heat water solution

Journal of Cereal Science 72 (2016) 1e9

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Effect of native aggregation state of soluble wheat gluten on deamidation behavior in a carboxylic acid/heat water solution Lan Liao a, b, *, Xue-yue Han a, b, Mou-ming Zhao c, Li Ni a, b, Zhi-bin Liu a, b, Wen Zhang a, b a

Institute of Food Science and Technology, College of Biological Science and Technology, Fuzhou University, Fuzhou, Fujian 350108, People's Republic of China b Fujian Center of Excellence for Food Biotechnology, Fuzhou University, Fuzhou, Fujian 350108, People’s Republic of China c College of Light Industry and Food Sciences, South China University of Technology, Guangzhou, Guangdong 510640, People's Republic of China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 December 2015 Accepted 14 September 2016 Available online 15 September 2016

To understand the role of native aggregation state (NAS) of soluble wheat gluten and fractions during deamidation in a carboxylic acid/heat water solution, changes in conformation and deamidation behavior as function of protein concentration from dilute to semi-concentrated regimes to control NAS were investigated by physicochemical properties, SDS-PAGE, molecular force change, intrinsic fluorescence emission spectroscopy (IFES) and FTIR. Our data show that, in this solution, the deamidated proteins displayed features characteristic of more scattered and flexible polymer structure in dilute concentration than concentrated ones. Degree of deamidation (DD), HD and Zeta potential exhibited strongly oppositely with the decreasing concentration. HWM-GS, u-gliadins and LWM-GS degraded into smaller peptides with decreasing of NAS. FTIR and IFES displayed that improved molecular flexibility with decreasing of concentration as detected by the increasing content of b-turn and b-sheet, as well as the red-shift of wheat gluten and gliadins at the expense of a-helix. Hydrophobic and hydrogen bond increased gradually and were dominant in inter-molecule as function of increasing concentration. The above information demonstrated that NAS of soluble wheat gluten dominated changes of deamidation behavior and conformation in a carboxylic acid/heat water solution. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Wheat gluten Citric acid deamidation Native aggregation state Deamidation behavior

1. Introduction Wheat is the most important human food grain due to uniquely forming dough that exhibits important properties required for the wider diversity of well-known food products. It ranks second in total production as a cereal crop, with maize being the first. Wheat gluten proteins differ from zein and exhibit prominent elastomeric properties responsible for the remarkable viscoelastic properties of dough. Those properties, which are ascribed to the ability of gluten proteins to form very long polymers, can be regarded as a real asset in the optics of producing protein-based food-products and biomaterials. Wheat gluten proteins are largely insoluble in water, rendering their study difficult and precluding their usage in formulated food

* Corresponding author. Institute of Food Science and Technology, Fuzhou University, Xue Yuan Road, University Town, Fuzhou, People's Republic of China. E-mail address: [email protected] (L. Liao). http://dx.doi.org/10.1016/j.jcs.2016.09.011 0733-5210/© 2016 Elsevier Ltd. All rights reserved.

or un-food products. Difficulty in solubilising wheat gluten proteins arises predominantly from a lack of ionisable groups and the very high molecular weight of the glutenins. Wheat gluten proteins have number of non-polar side residues, for instance, glutamine, asparagine, tryptophan, isoleucine, tyrosine, phenylalanine, proline, leucine and valine residues. They are subdivided into two groups on the basis of their ability to form polymeric species by means of intermolecular disulfide bonds between protein subunits. Gliadins, which account for about half of the total gluten proteins, are monomeric proteins and have a molecular weight Mw in the range 30e80 kDa. The other half are polymeric glutenins, modeled as a linear backbone of glutenin subunits (with Mw ranging from 30 to 200 kDa) linked together by disulfide bonds. Solubility of wheat gluten proteins in water could be substantially altered in food grade solvents by forming wheat gluten polymer solutions (such as a aqueous ethanol solvent (water/ethanol (50/50 v/v)) (Dahesh et al., 2014, 2016) and a heated water/acid solvent (water/acids (>99/<1 w/w))). Dahesh et al. found that the supramolecular organization of wheat gluten in a aqueous ethanol (water/ethanol (50/50 v/v))

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L. Liao et al. / Journal of Cereal Science 72 (2016) 1e9

solvent undergone assemblies like a spontaneously self-healing gelation driven by hydrogen bondings from dilute to more concentrated regimes (Dahesh et al., 2014, 2016). Our previous studies indicated the heated water/acid (water/acids (>99/<1 w/ w)) solvent worked in a deamidation mechanism of non-polar side residues with the swelling behavior of gluten network conformation through the balance between intra-molecular electrostatic repulsion and the molecular aggregation force, such as noncovalent (hydrophobic interactions) and disulphide bonds (Liao et al., 2010a, 2010b). Due to the complexity of wheat gluten proteins and a lack of knowledge on the their structure, few works were focused on the scaling behavior of wheat gluten proteins in polymer solutions as a function of polymer concentration. When being hydrated with water, molecules in wheat gluten cross-link together and form networks as a result of the quick formation of a protein matrix (Lagrain et al., 2010). When wheat gluten proteins is in a heated water/acid solvent (water/acids (>99/ <1 w/w)), acid deamidation inevitably occurs up and converts amide groups, primarily from Gln and Asn residues in wheat gluten, to carboxyl groups, which have attracted a lot of research work during the last few decades (Matsudomi et al., 1982; Riha et al., 1996; Liao et al., 2010a, 2010b). This carboxylation reaction significantly reduced hydrogen bonding in wheat gluten, increased the protein's polyelectrolyte character, enhanced the electrostatic repulsion between protein molecules, and allowed wheat gluten molecules to dissociate from each other, thereby increasing the solubility of wheat gluten. Accordingly, exposure extent of amides in wheat gluten polymer solution a heated water/acid solvent (water/acids (>99/<1 w/w)) will affect the accessibility of Hþ to amides groups, which may dominate the competition between the deamidation of amides groups and peptide chain hydrolysis. Since much variables may affect the conformation of wheat gluten in polymer solutions, it is necessary to find some general rules regarding deamidation behavior of modified wheat gluten solutions in a heated water/acid solvent, which is a popular processing condition for food industry. In this optics, our strategy was undergone by selecting wheat gluten proteins according to the native aggregation state of wheat gluten in citric acid/heated water solution as a function of concentration from dilute to semi-concentrated regimes as a model system. A set of experiments combining SDS-PAGE, FTIR spectroscopy, intrinsic fluorescence scanning and physicochemical measurements were carried out to study the role of native aggregation state of wheat gluten on changes of deamidation behavior and resultant conformation of modified wheat gluten as a function of soluble wheat gluten concentration in a citric acid/heated water solution. We also probed native aggregation state of two compositions of wheat gluten (gliadins and glutenins) during carboxylic acid deamidation, which was used as references in understanding the structural and physicochemical variation of wheat gluten. The combined analysis of three protein experimental measurements and the resultant deamidation behaviors in polymer solutions have been thoroughly discussed.

2. Materials and methods 2.1. Materials Strong wheat cultivar Zhoumai 22 was courtesy of the farmers who specially plant it at Zhoukou with a mechanically grinding into powder (Zhoukou, Henan, China). All other chemicals and solvents were of the analytical grade or HPLC grade and obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China) and Sigma Chemical Co. Ltd. (St. Louis, USA), respectively.

2.2. Experimental 2.2.1. Sample preparation Wheat gluten powder (84.54 ± 0.73% protein (N  5.7%), dry basis) was prepared by hand-washing a doughball using the distilled water, then being freeze-dried and grinded in a coffee grinder. For the separation of gliadin and glutenin, they were prepared as described by Kieffer et al. (2007) with a slight modification. Non-defatted dried gluten (50 g) was successively extracted with 70% (v/v) aqueous ethanol (3  500 mL) by means of an Ultra Turrax (IKA, Staufen, Germany) homogeniser (1 min) and continuous rotating agitation (30 min) at room temperature at least three time until the supernatant was clear after the suspensions were centrifuged at 10, 000g, 4  C for 10 min. Combined supernatants (gliadins) were submitted to a rotary evaporator to get rid of part of ethanol. Concentrated supernatants (gliadins) and combined sediment (glutenins) were freeze-dried respectively and milled for the further use. The protocol of wheat gluten, gliadin and glutenin citric acid/heated solvents as a function of concentration were derived from according to our previous method reported (Liao et al., 2011) with a slight modification. In brief, 5% (w/v) dried wheat gluten, gliadins and glutenins were first prepared rehydrated with a citric acid (0.024 M) distilled water and incubated for 2 h in a water bath shaker at room temperature, respectively. After 10 min centrifugation at 10, 000g at 4  C, soluble fractions were collected and determined the concentration of protein content according to the method of Lowry (Schacterle and Pollack, 1973) followed the dilution of four levels of 0.01, 0.10, 1.00 and 10.0 mg/mL concentration at least. Thereafter, aliquots were heated at 121  C for 10 min and immediately held in an ice water bath for 5 min to inactivate reaction, before being dialyzed in deionized water at 4  C for 24 h to remove ammonium and freeze-dried for the following determinations. These resultant modified wheat gluten as function of concentration from dilute to semi-concentrated regimes were abbreviated into CDWG-0.01, CDWG-0.1, CDWG-1 and CDWG-10, respectively. 2.2.2. Degree of deamidation (DD) Assessment for degree of deamidation was based on the ratio of the ammonia amount released from modified proteins to the total ammonia from the action proteins according to the method reported by Kato et al. (1987). 0.5 g of untreated wheat gluten was dissolved in 5 mL of 3 M HC1, then sealed in a 10 mL glass ampoule and heated at 121  C for 3 h to deamidate completely. The assay of ammonia amount released from modified proteins and total ammonia was analyzed according to ammonia by an ammonia electrode (Orion Research Inc., Boston, MA) with a reference curve using standard ammonium chloride solutions (104 M to 102 M) as described by the method of Shih (1990). 2.2.3. Degree of hydrolysis (DH) Degree of hydrolysis was defined as the percentage of peptide bonds cleaved during the reaction, which was calculated from the ratio of free amino groups to the total number of peptide bonds in soluble factions during deamidation, according to the method described by Adler-Nissen (1976). The determination of free amino groups in samples was carried out as follows: 0.25 mL of soluble fractions obtained from centrifugation was mixed with 2 mL of 0.2 M sodium phosphate buffer (pH 8.0) in the presence of 1% SDS and 2 mL of 0.1% trinitrobenzenesulfonic acid, followed by incubation in the dark for 60 min at 50  C. The reaction was quenched by adding 4 mL of 0.1 M HCl and the absorbance was read at 340 nm. L-leucine solutions of 0.15 - l.5 mM were used as standard. The total number of peptide bonds was obtained by the hydrolysis of relevant soluble factions under vacuum in 6 M HCl for 24 h at

L. Liao et al. / Journal of Cereal Science 72 (2016) 1e9

110  C in sealed tubes. 2.2.4. Zeta potential Zeta potential of samples as a function of concentration was measured by a laser doppler velocimetry and phase analysis light scattering technique (M3-PALS) using a Malvern Zetasizer Nano ZS (ZEN 3600) instrument (Malvern Instruments Ltd., Malvern, Worcestershire, UK) according to the procedure of Benelhadj et al. (2016) with some modifications. The data was the average values of three measurements performed with three individually prepared protein dispersions. 2.2.5. SDS-PAGE experiments SDS-PAGE of freeze-dried samples prepared at varied concentration was carried out according to the method of Laemmli et al. (1970) using 12% acrylamide separating gel and 5% acrylamide stacking gel. 2.2.6. Molecular force The solubility of samples as a function of concentration in different reducing solvents was used to evaluate changes of molecular forces of proteins at varied concentration. It was determined according to the procedure of Roussel and Cheftel (1990) with slight modifications. Reducing solvents used 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 6M urea (S3); 0.05 M PBS, pH 4.44 containing 2% (v/v) b-mercaptoethanol (S4); 0.05 M PBS, pH 4.44 containing 1% (w/v) SDS and 2% (v/v) b-mercaptoethanol (S5); 0.05 M PBS, pH 4.44 containing 1% (w/v) SDS and 6M urea (S6). All determinations were conducted in triplicate. 2.2.7. Intrinsic fluorescence emission spectroscopy Intrinsic emission fluorescence spectra of the protein samples were obtained in an F-7000 fluorophotometer (Hitachi Co., Hitachinake, Japan) as described by Chang et al. (2010). Protein solutions were prepared in 10 mmol/L phosphate buffer (pH 4.4). To minimize the contribution of tyrosyl residues to the emission spectra, the protein solutions were excited at 290 nm, and emission spectra were recorded from 300 to 400 nm at a constant slit of 5 nm for both excitation and emission. All determinations were conducted in triplicate. 2.2.8. Statistical analysis All the tests were carried out in triplicate. Analysis of variance and significant difference tests was 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. Chemical characterization of deamidated wheat gluten Results of degree of deamidation (DD), degree of hydrolysis (DH) and Zeta potential of deamidated samples as a function of concentration (from semi-dilute to semi-concentrated regimes) were summarized in Fig. 1A (wheat gluten), Fig. 1B (glutenin) and Fig. 1C (gliadin). As shown in Fig. 1A, features of DD and HD were characterized by a fast decrease rate reaching to the lower value when the protein concentration decreased to 0.1% from the maximum (DD, from 24.25% at 0.01% concentration to 2.80% at 0.1% concentration; DH, from 40.63% at 0.01% concentration to 11.79% at 0.1% concentration), followed by a generally leveling off to the minimum at 10% concentration with the raising of the reaction concentration.

3

For example, the DD and HD of CDWG-0.01 reached 24.25% and 40.64% after deamidation respectively, while those of the CDWG-10 were only 1.26% and 1.99%, respectively. Moreover, when the concentration of soluble wheat gluten increased from 0.01% to 1%, DD and HD decreased each other in a positively linear way (In fact, the concentration scale we had measured lowed to 0.0001% in this section for DD, DH and Zeta potential determinations, however, it was difficult to concentrate enough amount of samples of 0.0001% and 0.001% concentration by rotary evaporator without any thermal denaturation to meet the following determinations; DD and DH of the sample of 0.001% concentration were 90.29% and 93.28% respectively; DD and DH of the sample of 0.0001% concentration were 95.29% and 97.64% respectively; Data not shown). The correlation coefficient (R2) of DD and HD of wheat gluten solution as a function of concentration was up to 0.98 (data not shown), illustrating that there was a strong relationship between native aggregation state of soluble wheat gluten and rate of deamidation in a carboxylic acid/heat water solution. Lagrain et al. (2010) has reported a quick formation of the molecules aggregates of a protein matrix in wheat gluten by cross-linking together and networking when being hydrated with water. Our previous works also showed that an intense entanglement of the aggregate chains with a bulk network complicated by island-like and striped aggregates during hydrating without any treatment by applying AFM to investigate the fine structure of the macromolecules (Liao et al., 2010b). According to these reports, we could speculate that native conformation of wheat gluten in polymer solution had a marked effect on the exposure of amide groups in it, further affecting the ability of deamidation in modification processing of wheat gluten. The reasons are on account of that: (1) In the water hydrated state, wheat gluten forms a cohesive viscoelastic mass (Dahesh et al., 2016). The process of hydrating wheat gluten with water is itself the aggregation of wheat gluten molecules by forming network; (2) The higher concentration of wheat gluten molecules, the more compact conformation of soluble wheat gluten, (3) leading to the decrease of degree of exposure of amide groups, with inhibiting the accessibility of Hþ to amide groups in the interior of network. Surprisingly, similar to the performance of degree of deamidaiton, peptide bonds hydrolysis in current study was also observed dependent on the concentration of protein even in this citric acid/heated water solution (carboxylic acid deamidation upon hydrothermal treatment), which is rather unexpected in view of amide groups deamidation of the proteins in the sample. This feature is the sign that, in addition to deamidation, other mechanisms occurred, leading to a good correlation coefficient. Actually, peptide bonds hydrolysis and the deamidation of amide groups have mutual reciprocity and benefit. Peptide bonds hydrolysis definitely make the contribution to expand the conformation of protein and to expose more amide groups. More deamidation of amide groups is in favor of exposing peptide bonds by unfolding the conformation (Riha et al., 1996). From this point of view, it could be further speculated that native aggregation state of soluble wheat gluten might be the determinant factor for the unfolding of molecular structure and for determining the change trend of deamidation. With respect to zeta potential of wheat gluten as shown in Fig. 1A, the data of surface charge (17.9 mv to 18 mv) displayed a significantly (p < 0.05) stronger dependence of the concentration raised from 0.01% to 10%, that was in well accordance with the change of degree of deamidation. It suggested that the conformation of wheat gluten in the semi-dilute concentration was well unfolded and enough carboxyl groups was made from amides groups by deamidation, even after some of them were protonated in this acid solution. Interestingly, the value of surface charge of CDWG-1 was maximum, higher than the other concentrations of

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L. Liao et al. / Journal of Cereal Science 72 (2016) 1e9

A

Degree of deamidation(%) Degree of hydrolysis(%) Zeta potential(mv)

30

45

30

40 25 35 20

30

20

10

25 15 20

0

15

10

10

-10

5 5 0

0.01

0.1

1

10

-20

0

Concentration of deamidated wheat gluten(mg/mL)

B

Degree of deamidation(%) Degree of hydrolysis(%) Zeta potential(mv)

60 50 40

70

20

60

15

50

10

40

5

30 30 20

20

10

10

0 -5 -10 -15

0

0.01

0.1

1

10

0

Concentration of deamidated glutenin(mg/mL)

C

3.2. SDS-PAGE

Degree of deamidation(%) Degree of hydrolysis(%) Zeta potential(mv)

50

30 60

40

50

30

10

30

0

20 10

20

40

20

-10

10 -20

0

0.01

0.1

concentration, ulteriorly proving that the native aggregation state of soluble wheat gluten had a prominent impact on exposure of amide groups and peptide bonds in them. What is more, it was noteworthy that HD of glutenins were higher than those of wheat gluten and gliadins respectively when the concentration of protein was similar. For instance, when the concentration was at 0.01%, HD were 40.63% for wheat gluten, 55.88% for gliadin and 66.04% for glutenin. These observations suggested that the role of glutenin was dominant during the deamidation procedure of wheat gluten due to its special net structure. With respect to R2 of glutenins, it was minimum, lower than wheat gluten and gliadin (The correlation coefficient (R2) between DD and HD of gliadin and glutenin were 0.84 and 0.80, respectively, data not shown). It was probably related to the special structural characteristics of wheat gluten, glutenin and gliadin. Wheat gluten is special from other plant proteins in the aspect of structural properties of its molecules (Pouplin et al., 1999). It is proposed to have a loop and web-like three-dimensional structure (Shewry et al., 2000) and comprises mainly glutenin (network polypeptides), gliadin (single chain polypeptides), and low molecular weight albumins and globulins (Shewry et al., 2002). SeS bonds are predominantly inter-molecular in glutenins, which cross-link polypeptide chains, leading to the formation of a network structure of glutenins. In contrast, SeS bonds are intra-molecular in gliadins, resulting in globular polypeptide chains (Lasztity, 1986). From this point of view, it could be supposed that after the removal of gliadins, three-dimensional network of glutenins might become spongy. It might be easy for glutenins to penetrate by water without an excess of the unfolding of conformation, disliking wheat gluten and gliadins, which were of an entangled, tense and compact conformation. This presumable view fully corresponded to on the structure of glutenin gels described in the literature referring structural information from the viscoelatic response of gluten, which was that dense aggregates of glutenin proteins form a percolated network as would attractive colloidal particles (Dahesh et al., 2016).

1

10

0

Concentration of deamidated gliadin(mg/mL) Fig. 1. Degree of deamidation and hydrolysis and zeta potential of wheat gluten (A), glutenin (B) and gliadin (C) modified with critic acid deamidation upon hydrothermal treatment as a function of concentration.

protein, but not significant (p < 0.05). The possible reason was that carboxyl groups of CDWG-1.0 might be over protonated and the cationic amino groups dominated the surface of wheat gluten (Zhao et al., 2011). As shown in Fig. 1B and C, similar to the performance of wheat gluten, zeta potential, DD and HD of glutenins and gliadins increased significantly (p < 0.05) as the decrease of protein

SDS-PAGE (Fig. 2) were undertaken fundamental analysis to obtain the molecular weight distribution of wheat gluten, gliadins and glutenins during citric acid deamidation upon hydrothermal treatment from dilute to semi-concentrated regimes. As shown in lane 15 for native wheat gluten, subunits bands in wheat gluten isolate that were identified mainly as HMW-GS, LMW-GS (group B&C), a, b, g and u-gliadins and a-amylase trypsin inhibitors, which was with a distribution similar to that of a spring wheat gluten like the cultivar Jinan17 (DuPont et al., 2008). Compared to subunit bands of it (lane 15), the intensity of subunit bands (lane 11e14) of HWM-GS, u-gliadins, and LWM-GS (group B&C) (over 20 kDa) for CDWGs was observed generally gradually weakening (see lane 11e13), even disappeared (lane 14) from semi-concentrated to dilute regimes. This observation fully verified the speculation from the results of degree of hydrolysis from Fig. 1, suggesting that the open aggregation state of wheat gluten strikingly boosted degree of hydrolysis and deamidation. Interestingly, five newly formed bands (lane 13; see the arrows beside lane 13 in Fig. 2), which did not appear in lane 11 and lane 12, exhibited notably at the subunits bands of u-gliadins. However, the intensity of them by contrast thoroughly attenuated with decreasing of concentration. A tentative explanation might be that HWM-GS dissociated into smaller molecular weight peptide or the aggregation of the smaller peptides (LMW-GS (group B&C), a, b and g-gliadins) due to the thermal effect (Liao et al., 2010b; Shewry et al., 2002). This point however certainly need further investigation. Moreover, it was apparent that deamidated gliadins showed

L. Liao et al. / Journal of Cereal Science 72 (2016) 1e9

more markedly shifting bands than glutenins toward the underneath of gel from semi-concentrated to dilute regimes (lanes 1e5 for gliadins, lane 6e10 for glutenins). For example, the subunits band of deamidated u-gliadins disappeared gradually with a enhanced intensity of bands around 20.1 kDa with the decrease of concentration. By contrast, the intensity of subunits bands of deamidated glutenins ranging from 66.4 to 116 kDa, which exhibited notably in nonmodified glutenin (lane 10), attenuated slightly around this regions when the concentration decreased from 10% to 0.1%, finally disappeared thoroughly with decreasing concentration to 0.01%. This observation illustrated that gliadins were more susceptible to its native aggregation state than glutenins in this system. 3.3. Evaluation of secondary conformation Generally speaking, aggregation behavior of protein was associated with conformational and structural changes that determines the exposure of active residues. To get more insight into conformation of wheat gluten, gliadins and glutenins by deamidation at diffident concentration, fouried transform infrared (FTIR) analysis was performed to investigate the secondary structural differences. The amide I absorption occurs in the region 1600e1700 cm1. Deconvolution of amide I region of the samples indicated that they were composed of five components located at 1604, 1631, 1652,1679 and 1693 cm1, representing intermolecular b-sheets of protein aggregation, intra-molecular aggregation extended b-sheet (hydrated), a-helix, b-turn and extended b-sheet, respectively. Table 1 gathers five second conformation assignments information after second-derivative and deconvolviation from FTIR spectra of wheat gluten, gliadin and glutenin after carboxylic acid deamidation from semi-concentrated to dilute regimes. As shown in Table 1, the modified wheat glutens reduced in ahelix (from 38.22% to 31.08%) and generally increased in b-turn (22.89%e30.46%) with decreasing concentration of wheat gluten. Moreover, the variation in the percentage of b-turn was observed generally dependent on the concentration (Table 1). It indicated that Glu and Asp were gradually exposed when Gln and Asn in wheat gluten were deamidated by citric acid, since Glu and Asp are the main constituents of b-turn which located at the surface of protein (Chou and Fasman, 1977). Additional information can be obtained on the molecular flexibility of samples, estimated by the ratio of a-helix to b-sheet. We observed that although the content of b-sheet increased slightly from 38.89% at 10% concentration of protein solutions to 43.75% at 1%, and then declined substantially to 38.46% at 0.01%. However, the molecular flexibility of CDWG still increased generally (from 0.98 (10%) to 0.81 (0.01%)). Consistent conclusions are derived from here and demonstrates that the lower concentration of wheat gluten polymer solution, more flexible of modified wheat gluten after deamidation. As for deamidated glutenins and gliadins, generally, it was observed a significant decrease of the a-helix and an appreciable increase of b-sheet and b-turn content as increasing of protein concentration from semi-concentrated to dilute regimes after deamidation. This transformation of a-helix to b-sheet further verified a more flexible or extended conformation of deamidated wheat glutenins as a result of the decrease of concentration. Especially, those changes in the secondary structure of gliadins by citric acid deamidation were somewhat similar to the previous report by Wong et al. (2012), who reported that wheat gliadin deamidated by HCl exhibited an increase of b-sheet while a decrease of the a-helix. As explained above, the ratio of a-helix to b-sheet represents the molecular flexibility of protein. The values of a-helix to b-sheet of deamidated wheat gluten with the decreasing of protein

5

concentration became apparently smaller (from 0.98 to 0.81) as expected. This change is consistent with the speculation derived from Figs. 1 and 2, strongly supporting that the molecular structure of deamidated wheat gluten was more flexible, intrinsically due to more open conformation of substrate protein. With the enhancement of molecular flexibility, it strengthened the accessibility of Hþ to amide groups and peptide bonds, leading to the production of shorter peptides. It was the reason that the deamidated wheat gluten with lower concentration showed more molecular flexibility than that with higher concentration. Similarly, The values of a-helix to b-sheet of deamidated glutenin and gliadin concentration were also dependent with the decreasing of protein concentration from dilute to semi-concentrated regimes (from 1.17 to 1.04 for glutenin and from 0.87 to 0.74 for gliadin), puissantly proving that native protein aggregation state had markedly effect on deamidation rate of protein by the contribution of improving the molecular flexibility of acting protein. 3.4. Molecular force change In order to further investigate the effect of native protein aggregation state on deamidation behavior of protein during deamidated procedure, molecular force changes of modified protein with concentration from semi-concentrated to dilute regimes were detected after deamidation. Six reagents with specific chemical actions on protein were used to investigate the types of forces of deamidated protein. Hydrogen bonds was disrupted by Urea. Hydrogen bonds and hydrophobic interactions was disrupted by SDS. b-mercaptoethanol could cleave disulfide bonds to sulfhydryl groups (Liao et al., 2011). Molecular forces profiles of deamidated wheat gluten at different concentration were shown in Fig. 2A. The characteristic of intermolecular forces of protein were observed susceptible and dependent with increasing concentration of protein. These observation illustrated that degree of aggregation of wheat gluten enlarged with increasing of concentration, confirming the availability of our approach, which was an effective method to determine degree of aggregation of soluble wheat gluten polymer solution by the control of protein concentration from semiconcentrated to dilute regimes. We founded that, at the same concentration, the extent of increase in the solubility of deamidated wheat gluten using the denaturing agents were as follow: SDS and Urea (S6) > SDS (S2) > Urea (S3) > ME (S4) > PBS (S1) > SDS and ME (S5), indicating that hydrogen bonds and hydrophobic interactions were dominant in intermolecular force of wheat gluten during citric acid deamidation upon hydrothermal treatment. Furthermore, it was observed the significant (p < 0.05) decrease in the solubility of deamidated wheat gluten in the presence of b-mercaptoethanol (S4) and in the presence of SDS and b-mercaptoethanol (S5) at the same concentration, compared to the other reagents. It demonstrated that the contribution of disulfide bonds to stabilize wheat gluten network lowered during processing due to the enhancement of electrostatic repulsion by deamidation as well as hydrogen bonds and hydrophobic interactions by hydrothermal treatment. Upon deamidated processing, the solubility of deamidated glutenin in the presence of Urea (S3) had the largest values in comparison with other reagents at the same concentration of protein (Fig. 2B). It revealed that hydrogen bonds were major in glutenin structure. Additional information were obtained by disulfide bonds which was observed to make a contribution of reinforcing the deamidated glutenins network by a marked increase in the solubility of protein in the presence of SDS and b-mercaptoethanol (S5), compared with that in presence of b-mercaptoethanol (S4). This change in the disulfide bonds of glutenins by citric acid

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L. Liao et al. / Journal of Cereal Science 72 (2016) 1e9

Fig. 2. SDS-PAGE of wheat gluten, glutenin and gliadin modified with critic acid deamidation upon hydrothermal treatment as a function of concentration. The lanes were divided into four groups, which are MW standards (the leftmost lane), gliadin group, glutenin group and wheat gluten group. The lanes of the gliadin group from left to right were samples of 10% (lane 1), 1% (lane 2), 0.1% (lane 3) and 0.01% (lane 4) concentration and untreated gliadin (lane 5). The lanes of the glutenin group from left to right were samples of 10% (lane 6), 1% (lane 7), 0.1% (lane 8) and 0.01% (lane 9) concentration and untreated glutenin (lane 10). The lanes of the wheat gluten group from left to right were samples of 10% (lane 11), 1% (lane 12), 0.1% (lane 13) and 0.01% (lane 14) concentration and untreated wheat gluten (lane 15).

Table 1 Secondary structure of wheat gluten, glutenin and gliadin modified with critic acid upon hydrothermal treatment as a function of concentration. Samples

Concentration (mg/mL)

Intermolecular b-sheet aggregation extended

Intramolecular a-helix aggregation extended bsheet (hydrated)

b-turn

Extended b-sheet

a-turn/bsheet

Frequency (cm1)

Contenta Frequency (%) (cm1)

Contenta Frequency (%) (cm1)

Contenta Frequency (%) (cm1)

Contenta Frequency (%) (cm1)

Conte ta (%)

Wheat gluten Wheat gluten Wheat gluten Wheat gluten

10

1604.5

9.55

1629.6

27.56

1654.7

38.22

1679.7

22.89

1693.2

1.78

0.98c

1

1604.5

11.54

1631.5

30.77

1652.7

36.41

1679.7

19.74

1693.2

1.44

0.83b

0.1

1604.5

11.49

1631.5

29.94

1652.7

36.35

1679.7

21.28

1693.2

0.94

0.86b

0.01

1604.5

10.15

1629.6

26.05

1658.5

31.08

1679.7

30.46

1693.2

2.26

0.81

Glutenin Glutenin Glutenin Glutenin

10 1 0.1 0.01

1604.5 1604.5 1604.5 1604.5

9.37 9.20 10.83 12.61

1627.7 1627.7 1627.7 1627.7

24.48 24.71 25.22 23.31

1658.5 1656.6 1656.6 1656.6

41.58 39.23 39.69 39.48

1679.7 1679.7 1679.7 1679.7

22.84 22.96 22.53 22.83

1693.2 1693.2 1693.2 1693.2

1.73 1.90 1.73 1.79

1.17e 1.09c 1.05d 1.04d

Gliadin Gliadin Gliadin Gliadin

10 1 0.1 0.01

1604.5 1604.5 1606.4 1604.5

12.06 11.37 14.31 9.43

1629.6 1631.5 1633.5 1629.6

27.63 30.77 30.68 25.72

1658.5 1654.7 1654.7 1658.4

35.88 36.24 35.87 27.64

1679.7 1679.7 1681.7 1677.8

22.67 20.08 17.42 34.98

1693.2 1693.2 1693.2 1693.2

1.76 1.54 1.72 2.23

0.87b 0.83b 0.77 ab 0.74a

The data with different letters in the same column are significantly different (p < 0.05). a The percentage of secondary structure content is estimated as corresponding area as a percentage of total amide I band area.

deamidation at the same concentration were similar to our previous report by Liao et al. (2011), who also showed a significant increase of disulfide bonds upon wheat gluten deamidation. Fig. 2C illustrated that characteristic of molecular forces of deamidated gliadins was susceptible with the increase of gliadin concentration upon deamidation. Upon the same concentration of gliadin, the extent of increase in the solubility of deamidated gliadins using the denaturing agents were as follow: SDS and Urea (S6) > Urea (S3) > SDS (S2) > SDS and ME (S5) > ME (S4) > PBS (S1), suggesting that hydrogen bonds and hydrophobic interaction were

also dominant in stabilizing the gliadin structure. Moreover, like deamidated glutenins, the significant increase in the solubility of gliadins in the presence of SDS and b-mercaptoethanol (S5) was observed compared to that in presence of b-mercaptoethanol (S4). The above observations of molecular force of three proteins indicated that molecular force of wheat gluten, glutenin and gliadin corresponded to the growing of native protein aggregation state. This result was in excellent consistent with results from degree of deamidation and hydrolysis, SDS-PAGE and secondary conformation from FTIR. Hydrogen bonds and hydrophobic interactions were

L. Liao et al. / Journal of Cereal Science 72 (2016) 1e9

Concentration of protein(mg/mL)

0.25

7

0.01 mg/mL 0.1 mg/mL 1 mg/mL 10 mg/mL

A

0.20

0.15

0.10

0.05

0.00 S1

S2

S3

S4

S5

S6

Different modified reagents

Concentration of protein(mg/mL)

0.5

0.01 mg/mL 0.1 mg/mL 1 mg/mL 10 mg/mL

B

0.4

0.3

0.2

0.1

0.0

S1

S2

S3

S4

S5

S6

Different modified reagents

0.45

0.01 mg/mL 0.1 mg/mL 1 mg/mL 10 mg/mL

C

Concentration of protein(mg/mL)

0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00

S1

S2

S3

S4

S5

S6

Different modified reagents Fig. 3. Solubility profiles of aggregations in (wheat gluten (A), glutenin (B) and gliadin (C)) modified by critic acid deamidation upon hydrothermal treatment as a function of concentration. S1, 0.05 mol/LPBS, pH 4.44; S2, 0.05 mol/L PBS, pH 4.44 þ 1% (w/v) SDS; S3, 0.05 mol/L PBS, pH 4.44 þ 6 mol/L Urea; S4, 0.05 mol/L PBS, pH 4.44 þ 2% (v/v) ME; S5, 0.05 mol/L PBS, pH 4.44 þ 1% (w/v) SDS þ 2% (v/v) ME; S6, 0.05 mol/L PBS, pH 4.44 þ 1% (w/v) SDS þ 6 mol/L Urea.

Fig. 4. Endogenous fluorescence scanning of wheat gluten (A), glutenin (B) and gliadin (C)) modified by critic acid deamidation upon hydrothermal treatment as a function of concentration.

8

L. Liao et al. / Journal of Cereal Science 72 (2016) 1e9

observed dominant for wheat gluten and gliadins molecules, while hydrogen bonds and disulfide bonds for glutenins. Accordingly, we can speculated that with the decrease of concentration from semiconcentrated to dilute regimes, wheat gluten experienced the full unfolding of structure, leading to tempestuous damage to some of hydrogen bonds, hydrophobic interactions and disulfide bonds due to steric hindrance and balance of an awful lot of hydrogen bonds between protein and water. 3.5. Intrinsic fluorescence spectra Fig. 4A showed intrinsic fluorescence emission spectra of deamidated wheat gluten at each concentration. The fluorescence spectra reflected mostly the contribution of tryptophan residue, which had a high quantum yield and provided a sensitive means to monitor the conformational changes of protein in solution and protein at the interface (Agyare et al., 2008). The tryptophan fluorescence emission spectra of CDWG-0.01, CDWG-0.1, CDWG-1 had a lmax at 350.0 nm, 348.2 nm and 349.7 nm, respectively. Compared to the lmax of 344.6 nm of CDWG-10, this small red-shift towards longer wavelength implied that the tryptophan residue of deamidated wheat gluten at a lower concentration was exposed to a more hydrophilic environment due to the unfolding of the protein upon deamidation. Interestingly, in comparison with the lmax of 350.0 nm of CDWG-10.0 and 349.7 nm of CDWG-1, lmax shifted towards lower wavelengths for CDWG-0.1. This feature well corresponded to the transition of subunit bands obtained SDS-PAGE (Fig. 2) of wheat gluten sample at 0.1% concentration. It exhibited significantly newly formed subunit bands at the molecular weight higher than LWM-GS (group-B), which did not show in CDWG-0.01 (lane 11) and CDWG-0.1 (lane 12). Intrinsic fluorescence spectra of deamidated glutenins and gliadins at different concentration were presented in Fig. 4B and C, respectively. As shown in Fig. 3C, in well accordance to the change of wheat gluten, the lmax of deamidated gliadins shifted from 339.9 nm to 350.0 nm (red shift) as decreasing of gliadin concentration. This may be due to that there was more spare space in solution for protein rearrangement for wheat gluten or gliadin at the lower protein concentration (Liao et al., 2011). By contrast, it was observed that the lmax for glutenin shifted slightly from 350.3 nm to 349.7 nm (a blue shift) as the decrease of concentration (Fig. 4B). According to this result, we could confirm the speculation made above in the evaluation of DD and HD (see section 3.1), which was that it was easy for three-dimensional network of glutenins to dissolve and penetrate by water and Hþ without an excess of the unfolding of conformation after the removal of gliadins. Altogether, combined with DD, HD, Zeta potential, SDS-PAGE and secondary conformation evaluation, as well as the molecular force change, these results confirmed that native protein aggregation state had significant impact on conformational change of wheat gluten, glutenins and gliadins during carboxylic acid deamidation. At the lower protein concentration, it led to the increasing opportunity of Hþ contacting with amide bonds because of less entangled conformation, well unfolding of protein structure and more spare space to hydrophilic environment. 4. Conclusions In current study, we discussed the changes of molecular structure and deamidation behavior of wheat gluten, glutenin and gliadins in a citric acid/heat water solution by adjusting protein concentration from semi-concentrated to dilute regimes to control the degree aggregation of protein. Native aggregation state of protein had an significant influence on DD, HD, Zeta potential, which decreased markedly with the enhancement of the native

aggregation degree of protein. Furthermore, the lower concentration had positive effect on shearing protein structure, resulting in a greater molecular flexibility of three proteins. Higher concentration led to the gradual increase of intermolecular force in protein and the red shift in fluorescence spectra, indicating that the native aggregation state of protein also affected the tendency of rearrangement of protein molecules (such as deamidated rate) during the modification. In all, this work demonstrated that the native aggregation state had a strong relationship with deamidation behavior and it was the dominant factor for the unfolding extent of molecular structure and for determining the change trend of deamidation. It should be noted that the concentration scale we reported are limited (even though we had done lowed to 0.0001% only for DD, DH and Zeta potential determinations). We only probed the assembly structure of the polymer solution in a optics from deamidaiton. Works on viscoelastic properties of wheat gluten proteins and their assemblies as well as their dynamics in this reaction system are in progress. Acknowledgments This research was supported by the National Natural Science Foundation of China (no. 31201287) and Natural Science Foundation of Fujian province (no. 2015J05067). References Adler-Nissen, J., 1976. Enzymatic hydrolysis of proteins for increased solubility. J. Agric. Food Chem. 24, 1090e1093. Agyare, K.K., Xiong, Y.L., Addo, K., 2008. Influence of salt and pH on the solubility and structural characteristics of transglutaminase-treated wheat gluten hydrolysate. Food Chem. 107, 1131e1137. Benelhadj, S., Gharsallaoui, A., Degraeve, P., Attia, H., Ghorbel, D., 2016. Effect of pH on the functional properties of Arthrospira (Spirulina) platensis protein isolate. Food Chem. 194, 1056e1063. Chang, C.C.Y., Miyazaki, A., Dong, R., Kheirollah, A., Yu, C., Geng, Y., Higgs, H.N., Chang, T.-Y., 2010. Purification of recombinant acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) from H293 cells and binding studies between the enzyme and substrates using difference intrinsic fluorescence spectroscopy. Biochemistry 49, 9957e9963. Chou, P.Y., Fasman, G.D., 1977. b-Turns in proteins. J. Mol. Biol. 115, 135e175. Dahesh, M., Banc, A., Duri, A., Morel, M.H., Ramos, L., 2014. Polymeric assembly of gluten proteins in an aqueous ethanol solvent. J. Phys. Chem. B 118, 11065e11076. Dahesh, M., Banc, A., Duri, A., Morel, M.-H., Ramos, L., 2016. Spontaneous gelation of wheat gluten proteins in a food grade solvent. Food Hydrocoll. 52, 1e10. DuPont, F.M., Samoil, V., Chan, R., 2008. Extraction of up to 95% of wheat (Triticum aestivum) flour protein using warm sodium dodecyl sulfate (SDS) without reduction or sonication. J. Agric. Food Chem. 56, 7431e7438. Kato, A.T., Lee, Yoshinori, Matsudomi, Naotoshi, Kobayashi, Kunihiko, 1987. Effects of deamidation with chymotrypsin at pH 10 on the functional properties of proteins. J. Agric. Food Chem. 35, 285e288. €hler, P., Wieser, H., 2007. Effect of hydrostatic pressure and Kieffer, R., Schurer, F., Ko temperature on the chemical and functional properties of wheat gluten: studies on gluten, gliadin and glutenin. J. Cereal Sci. 45, 285e292. Laemmli, U.K., Beguin, F., Gujer-Kellenberger, G., 1970. A factor preventing the major head protein of bacteriophage T4 from random aggregation. J. Mol. Biol. 47, 69e85. Lagrain, B., Goderis, B., Brijs, K., Delcour, J.A., 2010. Molecular basis of processing wheat gluten toward biobased materials. Biomacromolecules 11, 533e541. Lasztity, R., 1986. Recent results in the investigation of the structure of the gluten complex. Food/Nahrung 30, 235e244. Liao, L., Liu, T.-x., Zhao, M.-m., Zhao, H.-f., Cui, C., 2011. Aggregation behavior of wheat gluten during carboxylic acid deamidation upon hydrothermal treatment. J. Cereal Sci. 54, 129e136. Liao, L., Liu, T.-X., Zhao, M.-M., Cui, C., Yuan, B.-E., Tang, S., Yang, F., 2010a. Functional, nutritional and conformational changes from deamidation of wheat gluten with succinic acid and citric acid. Food Chem. 123, 123e130. Liao, L., Zhao, M., Ren, J., Zhao, H., Cui, C., Hu, X., 2010b. Effect of acetic acid deamidation-induced modification on functional and nutritional properties and conformation of wheat gluten. J. Sci. Food Agric. 90, 409e417. Matsudomi, N., Kato, A., Kobayashi, K., 1982. Conformation and surface properties of deamidated gluten. Agric. Biol. Chem. 46, 1583e1586. Pouplin, M., Redl, A., Gontard, N., 1999. Glass transition of wheat gluten plasticized with water, glycerol, or sorbitol. J. Agric. Food Chem. 47, 538e543.

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