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Comparison of the conformational and nutritional changes of deamidated wheat gliadin by citric acid and hydrochloric acid
Journal of Cereal Science xxx (2014) 1e8
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Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs
Comparison of the conformational and nutritional changes of deamidated wheat gliadin by citric acid and hydrochloric acid Chaoying Qiu a, Weizheng Sun a, Guowan Su a, Chun Cui a, Mouming Zhao a, b, * a b
College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 November 2013 Received in revised form 3 February 2014 Accepted 4 February 2014
Deamidated wheat gliadins were prepared using hydrochloric acid (HCl) and citric acid (HDWG and CADWG), respectively. Their secondary structure, protein molecular interaction, thermal properties and nutritional changes were compared by Fourier transform infrared spectroscopy (FTIR), Raman spectrum, atomic force microscopy (AFM), differential scanning calorimetry (DSC), and amino acid analysis, respectively. Secondary structures and molecular vibration model showed slight difference between HDWG and CADWG, but significant difference between control gliadin and deamidated wheat gliadins. HDWG and CADWG had different shapes on the mica surface that the former showed some extent of linear aggregates and fibrils while the latter mainly exhibited globular aggregates. This result was further supported by thermal characteristics that CADWG had higher denaturation temperature than control gliadin and HDWG. Citric acid deamidation could increase the Lysine content and better maintain the total essential amino acids of in vitro digests of gliadin compared with HCl. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Wheat gliadin Deamidation Conformation Amino acid composition
1. Introduction Wheat gluten is the main source of plant proteins and sufficiently produced worldwide. It is mainly composed of glutenins and gliadins according to their polymerization properties. Gliadins are one kind of monomeric globular protein with low molecular weight (30 kDae100 kDa), bonded by intra-molecular disulfide bonds. They are classified into a-, b-, g- and u-gliadins according to biochemical and genetic properties (Thewissen et al., 2011). Gliadins are formed by a non-repetitive domain rich in a-helix structure and by a heterogeneous repetitive domain rich in b-reverse turns (Secundo and Guerrieri, 2005). A solution of gliadins was more effective in lowering surface tension than glutenin (Thewissen et al., 2011). However, the use of wheat gliadins is mainly in film materials or encapsulation prepared in alcohol solution. Its use as a functional protein is limited by the low solubility due to many nonpolar amino acids and glutamines (Wang et al., 2006). Chemical and enzymatic modifications have been made to improve the foaming and emulsifying properties of gliadins (Thewissen et al., 2011; Majzoobi et al., 2012). Deamidation is an
* Corresponding author. State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China. Tel./fax: þ86 20 87113914. E-mail address: [email protected] (M. Zhao).
effective way to increase protein solubility and improve functional properties by reducing intra-intermolecular hydrogen bonding and enhancing electrostatic repulsion between protein molecules (Day et al., 2009; Qiu et al., 2013). The most common method for the deamidation is by HCl treatment. However, considerable hydrolysis of the peptide bonds is inevitable to produce bitter tasting peptides and also reduces the processing property (Liao et al., 2010). Carboxylic acid was also reported to be a better choice to deamidate, which can reduce the potential risk for celiac people and create little proteolysis (Qiu et al., 2013). Deamidation under acid condition and high temperature caused changes in protein conformation due to increase of electrostatic repulsion. The amphiphilic characteristics of protein increased as a result of increase of negatively charged polar groups. The structure and conformational properties of protein could affect the nutritional and functional properties of the processed foods. A better understanding of the physicochemical, conformational properties and thermal properties of deamidated wheat gliadin can enhance their potential utilization as a kind of new food ingredient. Although deamidated wheat gluten and gliadin by different acids and their functional properties have been reported (Qiu et al., 2013; Wong et al., 2012), the research for deamidated gliadins focuses little on their thermal characteristics and amino acid composition changes. In addition, molecular vibration reflected by Raman spectrum and nanometer images by AFM can offer more structural
http://dx.doi.org/10.1016/j.jcs.2014.02.003 0733-5210/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Qiu, C., et al., Comparison of the conformational and nutritional changes of deamidated wheat gliadin by citric acid and hydrochloric acid, Journal of Cereal Science (2014), http://dx.doi.org/10.1016/j.jcs.2014.02.003
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C. Qiu et al. / Journal of Cereal Science xxx (2014) 1e8
information of gliadin (McMaster et al., 1999). Most research focused on the individual purified gliadin (McMaster et al., 1999; Paananen et al., 2006). Although the crude gliadin samples are more practically used for application, the research on the conformation and characteristics of crude gliadins is still limited. In our recent work, we investigated the properties of the citric acid deamidated wheat gliadins and found that citric acid deamidation remarkably increased the protein solubility and emulsion stability at neutral pH (Qiu et al., 2013). Some differences were observed between the performance of HCl deamidated gliadin and citric acid deamidated gliadin in emulsions (Day et al., 2009; Qiu et al., 2013). The functional properties of the proteins are related with physicochemical and conformational properties. In this paper, citric acid and HCl deamidation were carried out to compare the structural characteristics of gliadin treated with different acids. 2. Materials and methods 2.1. Materials Wheat gluten isolate was prepare by hand washing dough using distilled water from the strong wheat cultivar Jinan17 supplied by Jinhe flour Co., Ltd. (Foshan, Guangdong, China) after extraction of the lipid with chloroform by overhead agitator at 1500 rpm for 30 min. Then the gluten was freeze-dried. Protein content was 82.5% as determined by the Kjeldahl method (N 5.7). Pepsin from porcine gastric mucosa (400 U/mg solid) and pancreatin from porcine pancreas (8 USP) were purchased from Sigma Chemical Co. Ltd. (St. Louis, USA). All other chemicals and solvents were of analytical grade. 2.2. Preparation of deamidated wheat gliadin Wheat gluten (8%, w/v) was mixed with HCl (0.09 M) or citric acid (0.2 M) to form suspensions, respectively. The suspensions were hydrated in a shaking water bath at 70 C for 10 h and 20 h to get deamidated samples with similar deamidation degree levels (20% and 40%) by the two acids. The deamidation degree was determined according to Kato et al. (1987). Then the suspensions were neutralized with sodium hydroxide (1 M), dialyzed for 36 h at 4 C against distilled water and freeze-dried. Gliadin and deamidated gliadins were extracted according to the method of Thewissen et al. (2011). Wheat gluten or deamidated wheat gluten was mixed with two quantities (ten-fold) of 70% (v/v) ethanol. After stirring (30 min at 20 C) and centrifugation at 10,000 g for 10 min at 20 C (Hitachi Koki Co. Ltd., Tokyo, Japan), the supernatants were pooled. Before the second extraction step, the cohesive glutenin residue was mechanically disrupted with a spatula. Ethanol in the supernatants was removed by rotary evaporation (50 C), and the gliadin fractions were freeze-dried. Control gliadin, HDWG and CADWG with deamidation degrees of 20% and 40% (C, H-1, H-2, CA1 and CA-2) were used for the following research. 2.3. Nitrogen soluble index (NSI) and protein surface hydrophobicity (S0) Protein dispersions (10 mg/mL, dissolved in deionized water) were adjusted to a specific value within the range of pH 3e10 by 1 M HCl or NaOH. The dispersions were agitated with a magnetic stirrer for 1 h at room temperature, and then centrifuged at 12,000 g for 20 min. Protein content of the supernatant was determined according to Lowry et al. (1951). Bovine serum albumin was used as the standard. Protein solubility was calculated as nitrogen solubility index (NSI) ¼ (protein content of supernatant/ amount of proteins added) 100%.
Surface hydrophobicity (S0) was determined by the hydrophobicity fluorescence probe 1-anilino-8-naphthalenesulfonate (ANS) using an F7000 fluorescence spectrophotometer (Hitachi Co., Japan). A series of dilutions of each sample were made with 10 mM phosphate buffer (pH 7.0) to obtain a range of protein concentrations at 0.05, 0.1, 0.2, 0.5, 1.0 mg/mL. Then, 4 mL protein dispersion was mixed with 20 mL of 8 mM ANS. Fluorescence intensity (FI) was measured at the wavelengths of 390 nm (excitation) and 470 nm (emission), with a constant excitation and emission slit of 5 nm. The FI for each sample was then computed by subtracting the FI attributed to protein in buffer without ANS. The initial slope of the FI versus protein concentration plot was calculated by linear regression analysis and used as an index of S0. 2.4. Size exclusion high performance liquid chromatography (SEHPLC) The molecular weight of control gliadin, HDWG and CADWG was analyzed using size exclusion chromatography conducted according to Wong et al. (2012) with modifications. The chromatographic apparatus consisted of a Waters HPLC 600 system (Waters, Division of Millipore, Milford, MA, USA). Samples were prepared in 50 mM sodium phosphate buffer (pH 7.2) containing 0.5 wt% SDS. These samples (20 mL) were then injected into a TSK-GEL G4000SW column (7.5 mm i.d. 600 mm length, Tokyo, Japan) and run at 1 mL/min in a 50 mM sodium phosphate buffer (pH 7.2) containing 200 mM NaCl. The elution profiles were monitored at 214 nm with a UV detector. Thyroglobulin (669 kDa), aldolase (158 kDa), conalbumin (75 kDa) and ovalbumin (43 kDa) (Sigma co. St. Louis, MO, USA) were used as the standard proteins. The estimation of molecular weight was based on the elution profiles of protein standards. 2.5. Differential scanning calorimetry (DSC) The thermal characteristics of proteins were determined by DSC measurements performed using a TA Q100-DSC thermal analyzer (TA Instruments, New Castle, DE 19720, USA) according to Falcão-Rodrigues et al., (2005). The calorimeter was calibrated using an indium standard. Samples were equilibrated at water activity values of 0.79 (about 50% moisture). Samples of 5.0 0.1 mg were weighed into aluminum pans and covers were hermetically sealed into place. An empty, hermetically sealed pan was used as reference. The samples were analyzed at a rate of 10 C/min from 30 to 100 C. Denaturation temperature (Td) and denaturation enthalpy (DH) were analyzed from the thermograms by the Universal Analysis 2000 software, Version 4.1D (TA Instruments-Waters LLC). 2.6. Fourier transform infrared spectroscopy (FTIR) FTIR spectra of samples were recorded using a Nicolet 8210E FTIR spectrometer (Nicolet, WI) equipped with a deuterated triglycine sulfate detector. The sample powder (maintained at ambient temperature) included 1 mg sample per 200 mg of KBr. FTIR spectra were obtained from wave numbers from 400 to 4000 cm1 during 128 scans, with 2 cm1 resolution (Paragon 1000, PerkineElmer, USA). Interpretation of the changes in the overlapping amide band (1580e1700 cm1) components was made possible by deconvolution using Peak-Fit v 4.12 software (SPSS Inc., Chicago, IL). Full-width at half-maximum (fwhm) was 16 cm1 and kept constant for all peaks during deconvolution. Protein secondary structures were determined as percentages of a-helix, b-sheets, bturn and random coils according to Secundo and Guerrieri (2005).
Please cite this article in press as: Qiu, C., et al., Comparison of the conformational and nutritional changes of deamidated wheat gliadin by citric acid and hydrochloric acid, Journal of Cereal Science (2014), http://dx.doi.org/10.1016/j.jcs.2014.02.003
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Fig. 1. Characteristics of control gliadin (C), HDWG (H-1 and H-2) and CADWG (CA-1 and CA-2): (a) Solubility; (b) SE-HPLC elution profiles; (c) Surface hydrophobicity. Different letters (aee) on the top of each column indicates significant difference (p < 0.05).
2.7. Raman spectroscopy Raman spectra with an excitation at 632.8 nm (laser He/Ne, less than 10 mW on the sample) were obtained at room temperature, using a LABRAM-Aramis spectrometer (Horiba Jobin-Yvon, France). Spectral repeatability was smaller than 0.2 cm1, and spectral resolution was better than 1 cm1. Protein samples were placed on microscope slides. The laser was then focused on the samples. Raman spectra of at least three different positions were collected from 400 to 4000 cm1. The spectral data from the scans of samples in the Raman spectrophotometer were baseline corrected and normalized according to the protein phenylalanine peak at 1003 1 cm1.
centrifuged at 11,000 g for 15 min to evaluate amino acids in the supernatant. Samples of control gliadin and deamidated gliadins with deamidation time of 20 h and corresponding digests were hydrolyzed in sealed, evacuated glass tubes with 6 M HCl at 110 C for 24 h. The analysis was performed with a Membrapure A-300 amino acid analyzer (Membrapure Co., Frankfurt, Germany) equipped with a C18 column (4.6 mm 125 mm) for amino acid separation. Postcolumn reaction with ninhydrin yielded amino acid derivatives. The concentrations of the amino acids were determined from their respective absorption intensities, which were calibrated to the known concentrations of amino acid standards. 2.10. Statistical analysis
2.8. Atomic force microscopy (AFM) analysis AFM imaging was made using Tapping mode on Nanoscope IIIa Multimode Scanning Probe Microscope (Veeco Instruments, Santa Barbara, CA, USA). Aliquots (2 mL) of dispersions (20 mg/mL with distilled water and filtered through a 0.22 mm filter) were placed on a freshly cleaved mica disk and air-dried for 10 min at ambient temperature. Approximately 5e10 images were taken for each preparation.
All experiments were conducted in triplicate. Statistical calculation was investigated using the statistical package SPSS 11.5 (SPSS Inc., Chicago, IL) for one-way ANOVA. A least significant difference (LSD) test with a confidence interval of 95% was used to compare the means. All the data were expressed as mean standard deviation of triplicate determinations. 3. Results and discussion
2.9. Amino acid analysis
3.1. Physicochemical properties of deamidated wheat gliadins
In vitro digests of protein samples were carried out according to Zhu et al. (2008). Gliadin and deamidated gliadins (3% w/v, in MilliQ water) were adjusted to pH 2.0 with 1 M HCl and pepsin was added (4% w/w, protein basis). After incubation at 37 C for 1 h, the solution was adjusted to pH 5.3 with 0.9 M NaHCO3. Pancreatin (4% w/w, protein basis) was then added, and the pH was adjusted to 7.5 with 1 M NaOH. The solution was incubated at 37 C for 2 h. The digests were neutralized, heating at 90 C for 10 min and
3.1.1. Solubility Solubility is a physical characteristic closely related to its functional properties. Fig. 1a shows that the solubility of gliadin significantly increased and the isoelectric point shifted to lower pH values after deamidation. Gliadin in its natural form has low solubility near neutral pH values for it has a large amount of amide groups (Gln and Asn). The substitution of amide groups by carboxylic groups after deamidation can increase the net negative
Please cite this article in press as: Qiu, C., et al., Comparison of the conformational and nutritional changes of deamidated wheat gliadin by citric acid and hydrochloric acid, Journal of Cereal Science (2014), http://dx.doi.org/10.1016/j.jcs.2014.02.003
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Fig. 2. (a) Deconvoluted FTIR spectra in the amide I region of control gliadin (C), HDWG (H-1 and H-2) and CADWG (CA-1 and CA-2). (b) Relative areas of the bands fitted to the Fourier-deconvoluted spectra. (NH2)þ hydrated extended chains at 1596 cm1 and 1608 cm-1 a-helix: band at 1652e1654 cm1 b-sheets: bands at 1619e1620 cm1, 1630e 1632 cm1, 1677 cm1 and 1688 cm1 b-turn: band at 1664e1666 cm1. Random coil: band at 1643 cm1. Different letters (aed) on the top of each column (the same band) indicates significant difference (p < 0.05).
charge, thus can increase the solubility of gliadin at neutral pH (Qiu et al., 2013). Wheat gliadin deamidated by citric acid and HCl showed the isoelectric point around 5 and 4, respectively. 3.1.2. Molecular weight distribution determination by SE-HPLC Size exclusion chromatography analysis was carried out to examine changes of the molecular size of gliadin and deamidated gliadins (Fig. 1b). It seems that deamidation caused the two peaks of gliadin to merge. This phenomenon might be caused by the conformational changes of proteins. In Fig. 1b, both HCl and citric acid deamidation caused little hydrolysis and deamidated gliadins showed similar molecular weight distribution. This was because the acid concentration used here was relatively low and the temperature of 70 C was selected to minimize protein aggregation or hydrolysis. It is known that the effect of high temperature on protein structure is the irreversible unfolding of protein, aggregation and proteolysis (Liao et al., 2010). 3.1.3. Surface hydrophobicity (S0) Fig. 1c shows that S0 of the deamidated gliadins was significantly higher than the control gliadin. Deamidation caused unfolding of the protein and rearrangement of hydrophobic forces, so the
hydrophobic zones hidden inside the protein were exposed. Surface hydrophobicity is an important factor in determining the emulsifying properties. The increased hydrophobicity in protein would cause a better molecular arrangement at the oil-water interface, giving more stable emulsion (Matsudomi et al., 1985). As shown in Fig. 1c, S0 was higher in the CADWG. Gliadins further deamidated for 20 h (CA-2) exhibited a slight decrease of S0. Heating during deamidation could promote the unfolded protein to reassociate by hydrophobic interaction between exposed hydrophobic amino acid residues, which reduced hydrophobic moieties that could bind to the fluorescence probe (Zhao et al., 2011). 3.2. Conformational characteristics 3.2.1. Second structure characterization by FTIR The spectra in the amide I region were deconvoluted. Band fitting with Gaussian band shapes was performed on the deconvoluted spectra to estimate secondary structure content. Nine major bands were distinctly observed in the amide I bands region of the deconvoluted curve of control gliadin and deamidated gliadins (Fig. 2a). The amide I band mainly originated from C]O stretching with some contribution of the NeH vibration (Secundo and
Please cite this article in press as: Qiu, C., et al., Comparison of the conformational and nutritional changes of deamidated wheat gliadin by citric acid and hydrochloric acid, Journal of Cereal Science (2014), http://dx.doi.org/10.1016/j.jcs.2014.02.003
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Fig. 3. (a) Raman spectra of control gliadin (C), HDWG (H-1 and H-2) and CADWG (CA-1 and CA-2); (b) Normalized intensity of amide I and amide III Raman bands; (c) Normalized intensity of 1780 cm1 Raman bands; (d) Normalized intensity of 1340 cm1 and 757 cm1 Raman bands; (e) Normalized intensity of 1448 cm1 and 2934 cm1 Raman bands; (f) Tyr doublet around 854 and 830 cm1 (854 cm1/830 cm1). Different letters (aee) on the top of each column (the same band) indicates significant difference (p < 0.05).
Guerrieri, 2005). The strong absorption at 1652 cm1 indicated that the content of a-helix was high in gliadins and this component might also contain some random coil conformation and glutamine side chain (Secundo and Guerrieri, 2005). The spectral position of the components of amide I band in deamidated gliadins showed several differences of Fourier deconvoluted spectra with respect to the control gliadin. This indicated that the protein conformation was modified. Amide I band of deamidated gliadins showed an obvious peak at 1643 cm1. However, this peak was evident only by the curve-fitting analysis in control gliadin (Fig. 2a and b). This suggested that protein was unfolded and the intensity of random coil increased. Fig. 2b shows the relative proportion of the bands, which are assigned to the structure components according to the previous references (Secundo and Guerrieri, 2005; Ukai et al., 2008). Control gliadin contained similar structure position and secondary structure content compared with the result of Secundo and Guerrieri (2005). The intensity of the band assigned to random coil (1643 cm1) was evidently increased and the band assigned to bturn (1665 cm1) was slightly increased after deamidation. Deamidation turned glutamine residues to negatively charged glutamic acid residues, and thus led to the decrease in hydrogen-bonded bsheets structures and increase in more flexible structures like bturn and random coil (Wong et al., 2012). The band intensity of bsheets at 1619, 1677 and 1690 cm1 decreased while at 1632 cm1, slightly increased after deamidation (Fig. 2b). The slight shift of band at 1632 cm1 to 1630 cm1 (Fig. 2a) and increase of intensity suggested the increase in extended b-sheets structures at the expense of antiparallel b-sheets structure which features protein
aggregates at 1619 cm1 and 1690 cm1 (Choi and Ma, 2005; Wang et al., 2011). The NH2 scissoring of the glutamine side chains (1596 cm1 and 1608 cm1) showed significant decrease as a result of the deamidation process. Both HDWG of the two deamidation degrees showed similar deconvoluted spectra shape which was different from CADWG. Compared with control gliadin, CADWG had higher content of a-helix while HDWG had lower content. The b-turn content of CADWG (1665 cm1) was also higher than HDWG (Fig. 2b). 3.2.2. Raman spectra and molecular vibration Raman spectroscopy is a direct and a non-invasive technique for studying structural changes of proteins in solid states without destroying samples (Wong et al., 2009). Fig. 3a shows the typical Raman spectra and normalized intensity of several bands of gliadin and deamidated gliadins. Proteins with a high proportion of a-helical contents show an amide I band centered at around 1650e 1660 cm1. The amide I and amide III bands of all the samples were centered at 1656 and 1248 cm1, respectively (Fig. 1a), indicating that the content of a-helical and random coil was high in the protein (Herrero et al., 2008). The intensity and location of the phenylalanine band (1003 cm1) is not sensitive to protein conformation or to the microenvironment, so it was used as an internal standard to normalize the molecular vibration intensity (Herrero et al., 2008). Raman bands corresponding to amide I and III can be used to characterize protein backbone conformation (Wong et al., 2009). As shown in Fig. 3b, after deamidation, the intensity of amide I decreased while amide III increased. This indicated protein
Please cite this article in press as: Qiu, C., et al., Comparison of the conformational and nutritional changes of deamidated wheat gliadin by citric acid and hydrochloric acid, Journal of Cereal Science (2014), http://dx.doi.org/10.1016/j.jcs.2014.02.003
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unfolding (Gómez et al., 2013). The band at 1780 cm1 was attributed to the carboxylic acid vibration (Wong et al., 2009). In Fig. 3c, the intensity of the 1780 cm1 band of HDWG and CADWG increased, indicating the deamidation process. The bands near 757 cm1 and 1340 cm1 provide information about the microenvironment of the tryptophan residues. Decreased intensity at 1340 cm1 indicated more exposed tryptophan residues (Sun et al., 2011). The intensities of 854 cm1/830 cm1 was proposed as a means of determining whether the Tyr residue is solvent exposed or buried. When the intensity of the band at 854 cm1 is higher than the band at 830 cm1, the Tyr residues are exposed and when the intensity of the band at 854 cm1 is lower than the band at 830 cm1, the Tyr residues are buried within the protein network (Sun et al., 2011). The Tyr doublet ratio (854 cm1/830 cm1) of HDWG did not change significantly while the intensity first decreased then increased for CADWG at 10 h and 20 h. The ratio was always lower than 1, suggesting Tyr residues were mainly buried even after deamidation (Sun et al., 2011). The CeH stretching vibrations were represented by Raman bands at 1448 cm1 and 2934 cm1. As shown in Fig. 3e, the intensity of the CeH bending vibration band was evidently increased after deamidation, indicating protein unfolding and exposure of aliphatic side chains (Zhao et al., 2004). 3.2.3. Morphology analysis by AFM AFM is a nondestructive method to characterize the three dimensional structure of materials of single molecules as well as complex composite structures in the nanometer to 100 mm range (Guo et al., 2005). Fig. 4 shows the AFM images of gliadins and deamidated gliadins. Control gliadin exhibited monodisperse close packed globular shapes. This indicated the strong proteineprotein interaction while weak interactions with the hydrophilic mica (Paananen et al., 2006). Gliadins are intrinsically cohesive as they have a large number of glutamine residues in the polypeptide chains, causing aggregation through hydrogen bonding.
Fig. 5. DSC thermograms of control gliadin (C), HDWG (H-1 and H-2) and CADWG (CA1 and CA-2).
Deamidated gliadins exhibited significant difference compared with control gliadin for their unfolding of structures. For HDWG and CADWG at 10 h (Fig. 4b and d), there were some big clusters of protein formed and proteins still maintain globular form. At 20 h, HDWG showed some extended linear fibrillar structure (Fig. 4c) while CADWG showed mainly dispersed globular aggregates though a few fibrils can also be observed (Fig. 4e). These fibrils were reported to be assembled from monomer or dimer units of gliadin by ioneprotein interaction (McMaster et al., 1999). These results indicated that HDWG formed more flexible aggregates than CADWG. The difference in the tertiary structure of the protein probably induced the difference of proteineprotein interaction and protein-surface interaction, causing their different shapes.
Fig. 4. Tapping mode AFM images of (a) control gliadin (C), HDWG and CADWG of 10 h (b, d) and 20 h (c, e). The scan size is 3.00 mm 3.00 mm, and the z scale is 20 nm.
Please cite this article in press as: Qiu, C., et al., Comparison of the conformational and nutritional changes of deamidated wheat gliadin by citric acid and hydrochloric acid, Journal of Cereal Science (2014), http://dx.doi.org/10.1016/j.jcs.2014.02.003
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Table 1 Amino acids composition (mol%) of control gliadin, HDWG (H-2) and CADWG (CA-2) and their digests. Samples
Digested samples
C Asp þ Asn Thr Ser Glu þ Gln Gly Ala Cys Val Met Ile Leu Tyr Phe His Lys Arg Pro Trp Essential AA
2.24 2.04 5.09 30.27 2.66 2.64 2.34 3.98 1.12 3.87 7.15 2.03 5.01 3.09 1.79 4.51 19.49 0.66 25.63
H-2
a
0.05 0.04a 0.31a 1.07a 0.12a 0.15a 0.20a 0.05a 0.02a 0.32a 0.13a 0.11a 0.31a 0.15a 0.02b 0.22a 0.65a 0.02a 0.91a
1.91 2.01 4.91 33.35 4.44 2.96 2.43 3.48 0.49 3.36 6.86 2.24 4.26 2.62 1.66 5.21 17.21 0.61 22.72
CA-2
b
0.03 0.06a 0.10a 2.10a 0.23b 0.13a 0.12a 0.08b 0.06b 0.04b 0.09b 0.20b 0.34b 0.19b 0.05a 0.23b 0.33b 0.04ab 0.76b
2.16 2.32 5.38 31.92 4.76 2.81 2.32 3.45 0.47 3.31 6.51 2.30 4.17 2.86 1.90 4.15 18.61 0.60 22.73
C
a
0.11 0.10b 0.35a 1.33a 0.28b 0.10a 0.16a 0.21b 0.07b 0.15b 0.29b 0.14b 0.25b 0.09ab 0.06c 0.36a 0.26a 0.02b 1.15b
2.74 2.21 5.24 31.14 3.58 2.81 2.27 3.66 1.02 3.57 6.71 2.51 4.68 2.01 0.93 4.00 20.45 0.49 23.26
H-2
a
0.06 0.04a 0.11a 1.29a 0.15a 0.16a 0.14a 0.10a 0.03a 0.24a 0.20a 0.21a 0.11a 0.06a 0.03a 0.15a 1.14a 0.03a 0.78a
2.65 2.40 5.34 32.26 5.74 3.18 2.44 3.65 0.12 3.51 6.60 2.37 4.30 2.00 0.90 2.37 19.53 0.51 22.09
CA-2
ab
0.10 0.07b 0.08a 1.15a 0.31b 0.02b 0.10a 0.04a 0.02c 0.08a 0.13a 0.13a 0.06b 0.24a 0.02a 0.10b 1.06ab 0.02a 0.45b
2.55 2.46 5.47 31.46 5.25 3.10 2.38 4.16 0.27 3.34 6.50 2.41 4.33 3.04 1.47 4.21 17.21 0.64 22.93
0.07b 0.05b 0.14a 2.30a 0.27b 0.12b 0.04a 0.03b 0.02b 0.05a 0.06a 0.05a 0.04b 0.18b 0.04b 0.07a 1.51b 0.02b 0.31ab
aed
Different letters in the same row of the samples or digested samples indicate significant difference (p < 0.05).
Deamidated gliadin with higher negative charge had higher adsorption ability on the hydrophilic mica, revealing different aggregation structures on the substrate. 3.2.4. Thermal characters of gliadin and deamidated gliadins DSC is a pertinent technique for evaluating the state and nature of mainly tertiary conformations of food proteins and the role of chemical forces in stabilizing the protein conformation (Yin et al., 2011). Fig. 5 shows that control gliadin exhibited a Td value of 54.5 C and the enthalpy (DH) was 2.41 J/g. This Td value was a little higher than a previous report of gliadin by Falcão-Rodrigues et al. (2005) while a little lower than the result of León et al. (2003). This might be caused by the different wheat varieties or protein purity. The narrow shape of the peak of deamidated gliadins suggested that they denatured in a more cooperative way (Yin et al., 2011). HDWG exhibited Td values of 64.96 C and 63.80 C and DH of 4.81 J/g and 5.80 J/g at deamidation times of 10 h and 20 h, respectively, which was different from previous reports that HCl deamidated wheat protein showed no obvious endothermic peak during heating (Day et al., 2009; Friedli and Howell, 1996). This might be explained by the different DSC test conditions. It was reported that water content played a role in endothermic transition detection and water content around 50% was the most suitable condition to detect the endotherm (Falcão-Rodrigues et al., 2005; León et al., 2003). CADWG showed Td at 73.58 C and 75.57 C and DH of 5.79 J/g and 7.65 J/g at deamidation times of 10 h and 20 h, respectively. Unfolding of native protein after deamidation might cause the exposure of buried apolar groups, improving proteineprotein interactions by hydrophobic association. Thus, heating the unfolding gliadins would require the rupture of more hydrophobic groups than the native protein, resulting in higher Td (Choi and Ma, 2005). The higher Td value suggested more compact tertiary conformation of the polypeptides. Protein denaturation involves conformational changes from the native structure by disruption of chemical forces that maintain the structural integrity of the protein molecules like hydrogen bonds, hydrophobic bonds, ionic interactions and covalent disulfide bonds (Falcão-Rodrigues et al., 2005). AFM results (Fig. 4) showed that HDWG exhibited fibrillar aggregates which were more flexible. It was different from CADWG with mainly globular shapes. This could explain why HDWG exhibited lower denaturation temperatures than CADWG.
3.3. Amino acids composition Table 1 lists the amino acid composition of wheat gliadin and deamidated gliadins. Glu and Pro are the most abundant amino acids of wheat gliadin. The content of Lys in HDWG and its digest was decreased. However, it was increased in the digest of CADWG. Proteineprotein interactions involving ε-NH2 groups with the amide groups of Asp and Glu that resulted in cross-linking tend to cause destruction of Lys (Guo et al., 1999). Lys is considered to be the most important essential amino acid in cereal protein. This result suggested that the citric acid deamidation process was milder and more suitable for preparing deamidated gliadin with higher nutritional properties. Moreover, the digests of CADWG had higher amount of essential amino acids compared with HDWG. This indicated that citric acid deamidation can better maintain the essential amino acids of wheat gliadin. In addition, the loss of Met might be due to damage of the methyl-thiol group during the deamidation process (Guo et al., 1999). 4. Conclusions This work demonstrated the effects of HCl and citric acid deamidation on the structure characteristics of wheat gliadin. For these two acids, deamidation led to a slight difference of secondary structure change of wheat gliadin and side chain vibration models. Gliadin deamidated by HCl exhibited mainly flexible associated fibrils while globular aggregates by citric acid. The thermal properties indicated that CADWG had higher thermal stability than HDWG. The difference was supposed to exist in the way of protein aggregation and proteineprotein interaction of the two acids. In addition, citric acid deamidation was a better way to increase the Lys content and maintain the essential amino acids of gliadin after digestion. These results will be useful for predicting the structuree function relationships of wheat gliadins. Acknowledgments The authors are grateful to the National High Technology Research and Development Program of China (863 Program) (No. 2013AA102201), the National Science-Technology Supporting Project for 12th Five-Year Plan (2012BAD37B08) and the National
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Natural Science Foundation of China (No. 31171783 and No. 31201416) for their financial supports. References Choi, S.M., Ma, C.Y., 2005. Conformational study of globulin from common buckwheat (Fagopyrum esculentum Moench) by Fourier transform infrared spectroscopy and differential scanning calorimetry. J. Agric. Food Chem. 53, 8046e 8053. Day, L., Xu, M., Lundin, L., Wooster, T.J., 2009. Interfacial properties of deamidated wheat protein in relation to its ability to stabilize oil-in-water emulsions. Food Hydrocoll. 23, 2158e2167. Falcão-Rodrigues, M.M., Moldão-Martins, M., Beirão-da-Costa, 2005. Thermal properties of gluten proteins of two soft wheat varieties. Food Chem. 93, 459e 465. Friedli, G.L., Howell, N., 1996. Gelation properties of deamidated soluble wheat proteins. Food Hydrocoll. 10, 255e261. Gómez, A.V., Ferrer, E.G., Añón, M.C., Puppo, M.C., 2013. Changes in secondary structure of gluten proteins due to emulsifiers. J. Mol. Struct. 1033, 51e58. Guo, M., Flynn, A., Fox, P., 1999. Heat-induced changes in the nutritional properties of sodium caseinate. Int. Dairy J. 9, 243e247. Guo, Y.C., Liu, Z.D., An, H.J., Li, M.Q., Hu, J., 2005. Nano-structure and properties of maize zein studied by atomic force microscopy. J. Cereal Sci. 41, 277e281. Herrero, A.M., Carmona, P., Cofrades, S., Jiménez-Colmenero, F., 2008. Raman spectroscopic determination of structural changes in meat batters upon soy protein addition and heat treatment. Food Res. Int. 41, 765e772. Kato, A., Tanaka, A., Matsudomi, N., Kobayashi, K.J., 1987. Deamidation of food proteins by protease in alkaline pH. J. Agric. Food Chem. 35, 224e227. León, A., Rosell, C.M., Barber, C.B., 2003. A differential scanning calorimetry study of wheat proteins. Eur. Food Res. Technol. 217, 13e16. 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 Chem. 123, 123e130. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265e275. Majzoobi, M., Abedi, E., Farahnaky, A., Aminlari, M., 2012. Functional properties of acetylated glutenin and gliadin at varying pH values. Food Chem. 133, 1402e 1407. Matsudomi, N., Sasaki, T., Tanaka, A., Kobayashi, K., Kato, Akio., 1985. Polymerization of deamidated peptide fragments obtained with the mild acid hydrolysis of ovalbumin. J. Agric. Food Chem. 33, 738e742.
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Comparison of the conformational and nutritional changes of deamidated wheat gliadin by citric acid and hydrochloric acid Chaoying Qiu a, Weizheng Sun a, Guowan Su a, Chun Cui a, Mouming Zhao a, b, * a b
College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 November 2013 Received in revised form 3 February 2014 Accepted 4 February 2014
Deamidated wheat gliadins were prepared using hydrochloric acid (HCl) and citric acid (HDWG and CADWG), respectively. Their secondary structure, protein molecular interaction, thermal properties and nutritional changes were compared by Fourier transform infrared spectroscopy (FTIR), Raman spectrum, atomic force microscopy (AFM), differential scanning calorimetry (DSC), and amino acid analysis, respectively. Secondary structures and molecular vibration model showed slight difference between HDWG and CADWG, but significant difference between control gliadin and deamidated wheat gliadins. HDWG and CADWG had different shapes on the mica surface that the former showed some extent of linear aggregates and fibrils while the latter mainly exhibited globular aggregates. This result was further supported by thermal characteristics that CADWG had higher denaturation temperature than control gliadin and HDWG. Citric acid deamidation could increase the Lysine content and better maintain the total essential amino acids of in vitro digests of gliadin compared with HCl. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Wheat gliadin Deamidation Conformation Amino acid composition
1. Introduction Wheat gluten is the main source of plant proteins and sufficiently produced worldwide. It is mainly composed of glutenins and gliadins according to their polymerization properties. Gliadins are one kind of monomeric globular protein with low molecular weight (30 kDae100 kDa), bonded by intra-molecular disulfide bonds. They are classified into a-, b-, g- and u-gliadins according to biochemical and genetic properties (Thewissen et al., 2011). Gliadins are formed by a non-repetitive domain rich in a-helix structure and by a heterogeneous repetitive domain rich in b-reverse turns (Secundo and Guerrieri, 2005). A solution of gliadins was more effective in lowering surface tension than glutenin (Thewissen et al., 2011). However, the use of wheat gliadins is mainly in film materials or encapsulation prepared in alcohol solution. Its use as a functional protein is limited by the low solubility due to many nonpolar amino acids and glutamines (Wang et al., 2006). Chemical and enzymatic modifications have been made to improve the foaming and emulsifying properties of gliadins (Thewissen et al., 2011; Majzoobi et al., 2012). Deamidation is an
* Corresponding author. State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China. Tel./fax: þ86 20 87113914. E-mail address: [email protected] (M. Zhao).
effective way to increase protein solubility and improve functional properties by reducing intra-intermolecular hydrogen bonding and enhancing electrostatic repulsion between protein molecules (Day et al., 2009; Qiu et al., 2013). The most common method for the deamidation is by HCl treatment. However, considerable hydrolysis of the peptide bonds is inevitable to produce bitter tasting peptides and also reduces the processing property (Liao et al., 2010). Carboxylic acid was also reported to be a better choice to deamidate, which can reduce the potential risk for celiac people and create little proteolysis (Qiu et al., 2013). Deamidation under acid condition and high temperature caused changes in protein conformation due to increase of electrostatic repulsion. The amphiphilic characteristics of protein increased as a result of increase of negatively charged polar groups. The structure and conformational properties of protein could affect the nutritional and functional properties of the processed foods. A better understanding of the physicochemical, conformational properties and thermal properties of deamidated wheat gliadin can enhance their potential utilization as a kind of new food ingredient. Although deamidated wheat gluten and gliadin by different acids and their functional properties have been reported (Qiu et al., 2013; Wong et al., 2012), the research for deamidated gliadins focuses little on their thermal characteristics and amino acid composition changes. In addition, molecular vibration reflected by Raman spectrum and nanometer images by AFM can offer more structural
http://dx.doi.org/10.1016/j.jcs.2014.02.003 0733-5210/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Qiu, C., et al., Comparison of the conformational and nutritional changes of deamidated wheat gliadin by citric acid and hydrochloric acid, Journal of Cereal Science (2014), http://dx.doi.org/10.1016/j.jcs.2014.02.003
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information of gliadin (McMaster et al., 1999). Most research focused on the individual purified gliadin (McMaster et al., 1999; Paananen et al., 2006). Although the crude gliadin samples are more practically used for application, the research on the conformation and characteristics of crude gliadins is still limited. In our recent work, we investigated the properties of the citric acid deamidated wheat gliadins and found that citric acid deamidation remarkably increased the protein solubility and emulsion stability at neutral pH (Qiu et al., 2013). Some differences were observed between the performance of HCl deamidated gliadin and citric acid deamidated gliadin in emulsions (Day et al., 2009; Qiu et al., 2013). The functional properties of the proteins are related with physicochemical and conformational properties. In this paper, citric acid and HCl deamidation were carried out to compare the structural characteristics of gliadin treated with different acids. 2. Materials and methods 2.1. Materials Wheat gluten isolate was prepare by hand washing dough using distilled water from the strong wheat cultivar Jinan17 supplied by Jinhe flour Co., Ltd. (Foshan, Guangdong, China) after extraction of the lipid with chloroform by overhead agitator at 1500 rpm for 30 min. Then the gluten was freeze-dried. Protein content was 82.5% as determined by the Kjeldahl method (N 5.7). Pepsin from porcine gastric mucosa (400 U/mg solid) and pancreatin from porcine pancreas (8 USP) were purchased from Sigma Chemical Co. Ltd. (St. Louis, USA). All other chemicals and solvents were of analytical grade. 2.2. Preparation of deamidated wheat gliadin Wheat gluten (8%, w/v) was mixed with HCl (0.09 M) or citric acid (0.2 M) to form suspensions, respectively. The suspensions were hydrated in a shaking water bath at 70 C for 10 h and 20 h to get deamidated samples with similar deamidation degree levels (20% and 40%) by the two acids. The deamidation degree was determined according to Kato et al. (1987). Then the suspensions were neutralized with sodium hydroxide (1 M), dialyzed for 36 h at 4 C against distilled water and freeze-dried. Gliadin and deamidated gliadins were extracted according to the method of Thewissen et al. (2011). Wheat gluten or deamidated wheat gluten was mixed with two quantities (ten-fold) of 70% (v/v) ethanol. After stirring (30 min at 20 C) and centrifugation at 10,000 g for 10 min at 20 C (Hitachi Koki Co. Ltd., Tokyo, Japan), the supernatants were pooled. Before the second extraction step, the cohesive glutenin residue was mechanically disrupted with a spatula. Ethanol in the supernatants was removed by rotary evaporation (50 C), and the gliadin fractions were freeze-dried. Control gliadin, HDWG and CADWG with deamidation degrees of 20% and 40% (C, H-1, H-2, CA1 and CA-2) were used for the following research. 2.3. Nitrogen soluble index (NSI) and protein surface hydrophobicity (S0) Protein dispersions (10 mg/mL, dissolved in deionized water) were adjusted to a specific value within the range of pH 3e10 by 1 M HCl or NaOH. The dispersions were agitated with a magnetic stirrer for 1 h at room temperature, and then centrifuged at 12,000 g for 20 min. Protein content of the supernatant was determined according to Lowry et al. (1951). Bovine serum albumin was used as the standard. Protein solubility was calculated as nitrogen solubility index (NSI) ¼ (protein content of supernatant/ amount of proteins added) 100%.
Surface hydrophobicity (S0) was determined by the hydrophobicity fluorescence probe 1-anilino-8-naphthalenesulfonate (ANS) using an F7000 fluorescence spectrophotometer (Hitachi Co., Japan). A series of dilutions of each sample were made with 10 mM phosphate buffer (pH 7.0) to obtain a range of protein concentrations at 0.05, 0.1, 0.2, 0.5, 1.0 mg/mL. Then, 4 mL protein dispersion was mixed with 20 mL of 8 mM ANS. Fluorescence intensity (FI) was measured at the wavelengths of 390 nm (excitation) and 470 nm (emission), with a constant excitation and emission slit of 5 nm. The FI for each sample was then computed by subtracting the FI attributed to protein in buffer without ANS. The initial slope of the FI versus protein concentration plot was calculated by linear regression analysis and used as an index of S0. 2.4. Size exclusion high performance liquid chromatography (SEHPLC) The molecular weight of control gliadin, HDWG and CADWG was analyzed using size exclusion chromatography conducted according to Wong et al. (2012) with modifications. The chromatographic apparatus consisted of a Waters HPLC 600 system (Waters, Division of Millipore, Milford, MA, USA). Samples were prepared in 50 mM sodium phosphate buffer (pH 7.2) containing 0.5 wt% SDS. These samples (20 mL) were then injected into a TSK-GEL G4000SW column (7.5 mm i.d. 600 mm length, Tokyo, Japan) and run at 1 mL/min in a 50 mM sodium phosphate buffer (pH 7.2) containing 200 mM NaCl. The elution profiles were monitored at 214 nm with a UV detector. Thyroglobulin (669 kDa), aldolase (158 kDa), conalbumin (75 kDa) and ovalbumin (43 kDa) (Sigma co. St. Louis, MO, USA) were used as the standard proteins. The estimation of molecular weight was based on the elution profiles of protein standards. 2.5. Differential scanning calorimetry (DSC) The thermal characteristics of proteins were determined by DSC measurements performed using a TA Q100-DSC thermal analyzer (TA Instruments, New Castle, DE 19720, USA) according to Falcão-Rodrigues et al., (2005). The calorimeter was calibrated using an indium standard. Samples were equilibrated at water activity values of 0.79 (about 50% moisture). Samples of 5.0 0.1 mg were weighed into aluminum pans and covers were hermetically sealed into place. An empty, hermetically sealed pan was used as reference. The samples were analyzed at a rate of 10 C/min from 30 to 100 C. Denaturation temperature (Td) and denaturation enthalpy (DH) were analyzed from the thermograms by the Universal Analysis 2000 software, Version 4.1D (TA Instruments-Waters LLC). 2.6. Fourier transform infrared spectroscopy (FTIR) FTIR spectra of samples were recorded using a Nicolet 8210E FTIR spectrometer (Nicolet, WI) equipped with a deuterated triglycine sulfate detector. The sample powder (maintained at ambient temperature) included 1 mg sample per 200 mg of KBr. FTIR spectra were obtained from wave numbers from 400 to 4000 cm1 during 128 scans, with 2 cm1 resolution (Paragon 1000, PerkineElmer, USA). Interpretation of the changes in the overlapping amide band (1580e1700 cm1) components was made possible by deconvolution using Peak-Fit v 4.12 software (SPSS Inc., Chicago, IL). Full-width at half-maximum (fwhm) was 16 cm1 and kept constant for all peaks during deconvolution. Protein secondary structures were determined as percentages of a-helix, b-sheets, bturn and random coils according to Secundo and Guerrieri (2005).
Please cite this article in press as: Qiu, C., et al., Comparison of the conformational and nutritional changes of deamidated wheat gliadin by citric acid and hydrochloric acid, Journal of Cereal Science (2014), http://dx.doi.org/10.1016/j.jcs.2014.02.003
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Fig. 1. Characteristics of control gliadin (C), HDWG (H-1 and H-2) and CADWG (CA-1 and CA-2): (a) Solubility; (b) SE-HPLC elution profiles; (c) Surface hydrophobicity. Different letters (aee) on the top of each column indicates significant difference (p < 0.05).
2.7. Raman spectroscopy Raman spectra with an excitation at 632.8 nm (laser He/Ne, less than 10 mW on the sample) were obtained at room temperature, using a LABRAM-Aramis spectrometer (Horiba Jobin-Yvon, France). Spectral repeatability was smaller than 0.2 cm1, and spectral resolution was better than 1 cm1. Protein samples were placed on microscope slides. The laser was then focused on the samples. Raman spectra of at least three different positions were collected from 400 to 4000 cm1. The spectral data from the scans of samples in the Raman spectrophotometer were baseline corrected and normalized according to the protein phenylalanine peak at 1003 1 cm1.
centrifuged at 11,000 g for 15 min to evaluate amino acids in the supernatant. Samples of control gliadin and deamidated gliadins with deamidation time of 20 h and corresponding digests were hydrolyzed in sealed, evacuated glass tubes with 6 M HCl at 110 C for 24 h. The analysis was performed with a Membrapure A-300 amino acid analyzer (Membrapure Co., Frankfurt, Germany) equipped with a C18 column (4.6 mm 125 mm) for amino acid separation. Postcolumn reaction with ninhydrin yielded amino acid derivatives. The concentrations of the amino acids were determined from their respective absorption intensities, which were calibrated to the known concentrations of amino acid standards. 2.10. Statistical analysis
2.8. Atomic force microscopy (AFM) analysis AFM imaging was made using Tapping mode on Nanoscope IIIa Multimode Scanning Probe Microscope (Veeco Instruments, Santa Barbara, CA, USA). Aliquots (2 mL) of dispersions (20 mg/mL with distilled water and filtered through a 0.22 mm filter) were placed on a freshly cleaved mica disk and air-dried for 10 min at ambient temperature. Approximately 5e10 images were taken for each preparation.
All experiments were conducted in triplicate. Statistical calculation was investigated using the statistical package SPSS 11.5 (SPSS Inc., Chicago, IL) for one-way ANOVA. A least significant difference (LSD) test with a confidence interval of 95% was used to compare the means. All the data were expressed as mean standard deviation of triplicate determinations. 3. Results and discussion
2.9. Amino acid analysis
3.1. Physicochemical properties of deamidated wheat gliadins
In vitro digests of protein samples were carried out according to Zhu et al. (2008). Gliadin and deamidated gliadins (3% w/v, in MilliQ water) were adjusted to pH 2.0 with 1 M HCl and pepsin was added (4% w/w, protein basis). After incubation at 37 C for 1 h, the solution was adjusted to pH 5.3 with 0.9 M NaHCO3. Pancreatin (4% w/w, protein basis) was then added, and the pH was adjusted to 7.5 with 1 M NaOH. The solution was incubated at 37 C for 2 h. The digests were neutralized, heating at 90 C for 10 min and
3.1.1. Solubility Solubility is a physical characteristic closely related to its functional properties. Fig. 1a shows that the solubility of gliadin significantly increased and the isoelectric point shifted to lower pH values after deamidation. Gliadin in its natural form has low solubility near neutral pH values for it has a large amount of amide groups (Gln and Asn). The substitution of amide groups by carboxylic groups after deamidation can increase the net negative
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Fig. 2. (a) Deconvoluted FTIR spectra in the amide I region of control gliadin (C), HDWG (H-1 and H-2) and CADWG (CA-1 and CA-2). (b) Relative areas of the bands fitted to the Fourier-deconvoluted spectra. (NH2)þ hydrated extended chains at 1596 cm1 and 1608 cm-1 a-helix: band at 1652e1654 cm1 b-sheets: bands at 1619e1620 cm1, 1630e 1632 cm1, 1677 cm1 and 1688 cm1 b-turn: band at 1664e1666 cm1. Random coil: band at 1643 cm1. Different letters (aed) on the top of each column (the same band) indicates significant difference (p < 0.05).
charge, thus can increase the solubility of gliadin at neutral pH (Qiu et al., 2013). Wheat gliadin deamidated by citric acid and HCl showed the isoelectric point around 5 and 4, respectively. 3.1.2. Molecular weight distribution determination by SE-HPLC Size exclusion chromatography analysis was carried out to examine changes of the molecular size of gliadin and deamidated gliadins (Fig. 1b). It seems that deamidation caused the two peaks of gliadin to merge. This phenomenon might be caused by the conformational changes of proteins. In Fig. 1b, both HCl and citric acid deamidation caused little hydrolysis and deamidated gliadins showed similar molecular weight distribution. This was because the acid concentration used here was relatively low and the temperature of 70 C was selected to minimize protein aggregation or hydrolysis. It is known that the effect of high temperature on protein structure is the irreversible unfolding of protein, aggregation and proteolysis (Liao et al., 2010). 3.1.3. Surface hydrophobicity (S0) Fig. 1c shows that S0 of the deamidated gliadins was significantly higher than the control gliadin. Deamidation caused unfolding of the protein and rearrangement of hydrophobic forces, so the
hydrophobic zones hidden inside the protein were exposed. Surface hydrophobicity is an important factor in determining the emulsifying properties. The increased hydrophobicity in protein would cause a better molecular arrangement at the oil-water interface, giving more stable emulsion (Matsudomi et al., 1985). As shown in Fig. 1c, S0 was higher in the CADWG. Gliadins further deamidated for 20 h (CA-2) exhibited a slight decrease of S0. Heating during deamidation could promote the unfolded protein to reassociate by hydrophobic interaction between exposed hydrophobic amino acid residues, which reduced hydrophobic moieties that could bind to the fluorescence probe (Zhao et al., 2011). 3.2. Conformational characteristics 3.2.1. Second structure characterization by FTIR The spectra in the amide I region were deconvoluted. Band fitting with Gaussian band shapes was performed on the deconvoluted spectra to estimate secondary structure content. Nine major bands were distinctly observed in the amide I bands region of the deconvoluted curve of control gliadin and deamidated gliadins (Fig. 2a). The amide I band mainly originated from C]O stretching with some contribution of the NeH vibration (Secundo and
Please cite this article in press as: Qiu, C., et al., Comparison of the conformational and nutritional changes of deamidated wheat gliadin by citric acid and hydrochloric acid, Journal of Cereal Science (2014), http://dx.doi.org/10.1016/j.jcs.2014.02.003
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Fig. 3. (a) Raman spectra of control gliadin (C), HDWG (H-1 and H-2) and CADWG (CA-1 and CA-2); (b) Normalized intensity of amide I and amide III Raman bands; (c) Normalized intensity of 1780 cm1 Raman bands; (d) Normalized intensity of 1340 cm1 and 757 cm1 Raman bands; (e) Normalized intensity of 1448 cm1 and 2934 cm1 Raman bands; (f) Tyr doublet around 854 and 830 cm1 (854 cm1/830 cm1). Different letters (aee) on the top of each column (the same band) indicates significant difference (p < 0.05).
Guerrieri, 2005). The strong absorption at 1652 cm1 indicated that the content of a-helix was high in gliadins and this component might also contain some random coil conformation and glutamine side chain (Secundo and Guerrieri, 2005). The spectral position of the components of amide I band in deamidated gliadins showed several differences of Fourier deconvoluted spectra with respect to the control gliadin. This indicated that the protein conformation was modified. Amide I band of deamidated gliadins showed an obvious peak at 1643 cm1. However, this peak was evident only by the curve-fitting analysis in control gliadin (Fig. 2a and b). This suggested that protein was unfolded and the intensity of random coil increased. Fig. 2b shows the relative proportion of the bands, which are assigned to the structure components according to the previous references (Secundo and Guerrieri, 2005; Ukai et al., 2008). Control gliadin contained similar structure position and secondary structure content compared with the result of Secundo and Guerrieri (2005). The intensity of the band assigned to random coil (1643 cm1) was evidently increased and the band assigned to bturn (1665 cm1) was slightly increased after deamidation. Deamidation turned glutamine residues to negatively charged glutamic acid residues, and thus led to the decrease in hydrogen-bonded bsheets structures and increase in more flexible structures like bturn and random coil (Wong et al., 2012). The band intensity of bsheets at 1619, 1677 and 1690 cm1 decreased while at 1632 cm1, slightly increased after deamidation (Fig. 2b). The slight shift of band at 1632 cm1 to 1630 cm1 (Fig. 2a) and increase of intensity suggested the increase in extended b-sheets structures at the expense of antiparallel b-sheets structure which features protein
aggregates at 1619 cm1 and 1690 cm1 (Choi and Ma, 2005; Wang et al., 2011). The NH2 scissoring of the glutamine side chains (1596 cm1 and 1608 cm1) showed significant decrease as a result of the deamidation process. Both HDWG of the two deamidation degrees showed similar deconvoluted spectra shape which was different from CADWG. Compared with control gliadin, CADWG had higher content of a-helix while HDWG had lower content. The b-turn content of CADWG (1665 cm1) was also higher than HDWG (Fig. 2b). 3.2.2. Raman spectra and molecular vibration Raman spectroscopy is a direct and a non-invasive technique for studying structural changes of proteins in solid states without destroying samples (Wong et al., 2009). Fig. 3a shows the typical Raman spectra and normalized intensity of several bands of gliadin and deamidated gliadins. Proteins with a high proportion of a-helical contents show an amide I band centered at around 1650e 1660 cm1. The amide I and amide III bands of all the samples were centered at 1656 and 1248 cm1, respectively (Fig. 1a), indicating that the content of a-helical and random coil was high in the protein (Herrero et al., 2008). The intensity and location of the phenylalanine band (1003 cm1) is not sensitive to protein conformation or to the microenvironment, so it was used as an internal standard to normalize the molecular vibration intensity (Herrero et al., 2008). Raman bands corresponding to amide I and III can be used to characterize protein backbone conformation (Wong et al., 2009). As shown in Fig. 3b, after deamidation, the intensity of amide I decreased while amide III increased. This indicated protein
Please cite this article in press as: Qiu, C., et al., Comparison of the conformational and nutritional changes of deamidated wheat gliadin by citric acid and hydrochloric acid, Journal of Cereal Science (2014), http://dx.doi.org/10.1016/j.jcs.2014.02.003
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unfolding (Gómez et al., 2013). The band at 1780 cm1 was attributed to the carboxylic acid vibration (Wong et al., 2009). In Fig. 3c, the intensity of the 1780 cm1 band of HDWG and CADWG increased, indicating the deamidation process. The bands near 757 cm1 and 1340 cm1 provide information about the microenvironment of the tryptophan residues. Decreased intensity at 1340 cm1 indicated more exposed tryptophan residues (Sun et al., 2011). The intensities of 854 cm1/830 cm1 was proposed as a means of determining whether the Tyr residue is solvent exposed or buried. When the intensity of the band at 854 cm1 is higher than the band at 830 cm1, the Tyr residues are exposed and when the intensity of the band at 854 cm1 is lower than the band at 830 cm1, the Tyr residues are buried within the protein network (Sun et al., 2011). The Tyr doublet ratio (854 cm1/830 cm1) of HDWG did not change significantly while the intensity first decreased then increased for CADWG at 10 h and 20 h. The ratio was always lower than 1, suggesting Tyr residues were mainly buried even after deamidation (Sun et al., 2011). The CeH stretching vibrations were represented by Raman bands at 1448 cm1 and 2934 cm1. As shown in Fig. 3e, the intensity of the CeH bending vibration band was evidently increased after deamidation, indicating protein unfolding and exposure of aliphatic side chains (Zhao et al., 2004). 3.2.3. Morphology analysis by AFM AFM is a nondestructive method to characterize the three dimensional structure of materials of single molecules as well as complex composite structures in the nanometer to 100 mm range (Guo et al., 2005). Fig. 4 shows the AFM images of gliadins and deamidated gliadins. Control gliadin exhibited monodisperse close packed globular shapes. This indicated the strong proteineprotein interaction while weak interactions with the hydrophilic mica (Paananen et al., 2006). Gliadins are intrinsically cohesive as they have a large number of glutamine residues in the polypeptide chains, causing aggregation through hydrogen bonding.
Fig. 5. DSC thermograms of control gliadin (C), HDWG (H-1 and H-2) and CADWG (CA1 and CA-2).
Deamidated gliadins exhibited significant difference compared with control gliadin for their unfolding of structures. For HDWG and CADWG at 10 h (Fig. 4b and d), there were some big clusters of protein formed and proteins still maintain globular form. At 20 h, HDWG showed some extended linear fibrillar structure (Fig. 4c) while CADWG showed mainly dispersed globular aggregates though a few fibrils can also be observed (Fig. 4e). These fibrils were reported to be assembled from monomer or dimer units of gliadin by ioneprotein interaction (McMaster et al., 1999). These results indicated that HDWG formed more flexible aggregates than CADWG. The difference in the tertiary structure of the protein probably induced the difference of proteineprotein interaction and protein-surface interaction, causing their different shapes.
Fig. 4. Tapping mode AFM images of (a) control gliadin (C), HDWG and CADWG of 10 h (b, d) and 20 h (c, e). The scan size is 3.00 mm 3.00 mm, and the z scale is 20 nm.
Please cite this article in press as: Qiu, C., et al., Comparison of the conformational and nutritional changes of deamidated wheat gliadin by citric acid and hydrochloric acid, Journal of Cereal Science (2014), http://dx.doi.org/10.1016/j.jcs.2014.02.003
C. Qiu et al. / Journal of Cereal Science xxx (2014) 1e8
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Table 1 Amino acids composition (mol%) of control gliadin, HDWG (H-2) and CADWG (CA-2) and their digests. Samples
Digested samples
C Asp þ Asn Thr Ser Glu þ Gln Gly Ala Cys Val Met Ile Leu Tyr Phe His Lys Arg Pro Trp Essential AA
2.24 2.04 5.09 30.27 2.66 2.64 2.34 3.98 1.12 3.87 7.15 2.03 5.01 3.09 1.79 4.51 19.49 0.66 25.63
H-2
a
0.05 0.04a 0.31a 1.07a 0.12a 0.15a 0.20a 0.05a 0.02a 0.32a 0.13a 0.11a 0.31a 0.15a 0.02b 0.22a 0.65a 0.02a 0.91a
1.91 2.01 4.91 33.35 4.44 2.96 2.43 3.48 0.49 3.36 6.86 2.24 4.26 2.62 1.66 5.21 17.21 0.61 22.72
CA-2
b
0.03 0.06a 0.10a 2.10a 0.23b 0.13a 0.12a 0.08b 0.06b 0.04b 0.09b 0.20b 0.34b 0.19b 0.05a 0.23b 0.33b 0.04ab 0.76b
2.16 2.32 5.38 31.92 4.76 2.81 2.32 3.45 0.47 3.31 6.51 2.30 4.17 2.86 1.90 4.15 18.61 0.60 22.73
C
a
0.11 0.10b 0.35a 1.33a 0.28b 0.10a 0.16a 0.21b 0.07b 0.15b 0.29b 0.14b 0.25b 0.09ab 0.06c 0.36a 0.26a 0.02b 1.15b
2.74 2.21 5.24 31.14 3.58 2.81 2.27 3.66 1.02 3.57 6.71 2.51 4.68 2.01 0.93 4.00 20.45 0.49 23.26
H-2
a
0.06 0.04a 0.11a 1.29a 0.15a 0.16a 0.14a 0.10a 0.03a 0.24a 0.20a 0.21a 0.11a 0.06a 0.03a 0.15a 1.14a 0.03a 0.78a
2.65 2.40 5.34 32.26 5.74 3.18 2.44 3.65 0.12 3.51 6.60 2.37 4.30 2.00 0.90 2.37 19.53 0.51 22.09
CA-2
ab
0.10 0.07b 0.08a 1.15a 0.31b 0.02b 0.10a 0.04a 0.02c 0.08a 0.13a 0.13a 0.06b 0.24a 0.02a 0.10b 1.06ab 0.02a 0.45b
2.55 2.46 5.47 31.46 5.25 3.10 2.38 4.16 0.27 3.34 6.50 2.41 4.33 3.04 1.47 4.21 17.21 0.64 22.93
0.07b 0.05b 0.14a 2.30a 0.27b 0.12b 0.04a 0.03b 0.02b 0.05a 0.06a 0.05a 0.04b 0.18b 0.04b 0.07a 1.51b 0.02b 0.31ab
aed
Different letters in the same row of the samples or digested samples indicate significant difference (p < 0.05).
Deamidated gliadin with higher negative charge had higher adsorption ability on the hydrophilic mica, revealing different aggregation structures on the substrate. 3.2.4. Thermal characters of gliadin and deamidated gliadins DSC is a pertinent technique for evaluating the state and nature of mainly tertiary conformations of food proteins and the role of chemical forces in stabilizing the protein conformation (Yin et al., 2011). Fig. 5 shows that control gliadin exhibited a Td value of 54.5 C and the enthalpy (DH) was 2.41 J/g. This Td value was a little higher than a previous report of gliadin by Falcão-Rodrigues et al. (2005) while a little lower than the result of León et al. (2003). This might be caused by the different wheat varieties or protein purity. The narrow shape of the peak of deamidated gliadins suggested that they denatured in a more cooperative way (Yin et al., 2011). HDWG exhibited Td values of 64.96 C and 63.80 C and DH of 4.81 J/g and 5.80 J/g at deamidation times of 10 h and 20 h, respectively, which was different from previous reports that HCl deamidated wheat protein showed no obvious endothermic peak during heating (Day et al., 2009; Friedli and Howell, 1996). This might be explained by the different DSC test conditions. It was reported that water content played a role in endothermic transition detection and water content around 50% was the most suitable condition to detect the endotherm (Falcão-Rodrigues et al., 2005; León et al., 2003). CADWG showed Td at 73.58 C and 75.57 C and DH of 5.79 J/g and 7.65 J/g at deamidation times of 10 h and 20 h, respectively. Unfolding of native protein after deamidation might cause the exposure of buried apolar groups, improving proteineprotein interactions by hydrophobic association. Thus, heating the unfolding gliadins would require the rupture of more hydrophobic groups than the native protein, resulting in higher Td (Choi and Ma, 2005). The higher Td value suggested more compact tertiary conformation of the polypeptides. Protein denaturation involves conformational changes from the native structure by disruption of chemical forces that maintain the structural integrity of the protein molecules like hydrogen bonds, hydrophobic bonds, ionic interactions and covalent disulfide bonds (Falcão-Rodrigues et al., 2005). AFM results (Fig. 4) showed that HDWG exhibited fibrillar aggregates which were more flexible. It was different from CADWG with mainly globular shapes. This could explain why HDWG exhibited lower denaturation temperatures than CADWG.
3.3. Amino acids composition Table 1 lists the amino acid composition of wheat gliadin and deamidated gliadins. Glu and Pro are the most abundant amino acids of wheat gliadin. The content of Lys in HDWG and its digest was decreased. However, it was increased in the digest of CADWG. Proteineprotein interactions involving ε-NH2 groups with the amide groups of Asp and Glu that resulted in cross-linking tend to cause destruction of Lys (Guo et al., 1999). Lys is considered to be the most important essential amino acid in cereal protein. This result suggested that the citric acid deamidation process was milder and more suitable for preparing deamidated gliadin with higher nutritional properties. Moreover, the digests of CADWG had higher amount of essential amino acids compared with HDWG. This indicated that citric acid deamidation can better maintain the essential amino acids of wheat gliadin. In addition, the loss of Met might be due to damage of the methyl-thiol group during the deamidation process (Guo et al., 1999). 4. Conclusions This work demonstrated the effects of HCl and citric acid deamidation on the structure characteristics of wheat gliadin. For these two acids, deamidation led to a slight difference of secondary structure change of wheat gliadin and side chain vibration models. Gliadin deamidated by HCl exhibited mainly flexible associated fibrils while globular aggregates by citric acid. The thermal properties indicated that CADWG had higher thermal stability than HDWG. The difference was supposed to exist in the way of protein aggregation and proteineprotein interaction of the two acids. In addition, citric acid deamidation was a better way to increase the Lys content and maintain the essential amino acids of gliadin after digestion. These results will be useful for predicting the structuree function relationships of wheat gliadins. Acknowledgments The authors are grateful to the National High Technology Research and Development Program of China (863 Program) (No. 2013AA102201), the National Science-Technology Supporting Project for 12th Five-Year Plan (2012BAD37B08) and the National
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Please cite this article in press as: Qiu, C., et al., Comparison of the conformational and nutritional changes of deamidated wheat gliadin by citric acid and hydrochloric acid, Journal of Cereal Science (2014), http://dx.doi.org/10.1016/j.jcs.2014.02.003