3.1.1 Dough Rheology
Frequency scanning of dough is one of the main methods to determine the dynamic oscillation characteristics of dough. The energy G' and the G" are two important indicators of frequency scanning, which can fully reflect the viscoelastic properties of the dough. and their ratio is the tanδ. The G ' is always much larger than the G", and tanδ is always less than 1 which indicates the dough mainly undergoes elastic deformation. The G' and G" of URD, MRD, and WD varied with frequency sweep from 0.1~20 Hz. As shown in Figure 1 that G' and G" increase with increasing frequency for all samples, meanwhile G' is greater than G" indicating the elastic nature of the dough. It is consistent with Jelena's experimental findings [15]. The G' changes more gently with frequency, while G" more significantly. It is due to that more and more number of times of cycle scans was required when frequency increased. The dough is a viscoelastic body with a gel network structure. Namely the higher the frequency, the shorter the interval in frequency sweeps, which resulting in a shorter time for the dough to recover. Therefore, the dough exhibits a property similar to that of a solid state due to the lack of time to recover [16]. Compared with the three samples, both the G' and the G" showed that URD was higher than MRD and MRD was higher than WD at all the frequency range tested, which indicate that the rheological properties of MRD have been changed. The G' and G" of MRD and URD are higher than that of WD, which should be related to the properties of the protein and starch in raw materials. Tanδ the viscoelastic ratio, the larger it is, the greater viscosity and smaller elasticity the sample behaves. The tanδ of MRD and URD is 0.44 and 0.35 respectively, which is far lower than 0.8 of WD. This indicates that compared with WD the viscosity is smaller and the elasticity is stronger for URD. This runs counter to our previous understanding that rice proteins in URD are not gluten proteins and are not able to form gluten network structures [17], so the elasticity of URD should be smaller than that of WD. After analyzing the reasons, the author believes that the protein content in rice is around 9% and rice glutenin accounts for about 80% of rice protein, which content a large number of intramolecular and intermolecular disulfide bonds (most glutenins in cereals have this characteristic) [18], so the glutenin in rice is not less than that in wheat flour quantitatively. The glutenin in wheat endows WD with good elasticity and glutenin in URD can also endow URD with certain elastic toughness. However, the structure of rice gluten is different from that of wheat gluten, and the content of gliadin in rice is less and the viscosity absent, so it is impossible to form a good viscosity ratio under the action of wheat gluten and wheat glutenin in wheat flour, Therefore, the viscosity of URD is small and the elastic toughness is strong. This observation is similar to the results obtained by Sivaramakrishnan et al [19]. The G" of MRD was slightly higher than that of URD and it was similar to the tanδ of WD. This indicates that the modification method improves the viscoelastic ratio of URD, leading to an increase in tanδ, an increase in viscosity and a decrease in elasticity, which is closer to the viscoelastic ratio of WD.
3.1.2 Texture properties
Table 1 Effect of modifier on texture properties of dough
Sample
|
Hardness/g
|
Springiness
|
Chewiness/g
|
Resilience
|
URD
|
1062.04±58.55a
|
0.15±0.01b
|
20.74±2.60a
|
0.05±0.01b
|
MRD
|
865.81±53.71b
|
0.15±0.01 b
|
17.67±2.21a
|
0.05±0.01b
|
WD
|
158.29±11.0c
|
0.33±0.03a
|
18.38±4.13a
|
0.06±0.01a
|
Note: Different lowercase letters indicate significant difference between data(P<0.05)。
Texture analysis is a large deformation method to explore non-linear behaviour of dough. From table 1, it can be seen that the hardness of MRD is smaller than that of URD, the springiness and resilience were hardly changed, and the chewiness becomes smaller, which is similar to WD. The overall trend of the indicators of MRD texture characteristics shifted to WD, and the chewiness was slightly lower than WD, indicating that the addition of improvers caused structural changes in the dough. The hardness of MRD becomes lower, which may be caused by the addition of sodium metabisulfite. Disulfide bond of protein can be broken by sodium pyrosulfite in powder to enhance the softness and malleability of dough, which is mainly used in ramen dough [20]. The reason for the high hardness of URD should be inseparable from its protein and disulfide bond. Honda [21] and others have studied reducing the rigidity of URD by adding protease, so as to improve the quality of rice bread. On the other hand, since rice protein contains more gluten, TGase can induce gluten cross-linking in rice protein to form a larger polymeric protein [22]. Some literatures showed that adding TGase to gluten free bread and rye dough could increase the hardness of dough [23-24]. The addition of sodium metabisulfite is accompanied by the action of stirring, and the molecular chain expands along the direction of force, making the protein with disulfide bond broken more orderly. The structure diagram is shown in the following figure. Under the action of TGase, forming a more ordered network structure, making the texture characteristics of MRD closer to WD, which corresponds to the results of frequency scanning.
3.1.3 LF-NMR
Table 2 Effect of modifiers on low-field NMR of dough
Samples
|
T21
|
T22
|
T23
|
T24
|
A21
|
A22
|
A23
|
A24
|
URD
MRD
WD
|
0.16
0.14
0.12
|
1.00
0.57
0.76
|
8.11
5.34
4.64
|
114.98
75.65
65.79
|
3.56
1.40
3.72
|
35.38
31.35
19.63
|
60.41
66.54
75.55
|
0.65
0.71
1.09
|
Water plays a pivotal role in dough processing. Distribution of water in dough and interaction between dough components will affect the quality of dough [25]. The transverse relaxation time T2 reflects the fluidity of different forms of water in dough, mainly divided into T21 and T22, on behalf of the water tightly bound to gluten protein through hydrogen bond. T21 and T22 are called strong combined water and weak combined water respectively. The poor fluidity is bound water. T23 is adsorbed water. Its main form in dough is water that is not easy to flow, and its mobility is between bound water and free water. T24 represents the water that has a weak effect on the non water components in the dough. It is free water with the greatest fluidity.
Fig. 2 shows that T21 and T22 (bound water) of all dough have no obvious change. Compared with the URD, the peaks of the MRD T23 and T24 moved forward, indicating that the water in the MRD moved with the addition of the modifier. The relaxation time T21, T23 and T24 of WD were lower than those of URD and MRD. Only T22 MRD is close to WD. The shorter T2 time is, the smaller the fluidity of water molecules is, and the closer the binding degree is. It can be seen that the water molecules in WD were more closely bound than those in rice dough, but the water state of MRD is close to that of WD, which is consistent with Ozel [26] research results.
A21, A22, A23 and A24 are the peak areas corresponding to the relaxation time. The size of the relative peak area expresses the moisture content in dough. A21 and A22 are bound water. The content of strong bound water in all dough samples is low, while the content of weak bound water is high. A23 and A24 account for more in the dough, while A21 and A22 account for less in the dough. Compared with WD, the content of A21 and A22 (bound water) in URD is higher, while the content of A23 and A24 is lower. The water content of MRD is closer to that of WD in different states. The experimental results showed that the addition of sodium metabisulfite and TGase composite modifier reduced the bound water in MRD, and increased the migration of water. Part of the combined water in MRD migrates to the adsorbed water, resulting in enhancment of adsorbed water and free water. The reason may be that glutenin and gliadin in WD form a dense network structure in the dough which can wrap water molecules in it. The internal water molecules are mainly expressed in the form of adsorbed water [27]. However, due to the low content of gliadin in URD and the different structure of rice glutenin and glutenin, water molecules cannot wrap more water molecules as WD. Water molecules combine with other components. Therefore, there is more bound water and less adsorbed water and free water [28]. However, the introduction of modifier has made the protein cross linked. The network structure formed in MRD makes the water molecules more wrapped in the structure, increasing the adsorbed water, and making its structure more compact compared with the URD.
3.2 Effects of modifiers on the structure and properties of rice protein
3.2.1 Thermogravimetric
Table 3 Weight loss rate and degradation temperature of protein before and after modification
Sample
|
Weight loss rate(%)
|
Degradation temperature
Td(℃)
|
URP
|
74.46
|
451.70
|
MRP
|
73.37
|
354.95
|
WP
|
77.51
|
300.32
|
Thermogravimetric analysis is a technique for measuring the mass and temperature of substances, and to observe the change of sample weight with the increase of test temperature. After measurement information such as sample weight loss percentage, initial decomposition temperature (Tp), termination temperature (Td) and sample reaction rate can be obtained [29]. It can be seen from figure 3 that the thermogravimetric curve revealed the weight loss change of the protein as the temperature went up which can provide auxiliary information for the thermal stability of protein network structure. The two stages of weight loss are shown in the figure. The corresponding two peak temperatures were the temperatures at the maximum reaction rate of the two stages respectively. The initial weight loss at 50 ℃~150 ℃ is related to the loss of free water and combined water during heating [30]. When the temperature was further increased from 250℃ to 450℃, the peptide bond in protein would dehydrate rapidly, which will cause the breaking of covalent peptide bond, disulfide bond, O-N bond and O-O bond in the protein. The degradation temperatures of URP, MRP and wheat protein (WP) were 415.7 ℃, 354.95 ℃ and 300.32 ℃ respectively. The structure of URP is different from that of WP, so the degradation temperature was significantly different. However, the degradation temperature of MRP changed significantly, which was close to that of WP. The results were consistent with those of water migration. Some studies have shown that the higher the weight loss rate is, the more open and the weaker the network structure is, while the decrease indicates the more compact and stronger structure [31]. It can be seen from Table 3 that the weight loss rate of WP is higher than the other two proteins. It may be due to the different structural of wheat and rice protein. Wheat protein has a more open network structure, while rice protein has a more compact structure, which should also be the main reason for its strong toughness.
3.2.2 Particle size
Table 4 Effect of modifier on protein particle size and polydispersity coefficient
Samples
|
Average particle size/nm
|
PDI
|
URP
|
73.61±12.85a
|
0.37±0.05a
|
MRP
|
227.10±94.57a
|
0.28±0.02b
|
WP
|
136.00±28.82a
|
0.32±0.03b
|
Note: Different lowercase letters indicate significant difference between data(P<0.05).
The average particle size can indirectly reflect the interaction among particles in the solution. It can be seen from Fig.4 and Table 4 that the average grain size of WP was larger than that of URP, indicating that WP formed aggregates, while URP had a low degree of aggregation. This should also be one of the reasons why rice protein cannot form a good gluten network. After improvement, the grain size of rice protein tended to move to the right, and the width of the volume peak decreased, indicating that the addition of modifiers caused aggregation of MRP, increased the average grain size, and decreased the particle size distribution range [32]. The principle involved in this study is to use sodium metabisulfite to break some disulfide bonds. Because of inappropriate position of these disulfide bonds, the elasticity and toughness of rice balls are poor and the rigidity are strong. Then the oxygen in the air is used to oxidize the sulfhydryl group into disulfide bonds (this reaction has been proved to be weak later). Meanwhile new protein aggregates were formed by modifier TGase. According to the particle deformation principle of non Newtonian fluid in rheology [33], the newly formed bonding position is conducive to the formation of good order of rice clusters on account of stirring, which is also reflected in the basic rheology of rice clusters. It is also believed that TGase induces the cross-linking of gluten to form large aggregates. Low molecular weight proteins, i.e. albumin and globulin, cannot participate in aggregation because they are trapped in the polymerized gluten matrix. Polymerization promotes the hydrophobic interaction between proteins, and increases the number of surface drainage points. The probability of protein collision is increased, and the phenomenon of protein molecule aggregation is increased [34]. Based on the above theory, the particle size molecule of the improved protein is larger than that of the other two proteins in the refractometry process, which is consistent with Renzetti's [35]. The polydispersity coefficient (PDI) reflects the degree of particle size uniformity, and is an important indicator of particle size characterization [36]. The larger the PDI value, the larger the particle size distribution interval. PDI < 0.05, monodisperse systems, such as some emulsion standard samples. PDI < 0.08, nearly monodisperse systems. PDI = 0.08~0.7, moderate dispersion systems. It can be seen from Table 2 that the PDI values corresponding to the average particle size of three samples measured are between 0.08 and 0.7, indicating that the particle size distribution of the measurement system is uniform.
3.2.3 SEM
A URP, B MRP, C WP subscript 1-x1000 microscope; subscript 2-x5000 microscope
The scanning electron microscope diagram of MRP is illustrated in Fig. 5, which shows that obvious changes have taken place in the microstructure surface of URP and MRP, and the protein structure of rice without modifier treatment is shown in Fig. 5-A1. The protein structure is compact, showing bulk accumulation, and the particle size is uniform, which may be for this the reason why rice is too rigid when forming pellets. As shown in Fig. 5-A2, the surface of URP magnified by 5000 times is uneven, with uneven cracks and smaller pores. The structure of MRP with modifier changed obviously, as illustrated in Fig. 5-B1, forming obvious protein aggregates and connecting between aggregates. Compared with Fig. 5-C1, the electron microscope diagram of MRP was similar to that of WP. Both show the shape of aggregates. As showed in Fig. 5-B2, compared with Fig. 5-B1, the MRP seems to have a larger and more interconnected block structure, a smoother surface and fewer stomatal structures. These observations are consistent with Hu [37]. MRP changes obviously, showing flake structure and hierarchical stacking. Sodium metabisulfite can break the disulfide bond in URP, so that the structure of the protein is loose. TGase can bind with URP more easily and induce glutenin cross-linking in protein to form a large polymer, which is comparable to that of WP (Fig. 5-C2). And the pore structure appeared on the surface of WP.
3.2.4 SDS-PAGE
The main component of URP is glutenin, which is composed of several subunits connected by disulfide bonds. the molecular weights of three main subunits are 38, 25, 16kDa (or 33, 22, 14kDa), respectively, 16kDa is related to gliadin, two high molecular weight peptides are linked in the form of disulfide bond [38], and the range of natural rice proteins is between 9.17~74.85kDa. Explain the accuracy of the molecular weight of the standard sample in figure 6 (consistent with the specification) and URP, thus proving the accuracy of the software analysis. It can be seen from figure 6 that the color near the 21kDa and 29kDa bands of MRP is darker than that of URP, and there is no obvious change near 12kDa and 13kDa compared with URP, indicating that the composition of URP subunits has not changed, but the content of URP subunits has changed. The bands formed near 21kDa (glutenin basic subunits) and 29kDa (glutenin acidic subunits) in MRP are darker than the entirebands [39], which may be linked to the disulfide bond breakage in URP. The introduction of sodium metabisulfite reduced most disulfide bonds to sulfhydryl groups, and the effect was better than that of other proteins treated with β-mercaptoethanol. After TGase cross-linking, the loose protein structure interrupted by sodium metabisulfite produces intermolecular and intramolecular cross-linking, forming new aggregation forms, macromolecular groups and small molecular groups. This is consistent with the results of Huang [40].
3.2.5 FTIR
Table 5 Effects of amendments on secondary structure of rice protein
Sample
|
α-helix(%)
|
β-sheet(%)
|
β-turn(%)
|
Random coil(%)
|
URP
MRP
WP
|
13.67±0.10c
14.22±0.07b
14.54±0.08a
|
42.19±0.16b
39.28±0.23c
44.44±0.17a
|
29.87±0.10b
32.06±0.24a
25.62±0.14c
|
14.27±0.07b
14.43±0.05b
15.40±0.07a
|
Note: Different lowercase letters indicate significant difference between data(P<0.05).
Fourier transform infrared spectroscopy is an important method to effectively analyze the changes of protein secondary structure. The spectrum of amide I band in Fourier infrared spectrum is 1600~1700cm-1. The relationship between absorption peak and secondary structure is as follows: α-helix is 1650~1660 cm-1, β-sheet is 1610~1640cm-1 and 1670~1690cm-1, β-turn is 1660~1670cm-1 and 1690~1700cm-1, and random coil is 1640~1650cm-1 [41]. The secondary structures between polypeptide chains in protein molecules are mainly α-helix、β-sheet、β-turn and Random coil. As shown in Table 5, among URP α-helix、β-sheet、Random coil were slightly smaller than that of WP, while β-turn was slightly larger than that of WP. Compared with URP the α-helix content of MRP tended to be equal to WP. It has been reported that the hydrogen bond content in protein had a certain effect on α-helix content [42]. Therefor this may be the reason for the increase of α-helix. The increment of hydrogen bonding was confirmed by subsequent studies. The change of dough viscoelasticity is closely related to its secondary structure. Content of α-helix has an important influence on the hardness and elasticity of gluten because. α-helix structure is stable, tough and elastic [43]. The content of β-turn has an important influence on the viscosity of gluten [44]. β-sheet is a kind ofstretching structure composed of peptide chains. In the β-sheet structure, a hydrogen bond is formed between C=O and N-H on the main chain of the adjacent peptide chain, with intermolecular folding formed by strong hydrogen bond and antiparallel β-folding formed by weak hydrogen bond. All peptide bonds are involved in the formation of hydrogen bonds between chains, thus maintaining the stability of the β-sheet structure. The ratio of α-helix/ β-sheet is related to the flexibility of gluten protein. The smaller the α-helix/ β-sheet ratio, the more flexible the protein is[43]. The ratios of URP, MRP, WP α- helix/ β- sheet were 0.32, 0.36 and 0.33 respectively, indicating that the flexibility of URD was reduced. This may be due to the subsequent TGase induced cross-linking of URP. The addition of sodium metabisulfite broke the disulfide bond in the protein which leading to the extension of the protein structure, reduction of toughness and the exposure of hydrophobic amino acid residues [45]. Therefore, when the β-sheet content of MRP decreased, more hydrophobic sites were exposed which increased the surface hydrophobicity of the protein [46]. This was proved in the later study of chemical interactions. The increment of β-turn content in MRP indicates that the introduction of TGase recombined the dispersed amino acid residues disconnected by sodium metabisulfite, and the reaction sites of TGase were easier to bind. So that the hydrophobic groups exposed to the surface inside can regroup and form new aggregates. Thus, MRP had better network structure. (Fig. 8)[47].
3.3 Effect of improver on protein bonding in rice protein
3.3.1 Sulfhydryl and disulfide bonds
The content of sulfhydryl group and disulfide bond in the protein will have a significant impact on the structure and functional properties of the protein. Disulfide bond is a covalent bond connecting two semideaminate residues, which plays an important role in stabilizing protein conformation and maintaining protein activity. It can be seen from Fig. 9 that the content of total sulfhydryl group and disulfide bond in URP is significantly lower than that in WP. However URD shows strong rigidity and tenacity, which makes its viscoelasticity seriously unbalanced. This is why first its disulfide bond was broken, and then constructs the network through biological enzymes in our experiment. The content of total sulfhydryl, free sulfhydryl and disulfide bond of MRP was higher than that of URP and WP. This is because the sodium pyrosulfite in the improver can loosen the structure of URP, denature and stretch the internal structure of the protein, expose the sulfhydryl group, and significantly increase the total sulfhydryl group content. In addition, sodium metabisulfite broke the disulfide bond in URP to form sulfhydryl group, leading to the reduction of disulfide bond to sulfhydryl group, which increased the content of free sulfhydryl group. It was consistent with the conclusion obtained by Zhu [48] in the article. Under the condition of natural oxidation, the sulfhydryl group was largely converted into disulfide bond, which increases its content. The TGase in the modifier catalyzes the intermolecular ε – (γ- Glutamyl) - lysine covalent bond, forming the network structure, but independent of disulfide bond [49]. WP is a gluten structure formed by the disulfide bond within and between molecules [50], so the content of disulfide bond in WP is higher than free sulfhydryl group.
3.3.2 Analysis of chemical interactions
Protein is a biological macromolecule with spatial structure composed of various amino acids. Conformation is maintained by hydrogen bond, disulfide bond, ionic bond, hydrophobic interaction, Van der Waals force and ε – (γ- Glutamyl) - lysine covalent bond and other chemical forces. However ionic bond, hydrogen bond and hydrophobic interaction are the main forces to maintain the tertiary structure of rice protein. As shown in Fig. 10, hydrophobic interaction was the highest in the three protein samples which played a very important role in forming high-quality dough. When the dough was finished, the hydrophobic aggregates with hydrophobic regions were formed outside the gluten protein. At the same time, a hydrophilic area would be formed inside, and a large amount of water would be maintained [51]. The hydrogen bond, ionic bond and hydrophobic interaction force in URP and MRP were higher than that in WP. Combined with disulfide bond content, the reason may be due to that the main forced in URP are hydrogen bond, ionic bond and hydrophobic interaction, while the disulfide bond in WP has relatively more effects. The contents of hydrogen bond, ionic bond and hydrophobic bond in the MRP were higher than that in the URP, indicating that the tertiary structure of the protein had changed significantly under the action of the modifier. This may be because that the addition of sodium metabisulfite promoted the exposure of hydrophobic amino acid residues inside the protein and increased the hydrophobic interaction and hydrogen bond [52]. Whereas the addition of TGase not only catalyzes the intermolecular ε – (γ- The glutamyl) – lysine covalent bond but also promoted the reaction of glutamine with water to generate glutamic acid and ammonia.Thus the water molecules in hydrophobic interaction were reduced. Therefore, the increment of hydrophobic interaction and hydrogen bond content was not significant like that of ionic bond. Compared with URP, the ionic bond content of MRP increased significantly (P<0.05). This may be due to the addition of sodium metabisulfite, which affected the surface charge of protein molecules, leading to the introduction of salt ions, resulting in significantly increase of ionic bond content of MRP. Which was consistent with the conclusion reached by Li [53]. The results show that the modifier affected the interaction between protein molecules. The introduction of sodium metabisulfite increased the content of ionic bonds in the protein, and made the protein structure loose and easier to combine. The original loosely extended protein structure was joined by TGase, forming a network structure which had good hydrophobic interaction. This conclusion agree with the result of particle size.