Heat-induced polymerization reaction of wheat gluten proteins

Gluten is the heterogeneous mixture of the wheat storage proteins, consisting of monomeric gliadin and polymeric glutenin. Its heat-induced network formation is essential for many wheat-based applications. The crumb structure of bread for instance, and the swelling behavior and cooking losses of pasta, are greatly affected by the gluten networks. Furthermore, wheat gluten can serve as raw material for e.g. packaging materials. A complete understanding of the gluten cross-linking mechanism is of great value for controlling end-use quality of wheat- or gluten-based products. Gluten network formation is mainly ascribed to reducible disulfide cross-links, but the occurrence of non-reducible cross-links has also been suggested. The main objective of this doctoral dissertation was to investigate the potential importance of non-disulfide cross-links for heat-induced network formation of wheat gluten. The required method optimization was carried out and heat-induced gluten cross-linking was evaluated in both model and real systems.In a first part, amino acid analysis was optimized for wheat gluten. The procedure consists of hydrolysis of the peptide bonds in 6.0 M HCl at 110 °C for 24 h, followed by separation and quantification of the free amino acids by high-performance anion-exchange chromatography with integrated pulsed amperometric detection. In contrast to most conventional methods, this protocol requires neither pre- or post-column derivatization, nor a time-consuming oxidation step prior to hydrolysis. Correction factors were determined to account for incomplete release of gluten-bound valine and ileucine after hydrolysis for 24 h. Furthermore, it was illustrated that levels of all amino acids but serine remain constant during evaporation (110 °C, 3 h) of the hydrolysis mixtures. Gradient conditions used for chromatographic separation were modified to allow multiple sequential sample analyses without risk of column blockage as a result of high glutamine levels in gluten proteins. This accurate and user-friendly method determines, in addition to amino acids, cross-links derived from dehydro amino acids, e.g. lysinoalanine and lanthionine, and was of great value to study cross-linking reactions in gluten proteins. In a second part, gluten, or fractions thereof, were heated under different time, temperature, pH, and moisture content conditions and network formation was evaluated based on extractability in sodium dodecyl sulfate containing buffer, and levels of precursors, intermediates and end products of cross-linking reactions. Heat/alkali treatment (pH 8.0, 110-130 °C, 0-120 min) of gluten induced beta-elimination of SS, which released thiol groups and dehydroalanine, and further led to (i) disulfide cross-links by thiol-disulfide interchange and thiol oxidation, and (ii) the dehydroalanine derived cross-links lanthionine and lysinoalanine. In general, longer heating time, higher temperature, and more alkaline pH resulted in greater extractability loss. However, prolonged heating (e.g. pH 8.0, 130 °C, >15 min) led to protein degradation. Heating gluten at low moisture contents induced the formation of non-reducible cross-links without involvement of disulfide or thiol. Further research suggested the formation of isopeptide cross-links. Such cross-links were then identified using mass spectometry in heated gluten model peptides. In a third part, lanthionine and -to a lesser extent- lysinoalanine levels were found to increase during the production of hard pretzels. These wheat-based snacks thank their unique taste and texture to a dip in a hot alkaline solution prior to baking to gelatinize the starch. In conclusion, although disulfide cross-links play a key role during heat-induced network formation of gluten, cross-links derived from dehydro amino acids contribute to the gluten network during heating at alkaline pH, and isopeptide cross-linking occurs at high temperatures and low moisture contents. Non-disulfide cross-links may well be relevant for several wheat-based applications, as demonstrated here for pretzels.nrpages: 125status: publishe

Study of nonenzymic browning in alpha-amino acid and gamma-aminobutyric acid/sugar model systems

The reactivities, in nonenzymic browning, of gamma-aminobutyric acid (GABA), a non-protein amino acid with wide natural occurrence and potential health benefits as it occurs in foods, and of the alpha-L-amino acids arginine, glutamic acid, glutamine, leucine, lysine, and phenylalanine were investigated by heating equimolar mixtures of glucose and the cited amino acids at 110 degrees C at pH 6.0 for different times (0-4 h). Linear regression analysis indicated that the colour development in a GABA/glucose mixture was slower than that of a lysine/glucose mixture and comparable to that of a phenylalanine/glucose mixture. High-performance anion-exchange chromatography (HPAEC) with integrated pulsed amperometric detection (IPAD) showed that the decrease in GABA levels (ca. 10% after heating for 4 h) as a function of heating time was smaller than that of glucose (ca. 30% after heating for 4 h). At the same time, glucose to fructose isomerisation took place. After 20 min of heating at pH 6.0, all mixtures showed a fructose peak, the area of which increased with heating time. However, after correcting for fructose isomerisation, glucose losses were still higher than amino acid losses. In contrast to its precursor glutamic acid, GABA was stable during heating of a solution containing it alone. Heating of GABA-containing D-sugar solutions (xylose, fructose, glucose, maltose and sucrose) showed that the relative order of colour yield was pentose > hexose > disaccharides. As well as glucose to fructose isomerisation, HPAEC-IPAD allowed monitoring of the different isomerisation reactions occurring, and also disaccharide hydrolysis in the different GABA/sugar mixtures. (c) 2008 Elsevier Ltd. All rights reserved.status: publishe

Denaturation and covalent network formation of wheat gluten, globular proteins and mixtures thereof in aqueous ethanol and water

Food processing often includes heating and/or exposure to solvents as unit operations. Here, the impact of heating at 100 °C in water or aqueous ethanol [10 or 50% (v/v)] on denaturation and covalent network formation of three model proteins [bovine serum albumin (BSA), soy glycinin and wheat gliadin] was examined. Already at room temperature 50% (v/v) ethanol induced disulfide cross-linking between BSA proteins. Increased ethanol concentrations reduced heat-induced polymerization of soy glycinin and wheat gliadin. The use of aqueous ethanol limited the extent of β-elimination, sulfhydryl-disulfide exchange reactions and sulfhydryl oxidation. Gliadin and soy glycinin had higher colloidal stability in 50% (v/v) ethanol than in water. The conformation of BSA and soy glycinin already changed at lower temperatures in 50% (v/v) ethanol than in water. In all media, different proteins influenced each other’s denaturation and/or polymerization. During heating in water but not in 50% (v/v) ethanol, gliadin-BSA and gliadin-soy glycinin mixtures polymerized more than expected than the isolated proteins. Thus, phase-separation of proteins did not limit intermolecular disulfide formation. Pretreatment of proteins with aqueous ethanol did not substantially influence their subsequent polymerization during prolonged heating in water. However, ethanol pretreatment of gluten impacted heat-induced polymerization of BSA in gluten-BSA mixtures.status: publishe

Impact of extraction and elution media on separation of proteins by size exclusion high performance liquid chromatography

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Heat-Induced Cross-Linking and Degradation of Wheat Gluten, Serum Albumin, and Mixtures Thereof

Some wheat-based food systems, such as cakes, cookies, and egg noodles, contain mixtures of animal and plant (gluten) proteins and are processed under (mildly) alkaline conditions. Although changes in these proteins during processing can affect end product quality, they have seldom been studied. This study investigated protein cross-linking and degradation during heating (0-120 min, pH 8.0, 50-130 °C) of (mixtures of) wheat gluten and bovine serum albumin (BSA). The decrease in protein extractabilities in sodium dodecyl sulfate containing buffer under (non)reducing conditions and the levels of (cross-linked) amino acids were measured. No indications for polymerization at 50 °C were found. Below 100 °C, BSA polymerized more readily than wheat gluten. Above 100 °C, the opposite was observed. The kinetics of heat-induced polymerization of a 1:1 gluten-BSA mixture were similar to that of isolated gluten, implying that gluten decelerated BSA denaturation. Severe heating (130 °C, >15 min) induced degradation reactions in gluten but not in BSA. At all conditions used in this study, disulfide (SS) bonds contributed to the extractability loss. In addition, above 110 °C, β-elimination of cystine led to non-SS cross-links. Intramolecular SS bonds more often transformed in intermolecular non-SS bonds in BSA than in gluten.status: publishe

The impact of protein characteristics on the protein network in and properties of fresh and cooked wheat-based noodles

The relation between protein characteristics, protein network formation and wheat noodle properties was studied. Bovine serum albumin (BSA), soy glycinin, ovalbumin, S-ovalbumin and lysozyme were included in the recipe of wheat-based noodles. The characteristics of these non-wheat proteins impacted the type, rate and extent of protein network formation during noodle dough preparation and cooking, and thereby the properties of fresh and cooked noodles. None of the added proteins enhanced the properties of fresh noodles. BSA and soy glycinin enhanced Kieffer-rig extensibility parameters of cooked noodles. Addition of ovalbumin or S-ovalbumin led to excessive protein polymerization in cooked noodles and lowered their quality. Inclusion of lysozyme lowered the rate and extent of polymerization during cooking. Experiments in which urea, olive oil or urea were added in the recipe showed that non-covalent interactions dominate the properties of fresh noodles while covalent cross-links and hydrogen bonds mainly impact the properties of cooked noodles.status: publishe

Identification of intact high molecular weight glutenin subunits from the wheat proteome using combined liquid chromatography-electrospray ionization mass spectrometry.

The present paper describes a method for the identification of intact high molecular weight glutenin subunits (HMW-GS), the quality determining proteins from the wheat storage proteome. The method includes isolation of HMW-GS from wheat flour, further separation of HMW-GS by reversed-phase high-performance liquid chromatography (RP-HPLC), and their subsequent molecular identification with electrospray ionization mass spectrometry using a quadrupole-time-of-flight mass analyzer. For HMW-GS isolation, wheat proteins were reduced and extracted from flour with 50% 1-propanol containing 1% dithiothreitol. HMW-GS were then selectively precipitated from the protein mixture by adjusting the 1-propanol concentration to 60%. The composition of the precipitated proteins was first evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie staining and RP-HPLC with ultraviolet detection. Besides HMW-GS (≥65%), the isolated proteins mainly contained ω5-gliadins. Secondly, the isolated protein fraction was analyzed by liquid chromatography-mass spectrometry. Optimal chromatographic separation of HMW-GS from the other proteins in the isolated fraction was obtained when the mobile phase contained 0.1% trifluoroacetic acid as ion-pairing agent. Individual HMW-GS were then identified by determining their molecular masses from the high-resolution mass spectra and comparing these with theoretical masses calculated from amino acid sequences. Using formic acid instead of trifluoroacetic acid in the mobile phase increased protein peak intensities in the base peak mass chromatogram. This allowed the detection of even traces of other wheat proteins than HMW-GS in the isolated fraction, but the chromatographic separation was inferior with a major overlap between the elution ranges of HMW-GS and ω-gliadins. Overall, the described method allows a rapid assessment of wheat quality through the direct determination of the HMW-GS composition and offers a basis for further top-down proteomics of individual HMW-GS and the entire wheat glutenin fraction

Globular proteins influence protein network formation in and quality of wheat-based noodles

To address the demand for healthy protein-rich food, nutritious protein sources (e.g. egg, soy or milk) are sometimes included in wheat noodle recipes. Interactions and reactions between different types of proteins can impact complex food systems and are referred to as co-protein effects. They can be synergistic or antagonistic. The relation between protein characteristics, protein network formation and wheat noodle properties was studied. Globular model protein such as bovine serum albumin (BSA), soy glycinin, hen egg ovalbumin, S-ovalbumin and lysozyme were included in the recipe of wheat-based noodles. The characteristics of these non-wheat proteins impacted the type, rate and extent of protein network formation during noodle dough preparation and boiling, and thereby the properties of fresh (i.e. raw) and boiled noodles. None of the added proteins enhanced the properties of fresh noodles. BSA and soy glycinin enhanced Kieffer-rig extensibility parameters of cooked noodles. Addition of ovalbumin or S-ovalbumin led to excessive protein polymerization in cooked noodles and lowered their extensibility and cooking quality. Inclusion of lysozyme lowered the rate and extent of polymerization during boiling. Experiments in which salt, olive oil or urea were added in the recipe showed that non-covalent interactions dominate the properties of fresh noodles while covalent cross-links and hydrogen bonds mainly impact the properties of boiled noodles. In addition, synergistic or antagonistic co-protein effects of binary proteins mixtures in model systems were linked to the rate/extent of polymerization, optimal cooking time, cooking quality and Kieffer-rig extensibility of wheat-based noodles. Proteins from different sources cannot only enhance the nutritional value but also the quality of wheat-based noodles.status: publishe

Identification of lanthionine and lysinoalanine in heat-treated wheat gliadin and bovine serum albumin using tandem mass spectrometry with higher-energy collisional dissociation

The present manuscript reports on the identification of various dehydroamino acid-derived bonds and cross-links resulting from thermal treatment (excess water, 240 min, 130 °C) of two model food proteins, bovine serum albumin, and wheat gliadin. S-Carbamidomethylated tryptic and chymotryptic digests of unheated (control) and heated serum albumin and gliadin, respectively, were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC–ESI–MS/MS) with higher-energy collisional dissociation (HCD). Heat-induced β-elimination of cystine, serine and threonine, and subsequent Michael addition of cysteine and lysine to dehydroalanine and 3-methyl-dehydroalanine were demonstrated. Lanthionine, lysinoalanine, 3-methyl-lanthionine, and 3-methyl-lysinoalanine were identified. The detection of inter-chain lanthionine in both bovine serum albumin and wheat gliadin suggests the significance of these cross-links for food texture.status: publishe

Impact of casein and egg white proteins on the structure of wheat gluten-based protein-rich food

BACKGROUND: There is a growing interest in texturally and nutritionally satisfying vegetable alternatives for meat. Wheat gluten proteins have unique functional properties but a poor nutritional value in comparison to animal proteins. This study investigated the potential of egg white and bovine milk casein with well-balanced amino acid composition to increase the quality of wheat gluten-based protein-rich foods. RESULTS: Heating a wheat gluten (51.4 g) - water (100.0 ml) blend for 120 minutes at 100 °C increased its firmness less than heating a wheat gluten (33.0 g) - freeze dried egg white (16.8 g) - water (100.0 ml) blend. In contrast, the addition of casein to the gluten-water blend negatively impacted firmness after heating. Firmness was correlated to loss of protein extractability in sodium dodecyl sulfate containing medium during heating, which was higher with egg white than with casein. Even more, heat-induced polymerization of the gluten-water blend with egg white but not with casein was larger than expected from the losses in extractability of gluten and egg white on their own. CONCLUSION: Structure formation was favored by mixing gluten with egg white but not with casein. These observations were linked to the intrinsic polymerization behavior of egg white and casein, but also to their interaction with gluten. Thus, not all nutritionally suitable proteins can be used for enrichment of gluten-based protein-rich foods.status: publishe