Modifications of wheat flour proteins during in vitro digestion of bread dough, crumb, and crust: an electrophoretic and immunological study

The proteins of wheat flour have several biological activities that can affect human health and physiology when wheat-based foods are consumed. The modifications of bread crumb and crust proteins during an in vitro peptic/pancreatic digestion process were studied by electrophoresis and immunoblotting with polyclonal antibodies specific for single proteins or groups of homologous proteins of the wheat flour, and the results were compared to those obtained for an unheated dough sample. The results show that baking affects the extent of proteolysis and the immunological and physicochemical features of the digestion products in relation to the level of the heat treatment. Therefore, the results concerning the digestion of the unheated wheat flour or dough are not representative of what happens when baked products enter the human digestive tract.     

Localization of puroindoline-a and lipids in bread dough using confocal scanning laser microscopy

Puroindolines are lipid-binding proteins from wheat flour that play a significant role in bread crumb texture. The localization of wheat flour lipids and puroindoline-a (PIN-a) in bread dough was studied by confocal scanning laser microscopy (CSLM). Wheat lipids were located around gas cells (GC) and embedded within the protein-starch matrix (SPM) of the dough. PIN-a was mainly located in the matrix of dough, where it was associated with lipids. In contrast, in defatted dough, PIN-a was found around GC. Addition of puroindolines in bread dough induced a defatting of the gas bubble surface and a decrease of the lipid vesicles and/or droplet size embedded within the SPM. Therefore, puroindolines control the lipid partitioning within the different phases of dough, a phenomenon that should have important consequence on the gas bubble expansion and GC formation in the further stages (fermentation, baking) of the bread-making process.     

不同种类大豆蛋白粉对面包加工特性的影响

为探索大豆蛋白作为营养补充剂在面包中应用时,对面团物理特性和焙烤特性产生的影响,该文考察了不同种类的大豆蛋白制品,包括大豆分离蛋白、灭酶全脂粉、活性全脂粉、活性脱脂粉、灭酶脱脂粉对面团粉质特性、拉伸特性和焙烤特性的影响。结果表明,面粉的吸水率与大豆蛋白粉氮溶解指数显著相关,面团的抗拉阻力受大豆蛋白添加量的影响明显。大豆蛋白粉的加入,对面包比体积产生不利影响,下降趋势与大豆蛋白粉对面团拉伸特性的影响显著相关。大豆蛋白粉有软化面包质地的作用,活性全脂粉表现最为明显。大豆蛋白粉的加入量占面粉质量分数的3%时,对面包口感影响不明显,当加入量超过面粉质量分数的7％时,容易出现发粘和豆腥味等现象。     

Structural and functional characterization of pronyl-lysine,a novel protein modification in bread crust melanoidins showing in vitro antioxidative and phase I/II enzyme modulating activity

Application of an in vitro antioxidant assay to solvent fractions isolated from bread crust, bread crumb, and flour, respectively, revealed the highest antioxidative potential for the dark brown, ethanol solubles of the crust, whereas corresponding crumb and flour fractions showed only minor activities. To investigate whether these browning products may also act as antioxidants in biological systems, their modulating activity on detoxification enzymes was investigated as a functional parameter in intestinal Caco-2 cells. The bread crust and, in particular, the intensely brown, ethanolic crust fraction induced a significantly elevated glutathione S-transferase (GST) activity and a decreased phase I NADPH-cytochrome c reductase (CCR) activity compared to crumb-exposed cells. Antioxidant screening of Maillard-type model mixtures, followed by structure determination, revealed the pyrrolinone reductones 1 and 2 as the key antioxidants formed from the hexose-derived acetylformoin and N(alpha)-acetyl-L-lysine methyl ester or glycine methyl ester, chosen as model substances to mimic nonenzymatic browning reactions with the lysine side chain or the N terminus of proteins, respectively. Quantitation of protein-bound pyrrolinone reductonyl-lysine, abbreviated pronyl-lysine, revealed high amounts in the bread crust (62.2 mg/kg), low amounts in the crumb (8.0 mg/kg), and the absence of this compound in untreated flour. Exposing Caco-2 cells for 48 h to either synthetically pronylated albumin or purified pronyl-glycine (3) significantly increased phase II GST activity by 12 or 34%, respectively, thus demonstrating for the first time that "pronylated" proteins as part of bread crust melanoidins act as monofunctional inducers of GST, serving as a functional parameter of an antioxidant, chemopreventive activity in vitro.     

Differential recovery of lupin proteins from the gluten matrix in lupin-wheat bread as revealed by mass spectrometry and two-dimensional electrophoresis

Bread made from a mixture of wheat and lupin flour possesses a number of health benefits. The addition of lupin flour to wheat flour during breadmaking has major effects on bread properties. The present study investigated the lupin and wheat flour protein interactions during the breadmaking process including dough formation and baking by using proteomics research technologies including MS/MS to identify the proteins. Results revealed that qualitatively most proteins from both lupin and wheat flour remained unchanged after baking as per electrophoretic behavior, whereas some were incorporated into the bread gluten matrix and became unextractable. Most of the lupin α-conglutins could be readily extracted from the lupin-wheat bread even at low salt and nonreducing/nondenaturing extraction conditions. In contrast, most of the β-conglutins lost extractability, suggesting that they were trapped in the bread gluten matrix. The higher thermal stability of α-conglutins compared to β-conglutins is speculated to account for this difference.     

Fermented Sorghum as a Functional Ingredient in Composite Breads

Whole sorghum flour was fermented (a five‐day natural lactic acid fermentation) and dried under forced draught at 60°C, and evaluated for its effect on sorghum and wheat composite bread quality. In comparison with unfermented sorghum flour, fermentation decreased the flour pH from 6.2 to 3.4, decreased total starch and water‐soluble proteins, and increased enzyme‐susceptible starch, total protein, and the in vitro protein digestibility (IVPD). Fermentation and drying did not decrease the pasting temperature of sorghum flour, but slightly increased its peak and final viscosity. In comparison with composite bread dough containing unfermented sorghum flour, fermented and dried sorghum flour decreased the pH of the dough from 5.8 to 4.9, increased bread volume by ≈4%, improved crumb structure, and slightly decreased crumb firmness. IVPD of the composite bread was also improved. Mixing wet fermented sorghum flour directly with wheat flour (sourdough‐type process) further increased loaf volume and weight and reduced crumb firmness, and simplified the breadmaking process. It appears that the low pH of fermented sorghum flour inactivated amylases and increased the viscosity of sorghum flour, thus improving the gas‐holding capacity of sorghum and wheat composite dough. Fermentation of sorghum flour, particularly in a sourdough breadmaking process, appears to have considerable potential for increasing sorghum utilization in bread.     

Baking Performance of Durum and Soft Wheat Flour in a Sponge-Dough Breadmaking Procedure

Breadmaking properties were determined for formulations that included durum, soft, and spring wheat flour, using a pound-loaf sponge-dough baking procedure. Up to 60% durum or soft wheat flour plus 10% spring wheat flour could be incorporated at the sponge stage for optimum dough-handling properties. At remix, the dough stage required 30% spring wheat flour. Bread made with 100% spring wheat flour was used as a standard for comparison. Bread made with 60% durum flour exhibited internal crumb color that was slightly yellow. When storing pound bread loaves for 72 hr, crumb moisture content remained unchanged. Crumb firmness and enthalpy increased the most in bread made with 60% soft wheat flour. Crumb firmness increased the least in bread made with 100% spring wheat flour. Enthalpy changed the least in bread made with 60% durum flour. Crumb moisture content was significantly correlated with crumb firmness (r = -0.82) and enthalpy (r = -0.65). However, crumb moisture content was specific for each type of flour and a function of flour water absorption; therefore, these correlations should be interpreted with caution. Crumb firmness and enthalpy were significantly correlated (r = 0.65). Ball-milling flour resulted in an increase in water absorption of ≈2% and in crumb moisture content of ≈0.5% but had no effect on either crumb firmness or enthalpy.     

Effects of Flour Strength,Baking Absorption,and Processing Conditions on the Structure and Mechanical Properties of Bread Crumb

The objective of this study was to determine the effects of flour type, baking absorption, variation in sheeting, and dough proofing time on the density, crumb grain (visual texture), and mechanical properties (physical texture) of bread crumb. All response variables were measured on the same bread crumb specimens. Bread loaves were prepared by a short‐time bread‐making process using four spring wheat flours of varying strength. After crumb density measurement, digital image analysis (DIA) was used to determine crumb grain properties including crumb brightness, cell size, cell wall thickness, and crumb uniformity. Tensile tests were performed on bone‐shaped specimens cut from the same bread slices used for DIA to obtain values for Young's modulus, fracture stress, fracture strain, and fracture energy. Proof time had the most profound influence on the bread with substantial effects on loaf volume, crumb density, crumb brightness, and grain, as well as crumb mechanical properties. Increasing proof time resulted in higher loaf volume, lower crumb density and brightness, coarser crumb with fewer and larger cells with thicker cell walls, and weaker crumb tensile properties. Varying flour type also led to significant differences in most of the measured crumb parameters that appeared to correspond to differences in gluten strength among the flour samples. With increasing flour strength, there was a clear trend to increasing loaf volume, finer and more uniform crumb grain, and stronger and more extensible bread crumb. Increasing baking absorption had virtually no effect on crumb structure but significantly weakened crumb strength and increased fracture strain. In contrast, varying the number of sheeting passes had a minor effect on crumb cellular structure but no effect on mechanical properties. The experimental data were consistent with a cause‐effect relationship between flour strength and the tensile strength of bread crumb arising as a result of stronger flours exhibiting greater resistance to gas cell coalescence, thereby having fewer crumb defects.     

Effects of asparagine, fructose, and baking conditions on acrylamide content in yeast-leavened wheat bread

A repeatable procedure for studying the effects of internal and external factors on acrylamide content in yeast-leavened wheat bread has been developed. The dough contained wheat endosperm flour with a low content of precursors for acrylamide formation (asparagine and reducing sugars), dry yeast, salt, and water. The effects of asparagine and fructose, added to the dough, were studied in an experiment with a full factorial design. More than 99% of the acrylamide was found in the crust. Added asparagine dramatically increased the content of acrylamide in crusts dry matter (from about 80 microg/kg to between 600 and 6000 microg/kg) while added fructose did not influence the content. The effects of temperature and time of baking were studied in another experiment using a circumscribed central composite design. Mainly temperature (above 200 degrees C) but also time increased the acrylamide content in crust dry matter (from below 10 to 1900 microg/kg), and a significant interaction was found between these two factors. When baked at different conditions with the same ingredients, a highly significant relationship (P < 0.001) between color and acrylamide content in crust was found. Added asparagine, however, did not increase color, showing that mainly other amino compounds are involved in the browning reactions.     

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.     

Effect of cyclodextrin glycosyl transferase [corrected] on dough rheology and bread quality from rice flour

Gluten-free breads are usually characterized by deficient quality characteristics as compared to wheat breads. Problems related to volume and crumb texture are associated with gluten-free breads even when rice flour is used, which seems to be the best raw material for this type of bread. The potential use of cyclodextrin glycosyl transferase (CGTase) as a rice bread improver is presented. The effect of CGTase addition to rice flour on dough rheology and bread quality was investigated. In addition, an experimental design was developed to optimize the levels of CGTase, hydroxypropylmethylcellulose (HPMC), and oil. The addition of CGTase produced a reduction in the dough consistency and also in the elastic modulus. With regard to the rice bread quality, better specific volume, shape index, and crumb texture were obtained. The amount of cyclodextrins in the bread crumb was quantified to explain the action of this enzyme. The data indicate that the improving effect of the CGTase results from a combination of its hydrolyzing and cyclizing activities, the latter being responsible for the release of cyclodextrins, which have the ability to form complexes with lipids and proteins.     

Influence of baking conditions and precursor supplementation on the amounts of the antioxidant pronyl-L-lysine in bakery products

The influence of baking conditions and dough supplements on the amounts of the antioxidant and Phase II-Enzyme modulating, protein-bound 2,4-dihydroxy-2,5-dimethyl-1-(5-acetamino-5-methoxycarbonyl-pentyl)-3-oxo-2H-pyrrol (pronyl-L-lysine) in bakery products was investigated in quantitative studies. These studies revealed high amounts of the antioxidant in bread crust, only low amounts in the crumb, and the absence of this compound in untreated flour. The amounts of pronyl-L-lysine were found to be strongly influenced by the intensity of the thermal treatment. For example, increasing the baking time from 70 to 210 min or increasing the baking temperature from 220 to 260 degrees C led to a 5- or 3-fold increase in the concentrations of this antioxidant in the crust, respectively. In addition, modifications in the recipe showed to have a major impact on pronyl-L-lysine formation. For example, substituting 5% of the flour with the lysine-rich protein casein or with 10% of glucose increased the amounts of the antioxidant by more than 200%. Quantitative analyses of commercial bread samples collected from German bakeries revealed the highest amount of 43 mg/kg for a full grain bread, followed by a rye/wheat bread, both of which have been sourdough fermented. A mixed-grain bread as well as pale wheat bread, both prepared without sourdough fermentation, contained significantly lower amounts of pronyl-L-lysine, and German pretzels, which are treated with a dilute sodium hydroxide solution prior to baking, contained only trace amounts of pronyl-L-lysine (e.g., less than 5 mg/kg were detectable in pretzels). Systematic studies revealed that the decrease of the pH value induced by microbial acid formation during sourdough fermentation is the clue for producing high amounts of pronyl-L-lysine in baking products. These data clearly demonstrate for the first time that the amounts of the antioxidant and chemopreventive compound pronyl-L-lysine in bakery products is strongly dependent on the manufacturing conditions as well as the recipe.     

Semicarbazide formation in flour and bread

Azodicarbonamide, an approved food additive, is commonly used as a flour additive and dough conditioner in the United States and Canada. A number of researchers have clearly established a link between the use of azodicarbonamide and semicarbazide contamination in commercial bread products. However, all of these studies have primarily focused on the final baked product and have not extensively investigated the processing and conditions that affect the final semicarbazide levels. In this study, a previously developed method for measuring free semicarbazide in bread was applied to dough samples during the mixing and kneading process. Additionally, flour and bread samples were spiked with biurea or azodicarbonamide to help elucidate semicarbazide formation pathways. The results showed that semicarbazide was not formed as a byproduct of azodicarbonamide decomposition to biurea, which occurs upon the addition of water. Indeed, semicarbazide was not detected after room temperature or elevated temperature dough maturation, but only after baking. It was concluded that although azodicarbonamide is the initial starting material, semicarbazide formation in bread occurs through a stable intermediate, biurea.     

Phytochemical Profile and Antiproliferative Activity of Dough and Bread Fractions Made from Refined and Whole Wheat Flours

Phytochemical profile (phenolic acids, carotenoids, and tocopherols) and antiproliferative properties of bread processing fractions, including the dough, crumb, and upper crust made from refined wheat and whole wheat flours were analyzed for two wheat cultivars. Ferulic acid, lutein, and α‐tocopherol were the predominant phenolic acid, carotenoid, and tocopherol, respectively, extracted from all fractions. The levels of all phytochemicals in whole wheat samples were over eightfold higher than their corresponding refined wheat samples. The concentrations of total phenolic acids (soluble and insoluble bound) were higher in the upper crust of refined (∼60–90%) and whole wheat (∼15–40%) breads than their corresponding dough fractions. However, the dough of whole wheat had higher levels of tocopherols and carotenoids compared with the crumb and upper crust, suggesting that phenolic acids were relatively stable during baking, whereas tocopherols (∼25–80%) and carotenoids (∼20–80%), were partially degraded. The antiproliferative activity of whole wheat bread extracts against HT‐29 cancer cells was weakly correlated with total phenolic acids but showed no correlations with total carotenoid and total tocopherol contents.     

Effect of bread baking on the bioavailability of hydrogen-reduced iron powder added to unenriched refined wheat flour

Elemental iron powders are widely used to fortify flour and other cereal products. Our objective was to test the hypothesis that baking enhances the bioavailability of elemental iron powders by oxidizing Fe(0) to Fe(2+) or Fe(3+). An in vitro digestion/Caco-2 cell culture model and a piglet model were used to measure bioavailability. Bread flour, either unfortified or fortified with hydrogen-reduced (HR) iron powder or FeSO(4) (300 mg Fe/kg flour), was baked into bread. For the in vitro studies, bread samples were treated with pepsin at pH 2, 3, 4, 5, 6, or 7 and subsequently incubated with pancreatic enzymes at pH 7 in a chamber positioned above monolayers of cultured Caco-2 cells. Ferritin formation in the cells was used as an index of iron bioavailability. Ferritin formation in cells fed HR Fe bread was similar to cells fed FeSO(4) bread when the peptic digestion was conducted at a pH 2 but lower when the peptic phase was conducted at pH 3, 4, 5, 6, or 7 (P < 0.05). Pig diets containing 35% dried bread were prepared and fed to cross-bred (Hampshire x Landrace x Yorkshire) anemic pigs in two studies. The rate of increase in hemoglobin Fe over the feeding period was used to calculate relative biological value (RBV), an index of iron bioavailability. In the first pig study, RBV of HR Fe added to flour prior to baking was 47.9% when compared to FeSO(4) fortified flour (P < 0.05). In the second pig study, a third treatment consisting of unfortified bread with HR iron added during diet mixing (after bread baking) was included. RBVs of the HR Fe diet (Fe added after baking) and HR Fe diet (Fe added before baking) were 40.1% and 53.5%, respectively, compared to the FeSO(4) diet. Differences in RBV between the HR Fe (before and after baking) and FeSO(4) (before baking) treatment groups were significant, but the difference between the before and after HR treatment groups was not significant. We conclude that bread baking does not enhance the bioavailability of elemental iron powders.     

Pyranose Oxidase from Trametes multicolor Impacts Dough and Bread Microstructure

Pyranose oxidase (P₂O) improves wheat flour dough stability and bread quality. We related its effect on dough spread behavior to that on dough and bread crumb structure. Increasing P₂O addition levels gradually reduced dough flow. High P₂O addition levels further increased dough strength, significantly increased dough cell wall thickness, and decreased bread loaf volume. Taken together, affecting dough spread behavior impacts dough and bread (crumb) structure, and dough structure largely determines bread crumb structure.     

Reaction kinetics of gliadin-glutenin cross-linking in model systems and in bread making

The gluten proteins gliadin and glutenin are important for wheat flour functionality in bread making, where, during baking, they polymerize through a heat-induced sulfhydryl-disulfide exchange mechanism. A model system was used to study the kinetics of this reaction. Thus, gluten was subjected to hydrothermal treatment with the rapid visco analyzer (RVA) with holding temperatures of 80, 90, and 95 degrees C. At these temperatures, omega-gliadin solubility did not change, but the solubilities of alpha- and gamma-gliadin in 60% ethanol decreased according to first-order reaction kinetics. All reaction rate constants increased with temperature. The activation energies for the heat-induced exchange reaction were 110 and 147 kJ/mol for alpha- and gamma-gliadin, respectively. Starch did not influence the reaction rates of the association of alpha- and gamma-gliadin with glutenin. During gluten-starch model bread baking, glutenin oxidized first, and when the internal crumb temperature reached 100 degrees C, alpha- and gamma-gliadin cross-linked to glutenin, again following first-order reaction kinetics. The experimental findings and similarities in temperature conditions and reaction kinetics suggest that the RVA system can be instrumental in understanding gluten behavior in concentrated food systems, such as bread making.     

Use of fungal phytase to improve breadmaking performance of whole wheat bread.

The possible use of phytase as a breadmaking improver has been tested in whole wheat breads by adding different amounts of fungal phytase. The effect of phytase addition on the fermentation stage and the final bread quality was analyzed. The phytase addition shortened the fermentation period, without affecting the bread dough pH. Regarding the whole wheat bread, a considerable increase of the specific bread volume, an improvement of the crumb texture, and the width/height ratio of the bread slice were obtained. An in vitro assay revealed that the improving effect of phytase on breadmaking might be associated with the activation of alpha-amylase, due to the release of calcium ions from calcium-phytate complexes promoted by phytase activity. As a conclusion, phytase offers excellent possibilities as a breadmaking improver, with two main advantages: first, the nutritional improvement produced by decreasing phytate content, and second, all the benefits produced by alpha-amylase addition can be obtained by adding phytase, which promotes the activation of endogenous alpha-amylase.     

Dough and Baking Properties of High‐Amylose and Waxy Wheat Flours

The dough properties and baking qualities of a novel high‐amylose wheat flour (HAWF) and a waxy wheat flour (WWF) (both Triticum aestivum L.) were investigated by comparing them with common wheat flours. HAWF and WWF had more dietary fiber than Chinese Spring flour (CSF), a nonwaxy wheat flour. Also, HAWF contained larger amounts of lipids and proteins than WWF and CSF. There were significant differences in the amylose and amylopectin contents among all samples tested. Farinograph data showed water absorptions of HAWF and WWF were significantly higher than that of CSF, and both flours showed poorer flour qualities than CSF. The dough of WWF was weaker and less stable than that of CSF, whereas HAWF produced a harder and more viscous dough than CSF. Differential scanning calorimetry data showed that starch in HAWF dough gelatinized at a lower temperature in the baking process than the starches in doughs of WWF and CSF. The starch in a WWF suspension had a larger enthalpy of gelatinization than those in HAWF and CSF suspensions. Amylograph data showed that the WWF starch gelatinized faster and had a higher viscosity than that in CSF. The loaves made from WWF and CSF were significantly larger than the loaves made from HAWF. However, the appearance of bread baked with WWF and HAWF was inferior to the appearance of bread baked with CSF. Bread made with WWF became softer than the bread made with CSF after storage, and reheating was more effective in refreshing WWF bread than CSF bread. Moreover, clear differences in dough and bread samples were revealed by scanning electron microscopy. These differences might have some effect on dough and baking qualities.     

Model approach to starch functionality in bread making

We used modified wheat starches in gluten-starch flour models to study the role of starch in bread making. Incorporation of hydroxypropylated starch in the recipe reduced loaf volume and initial crumb firmness and increased crumb gas cell size. Firming rate and firmness after storage increased for loaves containing the least hydroxypropylated starch. Inclusion of cross-linked starch had little effect on loaf volume or crumb structure but increased crumb firmness. The firming rate was mostly similar to that of control samples. Presumably, the moment and extent of starch gelatinization and the concomitant water migration influence the structure formation during baking. Initial bread firmness seems determined by the rigidity of the gelatinized granules and leached amylose. Amylopectin retrogradation and strengthening of a long-range network by intensifying the inter- and intramolecular starch-starch and possibly also starch-gluten interactions (presumably because of water incorporation in retrograded amylopectin crystallites) play an important role in firming.