Functional, nutritional and conformational changes from deamidation of wheat gluten with succinic acid and citric acid

Food Chemistry 123 (2010) 123–130

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Functional, nutritional and conformational changes from deamidation of wheat gluten with succinic acid and citric acid Lan Liao, Tong-Xun Liu, Mou-Ming Zhao *, Chun Cui, Bo-En Yuan, Sheng Tang, Fei Yang College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, Guangdong, PR China

a r t i c l e

i n f o

Article history: Received 5 October 2009 Received in revised form 12 March 2010 Accepted 13 April 2010

Keywords: Wheat gluten Deamidation Succinic acid Citric acid Functional properties Protein conformation Nutritional property

a b s t r a c t Changes in deamidation degree, hydrolysis degree, nitrogen soluble index, the foaming and emulsification properties, the tertiary and secondary conformation and nutritional property of wheat gluten deamidated with succinic acid and citric acid were identified. Succinic acid and citric acid were found to effectively deamidate the amides in wheat gluten proteins into carboxyl groups, which resulted in a significant increase of the nitrogen soluble index of wheat gluten. Deamidation of wheat gluten by succinic acid was found to be more efficient than that with citric acid, although wheat gluten treated with succinic acid exhibited less improvement in the foaming capacity and stability and experienced inhibition in the emulsification activity compared with the gluten treated with citric acid. Wheat gluten deamidated with citric acid exhibited more flexible protein molecules, greater changes in the tertiary and secondary structures and better nutritional characteristics. These results may be useful to the food processing industry. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Wheat gluten, a byproduct of wheat starch industry, is an abundant source of plant proteins and is potentially very useful in food production because it is a ubiquitous and relatively inexpensive source of nutrition. Over the past few decades, wheat gluten has been readily used in food and non-food industries. However, the solubility, the most important property of proteins, in wheat gluten is limited, which makes its use in food processing limited as well. The hydrophobic nature of wheat gluten and its hydrogen bonds, as indicated by the large percentage (around 50%) of uncharged amino acid residues (glutamines (Gln) and asparagines (Asn)) in gliadin and glutenin, are the main reasons for its waterinsoluble characteristics. By transforming the amides of Gln and Asn into carboxyl groups through deamidation, the solubility of the proteins in wheat gluten could be transformed with such effects as changing the charge density, increasing the electrostatic repulsion and breaking the hydrogen bonds (Riha, Izzo, Zhang, & Ho, 1996). Considerable research has focused on catalysing the Abbreviations: SA, succinic acid; CA, citric acid; DWG, deamidated wheat gluten; S-DWG, succinic acid deamidated wheat gluten; C-DWG, citric acid deamidated wheat gluten; NSI, nitrogen soluble index; SDS–PAGE, sodium dodecyl sulphate– polyacrylamide gel electrophoresis; FTIR, Fourier transform infrared spectroscopy; EAI, emulsion activity index; ESI, emulsion stability index. * Corresponding author. Tel./fax: +86 20 87113914. E-mail address: [email protected] (M.-M. Zhao). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.04.017

deamidation of proteins by acids, bases and enzymes under certain physical conditions with different reaction mechanisms to improve their functional properties (Aranyi & Hawrylewicz, 1972; Berti et al., 2007; Chan & Ma, 1999; Kato, Tanaka, Lee, Matsudomi, & Kobayashi, 1987; Matsudomi, Sasaki, Kato, & Kobayashi, 1985; Wu, Nakai, & Powrie, 1976). Shih (1992, chap. 7) showed that the solubility, emulsification and foaming properties of proteins were enhanced after deamidation under mild acidic-heating conditions. In previous studies, hydrochloric acid has been the most frequently used acid catalyst for protein deamidation, in addition to sulphuric acid, phosphoric acid, formic acid, trichloroacetic acid and chlorosulfonic acid (Aranyi & Hawrylewicz, 1972; Chan & Ma, 1999; Matsudomi et al., 1985; Wu et al., 1976). However, deamidation with hydrochloric acid leads to certain problems, such as uncontrollable hydrolysis, the production of potentially carcinogenic compounds, including mono- and dichloropropanols and monochloropropanediols, and the isomerisation of certain amino acids, that have remain unresolved (Nagodawithana, 1994). Several studies have investigated the use of acetic acid in deamidation of proteins (Aranyi & Hawrylewicz, 1972; Berti et al., 2007; Liao et al., 2009; Wu et al., 1976). These studies showed that acetic acid-induced deamidation of proteins could be an economic and efficient way to improve the solubility, foaming and emulsification properties of proteins. However, limited data are available on the deamidation of proteins with other edible organic acids aside from acetic acid. Especially, their

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potential application as candidates for use in new product formulations and fortification, such as protein-fortified beverages, infant formulas, coffee whiteners, emulsifiers, and flavour enhancers, will require further investigation. In this study, succinic acid and citric acid, which contain the same carboxylic group as that in acetic acid, were tested for their ability to deamidate proteins in wheat gluten. Changes in the functional, conformational and nutritional properties of wheat gluten deamidated with succinic acid and citric acid were determined. 2. Materials and methods 2.1. Materials, chemicals and reagents Commercially produced wheat gluten with 74.95% (w/w, dry basis) crude protein, 6.68% moisture, 6.18% fat, and 9.51% carbohydrate was obtained from Lianhua Co. Ltd. (Zhoukou, China). The chemical l-anilino-8-naphthalene sulphonate (ANS) was obtained from Sigma Chemical Co. Ltd. (St. Louis, MO). All other chemicals and reagents were of analytical grade and obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). 2.2. Deamidation of wheat gluten To deamidate the proteins in the wheat gluten, 10% (w/v) wheat gluten was mixed with succinic acid (SA) (0.017–0.066 M) or citric acid (CA) (0.0095–0.038 M) to form suspensions. The suspensions were incubated for 2 h in a water bath shaker at room temperature and then heated at 121 °C for different time intervals (0–15 min). Acid-treated samples were withdrawn at a given time and immediately held in an ice water bath for 5 min to stop the reaction. The samples were centrifuged at 10,000g for 10 min at 4 °C, and soluble fractions were collected by using a syringe according to the method described by Aranyi and Hawrylewicz (1972). The collected soluble fractions were dialysed in deionised water at 4 °C for 24 h to remove the ammonium and then freeze-dried. Wheat gluten without acid treatment under the same conditions was used as the control sample.

of deamidation in the modified samples was determined from the ratio of the amount of ammonia released from the deamidated samples to the total amount of ammonia in wheat gluten. 2.4. Degree of hydrolysis (DH) and nitrogen soluble index (NSI) The degree of hydrolysis (DH) was defined as the percentage of peptide bonds cleaved during a reaction and calculated from the ratio of free amino groups to the total number of peptide bonds in soluble factions during deamidation, according to the method described by Adler-Nissen (1979). The nitrogen soluble index (NSI) was calculated as the ratio of the protein content in the collected soluble fractions to the total protein content of the suspension in the reaction, which was determined by Kjeldahl method with the nitrogen–protein conversion factor of 5.7 (AOAC, 1990). The protein content in the collected soluble fractions was determined according to the method described by Lowry, Rosebrough, Farr, and Randall (1951) using bovine serum albumin as the standard. 2.5. Evaluation of the emulsification characteristics The emulsification activity index (EAI) and the emulsion stability index (ESI) for the samples were determined by the turbidimetric technique described by Pearce and Kinsella (1978) with some modifications. Corn oil (10 ml) and protein solution (0.4% w/v sample in 0.01 M phosphate buffer, pH 7.0, 30 ml) were homogenised with an Ultra Turrax-homogenizer (model T25 Basic IKA Labortechnik homogenizer, IKA Works Inc., Wilmington, NC) at a speed 20,000g for 1 min at 30 °C. One ml of the emulsion was diluted serially by 1/50–1/500, using 0.1% sodium dodecyl sulphate (SDS). The absorbance was recorded at 500 nm (A500) with a Spectrumlab 22PC spectrophotometer (Shanghai Lengguang Technology Co. Ltd., Shanghai, PRC). EAI was calculated using the following equation:

n¼

2:303NA500 L

EAIðm2 =gÞ ¼ 2.3. Degree of deamidation The amount of ammonia produced from ammonium during the deamidation process before dialysis and freeze-drying was determined according to the method described by Kato et al. (1987). 0.5 g of untreated wheat gluten was dissolved in 5 ml of 3 M HC1, sealed in a 10 ml glass ampoule and heated at 121 °C for 3 h to reach complete deamidation. The amount of ammonia obtained in this step was determined to be the total amount of ammonia in the wheat gluten. The amount of ammonia released from the samples during the deamidation process was determined as follows: One millilitre of 2% boric acid solution, containing methyl red and bromocresol green, was put into the central chamber of a microdiffusion unit. To separate the NH3 gas in the outer chamber, 3 ml of saturated potassium metaborate was used. One millilitre of sample solution was placed in the outer chamber of the microdiffusion unit and mixed with the alkali solution previously placed in the chamber. The cover of microdiffusion unit was then immediately sealed with Arabia gum to prevent the ammonia gas from leaking out. Then, the alkali solution and the sample solution in the outer chamber were mixed to liberate the ammonia gas from the ammonium in the sample. The mixture was allowed to stand for 12 h at 30 °C to completely release the ammonia produced during the deamidation. The ammonia absorbed in the 4% boric acid solution in the central chamber was then titrated with 0.02 M HC1. The degree

2n104 ; C/

where A500 is the absorbance at 500 nm; / is the volume fraction of the dispersed phase; L is the light path in metres (1 cm); C is the concentration of the protein before the emulsion is formed (g/ml); N is the level of dilution; and f is the evaluation of the turbidity of the sample. ESI was determined by placing aliquots of the emulsion (10 ml) in 10.0 ml cylinders immediately after preparation. At 0 and 30 min following preparation of the emulsion, 0.1 ml samples were pipetted and diluted 100-fold prior to turbidity measurements. ESI was calculated as follows:

ESIðminÞ ¼

f0 DT ; Df

where Df is the change in turbidity after the time interval, DT (30 min). 2.6. Evaluation of the foaming properties The foaming properties were evaluated by the method described by Bernardi Don, Pilosof, and Bartholomai (1991) with modifications. A protein solution (1% w/v sample in 0.01 M phosphate buffer, pH 7.0) was homogenised with an Ultra Turraxhomogenizer (model T25 Basic IKA Labortechnik homogenizer, IKA Works Inc., Wilmington, NC) at a speed of 20,000g for 1 min at 30 °C. The foaming capacity was calculated as the percentage

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of increase in volume of the protein dispersion upon mixing, while foaming stability was estimated as the percentage of foam volume remaining after 30 min.

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der the following conditions: 80 scans, 2 s exposure time, 2 cm1 resolution. The averaged spectral data from the scans of samples in the Raman spectrophotometer were baseline corrected and normalised against the phenylalanine band at 1003 cm1.

2.7. Evaluation of the surface hydrophobicity The surface hydrophobicity was evaluated using l-anilino-8naphthalene sulphonate (ANS) as a fluorescence probe as described by Haskard and Li-Chan (1998) with modifications. Four millilitres of protein soluble fractions at successive concentrations (0.005%, 0.01%, 0.02%, 0.05%, 0.1%) (w/v) were prepared in 10 mM phosphate buffer (pH 7.0). To each protein fraction, 20 ll of 8  101 M ANS stock solution was added. The mixtures were shaken in a vortex mixer for 5 s. The fluorescence intensity (FI) was measured at wavelengths of 390 nm (excitation) and 470 nm (emission) using a RF-5301 PC spectrofluorometer (Shimadzu Corp., Kyoto, Japan) at 26 ± 0.5 °C, with a constant excitation and emission slit of 5 nm. The FI for each sample was then computed by subtracting the FI attributed to the protein in the buffer. The initial slope of the FI versus protein concentration was calculated by linear regression analysis and used as an index of the surface hydrophobicity. 2.8. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) Untreated wheat gluten and freeze-dried modified samples were dissolved in distilled water to 2 mg/ml. A small portion (20 ll) of each sample was mixed with 10% (w/v) SDS and 1% (v/ v) b-mercaptoethanol, denatured in boiling water for 10 min, and centrifuged at 10,000g for 3 min. Then, 10 ll of the denatured sample was loaded onto a homogeneous phastgel (DYCZ-30, Beijing Liuyi Instrument Factory, PRC) and tested according to Laemmli’s (1970) method using 12% acrylamide separating gel and 5% acrylamide stacking gel. Samples were prepared in Tris–glycine buffer (pH 8.8) containing 1.5% SDS and the gel sheets were stained with Coomassie brilliant blue R-250 for 1 h at 30 °C. The molecular weight standards (Bio-Rad, Hercules, CA) were as follows: phosphorylase (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa) and lysozyme (14.4 kDa). 2.9. Fourier transform infrared (FTIR) spectroscopy FTIR spectra of untreated wheat gluten and freeze-dried modified samples were recorded using a Nicolet 8210E FTIR spectrometer (Nicolet, WI) equipped with a deuterated triglycine sulphate detector. The spectrometer was continuously purged with dry air from a Balston dryer (Balston, MA). The sample powder (maintained at ambient temperature) included 1 mg sample per 200 mg of KBr. After homogenising with an agate mortar and pestle, the powder was pressed into pellets (1–2 mm thick films) with a 15-ton hydraulic press. FTIR spectra were obtained of wave number from 400 to 4000 cm1 during 128 scans, with 2 cm1 resolution (Paragon 1000, Perkin–Elmer, USA). Interpretation of the changes in the overlapping amide I band (1600–1700 cm1) components was made possible by deconvolution using Peak-Fit v4.12 software (SPSS Inc.). 2.10. 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). Solid samples obtained from the freeze-dried soluble deamidated fractions were placed on microscope slides. The laser was then focused on the samples. Each spectrum was obtained un-

2.11. Amino acid analysis Untreated wheat gluten and samples deamidated with succinic acid and citric acid were dissolved in water and then adjusted to pH 7.0 with 2 M NaOH followed by centrifugation at 10,000g for 10 min at 4 °C. The amino acid composition was determined by high-performance liquid chromatography (Waters, Milford, MA) equipped with a PICO.TAG column. The amino acid profile of the supernatant was determined according to the methods of Bidlingmeyer, Cohen, Tarvin, and Frost (1987) and Aaslyng, Martens, and Poll (1998). The total concentration of amino acid was determined after hydrolysis at 110 °C for 24 h with 6 M hydrochloric acid prior to derivatisation with phenyl isothiocyanate. Alkaline hydrolysis was also used for determination of the tryptophan level. 2.12. Statistical analysis All experiments were carried out in triplicate. Data were reported as mean ± standard deviation (SD). Analysis of variance and significant difference tests were conducted to identify differences among means by LSD’s multiple-range test using the SPSS software package (version 13.0 for Windows, SPSS Inc., Chicago, IL). p < 0.05 was considered statistically significant. 3. Results and discussion 3.1. Deamidation of wheat gluten with succinic acid and citric acid According to Raman spectroscopy, the carbonyl stretching frequencies of the carboxylic acid monomer are located between 1740 cm1 and 1800 cm1 (Colthup, Daly, & Wiberley, 1990). A newly formed C = O stretching vibration band was observed at 1745 cm1, which was attributed to the c-carboxyl groups of aspartic and glutamic acids (Fig. 1C; some data not shown). This band indicates that glutamines and asparagines were indeed deamidated by –COOH from succinic acid or citric acid to form corresponding glutamic and aspartic carboxyl groups (Wong, Choi, Phillips, & Ma, 2009). As shown in our previous study (Liao et al., 2009), deamidation with succinic acid or citric acid occurred in samples at the beginning of the moisture-heating incubation. The requirement that the reaction time during the measurement procedure is up to 12 h in this study likely resulted in deamidation in the samples. The degree of deamidation and NSI as a function of the reaction time at various concentrations of succinic acid and citric acid are presented in Table 1. In the absence of acid, the degree of deamidation of untreated wheat gluten increased slowly from 0% to 9.6% after 15 min. In the acid-treated samples, the degree of deamidation increased significantly (p < 0.05). At a given reaction time, the degree of deamidation increased with an increased concentration of succinic acid and citric acid. And at a given acid concentration, the degree of deamidation was characterised by an initial fast rate of increase for the first 5 min, reaching the maximum (>60%), followed by a slow decrease with a prolonged reaction time. The rate of deamidation of glutaminyl and asparaginyl residues in proteins is determined by the primary and tertiary structures that govern the distribution of the amino acid residues surrounding the glutaminyl and asparaginyl residues in proteins (Wright & Urry, 1991). The rate of deamidation of hydrion by succinic acid, estimated from the ratio of the degree of deamidation (described in

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a 1000

b 100

900 800 80

Raman Intensity

Raman Intensity

700 600 500 400

60

40

300 20

200 100

0 0 480

500

520

2550

540

2560

2570

2580

2590

2600

Wavenumber (1/cm)

Wavenumber (1/cm)

c 1200

Raman Intensity

1000

800

600

400

200

0

-200 1580

1600

1620

1640

1660

1680

1700

1720

1740

1760

Wavenumber (1/cm) Fig. 1. Raman spectroscopy of S-DWG (solid line) and C-DWG (dash line) at similar deamidation degree of 61%, NSI of 90% in 10 min moisture-heating. (A) Profile of disulfide bonds content, (B) profile of sulfhydryl group content and (C) profile of amide I and the dissociated or ionised carboxyl (COO) groups.

Table 1 Deamidation degree and nitrogen soluble index of wheat gluten deamidated with succinic acid and citric acida. Acids and concentration

Degree of deamididation (%) Treating time (min)

Nitrogen soluble index (%) Treating time (min)

0

5

10

15

0

5

10

15

Succinic acid 0.017 M 0.042 M 0.066 M

23.46 ± 0.10cA 24.40 ± 1.13cdA 27.20 ± 0.10eA

57.18 ± 0.10cBC 65.04 ± 0.69dCD 69.96 ± 0.28eC

55.55 ± 0.10cB 61.10 ± 0.87dC 63.10 ± 1.26deB

51.24 ± 1.53cB 54.39 ± 1.35cB 59.12 ± 0.10dB

12.81 ± 0.63bA 13.44 ± 0.50bcA 13.56 ± 0.36bcA

53.10 ± 1.60cB 87.74 ± 1.59deC 89.92 ± 1.34eC

64.99 ± 0.98cC 89.10 ± 1.88dC 95.23 ± 3.62eD

57.18 ± 0.10cBC 65.04 ± 0.69dB 69.96 ± 0.28eB

Citric acid 0.0095 M 0.024 M 0.038 M Untreated wheat gluten

18.10 ± 0.57bA 20.80 ± 0.28bcA 28.00 ± 0.28eA 0aA

45.33 ± 0.56bC 62.67 ± 0.56dC 64.29 ± 0.61dC 4.80 ± 0.55aB

40.20 ± 1.12bBC 61.15 ± 0.67dBC 62.52 ± 1.07eB 6.40 ± 0.10aC

38.23 ± 1.67bB 57.75 ± 0.84cdB 59.06 ± 1.10dB 9.60 ± 0.10aD

12.70 ± 0.51bA 12.91 ± 0.23bA 14.03 ± 0.10cA 3.48 ± 0.23aA

42.01 ± 0.26bBC 81.78 ± 1.35dC 89.59 ± 1.34eC 3.94 ± 0.31aB

41.01 ± 1.88bB 90.59 ± 0.28dD 93.02 ± 0.62edC 4.11 ± 0.06aC

45.33 ± 0.56bC 62.67 ± 0.56dB 64.29 ± 0.61dB 4.17 ± 0.01aC

a The data with different lowercase letters in the same row are significantly different (p < 0.05). The values with different uppercase letters in the same column are significantly different (p < 0.05).

Section 2.3) to the treatment time, was somewhat higher than that of citric acid (Table 1). This was also confirmed by the higher content of free Glu and Asp (Table 4) in succinic acid deamidated

wheat gluten, which indicated that the rate of deamidation of glutaminyl and asparaginyl residues in proteins with succinic acid was higher than that with citric acid. Succinic acid and citric acid

L. Liao et al. / Food Chemistry 123 (2010) 123–130

are weak electrolytes with electrolytic dissociation constants of 6.9  103 and 8.7  104, respectively (Willard & Furman, 1943). Consequently, the electrolytic dissociation hydrion concentration of citric acid in the wheat gluten suspension was lower than that of succinic acid. In addition, Shih and Kalmar (1987) identified the mechanism of catalysis in deamidation from the increased basicity of the amide bonds resulting from the combination of these bonds with the large anion of the catalyst. Thus, the higher deamidation rate in samples treated by succinic acid was probably due to the fact that the size and form of the anions from succinic acid seemed to be more appropriate for catalysis. The degree of hydrolysis of deamidated samples, as shown in Fig. 2a and b, decreased as the treatment time and acid concentration increased. Wright and Urry (1991) reported that the accessibility of H+ to amides and peptide bonds affected the competition between deamidation and peptide chain hydrolysis in reaction systems. Compared with the untreated wheat gluten, the content of free amino acid in the samples deamidated with succinic acid and citric acid remained nearly unchanged (data not shown), indicating that the catalysis of succinic acid and citric acid generally favored deamidation over peptide bond hydrolysis. We can therefore assume that the decrease in the degree of hydrolysis in deamidation when the treatment time and acid concentration increased was probably due to the significant (p < 0.05) increase in the amount of soluble protein after deamidation (Table 1). The increased NSI in samples deamidated with succinic or citric acid makes them good candidates for use in high-protein beverages and for products requiring emulsification and foaming properties.

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3.2. Evaluation of functional properties The emulsification and foaming properties of deamidated wheat gluten after treatment in moisture-heating for 10 min are presented in Table 2. The samples deamidated with succinic acid and citric acid are abbreviated as S-DWG and C-DWG, respectively. 3.2.1. Foaming property The foaming capacity of C-DWG and S-DWG increased significantly (p < 0.05) from 90 (the foaming capacity of untreated wheat gluten, deamidation degree = 0%) to 335 and 270, respectively. The foaming capacity and the stability of the acid-treated samples decreased as the degree of deamidation increased. This observation corresponds to the results of Wu et al. (1976) who showed that their samples exhibited a higher foaming capacity at a lower degree of deamidation, suggesting that an excessive increase in the deamidation degree did not increase the foaming capacity of the treated samples. The foaming capacity of C-DWG was higher than that of S-DWG at the same degree of deamidation. For instance, at a degree of deamidation of 61%, the foaming capacity of C-DWG (270) was about double that of S-DWG (120), in spite of the fact that the surface hydrophobicity of C-DWG was significantly (p < 0.05) lower than that of S-DWG (Table 3). In addition to the surface hydrophobicity, other structural properties were involved in the enhancement of the foaming capacity of C-DWG and were attributed to the greater flexibility of C-DWG (Table 3). Because higher flexible protein enhances the adsorption and anchorage of proteins at the

C

Degree of Hydrolysis (%)

0.5

a

SA0.017M SA0.042M SA0.066M

0.4 0.3

0.2 0.1

0.0 0.9

Degree of Hydrolysis (%)

0.8

b

5

10

15 CA0.0095M CA0.024M CA0.038M

10

15

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 5

Treating time (min) Fig. 2. Hydrolysis degree and SDS–PAGE analysis of deamidated wheat gluten. (a) Hydrolysis degree of S-DWG, (b) Hydrolysis degree of C-DWG and (c) SDS–PAGE of S-DWG and C-DWG at similar deamidation degree of 61%, NSI of 90% in 10 min moisture-heating, in which Lane 1–4 were, respectively, corresponded to the standard molecule protein, control sample, the S-DWG and C-DWG.

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Table 2 Foaming and emulsification properties of wheat gluten deamidated with acids in the 10 min moisture-heatinga.

a

Samples

pH

Foam capacity (%)

Succinic acid 0.017 M 0.042 M 0.066 M

4.31 3.82 3.59

270 ± 0.5d 120 ± 4.1b 155 ± 6.0cb

Citric acid 0.0095 M 0.024 M 0.038 M Untreated wheat gluten

4.22 3.51 3.20 7.00

335 ± 6.0e 270 ± 4.1d 160 ± 0.5cb 90 ± 4.1a

EAI (m2g1)

ESI (min)

26.7 ± 4.2ab 33.3 ± 4.0bc 33.9 ± 2.4bc

25.1 ± 1.1a 63.3 ± 2.3cd 55.6 ± 1.8cb

71.2 ± 2.0d 46.2 ± 4.5b 48.1 ± 3.3c

41.45 ± 0.5d 25.9 ± 3.9ab 28.8 ± 1.8b 22.2 ± 3.5a

53.9 ± 0.8cb 84.6 ± 2.8e 94.8 ± 2.4f 72.3 ± 1.3d

46.1 ± 2.5b 44.5 ± 0.5ab 47.1 ± 3.8c 42.0 ± 3.9a

Foam stability (%)

The data with different lowercase letters in the same column are significantly different (p < 0.05).

Table 3 Determined frequencies of amide I component bands, relative assigned structures and their distributions for secondary structural contents, and surface hydrophobicity of the untreated wheat gluten, S-DWG and C-DWGe. Untreated wheat glutena

Assignment

b-Sheet

Intermolecular b-sheets aggregation extended b-sheet (hydrated) Extended b-sheet

a-Helix b-Turn a-Helix/b-sheet Surface hydrophobicitye a b c d e

S-DWGd

C-DWG

d

Frequency (cm1)

Structure distributionc (%)

Frequency shift (cm1)

Structure distributionc (%)

Frequency shift (cm1)

Structure distributionc (%)

1606.58 1635.51 1697.23 1652.87 1679.87 0.715 50.35 ± 18a

14.515 32.505 1.224 34.483 17.273

0 0 0 0 0 0.706 524.55 ± 22c

15.097 32.646 1.022 34.431 17.305

0 0 –b 0 0 0.694 400.43 ± 9b

16.222 32.874 –b 34.091 16.813

The extent of denaturation for the control was defined as 0%. ‘–’ indicates no spectra in the FTIR. The percentage of secondary structure content is estimated as the corresponding area as a percentage of the total amide I band area. S-DWG and C-DWG were at the same deamidation degree of 61%, NSI of 90% in 10 min moisture-heating. The data with different letters in the same row of surface hydrophobicity are significantly different (p < 0.05).

Table 4 Amino acid composition of native wheat gluten, S-DWG and C-DWG (mg/100 g protein solution). Amino acids

Asp Glu Ser Gly His Arg Thr Ala Pro Tyr Val Met Cys Ile Leu Try Phe Lys Total Essential amino acids a b

Untreated wheat glutena

S-DWGb

C-DWGb

Total amino acid/mg/ 100 g

Total amino acid/mg/ 100 g

Free amino acid/mg/ 100 g

Total amino acid/mg/ 100 g

Free amino acid/mg/ 100 g

50.23 ± 2.11 421.03 ± 19.70 55.73 ± 2.42 66.56 ± 3.04 36.21 ± 1.51 57.19 ± 2.48 41.21 ± 2.03 36.10 ± 1.65 127.65 ± 5.88 29.02 ± 1.40 41.52 ± 2.00 10.45 ± 0.05 1.58 ± 0.07 31.04 ± 1.45 54.39 ± 2.51 8.20 ± 0.36 33.68 ± 1.48 19.42 ± 0.89 1061.22 ± 50.12 277.12 ± 12.56

37.08 ± 1.65 451.47 ± 21.31 65.19 ± 3.15 80.61 ± 3.86 29.72 ± 1.37 56.46 ± 2.54 45.92 ± 2.12 36.56 ± 1.69 127.32 ± 6.03 41.50 ± 2.05 40.57 ± 1.78 14.71 ± 0.07 1.15 ± 0.05 29.59 ± 1.39 49.61 ± 4.62 10.55 ± 0.50 33.02 ± 1.65 20.00 ± 0.87 1171.04 ± 53.56 273.69 ± 12.81

1.16 ± 0.05 1.56 ± 0.04 23.95 ± 1.08 2.10 ± 0.11 7.09 ± 0.26 15.99 ± 0.07 5.49 ± 0.21 7.37 ± 0.26 1.45 ± 0.07 18.23 ± 0.86 9.94 ± 0.04 4.05 ± 0.18 0.40 ± 0.01 8.09 ± 0.37 20.18 ± 0.95 6.97 ± 0.28 12.60 ± 0.06 6.27 ± 0.30 152.87 ± 7.43 80.68 ± 3.88

45.14 ± 1.78 495.79 ± 22.74 72.61 ± 3.27 100.88 ± 4.04 40.46 ± 1.71 67.19 ± 2.85 52.02 ± 2.37 40.37 ± 1.98 164.56 ± 7.86 59.35 ± 2.47 52.06 ± 2.22 20.69 ± 1.01 1.44 ± 0.06 37.53 ± 1.79 58.92 ± 2.67 12.58 ± 0.59 43.61 ± 2.11 27.16 ± 1.25 1379.78 ± 61.67 332.45 ± 15.14

0.71 ± 0.03 1.11 ± 0.05 21.47 ± 1.02 1.56 ± 0.06 15.60 ± 0.70 19.77 ± 0.88 6.33 ± 0.32 7.08 ± 0.32 1.48 ± 0.07 21.11 ± 1.00 10.03 ± 0.04 3.87 ± 0.15 0.61 ± 0.02 7.37 ± 0.34 18.73 ± 0.86 7.55 ± 0.39 11.53 ± 0.55 6.05 ± 0.30 161.96 ± 8.00 87.06 ± 3.97

Free amino acids for native wheat gluten was defined as zero. S-DWG and C-DWG were at the deamidation degree of 60% and NSI of 90% in the 10 min moisture-heating.

air–water interface and is believed to improve foaming capacity of wheat gluten after citric acid deamidation (Wagner & Gueguen, 1999). On the contrary, the foaming stability of C-DWG was lower

than that of S-DWG, probably due to that the smaller molecular weight of C-DWG after deamidation (Fig. 1) could not be well maintained strong surface tension.

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3.2.2. Emulsification properties As shown in Table 2, C-DWG exhibited better emulsification activity than did S-DWG and untreated wheat gluten, especially at higher degrees of deamidation. Surprisingly, the emulsification activity of S-DWG at different degrees of deamidation was lower than that of untreated wheat gluten. There was no significant change (p > 0.05) in the emulsion stability index (ESI) after deamidation of wheat gluten with citric acid and succinic acid of wheat gluten, except for in the sample treated with succinic acid with the low degree of deamidation for 10 min moisture-heating. Although the increase in the solubility of both treated samples would improve ESI, an excessive increase in the net charge may weaken protein–protein interactions and prevent the formation of an elastic film at the oil–liquid interface. Furthermore, the detrimental effect of peptide bond hydrolysis is related to the loss of globular structure and optimum size of peptides, which in turn can result in the formation of a thinner protein layer around the oil droplets and hence less stable emulsion (Nielsen, 1997).

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molecular flexibility of C-DWG (0.694), estimated by the ratio of

a-helix to b-sheet, was lower than that of S-DWG (0.706). Frequency shifts in the samples treated with succinic acid or citric acid from the untreated wheat gluten samples were all zero, indicating that the secondary conformational changes are mainly caused by increased electrostatic repulsion and decreased hydrogen bonding as a result of deamidation and not by the cleavage of peptide bonds. Greater increases of both intermolecular b-sheets (by protein aggregation) and extended b-sheet were observed in C-DWG. However, S-DWG had a smaller reduction in a-helix and b-turn than did C-DWG, suggesting that more Glu and Asp were exposed when Gln and Asn were deamidated with succinic acid since Glu and Asp are the main constituents of b-turn (Chou & Fasman, 1977). This was in accordance with the intensity of the Raman peaks (1745 cm1). The attributed location of the c-carboxyl groups of aspartic and glutamic acids was much stronger for S-DWG than for C-DWG (Fig. 1C). 3.4. Evaluation of amino acid analysis in S-DWG and C-DWG

3.3. Evaluation of conformational changes 3.3.1. Tertiary conformation SDS–PAGE gives useful information on conformational changes in the tertiary structure of wheat gluten. As shown in Fig. 2c, at the same degree of deamidation (61%), the protein fractions of wheat gluten with high molecular weights (from 40 kDa to 100 kDa) were more susceptible to degradation into smaller fragments (less than 14 kDa) from deamidation with citric acid (lane 4). In contrast, the molecular weights of the major protein bands of S-DWG (lane 3) decreased less gradually. The results from SDS–PAGE of S-DWG and C-DWG agree with the results in Fig. 2a and b. They confirm that succinic acid has a weaker effect in cleaving the peptide bonds of wheat gluten. In previous work (Liao et al., 2009), we reported that the peptide bonds of the gluten molecules were less cleaved in acetic acid-modified gluten than in HCl-deamidated gluten. Comparison of the SDS–PAGE of HCl and the acetic acid deamidated samples at a degree of deamidation of 60% with our current results allowed us to conclude that the degree of cleavage of the peptide linkages in gluten molecules was positively related to the strength of the acid, that was H-DWG (HCl deamidated wheat gluten) > C-DWG > S-DWG > A-DWG (acetic acid deamidated wheat gluten). Furthermore, the tertiary conformational changes due to disulfide bond disruption, which play an important role in protein folding and assembly, may also occur during acid deamidation of wheat gluten. As shown in Fig. 1, the presence of Raman stretching at 521 cm1 (Fig. 1A) and 2570–2590 cm1 (Fig. 1B) clearly indicated the presence of disulfide bonds and sulfhydryl groups in wheat gluten deamidated with succinic acid and citric acid, respectively (Liao et al., 2004). The intensity of the Raman peaks in the area (521 cm1) was much stronger for S-DWG than for C-DWG, showing that there were more disulfide bonds in S-DWG than in C-DWG. Moreover, the intensity of the Raman peaks (2570– 2590 cm1) was much stronger for C-DWG than for S-DWG, indicating that more sulfhydryl groups were produced by the deamidation of wheat gluten with citric acid than with succinic acid. The Raman spectroscopy and SDS–PAGE results allow us to conclude that the tertiary structure of C-DWG was less compact than that of S-DWG at the similar deamidation degree of 61%. 3.3.2. Secondary conformation Fourier transform infrared (FTIR) analysis was conducted to investigate the difference in the secondary structures in the protein conformations (Table 3). The untreated wheat gluten contained 34.5% a-helix, 17.3% b-turn and 48.8% b-sheet, in agreement with the report of Wang, Zhao, and Yang (2006). The

From a nutritional viewpoint, wheat gluten is considered to be a poor source of protein than animal sources and soybean, primarily because it is slightly deficient in two essential amino acids, lysine and threonine. With regard to possible or particular nutritional benefits or defects, extensive research on modifying wheat gluten to include all essential amino acids is needed for us to fully understand the nutritional value of gluten protein. As shown in Table 4, the composition of the amino acid compositions was not the same before and after the acid treatment, probably because that changes in soluble wheat gluten present in the assay mixture or to a different exposition to the assay reagents. The lysine content in S-DWG did not change significantly (p > 0.05) (from 19.42 to 20 mg/100 g). However, there was a significant (p < 0.05) enhancement in the lysine content of C-DWG (from 19.42 to 27.16 mg/100 g). Lysine is considered to be the most important essential amino acid in cereal protein. Deamidation with citric acid could thus improve the nutritional characteristics of wheat gluten. Additionally, the amount of essential amino acids in C-DWG was higher than in S-DWG. The amount of essential free amino acids accounted for up to 53.75% of the total amount of essential amino acids in C-DWG, which was higher than that in S-DWG (52.77%). Our results suggest that the nutritional characteristics of wheat gluten are well maintained or improved as regarding to the dissolution of essential amino acids in wheat gluten deamidated with citric acid. 4. Conclusions Our results showed that succinic acid and citric acid effectively deamidated amides into carboxyl groups in wheat gluten. Carboxylic acid is more appropriate for deamidation than inorganic acid (HCl). Samples treated with succinic acid (S-DWG) showed less improvement in the foaming capacity and stability as well as the emulsification activity. Samples treated with citric acid (C-DWG) possessed higher molecular flexibility, greater changes in the tertiary and secondary structures of wheat gluten and better nutritional characteristics. C-DWG has potential applications in new product formulations and fortification, such as protein-fortified beverages, infant formulas, coffee whiteners, emulsifiers, and flavour enhancers. Acknowledgments The authors are grateful to the China National 863 Program (no. 2006AA10Z326) and the National Natural Science Foundation of China (no. 20676044) for financial support for this research. Special

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