 ± 0.5b'b
40% 999.0 ± 11.3d'a 978.0 ± 7.1d'a 21.0 ± 4.2f'b 1914.5 ± 46.0e'a 936.5 ± 38.9e'a 95.3 ± 0.1a'a
50% 852.0 ± 1.4f'a 813.0 ± 2.8e'a 39.0 ± 1.4e'a 1681.5 ± 3.5f'a 868.5 ± 6.4f'a 94.9 ± 0.5a'a
60% 706.0 ± 1.1 g'a 700.0 ± 2.8f'a 6.0 ± 2.8 g'c 1557.5 ± 23.3 g'a 857.5 ± 26.2f'a 93.3 ± 0.6b'a
70% 933.0 ± 2.3e'b 828.0 ± 3.2e'b 105.0 ± 0.6d'b 1949.5 ± 3.5d'b 1121.5 ± 3.5d'a 90.0 ± 0.6c'a
10 min
Raw 2380.5 ± 12.4a'a 1567.3 ± 90.0a'a 813.3 ± 4.0a'a 3354.5 ± 12.1a'a 1787.5 ± 4.5a'a 70.2 ± 0.2e'a
20% 1535.0 ± 4.2b'b 1312.5 ± 0.7b'b 222.5 ± 4.9b'b 2696.5 ± 13.4b'b 1384.0 ± 14.1b'b 91.3 ± 0.1c'b
30% 1240.0 ± 12.7c'b 1150.5 ± 16.3c'b 89.5 ± 3.5d'b 2220.5 ± 10.6c'b 1070.0 ± 5.7c'b 94.1 ± 0.6ab'b
40% 877.5 ± 4.9d'b 870.0 ± 4.2d'b 17.5 ± 0.7f'b 1612.5 ± 9.2e'b 742.5 ± 13.4e'b 94.9 ± 0.3a'a
50% 559.5 ± 2.1f'b 514.0 ± 7.1f'b 45.5 ± 4.9e'a 1244.0 ± 2.8f'b 730.0 ± 9.9e'b 95.2 ± 0.1a'a
60% 527.5 ± 0.7 g'c 489.5 ± 0.7 g'c 38.0 ± 1.9e'a 1163.0 ± 8.5 g'b 673.5 ± 7.8f'b 93.6 ± 0.1b'a
70% 848.5 ± 7.8e'c 739.5 ± 0.7e'c 109.0 ± 8.5c'b 1737.0 ± 2.8d'c 997.5 ± 2.1d'c 90.5 ± 0.1d'a
20 min
Raw 2380.5 ± 12.4a'a 1567.3 ± 90.0a'a 813.3 ± 4.0a'a 3354.5 ± 12.1a'a 1787.5 ± 4.5a'a 70.2 ± 0.2f'a
20% 1374.0 ± 8.5b'c 1222.0 ± 2.8b'c 152.0 ± 5.7b'c 2516.0 ± 21.2b'c 1294.0 ± 24b'c 92.1 ± 0.1d'a
30% 1136.0 ± 4.2c'c 1060.0 ± 4.2c'c 76.0 ± 0.1d'c 1970.5 ± 19.1d'c 910.5 ± 23.3d'c 95.3 ± 0.1a'a
40% 815.0 ± 1.4e'c 775.0 ± 2.8e'c 40.0 ± 1.4e'a 1368.0 ± 2.8e'c 593.0 ± 5.8e'c 94.9 ± 0.4ab'a
50% 388.0 ± 4.2 g'c 357.0 ± 5.7 g'c 31.0 ± 1.4f'b 788.0 ± 4.2 g'c 431.0 ± 1.4 g'c 94.7 ± 0.1b'a
60% 554.0 ± 5.7f'b 541.5 ± 0.7f'b 12.5 ± 4.9 g'b 1099.0 ± 8.5f'c 557.5 ± 9.2f'c 93.3 ± 0.5c'a
70% 1126.0 ± 8.5d'a 994.5 ± 0.7d'a 131.5 ± 9.2c'a 2050.5 ± 12.0c'a 1056.0 ± 11.3c'b 87.6 ± 0.6e'b

Values are means ± SD. The lowercase letters a0 , b0 and c0 represent significant differences between the data for each milled fraction heated with different water contents
(p < 0.05); The lowercase letters a, b and c represent significant differences between the data for samples at the same water content (p < 0.05).
PV, peak viscosity; TV, through viscosity; BV, breakdown viscosity; FV, final viscosity; SV, setback viscosity; PT, pasting temperature.

Fig. 3. In vitro starch digestion curves of raw and heated samples with various water contents for 5min (a), 10 min (b), 20 min (c) and with 20%water for 5, 10, 20 min (d). Values are
means ± SD.
 P. Guo et al. / Food Hydrocolloids 82 (2018) 370e378
formation during processing of actual food ingredients such as flour
(De Pilli, Derossi, Talja, Jouppila, & Severini, 2011; De Pilli et al.,
2008). Our study confirmed for the first time by XRD and DSC
that starch-lipid complexes are formed during cooking of wheat
flour-water mixtures.
An interesting finding was that duration of heating had small
effect on disruption of crystalline structure of starch, but had
considerable effects on pasting properties of starch in cooked
wheat flour. Longer duration of cooking resulted in lower peak,
trough, setback and final viscosities at water contents of 20e50%.
These results indicated that structural order, most probably in
amorphous rather than in crystalline regions, was further disrupted
during longer duration of heating. We proposed that the short-
range molecular order in amorphous starch, which is not detec-
ted by XRD, FTIR and Raman spectroscopy, has a key influence on
pasting properties of cooked starch.
5. Conclusions
The present study has shown the major difference between
heating flour and isolated starch systems was the formation of bi-
nary (starch-lipid) or ternary (starch, protein and lipid) complexes
during cooking. The multi-scale structural orders of starch during
cooking were not greatly affected by the non-starch components in
wheat flour which might compete with starch for water. Even after
the starch appeared to be fully gelatinized according to XRD and
DSC analyses, some remnant structures were still present that
exhibited swelling and pasting properties. The degree of short-
range molecular order in amorphous starch is therefore proposed
to play a key role in determining the pasting properties of cooked
starch. The susceptibility of starch to in vitro amylolysis was greatly
increased when only a small amount of structural disruption had
occurred. The information obtained from the present study may
provide a more useful guide to the processing properties of wheat
flour-based food products than might be obtained from studies of
starch gelatinization in isolation.
Acknowledgements
The authors gratefully acknowledge the financial support from
the National Natural Science Foundation of China (31522043) and
Tianjin Natural Science Foundation for Distinguished Young Scholar
(17JCJQJC45600).
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.foodhyd.2018.04.012.
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