Gluten-starch interactions in wheat gluten during carboxylic acid deamidation upon hydrothermal treatment

Food Chemistry 283 (2019) 111–122

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Gluten-starch interactions in wheat gluten during carboxylic acid deamidation upon hydrothermal treatment

T

⁎

Lan Liaoa,b, , Feng-li Zhangb, Wei-Jie Linb, Zhang-fa Lib, Jing-yi Yangb, Kwan Hwa Parkb,d, Li Nib, Peng Liuc a

Department of Food Science, Foshan University, Foshan, Guangdong 528000, People’s Republic of China College of Biological Science and Technology, Fuzhou University, Fuzhou, Fujian 350108, People’s Republic of China c School of Chemistry and Chemical Engineering, Guangzhou University, Guangzhou 510000, People’s Republic of China d Center for Agricultural Biomaterials, Department of Food Science & Technology, College of Agriculture and Life Science, Seoul National University, San 56-1, Shillimdong, Kwanak-gu, Seoul 151-921, Republic of Korea b

A R T I C LE I N FO

A B S T R A C T

Keywords: Wheat gluten Starch Deamidation Insoluble precipitate Conformation

After carboxylic acid deamidation upon heating (CADH), wheat gluten still contains a total of ∼10% insoluble fractions, of which ∼10% is starch, which depreciate the values of wheat gluten. To elucidate gluten-starch interactions and their role in the deamidation behavior of gluten, the macrostructural characteristics of gluten citric acid suspensions of different concentrations (1% and 10%, w/v) and with different types of residual starch chains (achieved by enzyme hydrolyzed by α-amylase and/or glucoamylase assisted by sonication) were investigated. We found the degradation of long starch chains and branched short chains induced dramatic bondcleavages in insoluble glutenins and gliadins. FTIR and SDS-PAGE analyses indicated that without these two types of chains in the precipitates, the insoluble deamidated wheat gluten exhibited minimal changes in the molecular force and the conformation. Their glycosylation, hydrophobic force and hydrogen bonds between amylopectin and small proteins, such as LMW-GS and α, β, γ-gliadins, were detected. FTIR suggested that the associations between gliadins and amylopectin were covalent. Gluten-starch interactions were likely to cause an incomplete dissolution of wheat gluten during CADH. A simple model was proposed to clarify the aggregation state and the relationships between starch granules and wheat gluten components during CADH.

1. Introduction

2016), nutrition, function (Lei, Zhao, Selomulya, & Xiong, 2015; Qiu, Sun, Zhao, Cui, & Zhao, 2013), and celiac-related characteristics (Gourbeyre et al., 2012; Malalgoda & Simsek, 2017) of proteins. However, even after the deamidation of wheat gluten, about a total of 10% insoluble fractions still remain, among which 10% is starch (Roels, Grobet, & Delcour, 1998). These residues may depreciate the additional values of wheat gluten. In this work, we focus on the gluten-starch interactions and their influence on the deamidation behavior of gluten. Wheat gluten is a non-pure protein system, composed of not only proteins, but also lipids (∼3.5–6.8%), minerals (∼0.5–0.9%), and starch or non-starch polysaccharides (NSP) (∼7.0–16.0%) (Roels et al., 1998). Moreover, wheat protein is more complex in conformation than other natural plant proteins (Day, Augustin, Batey, & Wrigley, 2006). Wheat gluten is subdivided into gliadins (monomeric proteins; Mw: 30–80 kDa) and glutenins (polymeric proteins; Mw: 30–200 kDa) according to their abilities to form polymeric species by inter/

Wheat gluten, the byproduct protein of wheat starch, has attracted increasing attention as an alternative source of protein to replace expensive animal proteins owing to its unique characteristics and low cost (O’Brien & Wang, 2008). However, the utilization of wheat gluten is limited in food processing status quo, because of its low solubility and unsatisfactory functional properties, which is due to its large content of non-polar amino groups (Gln & Asn) and super high molecular weight of glutenins (Singh & MacRitchie, 2001a). The carboxylic acid deamidation is a promising method to improve the functional properties of wheat gluten by converting the amide groups to carboxyl groups (Glu & Asp, respectively). Deamidation dissociates protein polymers by increasing the electrostatic repulsion among protein molecular chains and increases the surface hydrophobicity (Liao, Zhao et al., 2010), which can enhance the molecular flexibility (Jekle, Mühlberger, & Becker,

⁎ Corresponding authors at: Department of Food Science, Foshan University, Foshan, Guangdong 528000, People’s Republic of China (L. Liao). School of Chemistry and Chemical Engineering, Guangzhou University, Guangzhou, 510006, People’s Republic of China (P. Liu). E-mail addresses: [email protected] (L. Liao), [email protected] (P. Liu).

https://doi.org/10.1016/j.foodchem.2019.01.019 Received 22 September 2018; Received in revised form 10 December 2018; Accepted 3 January 2019 Available online 14 January 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.

Food Chemistry 283 (2019) 111–122

L. Liao et al.

Fig. 1. Process diagram of the preparation of insoluble deamidated parts (A) and the profiles of the reducing sugar contents in supernatants controlled by amylolytic enzymes hydrolysis of the insoluble deamidated parts (B).

by α-amylase and/or glucoamylase assisted by sonication (Singh & MacRitchie, 2001a). Specifically, α-amylase is an endoglycosidase acting at random on 1,4-glucosidic linkages between glucosides along the starch chain, and ultimately yielding soluble maltotriose and maltose, or insoluble “limit dextrin”, namely the short branched starch chains centered around 1,6-glucosidic linkages (van der Maarel, van der Veen, Uitdehaag, Leemhuis, & Dijkhuizen, 2002). Glucoamylase decomposes starch by 1,4-glucosidic linkages, 1,6-glucosidic linkages and 1,3-glucosidic linkages, and can hydrolyze starch chains into soluble glucose totally (Saulnier, Andersson, & Åman, 1997). Based on these, the correspondent specific associations between long starch or branched short chains and wheat gluten molecules could be used to indirectly elucidate the interactions between gluten and starch molecules during carboxylic acid deamidation upon heating. Moreover, since the insoluble fraction of deamidated wheat gluten account for up to ∼10% of the acting protein, a further aim was to explore the reasons for the incomplete dissolution of wheat gluten after carboxylic acid deamidation.

intramolecular disulfide bonds (Dahesh, Banc, Duri, Morel, & Ramos, 2014). The gluten-starch interactions are important during heating because they induce vital changes in the fabricated cereal-based products (Jekle et al., 2016). During heating, starch becomes gelatinization and gluten undergoes denaturation. The interactions between gluten and native/ gelatinized starch in dough and bakery foods have been widely studied (Wang et al., 2017). However, there have been limited reports on the deamidation by carboxylic acids in wheat gluten upon hydrothermal treatment. In a previous study (Aranyi & Hawrylewicz, 1972), in commercially vital wheat gluten samples that were treated by a combined HCl/acetic acid method, starch and lipid were insoluble in the digestion mixture and did not affect deamidation and hydrolysis. Starch could be eliminated as a sediment and lipid accumulated at the top of the centrifugal tubes (Aranyi & Hawrylewicz, 1972). On the other hand, starch-protein interactions existed irreversibly, which might be due to the attraction between positively and negatively charged colloids in acidic (acetic acid) environments (Mohamed & Rayas-Duarte, 2003). Notwithstanding the above, from the conformational point of view, the specific bondages of residual starch molecules with hydrophobic wheat gluten proteins and the stereo-hindrance effect thereof referring competing hydration should be the main interactions during carboxylic acid deamidation upon heating, because residual starch molecules is stubborn and cannot be removed by the strong mechanical damage during washing (Baldwin, 2001). As for these specific bondages, it is still unknown whether their possible associations are covalent. If so, taking into the stereo-hindrance effect referring competing hydration, to what extent the starch molecules can influence the agglomeration or the dissociation behaviour of gluten polymers, especially in acid deamidation upon hydrothermal treatment, is worth to be investigated. Against this background, a set of experiments were designed to clarify the solubility and conformation characteristics of the deamidated protein precipitates after step-by-step removal of residual starch

2. Materials and methods 2.1. Materials Strong wheat cultivar Zhoumai 28 (Triticum aestivum L.) was purchased from farmers directly, and ground into powder by a mechanical mill (Zhoukou, Henan, China). Wheat gluten powder (84.54 ± 0.73% protein (N × 5.7%), dry basis) was prepared by handwashing the dough ball with distilled water until the washing water was entirely clear, the removal of lipids with chloroform, and then freeze-drying and grinding with a coffee grinder. α-Amylase (food-grade enzyme, 1 × 104 U/mL, un-thermostable, the best working temperature range of 50−60 °C) and glucoamylase (food-grade enzyme, 1 × 105 U/mL) were obtained from Novozymes (Beijing, China). All other chemicals and 112

Food Chemistry 283 (2019) 111–122

L. Liao et al.

for 6 h (O’Brien & Wang, 2008). The precipitates after hydrolysis by the two enzymes were collected and freeze-dried for further use (abbreviated as IWG-L for α-amylase and IWG-S for glucoamylase & α-amylase). The ultrasonic treatment of different compositions of insoluble parts after citric acid deamidation was conducted according to the reported method of Singh and MacRitchie (2001b). Specifically, 1IWG, 1IGlu, 1IGli, 10IWG, 10IGlu and 10IGli (0.05 g) were soaked in 50 mL of phosphate buffer (pH 7.0) for 30 min in a conical flask, respectively. The ultrasonic treatment parameters were 120 w (ultrasonic power), 10 min and 44 °C. Aliquots were centrifuged at 10,000×g for 10 min at 4 °C and the precipitates were collected and freeze-dried for further use (abbreviated, respectively, as U-1IWG, U-1IGlu, U-1IGli, U-10IWG, U10IGlu, U-10IGli, U-IWG-L and U-IWG-S). The combined treatment was carried out with the sequence of ultrasonic treatment before enzymes treatment. The consequent sediment was collected and freeze-dried respectively (abbreviated, respectively, as UA-1IWG, UA-1IGlu, UA1IGli, UA-10IWG, UA-10IGlu, UA-10IGli, U-IWG-L, U-IWG-S).

solvents, of analytical grade or HPLC-grade, were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China) and Sigma Chemical Co. Ltd. (St. Louis, USA), respectively. 2.2. Experimental 2.2.1. Deamidation of wheat gluten and preparation of insoluble parts The preparation of acid-deamidated wheat gluten with citric acid under hydrothermal treatment is shown in Fig. 1A (blue arrows) (Liao, Liu, et al., 2010). Briefly, wheat gluten (1% and 10%, w/v) was mixed with a citric acid solution to form suspensions. The suspensions were hydrated in a shaking water bath at ambient temperature for 6 h. Then, the suspensions were deposited in an autoclave at around 95 °C, and the temperature was increased to 121 °C within 3 min. Deamidation was carried out upon hydrothermal treatment at 121 °C for 10 min in the autoclave, followed by fast cooling within 3 min from 121 °C to 95 °C by releasing the artificially stream. The deamidated suspensions were then moved into an ice-bath to inactivate deamidation. After centrifugation at 10, 000×g for 10 min at 4 °C, the precipitates were pooled and freeze-dried (The freeze-dried precipitates are abbreviated as 1IWG and 10IWG, respectively).

2.2.4. Reducing sugar content determination The reducing sugars in the supernatants after ultrasonic treatment and/or enzymes treatment were quantified by the DNS method (Miller, 1959) with a UV/vis spectrophotometer (Onlab Instrument Co., Ltd. Shanghai, China) at a wavelength of 540 nm. It was used to the qualitative determination for starch content left in the precipitates.

2.2.2. Isolation of glutenins and gliadins Glutenins and gliadins were extracted according to a reported method (Wong et al., 2012) with slight modification. 1IWG and 10IWG (50 g each) were mixed with two quantities (500 mL each) of 70% (v/v) aqueous ethanol. The suspensions were homogenized using an Ultra Turrax (BRT Co. Ltd., Shanghai, China) homogenizer (1 min) and were subject to continuous rotating agitation (60 min) at room temperature three times until the supernatant after centrifugation at 10,000×g, 4 °C for 10 min was clear. Then, the precipitates were collected and freezedried (abbreviated as 1IGlu and 10IGlu, respectively). The combined supernatants (gliadins) were submitted to 1.5 M ammonium chloride solution to precipitate the proteins overnight at 4 °C. Following centrifugation at 10,000×g for 45 min at 4 °C, the precipitates were washed with deionized water twice and then freeze-dried for further use (abbreviated as 1IGli and 10IGli, respectively). All the supernatants were collected to measure the amounts of reducing sugars.

2.2.5. Protein nitrogen amount determination The protein nitrogen contents of samples were determined by the Kjeldahl method with a protein conversion factor of 5.7 (Liao, Liu, et al., 2010). 2.2.6. SDS-PAGE experiments Freeze-dried samples were dissolved in distilled water at 2 mg/mL. 2 mL of each sample was mixed with SDS and β-mercaptoethanol, then denatured in boiling water for 10 min and centrifuged at 10,000×g for 3 min. 10 μL of the denatured sample was loaded onto a homogeneous phastgel (DYCZ-30, Beijing Liuyi Instrument Factory, PRC) and tested using 10% acrylamide separating gel and 5% acrylamide stacking gel (Laemmli, 1970). Samples were prepared in Tris-glycine buffer (pH 8.8) containing 1.5% SDS. The gel sheets were stained with Coomassie brilliant blue R-250 for 1 h at 30 °C and de-stained appropriately with 10% acetic acid and 10% methanol in deionized water. The molecular weight standards were as follows: 116 (β-galactosidase), 66.2 (bovine serum albumin), 45 (ovalbumin), 35 (lactate dehydrogenase), 25 (REase Bsp981), 18.4 (β-lactoglobulin), 14.4 (Lysozyme) kDa.

2.2.3. Removal of starch by α-amylase and/or glucoamylase assisted by sonication Amylase hydrolysis, sonication treatment, and the combined treatments on the residual starch in IWGs, IGlis and IGlus after deamidation were conducted by the protocol as demonstrated in Fig. 1A. We firstly used α-amylase to remove long starch chains in insoluble fractions to discuss their interactions with wheat gluten components after carboxylic acid deamidation (Fig. 1A, blue arrows). Afterwards, both α-amylase and glucoamylase were applied to completely hydrolyze starch chains to glucose (Fig. 1A, red arrows) (O’Brien & Wang, 2008). For α-amylase hydrolysis, the dried 1IWG, 1IGlu, 1Gli, 10IWG, 10IGlu and 10IGli (0.05 g) were soaked in 50 mL of phosphate buffer (pH 7.0) for 30 min in a conical flask and 2.0 mL of α-amylase solution (0.5% w/v) was added (Lanthong, Nuisin, & Kiatkamjornwong, 2006). Aliquots were incubated and shaken at a rotary water bath shaker (SY2230, Crystal Technology & Industries, Inc., US) at 55 °C for 1 h, then heated at 100 °C in a boiling water bath for 10 min to completely inactivate α-amylase. Aliquots were centrifuged at 10, 000×g for 10 min at 4 °C. The precipitates were collected and freeze-dried for further use (abbreviated, respectively, as A-1IWG, A-1IGlu, A-1IGli, A-10IWG, A10IGlu and A-10IGli). Afterwards, as illustrated in Fig. 1B, the residual starch from 10IWG was hydrolyzed initially by α-amylase under the same conditions above, except that the incubating time was increased to 6 h to eliminate long starch chains. Then, glucoamylase (2.0 × 104 U/mL) was applied to completely hydrolyze short branched starch chains at pH 4.0, 40 °C

2.2.7. Molecular force profiles The solubility of different insoluble samples in different reducing solvents was used to evaluate the changes in molecular forces of proteins according to Roussel and Cheftel (1990). The six reducing solvents included 0.05 M phosphate buffered solution (PBS), pH 4.44 (S1); 0.05 M PBS, pH 4.44 containing 1% (w/v) sodium dodecyl sulfate (SDS) (S2); 0.05 M PBS, pH 4.44 containing 6 M urea (S3); 0.05 M PBS, pH 4.44 containing 2% (v/v) β-mercaptoethanol (S4); 0.05 M PBS, pH 4.44 containing 1% (w/v) SDS and 2% (v/v) β-mercaptoethanol (S5); 0.05 M PBS, pH 4.44 containing 1% (w/v) SDS and 6 M urea (S6). 2.2.8. Fourier transform infrared (FTIR) spectroscopy The freeze-dried samples (2 mg) were mixed with KBr (200 mg) and ground into fine powder in an agate mortar incubated with infrared light, which was then pressed into a slice. Fourier transform infrared spectra were recorded using a Nicolet 380FTIR spectrometer (Thermo Nicolet Corporation, USA) from 400 to 4000 cm−1 wavenumbers with a resolution of 2 cm−1 for 128 scans. The overlapping amide I band (1600–1700 cm−1) components were further interpreted by deconvolution using Peak-Fit v4.12 software (Peak-Fit v4.12, Systat Software, 113

Food Chemistry 283 (2019) 111–122

L. Liao et al.

conformation was evaluated by FTIR. The tertiary conformation is evaluated by tryptophan fluorescence emission spectroscopy.

Inc. Chicago, USA). 2.2.9. Intrinsic fluorescence emission spectroscopy The intrinsic emission fluorescence spectra of samples were obtained by an F-7000 fluorescence spectrophotometer (Hitachi Co., Hitachinake, Japan) (Qiu et al., 2013). Specifically, protein solutions (0.04 mg/mL) were prepared in 10 mM phosphate buffer (pH 4.4). The fluorescence spectra of samples were all recorded in the front-face configuration using an excitation wavelength of 295 nm, recorded from 300 to 400 nm at a constant slit of 5 nm and a scanning speed of 240 nm/min.

3.2.1. SDS-PAGE As shown in Fig. 2A, for the native samples before treatment (C group), although 1% (w/v) and 10% (w/v) deamidated insoluble fractions varied significantly (p < 0.05) in the degree of hydrolysis (data not shown), they had similar electrophoretic patterns under all conditions. It is evident that the subunit bands of ω-gliadins were not observed in all precipitates, which is in accordance with our previous report because the ω-gliadins were proteins that were poor in disulfide bonds (Liao et al., 2010; Shewry, 1997). The molecular weight of 1IWG (lane 1), 1IGlu (lane 2), 10IWG (lane 4) and 10IGlu (lane 5) were from 25 to 105 kDa. 1IGli (lane 3) and 10IGli (lane 6) were between 35 and 50 kDa. After ultrasonic treatment (Fig. 2A, U group), the intensity of subunit bands of U-1IWG (lane 7) and U-1IGlu (lane 8) decreased slightly in the high molecular weight region (71–105 kDa) of each column, and increased in the low molecular weight region (25–50 kDa). This indicates that sonication caused the scission of large glutenins (Singh & MacRitchie, 2001a). In addition, after sonication, the intensity of subunit bands from 25 to 50 kDa of gliadins ((1IGli (lane 9) and 10IGli (lane 12), Fig. 2A, U group) exhibited a notable increase, in comparison to the subunit bands of gliadins before treatment ((1IGli (lane 3) and 10IGli (lane 6) (Fig. 2A, C group). For A group and U + A group as shown in Fig. 2A, after α-amylase hydrolysis and the combined treatment, all the subunit bands disappeared at 71–105 kDa of HMW-GS for IWGs and IGlus (A-1IWG (line 13), A-1IGlu (line 14), A-10IWG (lane 16), A-10IGlu (lane 17), UA1IWG (lane 19), UA-1IGlu (lane 20), UA-10IWG (lane 22) and UA10IGlu (lane 23)). This demonstrates that long starch chains had strong associations with the subunit of glutenins, which was in agreement with a previous report by Saulnier et al. (1997), who stated that starch binds glutenins. Moreover, the subunit bands of IWGs and IGlus exhibited a significant weakening from 25 to 50 kDa (Fig. 2A U + A groups), which shows that they were much more susceptible to the combinational treatment than to the α-amylase or ultrasonic treatment alone. This result indicates that ultrasonic treatment benefited for the unfolding of glutenins. Additionally, for all IGlis, the intensity of their subunit bands from 25 to 50 kDa markedly increased with the increased extent of treatment. In particular, new bands of IGlis at 71–105 kDa were notably formed after enzyme hydrolysis. These might result from the self-aggregation of gliadins or sheared glutenins due to the removal of long starch chains (Liao, Liu, et al., 2010).

2.2.10. Statistical analysis All the tests were carried out in triplicate. Analysis of variance and significant difference tests were conducted to identify differences among means by LSD’s multiple-range test using SPSS software (version 13.0 for Windows, SPSS Inc., Chicago, IL). The significance level (p) was set at 0.05. 3. Results and discussion 3.1. Influence of protein concentration on gluten-starch interactions after deamidation When hydrating with water, wheat gluten molecules experience crosslinking and form networks spontaneously (Lagrain, Goderis, Brijs, & Delcour, 2010). The protein concentration has a significant influence on the extent of this aggregation and the exposure extent of amides in wheat gluten polymer networks. It also determines the competition between the acid deamidation of amide groups and the hydrolysis of peptide chains (Liao et al., 2016). Hence, the variations in protein concentrations affect the relationship between wheat protein components and starch molecules in the insoluble part of wheat gluten. Taking this into account, we prepared wheat gluten suspension of two concentrations, 1% (w/v) and 10% (w/v), which did not show significant (p < 0.05) difference in the degree of deamidation but significantly (p < 0.05) varied degrees of hydrolysis. The protein nitrogen content and the fractioned components of insoluble wheat gluten after deamidation were analyzed and shown in Table 1 (1% of wheat gluten concentration (1IWG), with a 1.530 ± 0.052 degree of deamidation and a 9.703% ± 0.071 degree of hydrolysis; 10% of wheat gluten concentration (10IWG), with a 1.265 ± 0.005 degree of deamidation and a 1.996% ± 0.007 degree of hydrolysis. data not shown). As shown, the protein nitrogen amounts of 1IWG, 1IGlu and 1IGli were not significantly (p < 0.05) different from those of 10IWG, 10IGlu and 10IGli, respectively. However, 10IWG and 10IGlu contained lower contents of starch than 1IWG and 1IGlu respectively. One reason for this could be the concentration of critic acid for deamidation (2.86 × 10−2 M for 10IWG versus 2.86 × 10−3 M for 1IWG), namely the higher concentration of acid caused a higher hydrolysis degree of starch. Moreover, the residual starch was predominantly left in 1IGlu and 10IGlu respectively, which was in agreement with previous results (Saulnier et al., 1997).

3.2.2. Molecular force change After the combined treatment by hydrolysis and sonication, six reagents with specific chemical actions on proteins (described in Section 2.2.7) were used to investigate the types of aggregation forces of 1% (w/v) and 10% (w/v) insoluble deamidated parts and their fractions. Non-covalent bonds (hydrophobic force, electrostatic force and hydrogen bonds) and covalent bonds (disulphide bonds and peptide bonds) are the main interactions of proteins (Lagrain et al., 2010). In general, as shown for IWGs (Fig. 2B-1 & 4), the curves of samples after α-amylase hydrolysis and ultrasonic treatment located on the top two, then followed by the samples with or without ultrasonic treatment. Expectedly, the extractable protein content after α-amylase and ultrasonic treatments (Fig. 2B-2 & 5) was the lowest. These results demonstrate that the stereospecific blockade of long starch chains, which was initially entrapped by the glutenins protein matrix, influenced the aggregation of proteins. Specifically, the long starch chains had a large free volume (Chen et al., 2009) and could increase the distance of protein molecular chains. With the hydrolysis of long starch chains, this stereospecific blockade vanished, leading to a notably sheared conformation of glutenins and the interruption of non-covalent and covalent bonds of glutenins. This resulted in the lowest extractable protein content of insoluble deamidated glutenins. This speculation is

3.2. Removal of long starch chains by α-amylase During the deamidation of wheat gluten upon heating, the accompanied starch experienced gelatinization and acid hydrolysis. The residual starch in the precipitates after centrifugation at 10,000×g for 10 min at 4 °C were insoluble amylose molecules (existed as long chains in a single helical structure), insoluble amylopectin molecules, blocklets, and granular residues (Chen et al., 2009). To assess the interactions between fractioned components and residual starch, the insoluble aggregates were treated by sonication and α-amylase hydrolysis. The cross-linkages was analyzed by SDS-PAGE (Fig. 2A). The molecular force was analyzed by six denaturing reagents (Fig. 2B). The secondary 114

115

84.0 ± 0.4a – – –

83.1 ± 0.6a – – –

90.1 ± 0.8b – – –

6.52 ± 0.05b – – –

7.70 ± 0.033b – – –

1.58 ± 0.13a – – –

10IWG U-10IWG A-10IWG UA-10IWG

10IGlu U-10IGlu A-10IGlu UA-10IGlu

10IGli U-10IGli A-10IGli UA-10IGli

348.3 350.1 335.7 336.3

338.3 345.9 339.1 334.4

349.0 350.5 337.6 346.2

Apex (nm)

348.8 349.9 335.2 335.7 345.5 335.2 335.9 331.2 348.6 349.0 330.5 333.4

Apex (nm)

714.5 559.7 378.6 406.1

389.5 575.4 527.3 413.1

707.0 414.1 523.3 712.9

Height (a.u)

406.2 429.7 421.5 378.6 370.4 359.8 308.5 280.8 407.8 499.9 514.1 473.2

1602.6 1604.6 1602.6 1604.6 1602.6 1602.6 1602.6 1604.6 1606.5 1604.6 1606.5 1602.6

Frequency(cm−1)

1604.6 1604.6 1606.5 1604.6

1602.6 1606.5 1604.6 1604.5

1604.6 1604.6 1606.5 1606.5

Frequency(cm−1)

12.7c 10.8b 13.7c 10.6b

8.77a 10.2b 7.81a 8.30a

11.9bc 9.60ab 11.1bc 9.80b

Contenta (%)

8.62bc 9.60b 5.80a 9.90 8.25bc 7.30b 5.80a 8.50bc 15.40d 9.30b 10.50c 7.80b

Contenta (%)

Intermolecular β-sheet aggregation extended

Intermolecular β-sheet aggregation extended

Height (a.u)

28.2a 29.0ab 28.2a 26.4a 30.1b 28.0a 28.2a 24.3a 32.4b 24.3a 27.9a 24.4a

1631.6 1635.4 1635.4 1635.4

1627.7 1635.4 1631.6 1629.6

1633.5 1635.4 1629.6 1629.6

Frequency (cm−1)

28.3b 31.2bc 32.5c 31.5bc

29.9b 32.4c 30.5bc 24.5a

31.6bc 32.2c 29.0b 22.3a

Contenta (%)

1654.7 1650.8 1652.8 1658.6 1652.8 1654.7 1652.8 1658.6 1652.8 1658.6 1654.7 1658.6

1652.8 1652.8 1652.8 1652.8

1652.8 1652.8 1654.7 1660.5

1652.8 1652.8 1650.8 1660.5

Frequency(cm−1)

b,c

37.1b 36.2b 39.5c 37.0b 37.1b 37.6b 39.5c 38.0bc 33.8a 37.8b 36.1b 37.8b

34.8a 34.4a 34.1a 34.7a

36.5a 35.1a 36.9ab 38.4b

34.6a 35.2a 36.2a 37.3ab

1679.8 1679.8 1679.8 1679.8 1679.8 1679.8 1679.8 1679.8 1679.8 1679.8 1679.8 1679.8

Frequency (cm−1)

β-Turn

1679.8 1679.8 1679.8 1679.8

1679.8 1679.8 1679.8 1679.8

1679.8 1679.8 1679.8 1679.8

Frequency(cm−1)

β-Turn

Contenta (%)

Contenta (%)

Frequency(cm−1)

α-Helix

α-helix

Contenta (%)

Intramolecular aggregation extended β-sheet (hydrated)

1631.5 1629.6 1627.7 1629.6 1627.7 1631.6 1627.7 1629.6 1635.4 1629.6 1633.5 1629.6

Frequency (cm−1)

Intramolecular aggregation extended βsheet (hydrated)

The percentage of secondary structure content is estimated as corresponding area as a percentage of total amide I band area. The character ‘–’ indicates no determination. The data with different letters in the same column is significantly different (p < 0.05).

a

Protein nitrogen amount (%)

82.4 ± 0.5a –c –c –c 81.5 ± 0.6a –c –c –c 89.6 ± 0.8b –c –c –c

Content of starch (%)

9.50 ± 0.066b –b –b –b 10.40 ± 0.099b –b –b –b 0.93 ± 0.066a –b –b –b

1IWG U-1IWG A-1IWG UA-1IWG 1IGlu U-1IGlu A-1IGlu UA-1IGlu 1IGli U-1IGli A-1IGli UA-1IGli

Protein nitrogen amount (%)

Samples

Content of starch (%)

Samples

22.0ab 21.1a 18.0a 21.3ab

22.6ab 20.5a 22.7ab 26.7b

20.0a 21.1a 21.8a 28.4c

Contenta (%)

23.7b 23.0b 24.1b 24.5b 22.3b 24.7b 24.1b 27.0c 16.8a 26.2c 23.3b 27.2c

Contenta (%)

1695.2 1695.2 1693.2 1695.2

1695.2 1695.2 1693.2 1695.2

1695.2 1695.2 1693.2 1695.2

Frequency(cm−1)

Extended β-sheet

1695.2 1695.2 1693.2 1695.2 1695.2 1695.2 1693.2 1695.2 1695.2 1695.2 1693.2 1695.2

Frequency(cm−1)

Extended β-sheet

2.20c 2.50d 1.70a 1.90a

2.23b 1.80a 2.09ab 2.10b

1.90a 1.90a 1.90a 2.20b

Contenta (%)

2.38b 2.20b 2.40b 2.20b 2.25b 2.40b 2.40b 2.20b 1.60a 2.40b 2.20b 2.80c

Contenta (%)

0.81ab 0.77a 0.71a 0.79a

0.89ab 0.79a 0.91b 1.10c

0.76a 0.81ab 0.86ab 1.09c

αHelix/ βsheet

0.95c 0.89b 1.09c 0.96c 0.91ab 1.00c 1.09c 1.08c 0.68a 1.05c 0.89b 1.08c

αHelix/ β-sheet

Table 1 The content of the starch contents, the protein nitrogen amounts, the secondary structure compositions by FTIR spectra and the endogenous fluorescence scanning of the insoluble deamidated samples by partly hydrolysis of long starch chains by α-amylase with or without sonication.

L. Liao et al.

Food Chemistry 283 (2019) 111–122

Food Chemistry 283 (2019) 111–122

L. Liao et al.

Fig. 2. SDS-PAGE band patterns (A, C) and molecular forces change profiles (B, D) of different insoluble parts of the deamidated wheat proteins. A, SDS-PAGE band patterns of insoluble parts of deamidated wheat gluten and compositions thereof. The lane groups of insoluble wheat gluten compositions, from left to right, are the control (without any further treatments) (C), with ultrasonic treatment (U), with α-amylase hydrolysis of starch long chains (A) and with the combined treatments of ultrasonic treatment and α-amylase hydrolysis of starch (U + A). The lanes of the group C from left to right were MW standards, 1IWG (lane 1), 1IGlu (lane 2), 1IGli (lane 3), 10IWG (lane 4), 10IGlu (lane 5) and 10IGli (lane 6). The lanes of the group U from left to right are U-1IWG (lane 7), U-1IGlu (lane 8), U-1IGli (lane 9), U10IWG (lane 10), U-10IGlu (lane 11) and U-10IGli (lane 12). The lanes of the group A from left to right are A-1IWG (lane 13), A-1IGlu (lane 14), A-1IGli (lane 15), A10IWG (lane 16), A-10IGlu (lane 17) and A-10IGli (lane 18). The lanes of the group U + A from left to right are UA-1IWG (lane 19), UA-1IGlu (lane 20), UA-1IGli (lane 21), UA-10IWG (lane 22), UA-10IGlu (lane 23) and UA-10IGli (lane 24). B, molecular forces change profiles of the aggregates in insoluble parts (1IWG (B-1), 1IGlu (B-2), 1IGli (B-3), 10IWG (B-4), 10IGlu (B-5) and10IGli (B-6)) by reducing reagents. (S1), 0.05 M PBS, pH 4.44; (S2), 0.05 M PBS, pH 4.44 + 1% (w/v) SDS; (S3), 0.05 M PBS, pH 4.44 + 6 M Urea; (S4), 0.05 M PBS, pH 4.44 + 2% (v/v) ME; (S5), 0.05 M PBS, pH 4.44 + 1% (w/v) SDS + 2% (v/v) ME; (S6), 0.05 M PBS, pH 4.44 + 1% (w/v) SDS + 6 M Urea. U-1IWG, U-1IGlu, U-1IGli, U-10IWG, U-10IGlu and U-10IGli are samples obtained from 1IWG, 1IGlu, 1IGlu, 10IWG, 10IGlu and 10IGli treated by ultrasonic treatment, respectively; A-1IWG, A-1IGlu, A-1IGli, A-10IWG, A-10IGlu and A-10IGli are samples obtained from 1IWG, 1IGlu, 1IGlu, 10IWG, 10IGlu and 10IGli treated by amylase hydrolysis of starch, respectively; UA-1IWG, UA-1IGlu, UA-1IGli, UA-10IWG, UA-10IGlu and UA-10IGli are samples obtained from 1IWG, 1IGlu, 1IGlu, 10IWG, 10IGlu and 10IGli treated by amylase hydrolysis of starch combined with ultrasonic treatment, respectively. C, SDS-PAGE band patterns of insoluble deamidated wheat gluten after sequential α-amylase and glucoamylase hydrolyzation of starch with/without the assist of sonication. Lane 1–3 are U-IWG, U-IWG-L and U-IWG-S, respectively. Lane 4–6 are IWG, IWG-L and IWG-S, respectively. The criteria for the marks on the right are seven protein groups (from top to bottom): higher molecular glutenin subunits (HMW-GS), glucoamylase, ω-gliadin (> 60 kDa), α-amylase, lower molecular glutenin subunits (LMW-GS) (group-B), LMW-GS (group-C) & α, β, γ-gliadins, and trypsin inhibitors. Here, glucoamylase and α-amylase are highlighted with the blue and red lines respectively in the picture. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

of A-1IGlu and UA-1IGlu generally increased slightly in S4 and S6 and decreased in S3 and S5 in comparison with 1IGlu. These results illustrate that disulfide bonds and hydrophobic force were the aggregation force for A-1IGlu. As shown in Fig. 2B-3, S3 and S6, 1IGli, U-1IGli, A1IGli and UA-1IGli were more effective than the other four reducing solvents. This indicates that hydrogen bonds and hydrophobic interactions played the major role in maintaining 1IGli, U-1IGli, A-1IGli and UA-1IGli. In particular, the observed extractable content in S4 was the lowest, illustrating that disulfide bonds made little contribution to insoluble gliadins. For the 10% deamidated insoluble parts (10IWG), as exhibited in Fig. 2B-4, the extractable protein content of UA-10IWG was higher than those of U-10IWG, A-10IWG and 10IWG. After α-amylase treatment, the extractable amount of A-10IWG in S3 and S6 was higher than those in U-10IWG and 10IWG and close to that in UA-10IWG. This demonstrates that more hydrophobic force and hydrogen bonds were formed after the removal of long starch chains. For A-10IGlu in S2 and S6 in Fig. 2B-5, the observed extract amounts were higher than those in other reducing solvents, illustrating that hydrophobic force and hydrogen bonds were the main force for A-10IGlu aggregates, not disulfide bonds like A-1IGlu.

consistent with the decreasing intensity of subunit bands of insoluble deamidated glutenins with the increased extent of treatments in the Fig. 2A (lane 5,11,17 and 23). On the other hand, compared with 1Gli and 10Gli (Fig. 2B-3&6), UA-1Gli and UA-10Gli experienced a slightly increased inhibition of the molecular force of insoluble deamidated gliadins. In particular, UA-10IGli had a higher solubility than U-10IGli and A-10IGli, similar to the curves of 10IWG, which were subjected to ultrasonic treatment and α-amylase treatment alone, respectively. This suggests that there was a stronger synergistic effect between ultrasonic and α-amylase treatments to inhibit the dissociation of insoluble gliadins. Specifically, as shown in Fig. 2B-1, after α-amylase and ultrasonic treatment, the protein solubility of UA-1IWG aggregates substantially increased in the presence of SDS and urea (S6) and urea alone (S3). This suggests that the non-covalent inter-molecular interactions (hydrogen bonds and/or hydrophobic interactions) were dominant in UA-1IWG aggregates. Additionally, the solubility of 1IWG, U-1IWG, A-1IWG and UA-1IWG aggregates increased in the presence of SDS and β-mercaptoethanol (S5), compared with those in the presence of β-mercaptoethanol (S4). This further implies the reinforcement of hydrophobic force on the aggregated network. As for insoluble glutenins, Fig. 2B-2 demonstrates that 1IGlu proteins solubilized much easier in the SDS-ME system than in the 2-ME, SDS, urea or sodium phosphate systems alone. Therefore, the decrease in protein solubility of 1IGlu might be caused by the combined effect of intermolecular disulfide bonds and hydrophobic interactions of glutenins during deamidation, but not by the hydrogen bonds. After sonication, comparing with the performance of 1IGlu, the extractable protein content of U-1IGlu increased in S2 and clearly decreased in S5 and S6. This verifies that the hydrophobic force was the main force to maintain U-1IGlu. After α-amylase hydrolysis with or without ultrasonic treatment, the extractable protein contents

3.2.3. FTIR spectroscopy To understand the unfolding and aggregation behaviour of deamidated insoluble parts, Fourier-transform infrared (FTIR) spectroscopy was used to investigate differences in their secondary structures (Zhao et al., 2012). The secondary structural compositions of 1% (w/v) and 10% (w/v) deamidated wheat gluten insoluble parts were shown in Table 1 and Fig. 1 in Supplementary Materials. To emphasize the main spectral features, the spectra were Fourier-deconvoluted by a computational technique. The Fourier-deconvolution reveals five bands at 116

Food Chemistry 283 (2019) 111–122

L. Liao et al.

Fig. 2. (continued)

approximately 1605, 1632, 1652, 1680 and 1695 cm−1. The 1632 cm−1 feature is the characteristic of aggregations with an extended β-sheet structure (Surewicz & Mantsch, 1988), while the lowfrequency amide I band at about 1605 cm−1 has been associated with

the β-sheet conformation. The prominent peak at 1652 cm−1 is assigned to the α-helical conformation, although the spectral contribution from the unordered conformation cannot be completely ruled out at this frequency (Surewicz & Mantsch, 1988). The 1680 cm−1 band is 117

Food Chemistry 283 (2019) 111–122

L. Liao et al.

the extended β-sheet structure and the transformation from β-sheets to α-helices. It indicates that the content of intramolecular hydrogen bonds in the α-helices increased and more compact conformations were achieved by the removal of long starch chains, which is in consistence with structural changes of wheat gluten during extrusion according to Mitchell and Areas (1992). 3.2.4. Tryptophan fluorescence emission spectra Table 1 and Fig. 2 in Supplementary Materials display the intrinsic fluorescence emission spectra of 1% (w/v) and 10% (w/v) deamidated insoluble parts. The intrinsic fluorescence spectrum is determined largely by the change in polarity of the environment of tryptophan (Try) residues and provides a sensitive means to monitor the tertiary conformation changes of protein in solution and at the interface (Pallarès, Vendrell, Avilés, & Ventura, 2004). The polarity of the environment surrounding tryptophan residues affects the fluorescence emission maximum (λmax) (Lakowicz, 2013). The λmax values of the samples (1% (w/v) and 10% (w/v), except 1IGlu) after sonication shifted towards longer wavelengths. After α-amylase hydrolysis of long starch chains with or without ultrasonic treatment, the λmax of all samples significantly shifted towards lower wavelengths. This implies that the tryptophan residue of U-1IWG was exposed to a more hydrophilic environment due to the partial unfolding of the protein upon ultrasonic treatment. For example, the λmax of 1IWG in solution was around 349 nm, and U-1IWG had a λmax at around 350 nm, a small shift towards longer wavelengths. Additionally, there was a shift in λmax towards lower wavelengths of 335.2 nm for A-1IWG and 335.7 nm for UA-1IWG compared with a λmax of 348.8 nm for 1IWG in solution. This λmax blue-shift indicates that Try residues were in a more hydrophobic environment, which is agreed with the results from molecular force changes (Fig. 2B). In general, interestingly, there was significant inhibition of tertiary and secondary conformation for insoluble wheat gluten and their gliadins factions after the removal of long starch chains. For starch in gluten, former researchers stated that the hydrophobic proteins located on the starch granule surface (Singh & MacRitchie, 2001a) so that residual insoluble starch banded well with insoluble deamidated glutenins (Baldwin, 2001). This was also the case here. As for the residual starch in insoluble deamidated wheat gluten in this study, intramolecular hydrogen bonds in α-helices increased and more compact conformations for insoluble wheat gluten and their gliadins factions appeared after the removal of long starch chains. Taking into account the stereo-hindrance effect thereof referring competing hydration of residual starch and insoluble deamidated wheat gluten molecules, it was in fact a revelation to us that the gluten starch chains interactions may play a more important role for the incomplete dissolution of wheat gluten after carboxylic acid deamidation and that the specific relationships of glutenins/gliadins with starch chains may be underestimated.

Fig. 2. (continued)

Fig. 2. (continued)

assigned to the β-turn conformation, and the 1695 cm−1 band is assigned to the extended β-sheet (Liao et al., 2010) (Fig. 1 in Supplementary Material). Table 1 shows a relatively equivalent β-sheet (∼45%) and α-helix (∼37%) containing all insoluble proteins, followed by a higher content of β-turn (up to 23%) in comparison with the soluble deamidated wheat gluten we reported previously (Liao, Qiu, et al., 2010). Among β-sheets, up to ∼35% (34–39%) of the 10% (w/v) deamidated wheat gluten insoluble parts were intra-molecular β-sheets, which was significantly higher than the content of the 1% (w/v) parts (24–32%). The molecular flexibility of samples (except for 10% (w/v) deamidated wheat gluten insoluble parts), which was reflected by the ratio of α-helix to β-sheet, increased generally with the treatment. These observations suggest a general decrease in the molecular flexibility of samples after α-amylase treatment with or without ultrasonic treatment, which is in correspondence with the SDS-PAGE (Fig. 2A) and molecular force change (Fig. 2B) results of insoluble gliadins. Furthermore, α-amylase hydrolysis of long starch chains with or without ultrasonic treatment led to a decreased percentage of aggregations with

3.3. Complete removal of starch by α-amylase and glucoamylase Here, α-amylase and glucoamylase were used to completely hydrolyze starch chains step-by-step to further investigate to what extent starch can influence the agglomeration or dissociation behavior of the gluten polymers during carboxylic acid deamidation. As shown in Fig. 2C, it can be seen that the high molecular weight protein fractions (HWG), ω-gliadin (> 60 kDa), LMW-GS and α, β, γgliadins of insoluble deamidated wheat gluten were more susceptible to be progressively degraded into smaller fragments (less than 25 kDa) with the removal of long starch chains (lane 1 & 2 and lane 4 & 5). When starch branched short chains were completely removed (lane 3 and lane 6), the band patterns of IWG-S and U-IWG-S exhibited a notable disappearance in comparison with the subunit bands of LMW-GS, and α, β, γ-gliadins of samples. This demonstrates that starch branched short chains had a strong relationship with LMW-GS and α, β, γ118

Food Chemistry 283 (2019) 111–122

L. Liao et al.

Table 2 The content of the starch contents, the secondary structure compositions by FTIR spectra and the endogenous fluorescence scanning of the insoluble deamidated samples by the complete hydrolyzation of long starch chains with α-amylase and short branched starch chains with glucoamylase. Samples

Content of starch (%)

Apex (nm)

Height (a.u)

Intermolecular β-sheet aggregation extended

Intramolecular aggregation extended βsheet (hydrated)

α-Helix

β-Turn

α-Helix/ β-sheet

Frequency (cm−1)

Contenta (%)

Frequency (cm−1)

Contenta (%)

Frequency (cm−1)

Contenta (%)

Frequency(cm−1)

Contenta (%)

IWG IWG-L IWG-S

100.0f 56.36 ± 1.23c 6.58 ± 0.23b

349.7 345.2 337.8

113.8 550.8 743.5

1604.6 1602.6 1602.6

11.01b 8.140a 11.12b

1635.4 1627.7 1627.7

33.90b 28.98a 30.71a

1652.8 1652.8 1652.8

36.50a 40.58b 39.32b

1679.8 1689.8 1677.8

18.58a 22.30b 18.84a

0.8127a 1.0932b 0.9400b

U-IWG U-IWG-L U-IWG-S

100.0f 53.74 ± 1.06c 7.660 ± 0.45b

338.2 350.1 338.2

387.5 671.9 743.2

1604.6 1602.6 1604.6

12.18b 7.830a 11.06b

1635.4 1627.7 1635.4

33.96b 30.08a 35.22c

1652.8 1652.8 1652.8

36.33a 40.01b 36.23a

1679.8 1689.8 1679.8

17.52a 22.08b 17.49a

0.7874a 1.0554b 0.7828a

a

The percentage of secondary structure content is estimated as corresponding area as a percentage of total amide I band area. The character ‘–’ indicates no determination. The data with different letters in the same column is significantly different (p < 0.05). b,c

around glycosidic bonds (Synytsya et al., 2009). Combined with the results of Fig. 2C & D above, Fig. 3 indicates that the interactions of short-branched starch chains and gliadins were α-pyran-glycosidic bonds. Wang et al. (2017) reported the formation of new disulphide bonds and non-covalent interactions as well as Maillard reaction products during twin-screw extrusion treatment played a primary role in interactions between starch and proteins. It could be speculated that the formation of α-pyran-glycosidic bonds was due to the Maillard reaction of insoluble short-branched starch chains and insoluble deamidated gliadins. From the results above, it could be concluded that gluten-starchchain interactions in insoluble deamidated wheat gluten were undervalued, and they played an important role for the incomplete dissolution of wheat gluten after carboxylic acid deamidation. In particular, the interactions between starch chains and insoluble deamidated gliadins were ignored for the modification of insoluble wheat gluten, the extent of which might be higher than that between starch chains and insoluble deamidated glutenins. 3.4. Simplified model As reported previously, most gliadins, which are hydrophobic, are present as monomers. While the glutenin fraction comprises aggregations by disulfide bonds and forms a network, in which starch granules are embedded (Kieffer, Schurer, Köhler, & Wieser, 2007). After citric acid deamidation, according to the results from Section 3.1, both long and short-branched starch chains showed clear and specific interactions with glutenins and gliadins altogether in insoluble deamidated wheat gluten residues by SDS-PAGE, FTIR and molecule force evaluations. In particular, for amylopectin and small proteins such as LMW-GS and α, β, γ-gliadins, their glycosylation, hydrophobic force and hydrogen bonds were underestimated and played an important role in glutenstarch interactions and the incomplete dissolution of wheat gluten. To support the above explanations, we speculated that starch granules, which were covered by water-insoluble gliadins by substantial strong bonding (glycosidic bonds, hydrophobic interactions and hydrogen bonds), were entrapped in the interior of the three-dimensional network of glutenins by direct interactions (glycosidic bonds, disulfide bonds, hydrogen bonds and hydrophobic interactions). Moreover, Roels et al. (1998) reported that gluten proteins agglomerated despite the presence of NSPs. In cooperation with NSPs (which covalently bonded with glutenins), starch granules (amylose and amylopectin), gliadins and glutenins, with specific associations with each other, formed an extremely compact conformation that inhibited the further hydration of wheat gluten protein molecules during deamidation. From this point of view, and combined with earlier studies (Shewry, Halford, Belton, & Tatham, 2002), as well as the quaternary and tertiary conformational

Fig. 3. FTIR spectroscopy of native wheat gliadin (CK) and deamidated wheat gliadin (DWG). The red vertical line displays the wavenumber at 852 nm of glycosidic bonds between gliadins and the branched short starch chains. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

gliadins. Fig. 2D displays the molecular force changes of the insoluble aggregates of deamidated wheat gluten by the combination of α-amylase and glucoamylase. Table 2 exhibits their secondary structure compositions by FTIR spectra and endogenous fluorescence scanning results. With the complete removal of long starch chains, the hydrophobic force and hydrogen bonds of aggregates were enhanced (including the samples after sonication). Surprisingly, U-IWG-L, which did not have long starch chains, exhibited the highest values of the molecular force, α-helix content, β-turn content, ratio of α-helix/β-sheet and λmax blue-shift extent. In contrast, the lowest values were observed for U-IWG-S, of which both long starch chains and branched short chains were all eliminated. This strongly indicates that the interactions of short-branched starch chains and small proteins such as LMW-GS and α, β, γ-gliadins were abundant at the gluten-starch interface, as indicated by SDS-PAGE results in Fig. 2C. To verify whether their possible associations between insoluble gliadins and starch molecules are covalently linked, Fig. 3 displays the FTIR spectra of native wheat gliadin (CK) and deamidated wheat gliadin (DWG). The bands at ∼860 cm−1 (863 cm−1 for DWG and 858 cm−1 for CK) were sensitive to the anomeric structure 119

Food Chemistry 283 (2019) 111–122

L. Liao et al.

Fig. 4. Proposed simplified model for the interactions between long/branched short starch chains and gliadins and/or glutenins in wheat gluten during carboxylic acid deamidation and amylolytic enzymes hydrolysis.

hydrothermal treatment, the accompanied starch suffered from gelatinization and acid hydrolysis, and the residual starch in the precipitates were insoluble amylose molecules, amylopectin molecules, blocklets, and granular residues; 4) Due to the uneven coverage by gliadins, these hydrolysates of residual starch molecules had direct interactions (glycosidic bonds, hydrogen bonds and hydrophobic interactions) with glutenins, which limited the unfolding of glutenins; 5) After the hydrolysis of long starch chains by α-amylase, glutenins were unfolded due to their direct interactions (glycosidic bonds, disulfide bonds, hydrogen bonds and hydrophobic interactions), and finally sheared after the complete hydrolysis of long starch chains; 6) Gliadins at the surface

characteristics of wheat gluten (Shewry et al., 2002) and the internal architecture of starch granule organization (Gallant, Bouchet, & Baldwin, 1997), a simple model was proposed to clarify the aggregation state and the relationships between starch granules and wheat gluten components (visualized in Fig. 4): 1) Before deamidation, starch granules were encapsulated by water-insoluble gliadins at the surface by glycosidic bonds, hydrophobic interactions and hydrogen bonds to from a composite; 2) These composites were entrapped in the interior of a three-dimensional network of glutenins, some of which were covalently linked with NSPs; 3) During the hydration in a shaking water bath at ambient temperature for 6 h and carboxylic acid deamidation upon 120

Food Chemistry 283 (2019) 111–122

L. Liao et al.

of amylopectin molecules cross-linked with glutenins by disulfide bonds, hydrophobic interactions and hydrogen bonds; 7) A larger composite with an extremely compact conformation of insoluble protein was formed.

structural characteristics of transglutaminase-treated wheat gluten hydrolysate. Food Chemistry, 107(3), 1131–1137. Aranyi, C., & Hawrylewicz, E. J. (1972). Preparation and isolation of acid-catalyzed hydrolysates from wheat gluten. Journal of Agricultural and Food Chemistry, 20(3), 670–675. Baldwin, P. M. (2001). Starch granule-associated proteins and polypeptides: A review. Starch-Starke, 53(10), 475–503. Chen, P., Yu, L., Simon, G., Petinakis, E., Dean, K., & Chen, L. (2009). Morphologies and microstructures of cornstarches with different amylose–amylopectin ratios studied by confocal laser scanning microscope. Journal of Cereal Science, 50(2), 241–247. Cuq, B., Boutrot, F., Redl, A., & Lullien-Pellerin, V. (2000). Study of the temperature effect on the formation of wheat gluten network: Influence on mechanical properties and protein solubility. Journal of Agricultural and Food Chemistry, 48(7), 2954–2959. Dahesh, M., Banc, A., Duri, A., Morel, M. H., & Ramos, L. (2014). Polymeric assembly of gluten proteins in an aqueous ethanol solvent. The Journal of Physical Chemistry B, 118(38), 11065–11076. Day, L., Augustin, M. A., Batey, I. L., & Wrigley, C. W. (2006). Wheat-gluten uses and industry needs. Trends in Food Science & Technology, 17(2), 82–90. Gallant, D. J., Bouchet, B., & Baldwin, P. M. (1997). Microscopy of starch: Evidence of a new level of granule organization. Carbohydrate Polymers, 32(3), 177–191. Gourbeyre, P., Denery-Papini, S., Larré, C., Gaudin, J.-C., Brossard, C., & Bodinier, M. (2012). Wheat gliadins modified by deamidation are more efficient than native gliadins in inducing a Th2 response in Balb/c mice experimentally sensitized to wheat allergens. Molecular Nutrition & Food Research, 56(2), 336–344. Jekle, M., Mühlberger, K., & Becker, T. (2016). Starch-gluten interactions during gelatinization and its functionality in dough like model systems. Food Hydrocolloids, 54(Part A), 196–201. Kieffer, R., Schurer, F., Köhler, P., & Wieser, H. (2007). Effect of hydrostatic pressure and temperature on the chemical and functional properties of wheat gluten: Studies on gluten, gliadin and glutenin. Journal of Cereal Science, 45(3), 285–292. Laemmli, U. K. (1970). Cleavage of structural proteins during the sssembly of the head of bacteriophage T4. Nature, 227(5259), 680–685. Lagrain, B., Goderis, B., Brijs, K., & Delcour, J. A. (2010). Molecular basis of processing wheat gluten toward biobased materials. Biomacromolecules, 11(3), 533–541. Lakowicz, J. R. (2013). Principles of fluorescence spectroscopy. Springer Science & Business Media. Lanthong, P., Nuisin, R., & Kiatkamjornwong, S. (2006). Graft copolymerization, characterization, and degradation of cassava starch-g-acrylamide/itaconic acid superabsorbents. Carbohydrate Polymers, 66(2), 229–245. Lei, L., Zhao, Q., Selomulya, C., & Xiong, H. (2015). The effect of deamidation on the structural, functional, and rheological properties of glutelin prepared from Akebia trifoliata var. australis seed. Food Chemistry, 178(1), 96–105. Liao, L., Han, X.-Y., Zhao, M.-M., Ni, L., Liu, Z.-B., & Zhang, W. (2016). Effect of native aggregation state of soluble wheat gluten on deamidation behavior in a carboxylic acid/heat water solution. Journal of Cereal Science, 72(Supplement C), 1–9. Liao, L., Liu, T.-X., Zhao, M.-M., Cui, C., Yuan, B.-E., Tang, S., & Yang, F. (2010). Functional, nutritional and conformational changes from deamidation of wheat gluten with succinic acid and citric acid. Food Chemistry, 123(1), 123–130. Liao, L., Qiu, C.-Y., Liu, T.-X., Zhao, M.-M., Ren, J.-Y., & Zhao, H.-F. (2010). Susceptibility of wheat gluten to enzymatic hydrolysis following deamidation with acetic acid and sensory characteristics of the resultant hydrolysates. Journal of Cereal Science, 52(3), 395–403. Liao, L., Zhao, M., Ren, J., Zhao, H., Cui, C., & Hu, X. (2010). Effect of acetic acid deamidation-induced modification on functional and nutritional properties and conformation of wheat gluten. Journal of Science of Food and Agriculture, 90(3), 409–417. Malalgoda, M., & Simsek, S. (2017). Celiac disease and cereal proteins. Food Hydrocolloids, 68, 108–113. Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31, 426–428. Mitchell, J., & Areas, J. (1992). Structural changes in biopolymers during extrusion. Food Extrusion Science and Technology, 345–360. Mohamed, A. A., & Rayas-Duarte, P. (2003). The effect of mixing and wheat protein/ gluten on the gelatinization of wheat starch: Names are necessary to report factually on available data; however, the USDA neither guarantee nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may also be suitable. Food Chemistry, 81(4), 533–545. O’Brien, S., & Wang, Y.-J. (2008). Susceptibility of annealed starches to hydrolysis by αamylase and glucoamylase. Carbohydrate Polymers, 72(4), 597–607. Shewry, P. R., & Tatham, A. S. (1997). Disulphide bonds in wheat gluten proteins. Journal of Cereal Science, 25, 207–227. Pallarès, I., Vendrell, J., Avilés, F. X., & Ventura, S. (2004). Amyloid fibril formation by a partially structured intermediate state of α-chymotrypsin. Journal of molecular biology, 342(1), 321–331. Qiu, C., Sun, W., Zhao, Q., Cui, C., & Zhao, M. (2013). Emulsifying and surface properties of citric acid deamidated wheat gliadin. Journal of Cereal Science, 58(1), 68–75. Roels, S. P., Grobet, P. J., & Delcour, J. A. (1998). Distribution of carbohydrates in gluten Fractions isolated from european wheats (Triticum aestivum L.) in a batter system. Journal of Agricultural and Food Chemistry, 46(4), 1334–1343. Roussel, H., & Cheftel, J. C. (1990). Mechanism of gelation of sardine proteins - Influence of thermal-processing and of various additives on the texture and protein solubility of Kamaboko gels. International Journal Of Food Science And Technology, 25(3), 260–280. Saulnier, L., Andersson, R., & Åman, P. (1997). A study of the polysaccharide components in gluten. Journal of Cereal Science, 25(2), 121–127. Shewry, P., Halford, N. G., Belton, P. S., & Tatham, A. S. (2002). The structure and properties of gluten: An elastic protein from wheat grain. Philosophical Transactions of the Royal Society of London B: Biological Sciences, 357(1418), 133–142.

4. Conclusions In summary, this is an important investigation of the solubilisation of wheat gluten, which is the first and key step for its further usage in areas other than the baking industry and feed additives. Residual starch in wheat gluten is inevitable when producing wheat gluten, especially commercially vital wheat gluten, which contains approximately 5–8% starch. Substantial previous work has been performed towards maximising the solubilisation of wheat gluten, including deamidation (Agyare, Xiong, & Addo, 2007; Cuq, Boutrot, Redl, & Lullien-Pellerin, 2000; Yong, Yamaguchi, & Matsumura, 2006). However, remaining insoluble fractions still contain up to ∼10% of the acting proteins, of which ∼10% is starch, which depreciates additional values of wheat gluten. In current work, α-amylase and glucoamylase were used to hydrolyze starch chains step-by-step in insoluble deamidated wheat gluten. Moreover, assisted by sonication, which could unfold the conformation of proteins, the interactions between gluten starch chains were discussed during carboxylic acid deamidation. This work demonstrated that insoluble amylose and amylopectin molecules significantly inhibited the unfolding of the tertiary and secondary conformation of insoluble deamidated wheat gluten. This was reflected by the formation of an extremely compact conformation due to the strong specific covalent and un-covalent interactions (hydrophobic interactions, disulfide bonds and glycosidic bonds supported by SDS-PAGE and FTIR results) with glutenins and gliadins. We, therefore, speculated that amylose and amylopectin molecules might strongly bind with parts of the residual glutenins and gliadins after deamidation, then jointly play an important role. In particular, for amylopectin and small proteins such as LMW-GS and α, β, γ-gliadins, the glycosylation, hydrophobic force and hydrogen bonds were undervalued. Possibly, the insoluble starch chains might play a pivotal role in gluten-starch interactions and might be the reason for the incomplete dissolution of wheat gluten after carboxylic acids deamidation. Therefore, a gentle enzymatic modification with glucoamylase is an effective approach for maximizing the dissolution of wheat gluten. Further work on a coupling treatment of carboxylic acid deamidation and glucoamylase hydrolysis of wheat gluten is needed. Acknowledgement This research was supported by the “National Natural Science Foundation of China (no. 31201287), the “Science Foundation Program of Fujian province of China” (no. 2016LSX) and the “Outstanding Youth Research Teachers Program of Fujian province Education Department of the Year of 2017”. The research leading to these results has also received funding from the “Scientific-Technological Program of Fuzhou” (no. 2018-G-81), and the “Pearl River S&T Nova Program of Guangzhou” (no.201610010019). Special thanks to Dr. Feng-wei Xie in University of Warwick, WMG, International Institute for Nanocomposites Manufacturing (IINM) for language polishing. The authors have declared no conflict of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2019.01.019. References Agyare, K., Xiong, Y., & Addo, K. (2007). Influence of salt and pH on the solubility and

121

Food Chemistry 283 (2019) 111–122

L. Liao et al.

Wang, K., Li, C., Wang, B., Yang, W., Luo, S., Zhao, Y., ... Zheng, Z. (2017). Formation of macromolecules in wheat gluten/starch mixtures during twin-screw extrusion: Effect of different additives. Journal of the Science of Food and Agriculture, 97(15), 5131–5138. Wong, B. T., Zhai, J., Hoffmann, S. V., Aguilar, M.-I., Augustin, M., Wooster, T. J., & Day, L. (2012). Conformational changes to deamidated wheat gliadins and β-casein upon adsorption to oil–water emulsion interfaces. Food Hydrocolloids, 27(1), 91–101. Yong, Y. H., Yamaguchi, S., & Matsumura, Y. (2006). Effects of enzymatic deamidation by protein-glutaminase on structure and functional properties of wheat gluten. Journal of Agricultural and Food Chemistry, 54(16), 6034–6040. Zhao, Selomulya, C., Xiong, H., Chen, X. D., Ruan, X., Wang, S., Xie, J., Peng, H., Sun, W., & Zhou, Q. (2012). Comparison of functional and structural properties of native and industrial process-modified proteins from long-grain indica rice. Journal of Cereal Science, 56(3), 568–575.

Singh, H., & MacRitchie, F. (2001a). Application of polymer science to properties of gluten. Journal of Cereal Science, 33(3), 231–243. Singh, H., & MacRitchie, F. (2001b). Use of sonication to probe wheat gluten structure. Cereal Chemistry, 78(5), 526–529. Surewicz, W. K., & Mantsch, H. H. (1988). New insight into protein secondary structure from resolution-enhanced infrared spectra. Biochimica et Biophysica Acta (BBA)Protein Structure and Molecular Enzymology, 952, 115–130. Synytsya, A., Míčková, K., Synytsya, A., Jablonský, I., Spěváček, J., Erban, V., ... Čopíková, J. (2009). Glucans from fruit bodies of cultivated mushrooms Pleurotus ostreatus and Pleurotus eryngii: Structure and potential prebiotic activity. Carbohydrate Polymers, 76(4), 548–556. van der Maarel, M. J. E. C., van der Veen, B., Uitdehaag, J. C. M., Leemhuis, H., & Dijkhuizen, L. (2002). Properties and applications of starch-converting enzymes of the α-amylase family. Journal of Biotechnology, 94(2), 137–155.

122