3.1. In Vitro Digestibility of Recombinant Flours
The in vitro digestion curve of all recombinant flours showed faster digestion rates of starch within the first 30 min (
Figure 1). Comparing 200215 and 200315, significantly more RS but less RDS and SDS were found in 200115, indicating that stronger gluten strength could reduce the digestibility of starch. There were no significant differences in RDS, SDS, and RS between 200215 and 200315, indicating that the difference in HMW-GSs at
Glu-
B1 had little if any effect on starch digestion. For the 2001 samples, the proportional increase of glutenin in gluten protein (200131) had no significant effect on the content of RDS, SDS, and RS, however, a significant content decrease in RS and increase in RDS were found in the gluten containing more gliadin (200113). This difference in 2001 samples indicated that the barrier effect of strong-strength gluten protein on starch granules was weakened with the increasing proportion of gliadin, and the inhibiting effect of macromolecular protein network on starch digestibility was greater than that of monomeric protein. For 2002 samples, part of the RDS was converted to RS after the increase of glutenin (200231), while part of SDS was converted to RS after the increase of gliadin (200213). The difference between 200231 and 200213 indicated that for moderate-strength gluten protein, the enhanced coating effect of the stronger gluten network mainly inhibited the rapid digestion of starch, while the additional monomeric proteins were more prone to interacting with amylase or starch and reducing the amount of SDS. Although there was no difference in RS content between 200231 and 200213, more SDS and less RDS were detected in 200231 than those in 200213, which indicated that for the flour with moderate-strength gluten, compared with the proportional increase of monomeric gliadins, the improved strength of gluten containing more glutenins was more conducive to reducing the starch digestibility. For 2003 samples, part of RDS and SDS were converted into RS after the increase of glutenin, and part of RDS was converted to SDS with the increase of gliadin, which indicated that for the flour with weak-strength gluten, the enhancement of gluten strength through quantitative increase of glutenin was more effective to inhibit starch digestion than the proportional increase of monomeric gliadins. In brief, for the flour with moderate or weak-gluten strength, the proportional increase in glutenin and gliadin significantly decreased the starch digestibility. The inhibition effect of an enhanced macromolecular gluten network on starch digestion was stronger than that of gluten protein with more monomeric proteins. However, for the flour with high-strength gluten protein, further enhancement of macromolecular gluten network or incorporation of additional monomeric gliadins did not improve the resistance of starch to digestion.
3.3. Protein Behaviors of the Recombinant Flours
According to a previous study [
8], the polymeric proteins (LPP, MPP, SPP) in the recombinant flour are mainly composed of glutenin and gliadin, while monomeric gliadin is the main component of LMP, and SMP is mainly composed of albumin and globulin. As shown in
Table 2, in the recombinant flours prepared with starch and different natural gluten proteins from the three wheat NILs (200115, 200215, 200315), no significant difference in the contents of polymeric and monomeric proteins among the three samples were found, but the UPP% in 200115 was significantly higher than that in 200215 and 200315, which was consistent with the strong-strength gluten contributing to the formation of thermally stable protein polymers remained intact structure during cooking. For the wheat NILs of 2001 and 2003, the proportional increase of glutenin or gliadin significantly decreased the contents of LPP, MPP and UPP, and the high proportion of gliadin in gluten protein (200113 and 200313) led to greater reduction in LPP, MPP, and UPP compared to the gluten proteins with high percentage of glutenin (200131 and 200331), in contrast, the content of SPP and LMP increased significantly with the ratio variation of glutenin to gliadin, and the highest content of SPP or LMP was found in gluten with high percentage of gliadin (200113 and 200313). All these results showed that, for the flours with strong and weak-strength gluten proteins, the modification of glutenin-gliadin ratio (approximately 1:1 in natural gluten protein) led to a trend for damaging the stability and strength of the gluten networks. This tendency for reduced stability and strength of the gluten networks was attributed to the fact that the LPP, MPP, and UPP were partially depolymerized into SPP and LMP. Furthermore, compared with the gluten protein with high content of glutenin (200131, 200331), the proportional increase of gliadin caused much greater damage to gluten network. Different from 200131 and 200331, 200231 had the highest content of LPP, MPP, UPP, and the lowest content of LMP among all the recombinant flours. Therefore, we believe that the quantitative increase of glutenin in moderate-strength gluten is more conducive to the formation of a much stronger gluten network, and also promoted the binding of monomeric gliadin to amylose or short amylopectin, all these changes consequently led to the low content of RDS (
Figure 1B). Within each group of recombinant flours, the content of monomeric gliadin (LMP) was lowest when natural gluten was recombined with starch, probably because the separation of glutenin and gliadin disrupted the original protein network.
3.4. Comparison of Protein Secondary Structure of the Recombinant Flours
For the three wheat NILs, significant differences in the contents of α--helix and β-sheet under different glutenin-gliadin ratios were found (
Table 3). Quantitative increase of gliadin in the gluten protein of the three NILs always led to increased content of α-helix, which was consistent with the finding that gliadin was rich in α-helix [
28]. The change of α-helix content was opposite to the change of UPP% (
Table 2) in each group of the recombinant flours, which indicated that the quantity of α-helix was negatively correlated with the gluten strength during heat treatment. This assertion is supported by our previous finding that a lower content of α-helix may be attributed to the intermolecular disulfide bonds breaking and gluten rearrangement induced by heating treatment [
8]. The content of β-sheet was positively correlated with the stability of protein secondary structure [
8]. The increase in glutenin led to increased β-sheet content in the recombinant flours, which confirmed the report that glutenin contained higher content of β-sheet than that of gliadin [
29]. To further explore the potential relationship between the secondary structure characteristics and protein polymerization/depolymerization behavior in recombinant flours, we calculated the ratio of β-sheet to α-helix (β-sheet/α-helix). It can be found that when the β-sheet/α-helix was lower than 1.7, it has a linear, positive, correlation with the UPP% (
Table 2) which indicated that within a certain range the higher β-sheet/α-helix can be used to characterize the formation of strong and thermally stable gluten network. The same variation in the trend of disulfide bond and β-sheet/α-helix formation in the recombinant flours (
Table 3 and
Table 4) was consistent with this suggestion.
For the recombinant flours containing natural gluten protein and starch, 200115 had the optimal β-sheet/α-helix compared to 200215 and 200315, which contributed to the formation of more compact and stable gluten network that inhibited the enzymatic hydrolysis of starch and produced more RS and less RDS (
Figure 1B). For the recombinant flours containing higher content of glutenin, excessively high β-sheet/α-helix in 200131 increased the digestibility of starch slightly, although the RDS’s in 200231 and 200331 were still higher than in 200131, consistent with the increased β-sheet/α-helix content improving the starch digestion resistibility as reflected in their significantly increased RS (
Figure 1B). The increase of gliadin in gluten protein caused a significant decrease of β-sheet/α-helix in 200113 and 200213. The β-sheet/α-helix decrease in 200113 led to weakened gluten network, thus resulting in increased starch digestibility characterized by significant increase in RDS and decrease in RS compared to 200115. However, the reduced β-sheet/α-helix in 200213 caused decrease in SDS and increase in RS, indicating that additional gliadins added in moderate-strength gluten tended to enhance the interactions between monomeric gliadin and starch or amylase, thus converting part of SDS to RS. In 200313, the high proportion of gliadin led to significant increases in α-helix and β-sheet when compared to 200315, but no significant change in β-sheet/α-helix was found. The additional gliadins in weaker gluten protein (200313) appeared to convert partial RDS (from 67% to 60%) into SDS (from 11% to 19%) through the enhanced interactions between monomeric proteins and starch or amylase.
3.5. Quantitative Changes of Different Chemical Bonds within Protein of the Recombinant Flours
As a relatively stable covalent bonds, disulfide bonds play an important role in stabilizing the spatial structure of the peptide chain. As shown in
Table 4, under the same glutenin-gliadin ratio, the content of disulfide bond in recombinant flours was always ranked as 2001 > 2003 > 2002. According to the higher UPP% and β-sheet/α-helix in 2001 samples (
Table 2 and
Table 3), it is evident that the more sulfhydryl-disulfide bond exchange reaction and sulfhydryl oxidation occurred in the cooking of the recombinant flours from 2001 promoted the formation of a more extensive and compact gluten network [
30]. The content of disulfide bond in 2003 samples was higher than that in 2002 samples, which might be due to the greater contribution of subunits 7 + 8 than subunits 7 + 9 to the accumulation of gluten proteins in wheat kernel [
31]. In general, the disulfide bond content increased with the increase of glutenin-gliadin ratio in all the recombinant flours. This is consistent with the report that the disulfide bonds in gluten network are mainly formed through the oxidative crosslinking of cysteine residues located at the N- or C-terminal of HMW-GSs and LMW-GSs [
32], while fewer disulfide bonds between monomeric gliadin and glutenin were generated when the temperature was higher than 80 °C [
33]. The significantly lower quantity of disulfide bonds in the gluten proteins with increased gliadin further showed that the additional gliadins in gluten protein interrupted the formation of disulfide bonds. Notably, more disulfide bonds were detected in 200331 compared to 200315, but the content of polymeric proteins and UPP% in 200331 were significantly lower than those in 200315 (
Table 2), this contradiction implied that more intramolecular disulfide bonds or intermolecular disulfide bonds between glutenin and gliadin were formed in 200331, and impeded the development of extensive gluten networks. In contrast, 200231 had the highest UPP% in all the recombinant flours, but a lower quantity of disulfide bonds was detected. Considering the strongest tryptophan fluorescence intensity of 200231 among all the recombinant flours (
Figure 2), we suppose that a strong protein-starch interaction has occurred in 200231, and reduced the protein extractability significantly.
Hydrophobic interactions are the main driving force of protein folding. For the three NILs, the intensity of hydrophobic interaction was increased to varying degrees with the increase of glutenin content, indicating that higher glutenin content could promote the aggregation of hydrophobic residues and stabilize the structure of protein networks. For 2001 samples, the hydrophobic interaction was always weaker than most of the 2002 and 2003 samples, which may be attributed to the stronger protein-starch interaction in 2001 samples that hindered the aggregation of hydrophobic groups in protein. This prediction was also confirmed by the significantly higher hydrogen bond content in 2001 samples [
8]. Notably, protein in 200313 was characterized by weak hydrophobic interactions but high content of hydrogen bonds, indicating the relatively strong protein-starch interaction that resulted low content of RDS in 200313 (
Figure 1B).
The recombinant flours directly prepared by natural gluten and starch showed much higher content of hydrogen bonds compared to the flours with increased proportion of glutenin or gliadin, this difference may be due to the fact that some original hydrogen bonds existing in the gluten extractions were broken in the separation of glutenin and gliadin. Furthermore, when the proportion of glutenin or gliadin was increased, the hydrogen bond content decreased to different degrees and the lower content of hydrogen bond was accompanied by the higher content of gliadin. This phenomenon indicated that the incorporation of additional glutenin or gliadin led to the rearrangement of original gluten structure. As for the gluten with increased content of glutenin, the decreased hydrogen bond was accompanied by the significant increases in both hydrophobic interactions and disulfide bonds, resulting in a more extensive and stable gluten network, however, for the gluten with increased gliadins, significant decreases in disulfide bonds and hydrogen bonds indicated the destruction of gluten structure. The content of ionic bonds in gluten protein was significantly lower compared with the other chemical bonds. Moreover, regardless of the composition of gluten protein, the flours containing 5 + 10 subunits showed relatively high ionic bonds, which was consistent with our previous study [
8].
3.6. Long- and Short-Range Molecular Order of Starch in Recombinant Flours
X-ray diffraction (
Figure 3) showed that all the cooked recombinant flours showed typical V-shaped crystallization peaks with a strong diffraction peak at 19.95°, which was mainly due to the combination of amylose and a series of micromolecules (such as lipids or proteins) to form a single left--handed complexes [
17].
In the FTIR spectrum, the peak at 1538 cm
−1 was correlated with the presence of starch-protein complex, the peaks at 1714 cm
−1 and 2854 cm
−1 indicated the existence of lipid-starch complex, and the simultaneous appearance of the three peaks indicated the presence of starch-lipid-protein complexes in the samples [
34]. As shown in
Figure 4, all the cooked recombinant flours showed peaks with different intensities at 1538 cm
−1, indicating the formation of starch-protein complexes. No obvious peak was observed at 2854 cm
−1 and 1745 cm
−1, which possibly indicated that the low content of lipid in wheat flour and the further lipid loss during separating of gluten proteins and starch reduced the possibility for forming starch-lipid or starch-lipid-protein complexes. Compared with other flours, more obvious peaks at 1538 cm
−1 were observed in 200115, 200215, 200315, 200113, and 200231, in view of their lower LMP contents (
Table 2), we proposed that more starch-protein complexes were formed in these flours.
For all the recombinant flours, the relative crystallinity of starch increased significantly with the increase of glutenin content, while the high content of gliadin led to the significant reduction in relative crystallinity (
Table 5). This result indicated that the high proportion of glutenin contributed to a more extensive and compact gluten network that effectively prevented the swelling and gelatinization of starch, while additional monomeric gliadins had negative effect to the gluten strength and long-range molecular order of starch. Notably, the recombinant flours with relatively stronger gluten strength, such as 200115, 200231, and 200331, showed comparatively higher relative crystallinity that supposed to enhance the starch digestion resistibility and produce more RS in starch digestion (
Figure 1B).
In the FTIR spectrum, the absorbance at 995 cm
−1 and 1047 cm
−1 is related to the order degree of starch molecules, while the sensitivity at 1022 cm
−1 is linked to the amorphous structure of starch. Therefore, the degree of orderliness (DO) and the degree of double helix (DD) of starch can be characterized by 1047/1022 and 995/1022, respectively [
35]. Moreover, the full width at half-maximum (FWHM) of the band at 480 cm
−1 of LCM-Raman spectrum was also used to characterize the short-range molecular order of starch [
36]. As shown in
Table 5, the starch in 200215 and 200315 showed higher DO and DD values and lower FWHM than the other recombinant flours, indicating the natural moderate- or weak-strength gluten protein containing approximately equal amount of glutenin and gliadin could promote the formation of more ordered starch structure rich in double helices. For the 2001 samples, high proportion of gliadin in gluten was contributed to the high short-range molecular order of starch in 200113 that characterized by higher DO, DD, and lower FWHM. This result can be interpreted in two ways: firstly, the weak-strength gluten network of 200113 facilitated the pasting of starch, thus releasing more short-branch starch that incorporated into the formation of more double helix; secondly, additional monomeric gliadins enhanced the interaction between gliadin and short-chain starch, and eventually improved the short-range molecular order of starch. Notably, within the three recombinant flours prepared by natural gluten and starch (200115, 200215, 200315), the decrease in FWHM and increases in DO and DD were observed with the descend of the gluten strength, which may be due to the fact that the weak gluten network was more likely to break and depolymerized during cooking, thus facilitating the interactions between protein and starch fragments and improving the short-range molecular order of starch. The DO or DD of 200113 and 200213 was significantly higher than that of 200131 and 200231, respectively, which indicated that the incorporation of additional gliadins probably improved the short-range molecular order of starch in strong- and moderate-strength gluten flour by enhancing the interactions between monomeric gliadins and gelatinized starch.
Besides the non-covalent bonding of protein to starch, the covalent interaction between protein and starch was detected according to the shifts of the bands at ~860 cm
−1 that were sensitive to the anomeric structure around glycosidic bonds [
15]. The peak wavelength and transmittance changes of the recombinant flours were accompanied by the variation in glutenin-gliadin ratio (
Table 5), suggesting gluten strength and the ratio of glutenin to gliadin could significantly affect the binding between monomeric protein and starch. Notably, a peak wavelength migration and a relatively lower transmittances were found in the recombinant flours composed by natural gluten proteins and starch, indicating that stronger glycoside bonds between protein and starch were existed in these flours. The low amount of LMP in 200115, 200215 and 200315 (
Table 2) was consistent with this judgment. The decrease of gluten strength, related to a significant decreased in transmittances, which may be due to the fact that the protein network in 200315 was more damaged during cooking, thus forming stronger glycosidic bonds between gliadin and amylose/amylopectin. For the recombinant flours with high content of glutenin (200131, 200231, 200331), the peak deviation was only found in the weak-gluten flour (200331), which indicated the existence of strong protein-starch interaction. Although there was no change of wavenumber in 200131 and 200231, the transmittance in 200131 was significantly lower than that in 200231, indicating that the starch in the strong-gluten flour was less damaged and retained more glycoside bonds. For the recombinant flours containing high content of gliadin (200113, 200213, 200313), the shift extent of peak and transmittance were increased with the decrease of gluten strength, indicating weaker glycosidic linkage between monomeric gliadin and starch was more prone to from in the flours with weak-strength gluten.
NMR can be used to assess helix content in starch [
37]. The wide peak of C1 at 103 or 104 ppm corresponded to typical single helices organized as V-type crystalline phases or dispersed in amorphous phases, while the peak near 76 ppm was positively correlated with the content of double helix in the starch of the recombinant flours [
38]. As shown in
Figure 5, the V-type crystals formed in all recombinant flours. As shown in
Table 6, for 2002 and 2003 samples, an increase in content of single and double helices was observed after the quantitative increase of glutenin, suggesting the introduction of additional glutenin in moderate- and weak-strength gluten flours promoted the formation of stable protein network, thus reducing the degree of starch fragmentation during high-temperature cooking [
13]. For 2001 samples, the single helix was the highest in 200115, but the content of the double helix was the lowest, we assumed that the cooking promoted the unwinding of amylose/short amylopectin and facilitated the formation of more starch-lipid/protein complex [
39]. The lower LMP% in 200115 compared to 200131 and 200113 confirmed this indication of protein-starch complex formation (
Table 2). In addition, the lipid loss during the preparation of recombinant flours with high content of glutenin or gliadin may be impeded by the formation of V-type starch-lipid complex in 200131 and 200113, thus further increasing the content of single helix in 200115. For the weak-strength gluten flours, the content of PPA decreased after the proportion of glutenin or gliadin increased, which was consistent with the finding that the content of RDS in 200331 and 200313 was significantly lower than that in 200315 (
Figure 1B).
Combining the single helix content in the starch from different recombinant flours and the shift and intensity of the 860 cm
−1 peak from FTIR, we inferred that the covalent protein-starch complex accounted for a high proportion of the single helix in the recombinant flours containing natural gluten. In contrast, more non-covalent protein/lipid-starch complexes may be involved in the high content of single helix in 200231 and 200331. For the recombinant flours with increased gliadin, there were still more starch fragments linked to the protein (
Figure 4 and
Table 5) although the single helix was damaged greatly due to the decrease of the gluten strength.