Mechanisms of starch gelatinization during heating of wheat flour and its effect on in vitro starch digestibility

Accepted Manuscript Mechanisms of starch gelatinization during heating of wheat flour and its effect on in vitro starch digestibility Peng Guo, Jinglin Yu, Les Copeland, Shuo Wang, Shujun Wang PII:

S0268-005X(17)32098-2

DOI:

10.1016/j.foodhyd.2018.04.012

Reference:

FOOHYD 4379

To appear in:

Food Hydrocolloids

Received Date: 19 December 2017 Revised Date:

11 March 2018

Accepted Date: 6 April 2018

Please cite this article as: Guo, P., Yu, J., Copeland, L., Wang, S., Wang, S., Mechanisms of starch gelatinization during heating of wheat flour and its effect on in vitro starch digestibility, Food Hydrocolloids (2018), doi: 10.1016/j.foodhyd.2018.04.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Mechanisms of starch gelatinization during heating of wheat flour

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and its effect on in vitro starch digestibility

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State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science & Technology, Tianjin 300457, China

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Key Laboratory of Food Nutrition and Safety, Ministry of Education, Tianjin University of

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Science & Technology, Tianjin 300457, China

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School of Food Engineering and Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China

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The University of Sydney, Sydney Institute of Agriculture, School of Life and

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Environmental Sciences, NSW Australia 2006

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Peng Guoabc, Jinglin Yua, Les Copelandd, Shuo Wange*, Shujun Wangabc*

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Tianjin Key Laboratory of Food Science and Human Health, School of Medicine, Nankai University, Tianjin 30071, China

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* Corresponding authors: Dr. Shujun Wang or Dr. Shuo Wang

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Mailing address: No 29, 13th Avenue, Tianjin Economic and Developmental Area (TEDA), Tianjin

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300457, China

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Phone: 86-22-60912486

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E-mail address: [email protected] or [email protected]

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Abstract: This study aimed to understand mechanisms of starch gelatinization during 1

ACCEPTED MANUSCRIPT heating of wheat (Triticum aestivum L.) flour, and its effects on in vitro starch

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amylolysis of cooked wheat flour. At water content of 20%, cooking did not disrupt

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greatly the crystalline structures of starch in wheat flour. Considerable disruption of

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ordered structures of starch occurred with cooking at a water content of 30%, and

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starch was fully gelatinized at a water content of 40%. The transition of the X-ray

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diffraction pattern from A to V types with increasing water content suggested the

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formation of starch-lipid complexes during cooking of flour. Typical starch pasting

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profiles of cooked wheat flour were observed at water contents of 40 and 50%,

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whereas longer cooking time decreased the pasting viscosities of the flour. Cooking

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the flour for only 5 min with 20% water greatly increased the rate and extent of in

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vitro enzymatic digestion of starch, which were not increased further by longer

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cooking or higher water content. From this study, we conclude that the progression of

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starch gelatinization during cooking was not greatly affected by non-starch

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components in wheat flour, and that some structural order in amorphous regions of

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starch contributed to the development of pasting viscosities.

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Key words: Triticum aestivum; wheat flour; cooking; structural order; in vitro starch

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digestibility

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1. Introduction 2

ACCEPTED MANUSCRIPT Wheat (Triticum aestivum L.) is one of the most cultivated cereal grains and supplies

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the daily calories as an important staple food in many countries. Wheat is the most

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important cereal used in breadmaking and other cereal-based products (cookies, pasta,

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cakes, etc.) (Veraverbeke & Delcour, 2002). Wheat flour consists mainly of starch

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(about 70-75%) and proteins (about 8-14%). Some minor components such as lipids

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(about 2%), non-starch polysaccharides (about 2-3%), minerals, vitamins,

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antioxidants and other nutrients are also present in the whole wheat flour (Hemery,

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Rouau, Lullien-Pellerin, Barron, & Abecassis, 2007). Starch is the main source of

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glycemic carbohydrates in the human diet and also a major determinant of quality of

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wheat-based products (Wang & Copeland, 2013). The increasing incidence of type 2

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diabetes and cardiovascular diseases associated with consumption of excess starchy

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foods is of considerable nutritional concern (Taylor, Emmambux, & Kruger, 2014;

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Tian, Chen, Ye, & Chen, 2016; Willett, Manson, & Liu, 2002). This has led to much

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research on how the structural and functional properties of starch, interactions

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between starch and other food components, and properties of the food matrix affect

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the rate and extent of starch digestion (Singh, Dartois, & Kaur, 2010; Wang &

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Copeland, 2013). Moreover, there is growing interest in using wholemeal flours or

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adding bran fractions to refined flours in foods to help moderate post-prandial blood

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glucose response (Hemdane et al., 2016; Petitot, Barron, Morel, & Micard, 2010;

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Rosa-Sibakov, Poutanen, & Micard, 2015).

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Starch granules have a complex hierarchical structure, which is studied with multiple 3

ACCEPTED MANUSCRIPT techniques such as DSC, XRD, SAXS and SEM over a scale ranging from nano- to

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micrometers (Pérez & Bertoft, 2010; Wang, Li, Copeland, Niu, & Wang, 2015). Upon

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heating, starch granules are gelatinized causing disruption of multi-scale structures,

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with the extent of disruption depending on temperature, water content, and length of

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heating time and rate (Mariotti, Zardi, Lucisano, & Pagani, 2005; Wang & Copeland,

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2013). At a fixed heating temperature, water content plays a more important role in

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disruption of starch structures and increasing starch digestibility than length of

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heating time (Wang, Wang, Guo, Liu, & Wang, 2017b), whereas at a fixed water

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content, higher temperature results in greater disruption of starch structure (Pan et al.,

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2017; Wang, Sun, Wang, Wang, & Copeland, 2016a; Zhang et al., 2014).

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Use of purified starch as a model material has led to an understanding of the

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disassembly mechanisms of starch granules during thermal food processing. However,

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the presence of proteins and lipids in cereal-based food systems may affect the

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gelatinization behavior of starch during processing through the potential for complex

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formation with starch (Wang, Zheng, Yu, Wang, & Copeland, 2017d) or by changing

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water availability (Kim et al., 2008; Zou, Sissons, Gidley, Gilbert, & Warren, 2015).

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Interactions between starch, proteins and lipids during thermal processing have been

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studied extensively in model systems (Wang et al., 2017d; Zhang, Maladen, &

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Hamaker, 2003; Zhang, Maladen, Campanella, & Hamaker, 2010). Although the

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presence of other components in flour is assumed to affect the functionality of starch

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for food processing and human nutrition, there is little actual information on this. For

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ACCEPTED MANUSCRIPT example, how proteins in might influence starch gelatinization during cooking of

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doughs and batters is not understood. Studying the mechanism of starch gelatinization

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in wheat flour is an important step towards predicting and, in turn, controlling the

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processing and functional properties of more complex cereal-based food systems.

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Hence, in the present study, standard wheat flour was used as a model to study

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gelatinization properties of starch cooked with food components that might be present

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during processing.

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2. Material and methods

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2.1 Materials

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The standard wheat flour was purchased from local market (Tianjin, China). Total

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Starch kit, Glucose Oxidase/peroxidase (GOPOD) kit, Total Dietary Fiber kit,

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amyloglucosidase (AMG, 3260 U/ml) were purchased from Megazyme International

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Ireland Ltd. (Bray Co., Wicklow, Ireland). Porcine pancreatic α-amylase (PPA,

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A3176，EC 3.2.1.1, type VI-B, 16 units/mg), was purchased from Sigma Chemical Co.

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(St. Louis, Mo., USA). Other chemical reagents were of analytical grade.

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2.2 Flour analysis

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Total starch content of wheat flour was determined using a Megazyme Total Starch kit

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according to a modified assay procedure (DMSO format) (Edwards, Warren, Milligan,

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Butterworth, & Ellis, 2014). Total dietary fiber content was quantified according to

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AOAC Official Methods 991.43 (AOAC, 2012) using Megazyme K-TDFR assay kit. 5

ACCEPTED MANUSCRIPT The protein content (N×5.7) was determined according to the Kjeldahl method

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(AACC method 11-15.02) and the moisture content was measured by drying the

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samples in an oven at 105°C to a constant moisture content (AACC method 46-12.01).

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The crude fat content was determined by Soxhlet extraction utilizing petroleum ether

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as solvent (AACC method 30-25.01). The total starch, crude protein, crude lipid, total

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dietary fiber and moisture contents of wheat flour were 60.7, 12.4, 2.0, 9.4 and 9.9%,

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respectively.

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2.3 Heat treatment of wheat flour-water mixtures

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The heat treatment of wheat flour-water mixtures was performed by a method

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described elsewhere (Wang et al., 2017b). Briefly, wheat flour (25g) was weighed

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accurately into a polypropylene bag, and the requisite amount of distilled water was

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added to obtain water contents of 20, 30, 40, 50, 60 and 70% (w/w, dry flour basis).

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The components were mixed thoroughly during the addition of water. The bags were

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sealed by vacuum and allowed to stand for 12 h at room temperature before heating in

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a boiling water bath for 5, 10 and 20 min, respectively. After heating, the samples

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were frozen immediately in liquid nitrogen, freeze-dried, ground into powder and

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passed through a 60 mesh (250 microns) sieve for further structural and functional

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analysis.

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2.4 Attenuated Total Reflectance-Fourier transform infrared spectroscopy

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The attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectra of 6

ACCEPTED MANUSCRIPT raw and heated samples were determined using a Thermo Scientific Nicolet IS50

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FTIR spectrometer (Thermo Fisher Scientific, USA). Raw and heated samples (150

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mg) were pressed into round tablets and spectra were collected in the range of

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4000-400 cm-1. Each spectrum was collected at a resolution of 4 cm-1 with an

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accumulation of 64 scans against air as background. A minimum of six spectra were

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collected for each sample. The ratio of absorbances at 1022/995 cm-1 were obtained

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and used to determine the short-range molecular order of starch in samples (Wang,

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Zhang, Wang, & Copeland, 2016b).

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2.5 Laser confocal micro-Raman spectroscopy

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Laser confocal micro-Raman (LCM-Raman) spectra were obtained using a Renishaw

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Invia Raman microscope system (Renishaw, Gloucestershire, United Kingdom)

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equipped with a Leica microscope (Leica Biosystems, Wetzlar, Germany); the laser

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source was a 785 nm green diode. The spectra from 3200 to 100 cm-1 were collected

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from at least six different positions of each sample. The full width at half maximum

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(FWHM) of the band at 480 cm-1, which represents the short-range ordered structures

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in starch (Wang et al., 2015; Wang et al., 2016b), was obtained using the WIRE 2.0

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software.

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2.6 X-ray diffraction analysis (XRD)

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The X-ray diffractometer (D8 Advance, Bruker, Germany) was operated at 40 kV and

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40 mA with Cu Kα radiation (λ= 0.154 nm). Raw and heated samples were 7

ACCEPTED MANUSCRIPT equilibrated in a chamber with a saturated NaCl solution at 25 °C for 7 days prior to

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analysis. The samples were scanned from 4° to 40° (2θ) at a speed of 2°/min and a

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step size of 0.02°. The diffractograms were analyzed and the relative crystallinity was

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calculated using the software of TOPAS 5.0 (Bruker, Germany).

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2.7 Differential scanning calorimetry (DSC)

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Thermal properties of samples were measured using a Differential Scanning

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Calorimeter (200F3, Netzsch, Germany) equipped with a thermal analysis data station.

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Raw and heated samples were weighed accurately (approximately 3.0 mg, dry basis)

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into a 40 µl aluminum sample pan. Distilled water was added with a pipette to obtain

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a starch: water ratio (w/v) of 1:4 in the DSC pans. The pans were sealed and allowed

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to stand for 12 h at room temperature before analysis. The pans were heated from

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20 °C to 130 °C at a rate of 10 °C/min. An empty aluminum pan was used as the

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reference. The gelatinization enthalpy (∆H), onset temperature (To), peak temperature

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(Tp), and conclusion temperature (Tc) were obtained using data recording software.

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2.8 Scanning electron microscopy (SEM)

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The morphology of flour before and after hydrothermal treatment was observed using

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a scanning electron microscope (JSM-IT300LV, JEOL, Japan). The lyophilized

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powders were mounted on the stubs with double-sided adhesive tape, and coated with

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gold in a sputter coater (JEC-3000FC, Tokyo, Japan). Images were taken at an

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accelerating voltage of 10 kV. 8

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2.9 Rapid viscosity analysis (RVA)

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The pasting profiles of raw and heated wheat flour were determined using a Rapid

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Viscosity Analyser-Tecmaster (Perten Instruments, Australia). Raw and heated

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samples (3.0 g, dry basis) were weighed exactly into a RVA canister, and distilled

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water was added to obtain a total weight of 28.0 g. The standard profile, in which the

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sample is equilibrated at 50 °C for 1 min, heated at 12 °C /min to 95 °C, held at 95 °C

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for 2.5 min, cooled at 12 °C /min to 50 °C, and held at 50 °C for 2 min was used. The

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paddle speed was 960 rpm for the first 10 s, then 160 rpm for the remainder of the

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experiment. The peak, trough, breakdown, final and setback viscosities and pasting

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temperature were obtained using the Thermocline 3.0 software.

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2.10 In vitro starch digestion

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In vitro starch digestion was determined according to the method described elsewhere

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(Wang et al., 2016a). At specific time points (10, 20, 40, 60, 80, 100, 120 min) during

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the digestion, aliquots (50 µL) were taken and mixed with 950 µL 95% (v/v) ethanol

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to terminate the enzymatic reaction; the content of glucose released in the digestion

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solution was measured using the Meagazyme GOPOD kit. The percentage of starch

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hydrolysed was calculated by multiplying the glucose content with a factor of 0.9.

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The starch digestograms were plotted and fitted to the first-order rate equation (Goñi,

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Garcia-Alonso, & Saura-Calixto, 1997).

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Ct=C∞ (1-e-kt)

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ACCEPTED MANUSCRIPT where Ct is the amount of starch digested at time t (min), C∞ is the estimated amount

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of starch digested at the reaction end point, and k (min-1) is the first order rate

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coefficient. For ease of interpretation, Ct was expressed as the percentage of starch

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hydrolysed. The value of k can be calculated from the slope of a linear-least-squares

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fit of a plot of ln (1-Ct/C∞) against t.

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2.11 Statistical analysis

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Results are reported as the mean values and standard deviations. Statistical analysis

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was performed by one-way analysis of variance using the SPSS Statistical Software

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Program (version 19.0, IBM Corp., USA) and significant differences were detected by

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the Duncan’s test (p < 0.05).

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3. Results

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3.1 Thermal properties of raw and heated samples

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Thermal properties of raw and heated samples are summarized in Table 1. The

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gelatinization enthalpy of raw flour was 6.3 J/g. At water content of 20%, there were

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small decreases in enthalpy change of samples that were heated for 5, 10 and 20 min

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(5.5 to 5.8 J/g), but differences in enthalpy change between the different heating times

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were not significant. When the water content increased to 30%, a large decrease in

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gelatinization enthalpies of heated samples was observed, indicative of considerable

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disruption of starch structure during heating. Longer duration of heating (20 min)

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resulted in a further small decrease in enthalpy change (from 2.1 to 1.7 J/g). Similar

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ACCEPTED MANUSCRIPT results were also observed for pure wheat starch after heating for different lengths of

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time (Wang et al., 2017b). No gelatinization peaks were detected for samples that

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were heated with 40 to 70% water content for only 5 min, indicating complete

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gelatinization of starch had occurred under these conditions. To, Tp and Tc of starch in

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the pre-heated wheat flours increased with increasing water content or heating time.

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These increases in the thermal transition temperatures are likely to represent more

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stable starch crystallites that remain after less stable crystallites melted during

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pre-heating (Wang et al., 2016b).

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3.2 Short-range molecular order of starch

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The ratio of absorbances at 1022/995 cm-1 in ATR-FTIR spectra of raw and heated

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samples is summarized in Table 2. These ratios can be used to characterize the

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short-range molecular order of starch. Gelatinization disrupts crystalline structure and

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increases the proportion of amorphous regions in starch, leading to a decreased

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intensity of the band at 995 cm-1 ascribed to crystalline regions and an increased

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intensity at 1022 cm-1 associated with amorphous regions (Wang, Wang, Liu, Wang, &

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Copeland, 2017c). After heating the flour for 5, 10 and 20 min, the ratios of

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absorbances at 1022/995 cm-1 increased gradually with increasing water content to

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40%, above which no further significant increases in the ratio occurred (Table 2).

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This observation is consistent with the DSC results, in that long-range crystallinity

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was completely disrupted at water content of 40%. At each water content, length of

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heating time had little effect on the ratio of absorbances at 1022/995 cm-1 (Table 2).

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The FWHM of the band at 480 cm-1 is sensitive to the changes in the short-range

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molecular order of starch during gelatinization or retrogradation (Wang et al., 2016b).

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After heating for 5, 10 and 20 min, the FWHM of the band at 480 cm-1 increased

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gradually with increasing water content up to 40%, with only small increases being

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noted at higher water content (Table 2), consistent with the FTIR results. No

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significant changes were noted between the samples after heating for different lengths

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of time at the same water content.

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3.3 X-ray diffraction analysis

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The XRD patterns of raw and the heated samples are presented in Figure 1. The

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starch in raw wheat flour exhibited A-type diffraction pattern with strong diffraction

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peaks at 15.3°, 17.2°, 18.2° and 23.3°(2θ) (Figure 1a). The obvious diffraction peak

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at around 20.0° (2θ) was assigned to endogenous starch-lipid complex. No significant

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differences in diffraction patterns were observed between raw flour and samples

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heated with 20% water for 5, 10 and 20 min, although the relative crystallinity of the

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heated samples was slightly lower than that of raw flour (Figure 1a). After heating

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with 30% water, the diffraction peaks became weaker and the relative crystallinity

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decreased substantially (19.1, 19.3 and 17.9% after 5, 10 and 20 min, respectively)

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(Figure 1b). With further increases in water content (from 40% to 70%), the typical

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A-type diffraction peaks disappeared, indicating complete disruption of starch

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long-range crystallinity (Figure 1c-1f). Two prominent peaks at around 13° and 20°

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ACCEPTED MANUSCRIPT (2θ) emerged, especially at water contents of 30 and 40%, indicating the formation of

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starch-lipid or starch-protein-lipid complexes in heated samples (Wang et al., 2017d).

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Similar observations have already been reported on heat-moisture treated wheat flour

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(Chen, He, Fu, & Huang, 2015) and sorghum flour extrudates (Jafari, Koocheki, &

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Milani, 2017). A small peak at around 17° (2θ) was observed for heated samples at

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water contents of 60 and 70%, suggesting the retrogradation of starch in heated

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samples. At each water content, heating duration had little effect on crystallinity of

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samples.

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To further illustrate the effect of water content on the crystallinity of heated samples,

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the 2D views of XRD diffraction patterns are shown in Figure 1g, which represents a

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stack of XRD scans with a colored ruler indicating peak intensities from strong to

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weak. As similar patterns were observed for all the heated samples, only the 2D view

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of samples that were heated for 5 min are shown (Figure 1g). With increasing water

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content, the typical A-type crystalline peaks (denoted by arrows in Figure 1g)

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gradually weakened and disappeared at water content of 40%. For raw flour and flour

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that was heated with 20% water, no diffraction peak was observed at 13o (2θ). After

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heating at 30% water, the samples presented a weak peak at 13o (2θ), which became

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progressively stronger with increasing water content. In contrast, the peak at 20° (2θ),

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which was weak for raw flour, became increasingly obvious with increasing water

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content to 40~50%. These observations confirm that the V-type starch-lipid

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complexes were formed during heating of wheat flour.

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3.4 Morphology of starch

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The microstructure of raw and heated samples is shown in Figure S1. The raw wheat

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flour showed the typical large A-type and small B-type starch granules (Figure S1a).

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After heating with 20% water, mostly starch granules were intact, although some

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starch granules seemed to adhere to each other (Figure S1b-d). After heating with

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30% water, swelling and deformation of the starch granules was clearly apparent

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(Figure S1e-g), which increased considerably as the water content increased to

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40-50% (Figure S1h-m). It is interesting to note that while the starch granule

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contours could still be identified, the crystalline structure of starch was hardly

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detected by X-ray diffraction (Figure 1c and 1d). Similarly, complete disruption of

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crystalline structures during gelatinization while the contours of swollen starch

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granules are still evident has been reported in previous studies (Wang & Copeland,

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2012; Wang et al., 2016b). As the water content increased further (60-70%), the starch

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granules were disrupted completely. Increasing heating times from 5 to 10 or 20 min

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seemed to bring about few additional changes in granular morphology of starch in

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cooked flours.

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3.5 Pasting properties

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The pasting profiles of raw and heated samples are given in Figure 2 and the

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corresponding pasting parameters are summarized in Table 3. The samples heated

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with water contents of 20 to 50% exhibited typical viscosity profiles, although there 14

ACCEPTED MANUSCRIPT were gradual decreases in peak, trough, setback and final viscosities and increases in

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pasting temperature compared with the raw flour. While the overall crystallinity was

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completely disrupted after heating at water contents of 40 and 50%, the heated

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samples still developed the typical viscosity pattern, as was similarly observed for

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cooked rice flours (Wang et al., 2017a). Clearly, some ordered structures remain,

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which can swell and develop paste viscosity but are no longer detectable by XRD and

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DSC. Moreover, with water contents of 20 to 50%, the peak, trough, setback and final

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viscosities decreased as length of heating time increased, indicating structural order

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other than crystalline structures continued to be disrupted progressively. In contrast,

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the samples that were heated with 60 or 70% water displayed different pasting

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profiles that indicated the presence of cold water-swelling starch. Similar results were

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also reported for heated rice flours (Chung, Liu, Huang, Yin, & Li, 2010; Wang et al.,

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2017a).

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3.8 In vitro starch digestibility

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Digestograms of starch in raw and heated flour samples are shown in Figure 3a-c and

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the corresponding fit of the data to the equation for first order kinetics are also

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presented. Starch in raw wheat flour was hydrolyzed very slowly, with only about

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20% broken down to glucose after 2 h incubation. Similar digestion patterns were

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observed for the samples heated with 20 to 70% water. After heating for only 5 min

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with water content of 20%, the rate and extent of starch digestibility were increased

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greatly, with a final digestion percentage after 2 h of over 85%. Under these heating

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ACCEPTED MANUSCRIPT conditions, long-range structural order, as measured by XRD and DSC was almost

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unaffected. Further increases in water content or heating time resulted in only minor

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increases in digestion rate. The small differences in extent and rate of starch digestion

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in all heated samples were consistent with our previous findings that structural order

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in gelatinized starch is not the determinant for in vitro starch digestibility (Wang et al.,

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2017c).

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4. Discussion

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In the present study, gelatinization properties of starch in wheat flour during cooking

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with water were monitored by a combination of analytical methods. An advantage of

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using wheat flour as model system to study starch gelatinization rather than isolated

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starch during cooking or simulated food processing is that the effect of other

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components on starch gelatinization can be revealed. Thus, the present study has

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relevance to foods made from doughs (e.g., bakery products, noodles, pasta) and

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batters (e.g., pancakes, coatings for fried foods). The results showed the short- and

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long-range molecular orders of starch in wheat flour were disrupted progressively

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with increasing water content during cooking. The DSC transition temperatures

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increased and enthalpy change decreased with increasing water content. At a water

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content of 40% or higher, the overall loss of structural order in wheat flour indicated

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the starch was fully gelatinized during cooking. Nevertheless, remnant structures in

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the starch were still able to exhibit some swelling and pasting properties in samples

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heated with water content up to 50%. Length of heating duration had much less effect

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ACCEPTED MANUSCRIPT compared to water content on the disruption of long- and short-range molecular orders

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of starch in wheat flour, consistent with previous studies (Sui et al., 2015; Wang et al.,

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2017b). Heating for only 5 min at water content as low as 20% greatly increased the

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in vitro enzymatic digestibility of starch in wheat flour, as observed for isolated starch

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(Wang et al., 2017b). These results confirmed that loss of crystalline structure in

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gelatinized starch is not the rate-determining factor for starch digestion (Wang et al.,

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2017c). Moreover, our results are consistent with the proposal that molecular density

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of amorphous regions (Zhang, Dhital, & Gidley, 2015) or quantity of flexible

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α-glucan chains protruding from the granule surface (Baldwin et al., 2015) are key

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structural features that determine the rate of starch digestion, mostly by influencing

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the access/binding of alpha-amylase to starch. The presence of proteins and other

364

constituents in the flour did not seem to block this access.

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The present study of heated wheat flour-water systems showed that there was no

367

impediment to starch gelatinization or any lesser degree of disruption of starch

368

structures, compared with isolated starch in similar experimental systems (Wang et al.,

369

2017b). For example, the degree of gelatinization after heating with 30% water was

370

72-73% for wheat starch (Wang et al., 2017b) and starch in wheat flour, whereas at a

371

water content of 40% or higher, starch in both systems was almost completely

372

gelatinized. These results indicated that non-starch components, either in a free state

373

or after complexing with starch during heating, do not influence the progression of

374

starch gelatinization in wheat flour. In previous studies, proteins and non-starch

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17

ACCEPTED MANUSCRIPT polysaccharides have been shown to retard the progression of starch gelatinization in

376

model systems by reducing water availability for starch gelatinization or by

377

complexing with starch (Jekle, Mühlberger, & Becker, 2016; Mohamed &

378

Rayas-Duarte, 2003; Tester & Sommerville, 2003). The conclusion drawn from these

379

previous studies was based mainly on the increased gelatinization temperatures in the

380

presence of added proteins and non-starch polysaccharides. Our study provided solid

381

evidences that starch gelatinization in wheat flour was not affected obviously by the

382

presence of other components during cooking.

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A key difference between the flour and isolated starch systems demonstrated by the

385

present study was the formation of starch-lipid or starch-lipid-protein complexes

386

during heating of wheat flour-water mixtures. The clearly evident transformation of

387

the A to A + V, and finally to V type XRD trace, and accompanying DSC analyses,

388

confirmed that starch-lipid or starch-lipid-protein complexes are formed during

389

heating of wheat flour-water mixtures. The formation of amylose-lipid complexes has

390

been studied mostly in model mixtures, but relatively little is known about complex

391

formation during processing of actual food ingredients such as flour (De Pilli, Derossi,

392

Talja, Jouppila, & Severini, 2011; De Pilli et al., 2008). Our study confirmed for the

393

first time by XRD and DSC that starch-lipid complexes are formed during cooking of

394

wheat flour-water mixtures.

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An interesting finding was that duration of heating had small effect on disruption of 18

ACCEPTED MANUSCRIPT crystalline structure of starch, but had considerable effects on pasting properties of

398

starch in cooked wheat flour. Longer duration of cooking resulted in lower peak,

399

trough, setback and final viscosities at water contents of 20~50%. These results

400

indicated that structural order, most probably in amorphous rather than in crystalline

401

regions, was further disrupted during longer duration of heating. We proposed that the

402

short-range molecular order in amorphous starch, which is not detected by XRD,

403

FTIR and Raman spectroscopy, has a key influence on pasting properties of cooked

404

starch.

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5. Conclusions

407

The present study has shown the major difference between heating flour and isolated

408

starch systems was the formation of binary (starch-lipid) or ternary (starch, protein

409

and lipid) complexes during cooking. The multi-scale structural orders of starch

410

during cooking were not greatly affected by the non-starch components in wheat flour

411

which might compete with starch for water. Even after the starch appeared to be fully

412

gelatinized according to XRD and DSC analyses, some remnant structures were still

413

present that exhibited swelling and pasting properties. The degree of short-range

414

molecular order in amorphous starch is therefore proposed to play a key role in

415

determining the pasting properties of cooked starch. The susceptibility of starch to in

416

vitro amylolysis was greatly increased when

417

disruption had occurred. The information obtained from the present study may

418

provide a more useful guide to the processing properties of wheat flour-based food

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only a small amount of structural

ACCEPTED MANUSCRIPT 419

products than might be obtained from studies of starch gelatinization in isolation

420

Acknowledgements

422

The authors gratefully acknowledge the financial support from the National Natural

423

Science Foundation of China (31522043) and Tianjin Natural Science Foundation for

424

Distinguished Young Scholar (17JCJQJC45600).

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treatment reaction conditions on the physicochemical and structural properties

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of maize starch: Moisture and length of heating. Food Chemistry, 173, 1125.

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Tester, R. F., & Sommerville, M. D. (2003). The effects of non-starch polysaccharides

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on the extent of gelatinisation, swelling and α-amylase hydrolysis of maize

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and wheat starches. Food Hydrocolloids, 17(1), 41-54.

Tian, J., Chen, J., Ye, X., & Chen, S. (2016). Health benefits of the potato affected by domestic cooking: A review. Food Chemistry, 202, 165-175. Veraverbeke, W. S., & Delcour, J. A. (2002). Wheat protein composition and 23

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properties of wheat glutenin in relation to breadmaking functionality. Critical

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Reviews in Food Science and Nutrition, 42(3), 179-208.

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Wang, S., & Copeland, L. (2012). Phase transitions of pea starch over a wide range of water content. Journal of Agricultural & Food Chemistry, 60(25), 6439-6446.

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Wang, S., & Copeland, L. (2013). Molecular disassembly of starch granules during

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gelatinization and its effect on starch digestibility: a review. Food & Function,

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4(11), 1564-1580.

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Wang, S., Li, C., Copeland, L., Niu, Q., & Wang, S. (2015). Starch retrogradation: A

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comprehensive review. Comprehensive Reviews in Food Science & Food

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Wang, S., Li, P., Zhang, T., Yu, J., Wang, S., & Copeland, L. (2017a). In vitro starch

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digestibility of rice flour is not affected by method of cooking. LWT- Food

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Science and Technology, 84.

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Wang, S., Sun, Y., Wang, J., Wang, S., & Copeland, L. (2016a). Molecular

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disassembly of rice and lotus starches during thermal processing and its effect

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Wang, S., Wang, S., Guo, P., Liu, L., & Wang, S. (2017b). Multi-scale structural

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changes of wheat and yam starches during cooking and their effect on in vitro

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enzymatic digestibility. Journal of Agricultural & Food Chemistry, 65(1), 156-166.

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Wang, S., Wang, S., Liu, L., Wang, S., & Copeland, L. (2017c). Structural orders of

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wheat starch do not determine the in vitro enzymatic digestibility. Journal of 24

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Agricultural & Food Chemistry, 65(8), 1697-1706. Wang, S., Zhang, X., Wang, S., & Copeland, L. (2016b). Changes of multi-scale

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structure during mimicked DSC heating reveal the nature of starch

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gelatinization. Scientific Reports, 6, 28271.

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Wang, S., Zheng, M., Yu, J., Wang, S., & Copeland, L. (2017d). Insights into the

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formation and structures of starch-protein-lipid complexes. Journal of

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Agricultural & Food Chemistry, 65(9), 1960-1966.

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Willett, W., Manson, J., & Liu, S. (2002). Glycemic index, glycemic load, and risk of

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type 2 diabetes. American Journal of Clinical Nutrition, 76(1), 274. Zhang, B., Dhital, S., & Gidley, M. J. (2015). Densely packed matrices as rate

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determining features in starch hydrolysis. Trends in Food Science &

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Technology, 43(1), 18-31.

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Zhang, B., Zhao, Y., Li, X., Li, L., Xie, F., & Chen, L. (2014). Supramolecular

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structural changes of waxy and high-amylose cornstarches heated in abundant

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water. Food Hydrocolloids, 35(3), 700-709.

545 546

Zhang, G., And, M. D. M., & Hamaker, B. R. (2003). Detection of a novel three

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component complex consisting of starch, protein, and free fatty acids. Journal of Agricultural & Food Chemistry, 51(9), 2801-2805.

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Zhang, G., Maladen, M., Campanella, O. H., & Hamaker, B. R. (2010). Free fatty

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acids electronically bridge the self-assembly of a three-component

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nanocomplex consisting of amylose, protein, and free fatty acids. Journal of

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Agricultural & Food Chemistry, 58(16), 9164-9170. 25

ACCEPTED MANUSCRIPT Zou, W., Sissons, M., Gidley, M. J., Gilbert, R. G., & Warren, F. J. (2015). Combined

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techniques for characterising pasta structure reveals how the gluten network

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slows enzymic digestion rate. Food Chemistry, 188, 559-568.

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ACCEPTED MANUSCRIPT Figure captions

554 555

Figure 1. XRD patterns of raw and heated samples with 20-70% water content (RC,

557

relative crystallinity). a: raw and 20% water content; b: 30% water content; c: 40%

558

water content; d: 50% water content; e: 60% water content; f: 70% water content; g:

559

2D views of XRD diffraction patterns of raw and heated flour samples for 5 min at

560

various water contents.

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Figure 2. Pasting profiles of raw and heated flour samples for 5min (a), 10 min (b)

565

and 20 min (c).

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Figure 3. In vitro starch digestion curves of raw and heated samples with various

568

water contents for 5min (a), 10 min (b), 20 min (c) and with 20%water for 5, 10, 20

569

min (d). Values are means ± SD.

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27

ACCEPTED MANUSCRIPT Figure 1 30000

RC=32.3% 15000

RC=32.7% 10000

RC=32.4% RC=34.4%

5000

20000

RC=19.3% 15000

10

15

20

25

30

35

Diffraction angle (2θ)

5

40

30000

c

Diffraction intensity (counts)

40%-5min 40%-10min 40%-20min

15000

RC=7.3%

10000

RC=7.1%

5000

RC=7.1%

0

25000

10

15

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30

Diffraction angle (2θ)

20000

35

40

50%-5min 50%-10min 50%-20min

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10000

RC=6.6%

5000

0

5

10

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Diffraction angle (2θ)

35

40

60%-5min 60%-10min 60%-20min

17°

20000

RC=7.4%

15000

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30000

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Diffraction intensity (counts)

30000

Diffraction intensity (counts)

RC=17.9%

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10000

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25000

30%-5min 30%-10min 30%-20min

b

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20%-5min 20%-10min 20%-20min Raw

Diffraction intensity (counts)

Diffraction intensity (counts)

30000

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f

70%-5min 70%-10min 70%-20min

17°

20000

RC=8.2% 15000

RC=8.8%

10000

RC=8.6%

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Diffraction angle (2θ)

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ACCEPTED MANUSCRIPT Figure 2

a

80

RI PT

2000

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Temperature (°C)

3000

Viscosity (cP)

100

Raw flour 20%-5min 30%-5min 40%-5min 50%-5min 60%-5min 70%-5min

70

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3000

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4

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Temperature (°C)

0

SC

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10

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Time(min) 100

EP

Viscosity (cP)

AC C

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90

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60

50 0 0

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Temperature (°C)

c

ACCEPTED MANUSCRIPT Figure 3 100

100

b

a 80

40

10min-20% k=0.018± 0.002 10min-30% k=0.022± 0.001 10min-40% k=0.023± 0.001 10min-50% k=0.025± 0.001 10min-60% k=0.024± 0.001 10min-70% k=0.025± 0.001 Raw flour k=0.006± 0.001

60

40

20

20

0

0 0

20

40

60

80

100

120

0

20

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Time(min)

d

c 80

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Starch digested (%)

80

20min-20% k=0.018±0.002 20min-30% k=0.026±0.001 20min-40% k=0.027±0.001 20min-50% k=0.026±0.001 20min-60% k=0.027±0.001 20min-70% k=0.027±0.001 Raw flour k=0.006±0.001

100

120

60

40

20%-5min k=0.016± 0.001 20%-10min k=0.018± 0.002 20%-20min k=0.018± 0.002

20

20

0

0 0

20

40

60

80

100

0

20

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EP

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40

60

Time (min)

100

60

RI PT

5min-20% k=0.016± 0.001 5min-30% k=0.020± 0.001 5min-40% k=0.022± 0.001 5min-50% k=0.023± 0.001 5min-60% k=0.023± 0.001 5min-70% k=0.025± 0.001 Raw flour k=0.006± 0.001

60

Starch digested (%)

Starch digested (%)

80

31

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ACCEPTED MANUSCRIPT Table 1. Gelatinization transition properties of raw and heated samples To (oC)

Water content

Tp (oC)

Tc (oC)

∆H (J/g flour)

5 min 59.2±0.3c'a 60.8±0.2b'c 64.1±0.5a'b N.D. N.D. N.D. N.D.

65.1±0.5c'a 66.8±0.3b'c 71.3±0.4a'c N.D. N.D. N.D. N.D.

71.6±0.3c'a 72.5±0.2b'b 78.4±0.2a'b N.D. N.D. N.D. N.D.

6.3±0.1a'a 5.8±0.3b'a 2.1±0.2c'a N.D. N.D. N.D. N.D.

71.6±0.3c'a 73.4±0.3b'a 78.7±0.9a'b N.D. N.D. N.D. N.D.

6.3±0.1a'a 5.8±0.3b'a 2.0±0.1c'a N.D. N.D. N.D. N.D.

71.6±0.3c'a 74.1±0.7b'a 81.1±0.2a'a N.D. N.D. N.D. N.D.

6.3±0.1a'a 5.5±0.3b'a 1.7±0.1c'b N.D. N.D. N.D. N.D.

RI PT

Raw 20% 30% 40% 50% 60% 70%

59.2±0.3c'a 61.9±0.3b'b 65.7±0.5a'a N.D. N.D. N.D. N.D.

65.1±0.5c'a 67.5±0.3b'b 72.7±0.3a'b N.D. N.D. N.D. N.D.

M AN U

Raw 20% 30% 40% 50% 60% 70%

SC

10 min

20 min

59.2±0.3c'a 62.6±0.1b'a 66.4±0.2a'a N.D. N.D. N.D. N.D.

65.1±0.5c'a 68.3±0.2b'a 76.1±0.1a'a N.D. N.D. N.D. N.D.

TE D

Raw 20% 30% 40% 50% 60% 70%

EP

Values are means ± SD. The lowercase letters a′, b′ and c′ represent significant differences 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).

AC C

To, onset temperature; Tp, peak temperature; Tc, conclusion temperature; ∆H, gelatinization enthalpy. N.D., not detected.

32

ACCEPTED MANUSCRIPT Table 2. Short-range molecular orders of starch in raw and heated samples determined by ATR-FTIR and Raman spectra Length of heating time Water content 5 min

10 min

20 min

IR ratios of absorbances at 1022/995 cm-1

15.5±0.6c'a 15.7±0.6c'a 16.8±0.7bc'a 17.4±0.7ab'a 17.6±1.1ab'a 18.0±0.7a'a 18.2±1.5a'a

RI PT

Raw 20% 30% 40% 50% 60% 70%

1.104±0.016c'a 1.104±0.016d'a 1.217±0.027b'a 1.210±0.043c'a 1.322±0.035a'a 1.269±0.021bc'a 1.347±0.042a'a 1.350±0.01a'a 1.324±0.059a'a 1.324±0.011ab'a 1.328±0.013a'a 1.332±0.056ab'a 1.338±0.034a'a 1.343±0.035a'a FWHM of the band at 480 cm-1 15.5±0.6c'a 16.0±0.4bc'a 16.7±0.8b'a 17.8±0.7a'a 17.9±0.5a'a 18.2±1.1a'a 18.5±0.3a'a

SC

1.104±0.016c'a 1.215±0.027b'a 1.301±0.043a'a 1.358±0.056a'a 1.324±0.059a'a 1.346±0.054a'a 1.314±0.011a'a

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Raw 20% 30% 40% 50% 60% 70%

15.5±0.6c'a 16.0±0.6c'a 16.8±0.7bc'a 17.8±0.8ab'a 18.3±0.9a'a 18.4±1.1a'a 18.5±1.8a'a

Values are means ± SD. The different lowercase letters with apostrophe represent significant differences between the data in the same column (p < 0.05); and the lowercase letters represent

TE D

significant differences between the data in the same row (p < 0.05).

AC C

EP

FWHM: full width at half maximum.

33

ACCEPTED MANUSCRIPT

Table 3 Pasting parameters of raw and heated samples

Raw 20% 30% 40% 50% 60% 70%

2380.5±12.4a'a 1934.5±34.6b'a 1452.0±5.7c'a 999.0±11.3d'a 852.0±1.4f'a 706.0±1.1g'a 933.0±2.3e'b

1567.3±90.0a'a 1493.0±25.5b'a 1264.5±4.9c'a 978.0±7.1d'a 813.0±2.8e'a 700.0±2.8f'a 828.0±3.2e'b

813.3±4.0a'a 441.5±9.2b'a 187.5±10.6c'a 21.0±4.2f'b 39.0±1.4e'a 6.0±2.8g'c 105.0±0.6d'b

Raw 20% 30% 40% 50% 60% 70%

2380.5±12.4a'a 1535.0±4.2b'b 1240.0±12.7c'b 877.5±4.9d'b 559.5±2.1f'b 527.5±0.7g'c 848.5±7.8e'c

1567.3±90.0a'a 1312.5±0.7b'b 1150.5±16.3c'b 870.0±4.2d'b 514.0±7.1f'b 489.5±0.7g'c 739.5±0.7e'c

813.3±4.0a'a 222.5±4.9b'b 89.5±3.5d'b 17.5±0.7f'b 45.5±4.9e'a 38.0±1.9e'a 109.0±8.5c'b

Raw 20% 30% 40% 50% 60% 70%

2380.5±12.4a'a 1374.0±8.5b'c 1136.0±4.2c'c 815.0±1.4e'c 388.0±4.2g'c 554.0±5.7f'b 1126.0±8.5d'a

1567.3±90.0a'a 1222.0±2.8b'c 1060.0±4.2c'c 775.0±2.8e'c 357.0±5.7g'c 541.5±0.7f'b 994.5±0.7d'a

FV (cP)

RI PT

BV (cP)

5 min 3354.5±12.1a'a 3065.0±36.8b'a 2554.5±24.7c'a 1914.5±46.0e'a 1681.5±3.5f'a 1557.5±23.3g'a 1949.5±3.5d'b 10 min 3354.5±12.1a'a 2696.5±13.4b'b 2220.5±10.6c'b 1612.5±9.2e'b 1244.0±2.8f'b 1163.0±8.5g'b 1737.0±2.8d'c 20 min 3354.5±12.1a'a 2516.0±21.2b'c 1970.5±19.1d'c 1368.0±2.8e'c 788.0±4.2g'c 1099.0±8.5f'c 2050.5±12.0c'a

SC

TV (cP)

EP

TE D

M AN U

PV (cP)

AC C

Water content

813.3±4.0a'a 152.0±5.7b'c 76.0±0.1d'c 40.0±1.4e'a 31.0±1.4f'b 12.5±4.9g'b 131.5±9.2c'a

SV (cP)

PT (°C)

1787.5±4.5a'a 1572.0±11.3b'a 1290.0±29.7c'a 936.5±38.9e'a 868.5±6.4f'a 857.5±26.2f'a 1121.5±3.5d'a

70.2±0.2d'a 89.7±0.1c'c 93.3±0.5b'b 95.3±0.1a'a 94.9±0.5a'a 93.3±0.6b'a 90.0±0.6c'a

1787.5±4.5a'a 1384.0±14.1b'b 1070.0±5.7c'b 742.5±13.4e'b 730.0±9.9e'b 673.5±7.8f'b 997.5±2.1d'c

70.2±0.2e'a 91.3±0.1c'b 94.1±0.6ab'b 94.9±0.3a'a 95.2±0.1a'a 93.6±0.1b'a 90.5±0.1d'a

1787.5±4.5a'a 1294.0±24b'c 910.5±23.3d'c 593.0±5.8e'c 431.0±1.4g'c 557.5±9.2f'c 1056.0±11.3c'b

70.2±0.2f'a 92.1±0.1d'a 95.3±0.1a'a 94.9±0.4ab'a 94.7±0.1b'a 93.3±0.5c'a 87.6±0.6e'b

Values are means ± SD. The lowercase letters a′, b′ and c′ represent significant differences between the data for each milled fraction with different water contents (p < 0.05); The lowercase letters a, b and c represent significant differences between the data in the same water content samples (p < 0.05).

PV, peak viscosity; TV, through viscosity; BV, breakdown viscosity; FV, final viscosity; SV, setback viscosity; PT, pasting temperature.

34

ACCEPTED MANUSCRIPT Highlights Starch gelatinization studied by using wheat flour as a model material.

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Starch gelatinization not affected greatly by other components in wheat flour.

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Water played a more important role in starch gelatinization during cooking.

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V-type starch-lipid complex formed during heating of wheat flour.

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Ordered structure in starch proposed to determine viscosities of cooked flour.

AC C

EP

TE D

M AN U

SC

RI PT

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