The Combination of Rhizopus chinensis Lipase and Transglutaminase Affects the Rheology and Glutenin Macropolymer Properties of Frozen Dough

The combination of Rhizopus chinensis lipase (RCL) and transglutaminase (TG) was previously reported to improve the quality of frozen dough bread. In this study, the effects of RCL, TG, and their combination on the modification of glutenin macropolymer (GMP) and rheological properties of dough during frozen storage were investigated. Frozen storage changed both GMP and rheology properties of dough. TG treatment significantly decreased the ratio of high‐molecular‐weight glutenin subunits to low‐molecular‐weight glutenin subunits and GMP content in fresh dough, and GMP particle size increased. The effect of RCL on GMP properties was not significant, but its combination with TG dramatically increased the proportion of the larger particles and weighted average volume (D_4.3) in GMP. The treatment with the enzyme combination could have inhibited the depolymerization of GMP, which slowed down the decrease rate of some parameters such as GMP content, proportion of larger particles, D_4.3, and release of free amino and thiol groups during frozen storage. The modification of GMP properties by enzyme treatment weakened the effect of the freezing process on rheological properties of dough, especially TG treatment and its combination with RCL. Correlation between GMP particle size and dough properties (dough tensile force and elastic modulus) after freezing and enzyme treatment were confirmed.     

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.     

Effects of Genotype and Environment on Glutenin Polymers and Breadmaking Quality

Six genotypes of hard red spring (HRS) wheat were grown at seven environments in North Dakota during 1998. Effects of genotype and environment on glutenin polymeric proteins and dough mixing and baking properties were examined. Genotype, environment, and genotype‐by‐environment interaction all significantly affected protein and dough mixing properties. However, different protein and quality measurements showed differences for relative influences of genotype and environment. Total flour protein content and SDS‐soluble glutenin content were influenced more by environmental than genetic factors, while SDS‐insoluble glutenin content was controlled more by genetic than environmental factors. Significant genotypic and environmental effects were found for the size distribution of SDS‐soluble glutenins and between SDS‐soluble and SDS‐insoluble glutenins as well as % SDS‐insoluble glutenins. With increased flour protein content, the proportions of monomeric proteins and SDS‐insoluble glutenin polymers appeared to increase, but SDS‐soluble glutenins decreased. Flour protein content and the size distribution between SDS‐soluble and SDS‐insoluble glutenin polymers were significantly correlated with dough mixing properties. Environment affected not only total flour protein content but also the content of different protein fractions and size distributions of glutenin polymers, which, in turn, influenced properties of dough mixing. Flour protein content, % SDS‐insoluble glutenin polymers in flour, and ratio of SDS‐soluble to SDS‐insoluble glutenins all were highly associated with dough mixing properties and loaf volume.     

Effects of Transglutaminase Enzyme on Fundamental Rheological Properties of Sound and Bug‐Damaged Wheat Flour Doughs

Transglutaminase (TG) catalyzes the formation of nondisulfide covalent crosslinks between peptide‐bound glutaminyl residues and ∊‐amino groups of lysine residues in proteins. Crosslinks among wheat gluten proteins by TG are of particular interest because of their high glutamine content. Depolymerization of wheat gluten proteins by proteolytic enzymes associated with bug damage causes rapid deterioration of dough properties and bread quality. The aim of the present study was to investigate the possibility of using TG to regain gluten strength adversely affected by wheat bug proteases. A heavily bug‐damaged (Eurygaster spp.) wheat flour was blended with sound cv. Augusta or cv. Sharpshooter flours. Dynamic rheological measurements, involving a frequency sweep at a fixed shear stress, were performed after 0, 30, and 60 min of incubation on doughs made from sound or blended flour samples. The complex moduli (G* values) of Augusta and Sharpshooter doughs blended with 10% bug‐damaged flour decreased significantly after 30 min of incubation. These dough samples were extremely soft and sticky and impossible to handle for testing purposes after 60 min of incubation. To test the possibility of using TG to counteract the hydrolyzing effect of bug proteases on gluten proteins, TG was added to the flour blends. The G* values of TG‐treated sound Augusta or Sharpshooter doughs increased significantly after 60 min of incubation. The G* values of the Augusta or Sharpshooter doughs blended with bug‐damaged flour increased significantly rather than decreased after 30 and 60 min of incubation when TG was included in the dough formulation. This indicates that the TG enzyme substantially rebuilds structure of dough hydrolyzed by wheat bug protease enzymes.     

Glutenin Macropolymer in Salted and Alkaline Noodle Doughs

An attempt was made to understand the physicochemical attributes that are the basis of physical differences between alkaline and salted noodle doughs. Flour and dough properties of one soft and three hard‐grained wheat cultivars were observed. Doughs were made with either sodium chloride or sodium carbonate. Each formulation variant was tested at both high and low water additions. Samples for glutenin macropolymer (GMP) isolation were taken at selected noodle dough processing stages. When a 1.67% w/v Na₂CO₃ solution was used for mixograph testing, dough characteristics were radically altered and differences between cultivars were masked. In lubricated squeezing flow (LSF) testing, hard wheat noodle doughs had significantly (P < 0.01) longer relaxation times and higher % residual force values than soft wheat doughs in both the salted and alkaline variants. LSF maximum force and biaxial viscosity were significantly higher in alkaline doughs than salted. GMP extracted from alkaline doughs was gummy and sticky, and was more opaque than GMP from salted doughs. GMP weight decreased sequentially when extracted from samples taken in the active phase (mix, compound, sheet) of noodle dough processing and decreased more in alkaline doughs. GMP weight increased more after 24 hr of dough rest in salted doughs. GMP gel strength was noticeably higher in GMP extracted from alkaline doughs. After dough resting, alkaline GMP gel strength significantly increased, whereas it decreased in GMP from salted doughs, suggesting a role for GMP in the increased stiffness of alkaline noodle doughs.     

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.     

Effect of pentosanase and oxidases on the characteristics of doughs and the glutenin macropolymer (GMP)

Rheological characteristics of dough and glutenin macropolymer (GMP) extracted thereof were investigated. Three single enzymes, pentosanase (PP), glucoseoxidase (GLZ), and laccase (LAC), and their combinations were used. GLZ gave the least extensible and most resistant dough, and pentosanase/glucoseoxidase (PPGLZ) resulted in dough with improved extensibility. The enzymes improved gluten quality. The glutenin macropolymer (GMP) was characterized in terms of wet weight, protein content, pentosan association, and dynamic rheological properties. Enzymatic addition decreased the wet weight of GMP but increased the protein content. PP decreased the content of pentosans on the GMP, but single oxidases increased the content of pentosans associated with GMP. PP did not modify the elastic modulus (G') of the GMP, whereas GLZ increased G' by increasing the polymerization of proteins and LAC diminished G'. The combination PPGLZ produced a synergic increase of G'.     

Properties of Arabinoxylans in Frozen Dough Enriched with Wheat Fiber

The global market for frozen bread dough is rising; however, its quality could deteriorate during extended storage. Our previous study indicated that undesirable changes caused by freezing could be reduced by adding arabinoxylan‐rich fiber sources. The present study investigated the changes in arabinoxylan properties of yeasted dough during frozen storage. Dough samples made from refined, whole, and fiber‐enriched (15% either wheat aleurone or bran) flours were stored at –18°C for nine weeks, and structural properties of arabinoxylan were probed during storage. Water‐extractable arabinoxylan (WEAX) content in dough samples increased by about 19–33% during the first three weeks of storage. Prolonged storage of dough (weeks 6 and 9), however, correlated with a decline in WEAX content. Average molecular weight and intrinsic viscosity of WEAX decreased during storage for all frozen dough samples. Arabinose‐to‐xylose ratios also decreased by 11 and 6% for control and composite dough samples, respectively. There was a significant positive correlation (r = 0.89, P < 0.0001) between WEAX content of dough and bread quality throughout the storage period. The results demonstrated that changes in dough quality during frozen storage were related to changes in the content and structure of WEAX that took place during frozen storage.     

Effect of freezing and frozen storage of doughs on bread quality

The effects of freezing and storage in frozen conditions on bread quality, crumb properties, and aggregative behavior of glutenins were analyzed. The effect of different additives on bread quality was also studied. The results obtained showed that freezing and storage at -18 degrees C decreased the bread quality. Samples stored in frozen conditions supplemented with diacetyl-tartaric acid ester of monoglycerides, gluten, and guar gum produced breads of greater volume and more open crumb structure than those prepared with the base formulation (without additives). All additives analyzed increased the proof time. Crumb firmness increased with dough frozen storage and bread aging time at 4 degrees C. A decrease in the amount of glutenin subunits of high molecular mass was observed by electrophoresis analysis of the SDS-soluble proteins aggregates extracted from the frozen dough. This result suggested that the protein matrix of bread underwent depolymerization during storage in frozen conditions.     

Effects of Micronization on Protein and Rheological Properties of Spring Wheat

This research investigated the effects of micronization, at different moisture levels, on the chemical and rheological properties of wheat. A set of tests designed to analyze protein fraction characteristics and rheological behaviors were conducted on samples from four wheat cultivars (AC Karma, AC Barrie, Glenlea, and Kanata). After being subjected to infrared radiation at three moisture levels (as‐is, 16%, and 22%), the seeds were milled to produce straight‐grade flour. The protein fractionation test revealed significant decreases (P ≤ 0.01) in both monomeric proteins (from 54% of total protein in the control to 37% in the tempered micronized sample) and soluble glutenins (9.4–2.5%). There was a strong negative correlation (r = ‐0.98) between the percentages of monomeric proteins and insoluble glutenins. Total extractable proteins of micronized samples tempered to 22% moisture decreased 43.5% when compared with nonmicronized control samples using size‐exclusion HPLC (SE‐HPLC). Micronization had a significant effect on gluten properties, as seen from a decrease in water absorption (P ≤ 0.01) and dough development time (P ≤ 0.01). Results showed that micronization at 100 ± 5°C had detrimental effects on wheat flour gluten functionality, including a decrease in protein solubility and impairment of rheological properties. These phenomena could be due to the formation of both hydrophobic and disulfide bonds in wheat during micronization.     

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.     

Effect of Nitrogen Fertilization on Quantity of Flour Protein Components,Dough Properties,and Breadmaking Quality of Wheat

Three winter wheat varieties with differing breadmaking quality were grown at two locations in two years at 0 or 3 × 60 kg of nitrogen application. The effect of nitrogen on amount of different components of gluten proteins was determined by reverse-phase HPLC. A high amount of nitrogen led generally to a significant increase of total protein content. However, this increase was obvious only for the gluten proteins; albumins and globulins remained nearly unaffected. The effect of increased protein content on gliadin to glutenin (gli-glu) ratio was inconsistent. While increased protein content increased the gli-glu ratio in the variety Capo, the opposite was true for the variety Renan. Gli-glu ratio of the variety Lindos showed no discernible tendency. As total protein content increased, the ratio of low molecular weight (LMW) to high molecular weight (HMW) glutenins decreased consistently, i.e., in all varieties, in both years and locations. Change of LMW to HMW ratio showed a significant negative correlation to sedimentation value and bread volume. There was no consistent change in the ratio between x- and y-type HMW subunits due to fertilization, as could be shown by densitometric measurements on SDS-PAGE gels. This ratio appeared to be dependent on the genotype and has decreased with decreasing quality. The amount of x-type subunits correlated closely with sedimentation value and bread volume. These results suggest that ratio of HMW glutenins, especially x-type subunits, to total protein content could be the best early detectable parameter with high predictive value for breadmaking quality.     

Mixtures of Two Argentinean Wheat Cultivars of Different Quality: A Study on Breadmaking Performance

Commercial Argentinean wheat flours are commonly composed of at least two wheat cultivars to obtain products of constant quality. Flours from two different wheats (Buck Pronto [BP] and Klein Escudo [KE]) with different protein profiles and breadmaking performance were assayed. Glu‐1 alleles are expressed in BP with 2, 7+8, 5+10 HMW glutenins subunits, while in KE 2, 7+9, 5+10 HMW glutenins subunits are present. Different sample blends (BP and KE) of 100:0, 75:25, 50:50, 25:75, and 0:100 were analyzed to relate composition of blends to the characteristics of dough and breads obtained from them. Physicochemical assays, alveograms, farinograms, and the SDS sedimentation test (SDSS test) were applied to characterize pure flours and blends. Rheological behavior of dough was studied by texture profile analysis (TPA) and dynamic assays. Scanning electron microscopy (SEM) was used to analyze dough microstructure. Alveographic W and farinographic stability values of blends with ≤50% KE were higher than expected. The same behavior was observed in SDSS‐test values, hardness (TPA), and rheometric assays. These results indicate that the performance of a cultivar like BP, rendering strong flours, would not be affected when a less strong flour is incorporated up to a certain proportion. However, bread quality of BP was superior to that of KE and BP‐KE blends. The decrease in bread quality was particularly evident in the increase in the shape ratio (W/H). Correlations could be established among some textural parameters of dough (hardness and elasticity) and certain quality attributes of bread (crumb and crust hardness).     

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 Glycerol and Sorbitol on the Properties of Dough and White Bread

Polyols could prolong shelf life and improve the quality of white bread. But the effect of high contents of polyols on dough properties and bread qualities is not yet clearly known. Thus, the properties of dough and white bread with different addition of polyols were evaluated by means of selected physicochemical properties. Rheology experiment results showed that both glycerol and sorbitol decreased the G′ and G″ of the dough. The results of thermogravimetric analysis revealed that polyols hindered the evaporation of water and that glycerol had a greater capacity for water retention than did sorbitol. In the bread, they caused more water to be absorbed on the surface of the gluten–starch system. They decreased the water activity and mass loss of the bread, but the specific volume of the bread also decreased. We found when glycerol and sorbitol addition was higher than 8%, it could slightly increase the viscidity of dough, enhance the moisture content of bread, and reduce the water activity of bread. But the gluten strength of dough decreased, and shaping and proofing of dough were difficult, which resulted in the deterioration the quality of white bread. We conclude that the addition of glycerol or sorbitol below 8% would be beneficial to the properties of dough and white bread and that sorbitol is a better option than glycerol.     

Depolymerization of the Glutenin Macropolymer During Dough Mixing: I. Changes in Levels,Molecular Weight Distribution,and Overall Composition

Changes in the amounts, molecular weight distributions, and levels of major groups of subunits in the glutenin macropolymer (GMP) of doughs during mixing were investigated. The GMP (gel protein) is the unreduced fraction of gluten protein that remains as a layer on top of the starch after extraction of SDS-soluble proteins and centrifugation. Experiments involved doughs prepared from flours derived from one weak and one strong cultivar and lines derived from cv. Olympic that were null for specific high molecular weight glutenin subunits (HMW-GS). During mixing, the amount of GMP decreased; the major changes occurred before peak mixing time (MT, achievement of peak resistance). In addition, the average apparent molecular weight of GMP (determined by both size-exclusion HPLC and multilayer gel electrophoresis) decreased during mixing, but in this case, the major changes were seen later in the mixing process, during dough breakdown. Even after extensive mixing, polymers and oligomers were released, not free glutenin subunits. During dough breakdown, the composition of GMP also changed, such that the proportion of HMW-GS decreased but β-amylases/D low molecular weight glutenin subunits (LMW-GS) increased. Changes in the total amounts of other LMW-GS typically were smaller with a decrease in the proportion of B subunits and an increase in the proportion of C subunits. The major changes in GMP composition were observed after peak MT (peak resistance) occurring earlier and to a greater extent in the weaker dough. Our results suggest that dough breakdown during mixing may be triggered by loss of HMW-GS, leading to changes in the molecular weight distribution and composition of the disulfide-bonded GMP.     

Relationship between endogenous protein disulfide isomerase family proteins and glutenin macropolymer

The effects of endogenous protein disulfide isomerase (PDI) family proteins on the properties of gluten proteins in dough during breadmaking were determined using bacitracin, an inhibitor of PDI. Bread loaf volume in the presence of bacitracin was increased to 118% of that in the absence of bacitracin. The addition of bacitracin caused a decrease in the extension tolerance of the dough. The amount of sodium dodecyl sulfate (SDS)-insoluble glutenin macropolymer (GMP) in dough decreased to approximately 70% of that in flour during the 20 min of mixing for doughmaking. The addition of bacitracin to dough caused a dramatic GMP decrease, corresponding to ～20-30% of that in flour during the 20 min of mixing. The decrease in GMP was compensated by an increase in SDS-soluble glutenin polymer. Taken together, these results suggest that the endogenous PDI family proteins in flour suppress the depolymerization of GMP during dough mixing.     

Effects of Temperature and Mechanical Input on Semisweet Biscuit (Cookie) Quality and Dough Characteristics

The effects of specific mechanical energy (SME) and dough temperature at the end of mixing (T_f) on semisweet biscuit dough characteristics and biscuit quality were studied using an experimental mixer fitted with monitoring devices. The fluid circulating in the double jacket of the mixing bowl was regulated at variable temperatures and mixed dough samples were prepared at T_f of 23, 30, and 37°C for three levels of SME input (20, 60 and 120 kJ/kg). Correlation analysis showed that semisweet biscuit length and thickness were independent quality parameters, influenced respectively by the T_f of dough and SME. Biscuit thickness and volume increased with SME input, but SME had no significant influence on the physicochemical characteristics of the dough. Biscuit length was related to the density and stickiness of the dough and to rheological behavior as assessed by fundamental and empirical measurements. A rise in dough temperature >35°C induced a dramatic increase in viscoelastic properties, leading to biscuit shrinkage. The increase of dough density with T_f seemed to be related to the melting of solid fat in the dough recipe. Melting of fat during mixing could also be a source of viscoelastic changes in the dough at T_f.     

Correlation of Quality Parameters with the Baking Performance of Wheat Flours

The baking performance of a set of flours from 13 wheat cultivars was determined by means of two different microscale baking tests (10 g of flour each). In the micro‐rapid‐mix test the dough was mixed for a fixed time at a high speed, whereas the microbaking test used mixing to optimum dough consistency in a microfarinograph. Quality parameters such as sedimentation value, crude protein content, dough and gluten extension data, and microfarinograph data were also determined. Finally, quality‐related protein fractions (gliadins, glutenins, SDS‐soluble proteins, and glutenin macropolymer) were quantitated by extraction/HPLC methods with reversed‐phase and gel‐permeation columns. All quality parameters were correlated with the bread volumes of both baking tests. The results demonstrated that the microbaking test (adapted mixing time) was much more closely related to the quality parameters than the micro‐rapid‐mix test (fixed mixing time), which hardly showed any correlation. Among the standard quality parameters, only the crude protein content showed a medium correlation with the bread volume of the microbaking test (r = 0.71), whereas the contents of gliadins (r = 0.80), glutenins (r = 0.76), and glutenin macropolymer (r = 0.80) appeared to be suitable parameters to predict the baking performance of wheat flour. All other quality parameters were not or were only weakly correlated and unsuitable for predicting baking performance.     

Contribution of Hydrophobic Soluble Gluten Proteins,Fractionated by Hydrophobic Interaction Chromatography in Highly Acetylated Agarose,to Dough Rheological Properties

Hydrophobic interaction chromatography with highly acetylated agarose in 1‐mL columns was used to fractionate gliadins and acid‐soluble glutenins. Proteins were eluted in two fractions, the first with acetate buffer (pH 3.6) containing 35% propanol, and the second with Tris buffer in 8M urea. The proportion of eluted protein in the second fraction was called the surface hydrophobicity index. The study included 20 wheat samples of different baking qualities. Multiple regression analysis using the general linear model combined with the stepwise technique was used to relate the surface hydrophobicity index of soluble gluten proteins to specific dough rheological characteristics. Surface hydrophobicity index of gliadins and acetic acid soluble glutenins explained part of the variability of swelling index, extensibility, and work of deformation (dough strength) measured with the alveograph, and part of the farinograph water absorption variability, but showed no relationship to dough mixing characteristics. Hydrophobic soluble gluten proteins fractionated by hydrophobic interaction chromatography (HIC) explained a part of the variability of dough rheological properties.