Formation of Homopolymers and Heteropolymers Between Wheat Flour and Several Protein Sources by Transglutaminase‐Catalyzed Cross‐Linking

The effect of different protein sources (soy flour, lupin flour, egg albumin, gelatin powder, protein‐rich beer yeast flour) on wheat dough functionality was tested by determining gluten index, texture properties, and thermomechanical parameters. Transglutaminase (TG) was also added to improve the dough functionality by forming cross‐links. The presence of protein sources had a significant effect on the gluten index, with the exception of lupin flour. Gelatin and the presence of TG resulted in significant single effects on the texture properties of the wheat‐protein dough. All the protein sources significantly modified the mixing characteristics of the dough or the thermal behavior. Capillary electrophoresis studies of the water‐soluble, salt‐soluble, and glutenin proteins indicated that interactions were mainly within proteins, thus homologous polymers. Scanning electron microscopy studies of the doughs made from blends of wheat and protein sources doughs supported the formation of heterologous structures in the wheat‐lupin blends. The combination of TG and lupin would be a promising method to be used on the treatment of insect‐damaged or weak flours, to increase the gluten strength.     

Wheat Flour Proteins as Affected by Transglutaminase and Glucose Oxidase

Enzymes are good tool to modify wheat proteins by creating new bonds between the protein chains. In this study, the effect of the addition of glucose oxidase (GO) and transglutaminase (TG) on the wheat flour proteins is presented. The modification of wheat proteins was determined by analyzing the changes in gluten quality, alveograph parameters, and protein modifications. The amount of wet gluten increased with the addition of GO and TG, but the gluten quality was not improved in any case. Regarding the alveograph parameters, the effect of GO was readily evident obtaining wheat dough with higher tenacity and lower extensibility than the control, while TG led to doughs with lower tenacity and that were also less extensible. The protein modifications were characterized by free‐zone capillary electrophoresis (FZCE). FZCE data indicated that TG polymerizes mainly glutenins and, of those, the high molecular weight glutenin subunits were the most affected.     

Studies on Effects of Microbial Transglutaminase on Gluten Proteins of Wheat. I. Biochemical Analysis

The enzyme transglutaminase (TG) is known to have beneficial effects on breadmaking. However, only limited information is available on the structural changes of gluten proteins caused by TG treatment. The effect of TG has, therefore, been systematically studied by means of model peptides, suspensions of wheat flours and doughs. The treatment of synthetic peptides mimicking amino acid sequences of HMW subunits of glutenin with TG results in isopeptide bonds between glutamine and lysine residues. To study the effect on gluten proteins, different amounts of TG (0 to 900 mg enzyme protein per kg) were dissolved in a buffer and added to wheat flour. The flour suspensions were incubated and centrifuged and the residues were successively extracted with water, a salt solution, 60% aqueous ethanol (gliadin fraction) and SDS solution including a reducing agent (glutenin fraction). The characterization of the fractions by amino acid analysis, SDS‐PAGE, gel permeation HPLC and reversed‐phase HPLC has indicated that the quantity of extractable gliadins decreases by increasing TG amounts. Among gliadins, the ω5‐type was affected to the greatest extent by the reduction of extractability, followed by the ω1,2‐, α‐ and γ‐types. The oligomeric portion of the gliadin fractions (HMW gliadin) was strongly reduced when flour was treated with 450 and 900 mg TG per kg of flour, respectively. In the first instance, the quantity of the glutenin fractions increased by the treatment of flour with 90 and 450 mg TG per kg of flour, and significantly decreased by the treatment of flour with 900 mg TG per kg of flour. Parallel to an increase in TG concentration, the amounts of glutenin‐bound ω‐gliadins and HMW subunits were strongly reduced, whereas the LMW subunits reached a maximal amount after treatment with 450 mg TG per kg of flour. The insoluble residue was almost free of protein when flour was treated with lower amounts of TG. Higher amounts led to a great increase of protein in the residues. The effects of TG on doughs were similar to those of flour suspensions, but less strongly pronounced probably due to the lower water content of the dough system. Sequence analysis of peptides from a thermolytic digest of the insoluble residue revealed that HMW subunits of glutenin and α‐gliadins were predominantly involved in cross‐links formed by TG treatment.     

Rheology,Microstructure, and Baking Characteristics of Frozen Dough Containing Rhizopus chinensis Lipase and Transglutaminase

The beneficial effects of a new recombinant lipase (Rhizopus chinensis lipase [RCL]) and transglutaminase (TG) were investigated on frozen dough systems and their breadmaking quality. Rheological properties and microstructure of doughs were measured using a dynamic rheometer, rheofermentometer F3, and scanning electron microscopy (SEM). Measurements of viscoelastic properties showed that both G′ and G″ of dough containing RCL and TG were greater than those of the control after 35 days of frozen storage. The SEM micrographs showed that dough containing RCL and TG had the most starch granules embedded in or attached to the gluten network, and the gluten seemed more powerful and resilient than for the control dough after 35 days of frozen storage. Results of the gas production and dough development tests indicated that RCL and TG improved the rheofermentative characteristics of frozen dough. RCL and TG could improve water‐holding capacity and significantly increase the glycerol content of the control dough. Image analyses showed that bread crumbs containing RCL and TG had a more open network and uniform crumb structure, which resulted in higher specific volume. This combination also yielded a product with higher sensory scores for test breads.     

Effects of Ferulic Acid and Transglutaminase on Hard Wheat Flour Dough and Bread

The effects of ferulic acid and transglutaminase (TG) on the properties of wheat flour dough and bread were investigated. Ferulic acid and TG were blended with hard wheat flour at levels of 250 and 2,000 ppm of flour weight, respectively. The addition of ferulic acid reduced the mixing time and mixing tolerance. The addition of TG did not obviously affect the mixing properties. Significant effects of ferulic acid plus TG on the rested dough texture were observed for overmixed dough. The maximum resistance (Rmax) of the dough was significantly reduced with the addition of ferulic acid but increased with the addition of TG. The addition of TG with ferulic acid restored the Rmax reduced by ferulic acid alone. The proportion of SDS‐soluble high molecular weight proteins in the dough increased with the addition of ferulic acid and decreased with TG, when assessed with size‐exclusion HPLC fractionation. Although the addition of TG improved the handling properties of the dough made sticky with added ferulic acid, it did not improve the quality of the bread with added ferulic acid as measured by loaf volume and firmness.     

Effect of Physical State of Nonpolar Lipids on Rheology and Microstructure of Gluten‐Starch and Wheat Flour Doughs

To clarify the effects of solid fat and liquid oil on dough in more detail in a simpler system, gluten‐starch doughs with different gluten contents were investigated. The results from rheological measurements indicate that dough with a higher starch content has less resistance to strain and dough with a lower starch content has a rubber‐like structure. The effects of the physical state of nonpolar lipids such as fat and oil on gluten‐starch doughs and wheat flour doughs were investigated using rheological measurements and scanning electron microscopy. Fat‐containing dough had more gas cells and a very smooth gluten gel surface with few holes, which may provide higher tolerance to strain. Moreover, the fat seemed to uniformly distribute the gluten gel between the starch granules in the dough, which reduced the friction between starch granules and led to a lower storage modulus. A mechanism governing the effect of fats on loaf volume is proposed based on the phenomena observed in the fat‐containing dough.     

Evaluation of Various Baking Methods for Polished Wheat Flours

Flour qualities of polished wheat flours of three fractions, C‐1 (100–90%), C‐5 (60–50%), and C‐8 (30–0%), obtained from hard‐type wheat grain were used for the evaluation of four kinds of baking methods: optimized straight (OSM), long fermentation (LFM), sponge‐dough (SDM) and no‐time (NTM) methods. The dough stability of C‐5 in farinograph mixing was excellent and the maturity of polished flour doughs during storage in extensigraph was more improved than those of the commercial wheat flour (CW). There were no significant differences in the viscoelastic properties of CW dough after mixing, regardless of the baking method, while those of polished flour doughs were changed by the baking method; this tendency became clear after fermentation. The polished flours could make a better gluten structure in the dough samples after mixing or fermentation using LFM and SDM, as compared with other baking methods. Baking qualities such as specific volume and storage properties of breads from all polished flours made with SDM increased more than with other methods. In addition, viscoelastic properties of C‐5 and C‐8 doughs fermented by SDM were similar to those of CW, and the C‐5 breadcrumb showed softness similar to that of the CW. Also, SDM could make C‐5 bread with significantly higher elasticity and cohesiveness after storage for five days when compared with CW bread. Therefore, SDM with long fermentation, as compared with other baking methods, was considered suitable for use with polished flours to give better effects on dough properties during fermentation, resulting in more favorable bread qualities.     

Comparison of Small and Large Deformation Measurements of Whole Meal Rye Doughs

The rheological properties of rye flour-water-salt doughs prepared from different flour types (different falling number and coarseness) at different water levels were studied after mixing and after 90 min of incubation (30°C and 80% rh). Both the effect of water and the coarseness of the flour had significant effects on storage modulus (G′) measured by oscillatory test in the linear viscoelastic region and on compressional force measured at large deformation. The results of the two rheological methods correlated very well with each other (correlation coefficients varied in the different doughs at r = 0.975–0.999). Dough rheological measurements suggested that falling number did not have a statistically significant effect on dough rheology after mixing or incubation. Although the two rheological methods correlated well, the responses for incubation were different. In the small deformation method, the storage modulus of all doughs, independent of the falling number, decreased during incubation, whereas in the large deformation method, only the hardness of doughs made from flours with lower falling number decreased during incubation. The rheological measurements of doughs after mixing and the viscosity measurements of flourwater suspension at 30 and 40°C did not correlate with each other. Total pentosans have great effect on viscosity measurements of flour-water suspensions, whereas flour particle size and soluble pentosans correlated more with rheological properties of doughs (r = 0.851 between G′ and soluble pentosans).     

Relationships Between Gluten Rheological Properties and Hearth Loaf Characteristics

The rheological properties of fresh gluten in small amplitude oscillation in shear (SAOS) and creep recovery after short application of stress was related to the hearth breadbaking performance of wheat flours using the multivariate statistics partial least squares (PLS) regression. The picture was completed by dough mixing and extensional properties, flour protein size distribution determined by SE‐HPLC, and high molecular weight glutenin subunit (HMW‐GS) composition. The sample set comprised 20 wheat cultivars grown at two different levels of nitrogen fertilizer in one location. Flours yielding stiffer and more elastic glutens, with higher elastic and viscous moduli (G′ and G″) and lower tan δ values in SAOS, gave doughs that were better able to retain their shape during proving and baking, resulting in breads of high form ratios. Creep recovery measurements after short application of stress showed that glutens from flours of good breadmaking quality had high relative elastic recovery. The nitrogen fertilizer level affected the protein size distribution by an increase in monomeric proteins (gliadins), which gave glutens of higher tan δ and flatter bread loaves (lower form ratio).     

Microbial Transglutaminase as a Tool to Restore the Functionality of Gluten from Insect‐Damaged Wheat

In some wheat‐growing countries, considerable quantities of commercial wheat are rendered unusable in standard baking because of preharvest damage of the grain by protease‐injecting bugs. In the present study, we studied the ability of transglutaminase (TG) treatment of damaged wheat flour to return the functionality of the gluten network. To confirm the TG cross‐linking, the degree of protein hydrolysis, the amount of free thiol groups, and the electrophoresis properties of glutenin subunits were determined. The effectiveness of the TG treatment on insect‐damaged wheat was analyzed by measuring the dough mixing behavior and the gluten quality. A decrease in the degree of hydrolysis (or free amino groups), a reduction in thiol group concentration, and a decrease of extractable high molecular weight glutenin subunits (HMW‐GS) (measured by high‐performance capillary electrophoresis) confirmed the protein cross‐linking catalyzed by TG, the simultaneous formation of disulfide bonds by the proximity of the cross‐linked polypeptide chains, and the formation of aggregates of high molecular weight. The TG treatment of the damaged wheat flour led to a recovery of the consistograph parameters and gluten index value, and the covalent nature of the bonds ensured the stability of the protein changes.     

Characteristics of Perennial Wheatgrass (Thinopyrum intermedium) and Refined Wheat Flour Blends: Impact on Rheological Properties

Intermediate wheatgrass (IWG) (Thinopyrum intermedium) is a perennial grass with desirable agronomic traits and positive effects on the environment. It has high fiber and protein contents, which increase the interest in using IWG for human consumption. In this study, IWG flour was blended with refined wheat at four IWG‐to‐wheat ratios (0:100, 50:50, 75:25, and 100:0). Samples were analyzed for proximate composition, microstructure features, pasting properties (Micro Visco‐Amylo‐Graph device), protein solubility, and total and accessible thiols. Gluten aggregation properties (GlutoPeak tester) and mixing profile (Farinograph‐AT device) were also evaluated. IWG flour enrichment increased the pasting temperature and decreased the peak viscosity of blended flours. IWG proteins exhibited higher solubility than wheat, with a high amount of accessible and total thiols. The GlutoPeak tester highlighted the ability of IWG proteins to aggregate and generate torque. Higher IWG flour enrichment resulted in faster gluten aggregation with lower peak torque, suggesting weakening of wheat gluten strength. Finally, the addition of IWG to refined wheat flour resulted in a decrease in dough development time and an increase in consistency, likely because of the higher levels of fiber in IWG. The 50% IWG flour enrichment represents a good compromise between nutritional improvement and maintenance of the pasting properties, protein characteristics, and gluten aggregation kinetics.     

Wheat Intercultivar Differences in Susceptibility of Glutenin Protein to Effects of Bug (Eurygaster integriceps) Protease

Preharvest bug damage to wheat can cause significant losses in bread‐making quality. One of the most prevalent forms of bug damage which frequently occurs in most countries of the Middle East, Eastern Europe and North Africa can be attributed to Heteropterous insects, particularly Eurygaster spp. Intercultivar differences in the susceptibility of glutenin to proteolytic degradation by the bug Eurygaster integriceps were investigated using six breadwheat cultivars of Turkish origin. Crude enzyme extract was prepared with distilled water from bug‐damaged wheat. The freeze‐dried extract was blended with sound samples of ground wheat, and the mixture was incubated in distilled water for 30 and 60 min at 37°C and subsequently freeze‐dried. The proteolytic effects of bug damage were determined on large polymeric glutenin. The latter was measured as 50% 1‐propanol insoluble (50PI) glutenin extractable with 50% 1‐propanol in reductant dithiothreitol. The decreases in the amount of 50PI glutenin and the high and low molecular weight subunits were quantified using reversed‐phase HPLC. There was a substantial and progressive decrease in the quantity of 50PI glutenin and its subunits with increasing incubation time. Intercultivar differences were observed that were unrelated to intrinsic levels of proteolytic activity. After 60 min of incubation, the relative decrease in 50PI glutenin compared with control samples ranged from 43% (cv. Ankara) to 65 % (cv. Kirkpinar). Some cultivars (Lancer, Ankara and Gün) with similar levels of intrinsic proteolytic activity showed significantly different responses to bug protease. One cultivar (cv. Kirkpinar) with the lowest proteolytic activity was the most susceptible. High quality breadwheats (cvs. Bezostaya, Lancer, Kiraç and Gün) were generally more resistant to the bug protease, although Ankara, with both intermediate protease activity and breadmaking quality, was the most resistant cultivar. While the 50PI glutenin test was very effective in quantifying the damaging effects of bug protease on wheat protein quality, the nature of the intercultivar differences was unclear.     

Association of Glutenin Subunit Composition and Dough Rheological Characteristics with Cookie Baking Properties of Soft Wheat Cultivars

The effect of flour type and dough rheology on cookie development during baking was investigated using seven different soft winter wheat cultivars. Electrophoresis was used to determine the hydrolyzing effects of a commercial protease enzyme on gluten protein and to evaluate the relationships between protein composition and baking characteristics. The SDS‐PAGE technique differentiated flour cultivars based on the glutenin subunits pattern. Electrophoresis result showed that the protease degraded the glutenin subunits of flour gluten. Extensional viscosities of cookie dough at all three crosshead speeds were able to discriminate flour cultivar and correlated strongly and negatively to baking performance (P < 0.0001). The cookie doughs exhibited extensional strain hardening behavior and those values significantly correlated to baking characteristics. Of all rheological measurements calculated, dough consistency index exhibited the strongest correlation coefficient with baking parameters. The degradation effects of the protease enzyme resulted in more pronounced improvements on baking characteristics compared with dough rheological properties. Stepwise multiple regression showed that the dough consistency index, the presence or absence of the fourth (44 kDa) subunit in LMW‐GS and the fifth subunit (71 kDa) subunit in HMW‐GS were predominant parameters in predicting cookie baking properties.     

Baking Quality of Bread Wheat Cultivars Damaged by Nysius simulans

Wheat grain may be attacked by different insect species. Among them, some Heteroptera species (e.g., Aelia spp. and Eurygaster spp.) reduce wheat breadmaking quality; others, such as Nysius simulans , commonly extract water and nutrients from soy plants. The aim of this study was to assess the effect of N. simulans infestation on breadmaking quality of different bread wheat cultivars. Twelve wheat cultivars (damaged and undamaged by N. simulans ) were studied. Infested grain percentage varied between 51 and 78%, depending on cultivar. Protein and gluten quantity and quality were significantly reduced in damaged flours, as shown by gluten index, solvent retention capacity, and SDS sedimentation index. SDS‐PAGE from water‐extractable proteins evidenced an important proteolytic activity in damaged samples. Dough rheological properties showed a reduced dough viscoelasticity in damaged samples. Microbread specific volume changed from 3.26 cm³/g for samples made with undamaged flour to 2.77 cm³/g for bread made with damaged flour. No evidence for modification in starch properties was found. The infestation by N. simulans reduced wheat breadmaking quality in all cultivars studied, as a result of proteolytic activity occurring after dough hydration. Results suggest that the presence of N. simulans should be considered as a factor affecting wheat crops, mainly those located next to soy crop areas, which is the usual host for this insect.     

Effect of Physical States of Nonpolar Lipids on Rheology,Ultracentrifugation, and Microstructure of Wheat Flour Dough

In the previous study, we investigated effect of physical state of nonpolar lipids of gluten‐starch model dough. This experiment examined a real wheat flour dough system to assess the role of fat crystals in the breadmaking processes. These experiments were performed with a baking test and an investigation of wheat flour dough through rheological measurements (both large and small deformations), scanning electron microscopy, and ultracentrifugation. As a result, we found that the added oil was absorbed in the gluten structure, causing the aggregation of the gluten, which gave rise to more elastic behavior. In contrast, solid fat seemed to be distributed uniformly between the starch granules in the dough, reducing the friction between the starch granules and facilitating thin gluten gel layers. These properties lead to the lower G′ value and the increased viscous behavior, which yields an increase in loaf volume. In addition, the supposed mechanism behind the large loaf volume described in the previous study was that fat provides a uniform distribution of the dough components, and that the dough can thus expand easily, resulting in a larger loaf volume, which was supported in the wheat flour dough system. In conclusion, we found that thin, expandable gluten films and the uniform dispersion of gluten and starch granules in the dough are prerequisites for attaining better baking performance.     

Effect of Damaged Starch and NaCl Level on the Dough Handling Properties of a Canadian Western Red Spring Wheat

The effects of damaged starch and NaCl (1 and 2% w/w [flour weight]) on the dough handling properties of a wheat flour (Triticum asetivum L. ‘Roblin’) were investigated with rheological and textural methods. Damaged starch levels of the base flour and three remilled flours (using reduction rolls with decreasing gap sizes) were 5.42, 6.23, 7.30, and 8.43%. Rheological measurements on the dough showed that the complex modulus increased and the loss tangent (tan δ) decreased with increasing damaged starch levels in the flour, indicating that greater amounts of damaged starch produced stiffer dough. The base flour produced doughs with the highest creep compliance value (J _max), whereas the flour with the most damaged starch deformed the least. Higher levels of salt produced stiffer dough that deformed less, as evident by the higher complex modulus and lower creep compliance, compared with 1% NaCl. Damaged starch overall decreased dough stickiness (N), work of adhesion (N·s), and cohesiveness (mm). Increasing the salt content decreased the stickiness of the doughs. Increasing the damaged starch greatly increased dough extensibility at 1% NaCl. The greater amounts of damaged starch in the remilled flour mitigated some of the negative effects of reducing the salt content on the dough machinability.     

Biochemical Studies of Proteins in Nondeveloped,Partially Developed,and Developed Doughs

Nondeveloped, partially developed with shear and extensional deformations, and developed doughs represent different stages of dough development. To understand the relationship between gluten proteins and dough rheology, this study used disulfide‐sulfhydryl analyses, gel filtration chromatography, SDS‐PAGE, acid polyacrylamide gel electrophoresis (A‐PAGE), and densitometry to examine proteins in the four types of doughs mentioned. Free sulfhydryl content was the lowest in native flour and nondeveloped dough, and the highest in partially developed doughs, while a reverse trend was observed for disulfide content. For each flour sample, the protein elution profile from gel filtration chromatography shifted with the level of dough development. With respect to the smallest sized molecules, native flour had the most, followed by nondeveloped, partially developed, and then developed doughs. SDS‐PAGE and A‐PAGE exhibited similar protein patterns among the same chromatographed protein fractions of each native flour and its different doughs. Densitometric data showed that the amount of high molecular weight (HMW) glutenins increased and the amounts of low molecular weight (LMW) glutenins, gliadins, and albumins/globulins decreased with progressive stages of dough development. In conjunction with previously published results, indications are that the increase in the size and the amount of HMW glutenins is related to the strength of dough and the amount of protein matrix present in the dough.     

Effect of Mixing Time on Gluten Recovered by Ultracentrifugation Studied by Microscopy and Rheological Measurements

The effect of mixing time on gluten formation was studied for four commercial flour mixtures. The gluten phase was separated from dough using a nondestructive ultracentrifugation method. Small deformation dynamic rheological measurements and light and scanning electron microscopy were used. The recovered gluten was relatively pure with a small amount of starch granules embedded. The protein matrix observed by microscopy became smoother with prolonged mixing. No effect of overmixing was observed on the storage modulus (G′) of gluten for any of the flours. The amount of water in gluten increased from optimum to over‐mixing for most of the flours. Increased water content during prolonged mixing was not related to an effect on G′. The Standard flour resulted in the highest water content of gluten, which increased considerably with mixing time. The Strong flour had the lowest G′ of dough, a high G′ of gluten, and no increase in gluten water content from optimum to over‐mixing. The Durum flour did not show gluten development and breakdown similar to the other flours. The differences in gluten protein network formation during dough mixing are genetically determined and depend on the flour type.     

Potential of Triticale as a Substitute for Wheat in Flour Tortilla Production

The potential of triticale as a partial or total substitute for wheat in flour tortilla production was evaluated. Different mixtures of triticale and wheat flours were tested in a typical hot‐press formulation. Both grains yielded similar amounts of flour. Wheat flour contained 1.5% more crude protein, 1.6× more gluten, and produced stronger dough than triticale. Triticale flour significantly reduced optimum water absorption and mix time of blends. Flour tortillas with 100% triticale absorbed 8% less water and required 25% of the mix time of the control wheat flour tortilla. The yield of triticale tortillas was lower than the rest of the tortillas due to lower moisture content and water absorption. Triticale dough balls required less proofing and ruptured during hot pressing, thus producing defective tortillas. The 50:50 flour mixture produced doughs with acceptable rheological properties and good quality tortillas. Addition of 1% vital gluten to the 75:25 triticale‐wheat flour mix or 2% to the 100% triticale flour significantly increased water absorption and mix time and improved dough properties and tortilla yields. Textural studies indicated that increasing levels of triticale flour reduced the force required to rupture tortillas. For all tortilla systems, rupture force gradually increased, and extensibility decreased during seven days of storage at room temperature; the highest rate of change occurred during the first day. Sensory evaluation tests indicated that triticale could substitute for 50% of wheat flour without affecting texture, color, flavor, and overall acceptability of tortillas. For production of 100% triticale flour tortillas, at least 2% vital gluten had to be added to the formulation.     

Influence of Added Starch on Mixing of Dough Made with Three Wheat Flours Differing in High Molecular Weight Subunit Composition: Rheological Behavior

The effect of mixing time (6 and 20 min) and starch content were studied on doughs prepared with three wheat flours differing in high molecular weight subunit composition. Rheological measurements were performed in dynamic oscillation: frequency and strain sweeps, stress relaxation, and in large deformation viscosity measurements. The flours were diluted with starch to cover flour protein contents of 10–15%. Water was added to keep the starch‐water ratio constant when doughs were prepared with different protein contents. By increasing the starch content of the doughs, the rheological properties approached those of a starch‐water mixture prepared with the same starch‐water ratio as in the dough. The effect of the starch granules was reinforced by prolonged mixing. This may explain the higher values of the storage modulus and relaxation times observed after 20 min of mixing. Qualities related to gluten properties, appeared more clearly in large deformation viscosity measurements.