Food Chemistry 178 (2015) 96–105

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The effect of deamidation on the structural, functional, and rheological properties of glutelin prepared from Akebia trifoliata var. australis seed Li Lei a, Qiang Zhao a,⇑, Cordelia Selomulya b, Hua Xiong a,⇑ a b

State Key Laboratory of Food Science and Technology, Nanchang University, Jiangxi 330047, China Department of Chemical Engineering, Monash University, Clayton, Victoria 3800, Australia

a r t i c l e

i n f o

Article history: Received 14 September 2014 Received in revised form 15 January 2015 Accepted 17 January 2015 Available online 23 January 2015 Keywords: Akebia trifoliata var. australis seed Glutelin Deamidation Edible organic acid Functional properties Rheological properties

a b s t r a c t The characteristics of glutelin samples from Akebia trifoliata var. australis seeds (AG) that had been deamidated by malic acid (MDAG) and by citric acid (CDAG) were investigated. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis showed high-molecular-weight subunits that were degraded into smaller fragments, and FTIR indicated a decrease in the number of b-sheet groups and an increase in the amount of b-turns in the deamidated samples. These results could be caused by the cleaving of partial disulfide bonds to form new sulfhydryl groups during deamidation. Citric acid was found to be more effective at deamidation and hydrolysis, resulting in a higher solubility and emulsifying activity for CDAG, and MDAG also exhibited some improvement in terms of surface hydrophobicity and emulsion ability. Rheology showed that the gelation point for deamidated samples was increased, and the gel network was strengthened. The amounts of essential amino acids that were well-preserved and the improved solubility, emulsification, and rheology properties of AG after acid-heating deamidation show that this technique can be useful for treating other plant-based food ingredients in the future. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Plant proteins have played an increasingly important role as an alternative source of protein to replace the more expensive animal proteins in protein-rich foods in order to meet human dietary needs in developing countries. Thus, the search for new plant protein sources has attracted significant attention (Day, 2013). Akebia trifoliata (Thunb.) Koidz. var. australis (Diels) Rehd. is a liana commonly known as ‘‘Bai Mu Tong’’ in China (Gao & Wang, 2006). It is a fast-growing plant that bears many seeds (up to 200) and is widely available in Japan, China, and Korea (Liu, Ma, Zheng, Zhang, & Lin, 2007). Many parts of the plant are edible; for example, the fruits and stems are used as folk medicinal herbs for diuretic and antiphlogistic purposes (Kawata, Kameda, & Miyazawa, 2007), the dried young leaves are used as a tea substitute, and the fresh fruits can be directly consumed or processed into juice or fruit vinegar (Jiang et al., 2012). The oil pressed from A. trifoliata var. australis seeds (AS) is commonly used as edible oil in southern China (Kitaoka et al., 2009). The protein-rich AS meal left after oil extraction is a by-product for livestock, and it could be a potential new source of low-cost plant protein for human consumption. ⇑ Corresponding authors. Tel./fax: +86 791 86634810. E-mail addresses: [email protected], [email protected] (Q. Zhao), [email protected] (H. Xiong). http://dx.doi.org/10.1016/j.foodchem.2015.01.081 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

We previously reported that AS contains 38.83% oil and 17.23% protein, the primary fraction of which is glutelin and accounts for 46.40% of the protein fractions with great nutritional properties (Du et al., 2012). However, the poor solubility of glutelin in aqueous solution because of its high contents of glutamines, asparagine residues, and non-polar amino acid residues restricts its applications in food processing. It is thus necessary to improve the solubility and optimization of its functional properties in aqueous solutions by appropriate modification methods. Deamidation is a modification method for improving the solubility and other functional properties of proteins by getting rid of amide groups, originally on uncharged amino acids, to form acidic residues (Zhao, Tian, & Chen, 2011). This process can thereby dissociate protein polymers, increasing the electrostatic repulsion among protein molecular chains and the surface hydrophobicity and flexibility of the glutelin molecule (Matsudomi, Kaneko, Kato, & Kobayashi, 1981; Qiu, Sun, Cui, & Zhao, 2013). A number of studies have focused on the deamidation-induced modification of plant proteins, including wheat gluten (Qiu, Sun, Cui, et al., 2013), rice protein (Liu et al., 2011), oat protein (Mirmoghtadaie, Kadivar, & Shahedi, 2009) and barley glutelin (Zhao et al., 2011), to improve their functional properties with different reaction mechanisms by using acids, bases, and enzymes. Among the available acid catalysts for protein deamidation, hydrochloric acid is the most commonly used besides sulfuric acid, formic acid, phosphoric acid, chlorosul-

L. Lei et al. / Food Chemistry 178 (2015) 96–105

fonic acid, and trichloroacetic acid (Liao et al., 2010). However, deamidation by hydrochloric acid also produces potentially carcinogenic substances (such as chloropropanol), and causes the uncontrollable hydrolysis of peptide bonds and the isomerization of some amino acids (Liao et al., 2010). Several studies have demonstrated that the deamidation of gluten by organic acids could enhance the properties of gluten. Liao et al. (2010) found that wheat gluten that was deamidated by citric acid and succinic acid had a higher molecular flexibility and better nutritional characteristics because of structural changes. Qiu, Sun, Cui, et al. (2013) investigated the effect of deamidating with citric acid on the properties of wheat gliadin and the emulsion characteristics of D-gliadin. Their results showed that citric acid deamidation could effectively deamidate gliadin with little hydrolysis and markedly improved the solubility of wheat gliadin and the emulsifying properties of D-gliadin. Most importantly, organic acids could avoid excessive deamidation because of their mild characteristics (Liao et al., 2010; Qiu, Sun, Cui, et al., 2013). It is therefore of great interest to investigate the influence of acid-heating deamidation with edible organic acids on plant proteins, including glutelin from AS, about which very little information is currently available. Citric acid and malic acid are GRAS (Generally Recognized as Safe)-listed compounds and the most common organic acids used as preservatives, acidulants, or flavoring agents. Both acids play an important role in inhibiting the growth of pathogenic microorganisms that cause food spoilage. Citric acid has a mild refreshing acidity and is commonly used in a variety of food products, with almost 70% of the market share of organic acids. Citric acid is a weak organic acid, and malic acid is a better sour stimulus than citric acid. The US FDA (Food and Drug Administration) has limited the application of citric acid as an ingredient in foods for children and the elderly; therefore, L-malic acid has gradually replaced some citric acid applications in the food industry in recent years. Malic acid and citric acid, which are two types of edible organic acids with different carboxylic groups, were chosen to compare the structure and the functional and rheological properties of glutelin from AS after acid-heating deamidation. 2. Materials and methods 2.1. Materials Ripe A. trifoliata var. australis fruits were kindly provided by Jiujiang Regression Biological Technology Development Co., Ltd. (Jiujiang, China). The seeds were pulled out from the fresh fruits to remove the pulp that was adhering to the seeds, and the fruit was dried to a constant weight at 50 °C for 48 h, before storage at 20 °C. The canola oil used for the emulsification study was purchased from a local supermarket. An unstained standard protein molecule marker ranging from 10 to 170 kDa for the SDS–PAGE, an ammonia kit, 1-anilonaphthalene-8-sulfonic-acid (ANS), and 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) were purchased from Sigma–Aldrich (St. Louis, MO, USA). All other reagents were of analytical or chromatographic grade. 2.2. Preparing glutelin Glutelin that was isolated from AS was prepared according to the method in our previous study (Du et al., 2012) with slight modification. In brief, the AS was de-hulled, ground, and defatted to obtain the defatted flours (DAF). The DAF was then passed through a 100-mesh sieve (0.15 mm) before protein extraction. Glutelin isolation from DAF was performed by the sequential extraction of the albumin, globulin, prolamin, and glutelin fractions with deionized water, 0.5 M NaCl, 70% ethanol, and 0.1 M NaOH under mag-

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netic stirring for 2 h with a liquid:solid ratio of 15:1 at room temperature before centrifugation at 4800g for 15 min. To remove the other fractions, each extraction step was repeated twice. The glutelin fraction from the combined supernatants of the first and second extractions was isoelectrically precipitated at pH 4.5 with 1 M HCl and kept at 4 °C for 1 h. The precipitate was recovered by centrifugation at 4800g for 15 min and washed with deionized water twice. The pH value of the precipitate was adjusted to 7.0 with 1 M HCl and 1 M NaOH before dialysis with deionized water for 24 h at 4 °C. The sample was then freeze-dried for storage. The nitrogen content of glutelin was determined to be 87.9% by the Kjeldahl method at a nitrogen conversion factor of 6.25. 2.3. Glutelin deamidation The deamidation of the glutelin samples with malic acid (which is abbreviated as MDAG) and by citric acid (CDAG) under hydrothermal treatment was conducted according to the method described by Liao et al. (2010), Qiu, Sun, Cui, et al. (2013) with some modifications. In brief, 8% (w/v) glutelin was mixed with malic acid and citric acid (0.2 M) to form suspensions. The dispersions were hydrated in a water shaker for 12 h at 65 °C. After that, the acid-heating samples were immediately held in an ice water bath for 1 h to stop the reaction. The samples were then centrifuged at 10,000g for 10 min at 4 °C, and the soluble fractions were collected using a syringe according to the method described by Du et al. (2012). The collected soluble fractions were neutralized with 0.5 M NaOH and dialyzed in deionized water at 4 °C for 24 h to remove ammonium, and then freeze-dried. Glutelin without acid treatment was used under the same conditions as the control sample (AG). 2.4. Assessing the degree of deamidation and hydrolysis The degree of deamidation (DD) was determined according to Kato, Tanaka, Lee, Matsudomi, and Kobayashi (1987). DD was referred to as the ratio of ammonia produced from the deamidated sample to the amount of ammonia released from completely deamidated glutelin. Complete deamidation was reached by dissolving 0.8 g of glutelin in 10 ml of 3 M HCl, followed by heating for 3 h at 121 °C. The ammonia content was determined by using an ammonia kit according to the instruction provided. The degree of hydrolysis (DH) is referred to as the percentage of the free amino groups cleaved from protein, which was the ratio of a-amino nitrogen to total nitrogen by measuring the free amino groups by using TNBS, according to McKellar (1981). A complete hydrolysis was performed by using 3 M HCl at 121 °C for 24 h to determine the total number of amino groups. 2.5. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS– PAGE) SDS–PAGE was performed in a discontinuous buffering system with a 12% separating gel and a 3% stacking gel according to the Laemmli (1970) method by using a Bio-Rad Mini PROTEAN 3 system (Bio-Rad Laboratories, Hercules, CA, USA). AG, MDAG and CDAG were dissolved in deionized water to 5 mg/ml. The protein samples were mixed with loading buffer (0.125 M Tris–HCl (pH 6.8), 4% (w/v) SDS, 20% (v/v) glycerol, 0.5% 2-mercaptoethanol and 1% bromophenol blue (w/v)) and then denatured in boiling water for 10 min, and centrifuged at 10,000g for 3 min. After cooling, 10 ll of supernatant from each sample was loaded into a homogeneous PhastGel. Electrophoresis was performed at a constant current of 15 mA for 30 min followed by 25 mA until the tracking dye reached the bottom of the gel. After electrophoresis, the gel was stained for 1 h with the solution (0.25 g of Coomassie

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blue (R-250) dissolved in 100 ml of ice-cold acetic acid/ethanol/ water solution (18:91:91, v/v/v)) and then de-stained for 12 h in ethanol/ice acetic acid/water solution (50:75:875, v/v/v). 2.6. The structural characteristics of deamidated glutelin 2.6.1. Fourier transform infrared (FTIR) spectroscopy The dried control sample and deamidated samples (1 mg) were mixed with KBr (100 mg) and ground into a fine powder in an agate mortar incubated with infrared light, then pressed into a pellet. The Fourier transform infrared spectra were recorded by using a Nicolet 5700 FTIR spectrometer (Thermo Nicolet Corporation, USA) from 400 to 4000 cm1 with a 4 cm1 resolution and an accumulation of 32 scans. The band assignments to secondary structural components were performed for amide region I (1600– 1700 cm1). 2.6.2. Determining the sulfhydryl groups (ASH) and disulfide bond (SAS) contents The change in the sulfhydryl group contents during the deamidation reaction was determined by using Ellman’s reagent (DTNB) according to Zhao et al. (2013). In brief, a 30 mg protein sample was dissolved in 10 ml of Tris–glycine buffer (0.086 M Tris, 0.09 M glycine, 0.04 M EDTA, pH 8.0) with 8 M urea (total sulfhydryl) or without 8 M urea (exposed sulfhydryl) and then 100 ll of DTNB (4 mg/ml) was added. The slurry was incubated in a dark place at 25 °C for 1 h with constant stirring and then centrifuged at 4800g for 15 min. The absorbance of the supernatant was determined at 412 nm. The SH group level was calculated as follows:

lmolSH=g ¼ 73:53  A412  D=C

73:53  A412  D  SHTotal C

2.8. Amino acid analysis The amino acid profiles of AG, MDAG and CDAG were determined by hydrolyzing with 6 M HCl under a vacuum at 110 °C for 24 h. The hydrolyzed samples were then loaded into an L8800 automatic amino acid analyzer (Hitachi, Japan). The amino acid profiles were presented in g/100 g protein. 2.9. Determining the functional properties 2.9.1. Solubility profile The solubility profiles of AG, MDAG and CDAG were determined according to the Du et al. (2012) with some modifications. Protein samples (0.25 g) were dispersed in 25 ml of deionized water to form 1% (w/v) solution. The dispersions were magnetically stirred for 30 min at room temperature to distribute them well. The pH of the solution was then adjusted with 1 M HCI or 1 M NaOH by magnetic stirring for 30 min at 25 °C before centrifuging at 4800g for 15 min to collect the soluble fractions. After the appropriate dilution, the protein contents of the supernatants were measured by Lowry’s method (1951) with bovine serum albumin as the standard used to calculate the protein solubility.

ð1Þ

where A412 is the absorbance at 412 nm against the blank, D (1.01) is the dilution coefficient, and C is the protein concentration in the tested sample (mg/ml). For the disulfide bond content determination, a 30 mg protein sample was dissolved in 10 ml of Tris–glycine 10 M urea buffer to assay the disulfide bond content, then 0.5 ml of the solution was mixed with 0.03 ml of mercaptoethanol. After that, the mixture was incubated at 25 °C for 1 h in a dark place, and 5 ml of trichloroacetic acid (12%, w/v) was added. The solution was allowed to stand for 1 h prior to centrifugation at 4800g for 15 min. The process was repeated twice. The residues were suspended in 3 ml of Tris–Gly 8 M Urea and 0.04 ml of DTNB. The solution was incubated in a dark place at 25 °C for 30 min with stirring and centrifuged at 4800g for 15 min. The absorbance of the solution was determined at 412 nm against the blank, and the total disulfide content was calculated as follows:

lmolS  S=g ¼

10 min in the dark. The fluorescence intensity (FI) of each sample was measured at 390 nm (excitation) for emissions within the 300–800 nm range by using an F-4500 model fluorometer (Hitachi Co., Tokyo, Japan). The initial slope of the FI vs. the protein concentration (%, w/v) was calculated by a linear regression analysis and used as an index of H0.

ð2Þ

where A412 is the absorbance at 412 nm, D (6.08) is the dilution factor, C is the protein concentration in the tested sample (mg/ml), and SHTotal is the result calculated as formula (1). The results were expressed as lmol/g of protein.

2.9.2. Emulsifying activity and emulsion stability The emulsifying activity index (EAI) and the emulsion stability index (ESI) of deamidated glutelin and control samples were determined by following the procedure by Pearce and Kinsella (1978). Aliquots (16 ml) of 0.1% protein solution (10 mM phosphate buffer, pH 7.0) were thoroughly mixed with 4 ml of soybean oil by using a homogenizer (model T18 digital ULTRA-TURRAX, IKA, Germany) at 12,000 rpm for 1 min at room temperature. After homogenization for 0 and 10 min, an emulsion (50 ml) was pipetted at 0.5 cm from the bottom of the container and mixed with 5 ml of 0.1% SDS (w/v). The absorbance of the solution was measured at 500 nm against a blank solution (0.1% SDS). The absorbance was measured immediately (A0) and after 10 min (A10), and these measurements were used to calculate the EAI and ESI as follows:

EAI ðm2 =gÞ ¼

ESI ð%Þ ¼

4:606 C  ð1  UÞ  104

 A0  100

A10  100 A0

ð3Þ

ð4Þ

where A0 and A10 were the absorbances at 500 nm as measured after an emulsion formed at 0 and 10 min, respectively, C (g/ml) was the protein concentration before emulsification, and U (0.20) was the oil volume fraction (v/v) of the emulsion.

2.7. Surface hydrophobicity (H0) 2.10. Dynamic oscillation rheology The surface hydrophobicity of the protein samples was measured in 0.01 M PBS buffer (pH 7.0) according to the method described by Kato and Nakai (1980) by using the fluorescence probe 1-anilinonaphthalene-8-sulfonic acid (ANS). Eight mM ANS in 10 mM phosphate buffer at pH 7.0 was prepared before the measurements. Protein solutions (4 ml) of various concentrations from 0.005% to 0.025% (w/v) in 10 mM phosphate buffer at pH 7.0 were mixed with 20 ml of freshly prepared ANS. The mixtures were shaken vigorously for 5 s to be made homogeneous and stored for

Dynamic oscillation measurements were performed by using a stress-controlled rheometer (DHR-2, TA Instruments, American) with a parallel plate system (40 mm diameter, 1 mm gap). This dynamic oscillation analysis determines the network of the storage modulus (G0 ) and loss modulus (G00 ) components at a strain of 1% and a frequency of 1 Hz. The AG, MDAG and CDAG were dissolved in deionized water to 50 mg/ml. The samples (25 °C) were transferred to the bottom plate with a syringe and the surface was cov-

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ered with a thin layer of liquid paraffin to prevent evaporation. The samples were heated from 25 to 95 °C at a rate of 5 °C/min, and then maintained at 95 °C for 5 min. They were subsequently cooled down to 25 °C at a rate of 5 °C/min and kept at 25 °C for 5 min. The gel point was the temperature at which the storage modulus and loss modulus intersected, at which point the time (t gel) and temperature (T gel) at the point were evaluated. After this measurement, and before removing the sample from the system, a frequency sweep was performed at a strain rate of 1%, from 0.01 to 10 Hz. 2.11. Statistical analysis All measurements were performed on triplicate samples. The results were expressed as the means ± standard deviations unless otherwise stated. A statistical analysis was conducted by using the statistical package SPSS 16.0 (SPSS, Inc., Chicago, IL, USA) for one-way ANOVA. Significant differences were defined as p < 0.05 according to Duncan’s Multiple Range Test. 3. Results and discussion 3.1. The characteristics of deamidated glutelin The degree of deamidation (DD) and hydrolysis (DH) for AG (untreated glutelin), MDAG (glutelin treated with malic acid), and CDAG (glutelin treated with citric acid) are shown in Fig. 1A

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and B, respectively. Both the degree of deamidation (DD) and the degree of hydrolysis (DH) of deamidated glutelin by citric acid were higher than those of malic acid. This finding was contrary to that of Liao et al. (2010), who found that the rate of deamidation of hydrion by succinic acid was higher than that of citric acid. Thus, the anions from citric acid appeared to be more appropriate for catalyzing deamidation in glutelin than those from malic acid in this case. A DH value of less than 6 implies that deamidation was accompanied by a slight hydrolysis, suggesting that the free amino acids of glutelin that were deamidated by citric acid and malic acid nearly remained in their relatively low state. The slight hydrolysis of peptide bonds would not adversely affect the amount of soluble protein (Liao et al., 2010). 3.2. Sulfhydryl groups (ASH) and disulfide bond (SAS) contents The total and exposed sulfhydryl and disulfide bond contents play important roles in the structural stability (Bonander, Leckner, Guo, Karlsson, & Sjölin, 2000). As shown in Fig. 1C, the total and exposed sulfhydryl contents of AG were 3.37 and 2.34 lmol/g of protein, respectively, indicating that approximately one-third of the sulfhydryls are buried inside the original glutelin. For deamidated samples, the values of total and exposed sulfhydryl contents were significantly higher than the values for AG (p < 0.05) at 9.02 and 8.57 lmol/g of protein for MDAG, and 9.25 and 9.02 lmol/g for CDAG. Almost all the free ASH groups in deamidated glutelin were exposed. However, the disulfide bond contents of

Fig. 1. The degree of deamidation (A), the degree of hydrolysis (B), total sulfhydryl (total-SH), exposed sulfhydryl (exposed-SH), and disulfide bond (SAS) contents (C), the surface hydrophobicity (D) of AG, MDAG and CDAG. Different superscript letters (a  c, A  B, m  n) indicate significant differences at the p < 0.05 level.

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deamidated glutelin were lower than AG (p < 0.05). Deamidation with organic acids was thought to cause a portion of disulfide bonds to cleave and form new sulfhydryl groups in glutelin because of the unfolding and aggregation of protein (Mirmoghtadaie et al., 2009). 3.3. Surface hydrophobicity (H0) The H0 values of glutelin increased significantly (p < 0.05) after deamidation, as shown in Fig. 1D, suggesting that there were more hydrophobic regions exposed on the surface of the protein. CDAG had a lower H0 value than MDAG at a neutral pH, which was consistent with Zhao, Tian, and Chen (2010), who found that a further increase in the DD resulted in a significant decrease in the surface hydrophobicity of barley hordein. A higher degree of deamidation might lead to extensive protein hydrolysis (Fig. 1B) resulting in shorter peptides with a higher charge density (ANH+3 and ACOO groups) and subsequent protein re-aggregation, which folded the exposed portion of the hydrophobic groups and decreased the surface hydrophobicity. In addition, excessive deamidation can cause unfolded proteins to aggregate by hydrophobic and electrostatic forces. Thus, a lower H0 value was observed in CDAG in comparison with MDAG. The H0 value is a key factor in the solubility and emulsifying properties of protein as a surface behavior of protein (Zhao et al., 2013). The increased H0 values in MDAG and CDAG could lead to a better molecular arrangement at the oil–water interface, resulting in a better emulsifying property for food processing (Wong et al., 2012). 3.4. SDS–PAGE SDS–PAGE has been used to identify degradations and changes in the conformation of protein fractions (Liao et al., 2010; Zhao et al., 2011) during deamidation treatments. The molecular weight (MW) distribution of the protein samples (AG, MDAG, and CDAG) is shown in Fig. 2. The control sample (lane 1) showed four major bands at 43, 29–34, 19, and 16 kDa. In the deamidated glutelin fractions, the bands at approximately 29–34, 19, and 16 kDa had become weaker and moved to lower molecular weights, and the distinct band of approximately 43 kDa, as marked by an arrow, disappeared in MADG (lane 2) and CDAG (lane 3). It was simultaneously observed that two distinct subunits near 14 and 9 kDa, as marked by arrows, appeared in MDAG and CDAG samples, which might be related to the protein fractions of glutelin having higher molecular weight degradations into smaller fragments after deamidation with organic acids. These results were consistent with the report by Liao et al. (2010), who found that the deamidated of wheat gluten by organic acids displayed a similar degradation, with large molecular weights more susceptible to degradation into smaller fragments. In addition, the bands in lane 3 were similar or even lighter than the corresponding bands in lane 2. Overall, the patterns of SDS–PAGE for AG, MDAG, and CDAG were consistent with data from Fig. 1A and B, indicating that citric acid had a stronger effect on cracking the peptide bonds of glutelin than malic acid. 3.5. Fourier transform infrared (FTIR) spectroscopy To gain a deeper understanding of the unfolding and aggregation of deamidated glutelin, Fourier transform infrared spectroscopy (FTIR) was conducted to investigate differences in the secondary structures of AG, MDAG, and CDAG by analyzing the amide I band known as the stretching vibration of C@O, which pertains to in-plane NH bending and CN stretching in the 1700– 1600 cm1 region of the protein (Zhao et al., 2012). Secondary structural compositions of AG and deamidated glutelin by malic acid and citric acid were shown in Table 1. The amide I bands of

Fig. 2. SDS–PAGE band patterns, the lanes from left to right are MW standards, AG (1), MDAG (2) and CDAG (3), the arrows mark different bands that disappeared or were newly generated in other lanes.

glutelin were made up of six major absorbance peaks, which can be assigned to the b-sheet (1616.2, 1629.8, and 1673.7 cm1), bturn (1658.8 and 1687.5) and random coil (1644.3), according to previous references (Zhao et al., 2011, 2012). There were some differences between deamidated glutelin and control glutelin in the secondary structure component, which confirmed that the protein conformation changed after deamidation treatment. The b-sheet groups in deamidated glutelin at approximately 1613–1616 and 1627–1631 cm1 decreased, suggesting that the protein was hydrolyzed and unfolded because some peptide bonds were cleaved. Because of their polar nature, b-turns always occur at protein surfaces, connecting with solvent water directly (Rose, Young, & Gierasch, 1983). As the smallest among secondary structures, these turns are involved in the formation of other bigger secondary structures, favoring the conformational stability of protein (Trevino, Schaefer, Scholtz, & Pace, 2007). The b-turn structure in MDAG and CDAG was evidently increased after deamidation, indicating that deamidating glutelin with organic acids improved the conformational stability of proteins because of the exposure of buried residues and the formation of more electrostatic repulsion and hydrogen bonds. However, there were no significant differences in the composition of secondary structures between MDAG and CDAG. Thus, treatments with malic acid and citric acid caused negligible differences with regards to the change in glutelin secondary structure. 3.6. Solubility Solubility in the aqueous phase is a very important functional property for protein for food industry applications because it is

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L. Lei et al. / Food Chemistry 178 (2015) 96–105 Table 1 The secondary structural compositions (%) of AG, MDAG and CDAG as determined by FTIR spectra. Samples

b-Sheet 1613–1616 cm

AG MDAG CDAG

13.96 9.26 7.58

Random coil

b-Turn 1

1

1

1

1

1627–1631 cm

1673–1675 cm

In total

1659–1664 cm

1688 cm

In total

1645 cm1

23.22 20.57 20.66

12.98 15.48 15.46

50.16 45.31 43.70

20.38 21.81 22.52

6.21 8.79 9.08

26.59 30.60 31.60

23.25 24.09 24.70

related to the emulsifying, foaming, and rheological properties (Qiu, Sun, Zhao, Cui, & Zhao, 2013; Takeda, Matsumura, & Shimizu, 2001; Zhao et al., 2013). The effects of deamidation with different edible organic acids on glutelin solubility were investigated. The pH-dependent solubility profiles of AG, MDAG, and CDAG are shown in Fig. 3A. The solubility of control and deamidated samples showed a similar trend with decreasing solubility followed by a gradual increase in the pH between 2 and 12. The lowest solubility was at pH 4, which was at the approximate isoelectric point of protein. It could be observed that the solubility of deamidated proteins was significantly improved compared with the control glutelin, increasing from 7.79% for AG to 39.13% for MDAG and 26.06% for CDAG at pH 4, respectively. Wheat gliadin that was deamidated by citric acid showed similar protein solubility profiles (Qiu, Sun, Zhao, et al., 2013). The improved solubility might be related to the decrease in molecular size and the degree of increase in hydrolysis, resulting in the unfolding structure of protein during deamidation (Mirmoghtadaie et al., 2009; Qiu, Sun, Zhao, et al., 2013). The exposure of the hydrophobic regions and charged polar groups that were originally on the inside of the protein promoted protein–water interactions and increased solubility (Chan & Ma, 1999), as confirmed by the amino acid composition (Table 2). Table 2 showed that both uncharged polar and acidic amino acids increased after deamidation. The solubility profiles of glutelin were superior at acidic and alkaline pH values after deamidation. For CDAG, the solubility values were 85.70%, 84.86%, 88.8%, and 87.78% at pH values of 2, 8, 10, and 12, respectively, which were relatively higher than MDAG with corresponding values of 71.72%, 66.99%, 76.54%, and 78.42%. These values were significantly higher than those of control glutelin (p < 0.05). Hence, they suggest that deamidation with edible organic acids can be an effective way to improve glutelin solubility and thus broaden the potential functional applications of deamidated glutelin in food processing.

3.7. Emulsifying properties The effects of the pH on the emulsifying activity index (EAI) and the emulsion stability index (ESI) are presented in Fig. 3B and C, respectively. CDAG exhibited better emulsifying activity than MDAG, especially at alkaline pH. In addition, the EAI values of deamidated glutelin increased significantly (p < 0.05) in comparison with those of the control sample within a pH range from 2 to 11. The results were consistent with previous reports that found deamidation to improve the emulsifying activity of wheat gluten (Liao et al., 2010) and oat protein isolate (Mirmoghtadaie et al., 2009). Increasing the solubility and surface hydrophobicity of deamidated glutelin could be responsible for this improvement in the EAI. Acid deamidation generally increased the number of polar and hydrophobic groups on the surface of protein that promoted their binding to the oil phase. These proteins might aggregate with the help of hydrophobic groups to form films at the oil–water interface, thus improving emulsification by forming a better hydrophobic–lipophilic balance (Liao et al., 2010; Mirmoghtadaie et al., 2009; Zhao et al., 2011). The minimum EAI

Fig. 3. The effect of the pH on protein solubility (A), the emulsifying activity index (EAI, B) and the emulsion stability index (ESI, C) of AG, MDAG and CDAG. Different superscript letters at the same pH values indicate significant differences at the p < 0.05 level.

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Table 2 The amino acids composition of AG, MDAG and CDAG (g/100 g protein).

a b c d e

Amino acid

AG

Thr Val Ile Leu Lys His Trp Met Phe Tyr Cys Asp Ser Glu Gly Ala Arg Pro Hydrophobica Uncharged-polarb Basicc Acidicd Essential amino acidse

2.44 4.96 3.75 7.39 4.39 2.08 Not determined 0.80 4.57 3.64 0.30 3.10 3.28 13.11 2.20 2.77 7.38 19.44 45.87 9.66 13.85 16.21 34.32

Ala, Val, Ile, Leu, Phe, Pro, Gly and Met. Cys, Thr, Tyr and Ser. Arg, His and Lys. Asp and Glu. Thr, Val, Ile, Leu, Lys, His, Met, Phe, Tyr and Cys.

MDAG

CDAG

3.17 4.72 3.56 7.05 4.35 1.92

2.87 4.81 3.59 6.84 4.07 1.94

0.61 4.32 3.57 0.21 5.05 5.82 21.27 3.10 2.70 7.19 12.29 38.35 12.75 13.46 26.33 33.48

0.63 4.29 3.48 0.11 4.64 5.47 20.96 2.90 2.68 6.35 12.35 38.08 11.92 12.36 25.60 32.63

of AG, MDAG and CDAG were 2.42, 3.66 and 3.44 m2 g1, respectively, at approximately the isoelectric point of pH 5. The highest EAI value (72.67 m2 g1) was observed in CDAG at pH 11. The trends for EAI values in both the control sample and deamidated samples were similar to their solubility profiles. The increased EAI might be explained by the increase in protein solubility (Fig. 3A) because the hydrolysis of peptide bonds would allow more protein molecules to be available at the oil–water interface. It is precisely because of the formation and stability of protein emulsions, i.e., protein with hydrophilic residues oriented toward the aqueous phase and lipophilic residues toward the oil phase at the oil–water interface, that the surface tension is reduced (Mao & Hua, 2012). Deamidation would decrease the molecular weight and unfold the structure of the protein as seen from the SDS–PAGE and FTIR data, which would expose buried hydrophobic residues and other functional groups within the protein matrix. This exposure would enhance interactions at the protein–oil interface (Mirmoghtadaie et al., 2009). Therefore, structural factors other than surface hydrophobicity and solubility should be considered to elucidate the relations between the emulsifying properties and the structures of proteins. Surprisingly, the emulsion stability index (ESI) of glutelin as isolated from AS decreased after deamidation (Fig. 3C), especially for CDAG. No clear trend was observed between the ESI and pH. The ESI values of AG were significantly (p < 0.05) higher than those of MDAG at almost all pH values (2–11), and the ESI values of MDAG were higher than those of CDAG. The results were consistent with those of Mirmoghtadaie et al. (2009), who found that the ESI of the

Fig. 4. The storage modulus (G0 ) and loss modulus (G00 ) evolution vs. temperature and time of AG (A), MDAG (B) and CDAG (C).

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oat protein isolate decreased after deamidation and succinylation. The differences in the ESI of AG, MDAG, and CDAG can be explained by excessive increases in the net charge and electrostatic repulsion, reducing the interactions between proteins so that the formation of the elastic film at the oil–water interface was prevented. There are other factors such as the pH, net charge, interfacial tension, viscosity and protein conformation that may affect the ESI values, such that further study is needed to elucidate this phenomenon. 3.8. Rheological properties The rheological behavior is related to the gelling capacity, foaming properties, and other functional properties of protein. It can affect the textural qualities of food, such as the taste, mouth feel, and shelf-life stability (Spotti et al., 2014). Fig. 4 shows the evolution of the storage modulus (G0 ) and loss modulus (G00 ) with time and temperature for AG (Fig. 4A), MDAG (Fig. 4B), and CDAG (Fig. 4C). With increasing temperatures, both the G0 and G00 of AG, MDAG, and CDAG decreased. The intersection between G0 and G00 (the gelation point) of deamidated glutelin was higher than that of the original glutelin. The gelation points were 45.0, 60.5, and 84.8 °C from the test software for AG, MDAG, and CDAG, respectively. This finding suggested that deamidation could reduce the sensitivity of proteins to the temperature, possibly due to the deamidation-induced unfolding of glutelin because more sulfhydryl groups and hydrophobic sites were exposed, causing extensive intermolecular interactions. The increased surface hydrophobicity coupled with the presence of more negatively charged polar groups decreased the protein–protein interactions caused by electrostatic repulsion, and thus enhanced the protein solubility and delayed the gelation point (Matsudomi, Kato, & Kobayashi, 1982). At a constant temperature of 95 °C for 5 min, both the G0 and G00 values of CDAG were significantly higher than the values for AG, suggesting that the CDAG gel network was much stronger than that of the control sample, and a further frequency sweep test for the strength of deamidated glutelin gel was performed and discussed as follows. During the final cooling stage from 95 to 25 °C, increases in both G0 and G00 were observed. The same phenomenon has been found in other systems such as soy proteins (Renkema & van Vliet, 2002), whey protein, and dextran conjugates (Spotti et al., 2014). Storage modulus (G0 ) remained much higher than loss modulus (G00 ) for CDAG for the whole cooling stage. However, G0 surpassed G00 after 2.5 and 2.4 min, respectively, for the gel systems of AG and MDAG, indicating that an elastic gel was formed and glutelin that was deamidated by malic acid could form a gel network more quickly than glutelin deamidated by citric acid and the control sample. This phenomenon could be related to the non-covalent bonds between the denatured proteins, such as van der Waals forces and hydrogen bonds (Martinez, Farías, & Pilosof, 2010). In the process of keeping the temperature at 25 °C for 5 min, both the G0 and G00 values of AG, MDAG, and CDAG were almost the highest during the entire temperature sweep process, with a G0 value consistently higher than the G00 values, indicating that the gel system formed from the three samples created a strong elastic gel network structure. The G0 value of AG was constant, and yet the G0 values of MDAG and CDAG increased for the whole constant temperature stage, indicating that the gelation of deamidated glutelin was more stable than control glutelin. To investigate the viscoelastic properties of the deamidated glutelin and control samples, frequency sweep measurements were conducted from 0.01 to 10 Hz. The storage modulus (G0 ) and the loss modulus (G00 ) of the protein gel samples are shown in Fig. 5A and B. The frequency sweeps of three protein samples were characteristic of elastic gels with G0 > G00 at all frequencies, which indicated that a strong, stable gel network was formed. In

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general, the G0 value indicates the strength of a gel network, and it can be influenced by the compactness of the structure or the number of cross-links in a gel system (Clark & Ross-Murphy, 1987). A higher G0 value indicates a stronger inter-molecular network and increased inter-molecular protein–protein interactions (Jones, 1979; Murray, Myers, Barker, & Maurice, 1981). The G0 values of both MDAG and CDAG were much higher than those of the control sample (Fig. 5A and B), especially for CDAG, and they were significantly twenty times more than that of the control sample, which indicated that deamidation strengthened the gel network of glutelin. This finding may be attributed to protein unfolding and the exposure of hydrophobic regions and charged polar groups after deamidation (Bryant & McClements, 1998), which would increase the forces affecting the dimensional network, such as electrostatic, hydrogen and hydrophobic interactions. At the same time, it was easy to find that both the G0 and G00 values of protein gel samples were independent of the frequency almost along the entire frequency range, except for at the beginning, when the G0 values increased slightly with higher frequency, and the G0 values varied more in the MDAG gel compared with the AG and CDAG gels. According to Clark and Ross-Murphy (1987), the frequency behaviors of the protein samples suggested that AG, MDAG and CDAG formed covalent, strong gels. However, the G00 values of CDAG were contrary to those of AG and MDAG and decreased with the frequency sweep. Different non-covalent interactions, such as hydrophobic interactions, hydrogen bonding, elec-

Fig. 5. Frequency sweeps for the gel systems of AG and MDAG (A), CDAG (B).

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trostatic interactions and covalent bonds would influence the formation and structure of gels (Spotti et al., 2014). The decreased G00 values in CDAG might be explained by the extensive protein hydrolysis resulting in subsequent protein re-aggregation, which folded the exposed portion of the hydrophobic groups and decreased the surface hydrophobic group. These results suggest that the rheological properties of glutelin as prepared from AS can be affected by deamidation treatment. In this study, the factor that promotes strengthened gelation could be changes in native protein structures, primarily in their secondary structure (Spotti et al., 2014), such as the increase in sulfhydryl groups (Fig. 1C) and hydrophobic bonds (Fig. 1D). 3.9. Amino acid composition of AG, MDAG and CDAG Table 2 lists the amino acid compositions of deamidated glutelins and the control. The amino acid composition of control glutelin in this study was consistent with Du et al. (2012). Untreated glutelin exhibited high glutamic and aspartic acid contents, with limited amino acids in terms of threonine, cysteine, histidine, and lysine. In deamidated glutelins, the available acidic amino acids were increased significantly by acid deamidation treatment, as shown in Table 2, because the amide groups on uncharged amino acid glutamines and asparagine residues were eliminated and formed glutamic and aspartic acid residues. However, the contents of uncharged polar amino acids increased primarily by increasing serine, which could contribute to the formation of new hydrogen bonds and the increase of protein solubility in aqueous solution. In summary, deamidation can dissociate protein polymers, thus increasing electrostatic repulsion and surface hydrophobicity, in addition to the flexibility of glutelin molecules. The content of the first limited amino acid (threonine) was enhanced significantly in MDAG, but it showed only a slight increase in CDAG. Lysine is considered to be the most important essential amino acid in cereal protein. In this case, deamidation treatment caused negligible changes in the lysine and histidine contents. The amounts of essential amino acids in both MDAG and CDAG were quite similar to those of the control glutelin. The amino acid composition results suggested that edible organic acid deamidation was mild and could maintain the essential amino acids of glutelin. 4. Conclusions In this study, deamidation with edible organic acids caused a significant improvement in the solubility and emulsifying properties of glutelin isolated from AS. The degrees of deamidation and hydrolysis of MDAG were 46.87% and 3.80%, which were lower than the values for CDAG (56.4% and 5.1%, respectively). Glutelin deamidated by citric acid (CDAG) showed better solubility and emulsifying abilities, but less improvement in the disulfide bond contents and surface hydrophobicity than glutelin deamidated by malic acid (MDAG). Furthermore, deamidation with citric acid had a greater effect on the changing glutelin structure than malic acid, resulting in a stronger degradation of subunits. The effects of deamidation on rheological properties were the increase in the onset temperature for gelation to occur and an improvement in the gel network strength. The amounts of essential amino acids in glutelin after deamidation could be maintained. Our results showed that citric acid was more preferable than malic acid; however, because citric acid may be limited for some food applications, malic acid can be an alternative. These new characteristics of deamidated glutelin suggested that acid-heating treatment could be a potential way to enhance this substance’s usability as a food additive in plant

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The effect of deamidation on the structural, functional, and rheological properties of glutelin prepared from Akebia trifoliata var. australis seed.

The characteristics of glutelin samples from Akebia trifoliata var. australis seeds (AG) that had been deamidated by malic acid (MDAG) and by citric a...
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