Structural changes of proteins in fresh noodles during their processing

ABSTRACT

Structural changes of proteins in fresh noodles during mixing, resting, sheeting, and cooking were investigated. The protein structural changes were evaluated by measuring the protein content, molecular weight, thiol and disulfide contents, secondary structure, thermal properties, microstructure, and mean particle size. The protein content was not significantly changed during fresh noodle processing. The wheat protein molecular weight decreased from 66.2 to 22.0 kDa during the cooking process. The disulfide content increased during the mixing and cooking processes. Additionally, the cooking process caused an increase in the number of gluten protein absorption peaks in the FTIR analysis. The secondary structure was mainly composed of β-sheets, and their proportion decreased in the cooking process. Gluten proteins were demonstrated to have high thermal stability with an average denaturation temperature of 84.9°C. There was a significant relationship between the gluten protein properties and fresh noodle processing.

KEYWORDS:

• Cooking process
• Fresh noodle
• Gluten proteins
• Microscopic structure
• Protein secondary structure

Introduction

Fresh noodles, a traditional staple food, have been consumed for thousands of years in many Asian countries. Because of their convenience, high nutritional quality, and enjoyable flavor, fresh noodles are popular globally.^[1,2] Fresh noodles are raw, wet noodles in which moisture content ranges from 32% to 40%. Notably, the process of fresh noodle production changes the components within the noodles, playing an important role in improving their quality.

Fresh noodle processing typically comprises dough mixing, resting, sheeting, cutting, and cooking. After those steps, noodle strands can be packed and marketed directly.^[3] The process of mixing aims to diffuse the components and to hydrate the flour particles, which is the basic procedure in noodle production.^[4] During dough mixing and resting, SDS-unextractable polymers in flours have a well-organized structure and are further adjusted.^[5] The sheeting process aims to achieve a smooth dough sheet of perfect thickness and the best eating quality.^[3] However, the process of sheeting has nonreversible effects on dough extension properties.^[6] In addition, in terms of quality and texture, the interaction pattern between the starch and protein of fresh noodles could have changed during the cooking process.^[7]

Wheat proteins are typical cereal proteins. The wheat gluten proteins form three-dimensional structures and play an important role in wheat dough quality. Wheat proteins are sensitive during the fresh noodle making process.^[8] The protein content and adequate gluten strength are closely correlated with fresh noodle quality, and a suitable range of protein content is important for textural characteristics.^[3] The protein content can reflect the level of nutrition in wheat flour and can have a significant effect on dough rheological properties.^[9] Gluten proteins are divided into gliadins and glutenins based on their solubility in alcohols.^[10] The proteins in wheat flour are composed of gliadins and glutenins. Gliadin proteins are monogenic alcohol-soluble proteins with molecular weights ranging from 30 to 60 kDa and are broadly classified into four types (ω-, γ-, β-, and α-gliadins). Glutenins, a type of polymeric protein, are categorized into low-molecular-weight glutenin subunits (LMW-GS) and high-molecular-weight glutenin subunits (HMW-GS).^[11–13] The proportion of extracted glutenin proteins has a crucial effect on the strength of dough. Rheological properties of dough have been correlated with the secondary structure of gluten proteins.^[14] To study the relationship of gluten particle size and wheat product properties, reducing the gluten protein particle sizes was shown to decrease the development time by farinograph and mixograph.^[15] The smaller sizes of gluten protein particles also indicated increased starch damage in dough.

This study aimed to investigate the protein structural changes of fresh noodle processing, which is very important to comprehend the quality properties of the noodles. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was used to evaluate the protein molecular weight. The contents of thiol and disulfide groups were used to evaluate the formation of the gluten network structure. Fourier transform infrared (FTIR) spectroscopy was applied to measure the proportion changes in the secondary structure. The thermal properties of glutens were measured by differential scanning calorimetry (DSC); scanning electron microscopy (SEM) was employed to have a better understanding of the gluten microscopic structures. The mean particle sizes of gluten proteins were measured using a laser particle size analyzer.

Materials and methods

Materials

High protein wheat flour was procured from Lijin Foodstuff & Edible Oil Corporation, Ltd. (Tianjin, China). The percentage moisture, protein, and ash contents were 10.9%, 12.2%, and 0.41%, respectively. Refined iodized salt was obtained from Tianjin Changlu Salt Industry Co., Ltd. (Tianjin, China). All chemicals and reagents used were of analytical grade.

Preparation of noodles and gluten samples

The noodle formula consisted of 100 parts of flour and 38 parts of distilled water. On a flour weight basis, NaCl was added at 2.0% in distilled water first. The ingredients were mixed for 8 min and formed a crumbly dough. The prepared dough was placed to rest in a plastic bag for 30 min at room temperature. The crumbly dough was passed through a hand-operated extruder machine (Mode FK156-3, Beijing, China) 5 times with the roller gap reduced continuously to obtain dough sheets. Then, sheets were cut into noodles 2 mm in width and 1 mm in thickness. Fresh noodles (50 g) were cooked in 500 mL of boiling distilled water for the optimal cooking time.

The samples were prepared following the method of Qiong Wang et al.^[16] and the method of sampling shown in Fig. 1. Samples from different sampling points were divided into two parts; the first was immediately lyophilized to obtain freeze-dried dough or noodles, and the second was water-washed until the water ran clear of starch to obtain gluten and then lyophilized to obtain freeze-dried gluten. All the above samples were ground into powder in a mortar, sieved, and preserved until analysis.

Figure 1. Method of sampling and sample analysis in fresh noodle processing.

Protein content

The protein content was determined by the Kjeldahl method with a conversion factor of 6.25 using a TUOPU Nitrogen Determinator (ZDDN-II, TUOPU Zhejiang Pty Ltd.). Samples were subject to three replicate determinations.

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

SDS-PAGE of proteins was tested according to the method of Man Li et al.^[8] with some modifications. SDS–PAGE was performed using 12% separating gel (pH 8.8) and 5% stacking gel (pH 6.8). A 50 mg quantity of each sample was stirred in 1 mL of extraction buffer (pH 6.8, 0.01 M Tris–HCl, including 10% (w/v) SDS, 5% (v/v) 2-mercaptoethanol (2-ME), 10% (v/v) glycerol, and 0.1% (w/v) bromophenol blue). The dispersions were heated for 5 min at 100°C and then were centrifuged (8000 g, 5 min). Sample volumes of 10 µL were loaded into the instrument, and electrophoresis was performed at 100 V during the run. The gel used 0.25% w/v Coomassie brilliant blue R250 stain and was de-stained in 10% acetic acid.

Fourier transform infrared spectroscopy

FTIR of gluten samples in the fresh noodle production process was performed according to the method of Kaiqiang Wang et al.^[17] FTIR spectra were recorded on an IS50 FTIR spectrometer (Thermo Nicolet Inc, Waltham, MA, USA) equipped with a DTGS KBr detector. Each freeze-dried gluten had a 1 mg sample weighed and added to 150 mg of KBr. These ingredients were mixed with an agate mortar and pestle. After that, samples were formed into tablets by hydraulic pressure, and the tablets were suitable for FTIR experiments. FTIR spectra were recorded at absorbances from 4000 to 400 cm⁻¹.

The amide I band (1600–1700 cm⁻¹) in each spectrum was analyzed using the Omnic software (version 8.1) and Peakfit software (version 4.12, SPSS Inc., Chicago, IL, USA) to evaluate the secondary structure changes in wheat gluten. The targeted structures were β-sheet, 1610–1640 cm⁻¹; random coils, 1640–1650 cm⁻¹; α-helices, 1650–1670 cm⁻¹; and β-turns, 1670–1700 cm^−1[18,19]

Thiol and disulfide contents

The values of disulfide SS contents could reflect the aggregation levels between proteins. Contents of accessible thiol (SHfree), total thiol equivalents (SHeq), and disulfide groups (SS) were determined according to the method described by Wagner Magali et al.^[20]cm⁻¹). For measurement of the total thiol equivalent groups (SH_eq), 30 mg of samples was exhaustively reduced with dithioerythritol (40 mM DTE in 80 mM Tris/HCl, pH 8.5, 0.3 mL) for 2 h at 60°C and then washed two times with 1.6 mL of glacial (18°C) acetic acid (100 mM) in acetone. After centrifugation (5 min, 959 g), the pellet was finally suspended in 1.3 mL of thiol reaction buffer and treated as previously described for (SH_free) determination. The disulfide group (SS) content was calculated from the SH_free and SH_eq determinations (SH_eq = 2SS+SH_free). All results are expressed in μmol per g of protein.

Differential scanning calorimetry

DSC of gluten samples was performed according to the method of Yulan Wang et al.^[21] with some modifications. DSC was carried out using a DSC-60A (Shimadzu, Japan) instrument with TA60 thermal analysis software. Approximately 2 mg of lyophilized sample was weighed into a hermetically sealed sample pan. The reference was an empty pan. DSC was performed in the temperature range of 25–200°C at a heating rate of 10°C/min. After heating, the sample was cooled to room temperature, and the DSC curves were obtained for the next step of analysis.

Scanning electron microscopy

SEM was performed by the method of Yulan Wang et al.^[21] and Agnieszka Nawrocka et al.^[22] The freeze-dried gluten samples were mounted on specimen stubs using double-sided adhesive tape and coated with gold. Coated samples were photographed using a JSM-IT300LV SEM (JEOL Ltd., Japan) with 3000 × magnification at an accelerating voltage of 10 kV.

Gluten proteins’ particle size measurement

The size of gluten protein particles was determined using a BT-90 Mastersizer (Dandong, China).^[16] Lyophilized gluten protein powder was dissolved in isopropyl alcohol, and it was mixed using mechanical agitation and treated with ultrasound for 1 min to dissolve the gluten clots to obtain an obscuration of 15–20%. The size of gluten protein particles was expressed by the mean particle size.

Statistical analysis

The SPSS statistical software (Version 19.0, SPSS Inc.) was used to certify significant differences in protein properties of different fresh noodle production processes by one-way analysis of variance (ANOVA), where p < 0.05 was considered to be significant.

Results and discussion

Wheat protein content

To understand the protein losses in fresh noodles during processing, the protein contents of mixed dough, rested dough, sheeted noodles, and cooked noodles were evaluated. The change of protein content in the fresh noodles during processing is shown in Fig. 2. At the five sampling points, the protein contents were 12.2 ± 0.3%, 12.2 ± 0.3%, 12.5 ± 0.1%, 12.0 ± 0.2%, and 13.1 ± 0.2%, respectively. The cooked noodles showed the highest protein content value compared to those after the other processes, and heat treatment by cooking significantly increased the protein content of fresh noodles (p < 0.05). As noodles were cooked in boiling water, the hydrogen bonds between the gluten protein and starch in noodles were continuously breaking, prompting starch gelatinization along with protein coagulation.^[7] Additionally, a notable amount of amylopectin and amylose had been dispersed out of the starch granules, and these starches were dissolved in the water.^[23] Therefore, the proportion of protein in cooked noodles was higher than in the other processes.

Figure 2. Changes in the total protein content in the fresh noodle processing. Sampling point 1 means wheat flour; sampling point 2 means mixed dough; sampling point 3 means rested dough; sampling point 4 means sheeted noodles; sampling point 5 means cooked noodles. Superscripts denote significant difference (p < 0.05).

Electrophoretic profiles of wheat proteins

Wheat protein profiles from SDS-PAGE are shown in Fig. 3. The protein molecular weight ranges from 14.4 to 97.4 kDa. The results showed that there were no significant changes in wheat protein molecular weight during the mixing, resting and sheeting processes. However, it could be observed that the intensity of some bands (Part B, 14.4–22.0 kDa) had increased greatly after cooking, and the bands (Part A, 66.2–97.4 kDa) that are mainly glutenin had decreased or vanished. These changes may reflect the decomposition of high molecular weight proteins due to the heat effect of cooking, which suggested polymerization of glutenin. This is consistent with a previous work, which reported that the intensity of the bands of glutenin (66.2 –97.4 kDa) noticeably decreased at 95°C and that glutenins exposed their inner structure more easily than gliadins at high temperature.^[24] With the successive and intense heat treatments, damage to the intramolecular disulfide bonds and development of new intermolecular disulfide bonds and protein–protein interactions occurred in wheat gluten proteins.^[25,26]

Figure 3. SDS-PAGE profiles of the total protein in the fresh noodle processing.

Contents of thiol and disulfide groups of wheat proteins

Accessible thiol (SH_free) in wheat proteins contributed to the formation of disulfide groups (–S–S–) and thus the gluten three-dimensional network structure.^[8] Table 1 shows the contents of SH_free, SHeq, and SS of wheat protein in the fresh noodle production processes. A decrease in SH_free was observed after mixing (50.96 to 44.34 μmol/g protein, p < 0.05) along with an increase in SS (36.01 to 41.10 μmol/g protein, p < 0.05). The results likely indicate the formation of the gluten three-dimensional structure during flour hydration and mixing. It should be noted that there was a significant decrease in SH_free after cooking (37.94 to 13.20 μmol/g protein, p < 0.05) and a significant increase in SS in the same process (44.28 to 57.44 μmol/g protein, p < 0.05). This result indicated an increasing aggregation of gluten protein upon heat treatments by cooking. This observation was in accordance with the results of SDS-PAGE, confirming the polymerization of glutenin. In a previous study, Wagner Magali et al.^[20] also discovered that the content of SH_free was reduced significantly during the pasta overcooking process.

Table 1. The contents of accessible thiol (SH_free), total thiol equivalent (SH_eq), and disulfide groups (SS) in the fresh noodle processing.

	Accessible thiol [SHfree](μmol/g)	Total thiol equivalent [SHeq] (μmol/g)	Disulfide groups [SS] (μmol/g)
Wheat flour	50.96 ± 2.60^a	122.98 ± 0.16^b	36.01 ± 1.22^d
Mixed dough	44.34 ± 1.56^b	126.54 ± 2.39^a	41.10 ± 0.42^c
Rested dough	45.66 ± 0.10^b	125.77 ± 0.36^ab	40.06 ± 0.23^c
Sheeted noodles	37.94 ± 0.83^c	126.51 ± 0.68^a	44.28 ± 0.75^b
Cooked noodles	13.20 ± 0.26^d	128.09 ± 0.52^a	57.44 ± 0.13^a

Values in the same column followed by the same letter are not significantly different (p < 0.05).

Secondary structure analysis of gluten proteins

FTIR spectroscopy can be used to observe changes in the secondary structure of gluten proteins.^[27] The FTIR spectra of gluten proteins are depicted in Fig. 4I. As shown in region A, there were three apparent peaks at 1022, 1081, and 1157 cm⁻¹, which corresponded to the C–O–C stretching and CO (–COH) stretching of starch.^[10] There existed no clear differences in region A during the mixing, resting and sheeting processes. In contrast, the intensities of these three peaks were observed to have increased significantly in gluten proteins of cooked noodles, reflecting that there was residual starch in the samples. This is potentially induced by the increased connectivity between protein and starch during cooking. Two peaks with considerable intensities never observed in other samples existed in the cooked noodle gluten protein spectra, one peak at 855 cm⁻¹, which is characteristic of aromatic C–H out-of-plane deformation, and another peak at 761 cm⁻¹, which may correspond to the S–N stretching mode.^[28,29]

Region B (1600–1700 cm⁻¹) is the amide I region. The secondary structure analysis from amide I bands has been broadly described. The stable band near 1655 cm⁻¹ is assigned to α-helical structure.^[30] In region B, the peak at 1654 cm⁻¹ exhibits different absorptive intensities during the noodle processing. During mixing, resting and sheeting processes, the absorption intensity of amide I increased gradually, whereas the absorption intensity of amide I decreased dramatically in the cooking process. This result indicated that the secondary structure in gluten proteins was broken by heating reactions in cooked noodles.

The amide I peaks ranging from 1700–1600 cm⁻¹ mainly originate from C = O stretching vibrations, and this region is influenced by hydrogen bonding. The amide I band of proteins is formed of many folded composing bands, which indicate different protein secondary structural components such as α-helices, β-sheets, β-turns, and random coil structures.^[19,31] Figure 4(II) shows the proportions of the secondary structures of wheat proteins in the fresh noodle processing. The fraction of β-sheet structure increased slightly (from 30.32% to 30.75%) during the mixing process. In contrast, the β-sheet structure decreased (from 30.57% to 27.57%) during the sheeting and cooking processes. Previous work has reported that the β-sheet structure composed of hydrophobic interactions and hydrogen bonding acts as a dilatant in the gluten network.^[32] During the dough mixing, the flour powder forms viscous wet granules; after proper kneading it becomes a rubber-like dough.^[33] Heat treatment can alter the structure and physicochemical properties of a protein.^[34] First, wheat flour was hydrated with water, which is good for the gluten network formation. Second, the hydrogen bonding in the gluten network was broken, due to the mechanical and heat treatment from the sheeting and cooking processes.

Figure 4. The change in the wheat proteins secondary structures in the fresh noodle processing. (I) FT-IR spectrum of wheat proteins in different fresh noodles processing shown in the range 400–1800 cm^–1 after baseline correction. (II) Proportions of the secondary structures of wheat proteins in the fresh noodles processing. Values on the pie chart means the percentage of each secondary structure.

DSC analysis of gluten proteins

Thermal analysis is usually used to study the influence of thermal processing on cereal proteins.^[35] Figure 5 presents examples of thermograms for gluten proteins during the fresh noodle making process. Table 2 shows characteristic thermal parameters of gluten proteins during the fresh noodle processing. The gluten protein DSC thermograms showed only one endothermic peak, which averaged at 84.9°C with average enthalpies of 149.20 J/g. Similarly, in a previous study, the gluten proteins had an ordered structure based on the high enthalpy values.^[35] A higher Tp indicated that the gluten protein was more thermodynamically stable. In other words, proteins with higher Tp are not easy to denature at lower temperatures.^[36] Therefore, the thermodynamic properties of gluten are stable. There was no significant change in the Tp of gluten during fresh noodle processing.

Table 2. Thermal characteristic parameters of gluten proteins in the fresh noodle processing.

Gluten samples	Denaturation temperature Tp(°C)	Enthalpy values ΔH(J/g)
Wheat flour	86.08	159.40
Mixed dough	78.86	128.94
Rested dough	83.25	131.72
Sheeted noodles	90.14	143.45
Cooked noodles	86.12	182.50

Figure 5. DSC curves of gluten proteins in the fresh noodle processing.

SEM image analysis of gluten proteins

Scanning Electron Microscopy was used to monitor changes in the microstructure of gluten.^[22,37] Figure 6 shows SEM photos of gluten proteins during fresh noodle processing. Small granules visible in the images are starch molecules that were not washed out. The visible apertures of gluten in mixed dough and rested dough were observed to be much larger than those in wheat flour, sheeted noodles and cooked noodles. In addition, gluten in Fig. 6A, 6D and 6E presented a more laminar structure, while gluten in Fig. 6B and 6C presented a more fibrous structure. Gluten protein polymerization might occur upon sheeting and cooking (Fig. 6D and 6E). Therefore, the gluten three-dimensional network structure was formed by mixing, completed by resting, and finally modified by sheeting and cooking. This was probably due to the mechanical and thermal effects from fresh noodle processing.

Figure 6. SEM photos of gluten proteins in the fresh noodle processing. (A) Wheat flour gluten, (B) mixed dough gluten, (C) rested dough gluten, (D) sheeted noodle gluten, (E) cooked noodle gluten.

Mean particle size of gluten proteins

By taking particle diameter measurement as a tool, the aggregation of gluten proteins was evaluated.^[16,38] As showed in Fig. 7, the mean particle size of gluten proteins significantly increased from 1.85 to 2.77 μm during the resting process (p < 0.05). The results may be because the mean particle size of gluten proteins was directly dependent upon the degree of gluten network structure, and the gluten was hydrated in the process of mixing and resting. However, the mean particle size of gluten proteins in both sheeted and cooked noodles decreased significantly. This result was probably due to gluten proteins being modified as a laminar structure, showing low solubility in isopropyl alcohol. The mean diameter results are consistent with the results of gluten SEM image analysis, confirming the laminar structure of glutenin in the sheeting and cooking processes. Agnese Panozzo et al. discovered that the changes in gluten particle size distribution suggested the modification of intra- and inter-molecular protein–protein interactions, which include the contents of SH_free and disulfide bonds (–S–S–).^[39] Therefore, the results are also consistent with the results of the thiol and disulfide group contents.

Figure 7. The change in the mean particle size of gluten proteins in the fresh noodles processing. Sampling point 1 means wheat flour gluten; sampling point 2 means mixed dough gluten; sampling point 3 means rested dough gluten; sampling point 4 means sheeted noodles gluten; sampling point 5 means cooked noodles gluten. Superscripts denote significant difference (p < 0.05).

Conclusions

Fresh noodle manufacture induced a series of remarkable changes in the physicochemical properties of gluten proteins. In detail, the mixing process was effective in improving gluten aggregation and forming gluten network structure by increasing the content of disulfide groups (–S–S–), the intensity of the amide I band and the proportion of β-sheets. The resting process only slightly completed the gluten network structure compared to the other processes. The sheeting process changed the microstructure of gluten from fibrous to laminar by mechanical treatment, and the denaturation temperature of sheeted noodles was determined to be the highest among the noodle processes. Notably, the cooking process increased the content of disulfide groups (–S–S–) and the number of FTIR absorption peaks but decreased the molecular weight and the proportion of β-sheets in gluten proteins. These significant changes in the cooking process indicated that the gluten network structure was dramatically modified by heat treatments. In conclusion, the gluten three-dimensional network structure was formed, completed and further modified during the mixing, resting, sheeting, and cooking phases, especially in the cooking process. This study increased our insight into the behaviors of the gluten network during processing and provided references for evaluating the noodle quality.