Effects of Water-Extractable Arabinoxylan on the Physicochemical

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Effects of water-extractable arabinoxylan on physicochemical properties and structure of wheat gluten by thermal treatment Yunping Zhu, Yu Wang, Jinlong Li, Fang li, Chao Teng, and xiuting li J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 17 May 2017 Downloaded from http://pubs.acs.org on May 18, 2017

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Journal of Agricultural and Food Chemistry

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Effects of water-extractable arabinoxylan on physicochemical properties and

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structure of wheat gluten by thermal treatment

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Yunping Zhu1,3, Yu Wang2, Jinlong Li2, Fang Li3,Chao Teng1,3, and Xiuting Li1,2∗

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1. Beijing Advanced Innovation Center for Food Nutrition and Human Health,

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Beijing Technology and Business University (BTBU), Beijing 100048,China

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2. School of Food and Chemical Engineering, Beijing Technology and Business

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University, No.33, Fucheng Road, Beijing 100048,P.R. China

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3. Beijing Engineering and Technology Research Center of Food Additives, Beijing

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Technology and Business University (BTBU), Beijing 100048, China

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∗

Corresponding author: Tel. +86-10-68985378; Fax. +86-10-68985456; E-mail [email protected] or [email protected]

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ACS Paragon Plus Environment


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Abstract

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This study investigated the effects of water-extractable arabinoxylan (WEAX) on

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gluten by thermal treatment. Fourier transform infrared spectroscopy (FTIR) results

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showed that heating significantly decreased β-sheets and β-turn structures in gluten

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proteins between 25°C and 55 °C. The addition of WEAX caused a transition from

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β-sheets to β-turn at above 55 °C. The ratio of weakly hydrogen-bonded β-sheets to

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strongly hydrogen-bonded β-sheets demonstrated an increasing trend, but WEAX can

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hinder this process. FT-Raman results revealed that a hydrophobic environment was

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developed with 5% WEAX at 25 °C, and phenolic hydroxyl on ferulic acid can form

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new H-bonds with the phenyl groups of the non-dissociated TYR residues. A 5%

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WEAX content is helpful for gluten to maintain its original gauche–gauche–gauche

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conformation of disulfide bond upon heating. In addition WEAX can reduce the

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elasticity of gluten and form a soft texture at 25 °C, 55 °C, and 75 °C.

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Key words: Arabinoxylan, thermal treatment, structure, wheat gluten, dynamic

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oscillatory rheology

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2


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Introduction

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Whole wheat, the natural unrefined state of wheat, features a host of important

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nutrients. Arabinoxylan (AX), the main component of the dietary fiber fraction of

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whole wheat, is abundant in bran (43.1%) and germ (15.3%). Large-scale prospective

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studies reported that cereal fibers are associated with a reduced risk of chronic

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diseases,

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gastrointestinal cancers (stomach and liver).2 Water extractable arabinxylan(WEAX)

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are particularly related to the functional effects of anti-inflammatory activity3 and

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reduction of postprandial glucose response. 4

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Fibre is known to influence dough properties and bread quality. WEAX, which

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constitutes 25% of total AX in cell walls of wheat endosperm was found to be

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beneficial in dough properties like water absorption and dough development time.5-6

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However, some authors also found negative effects both on gluten quality and dough

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properties. Dough became less extensible and stronger, gluten particle size

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distribution shifted to a higher value and their tendency to aggregate was lower.7

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Several explanations proposed by previous authors are summarized. WEAX exerts a

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dilution effect on the gluten network, and the high viscosity of WEAX is likely to

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impair attraction between particles7. Regarding the mixing process, the presence of

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ferulic acid esterified to AX polymers may enable AX polymers to oxidize and

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crosslink that may cause a detrimental effect by hindering gluten agglomeration. 8-9 In

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order to obtain whole wheat baking-products with good quality, it is very important to

such

as

cardiovascular

disorders,1

colorectal cancer,

3


and

other


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verify the underlying mechanisms of the interactions between AX and gluten during

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mixing or baking/steam processes. But until now, the mechanisms of the interactions

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are still not clear.

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Gluten endows functions in wheat products by conferring the water-absorption

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capacity, cohesiveness, viscosity, and elasticity of the dough. Gluten is composed of

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two main fractions, namely, insoluble glutenins and soluble gliadins, which are

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classified on the basis of their solubility in aqueous alcohols. Polymeric glutenins, the

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prime contributors to dough properties, confer elasticity. Monomeric gliadins act as

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plasticizers to glutenins.10 The structure of the gluten network is mainly stabilized by

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noncovalent bonds (hydrogen bonds, ionic bonds, and hydrophobic bonds). Although

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this class of chemical bonds is less energetic than covalent bonds, noncovalent bonds

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are proven to be of considerable importance to protein aggregation and dough

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structure.10-11 Covalent bonds, such as disulfide bonds, also play an important role in

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baking. Glutenins can be linked to gliadins at temperature exceeding 90 °C through a

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heat-induced SH–SS exchange reaction, whereas generated free SH-groups can

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further react with either gliadins or glutenins.11-12 However, the influence of AX on

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the changes of the structure of the gluten network by thermal treatment is still unclear.

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Our present research mainly focused on the effects of AX on heat-induced changes of

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wheat gluten. Secondary structures, rheological properties, and morphology of gluten

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proteins are investigated by Fourier transform infrared spectroscopy (FT-IR),

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rheometry, and scanning electron microscopy (SEM). 4


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Materials and methods

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Materials

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Gluten from wheat (Lot number: MKBR8637V) was purchased from Sigma Chemical

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Co. (St. Louis, USA). Moisture and crude protein of gluten from wheat was

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performed in triplicate by using the approved methods of AACC International, 2000

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(Methods 44-19, 46–13), Starch of gluten was measured using total starch kit

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(Megazyme International Ireland Ltd. Lot number: 160422 3) and the results were

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expressed as average values. The moisture, crude protein and total starch presented

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6.34%, 74.96% and 7.51% of dry gluten mass, respectively. WEAX (Lot number:

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120707a) (>94 g arabinoxylan AX/100 g dry matter) was purchased from Megazyme

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International Ireland Ltd. (Bray, Ireland).It was prepared by controlled acid hydrolysis

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of wheat flour AX by Megazyme International Ireland Ltd. (Bray, Ireland). The

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contents of moisture, ash, crude protein, starch and ferulic acid were 2.2 g/100 g, 2.3

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g/100 g, 2.3 g/100 g, 0.2 g/100 g, 0.079g/100g (dry basis, w/w), correspondingly.

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Sample preparation

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Gluten powder was thoroughly blended with 0% and 5% (w/w, per gram of gluten)

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WEAX solution. A 1.0 g gluten sample with 5.0% WEAX solution was mixed

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manually in a 50 mL centrifuge tube, For control sample, 5.0 mL of distilled water

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was added. Excess water was added to determine the effect of WEAX on gluten,

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without water becoming a limiting factor.13,14 Then the samples were transferred in to 5



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glass tubes and were set in an electrical heater. All samples were heated at 55 °C,

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75 °C, and 95 °C for 10 min. Samples were cooled in a water bath (4 °C) immediately

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after heating. Control was also kept in water for 10 min at room temperature (25 °C).

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All samples were freeze dried, and the freeze-dried gluten was ground and stored in a

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Ziploc bag at 4 °C until further use.

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Structure of gluten proteins

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Infrared spectra of gluten samples were recorded using an iS50 FT-IR

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spectrophotometer (Thermo Nicolet Inc., Waltham, MA, USA) equipped with a

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horizontal multi-reflectance diamond accessory. Spectra were collected in the

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4000–400 cm−1 infrared spectral range at room temperature. For each sample, 64

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spectra at 4 cm−1 resolution were averaged, and a minimum of three replicates were

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obtained. A background spectrum without sample trough sampling plate was collected

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before each sample was run. The quantity of secondary structure of protein in the Amide I

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region (1700-1600 cm−1) of gluten was estimated using Omnic software (version 8.0,

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Thermo Nicolet Inc., Waltham, MA, USA). The individual peaks in the amide I region

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severely overlapped and were thus resolved with Fourier self-deconvolution by using

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the method described by others15 with an enhancement factor of 1.3 and a bandwidth

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of 30. Second derivative of Fourier self-deconvoluted spectra was then calculated to

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determine the positions of the absorbance peaks located in the amide I region. The

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intensity of absorbance peaks at 1683 cm−1, 1649 cm−1, 1665 cm−1, 1632 cm−1 and

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1613 cm−1 was manually calculated. Glutamine absorbance peak obtained at 1600 6


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cm−1 was also collected to normalize other five peaks at different wave numbers. The

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positions and assignments of amide bands have been summarized by previous

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authors15, 1683/1649, 1665/1649, 1665/1683, and 1632/1613 were calculated to

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evaluate the relative proportion of each secondary structure.

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The disulfide bridge region (490–550 cm−1), aromatic amino acid environment

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tyrosine doublet (I(850)/I (830)), and tryptophan band (I(760)) were analyzed using

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FT–Raman spectra. Raman spectra were collected on a BrukerMultiRAM FT-Raman

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spectrophotometer (Bruker Optics, Germany) equipped with Nd:YAG laser at 1064

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nm. Spectra were collected at room temperature with a laser power of 251 mW and

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spectral resolution of 4 cm−1. High signal-to-noise ratio was assured by recording

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each spectrum after averaging 256 scans. FT–Raman spectra were plotted as intensity

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(arbitrary units) against Raman shift in wave number (cm−1).The spectra were

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automatically baseline corrected in the whole range(3500–50 cm−1)in OMNIC

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software (version 8.2, Thermo Fischer Scientific Inc., USA).Spectral data from the

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sample scans were manually normalized against the phenylalanine band at 1003 cm−1

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by using Origin.16-17

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Dynamic oscillatory tests

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Dynamic rheological measurements of gluten with WEAX were determined using an

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AR1500ex rheometer (TA Instruments, New Castle, DE, USA) under strain control

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mode as described by Bárcenas et al.13 with some modifications. The measuring 7



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system included a cone and a plate (20 mm diameter, 1 mm gap) to eliminate slippage

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during tests. A 1.0 g gluten sample was added with 5.0 mL of 0%, 5% (w/w, per gram

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of gluten) WEAX solution and centrifuged at 2000 g for 10 min, and supernatant was

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discarded. After centrifugation, samples consisting of hydrated gluten reached the

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optimum water-binding capacity. The gluten dough was placed between the plates

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within 1 h after hydration, and the test was started after 15 min of resting so that

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residual stresses can relax. The rim of each sample was coated with silicon oil to

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prevent evaporation during measurements. Strain sweeps at 1 Hz frequency at 25 °C,

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55 °C, 75 °C, and 95 °C and a strain of 3×10−3 was selected within the linear

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viscoelastic region. A frequency sweep from 0.1 Hz to 10 Hz was performed at

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constant strain. Frequency sweep tests were conducted from 0.1 Hz to 10 Hz at 25 °C

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and then subjected to programmed heating up to 55 °C, 75 °C, and 95 °C at a heating

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rate of 3 °C/min.

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Scanning Electron Microscope (SEM)

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Heated samples were transferred into a 50 mL centrifuge tube and frozen to -80°C

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overnight followed by lyophilized for 24 hours. Samples for SEM were cutting using

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scissor from central region to avoid environmental interference during freeze-drying.

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Sections of central region of samples were pictured. Freeze-dried samples were

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mounted on a silver specimen holder and coated with gold for 120 s. The

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microstructure of each cross-section sample was observed by SEM (HITACHI, Japan)

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at a voltage of 15.0 kV. 8


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Statistical analysis

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The results were statistically analyzed using SPSS (Systat Software Inc., San Jose,

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USA). ANOVA was used to determine significant differences between the results, and

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Duncan’s test was used to compare the means with a significant difference at the level

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of 0.05.

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Results and discussion

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Effect of WEAX on secondary structure of gluten proteins studied by FTIR spectrum

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analysis

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The effect of WEAX addition on the FTIR spectrum of gluten in gluten dough at

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various temperature levels was assessed. The protein-repeating units give rise to nine

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characteristic IR absorption bands, namely, amide A, B, and I−VII. Amide I and

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amide III spectral bands were found to be the most sensitive to the variations in

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protein secondary structure folding among the spectral regions arising out of coupled

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and uncoupled stretching and bending modes of amide bonds.18 The amide I spectral

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region (1700–1600 cm−1), which is mainly caused by the C=O stretch vibrations of

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the peptide linkages, is used to estimate the secondary structure of gluten because of

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its high sensitivity and strong signal. Each type of secondary structure gives rise to a

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relatively different C=O stretching frequency because of the unique molecular

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geometry and hydrogen-bonding pattern.18

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For both glutenins and gliadins, α-helices are present in the N- and C-terminal 9



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domains with β-turns and intermolecular β-sheets in the central repeatitive domain.

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These central repeat domain containing proline and tyrosine residues could form

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hydrophobic interactions. A “loop and train” model has been proposed.19 according to

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this model, unordered structures in the glutenins result from low-moisture conditions,

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which are highly compacted and stabilized by hydrogen bonding. Under a

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intermediate moisture content environment, hydrogen bonding was formed with a

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competition between glutamine side chains and water and lead to the formation of

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“loops” segments which are composed of β-turn structure. “Trains” segments,

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attributed to inter-chain interactions, are formed by β-sheets structure. As water

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content increases, the equilibrium between β-turns and β-sheets shifts toward β-turns.

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As shown in Figs.1A and 1B, the ratios reflect the relative content of β-sheets and

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β-turn that dominantly affect the property of gluten. The value of 1683/1649 and

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1665/1649 means the β-sheets to α-helix + random coil ratio and β-turn to α-helix +

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random coil ratio. The most marked decrease of β-sheets and β-turn, which from 0.48

185

to 0.38 and from 0.58 to 0.47, respectively occurred between 25 °C and 55 °C and

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appeared to be involved in the loss of certain baking functionality,11 this finding

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indicates that heating causes a decrease in the ordered β-sheets and β-turn structures.

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As shown in Fig.1C, the value of 1665/1683 represent the ratio of β-turns to β-sheets

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ratio, increased from 1.15 to 1.40 as the temperature increased from 25 °C to 95 °C,

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indicating a shift of the β-sheets to β-turn structure. This may be ascribed to high

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temperature decreasing the quantity and strength of hydrogen bonding; thus, the 10


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intermolecular β-sheets were no longer stable and are converted to a looser β-turn

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structure. This hypothesis can be further confirmed by measuring the strength of

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hydrogen bonding, and the result is shown in Fig.1D. 1632/1613 refers to more

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weakly hydrogen-bonded β-sheets to strongly hydrogen-bonded β-sheets. Although

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intra-molecular β-sheets and intermolecular β-sheets can also cause absorption band at

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1632cm−1 and 1613cm−1, respectively, under heat treatment, the intermolecular

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β-sheets are the least probable to transform into an intra-molecular β-sheets.17 Thus,

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these two bands are mainly attributed to hydrogen bonding. The increasing value of

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1632/1613 indicates that the strongly hydrogen-bonded β-sheets transforms into a

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weakly hydrogen-bonded β-sheets. This transformation also indicates less interaction

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left under high temperature.

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When WEAX is taken into consideration, the properties of gluten proteins are mainly

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determined by the amount and balance of chain entanglements, hydrogen bonds,

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hydrophobic interactions, electrostatic forces, covalent bonds, and disulfide bonds in

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the dough system.20 Among these interactions between AX and gluten, the formation

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of hydrogen bonds evidently plays an important role. As shown in Figs.1A and 1B,

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5%WEAX alleviates the decrease in the ordered β-sheets and β-turn structures. The

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decrease of sample with WEAX at 25 °C might attributed to the highly branched

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backbone of WEAX show steric hindrance preventing gluten forming loose β-turn

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structure. Lower ratio can be observed of control sample at 55 °C might caused by

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hydrophobic interaction which lead a compact structure of gluten molecular chains.21 11



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Compact structure shows low absorbance. However, this dynamic process could also

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be hindered with steric effect of WEAX, thus the structure might not be compact as

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control which show a higher absorbance.

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As presented in Fig.1C, 5% WEAX is beneficial for gluten to keep its original β-turn

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to β-sheets ratio by enhancing the function of hydrogen bonding and retaining a

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compact structure. Wellner et al.22 reported that the elasticity of wheat gluten is

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attributed to the combination of “trains”, which is associated with β-sheets structures

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and “loops” identified with the formation of β-turn structures. Lower loop-to-train

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ratio translates to higher resistance of gluten to extension. As demonstrated by

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Nawrocka23 both hydrogen bonding and disulfide bonds are crucial factors for gluten

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properties; in addition, the intermolecular disulfide bonds associated with β-sheets act

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synergistically, thereby stabilizing the gluten polymers. Thus, we can conclude that

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WEAX can keep the resistance of gluten and stabilize the gluten structure during

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thermal treatment.24 In Fig.1D, we can observe a significant difference in the ratio of

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weakly hydrogen-bonded β-sheets to strongly hydrogen-bonded β-sheets between

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control gluten dough and WEAX-gluten from 25 °C to 75 °C which means β-sheets

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structure can be stabled with WEAX might due to highly branched WEAX show

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steric hindrance preventing gluten forming loose β- turn structure. When heating to

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95 °C, effect of WEAX on gluten didn’t show significant different might because that

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high temperature increased mobility of WEAX molecular. Flexible WEAX molecular

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couldn’t hinder structural changes of gluten. This result directly confirms that WEAX 12


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is beneficial to maintain the β-sheets structure by keeping the strength of the hydrogen

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bond until 75 °C.

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Changes in aromatic amino acids environment

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Various Raman bands resulted from vibrations of indole ring presented in tryptophan

238

residues in repetitive region of gluten, could provide valuable information on the

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tryptophan environment. A decrease intensity of this band indicates TRP residues are

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in a hydrophobic microenvironment, whereas an increasing value indicate that

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tryptophan is involved in the H-bonding of a hydrophilic environment. Changing in

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peak intensity at 760 cm−1 is a good indicator of microenvironment of tryptophan

243

residues in proteins by which modifications in tertiary structure of proteins can be

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detected. TRP residues occur periodically throughout the length of gluten proteins and

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are often found in repeats of pairs of TRP residues. 3% to 5% TRP residues are often

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be found in glutenin subunits. Although disulfide bonds, as the main covalent bonds

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responsible for formation of gluten network during dough mixing, are identified as

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relevant with functional properties, TRP bonds (H-bonds) also participate in this

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process. The addition of WEAX causes strong changes in the normalized intensity of

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the 760 cm−1 band. Figure 2A shows that heating does not cause significant changes in

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the intensity of this band, although heating can break some hydrogen bonding;

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however, this process did not involve tryptophan much, and the microenvironment of

253

tryptophan remained stable. At 25 °C, an increase in this band was observed, when

254

WEAX was added, indicating that a hydrophilic environment was developed with 5% 13



255

WEAX. WEAX is known as hydrophilic polysaccharide which contains lots of

256

hydroxyl groups, these polar groups force hydrophobic regions in gluten aggregate in

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order to reduce surface tension. Similar results were observed in the locust bean

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gum–gluten system.25 as well as the chokeberry-gluten and cacao–gluten systems

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which result in a more ordered structure according to the authors.17 However, this

260

increase has yet to be observed at a higher temperature, indicating that this effect of

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WEAX on tryptophan is weakened by heating.

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Hydrogen bonding of the phenolic hydroxyl group of TYR residues can be detected

263

by determining the ratio of the tyrosyl doublet at 850 and 830 cm−1 (Fig. 2B). The

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tyrosyl doublet ratio is proposed to determine whether the TYR residue is exposed or

265

buried;23 when the band at 850 cm−1 is more pronounced than the band near 830 cm−1,

266

the TYR residue is exposed to act as a positive charge acceptor, resulting in changes

267

in the tertiary structure of proteins. Meanwhile, it can be interpreted that, if the

268

intensity at 850 cm-1 is lower than the intensity at 830 cm-1, the tyrosine residues are

269

buried within the protein network involving in intermolecular or intramolecular

270

interactions. A significant decrease of this value was observed between 55 °C and

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75 °C, suggesting a possible involvement of TYR residues in intermolecular or

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intra-molecular interactions under high temperature. WEAX produced significant

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changes in gluten upon heating. A decrease relative to the control was observed at

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25 °C and 55 °C, indicating that WEAX causes more buriedness and suggesting the

275

possible involvement of TYR residues in intermolecular or intra-molecular 14


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interactions.17 In addition, the decrease in ratio value suggests the formation of new

277

hydrogen bonds; in comparison, the increase in ratio value may indicate that TYR

278

residues acted as positive charge acceptors, favoring local charge repulsion between

279

protein molecules.26 This behavior is related to the chemical structure of fiber, and the

280

residues are connected with oscillations of the phenolic hydroxyl on ferulic acid. The

281

phenolic hydroxyl in the fiber can be exposed to form new H-bonds with the phenyl

282

groups of non-dissociated TYR residues. At 75 °C, an increase was observed

283

compared with control gluten, suggesting that 5% WEAX protects hydrogen bonding

284

to a certain degree but finally loses its efficacy at 95 °C.

285

Changes in disulphide bridges (S–S) conformation

286

Disulfide bridges (S–S) critically maintain the tertiary structure of proteins. Cysteine

287

residues, located on the C- and N-termini, which only account for 2% of total amino

288

acid form disulfide bonds that are important for structure and functionality of gluten.

289

Most cysteines are present in an oxidized state and form either intra-chain disulfide

290

bonds within a protein or the inter-chain disulfide bonds between proteins.

291

Conformational changes can be evaluated by determining the disulfide stretching

292

vibration (490–550 cm−1) because the disulfide bridges are crucial for proteins to

293

maintain a particular tertiary structure. The symmetrical stretching vibration of the S-S

294

bond is influenced by the conformation of C-atoms in the disulfide bridge. The

295

disulfide S–S stretch is visible in the 490–550 cm−1 region, thus providing an approach

296

to study structural changes for the disulfide band. Depending on the different 15



297

conformations of the C–S–S–C atoms, the 500–550 cm−1range has been assigned to

298

gauche–gauche–gauche conformation (510 cm−1), gauche–gauche–trans (525 cm−1),

299

and trans–gauche–trans (540 cm–1).16 In the control spectra(Fig. 2C), the S–S band is

300

prominent and is located at approximately 510 cm−1, indicating that these bonds are

301

primarily in the gauche–gauche–gauche, which is a stable conformation. An increase of

302

the 540 cm−1 band can be observed at 75 °C and 95 °C. This increase in the number of

303

trans−gauche−gauche and trans−gauche−trans S−S bonds might contributed to the

304

displacement of polypeptide chain that appeared in abnormal protein folding and

305

subunits aggregation which might be caused by thermal treatment.16 WEAX altered

306

the conformation of S−S bonds from gauche–gauche–gauche transformation into less

307

stable conformations (trans–gauche–gauche and trans–gauche-trans) leads to a less

308

stable protein complex. With 5% WEAX, the band at 540 cm−1 at 75 °C and 95 °C

309

disappeared, suggesting that WEAX is helpful for gluten to maintain its original

310

gauche–gauche–gauche conformation (510 cm−1) and keep the protein stable. This

311

phenomenon might attribute to hydrophilic AX molecules absorb more water in

312

microenvironment, which help gluten network develop more adequately. According to

313

Zhou et al.,16 S-S bridge tends to adopt a more stable conformation (g-g-g) at a higher

314

water absorption level.

315

Dynamic oscillatory tests

316

The oscillatory frequency sweep measurements of all samples were conducted in

317

linear viscoelastic range. The dynamic shear storage modulus (G’) is a measure of the 16


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energy recovered per cycle of deformation and represents a solid or elastic character

319

of the material. The loss modulus (G’’) is an estimate of the energy dissipated as heat

320

per cycle of deformation, which can be regarded as an indicator of the viscous

321

properties of the material.27 The loss tangent (tan δ = G’’/G’) is a factor that indicates

322

elastic and viscous properties. For example, rheological characteristics with changes

323

in G’ and G’’ for each sample during holding (25 °C) and heating (at 55 °C, 75 °C, and

324

95 °C) are presented in Fig. 3A.

325

Generally, samples with or without WEAX presented elastic, solid-like behavior

326

because the elastic modulus (G’) far exceeds the viscous modulus (G’’) throughout

327

the frequency range at all temperature levels. Heating generally causes a decrease in

328

G’ and G’’, and a significant decrease was observed in both G’ and G’’ between 25 °C

329

and 55°C; Similar results can be observed from other research,28 which reported a

330

decrease in G’ and tan δ upon increase of the temperature from 20 °C and 55 °C.

331

According to Tsiami et al.,29 gluten proteins are associated with disulfide bonds,

332

hydrogen bonding, and other non-covalent interactions. At 55 °C, the shifting of

333

disulfide bonds is extremely minor; hence, shifting is not a main factor causing the

334

phenomenon. Therefore, non-covalent associations seem to be related to the degree of

335

cross-linking depending on temperature, which results in a decrease in modulus with

336

increasing temperature. From 75 °C to 95°C, G’’ decreases considerably, indicating

337

that gliadins, whose fraction in gluten mainly causes viscosity interaction with

338

glutenins involved in the mechanism of disulfide bonds shifting. With 5% WEAX, G’ 17



339

and G’’ at all temperature levels decrease. Theoretically, WEAX can reduce the

340

elasticity of gluten, and two mechanisms seem to be plausible. First, WEAX can

341

interact with gluten by non-covalent cross-links (hydrogen bonding and hydrophobic

342

interaction),30 whereby the gluten exhibits lower extensibility and slower rate of

343

aggregation. Another explanation can be related to the good water-holding capacity of

344

WEAX, which markedly limits gluten hydration and thus interferes with the ability of

345

gluten to form a network. As shown in Fig.4, the tan δ of both control and

346

WEAX–gluten dough decreases with increasing temperature, implying that the heating

347

process generally leading a more elastic gluten system. With regard to the influence of

348

WEAX, samples with WEAX demonstrated a biphasic effect on heat-induced changes,

349

that is, a substantial decrease on tan δ occurred at 25 °C and 55 °C, followed by a

350

increase at 95 °C compared with the control dough. Glutenins are associated with

351

elastic behavior and started to be denatured at about 55 °C, and gliadins confer

352

viscosity at the thermal denaturation temperature of approximately 90 °C.11-12 The

353

results above suggested that WEAX mainly interferes with the formation of glutenins

354

linkage at about 55 °C but acted predominantly on the gliadins fraction at about

355

95 °C.

356

The mechanical spectra were evaluated using the linear variation fitting of the

357

logarithmic plot of G’ versus frequency (Table 1). The slope data provide information

358

on frequency dependence, whereas intercept values correspond to the magnitude of

359

the elastic modulus.31 In theory, the slope is assumed to be 0 in the case of a perfectly 18


Page 18 of 34

Page 19 of 34


360

cross-linked network, whereas the increasing values of slopes demonstrate that the

361

sample contains increasing fractions of uncross-linked materials.32 With increasing

362

temperature, the slope of all gluten dough samples showed a downward trend,

363

indicating an increase in molecular correlation that may be ascribed to protein

364

cross-linking and aggregation.33

365

The current results indicated that the value of slope decreases when 5% WEAX is

366

present at 25 °C, 55 °C, and 75 °C; these conditions may indicate a elastic texture

367

based on the observation of Khatkar et al.;34 the results may be attributed to the good

368

water-holding capacity of WEAX, which kept the gluten dough moist at relatively

369

low temperature levels. When the gluten dough was heated at higher temperature

370

(95 °C), an increase in slope was observed, indicating the lower resistance to

371

deformation, that is, WEAX made gluten dough softer.35 This experimental outcome

372

can be ascribed to the weakened intermolecular interaction of WEAX at higher

373

heating temperature, possibly forming a softer protein–polysaccharide complex.

374

Microstructure of hydrated wheat gluten samples

375

SEM was performed to evaluate the effects of AX on the hydrated wheat gluten

376

network. Fig.5 shows the images of the protein network. The control wheat gluten

377

displayed a spongy structure with regular uniform pores until reaching 95 °C; at this

378

temperature, irregular pores were observed, and the frame of the network became

379

weaker. This finding may have resulted from gluten protein denaturation. 19



380

The 5% WEAX content resulted in damage to this spongy, polyporous structure, and a

381

schistose, weak frame is formed. This result might be ascribed to the negative effect

382

of WEAX on gluten protein; thus, gluten experiences difficulty to form a regular

383

network. According to rheology study with 5% WEAX, G’ showed decrease at all

384

temperature levels, indicating that gluten possesses lower extensibility and a lower

385

rate of aggregation with WEAX. Larger pores are also present in samples with

386

WEAX, indicating a high accessibility of water to the amorphous regions of the

387

protein/polysaccharide complex. The good water-holding capacity of WEAX

388

molecules results in more water in the protein/polysaccharide complex; after

389

lyophilization, large pores were left, which resulted from ice crystal formation.

390

Acknowledgements

391

This research was financially supported by the Program for the National Natural

392

Science Foundation of China (No.31571872, No. 31501487), The Development of

393

Innovative Teams and Teachers’ Occupation Advancement Project of Beijing

394

Municipal Universities and Colleges (No. IDHT20130506).

395

20


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396

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Figure captions

Fig. 1 Effect of WEAX on the secondary structure content of gluten: A (1683/1649),B(1665/1649), C (1665/1683),and D(1632/1613). Fig.2 Effect of WEAX on the tertiary structure content of gluten: A (760), B (850/830), and C (490–550).

Fig.3 Mechanical spectrum for gluten–WEAX dough heating at different temperature levels: A (25 °C), B (55 °C), C (75 °C), and D (95 °C).

Fig.4 Curves of tan δ for gluten–WEAX dough heating to different temperature. Fig.5 Scanning electron micrographs of heated gluten without WEAX at different temperature are presented as A (25 °C), C (55 °C), E (75 °C), and G (95 °C); Scanning electron micrographs of heated gluten with 5% WEAX at different temperature are presented as B (25 °C), D (55 °C), F (75 °C), and H (95 °C).

27



Page 28 of 34

Table 1 Slope value and coordination number obtained from the regression lines of elastic modulus versus the frequency for gluten dough in the presence of varying concentrations of WEAX

25°C

WEAX

55°C

level (%)

Intercept

Slope

R2

Intercept

Slope

R2

0.0

3.45

0.2392

0.9955

3.18

0.1808

0.9834

5.0

3.38

0.2339

0.9954

3.09

0.1696

0.9785

75°C

WEAX

95°C

level (%)

Intercept

Slope

R2

Intercept

Slope

R2

0.0

3.15

0.1304

0.9692

3.25

0.1149

0.9907

5.0

3.05

0.1223

0.9563

3.10

0.1179

0.9861

Slope of logG' againstlog frequency.

28


Page 29 of 34


Figure 1

29



Figure 2

30


Page 30 of 34

Page 31 of 34


Figure3

31



Figure 4

32


Page 32 of 34

Page 33 of 34


Figure 5

33



Graphic for manuscript

34


Page 34 of 34

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