2018 Volume 24 Issue 6 Pages 1049-1058
Wheat gluten prepared by dispersion in the presence of ammonia has unique dough properties similar to those of gliadin. In the present study, the mechanism of expression of these gliadin-like characteristics was investigated. In prolonged stirring with ammonia treatment, gluten gained even less elasticity, as shown by the marked decrease of dough resistance and bandwidth. Size-exclusion HPLC analysis showed that large-molecular sized polymeric glutenins decreased, but small-molecular sized polymeric glutenins increased with increases in ammonia dispersion time. SDS-PAGE of proteins labeled with monobromobimane revealed that the content of free sulfhydryl groups in the D-type of low-molecular weight glutenin subunits (LMW-GS) significantly decreased in ammonia-treated gluten to 56% of that in control gluten. We propose that during ammonia dispersion, free sulfhydryl groups in the D-type of LMW-GS attack the disulfide bonds in large polymeric glutenins and consequently, new small polymeric glutenins are generated by disulfide-sulfhydryl exchange reactions.
Wheat flour can form a viscoelastic dough when mixed with water. This property is attributed to gluten proteins in flour that hydrate and consequently form a network. The physical properties of gluten are so unique that no other plant protein possesses them (Day, 2013). Gluten is mainly composed of two types of protein: monomeric gliadins and polymeric glutenins. Polymeric glutenin is formed by linkage of glutenin subunits with disulfide bonds (Shewry, 2003). Monomeric gliadins and polymeric glutenins have different and unique characteristics. For example, monomeric gliadins are soluble in alcohol-water solvents and confer extensibility and viscosity to dough, while polymeric glutenins are insoluble in alcohol-water solvents and confer strength and elasticity to dough (Shewry et al., 2003; Wieser et al., 2006; Wrigley et al., 2006).
Gluten isolated from flour and subsequently dried is commercially available as an ingredient to improve the quality of a variety of foodstuffs (Ponte et al., 2000; Van Der Borght et al., 2005; Day et al., 2006). Gluten powder is used in the manufacture of breads, noodles, cakes, cookies, sausages, fish cakes, cream, and coatings of various foods to enhance their appearance, texture, workability, and yield. There are two main types of gluten powders utilized in the food industry worldwide: flash-dried gluten and spray-dried gluten. Flash-dried gluten is manufactured by drying wet gluten using a ring dryer, while spray-dried gluten is produced by dispersing wet gluten in either acidic or basic (ammonia) solutions and then drying the dispersed gluten using a spray dryer (Cornell and Hoveling, 1998). Spray-dried gluten prepared in the presence of either acid or ammonia exhibits dough properties that differ from those of flash-dried gluten. Spray-dried gluten is much more adhesive and extensible than flash-dried gluten. Moreover, the use of spray-dried glutens with interesting properties (as described above) has been increasing for a number of applications. Nonetheless, only a handful of studies have characterized the molecular basis of these compounds to date (Lusena, 1950; McConnell, 1955; Staff, 1959; Mori et al., 1974). Clearly, there is a need for further molecular characterization of spray-dried glutens to develop new applications for the food industry.
We hypothesized that the rheological properties of spray-dried glutens can be attributed to the molecular characteristics of glutens in acidic or basic dispersions used for the preparation of spray-dried glutens. In a previous study, we reported that gluten powders prepared by dispersion in the presence of acetic acid or ammonia had characteristics similar to those of gliadins (Murakami et al., 2015). Recently, we demonstrated that ionic repulsion induced by acid dispersion resulted in polymeric glutenins rich in high-molecular weight glutenin subunits (HMW-GS) disaggregate, and therefore act like gliadins (Murakami et al., 2016).
The objective of the present study was to elucidate the molecular characteristics of polymeric glutenins when gluten is dispersed in the presence of ammonia. Here, we determined that the mechanism of the characteristic changes occurring in polymeric glutenins after ammonia dispersion differed from that when acid dispersion is used. Moreover, we found that the structural changes in polymeric glutenins were induced by disulfide-sulfhydryl (SS-SH) exchange reactions.
Materials Wet gluten was extracted from Australian hard wheat flour and stored at −22°C in a freezer, as previously reported (Murakami et al., 2016). Wet gluten contained 66.5% moisture, 0.65% dry ash, and 80.4% dry protein (conversion factor N × 5.7). Frozen wet gluten was thawed before use. All chemicals were of analytical grade.
Preparation of gluten powders in ammonia with different stirring times Wet gluten (100 g) was dispersed in 200 mL of 0.5 M ammonia solution. Dispersion of wet gluten was carried out by homogenization at 10,000 rpm for 8 min at 25°C using a homogenizer (Polytron PT10-35GT; Kinematica AG, Luzern, Switzerland) equipped with a PT-DA 30/2EC-B250 shaft, as previously conducted at these premises (Murakami et al., 2016). Dispersed gluten was stirred for 0, 0.5, 1, and 2 h using a magnetic stirrer in a 35°C water bath, and subsequently lyophilized (Freeze-drier, FDU-2100; Tokyo Rikakikai Co., Tokyo, Japan). Next, freeze-dried gluten was pulverized at 10,000 rpm using a pin mill (Pulverisette-14; Fritsch GmbH, Idar-Oberstein, Germany) in the presence of frozen carbon dioxide (dry ice) to protect gluten proteins from heat damage. The obtained gluten powders were sifted using a 120-µm pore screen. In total, four types of ammonia-treated gluten powders were prepared: non-extra stirred gluten powder, and gluten powders stirred for 0.5, 1, and 2 h. Control gluten powder was also prepared by lyophilizing and pulverizing wet gluten using the aforementioned method.
Preparation of ammonia-treated gluten with addition of NaCl Wet gluten (100 g) was dispersed in 200 mL of 0.5 M ammonia solution by homogenization at 10,000 rpm for 8 min at 25°C, in the presence of 0.04, 0.10, or 0.20 M NaCl. After a further 1 min of homogenization, the dispersed gluten was lyophilized and pulverized using the above method.
Measurement of gluten dough properties The properties of gluten dough were measured using a mixograph (35-g Mixograph; National Manufacturing Division, TMCO, Lincoln, NE, USA), as carried out in the previous study at these premises (Murakami et al., 2015). Gluten powder was blended with starch powder so that 30 g of powder mixture (dry matter basis) contained 7.5 g of protein. Measurement with the mixograph was conducted by adding water (20°C) to a total volume of 33 mL, including endogenous water in the powder. The following curve parameters were used for the mixogram: time to peak dough resistance (min; peak time), dough resistance integral at 3 min after peak dough resistance (%tq×min; dough resistance), and bandwidth integral at 3 min after peak dough resistance (%tq×min; bandwidth).
Determination of hydrolysis degree Gluten powder (0.1 g as protein) was suspended in 10 mL of trichloroacetic acid (12% w/v) in a 50-mL tube. The suspension was vigorously mixed for 3 min using a vortex mixer (HM-10H; AS ONE Corporation, Osaka, Japan), cooled in ice water for 60 min, and centrifuged at 10,000 × g for 10 min at 4°C. The supernatant was decanted and 10 mL of acetone was added to the precipitate to remove the remaining trichloroacetic acid. Next, the suspension was vigorously mixed for 3 min using a vortex mixer, cooled in ice water for 60 min, and centrifuged at 15,000 × g for 10 min at 4°C. The precipitate containing insoluble protein was collected and the protein content (conversion factor N × 5.7) was measured using the improved Dumas method (TruMac N; Leco Corp., St. Joseph, MI, USA). The soluble protein content was calculated by subtracting the insoluble protein content from the total protein content. The degree of hydrolysis was obtained from the ratio of soluble protein content to total protein content.
Determination of content of free sulfhydryl groups Content of free sulfhydryl groups was determined colorimetrically by reaction with 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), according to Ellman's method (Ellman, 1959). The sample (0.04 g as protein) was suspended in 10 mL of 0.05 M sodium phosphate buffer (pH 6.2) containing 2% SDS, 8 M urea, and 1 mM tetrasodium ethylene diamine tetraacetate. The suspension was then homogenized at 10,000 rpm for 1 min at room temperature using a homogenizer (Polytron PT10-35GT) equipped with a PT-DA 12/2EC-B154 shaft, and was then mixed three times during 60 min using a vortex mixer. After centrifugation at 36,500 × g for 10 min at 20°C, the obtained supernatant was mixed with 0.4 mL of 0.2% DTNB dissolved in sample buffer, and absorbance at 412 nm was read after 45 min. Absorbance values were converted to content of free sulfhydryl groups using a calibration curve with reduced glutathione (Veraverbeke et al., 2000).
Determination of protein content extracted with 70% ethanol Protein was extracted from gluten powders with 70% ethanol using a slightly modified version of a method we previously reported (Murakami et al., 2016). Control gluten powder (0.5 g) was suspended in 50 mL of 70% ethanol and homogenized at 8,000 rpm for 10 min at 25°C using a homogenizer (Polytron PT10-35GT; Kinematica AG) equipped with a PT-DA 12/2EC-B154 shaft. The suspension had a pH value of 6.6. Gluten powder (0.5 g) prepared by ammonia dispersion was suspended in 50 mL of 70% ethanol containing 1 mM acetic acid and homogenized using the method mentioned above, so that the pH of the suspension was also 6.6. The suspension was centrifuged at 10,000 × g for 10 min at 25°C and the supernatant was collected. Protein content in the supernatant, that is, protein extracted with 70% ethanol, was measured using the improved Dumas method (TruMac N; Leco Corp.).
Determination of content of SDS-insoluble polymeric protein (IPP) Gluten powder (0.1 g as protein) was suspended in 20 mL of 0.1 M sodium phosphate buffer (pH 6.9) containing 2% SDS and shaken for 18 h at 60°C, as carried out in the previous study at these premises (Murakami et al., 2016). After standing at room temperature for 2 h, the suspension was centrifuged at 36,500 × g for 30 min at 20°C. Protein content in the precipitate was measured as IPP content by using the improved Dumas method.
Size-exclusion (SE-) HPLC Ethanol-extractable or SDS-soluble protein in gluten powders was analyzed by SE-HPLC, according to the method reported previously (Murakami et al., 2016). For the preparation of ethanol-extractable protein, the supernatant obtained from the aforementioned extraction with 70% ethanol was used. Three milliliters of the supernatant was mixed with 6 mL of 0.1 M sodium phosphate buffer (pH 6.9) containing 2% SDS, shaken for 4 h at 60°C, and then passed through a 0.45-µm filter. For the preparation of SDS-soluble protein, the supernatant obtained in the procedure for determination of IPP content was passed through a 0.45-µm filter.
For the SE-HPLC analysis, a TSKgel α-6000 column (7.8 × 300 mm; Tosoh Co., Tokyo, Japan) with a TSK guard column (6.0 × 40 mm) was used, with a flow rate of 0.5 mL/min at 35°C and a wavelength of detection of 214 nm. The elution reagent was 50% acetonitrile in water containing 0.05% trifluoroacetic acid.
Polymeric glutenins showed a continuous broad peak detected ahead of gliadins. The broad peak of polymeric glutenins was partitioned into four fractions for analysis of molecular size distribution. The four fractions with elution volumes of 6.5–8.5 mL, 8.5–9.5 mL, 9.5–10.0 mL, and 10.0–10.5 mL were named F1, F2, F3, and F4, respectively. The ratio of protein in each fraction to whole gluten protein was calculated from two factors: the protein content that was applied to the HPLC and the ratio of area in each fraction to total area in the chromatogram.
Labeling of free sulfhydryl groups with monobromobimane (mBBr) Free sulfhydryl groups in gluten samples were labeled with monobromobimane (mBBr) using a slightly modified version of the previous method (Kobrehel et al., 1992; Rhazi et al., 2003). Gluten powder (16 mg as protein) was suspended in 4 mL of 0.1 M sodium phosphate buffer (pH 6.9) containing 2% SDS and 1 mM mBBr in a 50-mL tube. The suspension was homogenized at 10,000 rpm for 1 min using a homogenizer (Polytron PT10-35GT; Kinematica AG) equipped with a PT-DA 12/2EC-B154 shaft, and shaken for 2 h at 60°C. Ten percent SDS (0.1 mL) and 0.1 mL of 100 mM 2-mercaptoethanol were added to 0.8 mL of suspension to stop the reaction (Gobin et al., 1997; Rhazi et al., 2003). After 30-min incubation at room temperature, 0.3 mL of sample buffer (62.5 mM of Tris-HCl (pH 6.8), 2% SDS, and 20% glycerol) and 0.1 mL of 2-mercaptoethanol were added to 1.0 mL of sample solution. The sample solution was then boiled for 5 min.
Fluorescence imaging of mBBr-labeled gluten proteins SDS-PAGE analysis of gluten samples labeled with mBBr was carried out as follows. mBBr-labeled protein (68.6 µg) was added to each lane on a 10% separating gel (e-PAGEL, Atto Co., Tokyo, Japan). SDS-PAGE analysis was conducted as described in the previous study at these premises (Murakami et al., 2016).
After SDS-PAGE analysis, the gel was fixed in 12% trichloroacetic acid and then immersed in 30% methanol containing 10% acetic acid to remove excess mBBr. The gel was visualized with an ultraviolet transilluminator at 365 nm and imaged at a wavelength of 537 nm using an imaging system (FluorChem 8800; Alpha Innotech Co., San Leandro, CA, USA). Afterward, the gel was stained with Coomassie Brilliant Blue (CBB) and photographed. The density of bands detected with mBBr or CBB was analyzed using Image J v1.51f 17 August 2016i).
Statistical analysis All experiments were carried out in triplicate. Analysis of variance (ANOVA) was conducted to analyze the data. Significant differences between the means (P < 0.05) were identified with Fisher's LSD test (JMP Software v11.2; SAS Institute, Tokyo, Japan).
pH of the dispersion treatment and prepared gluten powders Gluten dispersed in 0.5 M ammonia solution had a pH of 10.4–10.5. In contrast, gluten powder prepared by lyophilization of dispersed gluten and suspended in water at 10% concentration had a pH of 7.1–7.3. The obtained gluten powder had the same pH even though the gluten was dispersed in 0.5 M ammonia solution with different stirring times. Control gluten powder suspended in water at 10% concentration had a pH of 6.2.
Dough properties of gluten powders prepared by ammonia dispersion with different stirring times The present study showed that gluten powder treated with 0.5 M ammonia had dough properties significantly different from those of the control gluten powder. Ammonia-treated gluten without prolonged stirring (Non-extra stirred ammonia-treated gluten) reached peak dough resistance in a remarkably short period of time (2.72 min), while it took longer (15.42 min) for control gluten powder (Fig. 1A). Dough resistance of non-extra stirred ammonia-treated gluten was significantly weaker (201.4%tq×min) than that of control gluten powder (227.1%tq×min) (Fig. 1B). Furthermore, the bandwidth of non-extra stirred ammonia-treated gluten indicated that dough elasticity was much weaker (39.7%tq×min) than that of the control gluten powder (67.9%tq×min) (Fig. 1C).
(A) Time to peak dough resistance (min; peak time). (B) Dough resistance integral at 3 min after peak dough resistance (%tq×min; dough resistance). (C) Bandwidth integral at 3 min after peak dough resistance (%tq×min; bandwidth), in mixograms of gluten powders. Ctl: Control gluten. 0 h: Non-extra stirred ammonia-treated gluten. 0.5 h, 1 h, and 2 h: Ammonia-treated gluten with further stirring for 0.5, 1 or 2 h. Error bars represent the standard deviation. Data were analyzed using Fisher's LSD test. The means with different letters are significantly (P < 0.05) different from each other.
When gluten was further stirred in the ammonia dispersion for 0.5, 1 and 2 h, dough properties were significantly different among the various types of gluten powders. The obtained gluten powders attained peak dough resistance in a significantly shorter time when gluten was stirred in the ammonia dispersion for a longer time after homogenization (Fig. 1A). Gluten stirred for 2 h reached peak dough resistance after 0.93 min, while non-extra stirred gluten peaked after 2.72 min. As stirring time is prolonged during ammonia dispersion of gluten, dough resistance and dough elasticity were weakened (Fig. 1B and Fig. 1C). Dough resistance and bandwidth were 128.1%tq×min and 10.6%tq×min, respectively, for gluten further stirred for 2 h compared with non-extra stirred gluten, which had dough resistance and bandwidth of 201.4%tq×min and 39.7%tq×min, respectively.
Dough properties of ammonia-dispersed gluten prepared with added NaCl Dough properties were examined using a mixograph when gluten was prepared with added NaCl in the ammonia dispersion. The time to attain peak dough resistance remained unchanged for ammonia-dispersed gluten even with increasing concentrations of NaCl (Fig. 2A). Furthermore, for ammonia-dispersed gluten, dough resistance remained unchanged when the concentration of NaCl was increased to 0.1 M in the ammonia dispersion. However, dough resistance for ammonia-dispersed gluten was somewhat stronger when the NaCl concentration increased to 0.2 M in the ammonia dispersion (Fig. 2B). The bandwidth indicating elasticity did not significantly change in the dough of ammonia-dispersed gluten even though the concentration of NaCl increased (Fig. 2C). These results indicated that the dough properties of ammonia-treated gluten were not sensitive to NaCl.
(A) Time to peak dough resistance (min; peak time). (B) Dough resistance integral at 3 min after peak dough resistance (%tq×min; dough resistance). (C) Bandwidth integral at 3 min after peak dough resistance (%tq×min; bandwidth), in mixograms of ammonia-treated gluten with added NaCl. X-axis: Concentration (M) of NaCl added in the preparation of ammonia-treated gluten powder. Error bars represent the standard deviation. Data were analyzed using Fisher's LSD test. The means with different letters are significantly (P < 0.05) different from each other.
Ethanol-extractability of polymeric glutenins by ammonia treatment of gluten The present study showed that the content of ethanol-extractable proteins increased in gluten powder treated with 0.5 M ammonia. Indeed, the content of ethanol-extractable proteins in whole gluten proteins was 58.4% in non-extra stirred ammonia-treated gluten and 50.6% in control gluten (Fig. 3).
Content of 70% ethanol-extractable proteins in gluten powders. Ctl: Control gluten. 0 h: Non-extra stirred ammonia-treated gluten. 0.5 h, 1 h, and 2 h: Ammonia-treated gluten with further stirring for 0.5, 1 or 2 h. Error bars represent the standard deviation. Data were analyzed using Fisher's LSD test. The means with different letters are significantly (P < 0.05) different from each other.
As stirring time increased in the ammonia dispersion of gluten, the content of ethanol-extractable proteins also increased significantly to 61.0% in ammonia-treated gluten with further 2-h stirring compared to 58.4% in non-extra stirred gluten (Fig. 3).
The molecular size distribution of ethanol-extractable proteins was measured using SE-HPLC. The chromatograms indicate that polymeric glutenins that were extractable with 70% ethanol increased in ammonia-treated gluten with further 2-h stirring compared to control gluten (Fig. 4A). Based on the chromatographic results, the ratio of ethanol-extractable glutenins in each fraction to whole gluten proteins in the gluten powders was calculated to compare the molecular size distribution (Fig. 4B). Ammonia-treated glutens with prolonged stirring (0.5, 1 and 2 h) had a higher content of ethanol-extractable polymeric glutenins in all fractions (F1–F4) compared to control gluten. Polymeric glutenins in the F4 fraction with a detected smaller molecular size became more ethanol-extractable as the stirring time of the ammonia dispersion increased; however, the change was slight.
(A) Typical chromatogram of SE-HPLC analysis of gluten powders. Solid line: SDS-soluble protein in control gluten. Dashed line: SDS-soluble protein in ammonia-treated gluten with further 2-h stirring. Short dashed line: 70% ethanol-extractable proteins in control gluten. Dashed dotted line: 70% ethanol-extractable proteins in ammonia-treated gluten with further 2-h stirring. Polymeric glutenins were partitioned into four fractions according to elution volumes, F1: 6.5–8.5 mL, F2: 8.5–9.5 mL, F3: 9.5–10.0 mL, F4: 10.0–10.5 mL. (B,C) Distribution of the molecular size in polymeric glutenins. (B) 70% ethanol-extractable proteins and (C) SDS-soluble proteins in gluten powders. IPP: Insoluble polymeric proteins. Ctl: Control gluten, 0 h: Non-extra stirred ammonia-treated gluten. 0.5 h, 1 h, and 2 h: Ammonia-treated gluten with further stirring for 0.5, 1 or 2 h. Error bars represent the standard deviation. Data were analyzed using Fisher's LSD test. The means with different letters are significantly (P < 0.05) different from each other.
Status of hydrolysis and free sulfhydryl groups We examined if gluten was hydrolyzed by ammonia treatment (Table 1). Despite further stirring, all types of ammonia-treated glutens were subject to the same low degree of hydrolysis as for control gluten; however, significant differences were not detected.
| Type of gluten powder | Degree of hydrolysis (%) | Content of free sulfhydryl groups (µmoL/g of protein) |
|---|---|---|
| Control gluten | 3.1 ± 0.4 a | 6.2 ± 0.1 a |
| Non-extra stirred ammonia-treated gluten | 2.6 ± 0.5 a | 4.2 ± 0.1 b |
| Ammonia-treated gluten with further 0.5-h stirring | 2.5 ± 0.7 a | 4.1 ± 0.1 b |
| Ammonia-treated gluten with further 1-h stirring | 2.6 ± 0.2 a | 4.0 ± 0.1 b |
| Ammonia-treated gluten with further 2-h stirring | 2.8 ± 0.3 a | 4.1 ± 0.2 b |
Data were analyzed using Fisher's LSD test. The means with different letters are significantly (P < 0.05) different from each other.
The content of free sulfhydryl groups was measured to determine whether ammonia dispersion changed the status of sulfhydryl groups in the gluten. The contents of free sulfhydryl groups in ammonia-treated glutens with further stirring for 0, 0.5, 1, and 2 h were found to be 4.2, 4.1, 4.0, and 4.1 µmol/g protein, respectively, which were significantly lower compared with that in control gluten (6.2 µmol/g protein) (Table 1). These results indicate that the content of free sulfhydryl groups decreased by about two-thirds of that in control gluten, which may suggest that about one-third of free sulfhydryl groups was converted to disulfides when gluten was treated with 0.5 M ammonia.
Effect of ammonia treatment of gluten on the molecular size of polymeric glutenins The effect of treatment by ammonia dispersion on the molecular size of gluten was examined by solubility in SDS solution, followed by SE-HPLC analysis. The content of proteins that were insoluble in SDS solution at 60°C (IPP) was 3.2% in control gluten, while the content of IPP was 4.3–4.6% in the ammonia-treated gluten with further stirring for 0–2 h, which showed that the content of IPP increased in ammonia-treated gluten (Fig. 4C).
SE-HPLC analysis was carried out for proteins that were soluble in SDS solution at 60°C, which accounted for 95.4–96.8% of whole gluten proteins (Dupont et al., 2008). The chromatograms indicate that polymeric glutenins dramatically decreased in large molecular size, but increased in small molecular size in ammonia-treated gluten with further 2-h stirring, in comparison with control gluten (Fig. 4A). Based on the chromatographic results, the ratio of SDS-soluble glutenins in each fraction to whole gluten proteins in the gluten powders was calculated to compare the molecular size distribution in polymeric glutenins (Fig. 4C). When compared with control gluten, non-extra stirred ammonia-treated gluten had a markedly lower content of polymeric glutenins in the F1 fraction. In contrast, it had a significantly higher content of polymeric glutenins in the F3 and F4 fractions. Furthermore, as the stirring time in ammonia dispersion of gluten is prolonged, polymeric glutenins in F1 and F2 showing a larger molecular size significantly decreased and those in F4 showing a smaller molecular size significantly increased. These results suggest that large-sized polymeric glutenins can be downsized when gluten is treated using ammonia dispersion.
Effect of ammonia treatment of gluten on free sulfhydryl groups in polymeric glutenins We examined the behavior of free sulfhydryl groups labeled with mBBr when gluten was treated with ammonia. The left panel in Fig. 5A shows bands that were stained with CBB, while the right panel shows some proteins containing sulfhydryl groups labeled with mBBr that were fluorescently detected. When the ratio of band densities observed with mBBr relative to CBB was calculated (Fig. 5B), no significant differences in band density with mBBr were detected between control gluten and ammonia-treated gluten for HMW-GS (I), B-type of LMW-GS/α- and γ-gliadin (III) and C-types of LMW-GS (IV). However, the density of the band corresponding to the D-type of LMW-GS/ω-gliadin (II) in ammonia-treated gluten was significantly lower than that in control gluten. The band intensity of ammonia-treated gluten was about 56% that of control gluten.
(A) Bands were stained with Coomassie Brilliant Blue (CBB) or labeled with monobromobimane (mBBr) in SDS-PAGE under reducing condition. (B) Ratio of density of band labeled with mBBr to density of band stained with CBB in gluten powder. Ctl: Control gluten. AT: Non-extra stirred ammonia-treated gluten. I: HMW-GS. II: D-type of LMW-GS or ω-gliadin. III: B-type of LMW-GS. IV: C-type of LMW-GS or α- and γ-gliadin. Error bars represent the standard deviation. Data were analyzed using Fisher's LSD test. The means with different letters are significantly (P < 0.05) different from each other.
In the present study, we found that as the ammonia treatment time increased, the dough of ammonia-treated gluten formed in a shorter time, but was weaker in terms of both resistance and elasticity, compared to that of control gluten (Fig. 1A, 1B, and 1C). These results indicate that the dough properties of ammonia-treated gluten resembled those of gliadin. In our previous study (Murakami et al., 2016), we found that the gluten adopted gliadin-like characteristics due to ionic repulsion between polymeric glutenins, which was induced when the gluten was treated with acid. On the other hand, the dough properties of ammonia-treated gluten were not sensitive to NaCl, suggesting that ionic repulsion was less of a factor than in the acid-treated gluten (Fig. 2A, 2B, and 2C).
Gliadin has a number of characteristics, including extractability with 70% ethanol. Non-extra stirred ammonia-treated gluten had a higher content of proteins (58.4%) that were extractable with 70% ethanol compared to control gluten (50.6%), indicating that 7.8% of polymeric glutenins became ethanol-extractable, similar to gliadins. However, when gluten was further stirred for 2 h in ammonia solution, ethanol-extractable proteins increased by only 2.6% (58.4% to 61.0%), despite the fact that dough properties were largely different (Fig. 1 and Fig. 3). These results suggest that, when treated with ammonia, a mere increase in ethanol-extractable proteins is not sufficient for gluten to be conferred gliadin-like dough properties. Wieser et al. (2006) reported that the major portion (80%) of the gliadin fraction consists of monomeric proteins, while the smaller portion (20%) contains oligomeric proteins linked by interchain disulfide bonds. We theorize that some parts of polymeric glutenins might have become oligomeric proteins, which are extractable with 70% ethanol, upon exposure to ammonia.
In contrast, the molecular size of polymeric glutenins significantly changed in SDS-soluble proteins, which occupied more than 95% of whole proteins when gluten was treated with ammonia (Fig. 4C). Large-molecular sized polymeric glutenins decreased and small-molecular sized polymeric glutenins increased in gluten subjected to long-term ammonia treatment. In general, the molecular size of gliadins is smaller than that of polymeric glutenins. Therefore, an increase in small-molecular sized polymeric glutenins would indicate that there was an increase in the content of proteins with a molecular size closer to that of gliadins. It can be inferred, therefore, that changes in the molecular size of polymeric glutenins can cause gluten to express dough properties similar to those detected when gliadins are added to gluten.
Although no changes in the degree of hydrolysis of polypeptides were detected, the content of free sulfhydryl groups was decreased with ammonia treatment of gluten (Table 1). These results lead us to the hypothesis that downsizing of polymeric glutenins occurred not due to hydrolysis, but as a result of recombination and generation of disulfide linkages between glutenin subunits. As the content of free sulfhydryl groups in ammonia-treated gluten with further 2-h stirring was the same as that of non-extra stirred ammonia-treated gluten, new disulfide linkages would have been generated as soon as the gluten was dispersed in ammonia solution.
SDS-PAGE analysis of mBBr-labeled gluten showed a decrease in the content of free sulfhydryl groups of the D-type of LMW-GS or ω-gliadins (Fig. 5A and Fig. 5B). These results suggest that sulfhydryl groups may be used for the formation of disulfide linkages when gluten is treated with ammonia. The D-type of LMW-GS has been defined as modified ω-gliadins, whereas gliadins with the odd number of cysteine residues are referred to as modified gliadins (Wieser et al., 2006). It has been reported elsewhere (Payne et al., 1988; Masci et al., 1993, 2002; Juhász and Gianibelli, 2006) that, in the case of D-type of LMW-GS, which contain mutant forms of ω-gliadins, the presence of at least one free sulfhydryl group enables them to establish inter-molecular disulfide bonds and be incorporated into the glutenin polymer, while ω-gliadins that lack cysteine residues are able to form neither intra- nor inter-chain disulfide bonds. Thus, this evidence highlights the possibility that sulfhydryl groups decreased by ammonia treatment may be derived not from ω-gliadins, but from the D-type of LMW-GS. Indeed, it has been previously reported that modified gliadins are covalently bound to glutenins via an inter-chain disulfide bond and act as chain terminators to polymerize glutenins, while LMW-GS with two or more cysteine residues to form an inter-chain disulfide bond acts as chain extenders (Kasarda, 1989; Masci et al., 1998; Tamás et al., 1998; Greenfield et al., 1998; Juhász and Gianibelli, 2006; Wieser et al., 2006). Most modified gliadins acting as chain terminators, such as the D-type of LMW-GS, seem to be present in the oligomeric fraction of glutenins (Wieser et al., 2006).
Taking all the evidence together, it can be inferred that disaggregation of polymeric glutenins could occur due to ionic repulsion when gluten was dispersed in basic ammonia solution. The chains in polymers move flexibly and contact each other inside the polymers themselves. It is likely that as free sulfhydryl groups become reactive under an alkaline condition, disulfide linkages are newly generated inside polymeric glutenins. Furthermore, the SS-SH exchange reaction occurs randomly between polymeric glutenins. The content of free sulfhydryl groups would not change seemingly in each type of glutenin subunits because of the random exchange of subunits. Alpha/beta- and γ-gliadins have an even number of cysteine residues that form intrachain-disulfide bonds, and are not able to link with other subunits. Disulfide interchange reaction between gliadins and glutenins could not occur in the ammonia dispersion at 35°C, since Wieser et al. (2006) reported that it is postulated to be induced by heat. During the random SS-SH exchange reaction, free sulfhydryl groups in the D-type of LMW-GS act as chain terminators, likely attacking disulfide bonds in large polymeric glutenins and therefore, create new small polymeric glutenins. As a result, the content of free sulfhydryl groups in the D-type of LMW-GS would decrease. It is believed that the attack continues as long as free sulfhydryl groups are present in the D-type of LMW-GS and the size of polymeric glutenins becomes smaller. Downsizing of polymeric glutenin can be the key factor for ammonia-treated gluten to express gliadin-like characteristics.
Control gluten was prepared from wheat flour dough that was mixed at neutral pH. The left panel in Fig. 5A shows that the content of free sulfhydryl groups in the D-type of LMW-GS decreased significantly when the gluten was dispersed under an alkaline condition, compared to when it was mixed at neutral pH. The free sulfhydryl groups in the D-type of LMW-GS might not be highly reactive at neutral pH. We propose that the unique reaction of downsizing of polymeric glutenin would be caused by an alkaline condition, making free sulfhydryl groups in the chain terminators more reactive.
We obtained ammonia-treated gluten powder by dispersion of gluten in a basic ammonia solution, followed by lyophilization to produce gluten powder with neutral pH. When gluten was subjected to long-term ammonia treatment, ammonia-treated gluten displayed dough properties resembling those of gliadins. We found that large-molecular sized polymeric glutenins decreased and small-molecular sized polymeric glutenins increased due to SS-SH exchange reactions when long-term ammonia treatment was applied. These changes in the molecular size of polymeric glutenins are believed to cause gluten to express dough properties as if gliadins were added. Here, we propose that free sulfhydryl groups in the D-type of LMW-GS become activated and, by acting as chain terminators under an alkaline condition, attack disulfide bonds in large polymeric glutenins to downsize the polymeric glutenins.
Coomassie Brilliant Blue
DTNB5,5′-dithiobis (2-nitrobenzoic acid)
HMW-GShigh-molecular weight glutenin subunit
IPPSDS-insoluble polymeric protein
LMW-GSlow-molecular weight glutenin subunit
mBBrmonobromobimane
SS-SHdisulfide-sulfhydryl
