Effects of laccase and xylanase on the chemical and rheological properties of oat and wheat doughs

The effects of Trametes hirsuta laccase and Pentopan Mono BG xylanase and their combination on oat, wheat, and mixed oat-wheat doughs and the corresponding breads were investigated. Laccase treatment decreased the content of water-extractable arabinoxylan (WEAX) in oat dough due to oxidative cross-linking of feruloylated arabinoxylans. Laccase treatment also increased the proportion of water-soluble polysaccharides (WSNSP) apparently due to the beta-glucanase side activity present in the laccase preparation. As a result of the laccase treatment, the firmness of fresh oat bread was increased. Xylanase treatment doubled the content of WEAX in oat dough and slightly increased the amount of WSNSP. Increased stiffness of the dough and firmness of the fresh bread were detected, probably because of the increased WEAX content, which decreased the amount of water available for beta-glucan. The combination of laccase and xylanase produced slight hydrolysis of beta-glucan by the beta-glucanase side activity of laccase and enhanced the availability of AX for xylanase with concomitant reduction of the amount and molar mass of WSNSP. Subsequently, the volume of oat bread was increased. Laccase treatment tightened wheat dough, probably due to cross-linking of WEAX to higher molecular weight. In oat-wheat dough, laccase slightly increased the proportion of WSNSP between medium to low molecular weight and increased the specific volume of the bread. Xylanase increased the contents of WEAX and WSNSP between medium to low molecular weight in oat-wheat dough, which increased the softness of the dough, as well as the specific volume and softness of the bread. The results thus indicate that a combination of laccase and xylanase was beneficial for the textures of both oat and oat-wheat breads.     

Use of Two Endoxylanases with Different Substrate Selectivity for Understanding Arabinoxylan Functionality in Wheat Flour Breadmaking

A Bacillus subtilis endoxylanase (XBS) with a strong selectivity for hydrolysis of water‐unextractable arabinoxylan (WU‐AX) and an Aspergillus aculeatus endoxylanase (XAA) with a strong selectivity for hydrolysis of water‐extractable arabinoxylan (WE‐AX) were used in straight‐dough breadmaking with two European wheat flours. Dough, fermented dough, and bread characteristics with different levels of enzyme addition were evaluated with a strong emphasis on the arabinoxylan (AX) population. The WU‐AX solubilized by XBS during breadmaking were mainly released during mixing and had higher molecular weight, in contrast to their counterparts solubilized by XAA, which were mainly released during fermentation and had lower molecular weight. This coincided with increased loaf volume with XBS and a negative to positive loaf volume response with XAA. Bread firmness and dough extract viscosity also were affected by endoxylanase addition. Results confirmed that WU‐AX are detrimental for breadmaking, while WE‐AX and solubilized AX with medium to high molecular weight have a positive impact on loaf volume.     

Impact of wheat flour-associated endoxylanases on arabinoxylan in dough after mixing and resting

The impact of varying levels of endoxylanase activity in wheat flour on arabinoxylan (AX) in mixed and rested dough was studied using eight industrially milled wheat flour fractions with varying endoxylanase activity levels. Analysis of the levels of reducing end xylose (RX) and solubilized AX (S-AX) formed during mixing and resting and their correlation with the endoxylanase activity in the flour milling fractions showed that solubilization of AX during the mixing phase is mainly due to mechanical forces, while solubilization of AX during resting is caused by endoxylanase activity. Moreover, solubilization of AX during the dough resting phase is more outspoken than that during the mixing phase. Besides endoxylanase activity, there were significant xylosidase and arabinofuranosidase activities during the dough resting phase. The results indicate that wheat flour-associated endoxylanases can alter part of the AX in dough, thereby changing their functionality in bread making and potentially affecting dough and end product properties.     

Structural Modifications of Gluten Proteins in Strong and Weak Wheat Dough During Mixing

The network‐forming attributes of gluten have been investigated for decades, but no study has comprehensively addressed the differences in gluten network evolution between strong and weak wheat types (hard and soft wheat). This study monitored changes in SDS protein extractability, SDS‐accessible thiols, protein surface hydrophobicity, molecular weight distribution, and secondary structural features of proteins during mixing to bring out the molecular determinants of protein network formation in hard and soft wheat dough. Soft wheat flour and dough exhibited greater protein extractability and more accessible thiols than hard wheat flour and dough. The addition of the thiol‐blocking agent N‐ethylmaleimide (NEM) resulted in similar results for protein extractability and accessible thiols in hard and soft wheat samples. Soft wheat dough had greater protein surface hydrophobicity than hard wheat and exhibited a larger decrease in surface hydrophobicity in the presence of NEM. Formation of high‐molecular‐weight (HMW) protein in soft wheat dough was primarily because of formation of disulfides among low‐molecular‐weight (LMW) proteins, as indicated by the absence of changes in protein distribution when NEM was present, whereas in hard wheat dough the LMW fraction formed disulfide interaction with the HMW fraction. Fourier transform infrared spectroscopy indicated formation of β‐sheets in dough from either wheat type at peak mixing torque. Formation of β‐sheets in soft wheat dough appears to be driven by hydrophobic interactions, whereas disulfide linkages stabilize secondary structure elements in hard wheat dough.     

Measurement of Dynamic Dough Density and Effect of Surfactants and Flour Type on Aeration During Mixing and Gas Retention During Proofing

A new method for measuring dough densities is presented, based on weighing small dough samples in air and immersed in xylene. The method can be used to evaluate the air content of low‐density doughs and to follow the changing density of a proofing dough sample. The method is applied to evaluate the effect of flour strength and surfactant addition on dough aeration and subsequent proofing. Doughs were mixed in a high‐speed mixer from two flours, a strong breadmaking flour and a weak flour. Surfactants sodium stearoyl lactylate (SSL) and diacetyl tartrate esters of monoglyceride (DATEM) were added at three levels, and the air content, proofing dynamics, and baked loaf quality were evaluated. The air content of dough was proportional to headspace pressure in the mixer, while the strong flour occluded less air than the weak flour. Surfactants greatly improved the volume of baked loaves but appeared to have no significant effect on air incorporation during mixing. The addition of surfactants appeared to increase the rate of growth of the dough piece during proofing, possibly due to increased bubble breakup during mixing or to increased rates of mass transfer of CO₂ into bubbles during proofing.     

Effect of l-Ascorbic Acid on the Rheological Properties of Wheat Flour-Water Dough

L-Ascorbic acid (AsA) and its related compounds play an important role as improvers in bread production. Addition of AsA and its related compounds, such as dehydro-L-AsA (DHA) and 2,3-diketo-L-gulonic acid (DKG), affected the rheological properties of flour-water dough during mixing, especially hardness. Addition of 10 or 100 ppm AsA increased the dough hardness of samples as compared with the control dough. Addition of DHA or DKG to dough only slightly increased hardness. Addition of p-quinone significantly increased the hardness. Both glutathione (GSH) and its oxidized form (GSSG) drastically decreased the hardness. Contents of AsA in the treated dough decreased and contents of DHA increased during mixing, suggesting that oxidation occurred. The oxidation rate of AsA was influenced by the concentration of AsA added. The improving effect of AsA on the rheological properties of flour-water dough seemed to be mostly dependent on reactive intermediate oxidation products such as O₂^-, while the contribution of DHA was rather limited.     

Optimized Methods for Incorporating Glutenin Subunits into Wheat Dough for Extension and Baking Studies

In order to study the functional properties of glutenin subunits added to a dough, they must be incorporated into the glutenin polymer. This requires partial reduction to open up the polymer, followed by oxidation to incorporate the added monomer into the polymer. Existing methods for incorporating glutenin subunits were suitable only for studies on mixing properties and needed to be modified for use in studies on extension and baking. A range of concentrations and of reaction times was therefore tested for both the reductant and the oxidant. In addition, mixing time as well as relaxation time before extension were varied. Extension curves and loaf heights were used to evaluate the treatments. Optimum conditions were developed that provided extension curves of normal dimensions but with altered shape. The conditions were reduction with 0.2 mg/mL of dithiothreitol (DTT) solution for 1 min followed by oxidation with 5 mg/mL of KIO₃ solution, then mixing the dough to 70% of the peak dough development time. For microbaking, the conditions of 2 mg/mL of DTT for 1 min, 2.5 mg/mL of KIO₃ for 5 min, and mixing the dough to peak development time allowed loaf height to be retained. The size distribution of the glutenin polymer was analyzed using size‐exclusion HPLC and field‐flow fractionation methods. This showed that the monomers were incorporated into the polymer and that polymer size was restored to control levels following reduction and oxidation.     

Wheat gluten phenolic acids: occurrence and fate upon mixing

Ferulic acid (FA, 4.9-17.7 microg/100 mg), sinapic acid (SA, 1.4-3.5 microg/100 mg), and traces of p-coumaric acid and vanillic acid were detected after saponification of six wheat glutens from industrial and pilot-scale origins. FA and SA occurred mostly as soluble-bound and insoluble-bound forms according to their extractability by acetone/methanol/water (7:7:6, v/v/v). The major part of FA (50-95%) was found in the unextractable fraction, whereas SA was mostly extractable (64-85%). The carbohydrate contents of the glutens were determined also after acid hydrolysis. The highest levels of glucose, arabinoxylan, and FA were obtained from the unextractable fractions of the pilot-scale extracted glutens, probably in relation with a lower efficiency of washing during extraction compared to industrial processes. On the other hand, SA compounds were in similar concentrations in all samples, suggesting their involvement in specific interactions during gluten protein agglomeration. Saponification of the soluble-bound phenolic acids released mainly glucose, whereas a beta-glucosidase treatment had no effect. FA and SA extractability, especially that of soluble-bound ones, decreased strongly in overmixed gluten/water doughs. These low molecular weight conjugates of phenolic acids could be involved in the dough breakdown phenomenon.     

Elucidating the mechanism of laccase and tyrosinase in wheat bread making

Cross-linking enzymes generate covalent bonds in and between food biopolymers. These enzymes are interesting tools for tailoring dough and bread structures, as the characteristics of the biopolymers significantly determine the viscoelastic and fracture properties of dough and bread. In this study, the influence of oxidative cross-linking enzymes, tyrosinase from the filamentous fungus Trichoderma reesei and laccase from the white rot fungus Trametes hirsuta, on dough and bread were examined. Oxidation of low molecular weight phenolic model compounds of flour, cross-linking of gluten proteins, dough rheology, and bread making were characterized during or after the enzymatic treatments. In the dough and bread experiments, laccase and tyrosinase were also studied in combination with xylanase. Of the model compounds tyrosine, p-coumaric acid, caffeic acid, ferulic acid, and Gly-Leu-Tyr tripeptide, tyrosinase oxidized all except ferulic acid. Laccase was able to oxidize each of the studied compounds. The phenolic acids were notably better substrates for laccase than l-tyrosine. When the ability of the enzymes to cross-link isolated gliadin and glutenin proteins was studied by the SDS-PAGE analysis, tyrosinase was found to cross-link the gliadin proteins effectively, whereas polymerization of the gliadins by laccase was observed only when a high enzyme dosage and prolonged incubation were used. Examination of large deformation rheology of dough showed that both laccase and tyrosinase made doughs harder and less extensible, and the effects increased as a function of the enzyme dosage. In bread making, interestingly, the pore size of the breads baked with tyrosinase turned out to be remarkably larger and more irregular when compared to that of the other breads. Nevertheless, both of the oxidative enzymes were found to soften the bread crumb and increase the volume of breads, and the best results were achieved in combination with xylanase.     

Effects of Mixing Conditions and Wheat Flour Dough Composition on Lipid Hydrolysis and Oxidation Levels in the Presence of Exogenous Lipase

The lipid profiles of wheat flour doughs containing exogenous lipase were studied under different mixing conditions using a microscale mixer. An experimental design comparing the effects of dough water content (52–68%), the speed of mixing (50–100 rpm), and the mixer temperature (18–32°C) showed that the hydrolysis levels were positively influenced by temperature and speed of mixing and negatively influenced by water content. The positive effect of temperature was enhanced both by highspeed mixing and low water content. The lipid oxidation levels were positively influenced by the speed of mixing and negatively influenced by the water content. The positive effect of temperature on the oxidation levels was less important. A series of experiments conducted with different types of industrial and semi-industrial mixers with equal concentrations of lipase added to the dough showed large differences among the rates of lipid hydrolysis and oxidation. However, the mixing conditions proposed by bakers to obtain doughs with similar handling properties led to similar dough lipid profiles. Sodium chloride did not change the lipid profile when added to dough. Conversely, calcium chloride promoted a large increase of lipid hydrolysis and oxidation due to its activation of lipase activity. Addition of yeast increased the lipid hydrolysis and slightly decreased lipid oxidation.     

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.     

In Situ Production of Prebiotic AXOS by Hyperthermophilic Xylanase B from Thermotoga maritima in High‐Quality Bread

In situ enrichment of bread with arabinoxylan‐oligosaccharides (AXOS) through enzymic degradation of wheat flour arabinoxylan (AX) by the hyperthermophilic xylanase B from Thermotoga maritima (rXTMB) was studied. The xylanolytic activity of rXTMB during breadmaking was essentially restricted to the baking phase. This prevented problems with dough processability and bread quality that generally are associated with thorough hydrolysis of the flour AX during dough mixing and fermentation. rXTMB action did not affect loaf volume. Bread with a dry matter AXOS content of 1.5% was obtained. Further increase in bread AXOS levels was achieved by combining rXTMB with xylanases from Pseudoalteromonas haloplanktis or Bacillus subtilis. Remarkably, such a combination synergistically increased the specific bread loaf volume. Assuming an average daily consumption of 180 g of fresh bread, the bread AXOS levels suffice to provide a substantial part of the AXOS intake leading to desired physiological effects in humans.     

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.     

Kinetics of enzyme-catalyzed cross-linking of feruloylated arabinan from sugar beet

Ferulic acid (FA) groups esterified to the arabinan side chains of pectic polysaccharides can be oxidatively cross-linked in vitro by horseradish peroxidase (HRP) catalysis in the presence of hydrogen peroxide (H(2)O(2)) to form ferulic acid dehydrodimers (diFAs). The present work investigated whether the kinetics of HRP catalyzed cross-linking of FA esterified to α-(1,5)-linked arabinans are affected by the length of the arabinan chains carrying the feruloyl substitutions. The kinetics of the HRP-catalyzed cross-linking of four sets of arabinan samples from sugar beet pulp, having different molecular weights and hence different degrees of polymerization, were monitored by the disappearance of FA absorbance at 316 nm. MALDI-TOF/TOF-MS analysis confirmed that the sugar beet arabinans were feruloyl-substituted, and HPLC analysis verified that the amounts of diFAs increased when FA levels decreased as a result of the enzymatic oxidation treatment with HRP and H(2)O(2). At equimolar levels of FA (0.0025-0.05 mM) in the arabinan samples, the initial rates of the HRP-catalyzed cross-linking of the longer chain arabinans were slower than those of the shorter chain arabinans. The lower initial rates may be the result of the slower movement of larger molecules coupled with steric phenomena, making the required initial reaction of two FAs on longer chain arabinans slower than on shorter arabinans.     

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.     

Generation of monochloropropanediols (MCPDs) in model dough systems. 1. Leavened doughs

The effect of dough recipe ingredients and processing on the generation of monochloropropanediol isomers (MCPDs) in leavened wheat doughs has been investigated. Commercial ingredients having no effect on MCPD formation were acetic acid and baking fats (triacylglycerols). Ingredients making a significant contribution to MCPD levels were yeast and flour improver [ascorbic acid, diacetyl tartaric acid esters of mono- and diglycerides (DATEM), and soya flour]. The results showed that free glycerol is a key precursor of MCPDs in leavened doughs. This glycerol is primarily generated by the yeast during proving but is also present in the flour, the yeast, and the improver. Under conditions of high dough moisture content (45%), MCPD formation was approximately proportional to glycerol concentration but showed a weaker dependence on chloride level, suggesting that the mechanisms of formation involved at least some reversible stages. MCPD generation increased with decreasing dough moisture to a point where the formation reaction was limited by chloride solubility and competing reactions involving glycerol and key precursor intermediates. These results could be predicted by a kinetic model derived from the experimental data. Glycerol was shown to account for 68% of MCPDs generated in proved full recipe dough.     

Refrigerated dough syruping in relation to the arabinoxylan population

Refrigerated doughs develop syruping upon prolonged storage. To assess the role of arabinoxylans (AX), in this phenomenon, the evolution of the AX population and syruping in refrigerated doughs during storage were studied. When doughs were kept at 6 degrees C for up to 34 days of storage, dough syruping increased from 0% (fresh dough) to 22% of dough weight, reaching a plateau after 16 days of storage. High-performance size exclusion chromatography and gas-liquid chromatography showed hydrolysis of water-unextractable AX in the refrigerated dough, resulting in increased levels of solubilized AX in the first 2 days of storage. Longer storage resulted in further degradation of solubilized and water-extractable AX. Increased syruping was accompanied by a decrease in farinograph dough consistency. The results support the hypothesis that loss of water-holding capacity due to degradation of AX by endogenous xylanases is responsible for dough syruping.     

Preparative Method for In Vitro Production of Functional Polymers from Glutenin Subunits of Wheat

An in vitro method for preparative‐scale production of artificial glutenin polymers utilizes a controlled environment for the oxidation of glutenin subunits (GS) isolated from wheat flour to achieve high polymerization efficiency. The functionality of in vitro polymers was tested in a 2‐g model dough system and was related to the treatment of the proteins before, during, and after in vitro polymerization. When added as the only polymeric component in a reconstituted model dough (built up from gliadin, water solubles, and starch fractions), in vitro polymers could mimic the behavior of native glutenin, demonstrating properties of dough development and breakdown. Manipulating the high molecular weight (HMW)‐GS to a low molecular weight (LMW)‐GS ratio altered the molecular weight distribution of in vitro polymers. In functional studies using the 2‐g mixograph, simple doughs built up from homopolymers of HMW‐GS were stronger than those using homopolymers of LMW‐GS. These differences may be accounted for, at least in part, by different polymer size distributions. The ability to control the size and composition of glutenin polymers shows the potential of this approach for investigating the effects of glutenin polymer size on dough function and flour end‐use quality.     

Depolymerization of the Glutenin Macropolymer During Mixing: II. Differences in Retention of Specific Glutenin Subunits

The depolymerization of individual high and low molecular weight (HMW and LMW, respectively) glutenin subunits (GS) from the glutenin macropolymer (GMP) in doughs during mixing was investigated by reversed-phase (RP) HPLC and SDS-PAGE. Cultivars with different dough strengths, as well as lines null for specific HMW-GS and biotypes differing at individual HMW-GS and LMW-GS encoding loci, were studied. During mixing, the proportion of total HMW-GS in GMP decreased, and the ratios of different subunits in the GMP in doughs changed. There was a loss of chromosome 1B- and 1D-encoded x-HMW-GS, while the relative proportions of y-HMW-GS (among HMW-GS) increased. Changes in 1B subunits occurred first, while most of the changes in 1D HMW-GS content occurred during dough breakdown. Changes were more pronounced for doughs of weak to average strengths than for stronger doughs. RP-HPLC analysis demonstrated a consistent increase in the retention times (surface hydrophobicity) of chromosome 1D-encoded HMW-GS but not of other HMW-GS or LMW-GS during mixing. SDS-PAGE and RP-HPLC demonstrated that specific B subunits, typically those with lower hydrophobicity, were selectively depolymerized from the GMP during dough breakdown, while the proportions of specific C subunits, typically those with greater hydrophobicity, increased. Similar trends were seen in analyses of several pairs of biotypes differing at single LMW-GS encoding loci, although there were slight differences in the depolymerization behavior of wheats with different allelic compositions. The results suggest that dough breakdown may be triggered by the loss of specific HMW-GS from the GMP, and a structural hierarchy may exist for different LMW-GS within glutenin in doughs.     

Refrigerated Dough Quality of Hard Red Spring Wheat: Effect of Genotype and Environment on Dough Syruping and Arabinoxylan Production

Arabinoxylans (AX) are the main nonstarch polysaccharides found in wheat flour. Structural changes of AX in refrigerated dough are linked to deleterious effects on refrigerated dough quality during storage. The purpose of this research was to evaluate the effect of cultivar and growing environment on dough syruping during refrigerated storage in relation to apparent xylanase activity and AX chemistry in hard red spring (HRS) wheat. Eight HRS cultivars that were grown at six locations over two years in North Dakota were evaluated for dough syruping during 15 days of refrigerated storage. When compared with genotypic effect, growing environment had a greater impact on apparent xylanase activity and dough syruping; they were found to have significant associations by log‐linear regression analysis. Specifically, wheat samples produced in a dry environment had lower apparent xylanase activity and degree of dough syruping than those from a wet environment. Some HRS cultivars were identified to be consistently lower in apparent xylanase activity and dough syruping across all growing environments, indicating that those cultivars had more stability over growing environment than other cultivars. These results indicate that certain cultivars that are grown in relatively dry environments in North Dakota are more suitable for use in refrigerated dough formulations.