Abstract
The glutenins and gliadins were added to the base flour at varying concentrations of 2, 4, 6, 8, and 10 g/100 g flour, respectively. The addition of glutenins remarkably improved the mixing characteristics of the flour, while gliadins resulted in decreased dough stability and increased softening of the dough. The pasting characteristics varied with the varying concentrations of gliadins and glutenins. The peak viscosity decreased upon the addition of gliadins and glutenin, with gliadins being more effective in reducing the values of peak, final, breakdown, and setback viscosities. The hardness of the dough improved upon the addition of glutenins, while the gliadins resulted in dough with greater adhesiveness and cohesiveness. The gluten recovery increased by 98.79% upon addition of 10 g/100 g flour gliadins while the gluten quality measured in terms of gluten index was increased by the addition of glutenins.
INTRODUCTION
Wheat is undoubtedly the supereminent cereal crop in the world due to its widespread distribution and extensive utilization for baked food products.[Citation1] It is consumed as food and feed in different ways throughout the world.[Citation2] The unusual properties of the wheat is ascribed to the presence of gluten forming storage proteins of the endosperm which are further composed of two fractions- the alcohol soluble gliadins and the alcohol insoluble glutenins.[Citation3,Citation4] Gliadins contain intra-molecular disulfide bonds, the breaking of which causes unfolding of the protein molecule. They are apparently responsible for the cohesive property of gluten.[Citation1,Citation5,Citation6] The glutenin proteins are multi-chained and appear to be mainly polymerized by disulfide bonds.[Citation7] These proteins appear to give the gluten its elastic properties. Glutenin is predominantly responsible for the elastic properties of dough and gliadin contributes to dough extensibility.[Citation4,Citation8] The relative proportions of these components in gluten affect the dough rheological properties, with higher glutenin content, imparting greater gluten strength in wheat. Wheat dough is viscoelastic, which means that it has both viscous and elastic characteristics. Upon hydration, the gliadins behave as a viscous liquid[Citation9,Citation10] which imparts extensibility to the dough. If dough is too viscous and flows too much during sheeting, the dough does not maintain the desired final shape. If the dough lacks in its elastic properties, it is difficult to form into the desired shape with the result that the final products are not desired by the customers. Moreover, an inverse relationship exists between the gliadin/glutenin ratio and the elasticity of gluten. Therefore, a correct balance of viscoelastic properties is very important to a successful sheeting process. Thus, on one hand, it is necessary to control the extensibility of the dough, and on the other hand it is important to control the elasticity. If it is not sticky enough, the dough will not be sufficiently cohesive to be formed by sheeting, and the final product does not have the desired crumb structure. It is necessary to have an appropriate extent of stickiness to hold the folded layers of dough together and to prevent large holes in the final baked food. Viscoelastic properties of gluten affect the rheological and textural properties of wheat dough. When the dough is developed by mixing, the gluten proteins form continuous three-dimensional viscoelastic network throughout the dough with starch granules behaving as filler. The three-dimensional structure of gluten matrix is stabilized by covalent (disulfide), hydrogen and non-covalent ionic bonds, and hydrophobic interactions. The balance between gliadins and glutenins is responsible for important rheological properties such as viscosity and elasticity.[Citation11,Citation12] Khatkar et al.[Citation8] studied the effect of various gliadin subfractions on the mixograph properties and observed that the resistance breakdown (RBD) values increased with addition of gliadin and its subgroups, while Uthayakumaran et al.[Citation13] reported that addition of gliadin subgroups reduce peak dough resistance (PDR) and RBD. Thus, numerous studies have been carried out on the study of glutenins on the mixing characteristics of the flour,[Citation14] but very little and contradictory literature is available on the effect of gliadins on mixing behavior of dough. Moreover, the effect of these subfractions on the pasting properties and texture profile analysis (TPA) has not been reported in the literature so far. Thus, the study was aimed to investigate the effect of gliadins and glutenins on the rheological, pasting, and textural properties of the dough so as to deduce their effect on the end product quality.
MATERIALS AND METHODS
Flour Sample
Straight grade flour, milled from wheat variety HW 2004 on Chopin mill was used for the study. The flour had moisture 11.32 g/100 g flour, protein 10.06 g/100 g flour protein and 0.54 g/100 g flour as determined by AACC Approved methods 44–15A, 46–30.[Citation15] This flour was used for extraction of gliadins and glutenins fractions and as base flour for addition of these subfractions.
Preparation of Protein Fractions
HW 2004 flour (100 g) was gently shaken with chloroform (200 ml) at room temperature and then filtered through filter paper to obtain defatted flour. The defatted flour was then allowed to dry at room temperature. Gluten was extracted from the defatted flour with Perten Glutomatic (2200) and the resulting gluten was freeze dried. The freeze dried gluten samples were ground in a pestle mortar and dissolved in 200 ml of 70% ethanol. The mixture was stirred on a magnetic stirrer for 3 h at 25°C followed by centrifugation for 30 min at 1000 g at 4°C. Supernatant was collected and the pellet was again extracted with 70% ethanol. The supernatants were pooled and ethanol was removed from the gliadin extracts using rotary evaporator at 30°C. The gliadin and glutenin fractions, thus, obtained were freeze dried and powdered in pestle and mortar.
Gliadin and Glutenin Addition and Mixing Effects
Changes in the dough mixing properties of the base flour were determined by a 4 g micro doughlab. The base flour (HW 2004) was supplemented with increasing amount of gliadin and glutenin fractions, i.e., 2, 4, 6, 8, and 10 g/100 g, respectively. The gluten subfraction was added to the base flour and put into the mixing bowl of micro doughlab. The instrument allows the dry mixing of the flour for one minute followed by the addition of water as determined by the instrument to reach the 500 FU line in the chart. The mixing of the dough continued for 12 min for all the gliadin fortified flours. However, for the glutenin fortified flours, the mixing time was manually increased to 30 min as the dough became too strong and the departure time could not be determined in 12 min cycle. The different mixing parameters determined by the micro doughlab were peak (FU), arrival time (min), dough development time (min), dough stability (min), departure time (min), dough softening (FU), peak energy (Wh/Kg) and bandwidth at peak (FU), respectively.
Gliadin and Glutenin Addition and Pasting Characteristics
The changes in the pasting characteristics of the base flour were determined by Rapid Visco Analyzer-TecMaster (Perten Instruments)(RVA) according to the AACC approved method 76–21. The gliadin and glutenin fractions were added to the base flour at 2, 4, 6, 8, and 10 g/100 g level, respectively. The different parameters determined by the RVA were peak viscosity, breakdown, trough, setback, final viscosity, pasting temperature, (°C) and peak time (min). All the parameters were expressed in Rapid Visco Units (RVU).
Instrumental TPA of Gliadin and Glutenin Added Dough
Dough samples prepared in the 4 g micro doughlab were studied using TPA. Cylindrical samples of 2 cm diameter and height 1 cm were obtained from dough. Samples were compressed to 75% of their original height. A plate-plate sensor system with a stainless probe SMSP/75 was used at a constant rate of 0.5 mm/sec. The texture of the dough was determined by a uniaxial compression test of two cycles (TPA) using TA-XT2i Texture Analyzer. Parameters such as hardness, adhesiveness, cohesiveness, and consistency were analyzed. Hardness is the maximum force obtained during the first compression cycle. Adhesiveness is the negative area obtained during the first cycle. Cohesiveness was obtained as the ratio between the positive areas of the second cycle and the first cycle. Consistency is the sum of the positive areas of the first and the second cycles.
Recovery of Gluten and Determination of Gluten Index
The dough with and without the addition of gliadin and glutenin fractions were prepared in micro doughlab and rested in water for 30 min. The dough was then washed under running tap water to remove the water soluble components such as starch. The weight of the resulting wet gluten was noted. The wet gluten was then centrifuged in gluten centrifuge to obtain the gluten index of the gliadin and glutenin fortified gluten.
Statistical Analysis
The experimental data collected was analyzed for significant differences with the help of analysis of variance (ANOVA) and correlation analysis was conducted using SPSS 16.0 software.
Table 1 Effect of gliadins and glutenins on micro doughlab parameters
RESULTS AND DISCUSSION
Effect of Added Gliadin and Glutenin on Rheological Properties
The results of the addition of gliadin and glutenin fractions on the rheological properties of the dough have been presented in . The peak height of the base flour was reported to be 510 FU. The peak dough height decreased considerably upon addition of the gliadins at increasing concentrations from 2–10 g/100 g, respectively. On the other hand, addition of glutenins to the base flour increased its peak dough height value upto 573 FU for 10 g/100 g glutenin addition. The peak dough height is an indicator of dough strength, i.e., the ability of the proteins to withstand the stress of mixing. Gliadin fortification caused the decrease in the peak dough height indicating the weakening of the dough. Dough development time decreased significantly from 3.5 to 2.02 min upon the addition of 10 g/100 g gliadins. The addition of 10 g/100 g gliadin to the base flour caused the maximum reduction in the time required for the optimum dough development indicating the shorter mixing time requirement to form optimum dough. This could be due to the alteration in the gliadin-glutenin balance in the dough and interaction of gluten subfractions with the base flour components. However, the added glutenins increased the time required for the development of dough. Moreover the glutenin increased the dough stability to as long as 30 min (10 g/100 g addition) while the added gliadins lowered the dough stability considerably. The doughs with added glutenins showed longer dough development time and produced more stable doughs with lesser degree of softening than the doughs supplemented with gliadins. The glutenins were effective in delaying the optimum dough development time with 10 g/100 g glutenins being the most effective in delaying the mixing time from 3.50 to 4.67 min. The resistance to the mixing of flour was due to the differences in gliadin to glutenin ratio of the flours. The addition of glutenins greatly increased the departure time indicating that the dough became stronger upon the addition of the fraction. Stability of the dough, indicating flours tolerance to mixing is important in deciding the end use quality of the wheat flour. It is generally recommended that stronger doughs with higher stability (hard flour) perform well for bread making while flours of lower stability (soft flour) produce good cookies and cakes. In the present study, it was found that the dough stability can be improved by the addition of glutenin fraction to the flour. Similar results were reported by Uthayakumaran et al.[Citation16] who found that increasing the Glu:Gli ratio improves the dough strength. A correlation between dough strength and glutenin proteins has been previously reported by Primo-Martin et al.[Citation17] Barak et al.[Citation18] also reported that Gli:Glu ratio significantly affect the dough strength properties. Moreover, it can be suggested that flours with higher glutenin content produce highly stable doughs that perform well during bread making allowing the dough to rise during fermentation and improving the load volume. Softening of dough plays an important role in determining the machine ability of the dough. Doughs too soft make the dough handling difficult during the processing of dough for food product. Gliadins have been known to be responsible for the viscous nature of the dough. Similarly, in this study it was found that as the percentage of gliadins in the flour increased, the degree of softening increased significantly while the higher glutenin levels markedly decreased the degree of dough softening. The peak energy is the amount of energy required by the dough to reach the peak height. Peak energy values decreased with increment in the gliadin concentration in the flour. The gliadin fractions in combination with the base flour were ineffective in producing changes in the band width. However, the glutenin fraction mixed with HW 2004 base flour at higher percentage (6, 8, and 10 g/100 g) showed a significant increase in band width at peak.
Effect of Gliadin and Glutenin Addition on Pasting Characteristics
The rapid visco analyzer indicates starch viscosity by measuring the resistance of flour and water slurry to the stirring action of a paddle. The highest point during the heating cycle is the peak viscosity. The pasting behavior of the gluten fractions supplemented dough has been presented in . The highest peak viscosity (198.92 RVU) was shown by the base flour HW 2004. The peak viscosity decreased with the increase in percentage of gliadins and glutenins added. This decrease could be attributed to the fact that the peak viscosity reflects the resistance of the paste to the rotating paddle and is mainly attributed to the quantity and quality of the starch in a particular wheat variety. Moreover, it has been reported that higher content of starch in flours, to some extent, contributes to higher pasting viscosities.[Citation19] In the present study, a percentage of the flour was replaced with protein fraction (gliadin and glutenin). As a result, the peak viscosity showed a gradual decrease as the levels of the gliadin and glutenin in the flour increased. Also, peak viscosity indicates the maximum swelling of the starch granules which could be adversely affected by the presence of higher protein which competes for the water along with the starch granules. The lowest peak viscosity (140.25 RVU) was observed upon addition of 10 g/100 g gliadin fraction. Breakdown indicates the stability of the paste during cooking, with lower breakdown viscosity inferring better resistance to shear thinning of flour pastes. The base flour HW 2004 showed the highest value of breakdown viscosity (49.25 RVU). It was observed that upon supplementation of flour with gliadin and glutenin fractions the breakdown viscosity decreased indicating the increased resistance of the flour pastes to shear thinning. This could be due to interaction of the increased levels of gluten subfractions with the flour components. The increased protein levels seemed to provide some protection against the breakdown. Setback is the recovery of the viscosity during cooling of the heated flour suspension. The setback viscosity decreased with the increase in the percentage of gliadins and glutenins added to flour. Highest setback viscosity was reported by base flour HW 2004 (133.42 RVU) while lowest setback viscosity (105.83 RVU) was observed with 10 g/100 g addition of gliadins to the base flour. Final viscosity of the flour paste is dependent on the starch content, amylose, amylopectin, amylose/amylopectin ratio.[Citation20] Final viscosity of the flours increases due to the aggregation of the amylose molecules.[Citation21] The final viscosity decreased significantly from 283.08 RVU (base flour) to 213.58 RVU (10 g/100 g gliadin addition) upon replacing a particular percentage of flour with gliadin and glutenins. Pasting temperature indicates the minimum temperature required to cook as well as the temperature at which the viscosity increases during the heating process. The added fractions of gliadins and glutenin were ineffective in producing remarkable effect on the pasting temperature. The pasting time indicates the time at which the viscosity of the flour paste first begins to increase. Pasting time did not follow any specific pattern in the present study. The principal finding of the study was the varying effect of gliadin and glutenins on altering the pasting properties. Gliadins were more effective in decreasing the values of pasting properties than glutenins. The rapid visco analyzer measures the resistance of the flour and water slurry to the stirring action of a paddle. Gliadins are considered to have a compact globular structure as compared to glutenins, which have a β sheet structure and tends to form an entangled network upon hydration. So, when a part of the flour was replaced by the above proteins in the present study, the glutenins formed a network throughout the flour paste and provided greater resistance to the stirring blade than the gliadins and resulted in higher flour pasting properties value than those obtained for gliadins.
Table 2 Effect of gliadins and glutenins on pasting characteristics
TPA of Gliadin and Glutenin Added Dough
The textural characteristics of the dough prepared with and without the addition of gluten subfractions are presented in . Varying the percentage of gliadin and glutenin in the base flour changed the textural characteristics of dough significantly. The hardness increased significantly from 1482.3 g (base flour) to 2119.3 g (10 g/100 g glutenin addition). On the other hand the addition of gliadins significantly lowered the hardness value to as low as 1025.6 g (10 g/100 g gliadin addition). It could be due to the fact that addition of glutenin to the flour made it stronger and thus it required more force to compress it to 75% while addition of gliadins made the dough weaker lowering its hardness values. It is long accepted fact that gliadins make the dough viscous while glutenins make the dough elastic. Adhesiveness is termed as the ability of the dough to get separated from the probe. The addition of gliadin to the base flour at increasing levels increased the adhesiveness of the dough making it difficult for the dough to get separated from the testing probe. The adhesiveness increased remarkably from –998.3 gs (base flour) to –1702.3 gs (10 g/100 g gliadin addition). On the other hand, addition of glutenin to the flour decreased the adhesiveness of the dough and increased its elasticity property as a result of which the dough did not adhered to the probe. The adhesiveness decreased from –886.5 gs (2 g/100 g glutenin) to –625.4 gs (10 g/100 g glutenin). The gliadins are generally responsible for the cohesiveness and extensibility of the dough while the glutenins make the dough more rubbery and elastic.[Citation22] The cohesiveness of the dough increased with the increase in the concentration of gliadin proteins in the flour. The cohesiveness of base flour was reported to be 0.89 which increased to 0.98 upon the addition of 10 g/100 g gliadins. On the other hand, glutenins remarkably lowered the cohesiveness to 0.47 (10 g/100 g glutenin addition). Consistency is obtained from the combined areas under the two curves obtained from the TPA curve. Higher consistency values infer stronger dough. The higher gliadin levels in the dough decreased the consistency values while the presence of higher glutenin levels increased it.
Table 3 Texture profile analysis of gliadin and glutenin fortified dough
Gluten Recovery and Assessment of the Gluten Quality
To determine the role of added gliadins and glutenins on the gluten recovery, experiments were conducted on 4 g batches of HW 2004 base flour. Flour samples with and without the addition of 2 g/100 g, 4 g/100 g, 6 g/100 g, 8 g/100 g, and 10 g/100 g gliadins and glutenins were formed into dough in micro doughlab. The quantity of gluten recovered from the base flour and base flour fortified with different percentages of gliadin and glutenins are presented in . The addition of gliadins and glutenins increased the wet gluten recovered from the dough from 24.89% (base flour) to 49.48% (10 g/100 g gliadin addition) and 47.98% (10 g/100 g glutenin addition), respectively. The proportional increase in the amount of wet gluten recovered from the gliadin and glutenin supplemented flour demonstrate the involvement of these proteins in the formation of gluten network. However, the gluten recovery from the gliadin supplemented flours was greater than the glutenin supplemented flours because of the higher water binding capacity of gliadins as compared to glutenin. Base flour supplemented with 10 g/100 g gliadins showed the highest increase in percentage recovery of gluten (98.79%). The quality of the gluten recovered from the various proportions of flour was assessed by gluten index. The gluten index is calculated from the amount of gluten remaining on the sieve after centrifugation of the gluten. Greater amount of gluten on the sieve is directly proportional to better quality of the gluten. In the present study, it was found that the addition of gliadins to the flour greatly lowered the gluten index while the addition of glutenins improved it. The base flour had the gluten index of 56.80%. The supplementation of gliadins to the flour lowered the gluten index to as low as 2.76 (10 g/100 g addition), while the addition of glutenins increased the gluten index to 91.50 (10 g/100 g addition), respectively. The above results confirmed the role of glutenins in imparting strength to the dough and the higher levels of gliadins in a particular wheat variety resulting in weaker gluten quality and dough.
Table 4 Recovery of gluten and gluten index of gliadin and glutenin added doughs
Table 5 Correlations among dough rheological, pasting, and textural properties of dough
Correlations Among Various Dough Rheological, Pasting Characteristics, and Textural Quality of Dough
The results of the correlation analysis among various parameters have been presented in . The peak dough height obtained in the micro doughlab graph correlated positively with dough development time (0.958), dough stability (0.952), peak energy (0.951), and band width at peak (0.802). However peak dough height was weakly negatively associated with setback viscosity (–0.350). Moreover, dough softening and cohesiveness showed strong negative correlation with peak height. Dough development time was found to be negatively associated with dough softening, cohesiveness and setback viscosity. The peak energy was found to be positively correlated (0.681) with the band width at peak. Band width at peak was negatively associated with the pasting properties of the flour. However, it was positively associated with the dough hardness and cohesiveness. The peak viscosity was found to be positively correlated with breakdown, setback, and final viscosity and weakly negatively correlated with cohesiveness. Breakdown viscosity was positively correlated with all the textural properties of dough. Dough hardness was found to be positively correlated with gluten index indicating that flours with higher glutenin levels have higher dough hardness while dough adhesiveness and cohesiveness showed negative correlations, respectively. In the present study, the addition of gliadins to the base flour at increasing levels increased the adhesiveness and cohesiveness of the dough while lowering the gluten index. Gluten index was also found to be positively associated with the peak dough height (0.897), dough development time (0.964), and dough stability (0.744), respectively.
CONCLUSION
The results of the study revealed the influence of gluten subfractions on dough properties. The base flour fortified with different gliadins and glutenins concentrations showed different mixing characteristics. Gliadins decreased the dough development time and increased the softening of the dough. The peak dough height showed significant positive correlation with the dough hardness. Band width at peak was negatively associated with the pasting properties of the flour. Breakdown viscosity was positively correlated with all the textural properties of dough Peak energy was positively correlated to dough stability. Greater gluten was recovered from the gliadin and glutenin fortified flours. Gluten index was positively influenced by the presence of glutenin proteins. The results of the study clearly indicated the role of gliadins and glutenins on the flour properties. The findings of the study could help in improving the properties of the flour so as to enhance the end product quality.
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