1. Introduction
Bread-making involves various processes, such as mixing, fermentation, proofing, and baking. Dough mixing is an important process that can affect the quality of the bread substantially, as viscoelastic gluten is formed during the mixing process. Many studies have been conducted to elucidate the mechanisms underlying gluten formation and structure [
1,
2,
3]. Some early models emphasized the importance of intermolecular disulfide bonds [
4,
5,
6]. However, since the late 1990s, most proposed gluten structures, such as the loop-train model [
7] and hyper-aggregation model [
8], have focused on noncovalent bonds. A recent study proposed a model in which different bonds or interactions play a role depending on the ratio of glutenin to gliadin during mixing [
9]. Much debate remains in regard to the structure and formation of the gluten network [
1,
10,
11,
12]. This research theme has been one of the major issues that have continued until the present.
In terms of changes in protein during dough mixing, a decrease in glutenin macropolymer [
13,
14], a decrease in unextractable polymeric protein (UPP) by sodium dodecyl sulfate (SDS), and a decrease in the molecular weight of UPP have been reported [
15]. These results have been attributed to protein depolymerization [
13,
14,
15]. Additionally, a decrease [
16] or increase [
17,
18] in free sulfhydryl groups during mixing has been reported. In addition, recent studies have reported data showing that SS binding does not continue to increase or decrease during mixing but increases in the early stage and then decreases significantly [
9,
19] or slightly [
20] as the mixing intensity increases and then decreases. Thus, there were many reports on changes in SS bonds during dough formation. However, as no method has been established to measure noncovalent bonds directly, little remains known about changes in noncovalent bonds during mixing.
Noncovalent bonds include electrostatic interactions, hydrophobic interactions, and hydrogen bonds. In our previous investigation [
21], we reported that noncovalent bonds weaken and the molecular size of polymeric proteins decrease during dough mixing, while ω-gliadin aggregates with other proteins through noncovalent bonds. However, we did not elucidate the behavior of each type of noncovalent bond, so which noncovalent bond was responsible for the aggregation of ω-gliadin during mixing remained unclear. Therefore, in this study, we focused on hydrophobic interactions, which is a kind of noncovalent bond, and studied changes in hydrophobic interactions during dough formation.
Wheat gluten proteins contain few ionizing groups but more than 35% hydrophobic amino acids [
2] and, thus, promote hydrophobic interactions. The impact of salt on the properties of dough is often explained by gluten aggregation resulting from hydrophobic interactions [
22]. Kinsella et al. [
23] showed that, in the presence of F
− or Cl
−, the hydrophobic interaction becomes stronger, and the proteins remain aggregated, making hydration difficult. Melnyk et al. [
24] demonstrated that kosmotropes, such as NaCl, reduce the water absorption of gluten and strengthen hydrophobic interactions.
Assessing the strength of hydrophobic interactions among proteins remains very challenging. Chen et al. [
25] used the SDS-binding capacity [
26] as an indicator for evaluating hydrophobic interactions and examined the effect of a NaCl addition but did not find a significant difference. Hydrophobic interactions have also been evaluated based on data from dough surface hydrophobicity generated by the front-face fluorescence method [
27,
28]. Bonomi et al. [
27] pointed out that the variation in front-face fluorescence is large and discussed surface hydrophobicity without evaluating any significant differences, but Jazaeri et al. [
28] discussed the data at a significance level of 10% (
p < 0.1). In our previous report [
21], we evaluated the strength of noncovalent bonds by extracting proteins with a series of SDS solutions, which weaken noncovalent bonds, at various concentrations. In this study, we analyzed hydrophobic interactions by extracting proteins in different concentrations of 1-proponol solutions, which weaken hydrophobic interactions. Furthermore, we used the front-face fluorescence method to examine the changes in the hydrophobic interactions during dough formation.
2. Materials and Methods
2.1. Wheat Flour
In the present study, we used the same two commercial flour samples (Nisshin Flour Milling Inc., Tokyo, Japan) as those used in a previous report [
21]: one milled from hard wheat with a protein content of 14.5% (referred to as “HF” (high-protein flour)) and the other from semi-hard wheat with a protein content of 11.7% (referred to as “LF” (low- protein flour)) (dry basis).
2.2. Dough Sampling during Mixing
The dough was sampled as described in a previous report [
21]. First, flour (200 g), distilled water (HF: 128.8 mL, LF: 117.4 mL; mean water absorption for the farinograph), and NaCl (4 g) were mixed at a constant temperature of 27 °C using a Swanson mixer (National Mfg. Co., Lincoln, NE, USA). All dough samples were mixed at 120 rpm (Association for Cereal Chemistry International standard mixing method 54-70.01) [
29] for 20 min to reach overmixing conditions. A data logger (AF-1700; ATTO Co., Tokyo, Japan) was used to monitor the change in electric power during mixing.
Figure 1 shows the mixing curves. Dough samples were collected at the following time points: dough buildup (HF: 4 min, LF: 6 min), peak consistency (HF: 8 min, LF: 11 min), dough breakdown (HF: 12 min, LF: 16 min), and overmixing (20 min).
2.3. Protein Extraction
Regarding the protein extraction, 30% or 10% 1-propanol was used as a solvent to weaken the relatively strong or weak hydrophobic interaction, and the proteins were extracted as described in a previous report [
21]. Immediately after sampling, the dough sample (1 g) was homogenized with 20 mL of 30% or 10% 1-propanol (hereafter “extraction solvent”) at 10,000 rpm for 5 min (Ace-AM10; Nihonseiki Kaisha Ltd., Tokyo, Japan). After centrifugation at 5000×
g for 10 min, the supernatant was collected, and extraction solvent (1 mL) was added to the residue. The suspension was centrifuged after stirring, and the supernatant was collected. The two supernatants were then combined and mixed, and an automated protein analyzer (Kjeltech 8400; FOSS, Hillerød, Denmark) was used to determine the amount of protein (N × 5.7). Each extraction procedure was repeated six times.
2.4. Analysis of Protein Size Distribution by Size-Exclusion High-Performance Liquid Chromatography (SE-HPLC)
Size-exclusion high-performance liquid chromatography (SE-HPLC) was performed in accordance with a previous report [
21]. The extracted protein solutions were made up to 100 mL with extraction solvent and then diluted twice with extraction solvent and passed through a 0.45-μm filter. An SE-HPLC system composed of an HPLC system (Chromaster; Hitachi High-tech Science, Tokyo, Japan) consisting of an online degasser, gradient pump, auto injector, column oven, and ultraviolet (UV) detector was used to analyze the molecular size distribution of the proteins. A size-exclusion column (300 × 7.8 mm i.d., 3 μm, Yarra SEC-4000; Phenomenex, Torrance, CA, USA) was used for separation. Phosphate buffer (50 mM, pH 7.0) containing 0.5% SDS was used as the mobile phase. All samples (50 μL) were then eluted at 35 °C at a flow rate of 0.5 mL/min.
Open LAB software (Hitachi High-tech Science) was used to integrate the UV signal (214 nm). We then multiplied the amount of extracted protein by the area ratio in the HPLC chromatogram to calculate the amount of protein in each fraction.
2.5. Analysis of Protein Composition by SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
The individual protein fractions separated by SE-HPLC in four independent runs were then combined, and trichloroacetic acid was added to make a final concentration of 20%, followed by vortexing. After 30 min of immersion in ice, the sample was centrifuged at 12,000× g for 5 min to precipitate proteins. After, the supernatant was discarded, and 2 mL of cold acetone was added. Then, the mixture was vortexed and centrifuged at 12,000× g for 5 min to remove nonprotein contaminants. This operation was repeated. After discarding the supernatant, the precipitated protein was dissolved in the sample buffer (20 μL; 1% SDS, 0.1-M Tris HCl, 20% glycerol, and an appropriate amount of bromophenol blue) for SDS-PAGE. Both with and without 2-mercaptoethanol (3 μL), the samples were heated at 95 °C for 3 min, cooled at room temperature, and then analyzed by SDS-PAGE on a Mini Protean Tetra System (Bio-Rad Laboratories, Hercules, CA, USA) at 200 A for 30 min using a mini protean 7.5% TGX gel and electrophoresis buffer (25-mM Tris-HCl, 192-mM glycine, and 0.1% SDS).
The gel was removed from the plate after electrophoresis, dyed with Oriole Fluorescent Gel Stain (Bio-Rad Laboratories) for 90 min, and scanned (ImageQuant LAS 4000; Cytiva, Tokyo, Japan).
2.6. Protein Identification by Two-Dimensional Fluorescence Difference Gel Electrophoresis (2D-DIGE)
Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) was performed as described in a previous report [
21]. The mixtures of protein aggregates (A) or monomeric proteins (B) in
Figure 2 collected twice were concentrated to 200 μL using a 10-kDa or 3-kDa cutoff ultrafiltration column (Vivaspin 20; Sartorius Lab Instruments, Göttingen, Germany). Next, 1.8 mL of cold acetone containing 10% trichloroacetic acid was added to the concentrated protein solution, which was then stored at −20 °C for 4 h. Following centrifugation at 14,000×
g for 8 min to precipitate proteins, the supernatant was discarded. Then, 1 mL of cold acetone was added and stored at −20 °C for 10 min. Following centrifugation at 14,000×
g for 8 min, the supernatant was discarded, and the residue was air-dried for 5 min. Next, 15 μL of swelling solution in the Glycine system reagent set for Auto2D (Merck, Tokyo, Japan) was added, followed by vortexing for 10 min. Then, 1 μL of 200 pmol fluorescent dye (IC3-Osu; Dojindo Laboratories, Kumamoto, Japan) or 1 μL of 200 pmol fluorescent dye (IC5-Osu; Dojindo Laboratories) dissolved in N,N-dimethylformamide, and 0.5 μL of 1.5-M Tris-HCl buffer (pH 8.8) was added to 10 μL of each sample at each mixing time. As an internal standard, 2 μL of each sample at each mixing time was mixed, followed by the addition of 1 μL of 200 pmol cyDye DIGE Fluor Cy2 minimal dye (Cytiva) and 0.5 μL of 1.5-M Tris- hydrochloric acid buffer (pH 8.8). Each sample and the internal standard were stored in the dark at 4 °C for 1 h for labeling; after which, 1 μL of 10-mM lysine was added to each sample and the labeled internal standard, and the mixture was stored at 4 °C for 10 min. Then, 2 μL of internal standard labeled with Cy2, 2 μL of sample labeled with IC3, 2 μL of sample labeled with IC5, and 7 μL of swelling fluid (swelling fluid:1-M dithiothreitol:ampholyte = 113.4:6:0.6) were mixed. The prepared mixture was then loaded for 2D-DIGE using Auto-2D plus (Merck). The electrophoresis conditions were set based on the desalting mode recommended by the manufacturer. An isoelectric focusing chip (pH 3–10) and a 10% PAGE chip (Merck) were used for the Auto-2D electrophoresis. Next, the gel was removed from the plate and scanned using Amersham Typhoon (Cytiva), and then, an Ettan DIGE analysis was carried out using Melanie 9 software (Cytiva).
2.7. Front-Face Fluorescence Method
The water was replaced with 400-μM 8-anilinonaphthalene sulfonic acid (8-ANS) and 50-μM thioflavin T to analyze the dough surface hydrophobicity and protein aggregation, respectively. The dough used for the front-face fluorescence analysis was prepared as described in
Section 2.2. HF and LF samples were collected from the start of the mixing after 2, 4, 6, 8, 10, 12, 16, and 20 min. Immediately after sampling, the dough was covered with plastic wrap, and an optical fiber from the fluorescence spectrometer (F-7000; Hitachi High-tech Science) was pushed into the samples to measure the fluorescence intensity. Next, 8-ANS and thioflavin T fluorescence were monitored by the emission spectra from 350 to 600 nm, with excitation at 384 nm and 435 nm, respectively, and the height of the peaks in the emission spectra was assumed to correspond to the fluorescence intensity, which was then compared between samples.
2.8. Statistics
Tukey’s method was applied to determine significant differences in the SE-HPLC (p < 0.05, n = 6) and front-face fluorescence analyses (p < 0.1, n = 6) using JMP software (SAS Institute Inc., Cary, NC, USA), and analysis of variance was applied to determine significant differences (p < 0.05, n = 4) in the 2D-DIGE analysis using Melanie 9 software (Cytiva).
4. Conclusions
This study showed changes in the hydrophobic interactions among proteins during dough formation. The hydrophobic interactions among proteins weaken, and a part of the protein aggregates disaggregate to monomers (mainly ω-gliadin) up to the peak consistency. After the peak consistency, the exposed hydrophobic groups in the protein interact with each other, and ω-gliadin reaggregates.
In a previous study [
21], it was reported that the molecular size of proteins decreases, because noncovalent bonds weaken during dough formation, while ω-gliadin aggregates with other proteins. In the present study, we confirmed the same behavior in the hydrophobic interactions as in the noncovalent interactions. This suggests that the effect of noncovalent bonds during dough formation is mainly due to hydrophobic interactions.
These findings suggest that, to gain a better understanding of the mechanism underlying gluten formation, it is important to analyze disulfide and other noncovalent bonds to obtain a comprehensive picture of dough formation.